OF MICE, MEN AND MEMORIES: THE ROLE OF THE RODENT HIPPOCAMPUS IN OBJECT RECOGNITION. Sarah J. Cohen. A Dissertation Submitted to the Faculty of

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1 OF MICE, MEN AND MEMORIES: THE ROLE OF THE RODENT HIPPOCAMPUS IN OBJECT RECOGNITION by Sarah J. Cohen A Dissertation Submitted to the Faculty of The Charles E. Schmidt College of Science In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Florida Atlantic University Boca on, FL May 2016

2 Copyright 2016 by Sarah J. Cohen ii

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4 ACKNOWLEDGEMENTS I would first of all like to thank my advisor, Dr. Robert W. Stackman for giving me invaluable guidance towards designing and collecting experimental data and to completing this dissertation. Additionally, I would like to thank my committee members, Dr. Robert W. Stackman, Dr. Elan Barenholtz, Dr. Janet Blanks, Dr. Kathleen Guthrie, Dr. Larry Squire, and Dr. Robert Vertes for their invaluable input in the preparation of this document. I would like to express my sincere thanks and love to my husband, daughter, mother, Sharon and brothers for their unwavering encouragement. Also, I would like to thank my colleagues in Dr. Stackman s lab for their assistance and support through the good times and the bad. I would like to make special mention of Mrs. Alcira H. Munchow, whose tireless efforts and encouragement made this document possible. Lastly, I would like to thank the National Institutes of Health for the support provided by grant NIH: R01MHO86591 and FAU for a Dissertation Year Award. iv

5 ABSTRACT Author: Title: Institution: Sarah J. Cohen Of Mice, Men and Memories: The Role of the Rodent Hippocampus in Object Recognition Florida Atlantic University Dissertation Advisor: Dr. Robert W. Stackman, Jr. Degree: Doctor of Philosophy Year: 2016 Establishing appropriate animal models for the study of human memory is paramount to the development of memory disorder treatments. Damage to the hippocampus, a medial temporal lobe brain structure, has been implicated in the memory loss associated with Alzheimer s disease and other dementias. In humans, the role of the hippocampus is largely defined; yet, its role in rodents is much less clear due to conflicting findings. To investigate these discrepancies, an extensive review of the rodent literature was conducted, with a focus on studies that used the Novel Object Recognition (NOR) paradigm for testing. The total amount of time the objects were explored during training and the delay imposed between training and testing seemed to determine hippocampal recruitment in rodents. Male C57BL/6J mice were implanted with bilateral dorsal CA1 guide cannulae to allow for the inactivation of the hippocampus at discrete time points in the task. The results suggest that the rodent hippocampus is crucial to the encoding, consolidation and retrieval of object memory. Next, it was determined that v

6 there is a delay-dependent involvement of the hippocampus in object memory, implying that other structures may be supporting the memory prior to the recruitment of hippocampus. In addition, when the context memory and object memory could be further dissociated, by altering the task design, the results imply a necessary role for the hippocampus in the object memory, irrespective of context. Also, making the task more perceptually demanding, by requiring the mice to perform a two-dimensional to threedimensional association between stimuli, engaged the hippocampus. Then, in the traditional NOR task, long and short training exploration times were imposed to determine brain region activity for weak and strong object memory. The inactivation and immunohistochemistry findings imply weak object memory is perirhinal cortex dependent, while strong object memory is hippocampal-dependent. Taken together, the findings suggest that mice, like humans, process object memory on a continuum from weak to strong, recruiting the hippocampus conditionally for strong familiarity. Confirming this functional similarity between the rodent and human object memory systems could be beneficial for future studies investigating memory disorders. vi

7 DEDICATION This manuscript is dedicated to my family, particularly my daughter, Ella, and my extremely patient and understanding husband, Aaron, who have supported me through my many years of research and writing. I also dedicate this work to my mother, who believes in the pursuit of my dreams.

8 OF MICE, MEN AND MEMORIES: THE ROLE OF THE RODENT HIPPOCAMPUS IN OBJECT RECOGNITION LIST OF TABLES...xiv LIST OF FIGURES... xv PART I: INTRODUCTION-THE HIPPOCAMPUS AND LONG-TERM MEMORY Hippocampal Neuroanatomy Long-Term Potentiation (LTP) Memory Processes and Systems Multiple Memory Systems Implicit/Nondeclarative Memory Explicit/Declarative Memory Biological Basis for Episodic Memory Spatial Memory Non-spatial Memory Recollection vs. Familiarity Immediate Early Gene Expression as a Marker for Memory Animal Use in the Study of Memory Conclusions and Dissertation Organization PART II: ASSESSING RODENT HIPPOCAMPAL INVOLVEMENT IN THE NOVEL OBJECT RECOGNITION TASK. A REVIEW viii

9 2.1 Abstract Introduction Novel Object Recognition Task Procedures and Behavior Quantification Advantages of NOR Task Variability in Sample Session Exploration Criteria Protocol Modifications Effects of Permanent vs. Temporary Lesion of the Rodent Hippocampus Effects by Size of Permanent Lesion Effects of Temporary Pharmacological Inactivation of the Hippocampus Variability in Intersession Delay and Sample Session Object Exploration Criteria A Model of the Contributions of Perirhinal Cortex and Hippocampus to NOR Alternative Explanations for Differences in Results and New Method Implementations Conclusions PART III: THE RODENT HIPPOCAMPUS IS ESSENTIAL FOR NON- SPATIAL OBJECT MEMORY Abstract Introduction Materials and Methods Mice and Surgery ix

10 3.3.2 Novel Object Recognition Task Intrahippocampal Cannulation and Microinfusion Histology Data Analysis Results Exp t 1. Hippocampus is required for object memory encoding and consolidation Exp t 2-4. Hippocampus is required for object memory consolidation Exp t 5. Hippocampal inactivation during all memory stages impairs NOR performance Exp t 6. Inactivating hippocampus blocks retrieval of a strong object memory PART IV: DISSOCIATING OBJECT-IN-CONTEXT MEMORY: THE RODENT HIPPOCAMPUS PROCESSES SPATIAL AND OBJECT MEMORY SEPARATELY Abstract Introduction Materials and Methods Mice and Surgery Intrahippocampal Cannulation and Microinfusion Novel Object Recognition Task and Protocols Data Anaylsis Histology x

11 4.4 Results Discussion PART V: EVERY PICTURE TELLS A STORY: EVIDENCE FOR PICTURE- OBJECT EQUIVALENCE IN MICE Abstract Introduction Materials and Methods Mice and surgery Intrahippocampal cannulation and microinfusion Novel object recognition task and protocols Data analysis Histology Results Experiments 1-4: Recognition of a 3D Object from a 2D Picture is Hippocampal Dependent Regardless of Symmetry, and is Not Affected by Low-Level Visual Properties or Viewing Angle Experiments 5-6: Picture/Object Recognition is Limited to Composite Image Memory Retrieval Experiment 7: Evidence of Picture-Object Equivalence - Item Recognition of a Learned Silhouette Discussion PART VI: OBJECT RECOGNITION MEMORY: DISTINCT YET COMPLEMENTARY ROLES OF THE MOUSE HIPPOCAMPUS AND PERIRHINAL CORTEX xi

12 6.1 Abstract Introduction Materials and Method Mice Intrahippocampal cannulation and microinfusion Novel object recognition task and protocols for inactivation studies Data analysis Histology Immunohistochemistry Cell Counting Immunostaining Data Analysis Results Inactivation Findings Strong Object Memory-Hippocampal Inactivation Strong Object Memory-Perirhinal Cortex Inactivation Weak Object Memory-Hippocampal Inactivation Weak Object Memory-Perirhinal Cortex Inactivation Immunohistochemical Findings Test Session Object Discrimination Strong Object Memory Weak Object Memory Additional Analyses Discussion xii

13 PART VII: GENERAL DISCUSSION Lesion Size Determines Hippocampal Involvement State Dependency and Inactivation Techniques NOR is a Spatial Task Strong and Weak Memories Reflect Recollection and Familiarity Direct Connections to the Hippocampus from Perirhinal Cortex Conclusions PART VIII: APPENDICES PART IX: REFERENCES xiii

14 TABLES Table 1. Index of the experimental details and results from a subset of published NOR studies testing the involvement of the rodent hippocampus and which a sample session exploration criterion was imposed Table 2. Extensive review of published object recognition studies with pertinent details of task design and results xiv

15 FIGURES Figure 1. Location and shape of hippocampus Figure 2. Hippocampal information network Figure 3. Morris Water Maze Figure 4. The novel object recognition (NOR) task Figure 5. Publication rate of permanent or temporary hippocampal experiments in which a sample session exploration criterion was imposed Figure 6. Qualitative model depicting how the perirhinal cortex and hippocampus contribute to object recognition memory Figure 7. Encoding, consolidation and retrieval of object memory by C57BL/6J mice requires hippocampus Figure 8. Hippocampal inactivation impairs the retrieval of a strong object memory Figure 9. Schematic of protocols and histological verifications Figure 10. Discrimination ratios for sample session and test session for the varying protocols Figure 11. Protocols used to test picture-object equivalence in mice Figure Supplemental protocols used to test picture-object equivalence in mice xv

16 Figure Exp. S1 - Consolidation of picture recognition memory is dependent upon the dorsal hippocampus Figure 12. Successful object discrimination regardless of symmetry, likeness or viewing angle of the sample picture, is impaired when the dorsal hippocampus is inactivated post-training Figure Exp. S2 - Replication of Exp. 1 with an extended test session duration Figure Exp. S3 - Extending the duration of the traditional object recognition test session (3D stimuli for sample and test sessions) significantly increases discrimination between objects Figure Exp. S4A and S4B Access to tactile information supersedes novelty preference when the novel stimulus is a 2D picture and the 3D object is familiar Figure Exp. S5 - Replication of Exp. 4 with an extended test session duration Figure Exp. S6 - Discrimination of an individual object presented in both 2D and 3D forms Figure 13. Mice rely on composite images for subsequent object recognition Figure 14. Accurate object discrimination after viewing the familiar object as a 2D silhouette Figure 15. The novel object recognition (NOR) task Figure 16. Representative photomicrographs of infusion sites and immunostained tissue sections for hippocampus and perirhinal cortex xvi

17 Figure 17. Strong and weak object discrimination for intact and inactivated hippocampal and perirhinal cortex regions Figure 18. Object discrimination and protein expression for mice in the strong and weak immunohistochemistry protocols Figure 19. Summary of the direct and indirect anatomical connections between perirhinal cortex and hippocampus xvii

18 PART I: INTRODUCTION-THE HIPPOCAMPUS AND LONG-TERM MEMORY 1.1 Hippocampal Neuroanatomy. The brain is the primary organ of the body that defines who we are, the actions we make and the feelings we have. It is able to process external stimuli and record experiences that can later be used for a variety of purposes. It is these memories that allow us to interact with the world in a meaningful way. The hippocampus, a small structure located in the medial temporal lobe of the brain (for location in human brain see Figure 1), was originally believed to be involved in olfaction (Green, 1964). However it has since been attributed to the orchestration of memory processes needed to form, store and retrieve vital information about all of our experiences. The hippocampal formation is unique in that the information has a unidirectional flow throughout a number of distinct subregions. More specifically, cortical information will arrive in the hippocampus from highly associative regions of the perirhinal and parahippocampal cortices through the entorhinal cortex (see Figure 2, (Petrantonakis and Poirazi, 2014)). Once arriving in the hippocampus, the information will flow through the tri-synaptic circuit composed of the dentate gyrus, CA3, and CA1. Specifically, information flows from the entorhinal cortex to the dentate gyrus, then from there to CA3 and then to CA1. However, there are also direct connections from axons of layer 2 of the entorhinal cortex that terminate directly in CA3 through the perforant path, and layer 3 1

19 axons that directly connect to CA1 through the TA-CA1 pathway (Anderson et al., 2006). The cells of CA1 project to the subiculum and then the axons loop back to the entorhinal cortex (Witter et al., 2000). Pyramidal cells are the principal neurons of the CA regions, and the pyramidal cells of CA3 send their axons to CA1. CA1 cells function as the primary output for the structure to the entorhinal cortex and subiculum, differentiating them from CA3 neurons which have extensive intrinsic excitatory connections. The formation of long-term declarative memories within the hippocampus is thought to be dependent on the flow of multisensory information though this highly developed structure. Two types of information are routed from distinct sub-regions of the entorhinal cortex through the hippocampus. The lateral entorhinal cortex transmits what information, and the medial entorhinal cortex conveys where information, which the hippocampus can then associate. The lateral entorhinal cortex receives inputs from the olfactory, visual and auditory systems which take in the non-spatial information about an experience, while the medial entorhinal cortex will process the spatial aspects of that experience (Anderson et al., 2006). The structures and pathways discussed are the key regions responsible for processing declarative or episodic memories. Subsequently, it has been strongly suggested that the hippocampus does not act as one unified structure. her, the hippocampus functions as two different and independent structures with the dorsal/septal region and ventral/temporal region responsible for different capacities (Moser and Moser, 1998). This theory is based on evidence stating that there are distinct input and output connections of the dorsal and ventral regions (Swanson and Cowan, 1977), ventral regions alter emotional behaviors (Henke, 1990), and spatial memories 2

20 rely on the dorsal region (Moser et al., 1995). Several behavioral studies corroborate this theory clearly stating that the dorsal hippocampus is primarily concerned with navigational learning and exploration while the ventral hippocampus is involved in stress and emotional behaviors (Fanselow and Dong, 2010). Spatial memory and navigation are key functions of the hippocampus which will be discussed later. It is important to note that there are also sub-cortical innervations of the hippocampus. Although theta rhythm has been strongly suggested in processes of arousal (Green and Arduini, 1954), it is also thought to play a significant role in mnemonic processes of the hippocampus (Vertes and Kocsis, 1997; Burgess et al., 2002; Vertes et al., 2004), and more specifically, mechanisms of learning and memory (Hasselmo, 2005). Animal behavioral studies report that temporary or permanent s to the medial septum, eliminating theta in the hippocampus, leads to impaired spatial memory (Mizumori et al., 1990a; Mizumori et al., 1990b; Vertes et al., 2004; Buzsaki, 2005). These findings imply that theta, arising from the medial septal region, may enhance hippocampal inputs to allow for information encoding (Vertes et al., 1986). Additionally, it has been reported that theta stimulation enhances hippocampal LTP (Staubli and Lynch, 1987; Kocsis and Vertes, 1997; Hyman et al., 2003). Clearly, there is strong evidence to suggest that theta promotes hippocampal-dependent memory processing through modulations in the information transmitted through the entorhinal cortex. The large pyramidal neurons and small interneurons present within the hippocampal formation resemble other cortical regions of the brain. However, as previously stated, what sets the hippocampus apart is that the information tends to flow through it in only one direction. Additionally, the hippocampus is highly organized to 3

21 process multi-sensory stimuli from many different cortical regions. The integration of this information is unique to the hippocampus (Anderson et al., 2006). Damage to the hippocampus leads to severe impairments in declarative memory; a topic that will be further discussed in subsequent sections. The rodent hippocampus is organized in a similar fashion; however, it resembles an elongated banana shape as opposed to a seahorse shape seen in humans. Rodent studies have been conducted to better understand navigation, neural mechanisms of memory and long-term potentiation (LTP). For the purposes on this thesis, the focus of the discussion will concentrate on the rodent hippocampus Long-Term Potentiation (LTP). Behavior is altered through learning. It is thought that these changes in behavior are a result of alterations in the nervous system. If these changes persist, the result is a learned memory. First described over 70 years ago, molecular activity changes were identified to be the foundations for learning and subsequent memory (Hebb, 1949); however, it wasn t until several years later that long-lasting forms of synaptic plasticity were identified as LTP (for review, see Bliss and Collingridge, 1993). Since then, many comparisons have been made between memory and LTP. LTP, refers to long-lasting increases in signal transmission due to heightened patterns of recent synaptic activity. Synaptic plasticity is thought to occur through changes in LTP which subsequently allow for memories to be formed. Memory may be supported by three main properties of LTP. Cooperativity describes LTP induction through many pathways converging on the same postsynaptic membrane with low frequency stimulation (McNaughton et al., 1978). Associativity 4

22 refers to the induction of LTP in a neuron due to the coupling of strong and weak stimulation that leads to the weak stimulation strengthening (Barrionuevo and Brown, 1983). Lastly, input selectivity dictates that LTP at one synapse will propagate other synapses specifically as dictated by cooperativity and associativity (Dunwiddie and Lynch, 1978). It is through these laws of LTP that the same neurons can be activated in concert repeatedly to activate memory retrieval. These claims have been tested in several studies that record cells within the hippocampus during active behavioral tasks. Although beyond the scope of this document, it is important to mention that there are other candidates for the molecular mechanisms supporting learning and memory aside from LTP. However, LTP remains as the most likely contender supporting memory in the mammalian brain based on significant physiological evidence. 1.2 Memory Processes and Systems. There are many processes that are involved in the formation, storage and retrieval of memories. Encoding refers to the initial acquisition of a memory, which is then subsequently stored for later recall, through a process of consolidation. Retrieval of these stored memories occurs when consolidated memories are brought into conscious recollection. Finally, reconsolidation refers to the alteration of a previously consolidated memory after its retrieval. The specific brain locations that these processes occur have been debated for different memory types. One explanation for the seeming discrepancies within the literature is that multiple structures many work collectively to support different stages of memory processing. Regardless, it is accepted that all long-term memories are formed, stored and recalled in a similar manner through processes of encoding, consolidation, and retrieval. 5

23 The medial temporal lobe is the region of the brain necessary for some types of learning and memory (Zola-Morgan et al., 1986; Bertolucci et al., 2004). Numerous studies have been conducted in humans suffering from extensive seizure activity within the brain and the effects that excising the hippocampus has on such conditions. A consequence of this surgical procedure is memory loss. It is therefore understood that episodic memory is housed in the medial temporal lobe. Also, it has been determined that the amnestic effects following hippocampal damage is due to the fact that this structure is crucial for memory formation, storage and maintenance. Much of the initial evidence for the role of the medial temporal lobe in memory came from studies of patient, HM. HM, or Henry Gustav Molaison, had suffered severe epileptic episodes due to a childhood accident that left him with brain damage. He was incapable of functioning as a normal adult and to try and alleviate some of the symptoms, a neurosurgeon removed both the left and right medial temporal lobes. Luckily, the seizures were better controlled post-surgery; however, a significant consequence of surgical intervention was profound memory loss. In studying HM s brain functioning, specific functions were localized to discrete brain regions (Squire, 2009). Memories governed by the hippocampus fall into two categories, anterograde (formation of a new memory) and retrograde (memory of the past). Both of these memory types were impaired in patient HM. Additionally, much of HM s declarative memory (memories for events and facts) was impaired, while his procedural memory (recollection of how to perform a task) was unaffected (Squire, 2009). This remarkable dissociation between declarative and procedural memory types found in patients with medial temporal lobe trauma has driven memory research. In turn, animal models have 6

24 been required to mimic this type of damage in better understanding the multiple memory systems of the brain. 1.3 Multiple Memory Systems. As stated above, the growing desire to study the human memory system arose from observations of patients with medial temporal lobe damage. Given that those with medial temporal lobe damage displayed some intact and some impaired memory processes, it was determined that multiple memory systems must exist for various memory types. The Multiple Memory Systems Theory, developed by Squire (1992), states that many areas of the brain contain highly developed circuitry to maintain differing memory types. This concept is supported by studies of HM who lost declarative memory, while his non-declarative memory remained intact after surgery (Squire and McKee, 1993). Much of the work that assesses long-term memory focuses on the hippocampus and its relationship to other structures and other memory systems. Therefore, extensive work has been performed to characterize the functioning of the hippocampus relative to other memory systems. Since it is clear that information is processed in a parallel manner (McDonald et al., 2004), the different memory systems are able to generally adapt based on the demand for processing specific information types. From these notions, the theory of Multiple Parallel Memory Systems (White and McDonald, 2002) was proposed to describe how multiple memory systems can interact in parallel, and how the hippocampus can interact with systems outside the hippocampus. There are two types of long-term memory: implicit/non-declarative and explicit/declarative. Implicit memories are unconscious memories like those for skills and procedures. On the other hand, declarative memories are able to be consciously recalled 7

25 from when they were learned, like facts and events. Both of these memory classifications will be discussed in further detail below Implicit/Nondeclarative Memory. Implicit memory is defined as memories for skills that are learned and then can guide behaviors without the need for conscious recall. Procedural memory is one such memory type. For example, the ability to tie a shoe lace is not consciously recalled but is still the knowledge needed to perform the task. Like procedural memory, described above, these memories are stored outside of the medial temporal lobe. Procedural memories are therefore not vulnerable to medial temporal lobe ablations, and this is the reason HM was unaffected in this area. Numerous studies performed in non-human primates and other animals have demonstrated that these non-declarative memories are more cortically dependent (Squire, 1992). Although these memories are not consciously recollected, they are expressed through performance and action. Priming is a technique commonly used to demonstrate that implicit memory does exist (Schacter, 1987). In experimental setting, subjects can be subconsciously prepared for a test and then demonstrate that knowledge on the test without having consciously studied for it. In such situations, the effects of priming are longer-lasting and have a greater impact on subsequent choice behaviors. Patients with amnesias due to temporal lobe damage tend to perform just as well as healthy patients in perceptual priming experimentation (Schacter, 1987). Another aspect of implicit memory is the Illusion of Truth effect (Begg et al., 1992). Subjects were told many statements, both true and false and were asked to rate them in terms of their believability. Statements that subjects heard multiple times tended 8

26 to be rated much more believable than those only stated once, even if they were untrue. Even when subjects were told in the beginning that specific statements were false, if they were repeated multiple times in the trial, they tended to be rated as true (Begg et al., 1992) Explicit/Declarative Memory. As previously stated, declarative memories (explicit memories) are long-term memories that are consciously recalled and can be described through verbal language. For example, recalling what you had for dinner last night, or the animals that live at the zoo. Often times these memories are associated with numerous associated memories that make them very specific and detailed. These are the memories that rely upon the medial temporal lobe, and more specifically, the hippocampus (Squire, 1992). The content and depth in which these memories are processed is what sets them apart from implicit memories and priming. There are two types of declarative memories, semantic (consciously recalled memories independent of a specific episode) and episodic (memories that involve the original context that they were experienced). Semantic memory refers to textbook types of learning, or the general knowledge we have about the world. It is believed that these memories are processed throughout the medial temporal lobe, as well as the hippocampus. Although the view of distributed semantic knowledge states that some semantic information about sound is stored in the auditory cortex, and visual features in the visual cortex, etc (Small et al., 1995). Declarative memories generally range from strong to weak, but are not really susceptible to decay over time. Episodic memories are thought to rely on the hippocampus and frontal lobes to a large extent. These memories are for events including the time and place that it occurred and 9

27 the specific environment (Squire, 1992; Eichenbaum, 2000; Eichenbaum and Cohen, 2014). These autobiographical memories provide us with a record of all of our personal experiences and can range from weak to strong, as with semantic memories. These longterm, declarative memories, which are dependent upon the medial temporal lobe circuitry described above, have been studied in great detail since such forms of memory are compromised with age and in many forms of dementia. Spatial and non-spatial memories, discussed in detail later, are classified under declarative episodic memories Biological Basis for Episodic Memory. Although episodic memories are attributed to the hippocampus, it remains unclear as to how the information is represented within its neural populations. Human-based studies are limited in scope forcing the field to look toward laboratory animal models in understanding various forms of memory (Tulving, 2002; Suddendorf and Busby, 2003). 1.4 Spatial Memory. Spatial memory is a form of episodic memory that supports the ability to associate a specific event with a context where it occurred. Information that can be gleamed from interactions with the environment is necessary for processes of complex adaptation. In humans, spatial memory allows for the performance of such tasks as driving and navigating. Context is important on the process of learning and memory and therefore investigators began looking at its influence on learning performance (Balsam, 1985). However, even earlier, Tulving and Thomson (1973) believed that it was the context within the memory that allows for the memory to be subsequently recalled. Others have agreed and it is understood that context allows for accurate memory retrieval. It has also been stated that there are different representations of spatial information. The cognitive 10

28 map theory, proposed by O Keefe and Nadel (1978), states that there are both egocentric and allocentric representations of reference frames. Egocentric representations refer to the spatial information obtained based on viewpoint. So it is based on the perception of the individual within the space. On the other hand, allocentric representations are viewpoint independent and refer to the spatial information gleaned from the relationship between stimuli and the individual. Therefore, allocentric representations of the spatial information are independent of the individual. Nadel and Hardt (2004) assert that it is through both of these representational processes of spatial information that individual organisms are able to navigate in various environments. The contribution of the human and rodent hippocampus to spatial learning and memory has been well characterized. Classically, rodents have been tested in the Morris Water Maze to better understand the neural correlates of spatial memory (Morris et al., 1982). The water maze task involves training rodents to locate a hidden platform in a specific location in a circular opaque colored pool (see Figure 3). Rodents learn to adequately navigate to the platform using external cues located outside the pool. During subsequent platform-less probe tests, the well-trained rodent will search in the precise location of the pool where the platform was found during training. Using this task, it was found that accurate navigation by rodents is dependent on an intact hippocampus. However, if the hippocampus is compromised, rodents are incapable of completing the task (Eichenbaum et al., 1990). Numerous studies have reported that the hippocampus is required for the encoding, consolidation and retrieval of these spatial memories. Most of the prior work demonstrating hippocampal involvement in spatial memory is based on traditional studies. While useful in producing a rodent model 11

29 of human medial temporal lobe amnesia, this technique is not necessarily best suited for determining the function of specific regions of the medial temporal lobe memory circuit. Due to compensatory mechanisms, it is possible that removing a structure and looking at the animal s ability to perform a task may not be the most appropriate method to investigate structure-function relationships. Reorganization of neural circuitry after a permanent could restore functions that are normally mediated by that region. Additionally, it is impossible to learn about the specific stages of memory processing when a structure is permanently removed. Therefore, local transient inactivation techniques have risen in popularity. When the hippocampus was inactivated before the probe test in the water maze, rodents searched the wrong location of the pool (Riedel et al., 1999). These findings indicate that a functioning hippocampus is needed for the retrieval of spatial memory without affecting search strategy, which must be governed by other brain regions outside the hippocampus. Place cells, discovered by O Keefe and Dostrovsky (1971), are thought to assist organisms in navigation through spatial environments. These cells are located in the hippocampus and are thought to be influenced by grid cells located in the medial entorhinal cortex (Fyhn et al., 2007) and head direction cells of the anterior thalamus (Yoganarasimha and Knierim, 2005). The term, place cell refers to hippocampal pyramidal cells that discharge at peak firing frequency whenever the animal occupies a specific location within an environment. The specified zone within the environment associated with this high firing pattern is referred to as the cell s place field. A given place cell will only fire when the animal is in that zone or field and will be silent when the animal occupies all other locations. Generally, the cell will fire at the same frequency 12

30 each time the organism enters that field (O'Keefe and Dostrovsky, 1971). Extracellular recording of these cells has allowed for deeper understanding of these cells and an animal s ability to navigate within its spatial environment. In experimental settings, arrays of tetrodes can be surgically implanted into the hippocampus of an animal, allowing for the simultaneous recording of multiple individual neurons in the freely moving animal. As previously stated, these place cells rely on the inputs of grid cells and head direction cells. Grid cells fire in a similar fashion to place cells; however, they fire in many locations resembling a hexagonal grid throughout the environment. In new spatial environments, the grid cells will fire in response to the new geometrical dimensions of the context which then signals the place cells to remap (Moser et al., 2008). Head direction cells fire in response to the animal s angular head orientation in the horizontal plane and are completely independent of spatial location. However, head direction cells have been shown to influence the remapping of place cells when an animal was placed in a familiar environment after having been disoriented (Knierim et al., 1995). Taken together, the information acquired by place, grid and head direction cells assist in the ability to process and perceive a spatial environment, which subsequently allows for directional navigation within that environment. Therefore, once a map for an environment is created, and the animal can use it to maintain its orientation, attention can be allocated to novel items encountered within the environment. 1.5 Non-spatial Memory. One type of non-spatial memory is object recognition memory, which is the focus of this thesis. This type of declarative memory is what allows us to recognize previously 13

31 viewed items. Localization of this type of memory is largely debated, as there is a sizeable discrepancy within the literature. Several reports state that the perirhinal cortex is responsible for object recognition memory (Bussey et al., 1999; Bussey et al., 2005; Wixted, 2007; Winters et al., 2008; Watson et al., 2012), while others argue a significant role for the hippocampus (Clark et al., 2000; Hammond et al., 2004; Broadbent et al., 2010). The following chapters will address these, and other, conflicting reports in greater detail. Since the hippocampus and perirhinal cortex are strongly connected, parsing apart their individual contributions to object memory remains difficult. In order to use rodents as an appropriate model for the human memory system, it must be confirmed that the rodent hippocampus governs both spatial and non-spatial aspects of declarative memory. The Novel Object Recognition Task (NOR) is classically used in assessing non-spatial memory in rodents (Ennaceur and Delacour, 1988). The specific aspects of this test will be further discussed in the following chapters but they typically involve object exploration sessions in familiar arenas. Animals are able to freely explore the objects, and based on the time they spend exploring, assessments of memory formation and retention can be made. This task is only possible because of the natural proclivity rodents have for exploring novel stimuli over familiar ones. Chapter 2 will go into detail about the previous research that has been performed using this task and the neural correlates that govern it. 1.6 Recollection vs. Familiarity. Declarative memory, and more specifically recognition memory, can be divided into two respective parts, recollection and familiarity. Tulving (1999) describes this subdivision as the declarative memory distinction between conscious recall and the sense 14

32 of familiarity. As humans, we can describe declarative memories as knowing them or remembering them (Rugg and Yonelinas, 2003). It is this terminology, so common to human language, which guides choice behaviors that are dependent on a previously encoded memory. For example, if a test subject is asked if he recognizes a photograph of the Eiffel Tower, a positive response could be due to a feeling of familiarity, or based on a memory of having been to Paris previously. The latter judgment option is indicative of recognition of the stimulus along with the contextual details for the event. Generally, these responses are given with more confidence than those that are based on familiarity (Dewhurst and Hitch, 1999). However, the contributions of both recollection and familiarity to declarative memory can be understood through ROC (receiver operating characteristics) curves (Yonelinas, 1994). Based on the number of correct and incorrect responses plotted against the confidence of those responses, varying graph shapes appear. Asymmetric graphs for healthy humans have led to the development of the dual processes model of memory retrieval that states that declarative memory retrieval is made up of both recollection and familiarity (Yonelinas, 2001). It has been suggested that recollection is impaired in amnesic patients, while familiarity remains largely intact. Based on these studies, recollection and familiarity are believed to be separate and distinct memory processes, referred to as the dual process recognition memory model. Additionally, these data also imply that familiarity is closely linked to implicit memory, since both are spared in patients with memory loss (Rugg and Yonelinas, 2003). However, if there is extensive brain damage to the entire medial temporal lobe, even familiarity is impaired (Wixted and Stretch, 2004). It has been argued that recollection is hippocampal-dependent, while familiarity is reliant on cortical 15

33 regions within the medial temporal lobe, but outside of the hippocampus, like the perirhinal cortex. Conversely, the single process model of recognition memory states that recollection and familiarity are not distinct processes, but are remember/know judgments based on a gradient from strong to weak (Donaldson, 1996; Wixted, 2007; Wixted and Mickes, 2010). Therefore, the distinction between these processes is not the neural correlates that control them, but is the strength of the memory (Ingram et al., 2012). In this manner, recollection and familiarity can be viewed as a continuous process in which memories are graded from weak (familiarity) to strong (recollection). Many of the previous studies investigating the contributions of different anatomical structures involve temporary or permanent s to regions of interest. However, several newer techniques have evolved to investigate brain activity during a specific task. Differential expression of immediate early genes, or IEGs, reflect relative activation of a given brain region. Measuring IEG expression may be a good marker to investigate the neural structures involved in familiarity and recollection. 1.7 Immediate Early Gene Expression as a Marker for Memory. The synthesis of mrna (Igaz et al., 2002; Parsons et al., 2006) and subsequent protein production (Flexner et al., 1963; Dudai, 2004; Sutton and Schuman, 2006; Costa- Mattioli and Sonenberg, 2008) has been demonstrated to be essential to the formation and maintenance of long-term memory (Kang and Schuman, 1996; Igaz et al., 2002). Immediate early genes, or IEGs, are the key factors ensuring that memories are formed and stabilized. IEGs are the first genes to be transcribed when a neuron becomes active. Additionally, if protein translation of IEGs is inhibited, long-term memory processes are 16

34 also inhibited (Parsons et al., 2006; Katche et al., 2010). In order to investigate cells and brain areas active at a specific time point, immunohistological staining techniques have been designed. These methods have been utilized throughout the brain and notably in the hippocampus during object discrimination testing (Aggleton and Brown, 2005; Albasser et al., 2010b). Classically, studies have examined the IEG c-fos in determining what neurons are activated during a specific task, and the IEG Arc for better understanding of behaviorally-triggered synaptic plasticity. Mice engineered with genetic deletion of the gene Arc failed to retain long-term memories (Plath et al., 2006). Additionally, mice treated with local microinfusion of antisense oligonucleotides to inhibit the expression of Arc protein fail to maintain LTP, or to consolidate long-term memories (Guzowski et al., 2000). These results are not surprising since it is Arc proteins that are likely formed in the neurons that assist in the maintenance of a memory, and effectively creating strong connections with other neurons to stabilize the memory (Guzowski et al., 2000; Guzowski, 2002). Therefore, IEG protein analyses are effective in not only determining what areas of the brain are active during a specific task but also which neurons are undergoing plasticity for long term memory maintenance. 1.8 Animal Use in the Study of Memory. Human memory is traditionally studied in patients who have survived disease and trauma and exhibit various impairments in functioning. The most notable case is that of H.M. who suffered from severe epilepsy. In an effort to alleviate these symptoms, his bilateral medial temporal lobes were resected. By studying the effects of this surgery on H.M. s mental faculties, immeasurable understanding of how memory is processed within the brain has been gained. Regardless, numerous questions regarding the 17

35 anatomical pathways and cellular mechanisms involved in memory remain unanswered. Since invasive experimentation is deemed unethical, alternative methods have been developed to help answer these questions. Studies involving non-human primates and other animals have allowed for the furthering of our knowledge of how memory is processed in healthy brains and the consequences of these functions when the brain is damaged in specific regions. Specific animal species are chosen who have anatomical and behavioral similarities to that of humans. Rodents have become a widely-used model for the study of human memory on a cellular through behavioral level. Although rodents are frequently used, their appropriateness as a model of human memory has been largely debated. Further support for the similarities between the memory systems in rodents and humans are essential in order to continue using these animals as a representative model for human memory processes. 1.9 Conclusions and Dissertation Organization. The main focus of this dissertation is to demonstrate the important role that the mouse hippocampus plays in object recognition memory. The Novel Object Recognition Task (NOR) has been developed into a standard learning and memory protocol for exploring various facets of object memory irrespective of context. The experiments that are outlined in the following chapters investigate the involvement of the hippocampus in object memory and the differential roles that it and the perirhinal cortex play in this memory type. All of the experimental findings provide strong support for the idea that the hippocampus is essential for strong object memory formation and retrieval. To elucidate further, Chapter 2 presents an extensive review of the published studies that have tested the contribution of the rodent hippocampus to the NOR task. A 18

36 combination of and temporary inactivation studies performed on both mice and rats reveal a compelling role for the hippocampus in this task under specific circumstances. Chapter 3 presents data that detail the specific involvement of the dorsal hippocampus in the NOR task in the distinct stages of object memory, as operationally defined by the NOR task sessions. Clearly, the results demonstrate that the hippocampus has a significant role in the encoding, consolidation, and retrieval of object memory in the object recognition memory task. Chapter 4 describes in more detail the role of the hippocampus, in NOR, when context and object information is further dissociated. By developing variations on the classic task, context and object memories were dissociated to reveal the role the hippocampus plays in the object memory alone. Chapter 5 presents data describing the cognitive abilities of mice using a variation on the NOR task that correlates 2-dimensional (2D) and 3-dimensional (3D) representations of objects. Surprisingly, the results reveal that mice are able to perceive that 2D pictures of objects are representative of their 3D forms. Chapter 6 presents behavioral and immunohistological evidence for the differential roles of the hippocampus and perirhinal cortex in object memory. Lastly, Chapter 7 concludes by drawing conclusions about all of the experiments together within the current state of hippocampal memory research, and implications for the future of this work. Clearly the role of the hippocampus in nonspatial object memory is contentious; however, this thesis provides convincing evidence, using a variety of techniques and tasks, that the rodent hippocampus is essential for strong object memory formation, maintenance and retrieval. 19

37 PART II: ASSESSING RODENT HIPPOCAMPAL INVOLVEMENT IN THE NOVEL OBJECT RECOGNITION TASK. A REVIEW Abstract. The novel object recognition (NOR) task has emerged as a popular method for testing the neurobiology of nonspatial memory in rodents. This task exploits the natural tendency of rodents to explore novel items and depending on the amount of time that rodents spend exploring the presented objects, inferences about memory can be established. Despite its wide use, the underlying neural circuitry and mechanisms supporting NOR have not been clearly defined. In particular, considerable debate has focused on whether the hippocampus plays a significant role in the object memory that is encoded, consolidated and then retrieved during discrete stages of the NOR task. Here we analyzed the results of all published reports in which the role of the rodent hippocampus in object memory was inferred from performance in the task with restricted parameters. We note that the remarkable variability in NOR methods across studies complicates the ability to draw meaningful conclusions from the work. Focusing on twelve reports in which a minimum criterion of sample session object exploration was imposed, we find that temporary or permanent of the hippocampus consistently disrupts object memory when a delay of 10 min or greater is imposed between the sample and test 1 This chapter was originally published as a Research Report in Behavioural Brain Research: Sarah J. Cohen & Robert W. Stackman, Jr., May 15, 2015, Assessing rodent hippocampal involvement in the novel object recognition task. A review, Behavioural Brain Research, 285, Reproduced with permission from Elsevier Inc. 20

38 sessions. We discuss the significance of a delay-dependent role of the hippocampus in NOR within the framework of the medial temporal lobe. We assert that standardization of the NOR protocol is essential for obtaining reliable data that can then be compared across studies to build consensus as to the specific contribution of the rodent hippocampus to object memory. 2.2 Introduction. We can all recall a time when walking down a crowded corridor, we happen upon a person who looks familiar. While we are confident that we have encountered this person before, we are unable to remember how or when we previously met. It is only through the information gathered during interactive conversation that we are able to recall who this person is and where we encountered them for the first time. This uncomfortable, yet common, scenario depicts our ability to subjectively recall previous information through distinct memory processes. Memory can be divided into two distinct categories, declarative and nondeclarative forms. Declarative memory, or explicit memory, is the ability to recall personal history, facts and events, and is dependent on the interconnected structures of the medial temporal lobe. Recognition, a subtype of declarative memory, reflects that of people, objects, and experiences. Clearly, the example stated above illustrates the two forms of recognition memory that are commonly experienced during a test of information retrieval, that is, familiarity and recollection. Familiarity is the immediate feeling that an event, individual, or item was previously encountered. This experience, referred to as knowing, does not involve the conscious recollection of details from the prior experience. For example, I know I have seen that person (or item) before; I just don t 21

39 remember where or when. Recollection, or remembering on the other hand, involves a slower process whereby full attention to the present stimuli (if any) induces an intended or conscious recall of the contextual details of the prior event or experience that is, specific information as to where and when the original experience occurred (Yonelinas and Levy, 2002; Eichenbaum et al., 2007). For example, I remember you. We met at the 2012 Society for Neuroscience meeting; our posters were next to one another on the second day of that conference, and you commented on how well I coordinated my outfit with the color scheme of my poster. Originally defined by Tulving (Tulving, 1985), the remember/know distinction is considered by many to reflect separate underlying behavioral processes of recognition memory. Although the processes of recollection and familiarity are distinct in the manner that they are experienced, it remains unclear whether different neurobiological mechanisms support them. Dual-process models of recognition memory state that recollection and familiarity are functionally separate systems (Yonelinas, 2002; Norman and O'Reilly, 2003; Rugg and Yonelinas, 2003; Yonelinas et al., 2005; Curran et al., 2006). Studies of human amnesiacs have revealed selective impairment of recollection, while sparing familiarity, and numerous functional imaging studies have identified that the separate processes are associated with regionspecific activation patterns. These findings are largely considered support for the view that familiarity and recollection utilize different underlying systems (Aggleton and Brown, 1999; Squire et al., 2007; Hannula and Ranganath, 2009). On the other hand, single-process models view the two declarative memory forms as a part of one distinct category of recognition memory (Yonelinas, 2002; Squire et al., 2007; Wixted, 2007). Here, memories are represented along a scale that ranges from weak to strong. Studies 22

40 have demonstrated that these two processes have a significant structural commonality that would point to a single process model. Similar structural activation is observed with both familiarity and recollection (Hannula and Ranganath, 2009). Regardless of how these forms of memory are thought to function, the fundamental concepts derived from the distinction between familiarity and recollection are useful for improving understanding of recognition memory mechanisms in both humans and laboratory animals. The medial temporal lobe is organized in a manner that supports memory. Various sub-regions have been identified as the structures critical in supporting memory in a variety of species (Burwell, 2000). The perirhinal cortex, parahippocampal cortex, and entorhinal cortex are anatomical structures identified as components of the what and where streams of experience-dependent sensory inputs that converge within the hippocampus. Traditionally, it is believed that the "what" information is conveyed through the perirhinal cortex, while the "where" information is transmitted through the parahippocampal and entorhinal cortices. It is only in the hippocampus that the "what" and "where" information is associated (Eichenbaum et al., 2007). However, in recent years, debate over whether the hippocampus is directly involved in encoding memories of the "what" information has increased. Similarly, many studies claim that familiarity is structurally distinct from that of recollection, with familiarity attributed to the perirhinal cortex and recollection to the hippocampus (Ranganath et al., 2004). Nevertheless, it is apparent that during recollection, it is the "what" and "where" associations that are being recovered. 23

41 In general, memories are formed and stabilized through three distinct processes. Encoding refers to the initial acquisition of the memory. Then, through phases of consolidation, the memory is preserved and stored for later recall. Finally, retrieval is the process by which the previously stored memories are reactivated. Many different tasks have been developed to investigate the neural basis of memory and its distinct stages. However, it is important to note that all methodologies have limitations, which should be considered when analyzing outcomes. Human recognition memory is commonly tested in the visual paired comparisons task (Fagan, 1970; see review Burbacher and Grant, 2012), while a modified version of the task has been implemented for rodents (Ennaceur, 2010). Functional imaging studies, in humans, have identified patterns of region-specific neural activation associated with recollection and familiarity; however, animal models enable investigation of the neurobiological circuitry and cellular mechanisms of recognition memory, which are not possible in humans. 2.3 Novel Object Recognition Task Procedures and Behavior Quantification. Implicit to the animal model approach is the necessity that the behavioral constructs that are modeled in rodents match to a large extent, human recognition memory. To this end, the spontaneous novel object recognition (NOR) task has emerged as the most popular test for assessing a rodent s ability to recognize a previously presented stimulus (Ennaceur and Delacour, 1988). Describing the task as such is misleading since it is not theoretically possible to recognize a novel object since recognition reflects prior exposure. While some have begun to adopt the more accurate phrase, spontaneous object recognition (SOR), most investigators continue to use novel 24

42 object recognition or NOR in referring to the task. For the purposes of this review, we will refer to the aforementioned task as NOR; however, we assert that this designation does not adequately describe the object recognition memory that can be inferred from it. Regardless, the NOR task has become the hallmark method used in assessing non-spatial object memory in rodents. Although there is considerable variability across labs in the NOR procedures used, most conduct the test in a familiar square or rectangular highwalled arena lacking polarizing spatial cues (see schematic in Figure 4 for a depiction of the most commonly applied variation of the NOR task). In an effort to further reduce contextual and spatial information, a Y-maze arena has been used in several influential studies (Bussey et al., 2000a; Winters et al., 2004; Forwood et al., 2005; Winters et al., 2008). Although this novel design reduces contextual information, reports using square or rectangular arenas limit spatial cues by minimizing all visual, textural, and odor stimuli. During what is referred to as the training or sample session, the rodent explores two identical novel objects encountered in a familiar arena. Object memory encoding is operationally defined as occurring during the sample session. Upon completion of the sample session the animal is removed from the arena for some specified amount of time (i.e., retention delay), during which the object memory is consolidated. For the subsequent test session, the rodent is returned to the same arena, which now contains an exact replica of the familiar object and a novel object, as a test of object memory retrieval. Rodents are self-motivated to spontaneously approach items and explore using multiple senses. Object exploration behavior is easily quantifiable and allows for the study of episodic-like memories in rodents. Rodents exhibit a natural proclivity to explore novel, non-threatening objects, and therefore, during the test session rodents 25

43 exhibit a preference for exploring the novel object significantly more than the familiar one. Thus, sample object memory strength is inferred from the preference of the rodent to explore the novel object over the familiar object during the test session. Object memory is quantified by computing discrimination measures from scores of the amount of time during the test session that each animal explores the respective objects. Preference for the novel object, demonstrated by an increase in exploration time for that item, indicates that a memory trace for the familiar object was properly encoded, consolidated and then retrieved to guide the rodent s behavior during the test session (Hammond et al., 2004; Aggleton et al., 2010; Broadbent et al., 2010; Burke et al., 2010; Oliveira et al., 2010; Cohen et al., 2013b). There are two quantitative measures that are commonly used in assessing test session exploration performance. The novel object preference ratio is determined by dividing the total object exploration time from the exploration time of the novel object. A value above 0.5 suggests preference for the novel object, while a value below 0.5 is indicative of familiar object preference. Chance performance is represented by a preference ratio of 0.5 (Hammond et al., 2004). Alternatively, the discrimination ratio is calculated by determining the difference in exploration time between the novel and familiar objects and dividing it by the total object exploration time. Discrimination ratio ranges from-1 to +1, with negative scores indicating preference for the familiar object and positive scores signifying preference for the novel object. Chance performance, or null preference, is represented by a score of 0 (Aggleton et al., 2010; Burke et al., 2010; Oliveira et al., 2010; Cohen et al., 2013b). By factoring in total object exploration time, discrimination ratio has emerged as the preferred measure of object memory in studies of NOR. Regardless of the measurement strategy employed, 26

44 deciphering object memory is dependent on the rodent exploring each presented object, in both the sample and test sessions, for at least a minimal amount of time Advantages of NOR Task. The NOR task offers advantages over other object memory tests for assessing recognition memory because it does not require any external motivation, reward or punishment (Nemanic et al., 2004), such as that required by the delayed non-match to sample task, in which a reward is administered when the subject correctly chooses a novel item over one that is familiar. Additionally, while rodents tend to require extensive training to accurately perform the delayed non-match to sample task (Eacott and Norman, 2004), no training other than arena habituation is necessary to elicit object exploratory behavior in the NOR task. Finally, the NOR task procedures do not generate stressful conditions, while offering a robust test of nonspatial memory in rodents (Bowman et al., 2003; Maras et al., 2014) Variability in Sample Session Exploration Criteria. During the sample session, the rodent is placed into the familiar arena that typically contains two identical novel objects, which the animal is then permitted to freely explore. The duration of the sample session tends to vary considerably across published papers (from 2-15 min). Many studies have implemented a procedure in which the sample session is terminated once the rodent accumulates a specific criterion amount of object exploration. Rodents that do not meet this sample session criterion are excluded from further analyses. Although there is considerable variability with respect to this criterion (at least s of total sample object exploration), it has been a common practice to allot mice a longer exploration criterion over rat counterparts. On average, 27

45 mice are required to explore the sample objects for about 38 s while rats are generally allotted around 30 s of total object exploration (see Table 1). However, this may be a factor that contributes to differences in experimental outcomes, given that the amount of time spent exploring an object may be directly proportional to the strength and detail of the memory formed. Imposing a criterion on sample object exploration is advantageous because all of the subjects, regardless of time in the arena, are matched for the amount of object exploration or degree of object training experienced. In fact, we have previously reported preliminary data demonstrating that the amount of object exploration during the sample session directly relates to the recruitment of different brain structures (Cohen et al., 2013a); these results will be discussed in a later section. Therefore, it is extremely important that each experimental parameter, like exploration criteria, be explicitly considered when designing a specific task, as varying results are bound to occur. For those studies in which a fixed length sample session was imposed without the requirement of a sample object exploration criterion, one can only assume that the subjects had sufficiently explored the objects. Nevertheless, during the sample session of the NOR task, the animal is expected to devote equal time to exploring each of the presented objects since they should all be novel. During the test session, one of the familiar objects, from training, is replaced with a novel item. The presented familiar item is typically an exact replica of those explored during the sample session. However, in some cases the test session familiar object is one of the previously explored objects chosen at random for test session exploration. It is expected that rodents will preferentially explore the novel object, if the memory of the familiar object was successfully encoded, consolidated and then retrieved. Failure to exhibit novel object 28

46 preference during the test session would be expected if object exploration during the sample session was minimal; a memory that was never encoded cannot be retrieved. Furthermore, weak novel object preference during the test session is widely considered to be indicative of impaired object memory; however, it could also reflect insufficient exploration of sample objects, which would go unnoticed if a sample session object exploration criterion was not required. Alternatively, the weak preference for the novel object can be interpreted as the retrieval of a weak memory for the sample objects. Although without sufficient information about the amount of object exploration during the sample session, test session performance is difficult to assess. Therefore, we contend that imposing a sample session object exploration criterion is essential for properly interpreting test session behavior. Given that object exploration is spontaneous, and variable from experiment to experiment and across labs, we suggest that it is important to determine whether the observed mean discrimination ratio or preference ratio score of each respective group of rodents differs significantly from chance. For example, it is expected that untreated, or sham ed rodents, should demonstrate significant novel object preference, during the test session, for the reasons described above. This analysis provides a good determinant of a failure of memory retrieval vs. retrieval of a weak memory for the sample objects. Moreover, in interpreting an observed lack of significant difference in discrimination ratios between sham and hippocampal ed rodents, one should consider whether performance of both groups is above chance (i.e., by comparing each group's discrimination ratio scores to chance). Our review of the literature reveals that this additional analysis of performance compared to chance is not always pursued, and 29

47 thereby one's view of a particular result might be limited. For example, one might report a significant difference in mean discrimination ratio between two groups of rats (e.g., sham and of region A) and interpret the result as the leading to a failure of object recognition memory. However, if subsequent analyses against chance were to reveal that the mean discrimination ratios of both groups were significantly above chance, then one would have to have to consider that the of region A merely attenuated object recognition memory, or that another region (e.g., region B) may also contribute to task performance. As this scenario suggests, analysis of test session performance against chance can provide a more thorough appreciation of a manipulation's influence on object memory in the NOR task Protocol Modifications. To date, there have been numerous reports in which permanent or temporary manipulations are made to discrete brain structures in order to define the exact neural substrates of object memory. The results of these studies have been largely inconclusive, and one goal of the present review is to determine similarities and differences within the studies to bring about some clarity to the role of the rodent hippocampus in object memory. Although the NOR task has been widely applied to the neurobiological study of memory, and in particular, to the analysis of the neural circuitry that subserves object memory, there appears to be little consistency in task procedures. There are vast differences in the characteristics of the objects used, the type of arena, technique, size, degree of specificity, duration of the delay imposed and sample session training criterion (i.e., whether one was imposed or not, and if so, then the specific 30

48 criterion also varies). In addition, the effects of rodent hippocampal s on object recognition memory could be influenced by the species used. Typically mice are allotted a longer sample session exploration time (Hammond et al., 2004; Cohen et al., 2013b) over their rat counterparts; however, this difference in required object exploration could contribute to a stronger vs. weaker memory formed. The significant number of variations across NOR protocols represents a considerable challenge to any effort to compare and contrast studies of object memory based on NOR performance. Clearly, any change in the protocol, whether minor or major, can influence the results. This problem was potently demonstrated in studies in which an identical set of behaviors were assessed in several strains of inbred mice simultaneously in three distinct geographic locations, with all other experimental features (i.e., experimenters, arenas, objects, exploration parameters and mouse strains) held consistent. Inconclusive findings are evidence that even when standardizations to the protocols are attempted, slight differences are still possible, which can lead to even greater alterations in data outcomes and interpretations (Crabbe et al., 1999). Similarly, changes to experimental protocols that lead to differences in result outcome have been demonstrated with other behavioral tasks. It was found that monkeys with hippocampal s show a more profound impairment in a visual pairedcomparisons task over the delayed nonmatching-to-sample test (Zeamer et al., 2011). This finding was attributed to the fact that the encoding time used for the former task was longer than that of the latter. However, it was later discovered that in addition to the encoding times being different, the object stimuli also varied in terms of shape, color, size, brightness and texture. In turn, the reported findings may be confounded by the differences in procedure, leading to inconclusive data (Zeamer et al., 2011). Regardless 31

49 of the behavioral task employed, experimental parameters require consistency to elicit reliable and definitive findings. The NOR test may have significant advantages; however, it is the uniqueness and modifications of each experimental design, from lab to lab that make it hard to evaluate the growing amount of data. Therefore, this review will stress the need for consistency among NOR protocol parameters to ensure accurate findings. We are not asserting that there is one correct method in which NOR should be performed, we are primarily attempting to demonstrate that the vast contradictory findings in the literature may be merely due to variations in NOR task parameters employed. Specifically, we will highlight two specific parameters that when varied seem to elicit different structural involvement in the rodent brain. 2.4 Effects of Permanent vs. Temporary Lesion of the Rodent Hippocampus Effects by Size of Permanent Lesion. The NOR task has been applied extensively to decipher the differential contributions of rodent medial temporal lobe structures to object recognition memory. Permanent s have been traditionally used to investigate the role of a brain structure in a given behavior, or to establish brain region-behavioral relationships. The functions impaired, or the differences in behavior observed between sham and ed rodents, are then interpreted as those dependent upon the damaged or absent region. To take this position is to assume that all other brain regions are intact and functioning normally in the absence of the damaged region - even those regions deafferented from the ed structure. However, as others have argued in the past, this followed by test approach is best suited for studying the degree of compensation the CNS can achieve after the, or simply, what the brain-damaged rodent can learn and remember. The 32

50 large majority of published studies have compared the NOR performance of rodents after sham or permanent brain s. The present review focuses solely on the effects of permanent or temporary s of the hippocampus on object memory in the NOR task. The permanent studies tend to follow a common plan in which the anterograde effects of hippocampal s are examined on object memory. Specifically, rodents receive a partial or complete of the hippocampus and after a recovery period of ~14 days, are habituated to the testing arena. During the subsequent sample and test sessions, hippocampal-ed rodents generally exhibit object exploration behavior consistent with that of sham-ed rodents. However, the majority of studies (72% of experiments in multi-study papers) find that novel object preference is spared in the hippocampal-ed rodents during the test session, while the remaining 28% of experiments report impaired novel object preference during the test session (see Table 2). It has been suggested that this discrepancy in outcome depends upon the relative completeness of the hippocampal. Further, a few studies have examined retrograde effects of hippocampal s on object memory, by presenting the sample session, and then following some interval, s are made to the hippocampus of a subset of the rodents. Differences in test session performance between the sham and ed groups are interpreted as evidence that consolidation or retrieval of object memory is or is not dependent upon the hippocampus. Of the six studies that have examined retrograde effects of hippocampal s on NOR, two find that the hippocampal impair retrieval of object memory encoded before the. Such results provide support for the view that the hippocampus participates in the encoding and consolidation of object memory; the post-training likely disrupts ongoing 33

51 memory consolidation processes or interferes with the object memory retrieval. In one case (Gaskin et al., 2003), rats were found to exhibit impaired novel object preference when the hippocampus was ed post-training, an effect consistent with the view that object memory depends upon the hippocampus. However, these rats were later presented with a sample session of new objects and exhibited intact object memory during the subsequent test session. These results are consistent with our contention that object memory normally depends upon the hippocampus (Cohen et al., 2013b), and Gaskin et al. (2003) interpreted their observed lack of effects on anterograde object memory as evidence that when the hippocampus is not available, then other extrahippocampal regions compensate for the lost structure and support the ability of the animals to discriminate the objects. We completely agree with this interpretation. However, we contest that the -induced compensatory role for extrahippocampal structures to support object memory likely also explains the spared object memory performance of hippocampal-ed rodents observed in so many experiments (see Table 2). It is unclear why such an interpretation is largely ignored our analyses of the permanent of rodent hippocampus/nor literature found remarkably little attention given to the possibility that spared hippocampal tissue or -induced plasticity or covert pathology could influence the behavior expressed by rodents with permanent hippocampal s. Aggleton and colleagues have provided some of the most compelling evidence that permanent s of the rat hippocampus causes a marked decrease in immediate early gene expression in the retrosplenial cortex (Jenkins et al., 2006; Albasser et al., 2007; Aggleton, 2008). Such pathology is considered 'covert' since the authors found no detectable cell loss in the retrosplenial cortex. Similar covert 34

52 pathology in retrosplenial cortex has also been reported after s of the anterior thalamic nuclei (Jenkins et al., 2002; Jenkins et al., 2004). Thus, it is imperative that interpretations of behaviors spared or impaired in rodents after permanent hippocampal s account for possible covert or overt pathology. The notion that neural circuitry undergoes reorganization after peripheral or central damage has been fundamental to the explanation for partial or complete recovery of motor and cognitive functions after stroke or traumatic brain injury (Cramer, 2008; Kokotilo et al., 2009; Yamashita and Abe, 2012), and reorganization of the human sensory and motor cortices is a well accepted consequence of practiced use (May, 2011; Thomas and Baker, 2013). The phenomenon of phantom limb pain is considered to be one of the most compelling (and accepted) models of deafferentation-induced plasticity or reorganization of the somatotopic body surface map in human primary somatosensory cortex (Pons et al., 1991; Flor et al., 1995; Ramachandran and Hirstein, 1998; Karl et al., 2001). In each of these conditions, experience-dependent reorganization of neural circuitry enables recovery of function or new abilities. One could apply this notion to the spared object memory observed in hippocampal ed rodents. That is, spared object memory reflects the emerging significant participation of extrahippocampal regions, as the 'phantom hippocampus'. The availability of excitotoxins (Jarrard, 2002) and higher resolution stereotaxic manipulators (Coffey et al., 2013) have greatly improved the specificity and accuracy of the permanent technique. However, this approach is not appropriate for testing specific time-dependent processes, and the differential role of given brain structures in distinct stages of memory processing. Therefore, understanding the anatomical basis of 35

53 the discrete steps in memory encoding, consolidation and retrieval cannot be sufficiently addressed with a permanent. Further, as discussed above, potential compensatory mechanisms can govern the restructuring of the memory circuit in the post- animal. Newly developed temporary pharmacological and genetic inactivation techniques allow for more precise investigation of distinct stages of memory and the brain structures involved Effects of Temporary Pharmacological Inactivation of the Hippocampus. There has been a steady increase in the application of neuropharmacological and genetic tools, which do not impose permanent structural changes in hippocampal circuitry, to investigate influences on distinct object memory processes (see Figure 5). The NOR task permits a clear operational definition of memory encoding, consolidation and retrieval due to its typical sample, then delay, then test sequence of a given trial. For example, administration of a drug, with a short onset of effect, before the sample session would enable one to examine effects of that treatment on the encoding of object memory; administration immediately following the sample session would test the treatment s effect on consolidation of object memory; and, administration immediately before the test session would test the treatment s effect on the retrieval of object memory. In addition, unintentional effects of experimental manipulations on task performance can be distinguished from those on learning and memory. For example, if a given treatment increases anxiety as a side effect, thigmotaxis and other mobility impairing or enhancing responses can be quantified; if the treatment affects attention or motivation one can assess the time required for the subjects to acquire some criterion amount of object 36

54 exploration (although this measure is not always taken into consideration and could affect the interpretation of findings, as previously stated). Clearly, when determining the treatment or technique that will be employed in a study, it is important to consider a variety of analytical measures to ensure that the treatment or method is not causing any unintended effects that can alter the interpretation of the data. Many studies have utilized the permanent technique to test the role of the rodent hippocampus in NOR, and most report that non-spatial object memory is spared. Such results are then interpreted as evidence that object memory is independent of the rodent hippocampus. However, the previously stated drawbacks of the permanent method are likely to have contributed to these findings (e.g., -induced compensation). Therefore, it is possible that this strategy of permanently silencing a brain region is not adequate for addressing the underlying function of a structure, but rather, it is beneficial for studying the structures needed to support memory when the structures that normally support it are missing. On the other hand, temporary pharmacological inactivation techniques have emerged as an alternative method that may be better suited for understanding the roles of specific brain regions. Drug administration directly into a discrete brain structure can transiently inactivate the area of interest. For example, a variety of drugs are available to inactivate the hippocampus via blockade of excitatory neurotransmission or increasing inhibitory neurotransmission. The transient nature of these pharmacological approaches circumvents some of the issues that arise from permanent. However, the temporary inactivation technique is not without limitations. Specifically, intracranial drug administration is thought to lack regional specificity, although the same can be said about s (Clark et al., 2000). Many studies 37

55 have used intracranial muscimol, a GABA A agonist, as the inactivating pharmacological agent, because it can impair the function of a structure without affecting fibers of passage (Chrobak et al., 1989; Allen et al., 2008). The precise distribution of muscimol or other drugs after local infusion within the CNS appears to depend on a number of factors such as lipid solubility, presence of fiber tracts, etc. (Martin, 1991; Arikan et al., 2002). The development of a fluorophor-conjugated muscimol has aided in relating observed behavioral effects to the drug's distribution within and beyond its intended target (Allen et al., 2008; Stackman et al., 2012; Cohen et al., 2013b). Taken collectively, the large majority of published results indicate that temporary inactivation of the rodent hippocampus impairs object recognition memory (see Table 1). Our lab reported that bilateral intrahippocampal microinfusion of lidocaine before the sample session impaired novel object preference in mice during a test session 24 h later, but spared novel object preference during a test session 5 min after the sample session (Hammond et al., 2004). These results were interpreted as support for the view that the hippocampus is critical for the processes of object recognition memory encoding and consolidation. However, an influence of intrahippocampal lidocaine on neural activity in fibers of passage cannot be ruled out in interpreting these results. We recently reported the impairing effect that muscimol microinfusion into the CA1 region of the dorsal hippocampus has on the encoding, consolidation and retrieval of object memory in mice (Cohen et al., 2013b). Regardless of whether the treatment was administered prior to the sample session, immediately following the sample session, or before the test session, the mice that received intrahippocampal muscimol, exhibited significantly lower discrimination ratios compared to controls. It is also important to note that the behavior 38

56 of the muscimol-treated or anisomycin-treated (0 and 2 hr post sample) groups were not significantly different from chance performance (Cohen et al., 2013b). These results confirm our earlier conclusion based on our lidocaine results and support those of others (de Lima et al., 2006; Rossato et al., 2007). Furthermore, we reported that fluorophorconjugated muscimol impaired retrieval of object memory in mice after bilateral intrahippocampal infusion, a behavioral effect due to the drug diffusing within, but not beyond, the CA1 region of the dorsal hippocampus (Cohen et al., 2013b). Our finding that object memory processes depend on the dorsal hippocampus is in conflict with the spared object memory reported in rodents after permanent hippocampal. It is noteworthy that inactivation of the hippocampus also impaired a form of object memory encoded explicitly independent of the arena context using a modified NOR protocol in which mice explored the same sample objects for three sessions (1/day) with each session in a novel context (Cohen et al., 2013b). Thus, temporary inactivation studies reveal that the rodent hippocampus is necessary for distinct stages of object memory, and for an object memory independent of context. In this manner we have reduced the relevance of spatial and contextual cues that some have argued define the hippocampal involvement in the NOR task. Therefore, we have created task parameters that mirror those of Winters and Bussey's enclosed Y-maze (Winters et al., 2004; Winters et al., 2008), yet we find that the hippocampus is involved in the object aspects of the memory, while they report that only the perirhinal cortex is involved in object recognition memory. Certainly a clear picture of the neural substrates of object memory is difficult to determine from the conflicting findings that have arisen from the various methodologies applied to the question of the hippocampal role in object memory. 39

57 2.4.3 Variability in Intersession Delay and Sample Session Object Exploration Criteria. The relative difficulty, or memory demand, of the NOR task can be adjusted by increasing or decreasing the delay imposed between the sample and test sessions. In this way, NOR has been used to study short-term and long-term object recognition memory, as well as the rate of object memory degradation. With sufficiently long delays, the memory of the familiar item becomes progressively more vulnerable to decay, and, in this case, during the test session the animal will explore the familiar object and the novel object to an equal extent. It is this delay variable that can be widely manipulated to achieve vastly different results. The same holds true for sample session exploration criterion. Changes made to increase or decrease the time required to explore sample session objects can affect test session performance. Central to this review is our contention that the differential sensitivity of rodent object memory to hippocampal compromise when delays of varying length are imposed between sample and test sessions reveals a temporal specificity for hippocampal involvement in object memory. Additionally, our analysis of previously reported data indicates that the largest inconsistencies in results of hippocampal manipulation are found amongst the training criterion imposed during the sample session. Based on our analysis of the published reports, we suggest that the large variability of behavioral findings may be related to differences in the inter-session delay and the sample session object exploration criterion. However, it is important to note that even when efforts are made to reduce variability across testing procedures, contradictory findings may still arise (Crabbe et al., 1999). Due to the large number of published studies, and the sizable variability between each study 40

58 parameter, determining the neural structures that are needed for object recognition memory has been widely debated. As stated above, the predominant view is that the perirhinal cortex is responsible for rodent object memory, while a few other groups have argued for the critical contribution of the hippocampus. However, there is no indication in the literature that both structures are needed. This seeming contradiction is hard to reconcile with the current published studies, given all of the variations in task procedures that have been imposed. It is likely that this type of episodic memory depends upon both the perirhinal cortex and hippocampus, with each structure holding a specific unique contribution, irrespective of context. The present review focuses on those reports in which the role of the mouse or rat hippocampus on object memory was tested in the NOR task using traditional s (i.e., electrolytic, radiofrequency or excitotoxic means) or functional inactivations (i.e., local microinfusion of lidocaine, muscimol, or AMPA receptor antagonist). For an extensive review of the NOR task and its application to the neurobiological study of memory, see review by Dere et al. (Dere et al., 2007). A more complete list of rodent object recognition experimental findings can be found in Table 2. Limiting our scope to the subset of studies that imposed a specific sample session exploration criterion, we analyzed 12 peer-reviewed reports most of which were multi-experiment papers. Noticeable trends in the patterns of results can be gleaned from Table 1, which summarizes a number of experiment features and the results of the 132 experiments contained within those selected reports. Over the last 20 years there has been a marked escalation in the total number of NOR publications, with the largest increase over the last decade. It is interesting to note 41

59 that the number of studies using the temporary inactivation approach has progressively and consistently increased in recent years (see Figure 5). Thus, the steady increase in total publications likely reflects a converging acceptance of the NOR task as a method for assessing object memory and the interest in whether object memory is hippocampaldependent. The increase in the number of temporary inactivation papers may reflect the previously mentioned advantages of this technique over permanent s. Given the aforementioned variations in NOR task procedures used by distinct labs to meet experimental requirements, we assert that it is difficult to draw overall conclusions about these techniques for assessing the neural basis of object memory. Therefore, no distinct differences or trends could be made based on the specific technique, species of rodent studied, or on the type or extent of the. However, interesting developments were evident in terms of sample session object exploration criteria and intersession delay (Table 1). Across all experiments analyzed, in which a training criteria was imposed (63 experiments in total) and regardless of technique used to impair hippocampal function, object memory was never found to be impaired when a delay of less than 10 min was imposed between the sample and test sessions. However, when the delay was extended to 10 min or greater, the results of 25 of the 37 permanent hippocampal experiments indicated sparing of object memory, while 12 indicated significant impairment. The reasons for this considerable discrepancy in the data are not clear. A clearer picture emerges when one considers those studies in which temporary inactivation was used to test the role of the rodent hippocampus in object memory using the NOR task. Specifically, 6 of the 8 published experiments found a significant impairment in object memory when a sample session exploration criteria and a delay 42

60 greater than 10 min were both imposed. The two remaining studies had been designed as control experiments, and as such the functional inactivation method used was not anticipated to yield an impairment of object memory. More specifically, these control experiments were designed to demonstrate that in situations that are not expected to impair the hippocampus, hippocampal inactivation would not elicit changes in overall behavior. First, when mice were administered intrahippocampal anisomycin, a protein synthesis inhibitor, 2 h after the sample session, it was expected that enough time had passed for protein translation to occur, such that object memory consolidation would not be affected. Thus, the study was designed as a control measure ensuring that the impairment found when anisomycin was given immediately and 2 hrs post sample was truly a result of protein synthesis inhibition during a critical consolidation time window. The second control study was performed to confirm that mice explore familiar objects more when placed in a novel environment, as had been established in rats (Mumby et al., 2002b). Mice explored two sample objects in the same arena for 10 min over 3 consecutive days. The test session was then conducted in a novel context. Indeed, as expected in the novel context both intrahippocampal vehicle and muscimol treated mice explored the familiar and novel objects equally. In contrast, when the 3 sample sessions were each presented in different contexts, the intrahippocampal vehicle mice would show preference for the novel object during the test session, while the muscimol mice did not (Cohen et al., 2013b). In light of the motivation for these control studies, it is reasonable to assert that 100% of the hippocampal inactivation experiments found that object memory was impaired when a sample exploration criterion was imposed and a delay of 10 min or 43

61 greater was defined between sample and test. This pattern of results suggests that the involvement of the rodent hippocampus in object memory depends upon the interval between training and testing. Given that the hippocampus is not informed a priori as to the retention interval to be imposed (i.e., that it will be less than or greater than 10 min), it is more parsimonious to suggest that the delay-dependent involvement of the hippocampus reflects a delay in the hippocampus receiving and processing object information from the sample session, or that sufficient training or exploration of the sample objects is required before the hippocampus is engaged. Our interpretation of the discrepancy in results of the inactivation experiments with those of permanent experiments is multi-faceted. It is clear from both permanent and temporary experiments that the hippocampus is not required if the delay between sample and test is less than 10 min. In this case we infer that the test session is presented before the hippocampus has received the object information, or the episodic memory of the sample session object exploration has been encoded by the hippocampus. It is possible that for a short time, extrahippocampal structures (e.g., the perirhinal cortex) are responsible for the temporary maintenance of the object memory. This explanation fits the observed lack of behavioral impairment when the intersession delay was less than 10 min. However, when longer delays are imposed, our analysis reveals that hippocampal inactivation is more likely to impair object memory, while permanent hippocampal s are not. This inconsistency is possibly due to the aforementioned drawbacks of the permanent technique. As a corollary, the other trend that emerged from our analysis was the notion that imposing a sample session object exploration criterion affected the experimental outcome 44

62 after hippocampal inactivation or. Based on the studies examined, when analyzing delay, exploration times during the sample session seem to be important in determining if the test session would reveal behavioral impairments. Since there does not appear to be any consistency within the literature as to a standard and sufficient training criterion, the interpretation of these data is much more difficult. It is reasonable to state that less time spent exploring objects (e.g., less than s of total object exploration) would lead to a weaker object memory encoded, since only basic information about the items is learned in a short time frame, and the opposite would hold true for longer exploration times (e.g., greater than s of total object exploration) (Stackman et al., 2002). The more information and details that can be acquired about an item, the stronger that memory is expected to be. This notion lends support to the theory that the perirhinal cortex is involved in object familiarity while the hippocampus is needed for recollection of the object experience or event. As previously mentioned, we recently reported preliminary evidence that object memory reflecting a low level of sample object exploration is vulnerable to inactivation of the mouse perirhinal cortex, but not hippocampus (Cohen et al., 2013a). Specifically, mice that were required to accumulate a very short sample session object exploration criterion demonstrated weak object memory during the test session, 24 h later. However, if the perirhinal cortex were temporarily inactivated immediately after acquiring this shortened sample session criterion, then these mice were impaired in the test session. Conversely, if the infusion was directed into the dorsal hippocampus, then the mice performed equivalently to controls in the test session, indicating retrieval of a weak object memory. Alternatively, if the mice were required to accumulate a large sample session object exploration criterion, perirhinal cortex 45

63 inactivation post-sample did not impair test session performance, while direct infusion into dorsal hippocampus elicited significant impairments (Cohen et al., 2013a). Our interpretation is that imposing a low sample session object exploration criterion probably yields an object memory that is weak, with fewer details being encoded and retained. The perirhinal cortex would most likely support memory of objects explored for a brief or limited amount of time, or in the absence of robust contextual cues (Winters et al., 2008). Conversely, object memories that reflect significantly greater time spent exploring the sample session objects to reach a higher training criterion are more vulnerable to inactivation of the hippocampus than the perirhinal cortex. This result implies that stronger, more deeply encoded object memory is more likely to be supported by the hippocampus. This theory reasonably justifies the seeming discrepancy in the literature regarding the contributions of the hippocampus and perirhinal cortex to object memory A Model of the Contributions of Perirhinal Cortex and Hippocampus to NOR. Although the hippocampus and perirhinal cortex often function as two parts of an interacting memory system, their contributions are distinct and dissociable (Squire et al., 2007; Winters et al., 2008; Wixted and Squire, 2011; Clark, 2013). Generally, it is the belief that the hippocampus and perirhinal cortex are functionally distinct based on the information they process. As previous stated we hold that the sample session exploration criterion, or more specifically the amount of object information acquired, that is the deciding factor in when the perirhinal cortex and hippocampus are playing their respective roles in object recognition memory. Therefore, we assert that it is not the categorical information that these regions process that separates them, but the strength of 46

64 the memory formed. This gradient of memory strength between the two structures may account for some of the disagreements in the literature as to how these structures are involved in episodic memory. The theory that these memories can be formed along a continuum of memory strength lends itself to the notion that these two structures could be storing information based on a level of weak familiarity or strong recollection (for a schematic of the proposed theory, see Figure 6). As the theoretical model depicts, at the start of the sample session, object information will begin to "flow" into the perirhinal cortex. After a critical or threshold amount of object information is acquired, a process by which the information is "transferred" to the hippocampus commences. If this threshold is not reached, then the information will remain perirhinal cortex dependent as a weak object memory. Conversely, if the threshold is reached, then the information will become hippocampal dependent as a strong object memory Alternative Explanations for Differences in Results and New Method Implementations. Although the interpretation of the emergent trends is plausible, there are other possible explanations for the inconsistencies in the effects of hippocampal manipulation on object memory. For example, an overwhelming majority of the studies we reviewed used rats, while very few have used mice. Thus, it is reasonable that some of the irregularities between studies could be attributable to species differences. However, this does not seem likely, as few other behavioral differences between species have been reported within these studies. Beyond the NOR task, there are reports demonstrating that relative to rats, mice are impaired in hippocampal-dependent place and matching-to-place learning in swimming pools (Whishaw and Tomie, 1996; Frick et al., 2000), and that 47

65 mice demonstrate significantly more bouts of aggressive behavior to juvenile counterparts compared to rats (Engelmann et al., 2011). It is equally or even more plausible that the trends we have identified in our analysis of the hippocampal involvement in NOR, are due to the inconsistencies and variations in the NOR protocol itself. As previously stated, there is no standard method by which the NOR task is conducted across labs. The overwhelmingly large number of task variables and manipulations make comparing studies extremely difficult. In turn, it is nearly impossible to gain definitive global insights about brain structures from all of the data that has been previously collected. A standardization of the NOR protocol is essential before one can equate findings across studies accurately. This is especially true for sample session object exploration criterions imposed. Given that this is the pivotal stage for which all subsequent results are based, consistency of training protocols is paramount to accurate data interpretation. As stated above, the sheer amount of information attained from the training session could be the deciding factor determining which brain structure is responsible for maintaining that information. Additionally, due to the number of disadvantages attributed to the permanent and temporary inactivation approaches, other techniques are now being explored. The lack of specificity and the imposed duration of the /inactivation are key shortcomings of current procedures. Optogenetics and DREADDs are two new techniques that use viral vectors to infect discrete types of neurons or brain regions with non-native channels or designer G-protein coupled receptors, which permit subsequent exquisite temporal and regional control over the activity of the infected neurons. Optogenetics permits near immediate and reversible silencing or activation of neurons in a given brain region (Fenno et al., 2011; Packer et 48

66 al., 2013), features that make the technique well suited for defining the neuronal bases of discrete object memory processes. The DREADDS approach permits one to silence or activate a given brain region via systemic injection of a drug whose only target is the designer receptor (Rogan and Roth, 2011); by infecting all of the neurons in a given brain region, this strategy is particularly well suited to determining the behavioral roles of restricted brain regions of interest. These new methods prevail over the limitations and criticisms of the currently utilized techniques. It is the hope that with these new methods, investigating the roles of specific brain regions in the NOR task will provide clearer evidence as to how the hippocampus, specifically, is functionally involved in object memory. 2.5 Conclusions. In summary, the goal of our review was to evaluate the current literature regarding the role of the rodent hippocampus in object recognition memory as assessed with the NOR task. As discussed, this literature is divisive and replete with conflicting results. Our analysis of those 12 published reports that met our exclusion criteria and in which temporary or permanent s were employed, revealed remarkable differences in experimental outcomes depending on the method of hippocampal used. That is, the majority of experiments find object recognition memory to be unimpaired in rodents with permanent s of the hippocampus, while a majority of experiments find object recognition memory processes to be impaired after temporary inactivation of the hippocampus. The permanent approach has enjoyed a long history in behavioral neuroscience and is appropriate for developing rodent models of human amnesia. However, we caution that the marked differences in outcome of experiments using 49

67 permanent vs. temporary hippocampal should call into question the appropriateness of the permanent method for testing the involvement of a given brain region in time-dependent behavioral processes. Moreover, that behavior displayed by rodents after permanent hippocampal reflects compensation of neural circuitry that includes overt and covert pathology. In this light, we contend that spared object recognition memory so often reported in rodents after permanent s of the hippocampus is actually 'phantom hippocampal-dependent object memory'; in the same way that phantom limb pain arises after amputation-induced reorganization of the human somatosensory cortex. The spared object recognition memory in the hippocampaled rodent arises from extrahippocampal structures due to reorganization of the medial temporal lobe circuit. Although temporary inactivation methods are better suited for defining the neural substrates of such time-dependent memory processes, the current techniques are not free of limitations. The on-going development of optogenetic and chemogenetic tools appears to hold strong promise as such approaches offer considerable advantages in terms of regional specificity and temporal control over more traditional functional inactivation methods. Our analysis also provides a theory that dictates two clear predictions as to the precise NOR test conditions that engage the rodent hippocampus. First, that the hippocampus is necessary for the retention of object recognition memory when a delay greater than 10 min is imposed between the NOR sample and test sessions. If a delay less than 10 min is imposed, then interrupting hippocampal function does not impair NOR performance; however, under these conditions the object memory is sensitive to perirhinal cortex inactivation. This theory suggests a partnership of sorts in which the perirhinal cortex and the hippocampus both 50

68 participate in object memory processing. Second to this hypothesis is that the involvement of the hippocampus appears to depend upon the amount of time the rodents spend exploring the objects during the sample session. That is, a threshold amount of sample object exploration beyond ~30 s on each object appears to be a condition required to engage the hippocampus or, more specifically, to "move" neural control over the memory from the perirhinal cortex to the hippocampus. Interestingly, both predictions hold the assertion that neither the hippocampus nor perirhinal cortex is solely responsible for object memory as assessed by the NOR task. Instead, our interpretation is that episodic memory for objects explored during the NOR sample session emerges from interactions between the perirhinal cortex and the hippocampus. It will be of interest to test these predictions more thoroughly in order to understand the neural circuit that subserves object memory and to further identify the behavioral roles for the individual structures within the medial temporal lobe memory system. We propose that the key to further investigating the relationship between these structures, and the issues of memory strength, and familiarity vs. recollection, requires that the NOR protocol be standardized in order to enable meaningful interpretation across experiments. It is only in such a common-use framework that meta-analyses of the sort attempted here, might reveal the definitive roles of these structures in object memory. 51

69 PART III: THE RODENT HIPPOCAMPUS IS ESSENTIAL FOR NON-SPATIAL OBJECT MEMORY2 3.1 Abstract. Elucidating the role of the rodent hippocampus in object recognition memory is critical for establishing the appropriateness of rodents as models of human memory and for their use in the development of memory disorder treatments. In mammals, spatial memory (O'Keefe and Nadel, 1978; Morris et al., 1982; Riedel et al., 1999; Squire et al., 2004; Eichenbaum et al., 2007; Moser et al., 2008) and non-spatial memory (Clark et al., 2001; Ross and Eichenbaum, 2006) depend upon the hippocampus and associated medial temporal lobe (MTL) structures. Although well-established in humans (Eichenbaum et al., 2007; Squire et al., 2007), the role of the rodent hippocampus in object memory remains highly debated due to conflicting findings across temporary and permanent hippocampal studies (Duva et al., 1997; Clark et al., 2000; Mumby et al., 2002b; Gaskin et al., 2003; Broadbent et al., 2004; Hammond et al., 2004; Winters et al., 2004; Forwood et al., 2005; Mumby et al., 2005; Ainge et al., 2006; Broadbent et al., 2010; Oliveira et al., 2010; Barker and Warburton, 2011) and evidence that the perirhinal cortex may support object memory (Winters et al., 2004; Winters and Bussey, 2005; Good et al., 2007). In the current studies, we used intra-hippocampal muscimol This chapter was originally published as a Report in Current Biology: Sarah J. Cohen, Alcira H. Munchow, Lisa M. Rios, Gongliang Zhang, Herborg N. Asgeirsdottir & Robert W. Stackman, Jr., Sept , The Rodent Hippocampus Is Essential for Nonspatial Object Memory, Current Biology, 23/17, Reproduced with permission from Elsevier Inc. 52

70 microinfusions to transiently inactivate the male C57BL/6J mouse hippocampus at distinct stages during the novel object recognition (NOR) task: during object memory encoding and consolidation, just consolidation and/or retrieval. Our results reveal a clear and compelling role of the rodent hippocampus in non-spatial object memory. 3.2 Introduction. Object recognition memory is a commonly used test of declarative memory a form of memory that is supported by the mammalian medial temporal lobe (MTL) memory circuit (Eichenbaum, 2000; Squire et al., 2004). However, the precise MTL brain regions that are critical for object memory remain poorly defined. Lesions of the HPC formation (HPC proper, dentate gyrus, entorhinal cortex and subiculum) impair performance of primates and rodents on a broad range of learning and memory tasks. Lesions, or temporary inactivation, of the rodent HPC impairs spatial memory processes (Morris et al., 1982; Riedel et al., 1999) and individual HPC neurons exhibit locationspecific firing properties when recorded from freely moving animals (O'Keefe, 1976; Muller, 1996). These findings have established the obligatory role of the rodent HPC in spatial memory processes (O'Keefe and Nadel, 1978; McNaughton et al., 2006; Moser et al., 2008). Lesions of rodent HPC also impair non-spatial memory in a variety of tasks (Raffaele and Olton, 1988; Bunsey and Eichenbaum, 1995; Dusek and Eichenbaum, 1997; Quinn et al., 2002; Ross and Eichenbaum, 2006). However, a majority of studies using a spontaneous NOR or paired comparisons test find that permanent HPC s spare object recognition memory (Duva et al., 1997; Mumby et al., 2002b; Gaskin et al., 2003; Winters et al., 2004; Forwood et al., 2005; Mumby et al., 2005; Ainge et al., 2006; Barker and Warburton, 2011). A few studies report that extensive HPC damage (e.g., 53

71 HPC cell loss >75%) impairs object recognition memory (Clark et al., 2000; Broadbent et al., 2004; Broadbent et al., 2010), suggesting that a large volume of HPC is recruited for accurate object recognition. However, mice that received pre-training local intra-hpc lidocaine failed to discriminate a novel object from a familiar one during a test session (Hammond et al., 2004), although post-training HPC inactivation by muscimol enhanced NOR (Oliveira et al., 2010). Taken together, these results fail to establish a clearly defined role of the HPC in object memory. By comparison, permanent or temporary s of perirhinal cortex consistently disrupt object recognition memory, while sparing spatial memory (Winters et al., 2004; Winters and Bussey, 2005). Thus, the majority of data offer strong support for a functional and independent dichotomy with the MTL memory circuit: that the rodent HPC is specialized for spatial memory, with a very limited role in object memory, while the rodent perirhinal cortex is specialized for object memory, with a minimal contribution to spatial memory. Results from human fmri studies suggest that familiarity-based recognition memory depends upon the perirhinal cortex, while recollection-based recognition memory that includes contextual or spatial elements depends upon the HPC. However, an alternative view holds that familiarity and recollection reflect weak and strong forms of object memory and that object memory processes are dependent upon both the perirhinal cortex and HPC (Squire et al., 2007). Thus, the contribution of the rodent HPC to nonspatial object recognition memory is not clear and remains a matter of considerable debate. Resolving this debate regarding the role of the rodent HPC in object recognition memory is critical for the development of animal models of human declarative memory and treatments of memory disorders. 54

72 Variability in the effects of rodent HPC s on object memory may stem from the use of permanent vs. temporary, length of the retention delay, and differences in NOR task procedures (Ainge et al., 2006). We used a discrete transient inactivation method to dissect the contributions of the mouse HPC to object memory encoding, consolidation and retrieval. The inactivation technique was chosen to avoid problems associated with traditional permanent s, such as post- functional compensation. The results of these studies reveal a clear and compelling role of the rodent HPC in all aspects of object memory. 3.3 Materials and Methods Mice and Surgery. Male C57BL/6J mice (7-10 wk old; Jackson Labs) were housed in a 12 h light/dark cycle, temperature- and humidity-controlled vivarium 4/cage with ad libitum access to food and water. Behavioral testing was conducted during the light cycle. All procedures were conducted in accordance with NIH guidelines and were approved by the Institutional Animal Care and Use Committee Novel Object Recognition Task. The apparatus consisted of two open-top, high-walled square arenas made of white ABS (each: 37.5 x 37.5 x 50 cm). For all experiments, each mouse was habituated to one of the arenas for 10 min/day for 2 consecutive days. For all other behavioral experiments, except the retrieval of strong object memory experiment (Exp t 6), each mouse received one sample session and one test session in the habituated arena (see Figure 7A). During the sample session, each mouse was returned to the familiar arena that now contained two identical novel toy objects (stainless steel cabinet leveling feet,

73 cm dia and 6.0 cm tall). The two sample objects were positioned on the arena floor 2 cm from opposite corners (NW and SE). The mouse was removed from the arena upon accumulating 30 s of exploration of both objects or 38 s of either object within a 10-min session. This was referred to as the sample object exploration criterion and was imposed to ensure that all mice accumulated similar exploration time with the objects. During the test session 24 h later, each mouse was given a 5 min test session in the familiar arena, which contained one of the familiar objects and one novel object (plastic toy gorilla attached to a Plexiglas base). The objects and the arena floor and walls were cleaned with 10% ethanol after each session. All behavioral testing data was digitally acquired by the EthoVision XT (Noldus Inc.) software package. Object exploration was scored off-line from the digital video files by experimenters that were blind to the treatment condition of the mice. Object memory was inferred from the discrimination ratio calculated for each subject by subtracting the time spent exploring the familiar object from the time spent exploring the novel object and dividing the result by the total time spent exploring both objects. Discrimination ratios range from -1 to 1, with 0 indicating chance performance a lack of preference for one object over another, and positive ratios indicating novel object preference Intrahippocampal Cannulation and Microinfusion. Mice were implanted with chronic bilateral guide cannulae (Plastics One, Inc.) above the CA1 region of dorsal hippocampus (A/P mm, M/L ± 1.5 mm, D/V mm from bregma) (Franklin and Paxinos, 2008). Mock infusions were given each day for the 2 days prior to the actual intra-hippocampal microinfusion to habituate the mice to the microinfusion procedure. At the time of actual infusions, mice received bilateral (

74 µl/side, µl/min) intra-hippocampal muscimol (1 µg/µl in 0.9% saline, Tocris), fluorophore-conjugated muscimol (FCM, 1 µg/µl in PBS, BODIPY TMR-X, Molecular Probes), anisomycin (40 µg/µl dose in PBS, Sigma Aldrich) or the appropriate vehicle. For Exp t 1 (encoding), muscimol or saline was infused 20 min prior to the sample session. For Exp t 2 (consolidation) muscimol or saline was infused immediately after the sample session. Anisomycin or PBS was infused immediately and 2 h after the sample session (Exp t 3) or only 2 h after the sample session (Exp t 4) to test the dependence of object memory consolidation on protein synthesis within the hippocampus. For Exp t 5 (simulated permanent hippocampal ) muscimol or saline was infused 20 min before and 2 h after the sample session, and again 40 min before the test session. For Exp t 6 (retrieval of strong object memory), mice received intra-hippocampal FCM or vehicle 40 min before the test session. Since mice received pre-test FCM, in Exp t 6, mock infusions were given before daily sample sessions Histology. Cannulae placements were confirmed by examination of Cresyl violet stained 50- µm coronal sections with light microscopic methods. The data for any mice that were determined to have inappropriate placement were excluded from the analyses Data Analysis. Mice that exhibited motor impairments after muscimol infusion or inability to reach sample exploration criterion within 10 min were excluded from the analyses. To test the strength of the novel object preference during the test session, we analyzed the discrimination ratios between vehicle and respective muscimol, anisomycin or FCM group using Student s t-tests. To determine whether motivation to explore objects during 57

75 the sample session was affected by the respective treatment, latency to reach the sample session object exploration criterion measures were also analyzed using Student s t-tests. Total test session object exploration measures were also analyzed using Student s t-tests. For Exp t 6, sample session object exploration was analyzed according to future treatment groups using a two-factor [treatment, sample session (1-3)] repeated measures ANOVA. For all tests, differences were considered significant if P < Results. Mice were surgically implanted with intracranial infusion cannulae at least one week before the onset of behavioral testing. The dorsal hippocampus was bilaterally inactivated at discrete time points relative to the NOR task: before the sample session to affect encoding and consolidation, after the sample session (consolidation) or before the test session (retrieval) (Figure 7A). During the sample session, each mouse explored two identical objects until the object exploration criterion was reached: 30 s exploration of both objects or 38 s of either object within 10 min, except where otherwise noted. Similar latency to criterion between groups established equal motivation to explore objects. After 24 h, each mouse was given a 5-min test session with one familiar and one novel object. Preference for exploring the novel object was determined by calculating a discrimination ratio for each mouse (Discrimination io=t novel -T familiar /T novel +T familiar ). Discrimination ratios were analyzed for treatment differences in object memory. Cannula placements were verified histologically (Figure 7B) Exp t 1. Hippocampus is required for object memory encoding and consolidation. Naïve mice received intra-hippocampal muscimol or the saline vehicle 20 min before the sample session, ensuring hippocampal inactivation across encoding and 58

76 into the consolidation stage (Bonnevie et al., 2013). Both groups reached sample session exploration criterion in similar times [saline 448 s, muscimol 360 s; t(11.52)=1.489, n.s.] and spent similar total amounts of time exploring test session objects [t(15)=1.147, n.s.]. However, muscimol group discrimination ratios were significantly lower than those of the saline group [t(15)=2.47, P=0.026, Figure 7C], suggesting that inactivation of the hippocampus 20 min prior to the sample session prevents encoding and/or consolidation of object memory Exp t 2-4. Hippocampus is required for object memory consolidation. Naïve mice received intra-hippocampal muscimol or saline immediately after the sample session (Exp t 2). Sample session latency to criterion was similar between future treatment groups [saline 469 s, muscimol 459 s; t(21)=1.93, n.s]. However, discrimination ratios of the muscimol group were significantly lower than those of the saline group [t(21)=5.93, P < 0.001, Figure 7D]. Another cohort of mice received intrahippocampal anisomycin both immediately and 2 h after the sample session to disrupt hippocampal protein synthesis during consolidation (Exp t 3). Discrimination ratios of the anisomycin-treated mice were also significantly lower than those of the vehicle group: t(25)=6.51, P<0.001, Figure 7E], consistent with a prior report (Rossato et al., 2007). NOR was spared in mice that only received intra-hippocampal anisomycin 2 h post-sample (Exp t 4, Figure 7E). Interestingly, intra-hippocampal anisomycin given 3 h, but not 6 h, post-sample impaired NOR (Rossato et al., 2007), therefore, the precise dynamics of protein synthesis-dependent consolidation of object memory remain unclear. Together our results indicate that consolidation of object memory requires a functional hippocampus and hippocampal protein synthesis occurring <2 h after the sample session. 59

77 3.4.3 Exp t 5. Hippocampal inactivation during all memory stages impairs NOR performance. To test our hypothesis that the frequently reported spared NOR after permanent hippocampal s is due to compensatory changes within the MTL, we inactivated the hippocampus during encoding, consolidation and retrieval phases. Naïve mice received intra-hippocampal muscimol or saline 20 min before and 2 h after the sample session and 20 min before the test session. Sample session latencies to criterion were equivalent [saline 474 s, muscimol 404 s; t(14)=1.13, n.s]; however, discrimination ratios were significantly lower in muscimol-treated mice than in saline-treated mice [t(8.46)=7.241, P < 0.001, Figure 7F]. These results suggest that spared object memory in hippocampal-ed rodents is likely supported by compensatory changes Exp t 6. Inactivating hippocampus blocks retrieval of a strong object memory. Naïve mice received three 10-min sample sessions in the same arena (1/day, inset Figure 8A) to permit the encoding of a strong object memory. Mice received intrahippocampal fluorophore-conjugated muscimol (Figure 8B, FCM, Molecular Probes, Eugene, OR) or vehicle 20 min before the test session (Exp t 6). Groups exhibited similar object exploration across sample sessions [group x session, F 2,28 =1.46, n.s.] and in the test session [inset Figure 8B, t(14)=-0.09, n.s]; however, FCM group discrimination ratios were significantly lower than those of the vehicle group [t(14)=3.11, P = 0.008, Figure 8C]. Examination of tissue sections revealed that at 30 min post-infusion, FCM spread in approximately a 300-µm radius from the estimated center of each infusion, not beyond the CA1 region of hippocampus (Figure 7B). Assuming a spherical distribution at both infusion sites, FCM affected approximately 1% of the entire hippocampal volume (Peirce et al., 2003). These results indicate that the dorsal hippocampus is critical for retrieval of 60

78 object memory, and very limited hippocampal inactivation is sufficient to impair NOR. 3.5 Discussion. These behavioral results establish that the rodent hippocampus is obligatory in object memory, corroborating literature regarding its role in non-spatial memory (Fortin et al., 2002; Fortin et al., 2004; Squire et al., 2007). Our finding that disruption of approximately 1% of total hippocampal volume blocked object memory processes contradicts reports that permanent s of <75% of hippocampus spare NOR (Broadbent et al., 2004; Ainge et al., 2006). However, such studies test what a hippocampal-ed rodent is capable of remembering (hippocampal-independent NOR) rather than whether object memory normally recruits the hippocampus. We argue that traditional s provide an adequate model of human amnesia, but are ill suited for delineating the hippocampal role in healthy memory processing. If rats with permanent hippocampal s are repeatedly exposed to the same context, then extra-hippocampal structures can support contextual memory (Wiltgen et al., 2006; Piterkin et al., 2008). Here, object memory encoded over three 10-min sample sessions remained sensitive to pre-test hippocampal inactivation, implying that preserved NOR in permanently ed rodents is due to compensatory plasticity rather than to normal extra-hippocampal capabilities. This view is bolstered by findings of the hippocampal inactivation during all stages study (Exp t 5), which also confirms that our other findings are not due to state-dependent effects. The argument for a double dissociation of perirhinal cortex and hippocampus posits that memory for objects independent of place/context selectively engages perirhinal cortex (Winters et al., 2008), while the conjunctive memory for objects in 61

79 place/context depends on hippocampus (Bussey et al., 2000a; Winters et al., 2004). Thus familiarity, or knowing that an item was recently viewed, depends on perirhinal cortex, while recollection, or remembering distinct details about an episode, depends on hippocampus (Brown and Aggleton, 2001). This hypothesis predicts that perirhinal cortex could support NOR performance despite a hippocampal, but we found that NOR performance was impaired after hippocampal inactivation. Evidently, NOR was not supported by perirhinal-dependent familiarity. Alternatively, recognition memory may exist on a continuum from weak to strong, whereby the encoding, consolidation and retrieval of only strong memory (based on familiarity or recollection) is hippocampaldependent (Squire et al., 2007). Considering the sensitivity to hippocampal inactivation, the probed memory reported here appears to be a strong one. If a weak counterpart was available in perirhinal cortex, then it was too weak to influence behavior and was, therefore, negligible. Evidence supporting the role of rodent perirhinal cortex in object memory is convincing (Winters et al., 2004), but does not eliminate a role for the hippocampus. her, s of perirhinal cortex may disrupt NOR by interfering with the flow of information through the MTL circuit. Unimodal (what/item) and polymodal (where/context) information streams are routed through perirhinal and parahippocampal cortices, respectively, to hippocampus (Burwell, 2000), and are likely both critical for spatial and non-spatial memory functions of hippocampus. Consistent with this view, perirhinal cortex s disrupt the stability of rodent hippocampal place cells (Muir and Bilkey, 2001). Considering the MTL s dense interconnectedness (Squire et al., 2007), we propose that the labor of explicit memory is carried by collective participation of 62

80 hippocampus, perirhinal cortex and associated regions, but stress that normal object memory processing indeed requires the hippocampus. While numerous reports state that the hippocampus is not involved in NOR, several studies support our findings (Clark et al., 2000; Gaskin et al., 2003; Broadbent et al., 2004; Hammond et al., 2004; de Lima et al., 2006; Broadbent et al., 2010). Our results elaborate on the conclusions of these supporting studies by establishing the critical and independent contribution of dorsal hippocampal activity to discrete stages of object memory. Further, the finding that the rodent hippocampus is involved in NOR is compatible with prior studies of other species, such as those assessing visual recognition memory in primates (Pascalis and Bachevalier, 1999; Zola et al., 2000; Nemanic et al., 2004; Zeamer et al., 2011). Considering the known role of the human and non-human primate hippocampus in recognition memory, it is likely that the rodent hippocampus plays a similar role. Our findings support this conclusion: the rodent hippocampus isn t just for space anymore. 63

81 PART IV: DISSOCIATING OBJECT-IN-CONTEXT MEMORY: THE RODENT HIPPOCAMPUS PROCESSES SPATIAL AND OBJECT MEMORY SEPARATELY 4.1 Abstract. In humans, the medial temporal lobe is paramount to remembering and recalling the spatial and nonspatial aspects of episodic memories. Although it has been shown that the rodent hippocampus is essential in spatial memory, its role in nonspatial object memory has been highly debated. Using an established novel object recognition (NOR) task, numerous studies have demonstrated that the rodent dorsal hippocampus is recruited only for object-in-context memories. However, recent reports have demonstrated a significant role for the rodent CA1 region of the dorsal hippocampus in object memory during this traditional task. To further differentiate the object and context memories within this task, variations of the conventional NOR task were developed and implemented for C57BL/6J male mice. The results indicate that in these modified tasks, inactivation of the hippocampus after the sample session produced deficits in object discrimination during the test session. Our findings support the notion that the rodent hippocampus is essential for the consolidation of object memories independent of the context, in these modified NOR tasks. 4.2 Introduction. The medial temporal lobe, including the hippocampus and associated regions, is 64

82 essential for spatial and nonspatial aspects of episodic memory in mammals (Morris et al., 1982; Squire et al., 2004; Eichenbaum et al., 2007). Central to this memory circuit is the hippocampal cognitive map that represents locations where relevant nonspatial items or objects were encountered and where specific events occurred within a contextual or spatial reference frame (O'Keefe and Nadel, 1978). Temporary inactivation or permanent of rodent hippocampus impairs spatial memory and navigation (Morris et al., 1982; Riedel et al., 1999); establishing that a functioning hippocampus is required for encoding, consolidating, and retrieving location-specific information. Further, individual hippocampal CA1 neurons represent place by firing at high frequencies when the rodent occupies different spatial locations (O'Keefe and Dostrovsky, 1971; Wilson and McNaughton, 1993). Recent studies reveal that CA1 neurons represent configural associations of odor-in-location (Komorowski et al., 2009) and object-in-location (Komorowski et al., 2009; Burke et al., 2011; Deshmukh and Knierim, 2013); such event representations provide further support for the cognitive map view defined above. Temporary inactivation or permanent of rodent hippocampus also impairs object memory in the novel object recognition (NOR) task (Clark et al., 2000; Hammond et al., 2004; de Lima et al., 2006; Cohen et al., 2013b). Furthermore, when mice explore novel objects compared to familiar objects, CA1 neurons discharge at higher rates, and dorsal hippocampal glutamate efflux is increased (Cohen et al., 2013b). These findings demonstrate that nonspatial object memory is hippocampal-dependent although there is evidence to the contrary (Winters et al., 2004; Mumby et al., 2005; Winters and Bussey, 2005; Ainge et al., 2006; Oliveira et al., 2010). In the standard NOR task, rodents encode an object-in-context memory, and arguably the hippocampal dependence observed may 65

83 reflect the contextual attributes of the task. However, hippocampal inactivation also impairs object-independent-of-context memory, in a modified NOR task (Cohen et al., 2013b), suggesting that hippocampal processing of object and context information may be dissociable. In fact, a parallel map theory has been proposed in which a bearing (spatial/contextual) map and a sketch (local cues, objects) map are constructed in the hippocampus and transmitted to the subiculum (Chang and Huerta, 2012). Evidence for an integration of these disparate spatial and object recognition signals within the dorsal subiculum has been reported (Jacobs and Schenk, 2003). The present studies utilize variations of the NOR task to further assess the dependence of context on the hippocampal involvement in object memory. Impairments of object memory observed following hippocampal inactivation suggest that the hippocampal spatial representation of the environment does not change with the addition of physical elements to the arenas. Therefore, the hippocampal-dependent object memory formed during the NOR task is likely not an object-in-context representation, but rather an object memory and a context memory dissociated. 4.3 Materials and Methods Mice and Surgery. Male C57BL/6J mice (7-10 wk old; Jackson Labs) were housed 4 per cage with ad libitum access to food and water. All procedures were conducted in accordance with NIH guidelines and were approved by the Institutional Animal Care and Use Committee. Surgical implantation of guide cannulae (n = 80) was completed when all mice were 8-10 weeks old, one week after acclimatization to the vivarium. 66

84 4.3.2 Intrahippocampal Cannulation and Microinfusion. Mice were implanted with chronic bilateral guide cannulae (Plastics One, Inc., Roanoke, VA) above the CA1 region of dorsal hippocampus (A/P mm, M/L ± 1.5 mm, D/V mm from bregma; corresponding to intermediate CA1). The guide cannulae were mounted to the skull using skull screws ( , Antrim) and cold-curing dental acrylic (ColdPac, Chicago, IL). Each mouse was administered buprenorphine (0.5 mg/kg, IP) after the surgery was completed and triple antibiotic ointment was applied to the wound. Behavioral testing began 7-10 days later to permit postoperative recovery. Each mouse received a mock infusion each day for the 2 days prior to the actual intrahippocampal microinfusion, 20 min prior to or immediately after the arena habituation, to acclimate the mice to the microinfusion procedure. The mock infusion procedure involved a brief restraint of the mouse, during which the protective cap and dummy internal cannula were removed, and dummy infusion cannulae inserted into each guide cannula. These dummy infusion cannulae did not project beyond the tip of the implanted guide cannulae. Once the infusion cannulae were inserted, the mouse was released into an empty polycarbonate mouse cage for the 3 min duration of the infusion. Each mouse was again briefly restrained to remove the infusion cannulae, replace the dummy internal cannulae and protective cap, and then returned to the home cage. For the actual microinfusions, mice received bilateral (0.35 µl/side, µl/min) intra-hippocampal muscimol (1 µg/µl in 0.9% saline, Tocris) or saline immediately after the sample session. However, for the conventional 5 min NOR experiment, intrahippocampal saline or muscimol was infused 20 min prior to the sample session. The procedures for the actual bilateral microinfusion followed that described above for the 67

85 mock infusion; however, this time the inserted infusion cannulae penetrated 1 mm beyond the tip of the guide cannulae to achieve bilateral intrahippocampal infusion Novel Object Recognition Task and Protocols. The apparatus consisted of two open-top, high-walled square arenas made of white ABS (each: 37.5 x 37.5 x 50 cm). Each mouse was habituated to one of the arenas for two 10-min sessions. Then, each mouse received one sample session and one test session in the habituated (i.e., familiar) arena. During the sample session, each mouse was returned to the familiar arena that now contained two identical novel objects (stainless steel cabinet leveling feet, each attached to a Plexiglas base, 4.2 cm dia and 6.0 cm tall). The two objects were positioned on the arena floor 2 cm from opposite corners (NW and SE). For all studies, each mouse was removed from the arena upon accumulating 30 s of exploration of both objects or 38 s on either object. This sample object exploration criterion was imposed to ensure that all mice were matched for sample session performance. The data from two mice that failed to reach the sample session exploration criteria were removed from the analyses. During the test session, presented 5 min, 20 min, or 24 h later, the familiar arena contained one of the familiar objects and one novel object (plastic toy gorilla or metal spring attached to a Plexiglas base). The mouse was removed from the arena after 5 min (see Figure 9a for protocol schematics). The objects and the arena floor and walls were cleaned with 10% ethanol after each session. All behavioral testing data was digitally acquired by the EthoVision XT (Noldus Inc., Leesburg, VA) software package. Object exploration was scored off-line from the digital video files by experimenters that were blind to the treatment condition of the mice. Object memory was inferred from the discrimination ratio calculated for each mouse by 68

86 subtracting the time spent exploring the familiar object from the time spent exploring the novel object and dividing the result by the total time spent exploring both objects. Discrimination ratio scores range from -1 to 1, with positive scores indicating novel object preference, while a ratio = 0 indicating chance performance or a lack of preference for one object over another. Cue Card NOR protocol: This protocol was designed to test the influence of NOR task performance (i.e., encoding and retrieving hippocampal-dependent object memory) in the presence of a dark polarizing cue card (a 41.9 cm x 26.1 cm piece of gray ABS) that was affixed to the north wall of the arena throughout the entire protocol, reminiscent of place cell recording procedures (Muller and Kubie, 1987; Kentros et al., 2004; Muzzio et al., 2009). All other procedures were as described above. Conventional NOR protocol: The arena and stimuli used in this protocol match those used typically to study NOR in rodents (Clark et al., 2000; Fernandez et al., 2008; Cohen et al., 2013b); yet, these environmental conditions are relatively diminished compared to those typically present during place cell recordings. All protocol sessions were conducted in the white, otherwise empty arenas, precisely as described above. Drop-in NOR protocol: This protocol was designed to promote recognition of the memory of the arena context and the potential encoding of an orthogonal memory of the objects. Each mouse first explored the familiar, otherwise empty arena for a total of 2 min, and then the sample or test session began when the respective objects were lowered into the arena. No attempt was made to limit the animal s view of the objects as they were being placed into the arena; however, the mice were placed into the arena facing the NE corner which lacked object stimuli. All other procedures were as described above. 69

87 4.3.4 Data Anaylsis. For all studies, the discrimination ratio and latency (in s) to reach the sample object exploration criterion scores of vehicle- and muscimol-treated mice were compared using two-tailed Student s t-tests Histology. At the conclusion of behavioral testing, each mouse was deeply anesthetized with 5% isoflurane and brains were dissected and preserved in 4% paraformaldehyde. All brains were cryoprotected, then sectioned at 50 µm using a sliding microtome (Leica SM2010) with an automatically controlled freezing stage (Physitemp Instruments, Clifton, NJ). Cannulae placements were confirmed by examination of cresyl violetstained sections under a light microscope. The data for any mice that were determined to have inappropriate placement were excluded from the analyses (for representative photomicrographs depicting correct placement of cannulae, see Figure 9b). 4.4 Results. Cue Card NOR: Naïve mice (n = 16) received bilateral intra-hippocampal muscimol or vehicle immediately after the sample session. Sample session latency to criterion was similar between the future treatment groups [vehicle 498 s, muscimol 448 s; t(14) = 1.00, n.s.]. However, the discrimination ratio scores, computed from the test session exploration 24 h later, were significantly lower for the post-sample muscimol group compared to those of the post-sample vehicle group [t(14) = 3.33, P = 0.005, Figure 10a]. Despite the differences in discrimination, total object exploration was not different between the postsample treatment groups [vehicle 52 s, muscimol 46 s; t(14) = 1.096, n.s.]. These results 70

88 confirm that the consolidation of object memory acquired in the Cue Card NOR task is dependent upon the dorsal hippocampus, consistent with previous studies (Cohen et al., 2013b). The results also imply that the inclusion of a salient polarizing cue inside the arena, which presumably facilitates the recall of the familiar context during the sample and test sessions, is not sufficient to rescue object discrimination when the dorsal hippocampus is impaired during object memory consolidation. Conventional NOR: <5 min delay: Naïve mice (n = 19) received intra-hippocampal muscimol or vehicle 20 min prior to the sample session. Sample session latency to criterion was equivalent between the treatment groups [vehicle 523 s, muscimol 503 s; t(17) = 0.42, n.s.]. For the test session, the discrimination ratio scores of both the muscimol and vehicle groups were similar in their preference for the novel object [t(17) = 1.30, n.s., see Figure 10b] and in total object exploration [vehicle 44s, muscimol 48s; t(17) = -0.66, n.s.]. These results imply that object discrimination is spared in hippocampal-inactivated mice when a short 5 min delay is imposed between the sample and test session. 20-min delay: Naïve mice (n = 19) received intra-hippocampal muscimol or vehicle immediately following the sample session. Sample session latency to criterion was similar between the future treatment groups [vehicle 500 s, muscimol 436 s; t(17) = 0.95, n.s.]. However, for the test session, the discrimination ratio scores of the postsample muscimol group were significantly lower than those of the post-sample vehicle group [t(17) = 5.41, P < 0.001, see Figure 10c]. Importantly, the total time the mice engaged in object exploration was equivalent between the two post-sample treatment conditions [vehicle 49 s, muscimol 45 s; t(17) = 0.57, n.s.]. These findings demonstrate 71

89 that the dorsal hippocampal function is critical for consolidation of object memory when a 20-min delay is imposed between the sample and test session stages of the task. We reported a similar effect from an experiment using identical post-sample hippocampal inactivation strategies, but with a 24-hr delay imposed between sessions. Specifically, the sample session latency to criterion was similar between the future treatment groups [vehicle 469 s, and muscimol 459 s; t(21) = 1.93, n.s.]. During the test session, the discrimination ratios of the post-sample muscimol mice were significantly lower than those of the post-sample vehicle mice [t(21) = 5.93, P < 0.001] (see chapter 3; Cohen et al., 2013b). These findings are equivalent to the discrimination ratios of the mice in the present Conventional NOR protocol when a 20-min delay was imposed. Together with results from the 5-min delay experiment, these findings are consistent with the view that the hippocampus plays a delay-dependent role in object memory. Drop-in NOR: Naïve mice (n = 19) received intra-hippocampal muscimol or vehicle immediately after the sample session. The sample session latency to criterion was similar between the future treatment groups [vehicle 356 s, muscimol 439 s; t(17) = -1.46, n.s.]; however, for the test session 24 h later, the discrimination ratio scores of the post-sample muscimol group were significantly lower than those of the post-sample vehicle group [t(17) = 2.77, P = 0.013, Figure 10d]. There was no difference in total object exploration between the two post-sample treatment groups [vehicle 57 s, muscimol 43 s; t(17) = 1.25, n.s.]. These results support the hippocampal-dependence of object memory consolidation and extend this to include conditions in which mice may encode an object memory distinct from the context. Also, these results imply that when mice are able to first recall the familiar 72

90 context, and only then given the opportunity to explore the new objects, a functional hippocampus is required during the consolidation phase of object memory for the mice to be able to demonstrate a preference during the test session. Additionally, these findings are consistent with a view that a pre-existing contextual memory may be modified to include object information. 4.5 Discussion. We previously found evidence that mice encode an object-in-context memory during the typical NOR task as mice fail to preferentially explore the novel object when the test session is presented in a novel context (Cohen et al., 2013b), likely because rodents exhibit increased exploration of familiar objects when presented in a novel context (Mumby et al., 2002b). However, the present experiments tested the involvement of the hippocampus in modified object recognition memory tasks, dissociating the objects from the context memories, using functional inactivation. Specifically, the results demonstrate that post-sample session inactivation of the hippocampus impaired object recognition memory during the test sessions regardless of the presence of a polarizing cue for orientation (Cue Card), or if time was provided for the mice to explore the arena before inserting objects (Drop-in). However, in the Conventional NOR protocol, when the delay between the sample and test sessions was reduced from 20 min to 5 min, novel object discrimination was equivalent between the intrahippocampal vehicle- and muscimol-treated mice. Thus, these results suggest a delay-dependent hippocampal involvement in object memory, consistent with previous studies (Clark et al., 2000; Hammond et al., 2004). Therefore, the findings suggest there is a post-sample session 73

91 time delay less than 20 min, before object information is processed within dorsal hippocampus. As described above, the results from our inactivation studies imply that the hippocampus is not engaged in object memory until a minimal amount of time has elapsed after the sample session. This result further supports simultaneous but independent processing of spatial and object information by the hippocampus. Other reports state that when a short delay is imposed between the sample and test session, the hippocampus is not needed for object memory retrieval (Clark et al., 2000; Hammond et al., 2004). However, based on our current data, by 20-min after the sample session, the hippocampus is already recruited for the spatial and the object memories, respectively. These data provide indirect support for the view that sufficient time is required for the object memory or information to become hippocampal-dependent, and therefore prior to the passage of such time, the hippocampus is completely devoted to spatial processing. However, when a 20-min delay is imposed, our inactivation results indicate that the object memory has now become hippocampal-dependent. Based on the presented data, it is plausible that it is due to a reallocation of resources once the hippocampus is engaged in processing both spatial and nonspatial information. Presumably, during the Drop-in NOR protocol (when no salient cue is available) the mice would have time to retrieve the map for the familiar context, before encoding a memory of the introduced sample objects. In this manner, the memory of the context and that of the objects would be encoded and retrieved independently or at distinct time points during the task. If, as many have proposed, nonspatial object recognition is supported by cortical structures, like the perirhinal cortex (Winters et al., 2004; Mumby 74

92 et al., 2005; Ainge et al., 2006), then intra-hippocampal muscimol should not impair object recognition in a version of the NOR task that explicitly dissociates the object and context memories. Our finding that intra-hippocampal muscimol impaired object memory in the Drop-in NOR protocol is consistent with our recent report that the hippocampus is required for retrieval of object memory independent of context (Cohen et al., 2013b), and both findings fail to support the interpretation that this form of object memory is dependent upon the perirhinal cortex. These results are consistent with the view that the hippocampal dependence of NOR is not explained by the spatial/contextual attributes of the task, but because the hippocampus is required for object memory processes. Together, these data suggest that a novel object-in-context representation is not created when the mice enter the familiar arena with objects during the sample and test sessions. Given that CA1 inactivation impaired object discrimination in each NOR protocol used (excluding the Conventional NOR protocol with <5-min delay; delaydependent hippocampal involvement in object memory), we conclude that it is not only the object-in-context memory that is impaired when hippocampal neuronal function is suppressed. Accordingly, these results demonstrate a compelling delay-dependent, contex-independent role of the rodent hippocampus in object memory. The present studies provide support for the cognitive map theory proposed by O Keefe & Nadel in 1978, stating that the hippocampus holds a general map of one s environment, while also significantly participating in object memory processing. The reported experiments offer invalidations of the object-in-context representation proposed to be a contributing factor for the role of the hippocampus during the novel object recognition paradigm. 75

93 PART V: EVERY PICTURE TELLS A STORY: EVIDENCE FOR PICTURE- OBJECT EQUIVALENCE IN MICE 5.1 Abstract. Picture-to-object equivalence, or the recognition of a three-dimensional (3D) object after viewing a two-dimensional (2D) photograph of that object, is a higher-order form of visual cognition demonstrated in primates and some birds, but thought to be beyond the perceptual ability of rodents. Behavioral and neurobiological mechanisms that support picture-to-object equivalence are not well understood. We used a modified visual recognition task, reminiscent of those used for primates, to test whether representational insight, necessary for picture-to-object equivalence, extends to mice. Mice that freely explored 2D photographs of an object during a sample session, were presented with the actual 3D object from the photograph and a novel 3D object during a test session 24 h later. Mice preferentially explored the novel 3D object, indicating recognition of the familiar 3D object. Post-sample inactivation of the hippocampus significantly impaired visual and object discrimination, likely by disrupting consolidation of memory for the 2D sample object. Accurate object discrimination was observed regardless of whether the explored 2D photographs depicted radially symmetric, radially asymmetric, visually similar, rotated or silhouetted objects, as well as when the novel test stimulus was presented in a 2D picture. Results also suggest that discrimination behavior is guided by a memory of the whole pictured object, rather than of its individual feature(s). Collectively, 76

94 we provide the first evidence that mice exhibit picture-to-object equivalence, and that this perceptual ability requires hippocampal-dependent visual recognition memory. These results offer strong support for the use of mice to investigate mechanisms and disorders of human memory and cognition. 5.2 Introduction. It is often said that a picture is worth a thousand words. Viewing a vacation photo, for example, can elicit full recollection of the when, where, why, and what was happening at the instant the photo was taken. The picture functions as a representation of that episodic memory. Photographs or pictures representing real-world physical items have been traditionally used to study visual recognition memory in primates, birds and rodents because they provide a consistent stimulus presentation regardless of viewing angle or orientation of the subject. However, it is not clear whether such studies elicit the perceptual inference in all species studied that relates a pictured object to its 3- dimensional (3D) physical form, known as picture-object equivalence (Bovet and Vauclair, 2000). Infants can differentiate 3D objects from their 2-dimensional (2D) picture representation (Hochberg and Brooks, 1962; Bower, 1972), yet the ability to form relationships between symbolic representations and their real-world references follows a developmental arc which may preclude younger infants from exhibiting representational inference (Pierroutsakos et al., 2005; DeLoache et al., 2010). With regard to non-humans, numerous animal species are able to adequately perform 2D picture recognition (Lubow, 1974; Prusky et al., 2004; Judge et al., 2012) and 2D picture to 3D object equivalence has been demonstrated in some nonhuman primates, and in pigeons (Rosenfeld and Van Hoesen, 1979; Savage-Rumbaugh et al., 1980; Watanabe, 1993). However, the extension 77

95 of these abilities to other species is not well established. Further, the neurobiological circuits that support picture-object equivalence have not been determined. Given the inherent difficulties in conducting mechanistic studies of the neural circuitry supporting higher-order cognition using primate models, there is pressure to define simpler, albeit appropriate, model systems. One animal model of particular interest is the rodent, which has proven effective in deciphering the dependence of long-term episodic memory on the medial temporal lobe (MTL), and the specific role for the hippocampus in spatial memory. Although rats can perform 2D picture recognition (Prusky et al., 2004; Clark et al., 2011), disagreement remains as to whether the neural circuitry supporting nonspatial, visual recognition memory in the rodent brain includes the hippocampus. Recent reports demonstrate a significant role of the mouse hippocampus in nonspatial object recognition memory (Hammond et al., 2004; Cohen et al., 2013b). However, the representational insight (Aust and Huber, 2010) that arises from perceiving that a 2D picture of an object corresponds to its 3D physical form may be considered to be beyond the ability of rodents, as suggested by an extensive review of the literature (Bovet and Vauclair, 2000). Therefore, studies of representational insight in picture-object recognition have largely overlooked rodents as experimental subjects, in favor of human and nonhuman primates (Hochberg and Brooks, 1962; Bower, 1972; Rosenfeld and Van Hoesen, 1979; Savage-Rumbaugh et al., 1980). The preference for higher-order subjects in studies of advanced visual processing of objects may be driven by the low visual acuity of rodents and that their visual cortex lacks a functional columnar organization typical of cats, tree shrews and primates (Hendry and Reid, 2000). 78

96 Nonetheless, the question of whether rodents are capable of representational insight in picture-object recognition remains unanswered. Here, we tested whether mice could perform tasks with visual perceptual demands similar to those typically presented to primates and birds. Our results provide compelling evidence that rodents are capable of representation insight in studies of picture-to-object recognition. Importantly, our results provide the first evidence that mice can spontaneously generalize behavioral responses for viewed pictures of objects to their actual 3D forms. We show that if mice spend sufficient time viewing pictures of an object, then they are able to subsequently discriminate between a familiar 3D physical object and a novel 3D physical object. Using a number of variations on a traditional object recognition task, we determined that mice are able to discriminate novel 3D objects based upon prior exposure to their 2D referents even when presented in different physical forms (2D or 3D). However, temporary inactivation of the mouse hippocampus obliterates this cognitive ability, indicating that the representational insight required for picture-object equivalence in mice is hippocampal dependent. Consequently, these surprising results provide support for the view that rodents are capable of advanced hippocampal-dependent visual perceptual capabilities, and indicate the appropriateness of rodents as models for mechanistic studies of object recognition. 5.3 Materials and Methods Mice and surgery. Male C57BL/6J mice (7-10 wk old; Jackson Labs) were housed 4 per cage with ad libitum access to food and water. All procedures were conducted in accordance with NIH guidelines and were approved by the FAU IACUC. For all inactivation experiments, surgical implantation of guide cannulae (n = 30) was 79

97 completed one week after acclimatization to the vivarium. For all other experiments, mice (n = 70) began testing at 8 weeks old, after one week of vivarium acclimatization Intrahippocampal cannulation and microinfusion. For the inactivation experiments (Exp. 1, 3, S1 and S2), mice were implanted with chronic bilateral guide cannulae (Plastics One, Inc., Roanoke, VA) above the CA1 region of dorsal hippocampus (A/P mm, M/L ± 1.5 mm, D/V mm from bregma; corresponding to intermediate CA1), as previously described (Cohen et al., 2013b). Behavioral testing began 7-10 days later to permit postoperative recovery. Each mouse received a mock infusion each day for 2 days, immediately after the arena habituation, to acclimate the mice to the microinfusion procedure, as previously described (Cohen et al., 2013b). For the actual microinfusions, mice received bilateral (0.35 µl/side, 0.33 µl/min) intrahippocampal muscimol (Tocris, 1 µg/µl in 0.9% sterile saline) or 0.9% sterile saline immediately after the sample session. For the actual bilateral microinfusion procedures see (Cohen et al., 2013b) Novel object recognition task and protocols. For all experiments, the apparatus consisted of two open-top, high-walled square arenas made of white ABS (each: 37.5 x 37.5 x 50 cm). For Exp. 3, 4, 6, S4B, S5 and S6, the mice were restricted to a zone delineated by a clear acrylic square arena insert (each: 25.4 x 25.4 x 50 cm) throughout all stages of testing. During day 1 and 2, each mouse was permitted to explore one of the arenas during a 10-min empty arena habituation session. On days 3 and 4, each mouse received one sample session and one test session, respectively, in the habituated (i.e., familiar) arena. During the sample session, each mouse was returned to the familiar arena that now contained identical novel 3D objects or 2D pictures of objects (stainless 80

98 steel cabinet leveling foot, or plastic toy gorilla, or a stainless steel spring, each attached to a Plexiglas base). The 3D objects were placed on the floor in the NW and SE corners. The two pictures were positioned on the arena walls, 2 cm from the floor (NW and SE). Picture/object exploration was defined as any time the mouse spent with its head oriented toward and within 2-3 cm of the stimuli. Each mouse was removed from the arena upon accumulating a minimum of 30 s exploration of each object/picture or 38 s on either object/picture within 10 min. This sample object exploration criterion was imposed to ensure that all mice were matched for sample session performance. The data from eight mice that failed to reach the sample session exploration criteria, within the 10 min session, were removed from the analyses. During the test session, presented 24 h later, the familiar arena contained combinations of familiar or novel 2D pictures along with familiar or novel 3D physical representations of 2D stimuli (see Figure 11 & Figure 11.1 for stimuli pairings and arena set-up). The mouse was removed from the arena after either 5 or 10 min (Exp. S2, S3, and S5). The pictures, objects, and arena (floor, walls and insert) were cleaned with 10% ethanol after each session. All behavioral testing data was digitally acquired by the EthoVision XT (Noldus Inc., Leesburg, VA) software package. Object exploration was scored off-line from the digital video files by experimenters that were blind to the treatment condition of the mice. Object memory was inferred from test session stimuli exploration and the discrimination ratio calculated for each mouse by subtracting the time spent exploring the familiar picture/object from the time spent exploring the novel picture/object and dividing the result by the total time spent exploring both items (Discrimination io = T novel T familiar / T novel + T familiar ). Discrimination ratio scores range from -1 to 1, with positive scores indicating novel stimulus preference, 81

99 while a ratio = 0 indicating chance performance or a lack of preference for one stimulus over another Data analysis. All data sets were normally distributed which allowed for analyses to be performed using parametric statistical tests. The discrimination ratio and latency (in s) to reach the sample object exploration criterion of saline- and muscimoltreated mice were compared using two-tailed Student s t-tests, paired t-tests were used to compare exploration times of each test session stimulus, and one-sample t-tests were used to compare the object discrimination behavior of the mice to chance performance. Interexperimental differences in discrimination ratio were assessed using two-tailed Student s t-tests. For Exp. 5, respective picture exploration times were analyzed by a withinsubjects ANOVA, followed by post-hoc Bonferroni pairwise comparisons using Holm- Sidak confidence-interval adjustments. For Exp. S2, discrimination ratios were analyzed by a within-subjects ANCOVA, with total exploration as a covariate Histology. Each mouse that had received intra-hippocampal microinfusions was deeply anesthetized with 5% isoflurane at the conclusion of the respective experiment, and brains were dissected and preserved in 4% paraformaldehyde. Cannulae placements were confirmed by examination of cresyl violet-stained coronal 50 µm brain sections under a light microscope (see Figure 11.2B). Data for any mice determined to have inappropriately placed cannulae were excluded from the analyses (n = 6). 5.4 Results. "These creatures you call mice, you see, they are not quite as they appear." - Douglas Adams 82

100 Recognition of a 3D object after viewing a 2D image of that object is a higherorder process of visual perception known as picture-object equivalence. This cognitive process has been demonstrated in primates, birds and other animal species; however, we provide the first evidence of this capacity in mice. For the following experiments, refer to Figure 11, Figure 11.1 and the Materials and Methods Section for specific methodological details. We first verified that naïve mice could successfully perform a 2D picture recognition memory task (Figure 11.1-Exp. S1), and demonstrated that consolidation of such picture memory is hippocampal-dependent (Figure 11.2A), confirming previous reports (Prusky et al., 2004; Forwood et al., 2007; Clark et al., 2011) Experiments 1-4: Recognition of a 3D Object from a 2D Picture is Hippocampal Dependent Regardless of Symmetry, and is Not Affected by Low-Level Visual Properties or Viewing Angle. We tested whether mice could spontaneously generalize recognition of actual 3D objects based on their memory of previously viewing a 2D picture of the object; behavior indicative of picture-object correspondence. Secondly, we tested whether that capacity required hippocampal-dependent memory (Figure 11, Exp. 1). During the sample session, mice explored 2D pictures of a radially symmetric object (foot). Latency to criterion was similar between future treatment groups indicating that all mice displayed similar motivation and attention to explore pictures [saline 587 s, muscimol 597 s; t(18) = -0.99, n.s.]. During the test session, mice were presented with 3D objects, the familiar, actual physical object that had been presented in picture form during the sample session (foot), and another that was novel (monkey). Mice that received intrahippocampal saline 83

101 immediately after the sample session, preferentially explored the novel monkey over the familiar foot during the test session [t(8) = -5.63, P < 0.001], while those that received intrahippocampal muscimol explored both objects equivalently [t(10) = -0.28, n.s.]; yielding significant differences in discrimination ratios [t(18) = 2.82, P < 0.05, Figure 12A] and no differences in total object exploration [saline 62 s, muscimol 56 s; t(18) = 1.15, n.s.]. These results suggest that post-sample hippocampal inactivation impaired memory consolidation for the pictured object, and thereby compromised the ability to mentally compare the 3D test objects to the sample session pictures. The exploration preference for the novel object over the familiar one is consistent with the representational inference necessary for picture-object correspondence. As compared to previously published data, these 2D-3D saline-treated mice and saline-treated mice tested in a standard object recognition task (3D objects for all sessions, 3D-3D mice (for data see Cohen et al., 2013b)) exhibit equivalent discrimination of the novel and familiar objects [t(15) = -1.16, n.s.]. Although both groups exhibit preference for the novel object, 2D-3D mice explore the familiar object longer [t(15) = 2.82, P = 0.01]. This additional exploration time may be required to associate the familiar 3D object with the recalled memory of the 2D picture. A follow-up 2D-3D experiment indicated that object discrimination improved when test session duration was doubled (Exp. S2), yet the muscimol-treated mice were, again, impaired (Figure 12.1). Similarly, for 3D-3D mice (for 5 min data see Cohen et al., 2013b), object discrimination improved when test session duration was doubled [Exp. S3; t(19) = -2.81, P = 0.01, Figure 12.2]. These findings indicate that unlike other animals (Humphrey, 1974), novelty-induced 84

102 exploration does not diminish over a short period, regardless of the form in which sample stimuli were presented. A follow-up picture-guided object discrimination experiment revealed that even under conditions in which objects were of similar size, color and luminance to account for salient low-level features, mice exhibit a preference for the test-session novel object (foot and spring; Figure 11, Exp. 2; t(9) = -3.85, P < 0.01). Evidently, mice are able to discriminate between test session familiar and novel objects regardless of visual similarities [t(9) = 3.71, P < 0.001, Figure 12B]. Successful picture-object recognition could have been a consequence of our pictured sample object having radial symmetry, making it invariant to viewing angle and easier to generalize to the actual object. Therefore, we permitted mice to view pictures of a radially asymmetric object from within a Plexiglas insert (monkey; Figure 11, Exp. 3). The use of the insert was necessary since the results of Exp. S4A and S4B (Figure 12.3) suggest that tactile information afforded by objects interferes with the expression of picture-object correspondence. Similar to Exp. 1, saline-treated mice preferentially explored the novel over the familiar object [t(8) = -3.69, P < 0.001], while muscimoltreated mice demonstrated equal preference [t(8) = 1.83, n.s.]; generating significant differences in discrimination ratio scores [t(16) = 4.49, P < 0.001, Figure 12C], but no difference in total object exploration [saline 51 s, muscimol 57 s; t(16) = -1.25, n.s.]. Correctly associating the internal representation of the picture to the actual object, regardless of its complexity, enables it to be identified as familiar, resulting in preferential exploration of the novel object. 85

103 Further, in a more challenging version of the task, sample pictures from Exp. 3 were replaced with pictures depicting a rotated/side-view of the monkey (Figure 11, Exp. 4). During the test session, mice discriminated between the familiar monkey, now presented from a different view than that of the sample picture, and the novel foot [t(9) = 4.07, P < 0.01, Figure 12D], with preference for the foot [t(9) = -3.51, P < 0.01]. Doubling test session duration (Exp. S5), increased subsequent object discrimination [t(9) = 4.86, P < 0.001, Figure 12.4], and novel object preference [t(9) = -5.12, P < 0.001]. Compared to the 5-min test, mice in the extended test demonstrated greater object discrimination [t(18) = 2.54, P < 0.05]. These results suggest that although some visual information about the monkey was absent from the sample picture, mice are likely performing a mental rotation of the remembered image to associate it with the 3D object during the test session. All above findings are contingent on a mouse s ability to perceive the difference between an actual object and a picture of that same object, as opposed to picture processing in a confusion mode (incapable of demonstrating equivalence since 2D and 3D stimuli are viewed as the same entity) (Fagot et al., 1999). To test this, mice visually explored the 3D monkeys, and then one monkey was replaced with a picture of that familiar monkey for the test session (Exp. S6). Object discrimination [t(8) = 7.80, P < , Figure 12.5] with preferential exploration of the familiar picture [t(8) = -6.79, P < 0.001] indicated that mice perceive that a picture of an object and the physical object are inherently different stimuli. 86

104 5.4.2 Experiments 5-6: Picture/Object Recognition is Limited to Composite Image Memory Retrieval. To determine if the recognition reported above reflected the memory of an individual feature of the sample pictured object, or of the composite sample image, mice explored pictures of the monkey (familiar) and a blank white picture, and 24 h later, explored the familiar picture, a scrambled version of the familiar picture, and a novel picture (Figure 11, Exp. 5). The novel and scrambled pictures were preferentially explored over the familiar [F(2, 27) = 10.03, P < 0.001; Figure 13A], with preferential exploration of the novel over the familiar (P < 0.001), the scrambled over the familiar (P = 0.001), and equivalent exploration of the novel and scrambled. These results indicate that preferential exploration of novel test stimuli is guided by memory of the composite image, rather than individual features. Complementary to Exp. 5, mice that explored scrambled pictures of the monkey (Figure 11, Exp. 6), failed to preferentially explore either the familiar or novel objects during the test session [t(7) = -0.35, n.s.], indicating non-discrimination [t(7) = 0.98, n.s., Figure 13B]. These results further suggest that mice can only identify objects previously viewed as pictures when the stimuli are recognizably similar Experiment 7: Evidence of Picture-Object Equivalence - Item Recognition of a Learned Silhouette. Matching a depicted abstract object to the actual object is a strong demonstration of cognition and equivalence. Similar to previous studies of higher-order species (Truppa et al., 2009), we tested item recognition when the pictures depicted less realistic images of the familiar object. Mice that explored silhouettes of the foot, discriminated between 87

105 the familiar foot and the novel spring during the test session [t(9) = 3.74, P < 0.01, Figure 14], preferentially exploring the spring [t(9) = -4.17, P < 0.01]. By modifying a primate task, our results strongly indicate that mice establish perceptual correspondence between pictorial representations of objects and their physical forms, indicative of picture-object equivalence. 5.5 Discussion. General knowledge about real-world items is largely acquired during indirect experiences with stimuli, which act as symbols of the actual physical item. For example, we can learn about the Eiffel Tower by repeatedly experiencing symbolic representations of it in the media; in doing so, we can deduce its actual structure. Upon our first visit to Paris, we recognize the tower from our memory of the previously viewed images; clearly, pictures are worth a thousand words. Pictures representing real-world physical items have been traditionally used to study visual recognition memory in primates, birds and rodents because they provide a consistent stimulus presentation regardless of viewing angle or orientation of the subject. However, it is unclear whether such studies elicit the perceptual inference, in all species studied, that relates a pictured object to its 3D physical form, known as picture-object equivalence (Bovet and Vauclair, 2000). Infants can differentiate 3D objects from their 2D picture representations (Hochberg and Brooks, 1962; Bower, 1972), yet the ability to form relationships between symbolic representations and their real-world references follows a developmental arc which may preclude younger infants from exhibiting representational inference (Pierroutsakos et al., 2005; DeLoache et al., 2010). Brief exposures to pictures of objects influence choice behaviors of preverbal infants when the representative physical objects 88

106 are subsequently introduced. Such results indicate that infants encode memories/representations of objects through experiences with pictures, which guide their preference for novel objects (Shinskey and Jachens, 2014). Also, numerous animal species adequately perform 2D picture recognition (Lubow, 1974; Prusky et al., 2004; Judge et al., 2012), and although primates and pigeons demonstrate picture-to-object equivalence (Rosenfeld and Van Hoesen, 1979; Savage-Rumbaugh et al., 1980; Watanabe, 1993), extension to other species is not well established. Moreover, the neurobiological circuitry supporting picture-object equivalence has not been determined. Given the inherent difficulties in conducting mechanistic studies of the neural circuitry supporting higher-order cognition using primate models, there is pressure to define simpler, albeit appropriate, model systems. Of particular interest is the rodent, which has proven effective in deciphering the dependence of long-term episodic memory on the medial temporal lobe (MTL), and the specific role for the hippocampus in spatial memory. Although rats can perform 2D picture recognition (Prusky et al., 2004; Clark et al., 2011), disagreement remains as to the contribution of the rodent hippocampus to nonspatial, visual recognition memory. We recently reported a significant role of the mouse hippocampus in nonspatial object recognition memory (Hammond et al., 2004; Cohen et al., 2013b). However, the representational insight (Aust and Huber, 2010) that arises from perceiving that a 2D picture of an object corresponds to its 3D physical form may be beyond the ability of rodents, as suggested by the literature (Bovet and Vauclair, 2000). Prior studies have largely overlooked rodents as experimental subjects, in favor of higher-order subjects (Hochberg and Brooks, 1962; Bower, 1972; Rosenfeld and Van Hoesen, 1979; Savage-Rumbaugh et al., 1980), likely due to their low visual 89

107 acuity and the lack of columnar organization in the rodent visual cortex, which is typical of cats, tree shrews and primates (Hendry and Reid, 2000). We designed a task of picture-object equivalence, reminiscent of those used in primates, to test memory in nonverbal animals by exploiting rodents natural preference for novelty. Specifically, we tested whether mice could make inferential judgments between an object and a recalled memory of a picture depicting that object. Our findings that mice preferentially explore a novel over a familiar object (previously experienced in picture form) imply that mice exhibit picture-to-object correspondence, regardless of object symmetry, likeness, viewing angle, composition and image realism. Importantly, we confirmed that mice perceive the inherent difference between a picture of an object and the actual 3D object itself. Further, we find that picture memory consolidation, required for such higher-order inference, depends upon the dorsal hippocampus. Recognition memory is well established in mice, yet our results are the first to indicate that rodents are capable of advanced visual recognition and learn indirectly about actual objects by viewing images. Additionally, consistent with studies in non-human primates (Truppa et al., 2009), Exp. 7 confirms that mice recognize a 3D object from a retrieved memory of it presented in silhouetted picture, providing the first compelling demonstration of picture-to-object equivalence in mice. Given that our task relies on spontaneous exploration, we maintain that there are few other explanations for the results presented. However, the extent to which mice, and other animals, are able to fully conceptualize that the image is a pictorial representation of a 3D object remains unclear (Judge et al., 2012). Further investigation is necessary to determine the neural mechanisms that underlie symbolic representations in the mouse brain. 90

108 There are two primary ways to conduct these picture-to-object equivalence studies. In one scenario, the sample session stimuli could have consisted of 3D objects, and the test session of 2D pictures of objects. However, we chose the alternate, more challenging, scenario in which the sample session consisted of 2D pictures and the test session consisted of 3D objects. We contend that restricting the mouse s view of the object during the sample session to a single angle afforded by the picture, placed a greater demand on the mice to subsequently relate that picture information to the actual object which could be viewed from multiple angles during the test session. Additionally, this more complex experimental design is reminiscent of those used in studies of pictureobject equivalence in primates (Truppa et al., 2009; Shinskey and Jachens, 2014). Visual recognition and representational insight were both impaired by hippocampal inactivation in mice, consistent with the notion that the hippocampus contributes to nonspatial aspects of declarative memory (Rudy and Sutherland, 1989; Alvarado and Rudy, 1995), while in contrast to the view that the perirhinal cortex governs these processes (Winters et al., 2008). Our task design may assess hippocampaldependent object familiarity, provided we define familiarity as recalling the object presented based on a stored representation of that object viewed from a single angle in picture form. Thus, the post-sample temporary inactivation of the hippocampus disrupted 2D picture memory consolidation, and mice subsequently failed to discriminate between the test session stimuli, indicating the necessity of the hippocampus in a test of familiarity. The hippocampus plays an essential role in declarative or explicit memory enabling an individual to replay a story of a previously encoded experience. We 91

109 suggest that that story enables one to recognize items learned in picture form when they are subsequently presented in 3D form. The rodent hippocampus likely encodes and consolidates the picture exploration as a story of that experience within a specific context as a form of explicit memory. Our findings provide the first indication that a functional rodent hippocampus is required for picture-to-object equivalence. Additionally, we provide the first evidence that mice make perceptual and conceptual judgments about presented task stimuli, which is surprising given that representational insight has been considered a defining capacity of primates. Taken together, our results provide convincing evidence that the mouse may serve as an effective model system to investigate higher-order sophisticated aspects of mammalian visual perception and recognition. 92

110 PART VI: OBJECT RECOGNITION MEMORY: DISTINCT YET COMPLEMENTARY ROLES OF THE MOUSE HIPPOCAMPUS AND PERIRHINAL CORTEX 6.1 Abstract. Although it is clear that an intact hippocampus is essential for spatial components of declarative memory in rodents, object recognition memory has been traditionally attributed to a functioning perirhinal cortex. However, several studies have demonstrated hippocampal involvement in the Novel Object Recognition Task, and it has been implied that these structures are differentially involved based on memory strength. Alterations made to the sample session exploration criteria, or the accumulation of object information, result in object memories of varying strength. In an attempt to resolve this controversy in the literature, the current studies employ temporary local inactivation techniques and immunohistochemistry in order to parse apart the contributions of these structures to object recognition. Temporary inactivation of the CA1 region of hippocampus immediately after sample session only impaired strong object memory, while transient inactivation of perirhinal cortex post-sample only impaired weak object memory. Additionally, the transcription and translation of proteins are required for the consolidation of episodic memory, and activation patterns of immediate early genes, like c-fos and arc, have been linked to neuronal activation and synaptic plasticity. Analyses of these proteins during various stages of the object recognition task suggest that strong 93

111 object memory is dependent on the dorsal CA1 region of the hippocampus, while weak object memory is perirhinal cortex dependent. Taken together, the results suggest that both structures are required for object memory under varying conditions. We assert that upon encountering a novel object, the perirhinal cortex begins to encode information about that item, forming a weak memory for the object. As a result of significant object exploration, a threshold amount of information is acquired, resulting in a strong hippocampal-dependent memory. These data serve to reconcile the dissension in the literature by demonstrating a functional role for both the hippocampus and perirhinal cortex in rodent object memory. 6.2 Introduction. Declarative, or explicit, memory is dependent on a number of structures within the mammalian medial temporal lobe (Eichenbaum, 2000; Squire et al., 2004). Unimodal sensory stimuli is sent from the perirhinal cortex to the hippocampus via the lateral entorhinal cortex (Burwell and Amaral, 1998; Witter and Amaral, 2004). Lesions to the hippocampus impair rodent performance on spatial learning and memory tasks (Morris et al., 1982; Riedel et al., 1999), establishing that the hippocampal formation is required for spatial memory processes (O'Keefe, 1976; Eichenbaum et al., 1990; McNaughton et al., 2006). Non-spatial memory has been attributed to the hippocampus, as demonstrated in studies (Cave and Squire, 1991; Bunsey and Eichenbaum, 1996; Eichenbaum et al., 1999; Alvarez et al., 2001). However, non-spatial object memory has been attributed to processes of the perirhinal cortex as demonstrated in non-human primates and rodents (Buffalo et al., 1999; Winters and Bussey, 2005). Yet, recent reports state that the rodent hippocampus is critical for object recognition memory (Cave and Squire, 1991; Clark et 94

112 al., 2000; Broadbent et al., 2010; Clarke et al., 2010; Cohen et al., 2013b). This apparent schism within the literature has yet to be reconciled. Perhaps both structures are necessary for object memory but in different capacities (Cohen and Stackman Jr, 2015). The functional dichotomy between the hippocampus and perirhinal cortex has been heavily studied, resulting in different theories for the types of non-spatial memory each processes. One theory posits that recollection is attributed to the hippocampus while familiarity is perirhinal cortex dependent. For example, reports state that rats with hippocampal s exhibit decreased recollection but increased familiarity, arguing that these structures are functionally independent with the hippocampus mediating recollection only (Sauvage et al., 2008). Similarly, it has been shown that local microinfusion of lidocaine into rat perirhinal cortex produces impairments in object memory, providing evidence that the perirhinal cortex is necessary for recollection (Winters and Bussey, 2005). An alternative view states that the functional difference between hippocampus and perirhinal cortex is with respect to strong and weak forms of non-spatial object memory (Squire et al., 2007). Accordingly, we assert that the functional difference between these structures is rooted in the gradient of object memory strength; strong object memory being hippocampal-dependent and weak object memory being perirhinal cortex dependent. The apparent variability in the perceived roles of these structures in object memory may stem from differences in experimental protocol. To investigate this notion in mice, we altered the sample session exploration criterion to produce either strong or weak memories. By controlling the duration of sample session object exploration to short or long time periods, memory strength was manipulated as demonstrated through test session object discrimination. Discreet temporary inactivation 95

113 of the CA1 region of the dorsal hippocampus or perirhinal cortex immediately after the sample session during either weak or strong object memory training, provided evidence for how these regions contribute to object recognition. Inactivation of CA1 after weak memory training did not affect test session performance; however, inactivation of CA1 after strong object memory training elicited significant impairments in object discrimination. In contrast, inverse findings were observed with the inactivation of perirhinal cortex, providing compelling evidence that the hippocampus processes strong object memory while the perirhinal cortex supports weak object memory. In addition, immunohistochemical techniques were employed to quantify the first proteins produced following neuronal activation, known as immediate early gene (IEG) proteins (Jones et al., 2001). Classically, analyses of c-fos and arc protein expression have been used in determining areas of the brain that are recruited at specific time points within a given task (Kubik et al., 2007; Albasser et al., 2010a; Katche et al., 2010). We designed the IEG behavioral protocol similar to the inactivation studies to permit protein expression comparisons between the CA1 region of hippocampus and perirhinal cortex under the different strong and weak memory testing conditions. Significant increases in IEG protein expression were only observed in CA1 following a strong object memory sample session. However, a weak object memory sample session elicited increased IEG protein expression only in perirhinal cortex. Based on our findings, we assert that strong object memory relies on the dorsal CA1 region of hippocampus and weak object memory on perirhinal cortex. Taken together, the present studies reaffirm that the functional distinction between these structures is based on memory strength. 96

114 6.3 Materials and Method Mice. Male C57BL/6J mice (7-10 wk old; Jackson Labs, Bar Harbor, ME) were housed 4 per cage with ad libitum access to food and water. The room temperature was maintained at 22 ± 4 C and humidity at 50 ± 5%. A 12-hour light/dark cycle was maintained beginning at 7:00 AM. All experimental procedures were conducted during the light period. All procedures were conducted in accordance with NIH guidelines and were approved by the IACUC. For the inactivation experiments, surgical implantation of guide cannulae (n = 90) was completed one week after acclimatization to the vivarium. Testing began 7 days post-operatively, when the mice were 9 weeks old. For the immunostaining experiments, testing began after one week acclimatization to the vivarium, when the mice were 8 weeks old (n = 66) Intrahippocampal cannulation and microinfusion. For the inactivation experiments, mice were implanted with chronic bilateral guide cannulae (Plastics One, Inc., Roanoke, VA) above the CA1 region of dorsal hippocampus (A/P mm, M/L ± 1.5 mm, D/V mm from bregma; corresponding to intermediate CA1), as previously described (Cohen et al., 2013b), or above the perirhinal cortex (A/P mm, M/L ± 4.0 mm, D/V mm from bregma). Behavioral testing began 7 days later to permit postoperative recovery. Each mouse received a mock infusion each day for 2 days, immediately after the arena habituation, to acclimate the mice to the microinfusion procedure, as previously described (Cohen et al., 2013b). For the actual microinfusions, mice received bilateral (0.35 µl/side, 0.33 µl/min) intra-hippocampal/intra-perirhinal cortex muscimol (Tocris, 1 µg/µl in 0.9% sterile saline) or 0.9% sterile saline immediately after the sample session. The procedures for the actual bilateral 97

115 microinfusion followed that described above for the mock infusion; however, this time the inserted infusion cannulae penetrated 1 mm beyond the tip of the guide cannulae to achieve bilateral intra-hippocampal/intra-perirhinal cortex infusion Novel object recognition task and protocols for inactivation studies. For all experiments, the apparatus consisted of two open-top, high-walled square arenas made of white ABS (each: 37.5 x 37.5 x 50 cm). For the inactivation experiments, during day 1 and 2, each mouse was habituated to one of the arenas for a 10 min empty arena session. On days 3 and 4, each mouse received one sample session and one test session, respectively, in the habituated (i.e., familiar) arena. During the sample session, each mouse was returned to the familiar arena that now contained two identical 3D objects (stainless steel cabinet leveling feet, each attached to a Plexiglas base, 4.2 cm dia and 6.0 cm tall, or plastic toy gorilla attached to a Plexiglas base) positioned in the northwest and southeast corners. To test strong object memory, each mouse was removed from the arena upon accumulating 30 s of exploration of each object or 38 s of either object, or 10 min had elapsed. To test weak object memory, each mouse was removed from the arena upon accumulating 10 s of exploration of each object or 13 s of either object, or 10 min had elapsed. This sample object exploration criterion was imposed to ensure that all mice were matched for sample session performance. The data from five mice that failed to reach the sample session exploration criteria were removed from the analyses. Microinfusions were administered immediately after the mouse was removed from the arena. During the test session, presented 24 h later, the familiar arena contained one of the familiar objects and one novel object (see Figure 15A). The mouse was removed from the arena after 5 min. The objects, arena floor and walls, were cleaned with 10% ethanol 98

116 after each session to remove olfactory cues. For the immunostaining experiments, variations to each daily protocol were instituted to ensure that behavioral comparisons could be made with yoked groups of mice (see Figure 15B). Groups of mice were perfused 90 min after each respective testing session. All behavioral testing data was digitally acquired by the EthoVision XT (Noldus Inc., Leesburg, VA) software package. Object exploration was scored off-line from the digital video files by experimenters that were blind to the treatment condition of the mice. Object memory was inferred from the discrimination ratio calculated for each mouse by subtracting the time spent exploring the familiar object from the time spent exploring the novel object and dividing the result by the total time spent exploring both items. Discrimination ratio scores range from -1 to 1, with positive scores indicating novel object preference, negative scores indicating familiar object preference and a ratio = 0 indicating chance performance or a lack of preference for one object over another Data analysis. The discrimination ratio and latency (in s) to reach the sample object exploration criterion scores of saline- and muscimol-treated mice were compared using two-tailed Student s t-tests. For the immunostaining experiments, strong and weak object memory protocol groups were compared using two-tailed Student s t- test Histology. For the inactivation studies, each surgically implanted mouse was deeply anesthetized with 5% isoflurane and brains were dissected and preserved in 4% paraformaldehyde. Cannulae placements were confirmed by examination of cresyl violet-stained coronal 50 µm brain sections under a light microscope. Data for any mice 99

117 determined to have inappropriately placed cannula were excluded from the analyses (n = 6). See Figure 16A for representative photomicrographs of cannula placement Immunohistochemistry. Ninety min following the conclusion of testing, mice were deeply anesthetized with sodium pentobarbitol. Each mouse was transcardially perfused with 0.1 M phosphate buffer, followed by 4% paraformaldehyde and brains were dissected and preserved in 4% paraformaldehyde for 5 days. Thirty µm thick slice sectioning was performed using a cryostat microtome. Sections were then stained using a standard immunohistochemical technique targeting the immediate early genes, c-fos or arc. On day 1, the tissue was quenched by incubation in 1% hydrogen peroxide. Endogenous peroxidase was then blocked with a solution of 0.3% triton and 5% normal goat serum solution in 0.1 M phosphate buffer. Next, the tissue was incubated in 0.1 M phosphate buffer containing c-fos or arc rabbit polyclonal antibody (1:500), and 3% normal goat serum and left to rotate overnight. On day 2, the tissue was washed with 0.1 M phosphate buffer and incubated in biotinylated goat anti-rabbit secondary antibody (1:200) and 3% normal goat serum. After 2 hrs the tissue was washed and processed with avidin-biotinylated peroxidase enzyme complex (ABS, 1:40) in 0.1 M phosphate buffer. The tissue was then quickly placed in diaminobenzidine (DAB), which allowed for visualization of the chemical reactions. Then the tissue was placed in a final wash of 0.1 M phosphate buffer. On days 2 and 3, the sliced sections of tissue were then mounted on gelatin-coated slides, counter stained using cresyl violet, coverslipped, and allowed 5 days to completely dry. Every third section (30µm) was taken during slicing such that each section was 90 µm apart from the previous. To quantify c-fos or arc-positive neurons in the hippocampus and perirhinal cortex, images were taken at the septal, 100

118 intermediate, and temporal levels of CA1. Bilateral images were matched from each respective level so that six sections were representing each animal. See Figure 16B for representative photomicrographs Cell Counting. The tissue sections were analyzed on a Nikon Eclipse 55i compound microscope and photographed using a Nikon DS-Fi1 camera at 100x magnification. Estimates of c-fos or arc-positive cells were made by the primary experimenters (to confirm unbiased counting), in CA1 and perirhinal cortex. The counters were blind to experimental condition by randomization of the order the tissue sections were presented. Nuclei in the target regions were counted if the color intensity of the label stain was greater than that of the background. Since each mouse was represented by three bilateral sections spanning the target region, the 6 counts were then averaged to provide one demonstrative count for each mouse. Various groups were then compared to determine significant differences in area activation Immunostaining Data Analysis. For the behavioral session of interest, the sample session, averaged regional counts were normalized according to matched pairs of animals for time in the arena (one AH3 to one sample session or one sample session 2 to test session). This procedure of normalization is needed to reduce the staining variability for each batch of mice and to ensure that any activation differences between mice were not a result of time in the arena. The normalization involved dividing the mean neuronal counts in each animal for a specified site by the combined means of both animals in the yoked pair. Normalized counts were then analyzed using a two-way ANOVA. For a significant region x memory strength interaction, Bonferroni post-hoc analyses were 101

119 performed. Group comparisons involving unyoked animals were analyzed by two-tailed Student s t-tests. 6.4 Results Inactivation Findings Strong Object Memory-Hippocampal Inactivation. Naïve mice received intra-hippocampal muscimol or saline immediately following the sample session. The sample session concluded for each mouse when the strong sample session exploration criterion was reached (30 s of exploration of each object or 38 s of either object, or 10 min had elapsed). Both groups reached sample session exploration criterion at similar times [saline 469 s, muscimol 459 s; t(21) = 1.93, n.s]. During the test session, 24 hr later, both groups spent equivalent times exploring the test session objects but the muscimol group discrimination ratios were significantly lower than those of the saline group [t(21) = 5.93, P < 0.001, see Figure 17A, reprint from (Cohen et al., 2013b)]. These findings suggest that inactivation of the CA1 region of the hippocampus after a sample session requiring considerable exploration of the objects, prevents strong object memory consolidation (Cohen et al., 2013b) Strong Object Memory-Perirhinal Cortex Inactivation. Naïve mice received intra-perirhinal cortex muscimol or saline immediately following the sample session. The sample session concluded for each mouse when strong sample session exploration criterion was reached. Both groups reached sample session exploration criterion at similar times [saline 565 s, muscimol 523 s; t(25) = 1.30, n.s]. During the test session, 24 hr later, both groups spent equivalent times exploring the test session objects and demonstrate similar discrimination of the novel object from that of 102

120 the familiar [t(25) = 0.62, n.s., see Figure 17A]. These results suggest that inactivation of the perirhinal cortex after a sample session requiring considerable exploration of the objects, has no impairing effects on strong object memory consolidation Weak Object Memory-Hippocampal Inactivation. Naïve mice received intra-hippocampal muscimol or saline immediately following the sample session. The sample session concluded for each mouse when the weak sample session exploration criterion was reached (10 s of exploration of each object or 13 s of either object, or 10 min had elapsed). Both groups reached sample session exploration criterion at similar times [saline 208 s, muscimol 153 s; t(18) = 0.05, n.s]. During the test session, 24 hr later, both groups spent equal times exploring the test session objects and the discrimination ratios were also equivalent [t(18) = -1.11, n.s., see Figure 17B]. These findings suggest that inactivation of the hippocampus after a sample session requiring minimal exploration of the objects has no impairing effects on weak object memory Weak Object Memory-Perirhinal Cortex Inactivation. Naïve mice received intra-perirhinal cortex muscimol or saline immediately following the sample session. The sample session concluded for each mouse when weak sample session exploration criterion was reached. Both groups reached sample session exploration criterion at similar times [saline 184 s, muscimol 182 s; t(18) = 0.07, n.s]. During the test session, 24 hr later, both groups spent equal times exploring the test session objects, however the discrimination ratios of the saline-treated mice were significantly greater than that of the muscimol-treated mice [t(18) = 2.73, P = 0.01, see Figure 17B]. These findings suggest that inactivation of the perirhinal cortex after a sample session requiring 103

121 minimal exploration of the objects significantly impairs weak object memory consolidation Immunohistochemical Findings Test Session Object Discrimination. During the test sessions for both the strong and weak memory protocols, mean discrimination ratio scores were calculated to confirm object memory was elicited with these variations in protocol testing sessions. Both groups discrimination ratio scores were significantly greater than chance [strong memory: t(5) = 8.53, P < 0.01; weak memory: t(3) = 6.71, P < 0.01], however the discrimination ratio of the strong object memory group was significantly greater than that of the weak object memory group [t(8) = 2.76, P = 0.03, see Figure 18A] Strong Object Memory. As a preliminary test of regional brain activation during the various stages of the NOR task in the CA1 region of the hippocampus, c-fos protein expression was initially quantified. Predictable activation patterns between groups of mice were analyzed for c-fos-positive neurons. Mice (n = 8) were sacrificed upon removal from the home cage to obtain a baseline level of c-fos expression in CA1. Comparisons were made to mice (n = 6) euthanized 90 min after an Arena Habituation 1 session (AH1). It can be assumed that upon entering a novel environment, the CA1 region of hippocampus should be engaged; leading to significant increases in c-fos expression from that of the home cage mice. Absolute c-fos counts for AH1 mice were significantly greater than that of home cage mice (t(12) = -7.15, P < 0.01), yielding anticipated patterns of activation. As another validation for the accuracy of the staining technique used, mice (n = 6) from AH1 and mice sacrificed 90 min after AH3 (n = 7) were compared. It was expected that the expression of c-fos would decrease 104

122 since there is no contextual novelty in AH3. C-fos positive neurons were significantly greater in AH1 mice as compared to mice in AH3 (t(11) = 3.47, P < 0.01). Finally, to investigate CA1 activity in the presence of only a novel object, normalized c-fos positive neurons were compared between mice that experienced a second sample session (S2; n = 8) and mice from a test session (n = 8). Interestingly, c-fos protein expression was significantly greater for test session mice than that of S2 (t(14) = -3.72, P < 0.01); likely reflecting the encoding of object novelty. As a better indication of the synaptic plasticity required for memory formation, arc protein was quantified for all further analyses. The specific sessions analyzed were AH3 and sample session to determine which structures are involved in object memory consolidation under a strong object memory protocol. The difference in protein expression between these groups is indicative of new object memory formation. Arc protein staining within the CA1 region, was significantly greater in the sample session group than the AH3 group (t(12) = 3.94, P < 0.01, see Figure 18B). We assert that this increased hippocampal activation was due to the exploration of the sample objects which were not present in the arena for AH3. The mean distance travelled by the mice in the AH3 group (2, cm) was not significantly different than that of mice in the sample session ( cm) (t(14) = 0.60, n.s.). This finding suggests that the mice in the sample session did not perceive the familiar context to be novel in the presence of novel objects; if they had, one would expect to see greater amounts of exploration during this session, and thus an overall greater distance travelled. Taken together, the significant increase in arc expression from AH3 to sample session is likely a result of CA1 plasticity, induced by object memory consolidation. 105

123 Conversely, analyses of arc protein expression in perirhinal cortex during strong AH3 and strong sample session demonstrate little involvement during these stages of the NOR task. Specifically, stained protein was not significantly different between AH3 and sample session mice (t(12) = 0.86, n.s., see Figure 18B). As expected, the lack of synthesized arc protein in the perirhinal cortex is attributed to the notion that strong object memory consolidation is not dependent on this structure Weak Object Memory. In testing weak object memory, hippocampal CA1 arc protein expression for the AH3 group was not significantly different from the sample group (t(12) = -1.07; n.s., see Figure 18C). This finding that the AH3 and sample groups are not different from one another in terms of arc-positive cells is indicative of no CA1 hippocampal involvement in weak object memory. This lack of group differences is probably due to the limited duration of object exploration dictated by the protocol. Conversely, in the perirhinal cortex, the sample group exhibited a significant increase in arc expression over that of the AH3 group (t(12) = 6.00, P < 0.001, see Figure 18C). The limited amount of exploration dictated by the protocol, elicited greater protein expression in the perirhinal cortex as compared to CA1 of hippocampus. This result is expected since object recognition memory has been traditionally attributed to processes of the perirhinal cortex Additional Analyses. To validate the accuracy of the staining technique used, groups of mice, with predictable activation patterns, were analyzed for arc-positive neurons just as had been done with c-fos (see section 2.2). Mice (n = 7) were sacrificed upon removal from the 106

124 home cage to obtain a baseline level of arc expression in CA1. Comparisons were made to mice (n = 7) euthanized 90 min after an Arena Habituation 1 session (AH1). Absolute arc counts for AH1 mice were significantly greater than that of home cage mice (t(12) = , P < 0.01); a predictable result. This finding confirmed that the staining protocol used yields appropriate patterns of arc activation. Since the CA1 region of the hippocampus processes context memory, an analysis of arc expression for AH3 groups in the strong and weak memory protocols should yield no difference. This comparison of AH3 groups in both protocols acts as a control providing confidence in the reliability of the data. Hippocampal CA1 protein levels were not significantly different between the two groups (t(12) = -0.93, n.s.). This result provides a baseline level of arc activity in the CA1 region of hippocampus within a familiar context, regardless of the time spent within it (i.e. strong and weak object memory protocols). Another interesting comparison in the CA1 region of hippocampus came from a separate subset of mice that experienced a second sample session, denoted S2 or a Test session in the strong object memory protocol, as reported in section 2.2. During the test session, the context and object positions were familiar to the mice; however, one of the objects was now novel. Arc-protein expression significantly increased during the test session (t(14) = -2.44, P < 0.05). The increase in arc expression from S2 to Test is likely due to the presence of a novel object, indicating a critical role for CA1 in object memory. 6.5 Discussion. The present set of experiments was designed to investigate the differential contributions of the hippocampus and perirhinal cortex to the classic novel object 107

125 recognition task in male C57BL/6J mice. By altering the amount of time the mice were allotted for sample session object exploration, weak or strong memories were formed. Using a temporary inactivation technique to block object memory consolidation and immunohistochemistry to stain for c-fos and arc proteins, the structures involved in object memory were evaluated. Two different sample session object exploration criteria were used to test whether the formation of strong object memory or weak object memory, respectively, differentially recruits CA1 or perirhinal cortex neurons. Firstly, the inactivation of CA1 during the consolidation of strong, but not weak, object memory impaired subsequent test session discrimination. Conversely, post-sample perirhinal cortex inactivation only impaired weak object memory discrimination during the test session, 24 hr later. These findings suggest that strong object memory is CA1 dependent, while weak object memory is perirhinal cortex dependent. As a corollary to the inactivation experiments, analyses of brain sections extracted from an additional cohort of mice showed that during the sample session of the strong memory protocol, a significantly larger ensemble of neurons was recruited in the CA1 region of the dorsal hippocampus when objects were present in the arena, as compared to when the mice were placed in the empty arena during AH3. This increase in active neurons, as reflected in arc-labelled neurons, during object exploration likely reflects the encoding of strong object memory within the hippocampus. This finding is in agreement with previous reports that when mice are allowed to explore each sample object for at least 30 seconds, the memory that is encoded for that object is dependent on an intact and fully functioning hippocampus (Cohen et al. 2013). However, when mice were trained in the weak object memory protocol there was no difference in neuronal activation between 108

126 the mice in the AH3 and the sample session groups. These findings indicate that strong object memory can be attributed to CA1 functioning, and not the perirhinal cortex, which is generally thought to govern object memory. Furthermore, the analyses of brain sections extracted from mice in the weak memory sample session group has shown that the ensemble of active neurons within the CA1 layer of the dorsal hippocampus was not significantly different in magnitude from the ensemble that was active in the hippocampus of mice in the weak memory AH3 group, during which mice were placed in an empty familiar arena. These results suggest that limiting the amount of novel object exploration results in a weak memory, reflected in weak object discrimination during the test session, which does not require the recruitment of the hippocampus. However, there was a significant increase in perirhinal cortex activation for mice trained in the weak object memory protocol. These results are in agreement with previous findings that have shown that when mice explored sample objects for a limited amount of time (in this case 10 seconds on each object), test session object recognition was by hippocampal or inactivation (Winters et al., 2008). These data imply that minimal exploration of novel objects requires the perirhinal cortex to maintain a weak memory for them; without recruiting the hippocampus. Lesion studies are not the most appropriate way to infer the function of a specific brain region, since the technique renders the region of interest destroyed and unavailable to process incoming information. Temporary inactivation is an alternative technique that avoids some of the pitfalls of the approach. Previous reports state that muscimol administration, directly to CA1, pre-sample session or pre-test, in a strong object memory protocol, leads to impairments in test session object memory (Cohen et al., 2013b). The 109

127 present studies provide additional inactivation data demonstrating a significant role for CA1 in strong object memory. Additionally, the current immunohistochemical findings provide further support by showing that a significantly greater number of CA1 neurons are active during a strong sample session as compared to perirhinal cortex. Alternatively, in a weak object memory protocol, only inactivation of perirhinal cortex led to impairments in test session object memory. These results are supported by the immunostaining findings showing increased neuronal activation in perirhinal cortex following a weak object memory sample session. While infusion techniques have proven to be an effective treatment to temporarily inactivate a given region of the brain, the technique is limited (for review, see Cohen and Stackman Jr, 2015). Future studies could employ optogenetics to selectively inactivate neurons in a given region for discreet time points. This technique would allow for cellular inactivation only while the mice are exploring the objects; better controlling the onset and offset of inactivation. The present studies lend support to the distinction of weak and strong memory demonstrating that the hippocampus supports strong memories, originally proposed by Dr. Larry Squire (2007). He suggested that the differences in memories supported by the hippocampus and perirhinal cortex were determined by memory strength, suggesting that medial temporal lobe structures neighboring the hippocampus were responsible for encoding weak memory, as opposed to familiarity. Furthermore, the present studies provide evidence for a phenomenon that has been well characterized on the behavioral level, by showing hippocampal activity on the molecular level. Increased hippocampal activation during the exploration of novel objects and imapirments in object discrimination following CA1 inactivation in the strong object memory protocol provides 110

128 a clear role for the hippocampus in strong object recognition memory. However, in agreement with much of the data, the inactivation and immunostaining findings from the weak object memory protocol provide support for a functional role of the perirhinal cortex in weak object recognition. This study provides evidence to suggest that object memory processing is completed by both the perirhinal cortex and the hippocampus in rodents, but that the recruitment of each structure depends on the strength of the memory, or alternatively, the amount of information gathered about the novel object. Our present findings provide a unifying theory on object memory processing, by providing evidence showing the necessity of both structures in object recognition memory. 111

129 PART VII: GENERAL DISCUSSION The neuroanatomical correlates of object recognition memory have been largely debated and the reported data has led to polarizing theories. The present set of experiments was designed with the intention of clarifying the role of the rodent hippocampus in non-spatial object memory. First, numerous studies were presented that clearly demonstrate a significant need for a functional hippocampus in object memory. Next, an attempt was made to reconcile the apparent schism in the literature by incorporating the present findings into the broader field. In doing so, a qualitative theory was developed for how the structures within the medial temporal lobe support object recognition memory in rodents. The decision to target the CA1 region of dorsal hippocampus in determining the regions directly supporting object memory is two-fold. First, the CA1 region is a reasonable target given that it represents the final output of the hippocampus. In addition, surgical manipulations guided toward CA1 are advantageous in that there is limited disturbance to overlying tissue. Also, there are direct projections from the perirhinal cortex that terminate in CA1, which will be discussed later (Naber et al., 1999; see Figure 19). The results of the current studies indicate that the the mouse hippocampus, and CA1 specifically, is necessary for object memory, particularly when the mice are trained in a strong object memory protocol. To confirm CA1 selectivity with these manipulations, mice inactivated with fluorophore-conjugated muscimol prior to the test session in a 112

130 strong object memory protocol displayed memory retrieval impairments (presented in chapter 3). These brains were processed and analyzed to determine the regional spread of the drug. Depicted in figure 8B is a representative example of the spread of FCM, which did not spread beyond the CA1 region of hippocampus, justifying the claims that these drug manipulations were reasonably specific. However, when considerably less time is allotted for object exploration training in a weak object memory protocol, the perirhinal cortex appears to be essential. These findings are extremely helpful in resolving the clear schism in the literature pertaining to the neural structures involved in object recognition memory in rodents (see Table 1 for a review of the previously reported studies). The studies clearly indicate that the mouse hippocampus is an integral structure in non-spatial, and not just spatial, memory. Employing a temporary inactivation technique in several variations of the traditional NOR task and immunohistochemistry staining to map behaviorally-triggered neuronal activation in distinct brain regions, revealed the differential roles of the hippocampus and perirhinal cortex in object memory. Additionally, the results assist in validating mice as a good animal model to study human related memory disorders. To summarize the studies presented, chapter 2 provides a review of the literature and alludes to a theory for how the hippocampus functions in object recognition memory. This bucket theory is then supported by the experiments presented in the subsequent chapters. The bucket theory states that as a mouse encounters a novel object, the perirhinal cortex begins to fill as new information about that item is acquired during its exploration. Once a critical threshold of information input has been reached, and after a specific amount of time has elapsed, the information spills out or is conveyed from 113

131 perirhinal cortical neurons to the hippocampus. If the critical threshold of information input is not achieved, then the information about the explored object remains within the perirhinal cortex, and is termed weak object memory. However, if the critical threshold is achieved, enabling sufficient information to be gleaned about the object, then the information about the explored object is transferred to the hippocampus, and is termed strong object memory. Further studies could attempt to elucidate the neural mechanisms by which the perirhinal cortex fills and spills into the hippocampus. Reportedly, long-term memory is dependent on a combination of glutamate receptors, CaMKII, PKA, PKC and ERK1/2 in the hippocampus; however, these pathways have also been implicated in memory formation in the perirhinal cortex (Izquierdo et al., 2006). An extensive analysis of how these pathways are activated or suppressed during strong and weak object memory training may be helpful in understanding the mechanisms underlying the filling of information within perirhinal cortex. Additionally, activitydependent activation of the Ras-MAPK pathway has been implicated in memory formation (Bolshakov, 2007). Analysis of NMDA glutamate receptors and calcium influx in hippocampal neurons, during NOR, may be beneficial in understanding the mechanisms by which information is transferred from the perirhinal cortex to the hippocampus. Chapter 3 provides the results of several experiments that demonstrate the hippocampal dependence of the encoding, consolidation and retrieval phases of strong object memory. Chapter 4 expands on the previous chapter s findings by presenting results from experiments using modified NOR protocols designed to dissociate object memory from already established context memories. The objective was to test whether such object from context dissociation affected the dependence of such object memory on 114

132 the hippocampus. Also, a delay-dependent recruitment of the hippocampus in the traditional NOR task was revealed, which is consistent with previous reports (Clark et al., 2000; Hammond et al., 2004). The current studies suggest the CA1 region of dorsal hippocampus is not recruited for a strong object memory task until at least 5, but less than 20, minutes has elapsed after object memory encoding. These results further demonstrate the critical delay-dependent hippocampal involvement in the NOR task. As the bucket theory illustrates, the object information is likely supported by other structures (i.e. the perirhinal cortex) during the period before the hippocampus is recruited. Chapter 5 presents data confirming mice are able to perform a purely visual picture recognition task and then asserts that mice can recognize 3D objects from learned 2D pictures. These findings are strengthened by the final study suggesting that mice recognize 3D objects from previously learned 2D abstract depictions. Additionally, these studies extend the prior chapters findings by indicating that picture-object correspondence, a perceptual task of object recognition, is also hippocampal dependent. Finally, chapter 6 provides results of experiments using functional inactivation and mapping of c-fos/arc protein expression, which demonstrate the functional dichotomy between the perirhinal cortex and hippocampus for object memory processes. The following sections examine how the present findings can be incorporated into the broader literature and how they help in reconciling the role of the hippocampus in object recognition memory. 7.1 Lesion Size Determines Hippocampal Involvement. The traditional approach has been extensively used in the investigation of the brain structures involved in the NOR task. Previous studies have reported that the extent of size is a main determinant of hippocampal involvement in object 115

133 memory. Lesions to less than 75% of the hippocampus have been ineffective in producing object memory impairments (Broadbent et al., 2004). Chapter 3 reports that a temporary inactivation of the hippocampus, with muscimol, produces object memory impairments even though less than 1% of total hippocampal volume was affected, as demonstrated in the FCM study. Clearly, there is a different explanation for why s do not produce the level of impairment seen with a temporary inactivation. As previously alluded to, likely the amount of time between and testing lends itself to compensatory mechanisms. It is possible that other regions within the medial temporal lobe support object memory when the hippocampus is taken offline for a significant amount of time greater than the half-life of a temporary drug inactivation. This calls into question the interpretations made about data collected from permanent studies. The permanent technique seems to be better suited to studying the degree to which the nervous system compensates for the loss of a specific brain structure. Spared object recognition memory elicited after a permanent is likely a result of extrahippocampal structures reorganizing to compensate for the loss. Temporary impairments may be a more appropriate technique for testing the involvement of a brain region in a time-dependent behavioral task (Redish, 2013). However, this technique also has limitations, which has increased the pressure to create better methods of investigating the neural correlates of different behavioral processes. These new techniques, including optogenetics and DREADDS, were briefly discussed in chapter State Dependency and Inactivation Techniques. Previous reports indicate that object memory relies on the hippocampus but other cortical structures within the medial temporal lobe can support these memories when the 116

134 hippocampus is impaired. Hippocampal s made after the encoding of object memory impair object discrimination; however, s made prior to encoding have no impairing effects (Mumby et al., 2007). These findings imply that when object memories are encoded in the presence of a functional hippocampus, the retrieval of that memory requires the hippocampus. However, if the object memory is initially encoded in the absence of a functional hippocampus, the retrieval of that memory also does not require a functional hippocampus. Therefore, it seems that hippocampal involvement is state dependent. Other studies report similar findings (Kesner et al., 1993; Winters et al., 2004; Forwood et al., 2005). All of these studies indicate that in the absence of a functional hippocampus during discrete stages of object memory tasks leads to impairments in a state dependent manner, while other times extra-hippocampal regions can compensate and sustain the memory. As previously described, s may not be an appropriate method to infer functionality of specific brain regions. Temporary pharmacological inactivation has arisen as a viable alternative to permanent s for studying brain function. Chapter 2 described the results of an experiment in which the hippocampus was inactivated during all memory processes (encoding, consolidation, and retrieval) to address the idea that the hippocampus is only involved in object memory in a state dependent manner. When the brain-state of the mouse was the same throughout the different stages of the task, hippocampal-dependence in object memory was observed. These findings are in stark contrast with the study previously discussed (Mumby et al., 2007), which implied that object memory initially encoded without a functional hippocampus can be accurately retrieved. 117

135 7.3 NOR is a Spatial Task. It has been argued that the NOR task is inherently spatial and will always recruit the hippocampus; making it difficult to study hippocampal involvement in object memory, irrespective of context. However, unlike the Morris Water Maze, the novel object recognition task does not require spatial navigation to elicit accurate behavioral performance. Yet, there are still aspects of the NOR task that contain spatial elements. The test session is conducted by replacing one of the familiar sample session objects with a novel object. Perhaps the novel object is viewed as novel because it is now in the location of a previously explored object. The hippocampus would be engaged by this object-in-location memory because it contains spatial aspects of context. It is not surprising that mice would show a preference for exploring the novel object since their memory of the spatial orientation of the arena has changed. To take this argument a step further, the mice treated with muscimol directly into the hippocampus demonstrate equal preference for the familiar and novel objects; likely as a result of impaired spatial and contextual memory for the arena. Therefore, it can be argued that the traditional NOR task cannot differentiate between context and object memories and therefore a majority of the reported studies likely test an object in context memory. However, there are several studies that better dissociate the two attributes of the object in context memory while still demonstrating hippocampal dependence of object memory. Similar to the use of a Y- maze (Winters et al., 2004), chapter 4 describes two different protocols meant to further dissociate the two memory types. The rationale was that by giving the mice a polarizing cue, or allowing them the time to recognize the arena prior to object exploration, the mice would more quickly determine that the arena was familiar, and have more time to focus 118

136 on the novel object(s). Likely, the impairments seen here, as opposed to the intact performance in the other studies, are due to protocol, i.e. technique or experimental design. In this sense, the notion that the NOR task is spatial in nature becomes irrelevant. 7.4 Strong and Weak Memories Reflect Recollection and Familiarity. Recognition memory is the ability to recognize previous events, items and experiences. There are two components of recognition memory that have been extensively studied, recollection and familiarity or remembering versus knowing. Research on episodic memory using rodents describes a functional and anatomical distinction between familiarity and recollection (Brown and Aggleton, 2001). Previous reports attribute familiarity to the perirhinal cortex (Winters and Bussey, 2005) and recollection to the hippocampus (Good et al., 2007; Sauvage et al., 2008). However, more recently, it has been proposed that familiarity and recollection describe the functional difference between weak and strong memories (Squire et al., 2007). Squire et al. (2007) report that perirhinal cortex neurons fire strongly to novel visual stimuli and less so once that stimulus is familiar (Squire et al., 2007). However, such novelty and recollection signals have also been detected in the hippocampus (Squire et al., 2007), supporting the view of a continuum of memory strength attributed to perirhinal cortex and hippocampus. The studies presented here support the notion that the difference between the perirhinal cortex and hippocampus is in terms of the strength of the memory formed. Proposed in chapter 2, the bucket theory implies that the initial weak information obtained through item exploration is perirhinal cortex dependent. However, once a critical threshold and time point is reached, the information strengthens to become hippocampal dependent. The fundamental quality of information is the same for both 119

137 structures; however, it is the quantity of information acquired that distinguishes the two structures. Less information is equivalent to a weaker memory, while more information/details is analogous to a stronger memory for that stimulus. 7.5 Direct Connections to the Hippocampus from Perirhinal Cortex. The lateral perforant path is largely discussed as communicating extrinsic efferents from the perirhinal cortex to the hippocampus indirectly through the entorhinal cortex (Hjorth-Simonsen, 1971; Hjorth-Simonsen and Jeune, 1972; Witter and Groenewegen, 1984; Liu and Bilkey, 1996). However, direct connections between the perirhinal cortex and hippocampus have also been reported (Knierim, 2015). Witter and Groenewegen (1984) demonstrated direct projections to the hippocampus from perirhinal cortex in the cat, and the same has been reported in the rat (Deacon et al., 1983; Kosel et al., 1983; Liu and Bilkey, 1996). Importantly, an interesting study reports that s to the entorhinal cortex of monkeys results in mild impairments in the delayed nonmatch to sample visual task (DNMS), while s to the perirhinal cortex results in severe impairments in the task (Meunier et al., 1993). It is understood that the communication between perirhinal cortex and hippocampus is essential in correctly performing a task of DNMS. Therefore, the previous findings, of differential impairments in the task elicited from s in the different regions, imply that direct connections between the perirhinal cortex and hippocampus must mediate correct performance in the task, and not the entorhinal cortex. Figure 19 illustrates the traditional indirect pathways of information flow from the perirhinal cortex to the hippocampus in solid lines, with the dotted lines representing the direct projections (Naber et al., 1999). The results of studies using electrophysiological stimulation and retrograde tracing procedures have demonstrated 120

138 that there are two main pathways sending information from perirhinal cortex to hippocampus: the indirect being, perirhinal cortex to entorhinal cortex to CA1/subiculum, and the direct being, perirhinal cortex to CA1/subiculum monosynaptically, which completely bypasses entorhinal cortex in the rat. Naber et al. (1999) studied these parallel pathways to determine the functional relevance of each and asserted that the functional difference between the direct and indirect pathways is in regard to the level of information processing required. Specifically, the direct pathway may send information as an identical copy to CA1, while the indirect pathway processes that information from the perirhinal cortex with converging inputs modulating the information before reaching CA1 (Naber et al., 1999). The present findings support the functional distinction made between the direct and indirect pathways if you consider that the weak and strong object memories are varied by their level of processing. Such varying levels of processing that may be a key feature in how the medial temporal lobe processes object information and in the processing of familiar and recollected information. 7.6 Conclusions. The results of the present set of experiments support the notion that the hippocampus and perirhinal cortex are both essential to object memory, but the contribution of these two regions can be functionally distinguished on the basis of memory strength. The compilation of studies presented here demonstrates the critical dependence of strong object memory upon the hippocampus, while weak object memory depends upon the perirhinal cortex. Further, the results of these studies support the view that the hippocampus is necessary for non-spatial object memory, while still maintaining a role for the perirhinal cortex. Thus, these results address a matter of much contention in 121

139 the literature on the neurobiological mechanisms of recognition memory. Using both temporary inactivation and immunohistochemical staining techniques, the results suggest a separate and important role for both of these medial temporal lobe structures, which help to address the inconsistencies within the literature while providing a unifying theory for object processing. The proposed bucket theory illustrates a possible functional mechanism by which the two structures work together to support memories of varying strength. Possibly, it is through these direct projections from perirhinal cortex to hippocampus that object memory is transferred to become stronger. Although they are generally overlooked, this direct pathway may be the key to better understanding the functional connections between these two structures. These data also lend support to the notion that mice can serve as appropriate animal model systems for studying human learning and memory disorders. 122

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144 127 Table 1. Index of the experimental details and results from a subset of published NOR studies testing the involvement of the rodent hippocampus and which a sample session exploration criterion was imposed. Abbreviations: HPC, hippocampus; antag, antagonist; diff, different; sig, significant; obj, object; PRH, perirhinal cortex; contra, contralateral; ipsi, ipsilateral; mpfc, medial prefrontal cortex; discrim, discriminate; exp, exploration; DR, discrimination ratio. Statistical analyses revealed no significant effect of treatment, arena characteristics, or rodent strain, on hippocampal involvement in the NOR task. Species & Strain Technique Structure Treatment Lesion size/ Time of Drug Administration Arena Criterion Delay Detailed Results Reference Summary of Findings Long-Evans inactivation HPC (dorsal) NMDA antag, APV (4 sites) pre-sample square 0.6m x 0.6 m 30-s sample 5-m not diff from sham (Baker and Kim, 2002) Long-Evans inactivation HPC (dorsal) NMDA antag, APV (4 sites) pre-sample square 0.6m x 0.6 m 30-s sample 3-h sig diff from sham (Baker and Kim, 2002) impaired Mouse C57BL/6J inactivation HPC (dorsal) muscimol pre-sample square 0.4m x 0.4m 30-s sample 24-h sig diff from sham (Cohen et al., 2013b) impaired Mouse C57BL/6J inactivation HPC (dorsal) muscimol post-sample square 0.4m x 0.4m 30-s sample 24-h sig diff from sham (Cohen et al., 2013b) impaired Mouse C57BL/6J inactivation HPC (dorsal) muscimol pre-, post-sample & pretest square 0.4m x 0.4m 30-s sample 24-h sig diff from sham (Cohen et al., 2013b) impaired Mouse C57BL/6J inactivation HPC (dorsal) anisomycin post-sample 0h and 2h square 0.4m x 0.4m 30-s sample 24-h sig diff from sham (Cohen et al., 2013b) impaired Mouse C57BL/6J inactivation HPC (dorsal) anisomycin post-sample 2h only (control study) square 0.4m x 0.4m 30-s sample 24-h not sig diff from sham (Cohen et al., 2013b)

145 128 Species & Strain Technique Structure Treatment Lesion size/ Time of Drug Administration Arena Criterion Delay Detailed Results Reference Summary of Findings Mouse C57BL/6J inactivation HPC (dorsal) muscimol pre-test (control study) novel context for test (square 0.2m x 0.2m) 30-s sample 24-h not sig diff from sham - controls fail to prefer novel obj (Cohen et al., 2013b) Mouse C57BL/6J inactivation HPC (dorsal) Lidocaine (0.5 µl of 4%/side) pre-sample square 0.4m x 0.4m 38-s sample 5-m not sig diff from sham (Hammond et al., 2004) Mouse C57BL/6J inactivation HPC (dorsal) Lidocaine (0.5 µl of 4%/side) pre-sample square 0.4m x 0.4m 38-s sample 24-h sig diff from sham (Hammond et al., 2004) impaired Lister hooded Lister hooded Lister hooded HPC Ibotenic acid Partial: 58% square 0.9m x 0.9 m HPC Ibotenic acid Partial: 58% square 0.9m x 0.9 m HPC Ibotenic acid Partial: 58% square 0.9m x 0.9 m 30-s sample 10-s not diff from sham - results differ if looking at first minute of test or 30-s exp. 30-s sample 1-m not diff from sham (Ainge et al., 2006) (Ainge et al., 2006) 30-s sample 10-m chance (Ainge et al., 2006) impaired Lister hooded Lister hooded Lister hooded HPC Ibotenic acid Partial: 58% square 0.9m x 0.9 m HPC Ibotenic acid Partial: 58% square 0.9m x 0.9 m HPC Ibotenic acid Complete: 98% square 0.9m x 0.9 m 30-s sample 1-h chance (Ainge et al., 2006) 30-s sample 24-h chance (Ainge et al., 2006) 30-s sample 10-s not diff from (Ainge et al., sham 2006) impaired impaired Lister hooded HPC Ibotenic acid Complete: 98% square 0.9m x 0.9 m 30-s sample 1-m not diff from sham (Ainge et al., 2006) Lister hooded HPC Ibotenic acid Complete: 98% square 0.9m x 0.9 m 30-s sample 10-m not diff from sham (Ainge et al., 2006) Lister hooded HPC Ibotenic acid Complete: 98% square 0.9m x 0.9 m 30-s sample 1-h not diff from sham (Ainge et al., 2006) Lister hooded HPC Ibotenic acid Complete: 98% square 0.9m x 0.9 m 30-s sample 24-h chance (Ainge et al., 2006) impaired Pigmented DA HPC NMDA Partial: average 58% rectangle 0.5m x 0.9m 40-s sample 5-m not diff from sham - HPC: 58% (40-93%) (Barker and Warburton, 2011)

146 129 Species & Strain Technique Structure Treatment Lesion size/ Time of Drug Administration Arena Criterion Delay Detailed Results Reference Summary of Findings Pigmented DA Pigmented DA Pigmented DA Pigmented DA Pigmented DA Pigmented DA Pigmented DA Pigmented DA Pigmented DA Pigmented DA Pigmented DA Pigmented DA Pigmented DA Pigmented DA HPC NMDA Partial: average 58% rectangle 0.5m x 0.9m HPC NMDA Partial: average 58% rectangle 0.5m x 0.9m PRH+HPC contra PRH+HPC contra PRH+HPC contra PRH+HPC ipsi PRH+HPC ipsi PRH+HPC ipsi mpfc+ HPC contra mpfc+ HPC contra mpfc+ HPC contra mpfc+ HPC ipsi mpfc+ HPC ipsi mpfc+ HPC ipsi NMDA PRH: average 85% HPC: average 54% NMDA PRH: average 85% HPC: average 54% NMDA PRH: average 85% HPC: average 54% NMDA PRH: average 86% HPC: average 61% NMDA PRH: average 86% HPC: average 61% NMDA PRH: average 86% HPC: average 61% NMDA mpfc: average 74% HPC: average 57% NMDA mpfc: average 74% HPC: average 57% NMDA mpfc: average 74% HPC: average 57% NMDA mpfc: average 69% HPC: average 54% NMDA mpfc: average 69% HPC: average 54% NMDA mpfc: average 69% HPC: average 54% rectangle 0.5m x 0.9m rectangle 0.5m x 0.9m rectangle 0.5m x 0.9m rectangle 0.5m x 0.9m rectangle 0.5m x 0.9m rectangle 0.5m x 0.9m rectangle 0.5m x 0.9m rectangle 0.5m x 0.9m rectangle 0.5m x 0.9m rectangle 0.5m x 0.9m rectangle 0.5m x 0.9m rectangle 0.5m x 0.9m 40-s sample 3-h not diff from sham 40-s sample 24-h not diff from sham 40-s sample 5-m not diff from sham 40-s sample 3-h not diff from sham 40-s sample 24-h not diff from sham 40-s sample 5-m not diff from sham 40-s sample 3-h not diff from sham 40-s sample 24-h not diff from sham 40-s sample 5-m not diff from sham 40-s sample 3-h not diff from sham 40-s sample 24-h not diff from sham 40-s sample 5-m not diff from sham 40-s sample 3-h not diff from sham 40-s sample 24-h not diff from sham (Barker and Warburton, 2011) (Barker and Warburton, 2011) (Barker and Warburton, 2011) (Barker and Warburton, 2011) (Barker and Warburton, 2011) (Barker and Warburton, 2011) (Barker and Warburton, 2011) (Barker and Warburton, 2011) (Barker and Warburton, 2011) (Barker and Warburton, 2011) (Barker and Warburton, 2011) (Barker and Warburton, 2011) (Barker and Warburton, 2011) (Barker and Warburton, 2011)

147 130 Species & Strain Technique Structure Treatment Lesion size/ Time of Drug Administration Arena Criterion Delay Detailed Results Reference Summary of Findings Pigmented DA Long-Evans HPC Radiofrequency Fornix Complete: ~80% square 1.0m x 1.0m HPC Ibotenic acid Complete: 89.8% square 0.9m x 0.9m 25-s sample 15-m not sig diff from sham - sig diff DR based on 1st min of test 30-s sample 10-s not sig diff from sham (Bussey et al., 2000a) (Clark et al., 2000) Long-Evans HPC Ibotenic acid Complete: 89.8% square 0.9m x 0.9m 30-s sample 1-m not sig diff from sham (Clark et al., 2000) Long-Evans HPC Ibotenic acid Complete: 89.8% square 0.9m x 0.9m 30-s sample 10-m sig diff from sham (Clark et al., 2000) impaired Long-Evans HPC Ibotenic acid Complete: 89.8% square 0.9m x 0.9m 30-s sample 1-h sig diff from sham (Clark et al., 2000) impaired Long-Evans HPC Ibotenic acid Complete: 89.8% square 0.9m x 0.9m 30-s sample 24-h sig diff from sham (Clark et al., 2000) impaired Long-Evans HPC Radiofrequency Complete: 71.2% square 0.9m x 0.9m 30-s sample 10-s not sig diff from sham (Clark et al., 2000) Long-Evans HPC Radiofrequency Complete: 71.2% square 0.9m x 0.9m 30-s sample 1-m not sig diff from sham (Clark et al., 2000) Long-Evans HPC Radiofrequency Complete: 71.2% square 0.9m x 0.9m 30-s sample 10-m sig diff from sham (Clark et al., 2000) impaired Long-Evans HPC Radiofrequency Complete: 71.2% square 0.9m x 0.9m 30-s sample 1-h sig diff from sham (Clark et al., 2000) impaired Long-Evans HPC Radiofrequency Complete: 71.2% square 0.9m x 0.9m 30-s sample 24-h sig diff from sham (Clark et al., 2000) impaired Long-Evans HPC Radiofrequency Fornix Complete: 100% square 0.9m x 0.9m 30-s sample 10-s not sig diff from sham (Clark et al., 2000) Long-Evans HPC Radiofrequency Fornix Complete: 100% square 0.9m x 0.9m 30-s sample 1-m not sig diff from sham (Clark et al., 2000)

148 131 Species & Strain Technique Structure Treatment Lesion size/ Time of Drug Administration Arena Criterion Delay Detailed Results Reference Summary of Findings Long-Evans HPC Radiofrequency Fornix Complete: 100% square 0.9m x 0.9m 30-s sample 10-m not sig diff from sham (Clark et al., 2000) Long-Evans HPC Radiofrequency Fornix Complete: 100% square 0.9m x 0.9m 30-s sample 1-h not sig diff from sham (Clark et al., 2000) Long-Evans HPC Radiofrequency Fornix Complete: 100% square 0.9m x 0.9m 30-s sample 24-h not sig diff from sham (Clark et al., 2000) Pigmented DA Pigmented DA Lister hooded PRH+HPC NMDA+ Radiofrequency PRH+HPC NMDA+ Radiofrequency Extensive Extensive square 1m x 1m square 1m x 1m 20-s (3 sessions) 40-s (3 sessions) HPC Ibotenic acid Complete Y-shaped arena 25-s sample (or 3 min) 15-m sig diff from sham - failed to discrim on all 3 sessions 15-m sig diff from sham - failed to discrim on 1st and 3rd sessions 15-m not sig diff from sham (Ennaceur and Aggleton, 1997) (Ennaceur and Aggleton, 1997) (Forwood et al., 2005) impaired impaired Lister hooded HPC Ibotenic acid Complete Y-shaped arena 25-s sample (or 3 min) 1-h not sig diff from sham (Forwood et al., 2005) Lister hooded HPC Ibotenic acid Complete Y-shaped arena 25-s sample (or 3 min) 24-h not sig diff from sham (Forwood et al., 2005) Lister hooded HPC Ibotenic acid Complete Y-shaped arena 25-s sample (or 3 min) 48-h not sig diff from sham (Forwood et al., 2005) Lister hooded Lister hooded HPC Ibotenic acid Complete: >70% square 1m x 1m HPC Ibotenic acid Complete: % cylinder 0.8m diameter 40-s sample 2-m not sig diff from sham - DR's = sham 0.63; HPC s sample 2-m not sig diff from sham (Good et al., 2007) (Langston and Wood, 2010) Lister hooded Lister hooded HPC NMDA (pretrain) HPC NMDA (pretrain) Extensive loss DG,CA3-1 Extensive loss DG,CA3-1 Y-shaped arena 25-s sample 0-s not sig diff from sham Y-shaped arena 25-s sample 15-m not sig diff from sham (Winters et al., 2004) (Winters et al., 2004) Lister hooded HPC NMDA (pretrain) Extensive loss DG,CA3-1 Y-shaped arena 25-s sample 1-h not sig diff from sham (Winters et al., 2004)

149 132 Species & Strain Technique Structure Treatment Lesion size/ Time of Drug Administration Arena Criterion Delay Detailed Results Reference Summary of Findings Lister hooded HPC NMDA (pretrain) Extensive loss DG,CA3-1 Y-shaped arena 25-s sample 24-h not sig diff from sham (Winters et al., 2004)

150 133 Table 2. Extensive review of published object recognition studies with pertinent details of task design and results Abbreviations: Antag, antagonist; HPC, hippocampus; diff, different; BLA, basolateral amygdala; Ctx, cortex; PRH, perirhinal cortex; obj, object; LEC, lateral entorhinal cortex; mpfc, medial prefrontal cortex; contra, contralateral; ipsi, ipsilateral; OL, object location; OIP, object in place; TO, temporal order; dhpc, dorsal hippocampus; NOPR, novel object preference ratio; DG, dentate gyrus; ego, egocentric; allo, allocentric; DNMS, delayed non-match to sample; DR, discrimination ratio; pref, preference; vca1, ventral CA1; nvl, novel; fam, familiar; dep, dependent; FNX, fornix; Co, cortex; reconfig, reconfigured; Acc, anterior cingulated cortex; RSc, retrosplenial cortex; Cg, cingulate ; CB, cingulum bundle; Pfc, prefrontal cortex, POR, postrhinal cortex; MWM, Morris Water Maze; RAM, radial arm maze. Species & Strain Long- Evans Long- Evans Technique & Treatment inactivation NMDA antag, APV (4 sites) inactivation NMDA antag, APV (4 sites) Structure HPC (dorsal) HPC (dorsal) Lesion size/ Time of Drug Administration pre-sample NOR square 0.6m x 0.6 m pre-sample NOR square 0.6m x 0.6 m Task Arena Criterion Delay Detailed Results 30-s sample criterion 30-s sample criterion Reference 5-m not diff from sham (Baker and Kim, 2002) 3-h sig diff from sham (Baker and Kim, 2002) Summary of Findings impaired Wistar Wistar inactivation anisomycin inactivation anisomycin HPC (dorsal) HPC (dorsal) post-sample NOR square 0.04m x 0.04m post-sample NOR square 0.04m x 0.04m 10-m sample 90-m not diff from sham (Balderas et al., 2008) 10-m sample 24-h not diff from sham (Balderas et al., 2008) Wistar inactivation anisomycin BLA post-sample NOR square 0.04m x 0.04m 10-m sample 90-m not diff from sham (Balderas et al., 2008)

151 134 Species & Strain Wistar Wistar Wistar Wistar Technique & Treatment inactivation anisomycin inactivation anisomycin inactivation anisomycin inactivation anisomycin Structure Lesion size/ Time of Drug Administration BLA post-sample NOR square 0.04m x 0.04m Insular Ctx post-sample NOR square 0.04m x 0.04m Insular Ctx post-sample NOR square 0.04m x 0.04m PRH post-sample NOR square 0.04m x 0.04m Task Arena Criterion Delay Detailed Results Reference 10-m sample 24-h not diff from sham (Balderas et al., 2008) 10-m sample 90-m not diff from sham (Balderas et al., 2008) 10-m sample 24-h sig diff from sham (Balderas et al., 2008) 10-m sample 90-m not diff from sham (Balderas et al., 2008) Summary of Findings impaired Wistar Wistar Wistar Wistar Wistar Wistar Wistar Wistar inactivation anisomycin inactivation anisomycin inactivation anisomycin inactivation anisomycin inactivation anisomycin inactivation anisomycin inactivation anisomycin inactivation anisomycin PRH post-sample NOR square 0.04m x 0.04m HPC (dorsal) HPC (dorsal) post-sample post-sample Object- Contex t Object- Contex t BLA post-sample Object- Contex t BLA post-sample Object- Contex t Insular Ctx post-sample Object- Contex t Insular Ctx post-sample Object- Contex t PRH post-sample Object- Contex t square 0.04m x 0.04m square 0.04m x 0.04m square 0.04m x 0.04m square 0.04m x 0.04m square 0.04m x 0.04m square 0.04m x 0.04m square 0.04m x 0.04m 10-m sample 24-h sig diff from sham (Balderas et al., 2008) 10-m sample 90-m not diff from sham (Balderas et al., 2008) 10-m sample 24-h sig diff from sham (Balderas et al., 2008) 10-m sample 90-m not diff from sham (Balderas et al., 2008) 10-m sample 24-h not diff from sham (Balderas et al., 2008) 10-m sample 90-m not diff from sham (Balderas et al., 2008) 10-m sample 24-h not diff from sham (Balderas et al., 2008) 10-m sample 90-m not diff from sham (Balderas et al., 2008) impaired impaired

152 135 Species & Strain Wistar Mouse C57BL/6J Technique & Treatment inactivation anisomycin inactivation muscimol Structure Lesion size/ Time of Drug Administration PRH post-sample Object- Contex t HPC (dorsal) Task Arena Criterion Delay Detailed Results square 0.04m x 0.04m pre-sample NOR square 0.04m x 0.04m Reference 10-m sample 24-h not diff from sham (Balderas et al., 2008) 30-s sample 24-h sig diff from sham (Cohen et al., 2013b) Summary of Findings impaired Mouse C57BL/6J Mouse C57BL/6J Mouse C57BL/6J Mouse C57BL/6J inactivation muscimol inactivation muscimol inactivation anisomycin inactivation anisomycin HPC (dorsal) HPC (dorsal) HPC (dorsal) HPC (dorsal) post-sample NOR square 0.04m x 0.04m pre-, postsample & pretest post-sample 0h and 2h post-sample 2h only NOR NOR NOR square 0.04m x 0.04m square 0.04m x 0.04m square 0.04m x 0.04m 30-s sample 24-h sig diff from sham (Cohen et al., 2013b) 30-s sample 24-h sig diff from sham (Cohen et al., 2013b) 30-s sample 24-h sig diff from sham (Cohen et al., 2013b) 30-s sample 24-h not sig diff from sham (Cohen et al., 2013b) impaired impaired impaired Mouse C57BL/6J Mouse C57BL/6J Mouse C57BL/6J inactivation FCM inactivation muscimol inactivation muscimol HPC (dorsal) HPC (dorsal) HPC (dorsal) pre-test NOR square 0.04m x 0.04m pre-test NOR novel context each day pre-test NOR novel context for test 3 x 10- m/day for 3d 3 x 10- m/day for 3d 24-h sig diff from sham (Cohen et al., 2013b) 24-h sig diff from sham (Cohen et al., 2013b) 30-s sample 24-h not sig diff from sham (controls fail to prefer novel obj) (Cohen et al., 2013b) impaired impaired Wistar inactivation muscimol HPC post-sample 0h NOR square 0.04m x 0.05m 5-m sample 1.5-h sig diff from sham (de Lima et al., 2006) impaired Wistar Wistar inactivation muscimol inactivation muscimol HPC post-sample 0h NOR square 0.04m x 0.05m HPC post-sample 3h NOR square 0.04m x 0.05m 5-m sample 24-h sig diff from sham (de Lima et al., 2006) 5-m sample 24-h sig diff from sham (de Lima et al., 2006) impaired impaired

153 136 Species & Strain Wistar Mouse C57BL/6J Mouse C57BL/6J Technique & Treatment inactivation muscimol inactivation Lidocaine (0.5 µl of 4%/side) inactivation Lidocaine (0.5 µl of 4%/side) Structure Lesion size/ Time of Drug Administration HPC post-sample 6h NOR square 0.04m x 0.05m HPC (dorsal) HPC (dorsal) pre-sample NOR square 0.04m x 0.04m pre-sample NOR square 0.04m x 0.04m Task Arena Criterion Delay Detailed Results Reference 5-m sample 24-h not sig diff from sham (de Lima et al., 2006) 38-s sample criterion 38-s sample criterion 5-m not sig diff from sham (Hammond et al., 2004) 24-h sig diff from sham (Hammond et al., 2004) Summary of Findings impaired Mouse C57BL/6J inactivation Lidocaine (0.5 µl of 4%/side) HPC (dorsal) pre-train MWM pool 109 cm dia sig diff from sham (Hammond et al., 2004) impaired intra-hpc lidocaine impairs spatial learning Long- Evans Long- Evans inactivation Lidocaine (0.8 µl of 4%/side) inactivation Lidocaine (0.8 µl of 4%/side) inactivation ZIP (inactivator of PKMzeta) mpfc pre-test NOR square 0.06m x 0.06m PRH pre-test NOR square 0.06m x 0.06m HPC (dorsal) 4-m sample 105-m not sig diff from sham (Hannesson et al., 2004b) 4-m sample 105-m sig diff from sham (Hannesson et al., 2004a) post-sample NOR 5-m sample 48-h not sig diff from sham (Hardt et al., 2010) impaired only looked at 1st min of test inactivation ZIP (inactivator of PKMzeta) HPC (dorsal) post-sample NORwhere 5-m sample 48-h sig diff from sham (Hardt et al., 2010) impaired inactivation ZIP (inactivator of PKMzeta) HPC (dorsal) post-sample NOR 5-m sample 7-days not sig diff from sham (Hardt et al., 2010) only looked at 1st min of test

154 137 Species & Strain Mouse C57BL/6J Technique & Treatment inactivation ZIP (inactivator of PKMzeta) inactivation muscimol Structure HPC (dorsal) HPC (dorsal) Lesion size/ Time of Drug Administration post-sample post-sample Task Arena Criterion Delay Detailed Results NORwhere Object- Place rectangle 0.06m x 0.05m Reference 5-m sample 7-days sig diff from sham (Hardt et al., 2010) 6-m sample 24-h non-displaced=displaced (Oliveira et al., 2010) Summary of Findings impaired impaired sample: 3 objects; test: 1 object displaced to new location Mouse C57BL/6J Mouse C57BL/6J inactivation muscimol inactivation muscimol HPC (dorsal) HPC (dorsal) 24-h pre-sample Object- Place rectangle 0.06m x 0.05m post-sample NOR rectangle 0.06m x 0.05m 6-m sample 24-h displaced>>non-displaced (Oliveira et al., 2010) 15-m sample 24-h sig diff from sham (Oliveira et al., 2010) enhanced very low object exploration in 15-m sessions! Wistar Wistar inactivation anisomycin inactivation anisomycin HPC (dorsal) HPC (dorsal) post-sample 0-m NOR square 0.05m x 0.05m post-sample 180- m NOR square 0.05m x 0.05m 5-m sample 24-h sig diff from sham (Rossato et al., 2007) 5-m sample 24-h sig diff from sham (Rossato et al., 2007) impaired impaired Wistar Wistar Pigmented DA Pigmented DA inactivation anisomycin inactivation anisomycin NMDA NMDA HPC (dorsal) HPC (dorsal) Rhinal+Te mporal Rhinal+Te mporal post-sample 360- m NOR square 0.05m x 0.05m post-sample 0-m NOR square 0.05m x 0.05m all of PRH, some LEC all of PRH, some LEC NOR NOR square 0.1m x 0.1m square 0.1m x 0.1m 5-m sample 24-h not sig diff from sham (Rossato et al., 2007) 5-m sample 3-h not sig diff from sham (Rossato et al., 2007) 3-m sample 15-m sig diff from sham (Aggleton et al., 1997) 25-s sample criterion 15-m sig diff from sham (Aggleton et al., 1997) impaired impaired

155 138 Species & Strain Lister hooded Technique & Treatment Ibotenic acid Structure Lesion size/ Time of Drug Administration Task Arena Criterion Delay Detailed Results Reference HPC Partial: 58% NOR 30-s sample 10-s not diff from sham (Ainge et al., 2006) Summary of Findings results differ if looking at first minute of test or 30s of exploration Lister hooded Ibotenic acid HPC Partial: 58% NOR 30-s sample 1-m not diff from sham (Ainge et al., 2006) Lister hooded Ibotenic acid HPC Partial: 58% NOR 30-s sample 10-m chance (Ainge et al., 2006) impaired Lister hooded Ibotenic acid HPC Partial: 58% NOR 30-s sample 1-h chance (Ainge et al., 2006) impaired Lister hooded Ibotenic acid HPC Partial: 58% NOR 30-s sample 24-h chance (Ainge et al., 2006) impaired Lister hooded Ibotenic acid HPC Complete: 98% NOR 30-s sample 10-s not diff from sham (Ainge et al., 2006) Lister hooded Ibotenic acid HPC Complete: 98% NOR 30-s sample 1-m not diff from sham (Ainge et al., 2006) Lister hooded Ibotenic acid HPC Complete: 98% NOR 30-s sample 10-m not diff from sham (Ainge et al., 2006) Lister hooded Ibotenic acid HPC Complete: 98% NOR 30-s sample 1-h not diff from sham (Ainge et al., 2006) Lister hooded Ibotenic acid HPC Complete: 98% NOR 30-s sample 24-h chance (Ainge et al., 2006) impaired Lister hooded Ibotenic acid HPC Partial: 63% NOR 3 x 5-m sample 10-m not diff from sham (Ainge et al., 2006)

156 139 Species & Strain Lister hooded Lister hooded Lister hooded Lister hooded Lister hooded Lister hooded Technique & Treatment Ibotenic acid Ibotenic acid Ibotenic acid Ibotenic acid Ibotenic acid Ibotenic acid Structure Lesion size/ Time of Drug Administration Task Arena Criterion Delay Detailed Results HPC Partial: 63% NOR 3 x 5-m sample HPC Partial: 63% NOR 3 x 5-m sample HPC Partial: 63% NOR 3 x 5-m sample HPC Complete:95% NOR 3 x 5-m sample HPC Complete:95% NOR 3 x 5-m sample HPC Complete:95% NOR 3 x 5-m sample Reference 1-h not diff from sham (Ainge et al., 2006) 4-h not diff from sham (Ainge et al., 2006) 24-h not diff from sham (Ainge et al., 2006) 10-m sig diff from sham (Ainge et al., 2006) 1-h sig diff from sham (Ainge et al., 2006) 4-h not diff from sham (Ainge et al., 2006) Summary of Findings Lister hooded Ibotenic acid HPC Complete:95% NOR 3 x 5-m sample 24-h not diff from sham (Ainge et al., 2006) Pigmented DA NMDA PRH Partial: 33-79% NOR square 1m x 1m 4-m sample 24-h Chance (Albasser et al., 2009) not diff from sham Pigmented DA NMDA PRH Partial: 33-79% NOR square 1m x 1m 6-m sample 24-h Chance (Albasser et al., 2009) not diff from sham Pigmented DA NMDA PRH Partial: 33-79% NOR square 1m x 1m 8-m sample 24-h chance (Albasser et al., 2009) Pigmented DA NMDA HPC Partial: average 58% NOR 40-s sample 5-m not diff from sham (Barker and Warburton, 2011)

157 140 Species & Strain Technique & Treatment Structure Lesion size/ Time of Drug Administration Task Arena Criterion Delay Detailed Results HPC: 58% (40-93%) Reference Summary of Findings Pigmented DA NMDA HPC Partial: average 58% NOR 40-s sample 3-h not diff from sham (Barker and Warburton, 2011) Pigmented DA NMDA HPC Partial: average 58% NOR 40-s sample 24-h not diff from sham (Barker and Warburton, 2011) Pigmented DA NMDA PRH Complete: ~84% NOR 40-s sample 5-m Chance (Barker and Warburton, 2011) impaired PRH: 84% (58-98%) Pigmented DA NMDA PRH Complete: ~84% NOR 40-s sample 3-h chance (Barker and Warburton, 2011) impaired Pigmented DA NMDA PRH Complete: ~84% NOR 40-s sample 24-h chance (Barker and Warburton, 2011) impaired Pigmented DA NMDA mpfc Partial: 79% NOR 40-s sample 5-m not diff from sham (Barker and Warburton, 2011) mpfc: 79% (72-86%) Pigmented DA NMDA mpfc Partial: 79% NOR 40-s sample 3-h not diff from sham (Barker and Warburton, 2011) Pigmented DA NMDA mpfc Partial: 79% NOR 40-s sample 24-h not diff from sham (Barker and Warburton, 2011) Pigmented DA NMDA PRH+HPC contra NOR 40-s sample 5-m not diff from sham (Barker and Warburton, 2011) Pigmented DA NMDA PRH+HPC contra NOR 40-s sample 3-h not diff from sham (Barker and Warburton, 2011)

158 141 Species & Strain Pigmented DA Technique & Treatment NMDA Structure PRH+HPC contra Lesion size/ Time of Drug Administration Task Arena Criterion Delay Detailed Results Reference NOR 40-s sample 24-h not diff from sham (Barker and Warburton, 2011) Summary of Findings Pigmented DA NMDA PRH+HPC ipsi NOR 40-s sample 5-m not diff from sham (Barker and Warburton, 2011) Pigmented DA NMDA PRH+HPC ipsi NOR 40-s sample 3-h not diff from sham (Barker and Warburton, 2011) Pigmented DA NMDA PRH+HPC ipsi NOR 40-s sample 24-h not diff from sham (Barker and Warburton, 2011) Pigmented DA NMDA mpfc+hp C contra NOR 40-s sample 5-m not diff from sham (Barker and Warburton, 2011) Pigmented DA NMDA mpfc+hp C contra NOR 40-s sample 3-h not diff from sham (Barker and Warburton, 2011) Pigmented DA NMDA mpfc+hp C contra NOR 40-s sample 24-h not diff from sham (Barker and Warburton, 2011) Pigmented DA NMDA mpfc+hp C ipsi NOR 40-s sample 5-m not diff from sham (Barker and Warburton, 2011) Pigmented DA NMDA mpfc+hp C ipsi NOR 40-s sample 3-h not diff from sham (Barker and Warburton, 2011) Pigmented DA NMDA mpfc+hp C ipsi NOR 40-s sample 24-h not diff from sham (Barker and Warburton, 2011) Pigmented DA NMDA other OL: only HPC impaired 5 m and 24 h, OIP: HPC, PRH, mpfc, H+mP c, H+P c impaired, TO: HPC, PRH, mpfc, H+mP c, H+P c impaired (Barker and Warburton, 2011) See detailed results

159 142 Species & Strain Long- Evans Technique & Treatment Ibotenic acid Structure HPC Lesion size/ Time of Drug Administration dhpc Partial: 50-75% Task Arena Criterion Delay Detailed Results Reference NOR 15-m sample 3-h not diff from sham (Broadbent et al., 2004) Summary of Findings dhpc 50-75%: NOPR = 71.1% Long- Evans Ibotenic acid HPC dhpc Complete: % NOR 15-m sample 3-h Chance (Broadbent et al., 2004) impaired dhpc %: NOPR = 55.9% Long- Evans Ibotenic acid HPC vhpc: 50% NOR 15-m sample 3-h not diff from sham (Broadbent et al., 2004) vhpc 50%: NOPR = 64.4% Long- Evans Ibotenic acid HPC dhpc Complete: % NOR 3 x 5-m/day for 4 d d post op not diff from sham (Broadbent et al., 2010) 1-d post-sample, NOPR = 61% Long- Evans Ibotenic acid HPC dhpc Complete: % NOR 3 x 5-m/day for 4 d d post op sig diff from sham (Broadbent et al., 2010) impaired 4-wk post-sample, NOPR = 55.9% Long- Evans Ibotenic acid HPC dhpc Complete: % NOR 3 x 5-m/day for 4 d d post op not diff from sham (Broadbent et al., 2010) 8-wk post-sample, NOPR = 64%

160 143 Species & Strain Long- Evans Technique & Treatment Ibotenic acid Structure HPC Lesion size/ Time of Drug Administration dhpc Complete: % Task Arena Criterion Delay Detailed Results Reference NOR 15-m sample 3-h sig diff from sham (Broadbent et al., 2010) Summary of Findings impaired Post op NOR (each study tested 4x) Pigmented DA Radiofrequency HPC Fornix Complete: ~80% NOR square 0.1m x 0.1m 25-s sample 15-m not sig diff from sham (Bussey et al., 2000b) sig diff DR based on 1st min of test Pigmented DA Pigmented DA NMDA Radiofrequency PRH HPC PRH+POR: almost all Fornix Complete: ~80% NOR Object- Place square 0.1m x 0.1m square 0.1m x 0.1m 25-s sample 15-m sig diff from sham (Bussey et al., 2000b) task-dep (Bussey et al., 2000b) impaired OL: Sham = PRH >> FNX, OIP: Sham >> FNX = PRH Pigmented DA NMDA PRH PRH+POR: almost all Object- Place square 0.1m x 0.1m task-dep (Bussey et al., 2000b) OL: Sham = PRH >> FNX, OIP: Sham >> FNX = PRH Long- Evans Long- Evans Ibotenic acid Ibotenic acid HPC HPC Complete: 89.8% Complete: 89.8% NOR NOR square 0.09m x 0.09m square 0.09m x 0.09m 30-s sample 10-s not sig diff from sham (Clark et al., 2000) 30-s sample 1-m not sig diff from sham (Clark et al., 2000) Long- Evans Ibotenic acid HPC Complete: 89.8% NOR square 0.09m x 0.09m 30-s sample 10-m sig diff from sham (Clark et al., 2000) impaired

161 144 Species & Strain Long- Evans Long- Evans Long- Evans Technique & Treatment Ibotenic acid Ibotenic acid Radiofrequency Structure HPC HPC HPC Lesion size/ Time of Drug Administration Complete: 89.8% Complete: 89.8% Complete: 71.2% Task Arena Criterion Delay Detailed Results NOR NOR NOR square 0.09m x 0.09m square 0.09m x 0.09m square 0.09m x 0.09m Reference 30-s sample 1-h sig diff from sham (Clark et al., 2000) 30-s sample 24-h sig diff from sham (Clark et al., 2000) 30-s sample 10-s not sig diff from sham (Clark et al., 2000) Summary of Findings impaired impaired Long- Evans Long- Evans Long- Evans Long- Evans Long- Evans Long- Evans Long- Evans Long- Evans Long- Evans Radiofrequency Radiofrequency Radiofrequency Radiofrequency Radiofrequency Radiofrequency Radiofrequency Radiofrequency Radiofrequency HPC HPC HPC HPC HPC HPC HPC HPC HPC Complete: 71.2% Complete: 71.2% Complete: 71.2% Complete: 71.2% Fornix Complete: 100% Fornix Complete: 100% Fornix Complete: 100% Fornix Complete: 100% Fornix Complete: 100% NOR NOR NOR NOR NOR NOR NOR NOR NOR square 0.09m x 0.09m square 0.09m x 0.09m square 0.09m x 0.09m square 0.09m x 0.09m square 0.09m x 0.09m square 0.09m x 0.09m square 0.09m x 0.09m square 0.09m x 0.09m square 0.09m x 0.09m 30-s sample 1-m not sig diff from sham (Clark et al., 2000) 30-s sample 10-m sig diff from sham (Clark et al., 2000) 30-s sample 1-h sig diff from sham (Clark et al., 2000) 30-s sample 24-h sig diff from sham (Clark et al., 2000) 30-s sample 10-s not sig diff from sham (Clark et al., 2000) 30-s sample 1-m not sig diff from sham (Clark et al., 2000) 30-s sample 10-m not sig diff from sham (Clark et al., 2000) 30-s sample 1-h not sig diff from sham (Clark et al., 2000) 30-s sample 24-h not sig diff from sham (Clark et al., 2000) impaired impaired impaired

162 145 Species & Strain Mouse C57BL/6 CR Technique & Treatment NMDA Structure Lesion size/ Time of Drug Administration HPC Partial: 54% NORwhat Task Arena Criterion Delay Detailed Results rectangle 0.04m x 0.02m Reference 5-m sample 50-m Chance (DeVito and Eichenbaum, 2010) Summary of Findings impaired sham = Obj A>>Obj B sample 1(A 4 copies); sample 2(B 4 copies); test(a and B) Mouse C57BL/6 CR NMDA mpfc Partial: 41% NORwhat rectangle 0.04m x 0.02m 5-m sample 50-m not sig diff from sham (DeVito and Eichenbaum, 2010) sham = Obj A>>Obj B Mouse C57BL/6 CR NMDA HPC where rectangle 0.04m x 0.02m 5-m sample 50-m Chance (DeVito and Eichenbaum, 2010) impaired sham = nvl place>>fam place sample 1(A 4 copies); sample 2(B 4 copies); test(a and B) Mouse C57BL/6 CR NMDA mpfc where rectangle 0.04m x 0.02m 5-m sample 50-m Chance (DeVito and Eichenbaum, 2010) impaired sham = nvl place>>fam place NMDA HPC when rectangle 0.04m x 0.02m 5-m sample 50-m Chance (DeVito and Eichenbaum, 2010) impaired sham = old>>recent

163 146 Species & Strain Technique & Treatment Structure Lesion size/ Time of Drug Administration Task Arena Criterion Delay Detailed Results sample 1(A 4 copies); sample 2(B 4 copies); test(a and B) Reference Summary of Findings Mouse C57BL/6 CR NMDA mpfc when rectangle 0.04m x 0.02m 5-m sample 50-m not sig diff from sham (DeVito and Eichenbaum, 2010) Wistar NMDA (posttrain) HPC Partial: most of CA1 DNMS runway s sham = old>>recent not sig diff from sham (Duva et al., 1997) displace novel object post-delay and receive food reward Wistar NMDA (pretrain) HPC Partial: most of CA1 DNMS runway s not sig diff from sham (Duva et al., 1997) displace novel object post-delay and receive food reward Agouti Radiofrequency HPC Fornix Complete: 100% whatwherewhen square 1m x 1m 2-m sig diff from sham (Eacott and Norman, 2004) impaired What (objects)-where (place)-when (context), 2-m, 5-m delays Agouti Agouti Agouti Radiofrequency Radiofrequency Radiofrequency PRH Complete whatwherewhen POR Extensive whatwhere- HPC Fornix Complete: 100% when whatwherewhen square 1m x 1m square 1m x 1m square 1m x 1m 2-m not sig diff from sham (Eacott and Norman, 2004) 2-m not sig diff from sham (Eacott and Norman, 2004) 5-m sig diff from sham (Eacott and Norman, 2004) impaired

164 147 Species & Strain Technique & Treatment Structure Lesion size/ Time of Drug Administration Task Arena Criterion Delay Detailed Results What (objects)-where (place)-when (context), 2-m, 5-m delays Reference Summary of Findings Agouti Agouti Pigmented DA Radiofrequency Radiofrequency Radiofrequency PRH Complete whatwherewhen POR Extensive whatwhere- HPC Fornix Complete: 100% when NOR square 1m x 1m square 1m x 1m square 0.1m x 0.1m 5-m not sig diff from sham (Eacott and Norman, 2004) 5-m not sig diff from sham (Eacott and Norman, 2004) 3-m sample 1-m not sig diff from sham (Ennaceur and Aggleton, 1994) Ttl Obj Exp. Fx>>Co Sample: A1 vs. A2; Test: A vs. B Pigmented DA Radiofrequency HPC Fornix Complete: 100% NOR square 0.1m x 0.1m 3-m sample 15-m not sig diff from sham (Ennaceur and Aggleton, 1994) Ttl Obj Exp. Fx>>Co Pigmented DA Radiofrequency HPC Fornix Complete: 100% NOR square 0.1m x 0.1m 3-m sample 240-m not sig diff from sham (Ennaceur and Aggleton, 1994) Ttl Obj Exp. Fx>>Co both sham and Fx groups at chance Pigmented DA Radiofrequency HPC Fornix Complete: 100% NORreconfi g1 square 0.1m x 0.1m 3-m sample 1-m Chance (Ennaceur and Aggleton, 1994) impaired

165 148 Species & Strain Technique & Treatment Structure Lesion size/ Time of Drug Administration Task Arena Criterion Delay Detailed Results Co & Fx: reconfiga>>a Configural1: Sample: A1 vs. A2; Test: A vs. reconfig A Reference Summary of Findings Pigmented DA Radiofrequency HPC Fornix Complete: 100% NORreconfi g1 square 0.1m x 0.1m 3-m sample 15-m not sig diff from sham (Ennaceur and Aggleton, 1994) both sham and Fx groups at chance Pigmented DA Radiofrequency HPC Fornix Complete: 100% NORreconfi g2 square 0.1m x 0.1m 3-m sample 1-m not sig diff from sham (Ennaceur and Aggleton, 1994) Co & Fx groups at chance Configural2: Sample: A1 vs. A2; Test: reconfig A vs. B Pigmented DA Radiofrequency HPC Fornix Complete: 100% NORreconfi g2 square 0.1m x 0.1m 3-m sample 15-m not sig diff from sham (Ennaceur and Aggleton, 1994) both sham and Fx prefer B to reconfig A Pigmented DA NMDA (pretrain) PRH Extensive NOR square 0.1m x 0.1m 3-m sample 1-m not sig diff from sham (Ennaceur et al., 1996) Pigmented DA Radiofrequency HPC Fornix Complete: 100% NOR square 0.1m x 0.1m 3-m sample 1-m not sig diff from sham (Ennaceur et al., 1996) Pigmented DA NMDA (pretrain) PRH Extensive NOR square 0.1m x 0.1m 3-m sample 15-m sig diff from sham (Ennaceur et al., 1996) impaired Pigmented DA Radiofrequency HPC Fornix Complete: 100% NOR square 0.1m x 0.1m 3-m sample 15-m not sig diff from sham (Ennaceur et al., 1996)

166 149 Species & Strain Pigmented DA Technique & Treatment NMDA (pretrain) Structure Lesion size/ Time of Drug Administration PRH Extensive NORreconfi g1 Task Arena Criterion Delay Detailed Results square 0.1m x 0.1m Reference 3-m sample 1-m not sig diff from sham (Ennaceur et al., 1996) Summary of Findings all groups prefer reconfig A to A Pigmented DA Radiofrequency HPC Fornix Complete: 100% NORreconfi g1 square 0.1m x 0.1m 3-m sample 1-m not sig diff from sham (Ennaceur et al., 1996) Pigmented DA NMDA (pretrain) PRH Extensive NORreconfi g1 square 0.1m x 0.1m 3-m sample 15-m not sig diff from sham (Ennaceur et al., 1996) all groups treat reconfig A and A as equal Pigmented DA Pigmented DA Radiofrequency NMDA (pretrain) HPC Fornix Complete: 100% NORreconfi g1 PRH Extensive NORreconfi g2 square 0.1m x 0.1m square 0.1m x 0.1m 3-m sample 15-m not sig diff from sham (Ennaceur et al., 1996) 3-m sample 1-m not sig diff from sham (Ennaceur et al., 1996) all groups treat B and reconfig A as novel Pigmented DA Pigmented DA Radiofrequency NMDA (pretrain) HPC Fornix Complete: 100% NORreconfi g2 PRH Extensive NORreconfi g2 square 0.1m x 0.1m square 0.1m x 0.1m 3-m sample 1-m not sig diff from sham (Ennaceur et al., 1996) 3-m sample 15-m not sig diff from sham (Ennaceur et al., 1996) all groups prefer B to reconfig A Pigmented DA Radiofrequency HPC Fornix Complete: 100% NORreconfi g2 square 0.1m x 0.1m 3-m sample 15-m not sig diff from sham (Ennaceur et al., 1996)

167 150 Species & Strain Pigmented DA Technique & Treatment NMDA (pretrain) Structure Lesion size/ Time of Drug Administration PRH Extensive NOR square 0.1m x 0.1m Task Arena Criterion Delay Detailed Results Reference 3-m sample 15-m sig diff from sham (Ennaceur and Aggleton, 1997) Summary of Findings impaired most caudal affected ventral CA1 too Pigmented DA NMDA+Radiofr equency PRH+HPC Extensive NOR square 0.1m x 0.1m 3-m sample 15-m not sig diff from sham (Ennaceur and Aggleton, 1997) Pigmented DA NMDA (pretrain) PRH Extensive NOR square 0.1m x 0.1m 3-m sample 1-h Chance (Ennaceur and Aggleton, 1997) but shams also failed to discriminate Pigmented DA NMDA+Radiofr equency PRH+HPC Extensive NOR square 0.1m x 0.1m 3-m sample 1-h Chance (Ennaceur and Aggleton, 1997) but shams also failed to discriminate Pigmented DA NMDA (pretrain) PRH Extensive NOR square 0.1m x 0.1m 20-s (3 sessions) 15-m not sig diff from sham (Ennaceur and Aggleton, 1997) failed to discriminate only on 1st session Pigmented DA NMDA+Radiofr equency PRH+HPC Extensive NOR square 0.1m x 0.1m 20-s (3 sessions) 15-m sig diff from sham (Ennaceur and Aggleton, 1997) impaired Pigmented DA NMDA (pretrain) PRH Extensive NOR square 0.1m x 0.1m 40-s (3 sessions) 15-m not sig diff from sham (Ennaceur and Aggleton, 1997)

168 151 Species & Strain Technique & Treatment Structure Lesion size/ Time of Drug Administration Task Arena Criterion Delay Detailed Results failed to discriminate only on 1st session Reference Summary of Findings Pigmented DA NMDA+Radiofr equency PRH+HPC Extensive NOR square 0.1m x 0.1m 40-s (3 sessions) 15-m sig diff from sham? (Ennaceur and Aggleton, 1997) impaired failed to discriminate on 1st and 3rd sessions Pigmented DA Pigmented DA Pigmented DA NMDA (pretrain) NMDA+Radiofr equency Radiofrequency PRH Extensive RAM not sig diff from sham (Ennaceur and Aggleton, 1997) PRH+HPC Extensive RAM sig diff from sham (Ennaceur and Aggleton, 1997) HPC Fornix Complete: 100% NOR square 0.1m x 0.1m 3-m sample 1-m not sig diff from sham (Ennaceur et al., 1997) impaired Pigmented DA Pigmented DA Pigmented DA NMDA (pretrain) NMDA (pretrain) Radiofrequency ACc Extensive (24a and 24b) RSc HPC Extensive (area 29) Fornix Complete: 100% NOR NOR NOR square 0.1m x 0.1m square 0.1m x 0.1m square 0.1m x 0.1m 3-m sample 1-m not sig diff from sham (Ennaceur et al., 1997) 3-m sample 1-m not sig diff from sham (Ennaceur et al., 1997) 3-m sample 15-m not sig diff from sham (Ennaceur et al., 1997) Pigmented DA Pigmented DA Pigmented DA NMDA (pretrain) NMDA (pretrain) Radiofrequency ACc Extensive (24a and 24b) RSc HPC Extensive (area 29) Fornix Complete: 100% NOR NOR NOR square 0.1m x 0.1m square 0.1m x 0.1m square 0.1m x 0.1m 3-m sample 15-m not sig diff from sham (Ennaceur et al., 1997) 3-m sample 15-m not sig diff from sham (Ennaceur et al., 1997) 3-m sample 15-m not sig diff from sham (Ennaceur et al., 1997)

169 152 Species & Strain Pigmented DA Pigmented DA Pigmented DA Pigmented DA Pigmented DA Pigmented DA Technique & Treatment NMDA (pretrain) Radiofrequency Radiofrequency NMDA (pretrain) Structure Pfc Lesion size/ Time of Drug Administration Extensive (area 32) Task Arena Criterion Delay Detailed Results NOR square 0.1m x 0.1m Cg Complete NOR square 0.1m x 0.1m CB Extensive NOR square 0.1m x 0.1m HPC Pfc Fornix Complete: 100% Extensive (area 32) NMDA (pretrain) NORwhere NMDA (pretrain) NORwhere Cg Complete NORwhere square 0.1m x 0.1m square 0.1m x 0.1m square 0.1m x 0.1m Reference 3-m sample 15-m not sig diff from sham (Ennaceur et al., 1997) 3-m sample 15-m not sig diff from sham (Ennaceur et al., 1997) 3-m sample 15-m not sig diff from sham (Ennaceur et al., 1997) 3-m sample 15-m sig diff from sham (Ennaceur et al., 1997) 3-m sample 15-m not sig diff from sham (Ennaceur et al., 1997) 3-m sample 15-m sig diff from sham (Ennaceur et al., 1997) Summary of Findings impaired impaired Pigmented DA Lister hooded Radiofrequency Ibotenic acid CB Extensive NORwhere square 0.1m x 0.1m HPC Complete DNMS cross maze 3-m sample 15-m not sig diff from sham (Ennaceur et al., 1997) ~5-s Chance (Forwood et al., 2005) trained to criterion of 75% correct (~8 trials/session) Lister hooded Lister hooded Ibotenic acid Ibotenic acid HPC Complete NOR Y- shaped arena HPC Complete NOR Y- shaped arena 25-s sample (or 3 min) 25-s sample (or 3 min) 15-m not sig diff from sham (Forwood et al., 2005) 1-h not sig diff from sham (Forwood et al., 2005) Lister hooded Ibotenic acid HPC Complete NOR Y- shaped arena 25-s sample (or 3 min) 24-h not sig diff from sham (Forwood et al., 2005)

170 153 Species & Strain Lister hooded Long- Evans Technique & Treatment Ibotenic acid NMDA (5-wk post-sample) Structure Lesion size/ Time of Drug Administration HPC Complete NOR Y- shaped arena HPC Extensive loss DG,CA3-1 Task Arena Criterion Delay Detailed Results NOR square 0.07m x 0.07m 25-s sample (or 3 min) 5-m sample/day x 5 Reference 48-h not sig diff from sham (Forwood et al., 2005) days postop Chance (Gaskin et al., 2003) Summary of Findings impaired sparing in vca1, no learning-surgery interval effect Long- Evans NMDA (1-wk post-sample) HPC Extensive loss DG,CA3-1 NOR square 0.07m x 0.07m 5-m sample/day x days post op Chance pref during 1st min of test slightly above chance (Gaskin et al., 2003) impaired Long- Evans Long- Evans Long- Evans NMDA (5-wk post-sample) NMDA (1-wk post-sample) NMDA (pretrain) HPC HPC HPC (dorsal) Extensive loss DG,CA3-1 Extensive loss DG,CA3-1 Complete: ~100% NOR NOR NOR square 0.07m x 0.07m square 0.07m x 0.07m square 0.07m x 0.07m 5-m sample (3 sessions) 5-m sample (3 sessions) 15-m not sig diff from sham (Gaskin et al., 2003) 24-h not sig diff from sham (Gaskin et al., 2003) 5-m sample 2-h sig diff from sham (Gaskin et al., 2010) impaired both groups significantly above chance Long- Evans NMDA (pretrain) HPC (dorsal) Complete: ~100% NOR square 0.07m x 0.07m 5-m sample (4 sessions) 24-h sig diff from sham (Gaskin et al., 2010) impaired sig diff from shams on 3 out of 4 sessions

171 154 Species & Strain Long- Evans Technique & Treatment NMDA (pretrain) Structure HPC (dorsal) Lesion size/ Time of Drug Administration Complete: ~100% Task Arena Criterion Delay Detailed Results NOR square 0.07m x 0.07m Reference 5-m sample 35-s sig diff from sham (Gaskin et al., 2010) Summary of Findings impaired at chance Long- Evans Lister hooded NMDA (pretrain) Ibotenic acid HPC (dorsal) Complete: ~100% NOR square 0.07m x 0.07m HPC Complete: >70% NOR square 0.1m x 0.1m 5-m sample (4 sessions) 40-s sample criterion 35-s sig diff from sham (Gaskin et al., 2010) 2-m not sig diff from sham (Good et al., 2007) impaired both groups sig above chance DR's = sham 0.63; HPC 0.6 Lister hooded Lister hooded Ibotenic acid Ibotenic acid HPC Complete: >70% NORtempor al HPC Complete: >70% NORwhere square 0.1m x 0.1m square 0.1m x 0.1m 5-m sample 2-m not sig diff from sham (Good et al., 2007) 5-m sample 2-m sig diff from sham (Good et al., 2007) impaired HPC = chance DR's = sham 0.67; HPC 0.43 Lister hooded Ibotenic acid HPC Complete: >70% NORspatiote mporal square 0.1m x 0.1m 5-m sample (1/pair) 2-m not sig diff from sham (Good et al., 2007) Sample 1(A and B); Sample 2(C and D)

172 155 Species & Strain Fisher 344 Technique & Treatment Electrolytic Structure Lesion size/ Time of Drug Administration HPC Complete NOR square 0.07m x 0.06m Task Arena Criterion Delay Detailed Results Reference 15-m sample 1-h sig diff from sham (Gould et al., 2002) Summary of Findings impaired HPC = chance Wistar Wistar Wistar Wistar Wistar NMDA (pretrain) NMDA (pretrain) NMDA (pretrain) NMDA (pretrain) NMDA (1-d post sample) HPC Moderate: 32-43% HPC Moderate: 32-43% Retrospleni al Retrospleni al Moderate: 32-43% Moderate: 32-43% HPC Moderate: 32-43% NOR NOR NOR NOR NOR square 0.05m x 0.05m square 0.05m x 0.05m square 0.05m x 0.05m square 0.05m x 0.05m square 0.05m x 0.05m 5-m sample 5-m not sig diff from sham (Haijima and Ichitani, 2012) 5-m sample 30-m not sig diff from sham (Haijima and Ichitani, 2012) 5-m sample 5-m not sig diff from sham (Haijima and Ichitani, 2012) 5-m sample 30-m not sig diff from sham (Haijima and Ichitani, 2012) 5-m sample 1-w sig diff from sham (Haijima and Ichitani, 2012) impairment only at 1 week compared to controls Wistar Wistar Wistar NMDA (1-d post sample) NMDA (1-d post sample) NMDA (1-d post sample) HPC Moderate: 32-43% Retrospleni al Retrospleni al Moderate: 32-43% Moderate: 32-43% NOR NOR NOR square 0.05m x 0.05m square 0.05m x 0.05m square 0.05m x 0.05m 5-m sample 4-w not sig diff from sham (Haijima and Ichitani, 2012) 5-m sample 1-w not sig diff from sham (Haijima and Ichitani, 2012) 5-m sample 4-w sig diff from sham (Haijima and Ichitani, 2012) Wistar Wistar NMDA (pretrain) NMDA (pretrain) HPC Moderate: 32-43% HPC Moderate: 32-43% NOR NOR square 0.05m x 0.05m square 0.05m x 0.05m 5-m sample 1-d sig diff from sham (Haijima and Ichitani, 2012) 5-m sample 4-w sig diff from sham (Haijima and Ichitani, 2012) impaired impaired

173 156 Species & Strain Wistar Technique & Treatment NMDA (pretrain) Structure Retrospleni al Lesion size/ Time of Drug Administration Moderate: 32-43% Task Arena Criterion Delay Detailed Results NOR square 0.05m x 0.05m Reference 5-m sample 1-d sig diff from sham (Haijima and Ichitani, 2012) Summary of Findings impaired Wistar Wistar Lister hooded NMDA (pretrain) Ibotenic acid Ibotenic acid Retrospleni al Moderate: 32-43% HPC Complete: % HPC Complete: % NOR NOR Object- Place square 0.05m x 0.05m cylinder 76 cm diameter cylinder 76 cm diameter 5-m sample 4-w sig diff from sham (Haijima and Ichitani, 2012) 15-s sample criterion 2-m not sig diff from sham (Langston and Wood, 2010) 2-m not sig diff from sham (Langston and Wood, 2010) impaired Lister hooded Lister hooded Lister hooded Lister hooded Lister hooded Sprague- Dawley Ibotenic acid Ibotenic acid Ibotenic acid Ibotenic acid Ibotenic acid Ibotenic acid HPC Complete: % HPC Complete: % HPC Complete: % HPC Complete: % HPC Complete: % Object- Contex t Obj- Place- Contex t Object swap Allo Obj- Place Ego Obj- Place cylinder 76 cm diameter cylinder 76 cm diameter cylinder 76 cm diameter cylinder 76 cm diameter cylinder 76 cm diameter HPC Complete NOR square 0.06m x 0.06m 2-m not sig diff from sham (Langston and Wood, 2010) 2-m sig diff from sham (Langston and Wood, 2010) 2-m sample 2-m not sig diff from sham (Langston and Wood, 2010) 2-m sample 2-m sig diff from sham (Langston and Wood, 2010) 2-m sample 2-m not sig diff from sham (Langston and Wood, 2010) 5-m sample 20-m not sig diff from sham (Liu and Bilkey, 2001) impaired impaired impaired but not sig diff from chance either Sprague- Dawley Ibotenic acid PRH Complete NOR square 0.06m x 0.06m 5-m sample 20-m sig diff from sham (Liu and Bilkey, 2001) impaired

174 157 Species & Strain Long- Evans Technique & Treatment Aspiration Structure Lesion size/ Time of Drug Administration HPC Complete: >70% DNMS elevated runway Task Arena Criterion Delay Detailed Results Reference 600-s sig diff from sham (Mumby et al., 1992) Summary of Findings impaired above chance Long- Evans Aspiration HPC Complete: >70% DNMS elevated runway 120-s not sig diff from sham (Mumby et al., 1992) delay-dependent deficit Long- Evans Aspiration HPC Complete: >70% DNMS elevated runway 60-s not sig diff from sham (Mumby et al., 1992) Long- Evans Aspiration HPC Complete: >70% DNMS elevated runway 15-s not sig diff from sham (Mumby et al., 1992) Long- Evans Aspiration HPC Complete: >70% DNMS elevated runway 4-s not sig diff from sham (Mumby et al., 1992) Long- Evans Long- Evans Long- Evans Long- Evans Aspiration (1-wk post sample) Aspiration (3-wk post sample) Aspiration (5-wk post sample) NMDA (pretrain) PRH Complete NOR square 0.07m x 0.07m PRH Complete NOR square 0.07m x 0.07m PRH Complete NOR square 0.07m x 0.07m HPC Extensive loss DG,CA3-1 NOR square 0.07m x 0.07m 1-wk sig diff from sham (Mumby et al., 2002a) 3-wk not sig diff from sham (Mumby et al., 2002a) 5-wk not sig diff from sham (Mumby et al., 2002a) 5-m sample 5-m not sig diff from sham (Mumby et al., 2002b) impaired 3 sessions Long- Evans HPC Extensive loss DG,CA3-1 NMDA (pretrain) Object- Place square 0.07m x 0.07m 5-m sample 5-m sig diff from sham (Mumby et al., 2002b) impaired 3 sessions

175 158 Species & Strain Long- Evans Technique & Treatment Structure HPC Lesion size/ Time of Drug Administration Extensive loss DG,CA3-1 Task Arena Criterion Delay Detailed Results NMDA (pretrain) Object- Contex t square 0.07m x 0.07m Reference 5-m sample 5-m sig diff from sham (Mumby et al., 2002b) Summary of Findings impaired chance, 3 sessions Long- Evans Long- Evans NMDA (pretrain) NMDA (pretrain) HPC HPC Extensive loss DG,CA3-1 Extensive loss DG,CA3-1 NOR NOR square 0.07m x 0.07m square 0.07m x 0.07m 5-m sample 24-h not sig diff from sham (Mumby et al., 2005) 5-m sample 1-wk not sig diff from sham (Mumby et al., 2005) Long- Evans Lister hooded Lister hooded NMDA (pretrain) NMDA (pretrain) NMDA (pretrain) HPC Extensive loss DG,CA3-1 NOR square 0.07m x 0.07m PRH Complete NOR Y- shaped arena PRH Complete NOR Y- shaped arena 5-m sample 3-wk not sig diff from sham (Mumby et al., 2005) 25-s sample criterion 25-s sample criterion 0-s not sig diff from sham (Winters et al., 2004) 15-m sig diff from sham (Winters et al., 2004) impaired but definitely above chance Lister hooded NMDA (pretrain) PRH Complete NOR Y- shaped arena 25-s sample criterion 1-h sig diff from sham (Winters et al., 2004) impaired but definitely above chance Lister hooded NMDA (pretrain) PRH Complete NOR Y- shaped arena 25-s sample criterion 24-h sig diff from sham (Winters et al., 2004) impaired but definitely above chance Lister hooded NMDA (pretrain) HPC Extensive loss DG,CA3-1 NOR Y- shaped arena 25-s sample criterion 0-s not sig diff from sham (Winters et al., 2004)

176 159 Species & Strain Lister hooded Technique & Treatment NMDA (pretrain) Structure HPC Lesion size/ Time of Drug Administration Extensive loss DG,CA3-1 Task Arena Criterion Delay Detailed Results NOR Y- shaped arena 25-s sample criterion Reference 15-m not sig diff from sham (Winters et al., 2004) Summary of Findings Lister hooded Lister hooded Mouse Swiss Mouse Swiss NMDA (pretrain) NMDA (pretrain) machr antag scopolamine (0.3 mg/kg) machr antag scopolamine (1 mg/kg) HPC HPC Extensive loss DG,CA3-1 Extensive loss DG,CA3-1 NOR Y- shaped arena NOR Y- shaped arena pre-sample NOR square 0.05m x 0.05m pre-sample NOR square 0.05m x 0.05m 25-s sample criterion 25-s sample criterion 1-h not sig diff from sham (Winters et al., 2004) 24-h not sig diff from sham (Winters et al., 2004) 10-m sample 3-h chance (Dodart et al., 1997) 10-m sample 3-h chance (Dodart et al., 1997) impaired impaired Mouse Swiss machr antag scopolamine (3 mg/kg) pre-sample NOR square 0.05m x 0.05m 10-m sample 3-h chance (Dodart et al., 1997) impaired

177 Figure 1. Location and shape of hippocampus. The hippocampus is located in the medial temporal lobe, within the limbic system, deep within the brain. A, Human brain. B, brain (Sokolowski and Corbin, 2012). Figure 1. Location and shape of hippocampus 160

178 Figure 2. Hippocampal information network. In rodents, the postrhinal cortex send spatial information to the hippocampus either directly or indirectly via the medial entorhinal cortex (MEC) and the perirhinal cortex sends object information to the hippocampus either directly or indirectly through the lateral entorhinal cortex (LEC). The entorhinal cortex sends projections via the perforant pathway (PP) to the dentate gyrus (DG) and via the mossy fibers (MF) on to CA3. Projections are then sent to CA1 via the Schaffer Collaterals (SC) and then onto the subiculum. Then the information will loop around back to the entorhinal cortex (Hartley et al., 2014). Specifically in terms of object information, the perirhinal cortex receives projections from visual association areas (like TE and TEO), V4 and parietal regions (including cingulate cortices). Figure 2. Hippocampal information network 161

179 Figure 3. Morris Water Maze. This task is conducted in a large circular pool containing opaque water and a hidden platform located below the water s surface. Rodents are released into the pool from a specific zone and are required to locate the platform using various distal cues. After a number of training trials, the platform is removed and swim path is analyzed. Intact spatial memory is infered from swim paths centered around the previous location of the platform (Image retrieved Aug. 2, 2015 from Figure 3. Morris Water Maze 162

180 Figure 4. The novel object recognition (NOR) task. The typical NOR protocol consists of a sample session (left) and a test session (right) conducted within a familiar cylindrical, rectangular, or y-shaped arena. During the sample session, the rodent is placed into the familiar arena to freely explore two presented novel objects for a specified amount of time. Upon completion of the sample session, the rodent is removed from the arena and is most often returned to its home cage. After a predetermined delay, the rodent is returned to the familiar arena for a time-bound test session in which one of the sample objects is replaced with a novel object. Task performance is assessed by analyzing the differences in time spent exploring both test session objects. These photographs depict an example of the arena and the objects our lab has used to test object memory in mice (Cohen et al., 2013b). Although our objects are placed into opposite corners of the arena; others have placed the objects into the center of the arena. Our typical sample session concludes once the mouse has accumulated 38 s of exploration on one object, or 30 s of exploration of both objects; our sample session exploration criterion. During the test session presented on Day 2, the arena includes a copy of the familiar object from the sample session and a novel object. The relative positions of the novel and familiar objects during the test session are counterbalanced to eliminate spatial bias in the task. Figure 4. The novel object recognition (NOR) task 163

181 Figure 5. Publication rate of permanent or temporary hippocampal experiments in which a sample session exploration criterion was imposed. The graph depicts the number of experiments testing the involvement of the hippocampus appearing within multiexperiment peer-reviewed object recognition reports published over the last 20 years (bars) and the specific number of hippocampal inactivation experiments (line). The time bins are defined as 3 to 5-year intervals. It is noteworthy that the publication rates of experiments that utilize the temporary inactivation technique have increased considerably in the last decade. Figure 5. Publication rate of permanent or temporary hippocampal experiments in which a sample session exploration criterion was imposed 164

182 Figure 6. Qualitative model depicting how the perirhinal cortex and hippocampus contribute to the objet recognition memory. A. Prior to the start of the NOR sample session, both structures lack sample object information. B. As object exploration commences during the sample session, the perirhinal cortex begins to accumulate with information. C. Until a minimum amount of sample object exploration has elapsed, the object information remains perirhinal cortex dependent. D. Once the critical threshold of object exploration is reached (perhaps 30/38 s of sample object exploration), a transfer of the object information to the hippocampus is initiated; this multi-synaptic transfer requires some time delay to be completed. Figure 6. Qualitative model depicting how the perirhinal cortex and hippocampus contribute to object recognition memory 165

183 Figure 7. Encoding, consolidation and retrieval of object memory by C57BL/6J mice requires hippocampus. a. Depiction of the NOR task sessions. Arrowheads indicate when intra-hippocampal infusions were given for specific experiments designated by lowercase letters corresponding to the respective graph (pre-sample, c, post-sample, d and e, or preand post-sample and pre-test, f). b. The distribution of intra-hippocampal infusion sites within the CA1 region of dorsal hippocampus for all experiments is depicted in gray shading against respective coronal plates from the Franklin & Paxinos atlas (Franklin and Paxinos, 2008) (numbers refer to mm from bregma). c. Intra-hippocampal infusion of muscimol c. pre-sample (saline, n = 8; muscimol, n = 9) or d. post-sample session (saline, n = 12; muscimol, n = 11) significantly impaired novel object preference (i.e., object memory) during the test session 24 h later. Mice exhibited similar levels of object exploration during the test session: pre-sample vehicle 45 s, muscimol 37 s.; post-sample saline 39 s, muscimol 41 s. e. Intra-hippocampal anisomycin immediately and 2 h after the sample session disrupted novel object preference (vehicle, n = 12; anisomycin, n = 15), although test session object exploration was similar: vehicle 45 s, anisomycin 38 s. However, object memory was spared in mice that received intra-hippocampal anisomycin only 2 h post-sample (vehicle, n = 12; anisomycin, n = 11), and again object exploration was similar: vehicle 45 s, anisomycin 37 s. f. Novel object preference was also impaired in mice given intra-hippocampal muscimol infusions pre-sample, post-sample, and pretest, simulating a permanent hippocampal (saline, n = 8; muscimol, n = 8). Test session object exploration was equivalent between the two groups: saline 40 s, muscimol 48 s. 166

184 Figure 7. Encoding, consolidation and retrieval of object memory by C57BL/6J mice requires hippocampus 167

185 Figure 8. Hippocampal inactivation impairs the retrieval of a strong object memory. Modified NOR task was designed to test the role of the hippocampus in a. contextdependent retrieval of strong object memory. Arrowhead indicates when the intrahippocampal infusion was conducted. b. Representative spread of pre-test intrahippocampal fluorophore-conjugated muscimol (FCM) within the dorsal hippocampus. c. Pre-test session infusion of FCM (Exp t 6) impaired object memory in mice that had received three 10-min sample sessions (1/day) in the same context (see photos in a), demonstrating hippocampal involvement in retrieving a strongly encoded object memory (saline, n = 8; FCM, n = 8). Figure 8. Hippocampal inactivation impairs the retrieval of a strong object memory 168

186 Figure 9. Schematic of protocols and histological verifications. a. Schematic depicting the functional inactivation protocols. The darker blue background highlights the specific sessions for the three different NOR protocols. Top- Cue Card NOR, white square arena with dark cue card on one wall throughout all sessions. Middle- Conventional NOR, white square arena (no polarizing cue). Bottom- Drop-in NOR, same arena as in Conventional NOR, except that objects were not introduced until after a 2 min free exploration of arena during object sessions. Mice received bilateral intra-hippocampal infusion of muscimol or saline immediately following the sample session. b. Top- Representative bilateral intra-hippocampal infusion sites within the CA1 region of the dorsal hippocampus for the Cue Card NOR experiment (black-filled circles). These sites are representative of the infusion locations for all of the inactivation experiments. Bottom- Characteristic photomicrograph of the intra-hippocampal microinfusion site into the CA1. Figure 9. Schematic of protocols and histological verifications 169

187 Figure 10. Discrimination ratios for sample session and test session for the varying protocols. a-d) Mean ± SEM discrimination ratios for object sessions in the Cue Card NOR, Conventional NOR (< 5-min and 20-min delays), and Drop-in NOR protocols. White and gray bars of each graph represent the vehicle and muscimol-treated groups, respectively. Within the bars, vertical lines represent object discrimination during the sample session, while diagonal lines indicate object discrimination during the test session. Object exploration during the sample session was equivalent across the treatment groups and mice did not discriminate between the two identical sample session objects. Significant differences between groups were found for the test session (all except Conventional NOR < 5-min delay), and these are designated with an asterisk. *, P < 0.05 vs. vehicle group. Each mouse received bilateral 0.35 µl microinfusion of vehicle or muscimol (1 µg/µl) immediately after acquiring the sample session exploration criterion (see Materials and Methods), or in the < 5-min delay of the Conventional NOR the treatments were administered 20-min prior to the sample session. 170

188 Figure 10. Discrimination ratios for sample session and test session for the varying protocols 171

189 Figure 11. Protocols used to test picture-object equivalence in mice. A, Conditions in the arena during each experiment. Each row offers a side view into the arena to depict stimuli presentation during the sample and test sessions, for each experiment. Mice explored pictures in the arena, during the sample session (left column). During the test session 24 h later (right column), mice explored the "familiar" physical object that had been presented as a picture during the sample session, and a novel object (Exp. 1-3); or the familiar object (sample session: rotated) and a novel object (Exp. 4); or the familiar picture, the "familiar" picture scrambled, and a novel picture (Exp. 5); or the familiar object (sample session: scrambled) and a novel object (Exp. 6); or the familiar object (sample session: silhouette) and a novel object (Exp. 7). Note, for Exp. 1 and 3, mice received a microinfusion of saline or muscimol (1 µg/µl) immediately following the sample session. During Exp. 3, 4 and 6, mice explored the stimuli from behind a clear Plexiglas insert, preventing access to tactile cues afforded by the stimuli. B, Close-up images of the six distinct pictures. 172

190 Figure 11. Protocols used to test picture-object equivalence in mice 173

191 Figure Supplemental protocols used to test picture-object equivalence in mice. This figure depicts the conditions in the arena during the respective sessions of each experiment. Each row represents the configuration of stimuli present in the arena from a side view for each experiment. Each experiment consisted of a sample session (left column) in which mice explored identical 2D pictures or 3D objects in a familiar arena, followed by a test session 24 h later (right column), in which mice explored: the 2D picture of the object that had been presented as a 2D picture during the sample session (the familiar picture), and a novel 2D picture (Exp. S1); the "familiar" 3D object (presented as a 2D picture during the sample session) and a novel 3D object (Exp. S2); the familiar 3D object (presented as a 3D object during the sample session) and a novel 3D object (Exp. S3); the familiar 3D object (presented as a 2D picture during the sample session) and a novel 2D picture (Exp. S4A and S4B); the familiar 3D object (presented as a rotated 2D picture during the sample session) and a novel 3D object (Exp. S5); or the familiar 3D object (presented as a 3D object during the sample session) and a novel 2D picture (Exp. S6). Note that for Exp. S4B-S6, mice explored the sample and test session stimuli from behind a clear Plexiglas insert that prevented access to tactile cues afforded by the stimuli. 174

192 Figure Supplemental protocols used to test picture-object equivalence in mice 175

193 Figure Exp. S1 - Consolidation of picture recognition memory is dependent upon the dorsal hippocampus. A, Each mouse explored two identical novel pictures of a radially symmetric metal leveling foot during the sample session. Upon acquiring sample session picture exploration criterion, the mouse was removed and received bilateral intrahippocampal saline or muscimol. The latency to reach the sample session exploration criterion was similar between the future treatment groups [saline 570 s, muscimol 565 s; t(16) = 0.15, n.s.]. During the test session 24 h later, a novel picture replaced one of the familiar pictures. The post-sample saline-treated mice explored the novel picture significantly more than they did the familiar picture during the test session [t(8) = -4.97, P = 0.001]; behavior consistent with visual recognition memory. However, post-sample muscimol-treated mice explored both pictures equivalently [t(8) = 0.89, n.s.], indicating a failure of recognition memory. Discrimination ratio scores were significantly different between the post-sample treatment groups [t(16) = 3.85, P = 0.001]. This difference between the groups was not due to a difference in overall object exploration during the test session [saline 16 s, muscimol 21 s; t(16) = -1.92, n.s.]. *, P = vs. the respective saline condition. B, Representative intrahippocampal infusion sites within the CA1 region of dorsal hippocampus for all infusion experiments (i.e., Exp 1, 3, S1, and S2) and representative photomicrograph of cannula placement (inset). Figure Exp. S1 - Consolidation of picture recognition memory is dependent upon the dorsal hippocampus 176

194 Figure 12. Successful object discrimination regardless of symmetry, likeness or viewing angle of the sample picture, is impaired when the dorsal hippocampus is inactivated posttraining. Test session performance of post-sample intrahippocampal saline- and muscimol-treated mice when the sample picture was of a symmetric or asymmetric object, and object exploration differences when session stimuli were visually similar, or the sample picture was presented as a rotated view of the test session familiar object (Exp. 1-4). A and C, Mice that received post-sample saline immediately after viewing pictures of a radially symmetric (A, Exp. 1) or a radially asymmetric object (C, Exp. 3), explored the novel object significantly more than the "familiar" during the test session; behavior consistent with picture-object correspondence. Mice that received post-sample muscimol explored both objects equivalently, irrespective of the pictured object s radial symmetry, implying that hippocampal inactivation impaired consolidation of memory for the pictured object, and consequently these mice failed to exhibit test session behavior consistent with picture-object correspondence. B, During the sample session, mice that viewed 2D pictures of a stimulus visually similar to the test session novel object from within the Plexiglas insert, 24 h later explored the familiar object significantly less than the novel object (Exp. 2). Inset, mice exhibited a significant discrimination of the novel object over the familiar object. D, Mice that viewed rotated pictures of the test session familiar object from within the Plexiglas insert, 24 h later explored the novel object significantly more than the (non-rotated) familiar object (Exp. 4). Inset, mice exhibited a significant discrimination of the novel object over the familiar (non-rotated) object. *, P < 0.05 vs. respective saline condition (A & C); vs. familiar object (B & D). 177

195 Figure 12. Successful object discrimination regardless of symmetry, likeness or viewing angle of the sample picture, is impaired when the dorsal hippocampus is inactivated posttraining 178

196 Figure Exp. S2 - Replication of Exp. 1 with an extended test session duration. This experiment tested whether extending the duration of the test session would improve the expression of picture-object equivalence in mice. Naïve mice (n = 20) received bilateral intra-hippocampal muscimol or saline immediately after the sample session. Sample session latency to criterion was similar between the future treatment groups [saline 575 s, muscimol 562 s; t(18) = 0.33, n.s.]. During the extended 10-min test session presented 24 h later, the saline-treated and muscimol-treated mice explored the novel object more than the familiar [t(10) = -9.91, P < and t(8) = -5.75, P < 0.001, respectively]. Although both groups performed above chance [t(18) = 5.96, P < 0.001], discrimination ratio scores were significantly lower for the post-sample muscimol mice as compared to the post-sample saline mice [t(18) = 5.56, P < 0.001]. There was no difference in overall object exploration during the test session [saline 118 s, muscimol 93 s; t(18) = 2.43, P = 0.026]. Further, an analysis of discrimination ratios, with total exploration as a covariate, preserved the significant treatment effect stated above [ANCOVA: F(1,17) = 54.48, P < 0.001). *, P < 0.05 vs. respective saline condition. Figure Exp. S2 - Replication of Exp. 1 with an extended test session duration 179

197 Figure Exp. S3 - Extending the duration of the traditional object recognition test session (3D stimuli for sample and test sessions) significantly increases discrimination between objects. This experiment tested whether extending the duration of the test session would affect the expression of object discrimination in mice, as observed with picture-object equivalence. The sample and test sessions both consisted of 3D objects, with a 24 h delay between sessions. Mice that explored test session objects for a 5-min test session (n = 12) exhibited a significant discrimination between the familiar and novel objects [for data see 5]. Similarly, mice given a 10-min test session preferred the novel object [t(8) = -7.34, P < 0.001], demonstrating clear discrimination between stimuli [t(8) = 14.35, P < 0.001]. However, the mice given a 10-min test session displayed a significantly greater discrimination between objects than those given a 5-min test session [t(19) = -2.81, P = 0.01]. These findings suggest that novelty-driven exploration follows a longer time course in mice than that described for other animals. *, P = 0.01 vs. 5 min test. Figure Exp. S3 - Extending the duration of the traditional object recognition test session (3D stimuli for sample and test sessions) significantly increases discrimination between objects 180

198 Figure Exp. S4A and S4B Access to tactile information supersedes novelty preference when the novel stimulus is a 2D picture and the 3D object is familiar. We tested whether the preference for exploring the novel stimulus would extend to a more challenging condition in which the mice were presented with a novel 2D picture stimulus and a familiar 3D object. In this case, the novel 2D picture was more similar to the sample stimulus based on its physical nature alone, which may have led mice to explore it less than the familiar 3D object. However, if the mice truly formed 2D to 3D equivalence, then it was predicted that the novel 2D picture would be preferred over the familiar 3D stimulus. A, Naïve mice (n = 10) explored 2D pictures during the sample session. Mice preferentially explored the "familiar" 3D object over the novel 2D picture during the test session 24 h after the sample session [t(9) = 7.02, P < 0.001], presumably because 3D objects are more appealing to mice than 2D pictures [discrimination ratio (see Inset), t(9) = , P < 0.001]. B, To test whether the substantial tactile information provided by the 3D object overrode the proclivity of mice to explore the novel 2D stimulus (Exp. S4A), we repeated the experiment, but the stimuli were placed outside of a clear Plexiglas insert within the arena to prevent access to tactile cues. This time, during the test session, the mice preferentially explored the novel 2D picture, over the familiar 3D object [t(9) = -5.50, P < 0.001]. Inset, mice exhibited a significant discrimination of the novel 2D picture over the 3D familiar object [t(9) = 5.80, P < 0.001]. This result indicates that when tactile information is unavailable, object exploration during the test session is guided by stimulus novelty rather than the physical characteristics of the objects. These results provide further support for the view that mice are capable of picture-to-object equivalence. *, P < vs. the familiar 3D object. Figure Exp. S4A and S4B Access to tactile information supersedes novelty preference when the novel stimulus is a 2D picture and the 3D object is familiar 181

199 Figure Exp. S5 - Replication of Exp. 4 with an extended test session duration. This experiment tested whether extending the duration of the test session would affect the expression of picture-object equivalence in mice. Naïve mice (n = 10) were exposed to rotated pictures of the familiar test session object during a sample session. They were then allotted 10 min to explore the test session objects, and demonstrated a significant preference for the novel 3D object [t(9) = -5.12, P < 0.001]. Inset, mice exhibited a significant discrimination of the novel object over the familiar object [t(9) = 4.86, P < 0.001]. Compared to the object discrimination elicited with a 5-min test session (Exp. 4), extending the duration of the test session increased object discrimination [t(18) = -2.54, P = 0.02]. These findings provide additional support that mice do not lose their proclivity to explore novel items after short intervals of time. *, P < 0.05 vs. 5-min test. Figure Exp. S5 - Replication of Exp. 4 with an extended test session duration 182

200 Figure Exp. S6 - Discrimination of an individual object presented in both 2D and 3D forms. It is possible that limitations of the mouse visual system may preclude mice from truly perceiving the difference between an actual 3D object and a 2D picture of that object. This experiment confirmed that the mice identify the 2D pictures and 3D objects as separate entities, as opposed to perceiving them as the same stimulus. During the sample session, naïve mice (n = 9) were placed within the Plexiglas arena insert for a maximum of 10 min where they visually explored two identical 3D objects. During the test session 24 h later, the mice were returned to the insert and allowed 5 min to visually explore the familiar 3D monkey and a familiar 2D picture of the monkey. The mice preferentially explored the familiar 2D picture over the familiar 3D object [t(8) = -6.79, P < 0.001]. This result indicates that rodents identify the 2D familiar picture as a stimulus visually distinct from that of the familiar 3D object. Inset, the mice exhibited a significant discrimination of the familiar 2D picture over the familiar 3D object; the discrimination ratio was significantly greater than chance performance [t(8) = 7.80, P < ]. *, P < vs. the familiar 3D object. Figure Exp. S6 - Discrimination of an individual object presented in both 2D and 3D forms 183

201 Figure 13. Mice rely on composite images for subsequent object recognition. Mice are incapable of matching scrambled images of an object to its actual 3D form or its holistic image. A, Mice explored pictures of an asymmetric object, and a blank picture, then 24 h later, explored both the novel picture and scrambled picture of the "familiar" object significantly more than the familiar picture (Exp. 5). This result suggests that mice likely recall the sample session picture as a composite image. B, Mice explored pictures of a scrambled object from within the Plexiglas insert, then 24 h later, visually explored the familiar and the novel objects equivalently (Exp. 6). Inset, mice did not discriminate between test objects, indicating that accurate identification of the familiar object is only possible when the picture representation is sufficiently similar to the actual object. *, P < 0.05 vs. familiar picture. Figure 13. Mice rely on composite images for subsequent object recognition 184

202 Figure 14. Accurate object discrimination after viewing the familiar object as a 2D silhouette. Mice explored abstract pictures of an object, and 24 h later, explored the novel object significantly more than the familiar object (Exp. 7). Inset, mice demonstrate object discrimination, indicating that the retrieved memory of the viewed silhouette conveyed enough information to permit recognition of the familiar object; furthering support for picture-object equivalence. *, P < 0.05 vs. familiar object. Figure 14. Accurate object discrimination after viewing the familiar object as a 2D silhouette 185

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