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1 ARTIFICIAL REARING ALTERS C-FOS EXPRESSlON IN THE BRAN OF JUVENILE FEMALE RATS Andrea Gonzalez A thesis submitted in conformity with the requirements For the degree of Master of Arts Graduate Department of Psychology University of Toronto QCopyright by Andrea Gonzalez 200 1

2 National Library I*l of Canada Acquisitions and Bibliographie Sewices Bibliothèque nationale du Canada Acquisitions et services bibliographiques 395 Weflington Street 395, rue Wellington Ottawa ON KiA ON4 ûttawaon K1AûN4 Canach Canada The author has granted a nonexclusive licence allowing the National Library of Canada to reproduce, loan, distri%ute or seu copies of this thesis in microform, paper or elecîronic formats. The author retains ownership of the copyright in this thesis. Neither the thesis nor substantial extracts f?om it rnay be printed or otherwise reproduced without the author's permission. L'auteur a accordé une licence non exclusive permettant à la Bibliothèque nationale du Canada de reproduire, prêter, distribuer ou vendre des copies de cette thèse sous la foxme de microfiche/film, de reproduction sur papier ou sur format électronique. L'auteur conserve la propriété du droit d'auteur qui protège cette thèse. Ni la thèse ni des extraits substantiels de celle-ci ne doivent être imprimés ou autrement reproduits sans son autorisation.

3 ARTIFICIAL REAR-iNG ALTERS C-FOS EXPRESSION [N THE BRAN OF JUVENILE FEMALE RATS Master of.arts Andrea Gonzalez Department of Psychology University of Toronto Abstract This study investigated the effects of early-rearing experiences on maternal and social behaviors and the pattern of c-fos activation in associated neural sites in juvenile female rats. From days 4 to 20 of life, female rats were reared artificially, without a mother or with their mothers. On postnatal day 20 a11 animals were randomly assigned to one of four juvenile conditions. The juvenile rats were assessed behaviorally, on matemal or social tests, and then sacrificed for imrnunohistochernical localization of c-fos. AR rats exposed to pups engaged in less pup-licking, and significantly more play behaviors. AR rats in social groups engaged in more agonostic behaviors towards playmates than 5IR rats. AR animals had significant reductions in c-fos immunoreaciivity in the medial preoptic area (MPOA), the parietal and piriform cortices. These results are discussed in terms of the various possible mechanisms that mediate the e ffec ts of early experience on juveni le behavior. Keywords: early expenence, c-fos, brain plasticity, artificial rearing, MPOA, juvenile maternal and social behaviors

4 Acknowledgements 1 would like to thank Professor Alison Fleming for her support, guidance, and infinite wisdom. I would also like to extend my thanks to Professor Scott MacDougalI-Shackelton for his insightful comments and his assistance with imrnunohistochemistry procedures. Many heartful thanks to other lab members and fnends that have supported me throughout the past two years, including, Alex Hemadez, Stephanie Rees, Irene Drmic, Radek Budin, and Ming Li. A special thanks to Emis Mohebat for assisting me with the imrnunohistochemistry during the most grueling moments and to Alison Diaz for her wonderful schematics. Thanks to Ann Lang. who is a wealth of knowledge and patience. I would like to thank Vedran, who is not only an inspiration to me professionally but also personally. Thank you Vcdran for your suppon. love and laughter. Finally. I would like to thank rny family for their encouragement and undying faith. 1 would like to especially thank my Mom who is my role model, my best friend and who taught me how to dream... iii

5 Table of Contents... Title Page Abstract Acknow ledgements... Table of Contents... introduction General Method... Subjects and Housing Apparatus... Procedure Surgery and Reanng... Rearing and Weaning... Weaning and Groups... Juveniie Materna1 Behavior Testing... Social Behavior Testing and Controls... Immunohistochemical Procedure... Quantification of Fos-lir... Statisical Analyses Results Juvenile Materna1 Behaviors: Cornparison of Rearing Conditions... Percent of Juvenile Animais Retneving...

6 Duration of Matemal Behaviors and Frequency of Retnevals... Juvenile Matemal Behaviors: 3-Group Cornparison... Percent of Juvenile Animals Retrieving... Duration of Materna1 Behaviors and Frequency of Retrievals... Juvenile Social Behavior: Comparison of Rearing Condition... Plus-Maze Test of Emotionality... Behavion Exhibited Dunng Reexposure... C-Fos immunohistochemistry... Reanng e ffects... Juvenile condition effects... Correlations Between Behavior and Density of Fos Labeiing... Brain schernatics and photomicrographs... Discussion... Re ferences... Figure Captions...

7 Anificial Rearing and Juvenile Materna1 Behavior 6 The purpose of the present study was to investigate the effects of early matemal deprivation on the later expression of rnaternal behavior and the pattern of c-fos activation in the brain of matemal juvenile female rats. A juvenile mode1 was chosen to initially study this question because juvenile rats exhibit parental behaviors towards foster pups in the absence of hormones and the behavior is regulated by the same neural systems as in the adult. Presently, there is a similar study underway that addresses the same question in adult postpartum rats. Mother rats respond to their newborn infants the moment they emerge from the birth canal (Rosenblatt& Lehrman, 1963). Immediately following parturition. female rats will display a standard set of rcsponses towards her Young. In her repertoire of behaviors, mother rats, for example, will build a nest, retneve pups to the nest, lick pups, especially the anogenital region. and adopt a crouching posture over the pups to promote feeding (Fleming & Rosenblatt. 1974: Rosenblatt & Lehrman. 1963; Weisner & Sheard. 1933). Circulating hormones towards the end of pregnancy and ai the time of parturition are thought to initiate the onset of matemal behavior. Specifically, this hormonal profile consists of decreasing progesterone levels, and increasing levels of estrogen, prolactin, oxytocin and other hormones (Bridges, 1990; Numan, 1994). Hormonal influences promote maternal responsiveness in a number of ways; they reduce female's neophobia, increase the attnctiveness of pup odors, and facilitates maternal leaming by promoting the pups' reinforcing value (Fleming, Cheung, Myhal. & Kessler, 1989; Fleming & Sarker, 1990; Fleming, Korsmit, & Deller, 1994). Hormones are essential for the initial activation of neural mechanisms that are involved in rnaternal

8 Artificial Rearing and luvenile Materna1 Behavior 7 behavior. Once the behavior is expressed, it no longer requires hormonal mediation of its maintenance (Fleming & Blass, 1994). The maintenance of matemal behavior over the next three weeks becomes less reliant on hormones and becornes dependent on incorning sensory information from the pups including somatosensory and chemosensory stimulation (Orpen & Fleming, 1987; Malenfant, Barry, & Fleming, 1991 ; Morgan, Fleming, & Stem, 1992) and most likely involves leaming and memory processes (Fleming & Blass, 1994). Unlike postpartum rat, virgin rats do not spontaneously behave matemally when presented with pups. In almost al1 cases virgin rats initially will exhibit avoidant responses, retrcating to the opposite side of the cage or in more extreme cases cannibalizing the pups (Fleming, Vaccaiino. & Luebke, 1980). If the exposure towards pups is maintained however, virgin rats will eventually become rnatemally responsive towards the pups, behaving in a similar fashion to that of the parturient female, within six to ten days (Rosenblatt, 1967). This process is commonly known as sensitization (Rosenblatt, 1967). Like virgin females, juvenile female rats can also be induced to exhibit components of materna1 behavior using a sensitization process. Several lines of research have shown that juveniles express many of the same cornponents of matemal behavior as adults show, through sensitization between ages of days. Juveniles are capable of building a nest, retrieving pups to the nest. and lying on top of the pups (hovering) and licking pups (Bridges. Zarrow. Goldrnan, & Denenberg, 1974; Bninelli & Ho fer, 1990; Brunelli, Shindledecker, & Hofer, 1985; Gray & Chelsey, 1984; Mayer & Rosenblatt, 1979). While juveniles exhibit components of matemal behavior it has been argued that

9 Artificial Rearing and Juvenile Materna1 Behavior 8 they do not display the full repertoire of behaviors seen in partunent females (Brunelli et al., 1985; Oxley & Fleming, 2000). Juveniles' matemal responsiveness has been referred to as having a "piecemeal" quality to it since they, a) are not always consistent in displaying matemal behavior, b) the maternal behaviors are not integrated well and c) the matemal responses are often mixed with play/social responses (Mayer, 1983). One criticism of these studies is that the researchers only investigated juvenile maternal behavior over a five-day pup-exposure pet-iod and had they extended the testing period they may have seen a more integrated and consistent pattern of behaviors from the juveniles. There is a large body of literature on the various neural substrates that are involved in matemal behavior. Two or the key structures involved in maternal behavior are the medial preoptic area (MPOA) and the bed nucleus of the stria terminalis (vbnst). Studies involving lesions or knife-cuts to the MPOA and vbnst have found disruptions in maternal responding (Jacoboson, Terkel, Bridges. & Sawyer, 1979; Numan, 1974; Numan, 1990; Numan & Numan, 1996). Lesions to the MPOA have also been found to disrupt instrumental responding for pup-delivery in an opennt conditioning paradigm (Lee, Clancy, & Fleming, 2000). Other studies have shown that hormonal stimulation; using implants of parturitional into the MPOA or vbnst, and electrical stimulation of the MPOA facilitate matemal behavior (Insel, 1990; Insel & Shapiro, 1992; Nurnan 1990; Morgan, Watchus, Milgram, & Fleming, 1999). Other neural stmctures implicated in matemal behavior include the amygdala and the ventromedial hypothalamus (VMH). both of which are suggested to inhibit maternal behavior. Lesions to the amygdala of virgin female rats result in short latencies to

10 Arti ficial Rearing and Juvenile Materna1 Behavior 9 become maternal, due to a release from 'inhibition'; behaviorally, these females are also less neophobic and show reduced aversive responses to pups (Fleming, et al., 1980). Such lesions are assumed to act by preventing aversive pup cues from affecting the amygdala (Fleming, et al., 1980). Conversely, electrical stimulation of the media1 arnygdala results in animals with longer latencies to become matemal (Morgan et al., 1999). Recent evidence has suggested that the VMH may be inhibitory to the expression of matemal behavior. Microinjections of tachykinin neuropeptide K into the VMH dclay the onsei of matemal behavior in females prirned to behave maternally (Sheehan & Numan, 1997). Furthemore, N-methyl-D-aspartic acid (NMDA) lesions of the VMH stimulated a rapid onsei of matemal bchavior in steroid-primed nulliparous femnles (Bridges, Mann, & Coppeta, 1999). There is increasing evidence that neural systems that underlie maternal responding by juveniles are similar to neural mechanisms that regulate adult matemal behavior. Lesion studies done in juvenile fcmale rats have supported the idea that bnin areas implicated in adult matemal behavior also play a role in juvenile maternal behavior. In juvenile rats, lesions of the MPOA disnipt pup retrieving and crouching (Kalinenchev, Rosenblatt, & Morrell, 2000; Oxley & Fleming, 2000); whereas lesions of' the media1 amygdala facilitate pup-retrieval and crouching (Oxley & Fieming, 2000). It is argued that only large lesions to the juvenile MPOA will produce such deficits (Kalinenchev, et al., 2000), and mal1 lesions will not disrupt behavior, as it will in the adult. Moreover, as in adult females, bilateral infusions of morphine disrupted parental behavior in both male and female juveniles, whereas morphine and naloxone did not produce any deficits (Kinsley, Wellman, Carr, & Graham, 1993; Wellrnan, Cam, Graham, Jones, Ruscio,

11 Arti ficial Rearing and Juveni le Materna1 Behavior 10 Billack, & Kinsley, 1997). Further research by Zaias, Okimoto, Trivedi, Mann, and Bridges (1996) has impiicated a role for opioids on parental behavior in juvenile rats. These researchers found that administration of naltrexone, an opiate antagonist, increased latencies in maternal responsiveness of juvenile rats. Taken together these studies illustrate a role for the MPOA and the amygdala in the developing circuitry of juvenile rats that exist pior to any expeiience or the onset of hormonal influences. A more recent method of investigating the neural circuitry underlying maternai invoives the use of early immediate genes, such as, c-fos. The proto-oncogene c-fos is a class of immediate early genes that producc a protein called Fos. which serves as a marker of neural activation which cnables bnin mapping of sites which are activated during various stimulus situations and behavioral States (Sagar, Sharp. & Curran, 1988). Studies using Fos as a marker of brain activation during matemal behavior has been uscd by many researchers (Fleming, Suh, Korsmit, & Rusak, 1994: Korsmit & Fleming, 19%; Lonstein, Simmons. Swann, & Stem, 1998; Lonstein & Stem, 1997; Numan. Numan, Marzella. & Palumbo, 1998; Sheehan, Cirrito, Numan & Numan, 2000). Rats that are matemally responsive show enhanced Fos-lir in a number of brain regions in the matemal circuit, including the MPOA, the arnygdala, and the ventral bed nucleus of the stria terminalis (vbnst), with higher numben of cells in each of the regions seen in mother rats exposed to pups (Fleming, Suh, Korsmit, & Rusak, 1994; Korsmit & Fleming, 1996; Lonstein. Simmons. Swann, & Stem, 1998; Numan, Numan, Marzella. & Palumbo, 1998). Few studies have looked at c-fos expression in the juvenile brain during the expression of matemal behavior in juvenile fernale rats. A recent study in our lab (Drmic

12 Artificial Rearing and luvenile Matemal Behavior 1 1 et al., submitted) found fernale juveniles that show maternal licking and crouching behavior when exposed to pups show an increase in Fos-lir in some of the same sites as found in adults. Juveniles that exhibited matemal responsiveness towards young pups had an increase in Fos-lir and PKC in the vbnst, similar to that of postpartum adults; and a decrease in the VMH in both pup-exposed and social groups; consistent with what bas been reported in the adult virgin (Numan & Numan, 1995; Drmic et al., submitted). Given that the VMH is involved in the inhibition of matemal behavior, it is possible that juvenile animals may be under inhibitory rnechanisms that decreases the likelihood that they will show matemal responsiveness towards young. Another study found that in femaie juvenile rats, the lateral habenula (LH) was the only area that had more c-fos expression in both matemal juveniles and adult rats compared to controls (Kalinichev, et al., 2000). Al1 other areas examined in juvenilcs did not produce significant results. They authors suggest that this lack of expression of c-fos in these brain regions in the juvenile may be due to immaturity of the system (Kalinichev, et al ). Based on the behavionl results, that juveniles were engaging in retrieval and other matemal-like behaviors, it is surprising that maternal behavior in the juvenile is not associated with an increase in Fos-lir in key matemal structures such as the MPOA. Although matemal behavior is quite stereotyped and species-characteristic, recent evidence indicates that the form it takes depends in part on the experiences that the animal has dunng its earlier life in the nest with mother and littermates. In a series of studies we have found, for instance, that if young are exposed to artificial odorants in the nest, while being nursed by their rnothen, in adulthood, they respond preferentially to pups with the same odor characteristics (Lovic, Shah, Oxley & Fleming, 1999; in prep).

13 Artificial Rearing and Juvenile Materna1 Behavior 12 As well, if young rats receive less licking while in the nest, they in tum, lick their own offspring less after the birth of their own litters (Caldji, Tannenbaum, Sharma, Francis. Plotsky & Meaney, 1997; Francis & Meaney, 1999; Gonzalez & Fleming, unpublished data; see also Moore, 1995). Finally, growing up in a large, as opposed to small litters, results in less licking stimulation received from own mother, which, in tum, translates into less licking of own offspring later on (Lovic, Shah, Oxley & Fleming, 1999). Al1 of these effects indicate the importance of varying the quality of the expenence while with mothcr and littermates. Another approach to the study of early experience, is to raise pups with reduced access to mother or in the absence of mother altogether (Gonzalez, Lovic, Ward, Wainwright & Fleming, ; Lovic, Gonzalez & Fleming, in press). Using this deprivation andior isolation paradigm, we have found. deficits in adult matemal responding to offspring, such as decreases in body licking and crouching over pups and an increase in inappropriate. non-pup directed behaviors (Gonzalez, Lovic, Ward, Wainwright & Fleming, 200 1; Lovic, Gonzalez & Fleming, in press). We have also found differences in artificially reared animals when iested as juveniles, such as, decreases in pup body licking and increases in social. agonistic behaviors towards same age conspecifics (Gonzalez, Riveros & Fleming, in prep). Taken together these studies highlight the importance of early experience and matemal care on subsequent social and materna1 behavior. There are a number of potential mechanisms thai could mediate these deprivationinduced deficits in materna1 behavior. Hypothesized mechanisms include. changes in the hypothalamic-pituitary-adrenal (HPA) axis (Kuhn & Schanenberg, 1998; Lui, Diorio.

14 Artificial Reanng and Juvenile Maternai Behavior 13 Tamenbaum, Caldj i, Francis, Freedman, S harma, Pearson, Plots ky, & Meaney, 1997; Suchecki, Rosenfeld, & Levine, 1993; Vasquez, van Oers, Levine, & Akil, 1996; van Oers, de Kloet, Whelan & Levine, 1998). A second possibility is changes in the dopamine reinforcement, which is known to play a role in materna1 behavior and is dysregulated as a function of early experience (Bridges. 1996; Fleming & Deller, 1994; Hall, Wilkinson & Robbins, 1999; Hansen, 1994; Hansen, Bergvall, & Nyiredi, 1993; Keer & Stem, 1999; Kehoe, Triano, Surcsh, Shoemaker, & Arons, 1998; Stem & Keer, 1999). A third possibility is changes in endocrinological receptor systems underlying matemal behavior, such as, oxytocin (Insel, 1997; Nelson & Panskepp, 1998; Noonan. Caldwell, Li, & Walker, 1994; Panskepp, Nelson, & Siviy, 1994; Uvans-Moberg, 1998). A final possibility is changes in the neural circuitry of matemal behavior, which is the hypothesis of the present study. The present study was designed to investigate the effects of early experiences, specifically, the amount of matemal care during the neonatal period, on: 1) female juvenile matemal and social behavior and, 2) brain changes, specifically, c-fos expression, that occur as a function of both early life experiences and expressed behaviors during the juvenile period. We hypothesized that rats that are isolated from their mothers would not only show deficits in their subsequent pup-directed behavior; they would also show reduced activation in the neural activity that underlies that behavior. Specifically, we hypothesized that in cornparison to mother-reared (MR) juvenile rats, juvenile rats that are artificially-reared (AR) would show reduced c-fos expression in response to pup-stimulation, in the media1 preoptic area (MPOA) and/or the bed nucleus of the stria terminalis (BNST), the amygdala, the lateral habenula and other

15 Artificial Rearing and Juvenile Materna1 Behavior 14 sites that are nonnally activated during the expression of matemal behavior. Moreover. AR anirnals that receive additional Iicking-Iike somatosensory stimulation are predicted to show a pattern of c-fos expression more sirnilar to MR animals. Finally, these group differences in c-fos expression are predicted to occur in response to a behavioral context that is not different between the groups. General Method Slbjects and Housing The subjects in this study were primiparous day old Sprague Dawley rats and four of their fernale offspring al1 of which were bom at University of Toronto at Mississauga, from a stock originally obtained from Charles Rivers Farms in St. Constant, Quebec. The animals were housed individually in clear, Plexiglas cages (22 x 44 x 30 cm). Animais were provided with woodshavings and had ad lib access to Purina Rat Chow food and water. The animals were maintained on a 12: 12-h light: dark cycle, with lights on at 0800h. The roorn temperature and humidity were maintained at 24OC and 40-50%, respectively. Animais used in this expenment were treated according to the standards set by the Canadian Council on Animal Care (CCAC). Apparatus During behavioral testing the animals were individually housed in clear, Plexiglas cages (22 x 44 x 30 cm). AI1 testing and subsequent behavioral measures were obtained using the subject's own cage as the testing apparatus. The elevated plus-maze was used as a test of emotionality. The plus maze was made of wood and painted gray. It consisted of four arms (1 O cm X 40 cm) raised from the floor (50 cm). Two of the arms

16 Artificial Reriring and Juveniie Materna1 Behavior 15 were open (i.e., no enclosing walls) and two were enclosed by three walls, 43 cm in height. Procedure Fernale dams gave binh, and on the day of parturition (PND O) their litters were cuiied to ten pups, four males and six females. On PND 4 four females were removed from the nest, three of the females undenvent a surgical procedure called a gastrostomy and a fourth was rnarked with coloring and returned to nest (intact control. motherreared). Two of the three females that undenuent surgery wcre raised artificially (experimental, minimally-reared and maxirnally-reared) and the third had the gastrostomy tube cut offjust outside the skin and was retumed to the nest aftcr being marked with a different color (surgical control, mother-reared). Experirnental rats were artificially reared frorn PND 4-20, with the exception of a suckle control and a sham surgery group which were reared with the dams. Hence, four neonatal groups were created, 1.artificially reared maximal stimulation (AR-MAX. n= 241, 2.artificially reared minimal stimulation (AR-MIN. n= 23). 3.surgicaVsham surgery, mother rearcd (MR-SHAM. n= 24), 4.intact control, mother reared (MR-CONTROL, n= 24). Sztrgey and Rearing: Al1 animals were weighed pior to surgery. The surgical animals were anesthetized in a bel1 jar with approximately 1-ZmL of methoxyflurorane (Metofane, CDMV Inc). The surgery involved inserting a leader wire (stainless steel, 0.25 mm in diameter) sheathed in Silastic tubing (Dow Coming, VWR Scientific) and PE- 10 (Clay Adams) tube into the pups' mouth and down the esophagus. Men the end of leader was visible (through the translucent skin of the pup) the pup was held firrnly and the leader

17 Artificial Rearing and Juvenile Materna1 Behavior 16 was pushed from within the stomach through the lateral wall of the stomach. The rest of the gastrostomy tube was lubricated with oil and was pulled gently through the pup until the Ranged end contacted the inside wall of the stomach. A washer was placed over the gastrostomy tube against the outer wall the pup and held in place with a small amount of Superglue (Hall, 1975; Diaz, Moore, Petracca, Schacher & Stamper, 1981). Neosporin, antibacterial cream was applied ropically at the site of penetration. The implantation usually took no more than 90 seconds and the pups awakened within 3-5 minutes. Rearing and Weaning: Following the gastrostomy, the pups were housed individually in plastic cups ( 1 1 cm in diameter x 1 jcrn deep) which Tir into a second weighted cup which floated in temperature-controlled water bath (aquarium filied with water maintained at 36 C; this temperature is decreased to approximately 27OC once the animais grow fur and can thermoregulate on their own). The cups were filled with corn-cob bedding (Bed O' Cobs) and the lids of the cups remained open to allow the gastrostomy tubing to emerge and to connect to nearby synnges containing milk formula. Syringes containing the formula diet (Messer diet, taken from the University of Iowa; Smart, Stephens & Katz, 1983; Messer, Thoman, Terrasa & Dallman, 1969) were mounted on timer-controlled infusion pumps (Harvard Apparatus Syringe Pumps Series PHD 2000). The pumps were programmed to infuse the diet for 10 minutes every hour, 24 hours daily. The amount of diet the pump was calibnted to deliver was based on a specified fraction of the mean pup weight for the pumps (for the first day, the amount was 33% of the mean body weight. This amount slowly increased up to a maximum of 47% of mean body weight).

18 ArtificiaI Rearing and Juvenile Maternai Behavior 1 7 Each morning the pups were removed From the cups, weighed and had their tubing flushed with O. lcc of distilled water. The infusion syringes were replaced with new syringes containing fresh diet and the pumps were recalibrated according to the new mean pup weight per pump. One group of artificially reared anirnals (minimal stimulation, MIN group) were swabbed twice a day (the required minimum) with a warm. wet paintbrush swiping their anogenital regions for approximately 45 seconds to stimulate urination and defecation. A second group of artificially reared pups (maximal stimulation. MAX group) were stroked five times a day with anogenital and ovenll body stimulation: the stimulation lasted two minutes per pup. This stimulation manipulation was camed out from the day the pups were placed on the pumps (PND 4) to the day of weaning (PND 20). Weaniizg and Grorrps: On PND 20 artificiaily reared animals were weaned off of the pumps and mothcr reared animals were weaned from the nest. At this time the survival rate for the artificially reared pups was 75%. Each animal was placed individually into a clear, Plexiglas cage (22 x cm) with regular Purina rat chow, a mashed version of rat chow mixed with the formula and fresh water. The subjects were given one shredded paper towel tom into strips that were 14 cm in length by 2cm in width for nest construction. The artificially reared subjects' tubing was cut offoutside the skin once the animal was determined to be eating well on its own. ARer weaning each set of siblings (one AR-MAX, one AR-MIN, one MR-SHAM and one MR-CONTROL) was randomly placed into one of five conditions: a pupexposeci group from PND (PUP4: n=20), a pup-exposed group from PND 22-29

19 Artificial Rearing and Juvenile Maternal Behavior 18 (PW8: n=20), a social group, exposed to a social conspecific from PND (SOC: n=20), a social group, exposed to a social conspecific from PND (SOC: n=15) and an isolated control group (CON: n=20). On PND 21 al1 animals were tested in the elevated plus maze. ARer an hour of habituation to the testing room, subjects were tested in the plus-rnaze for five minutes. At the start of the test the subjects were placed in the center of the maze and time spent in one of four arms (open or closed) was recorded for five minutes using an event recorder (NEC-PC 8300). Animals were said to be in either the closed or open arms of the maze, based on the location of their head and front extremities. Since the central area was not enclosed, it was included as part of the open am. For statistical purposes plus-maze measures were calculated as open am percent scores [(time spent in open armsbo0 seconds) x 100= % time spent in open arms]. Juvenile Maternul Behavior Tesring: In the pup-exposed group (PUP4) from PND females were tested for materna1 behavior each moming for four consecutive days, between 0900hr and 1300hr. In the pup-exposed group (PUPB) from PND females were tested for matemal behavior on eight consecutive days during the same time period. On the First test day four 1-5 day old pups were taken from a lactating 'donor' mother and placed into the cage of the subject on the opposite side to where the female juvenile was located or her nest was located. Maternal observations consisted of continuous recording of the frequency and duration of each behavior using a cornputer based event recorder (NEC PC-8300) in skewed time intervals ( x 1 O seconds). Testing was terminated after the tenminute interval and the foster pups were left with the female juvenile ovemight and

20 Artificial Rearing and Juvenile Maternal Behavior 19 removed the following moming. The foster pups were then retumed to their 'donor' mothen and a new recently fed set of four foster pups were placed in the cage to start the next 1 O-minute observation period. During the matemal observation period the following behaviors will be recorded: 1) retrieval- when the female picks up a pup in her mouth and carries it to her nest site, 2) pup licking which is subdivided into genital licking and body licking-which occurs when the female licks the pups' anogenital and body regions, 3) pup snifing-involves active sniffing of the pups by the female, 4) lying in contact with the pups or hovering- this occurs when the female is lying on top of or in contact with one or more of the pups, 5)nest building- this behavior occurs when the female brings the paper towel in to her nest or in the quadrant in which the pups are located, and 6) mouthing picking at the pup with an open mouth. In addition to materna1 behaviors, any play behaviors towards the pups were recorded. such as, jerking- when the animal twitches violently and repeatedly, runningkharging- when the animal mns energetically toward the pups or tears around the cage, playing- wrcstling with the paper towel and hanging from the top of the cage. Spot checks were done twice a day, hvo hours and four hours after the testing was completed. During the spot checks the location of the nest. the juvenile female and each of the four pups were recordrd on a sheet. These two pupexposed conditions were created because it was expected that the juveniles exposed to pups for only four days will be on the threshold of becoming matemal and the subjects exposed to pups for eight days would reach maternai criterion within this time period. Maternal criterion was defined as retrieving one or more pups to the nest. hovering over and licking the pups.

21 Artificial Rearing and Juvenile Matemal Behavior 20 After the last test was completed the pups remained with the juveniles until 2 100hr at which time the pups were removed and retumed to their mothers over night. The following rnorning between 0900hr and 1300hr four pups were reintroduced into the subject's cage. During reexposure another ten-minute behavioral test was recorded after which eight spot checks were done at 15-minute intervals. At the end of the two-hour reexposure period, the juvenile rats were overdosed with sodium pentobarbital in preparation for immunohistochernistry. Social Behavior Testing and Conrrols: Juvenile females in the social conditions were exposed to a sarne-age, same-sex conspecific for either four consecutive days (PND 22-25) or for eight consecutive days (PND 22-29). The subject's behavior was recorded on the first day that they were introduced to the conspecific (PND 22) for ten minutes. Social behaviors consisted of continuous recording of the frequency and duration of each behavior using the same method described above. Social behavion that were recorded included: 1) chargerunning after the playmate, 2) mount and pounce-grabbing the backside of the playmate or making contact by lunging at the playmate, 3) wrestling- rolling rough and tumble play, 3) hairpull- pulling or biting ai the hair of the playmate. 5) body sniffing- sniffing the playmate, 6) jerking- twitc hing violently, 7) grooming the partner or grooming self and 8) pinning partner- standing on top of the partner, holding thern down, 9) being pinned by partner and 10) huddle or settle with the partner in the corner 1 1) daning - ninning away from playmate, 12) running, jumping and climbing. The juvenile subject and their conspecific were left undisturbed for the following four or eight days depending on their experimental condition. On the last day of exposure, either PND 25 or PND 29

22 Artificial Rearing and Juvenile Materna! Behavior 2 1 at 2000hr the playmate was removed ovemight. The following moming between 0800hr hr the same playmates were reintroduced into the subject's home cage and the behavior of the subject was recorded for ten minutes. The playmate remained with the subjeci for the next two hours. At the end of the two-hour reexposure penod. the juvenile rats were overdosed with sodium pentobarbital in preparation for immunohistochemistry. The animals in the control group were ler undisturbed in their cage. Half of the control subjects were lefi undisturbed for four days (PND 22-25) and the other half were left alone for eight days (PND 22-29) to create two separate control groups matched for each of the pup-exposed and social conditions. Both the social and the control subjects were housed in a separate room (the pup deprived room) from the pup-exposed groups so as to eliminate any potential exposure to pup associated cues that these subjects may encounter. Inlnlttrtohistoc/iemicai Procedures: On the moming of the day of sacrifice. groups received two hours either with pups (PUPI and PUPB), with a same-age conspecific (social) or were left undisturbed (isolate control). At the end of the exposure period, the females were overdosed with 0.5cc of sodium pentobarbital (Somnotol, MTC Pharmaceuticals, MO). Subjects were perfused transcardially first with p hysiological saline followed by fresh, chilled 4% paraformaldehyde in O. 1 M phosphate buffer (ph=7.4). The brains were removed from the skulls and postfixed in a solution of 30% sucrose in 4% paraformaldehyde for cryoprotection until the brains sank (approximately 2 days). Coronal brain slices, 40 pmthick were sliced using a cryostat and the first slice was saved For immunohistochernistca1 analysis and a second slice was maintained as ertras as required. Slices were collected

23 Artificial Rearing and Juvenile Matemal Behavior 22 and stored in M phosphate-buffered saline treated with the antibacterial agent Triton-X (0.2%; PBSx; Dimension Labs, Mississauga, ON) until irnrnnohistochemical processing. Immunohistochemical procedures were perfonned in accordance to the protocols established by our laboratory (Dnnic, Oxley, O'Day & Fleming, submitted). The tissue was washed in a 0.3% hydrogen peroxide solution for 30 minutes. it was then rinsed in three changes of PBSx for 10 minutes each and then incubated for 18 hours in a sheep affinity-putified polyclonal antibody to Fos oncoproteins (Santa Cruz Biotech. Santa Cruz, CA) which \vas diluted to a concentration with 1 % normal rabbit serum (NRSPBSx; Vcctor Labs, Burlington. ON). Brain sections were cxposcd to the primary antibody ai 4OC for approximately 23 hours. Following incubation, the tissue was washed three times in PBSx and then placed in a 1500 dilution of secondary antibody (biotinylated anti-sheep irnmunoglobulin for Fos, obtained from Vector Laboratories) for 2 hours. Brain sections were then rinsed three times in PBSx for ten minutes each, and then placed for 1 hour in the Avidin-Biotin-Complex (Vector Laboratories). Next, the tissue was subjected to three IO-minute rinses in PBSx, and was placed in 0.05% diaminobenzidine (DAB/phosphate buffer solution) for five minutes. Glucose oxidase (0.006% salt) was then added to the tissue in the DAB solution and lefi for approximately 30-minutes or more depending on the strength of the chromogen reaction. The tissue was hed in three final washes of PBSx and then the brain sections were mounted on gelcoated slides and cover-slipped with Permount. In order to control for and assess the variability across immunohistochemical mns, brain sections of each set of experimenial siblings were represented in each run. To

24 Artificial Reanng and JuveniIe Materna1 Behavior 23 control for the specificity of the immunothistochemical staining, adsorption controls with the synthetic immunogen on brain sections were taken from altemate slices from two pup-exposed rats; no Fos-lir was detected in any brain region. Quanr13cation of Fos-lir: Analysis was conducted using previous methods established in our laboratory (Fleming et al., 1994; Fleming & Korsmit, 1996; Walsh et al., 1996). The neural sites that were examined for Fos-lir are sites that have been implicated in matemal behavior, social behavior, sensory processing and associative processes. All sites were examined at the anterior-posterior (AR, measured in millimeten) level in front of bregma. These sites included, the media1 preoptic area (MPOA; analyzed at -0.4 from bregma), the ventral bed nucleus of the stria terminalis (vbnst; -0.4), the ventromedial hypothalamus (VMH: -2.8 from bregma), the cortical. media1 and basolatenl amygdala (al1 analyzed at -2.8 from bregma), the paraventricular nucleus (PW; from bregma), the parietal and pirifonn cortex (-0.26 from bregma), the globus pallidus (-0.8 from bregma), the periaqueductal gray area (PAG; -5.2 from bregma), the lateral habenula (-2.8 from bregma) and the nucleus accumbens (0.70 from bregma). The number of cells showing Fos-lir were examined under a light microscope with a magnification of 10 X 15. A reticule containing a grid pattern measuring 10 x 10 squares ( 1 mm2) was inserted into the microscope. Using predetermined grid measurements for each neuroanatomical site based on the juvenile stereotaxic atlas of Shenvood and Timaras ( 1970) and based on the stereotaxic atlas of Paxinos and Watson (1998), the number of cells exhibiting Fos-lir in a given site was recorded. Counts were obtained using eye counts by a trained observed # blind to group condition. In order to determine consistency counts were tested for intn-

25 Artificial Rearing and Juvenile Maternal Behavior 23 observer reliability by being repeated on ten brains by the same investigator on two separate occasions. The intra-rater reliability for the 10 brains ranged from 0.76 to 0.92 for the different brain sites studied, with ratings for most sites showing ~ 0.84 (pco.01). Statistical Analyses Proportional data (percentage of anirnals retrieving) were analyzed with Chi square tests. Frequency and duration of behaviors for multi-group cornparisons were analyzed using a series of analysis of variance (ANOVA) followed by Tukey post-hoc tests. To determine whether there were differences between rearing conditions and juvenile conditions for cell numbers showing positive Fos-lir in each of the relevant neural sites, general linear rnultivariate ANOVAs were computed followed by Tukey post-hoc tests. The level of statistical signi ficance was ~~0.05. Pearson r correlations were contrasted for durations of maternal or social behaviors with plus-maze behavior and for number of fos-lir labeled cells and behavion recorded during reexposure phase. Data were analyzed using SPSS (SPSS 8.0 for Windows). RESULTS Juvenile Maternal Behaviors: Comparisons of Rearing Conditions, AR versus BIR To highlight the large effects of artificial rearing on juvenile maternal behavior, in the fint set of analyses the two mother reared groups were combined (MR=20) and the two artificially reared were combined (AR=20). The two mother reared groups did not differ from one another in any behavior, while the two artificially reared groups did differ on some behaviors, as will be described (below) when considering the effects of additional stimulation.

26 Artificial Reanng and Juvenile Maternal Behavior 25 Percent of juvenile materna1 anirnals retrieving The percent of animals retrieving were compared using a chi-squared test. As can be seen in Figure 1, the group showing the highest percent of animals retrieving pups were MR animals exposed to pups for 8 days (100%). while only 70% of the AR animals exposed to pups for 8 days retrieved. Animais from both rearing conditions with 4 days of exposure to pups showed the lowest percentage (30%) of retrieval during the juvenile period. This difference between the two groups, PUP4 and PUPI, for percent retrieving was significantly different within both the AR and MR groups (Chi-square, p c 0.0 1). This suggests that the longer the exposure to pups the more likely the animal is to retrieve. Insert Figure 1 about here Durations of Maternal Behnviors and Frequency of Retrievals As expected, the two pup-exposed groups, PUP4 and PUP8. did not differ from one another on any of the 'early' behaviors (days O and 1). Only 'late' behaviors. defined as the last two days of testing, (for PUP4= days 2 and 3; for PUP8= days 7 and 8) were analyzed to illustrate differences between rearing conditions. The duration and frequency of matemal behaviors were analyzed using a series of 2 (AR vs. MR) X 2 (PUP4 Vs PUP8) ANOVAs. The main finding was that artificial reanng reduces pup-body licking and pup anogenital licking in both juvenile groups and increases social directed behavior in the presence of pups. As can be seen in Figures 2 a-b, there was a significant effect for

27 Artificial Reanng and Juvenile Materna1 Behavior 26 rearing condition for pup body licking (F (1,36) = 10, p~o.0 1) and a marginal effect for pup anogenital licking (F (I,36) = 2.939, p=0.095). MR animals in both the PUP4 and the PUP8 groups engaged in more pup licking than did AR animals. There were no significant differences between rearing conditions for nest building, hovenng, pup body sniffing, or mouthing (see Figures Z c-f). There was a significant main effect for juvenile condition for pup body sniffing, (F (I,36) = 6.702, p<o.oz), and a marginal effect for mouthing, (F (1.36) = p=0.087), with PUPJ animals engaging in more body sniffing and PUP8 animals mouthing pups more. There were no differences between rearing conditions in latency to retrieve pups. There were no significûnt interactions Inscrt Figures 2 a-f about here There was a significant main effect for juvenile condition for frequency of retrievals, (F (1, 36) = 39.67, p<0.001), with PUP8 anirnals retrieving more pups than PUP4 animals (see Figure 3). which is consistent with the percent results reponed above. The frequency data for both materna1 and non-pup directed behaviors followed the same pattern of results as reponed for the duration data and are therefore not included as a separate set of analyses. Insert Figure 3 about here In addition to matemal behaviors, non-pup directed behaviors were recorded. There were significant main effects for rearing condition for hopping, (F (1.36) = 5.322,

28 Artificial Reanng and Juvenile Maternal Behavior 27 p<0.05, jerking, (F (1,36) = 7.684, p<o.oz) and running, (F (1, 36) = , ~ ), with AR animals showing increased durations of these behaviors than MR anirnals in both juvenile conditions (see Figures 4 a-c). These differences between rearing conditions highlight the increased overall hyperactivity of arti ficia!ly reared animals. There were no differences between groups for grooming, playing or digging. There was also no di fference between juvenile conditions for non-pup directed behaviors and there were no interactions. Insert Figure 4 a-c about here... Juvenile Maternal Behaviors: 3-Croup Comparison, MAX versus MIN versus MR In order to assess the efkcts of replacement somatosensory stimulation the two anificially reared groups were cornpared: AR-MAX, versus AR-MIN with the mother reared group. MR. Maternai behaviors were analyzed using a series 013 (MAX vs. MIN vs. MR) X 2 (PUP4 vs. PUPI) ANOVAs. These were followed by Tukey post hoc comparisons of pairs of groups. Percent of juvenile materna1 animals retrieving The percent of anirnals retrieving were compared using a chi-squared test. As cm be seen in Figure 5, the group showing the highest percent of animals retrieving pups were MR animals exposed to pups for 8 days (100%), an increase of 70% compared to the PUP4 day group. Only 60% of the MAX animals were retneving in both the 4 and the 8 day pup groups. MIN animals with 4 days of exposure to pups shocved the lowest

29 Artificial Rearing and luvenile Matemal Behavior 28 percentage (0%) of retrievals dunng the juvenile period and increased to 80% in the 8 day group (Chi-square, p < 0.05) Insert Figure 5 about here Durations of Materna1 Behaviors and Frequency of Retrievals Among arti ficially reared animals. maximal stimulation restores some of the deficits produced by artificial reanng, including pup licking. There was a significant main effect for rearing condition for pup body licking (F (2.34) = 5.940, p<0.02), hopping, (F(2, 31) = , p<o.o5), jerking, (F (2. 34) = , p<0.05), and running, (F (2,34) = 7.87, pco.02). as can be seen in Figure 6. Post hoc cornparisons show that the AR-MIN group was significantly different from the MR group for body licking and hopping and both the AR-MIN and the AR-MAX were significantly different from the MR group for running around the cage. As seen in Figures 6a, AR-MIN animals engaged in less pup body licking and the AR-MAX group fell between the AR-MM and MR animals. Significant main effects for juvenile condition for pup body snifiing (F(2.34) = 5.733, pco.05) and frequency of retrievals (F (?,34) = 33.72, pc ) were found (sec Figure 6). No other significant effects for hovering, nest building, mouthing, grooming, digging, and piaying were found. No significant interactions were found for any of these behaviors. Insert Figures 6 a-f about here

30 Artificial Re3sing and Juvenile Materna1 Behavior 29 Juvenile Social Behavior: Cornparison of Rearing Condition, AR versus MR The 4 and 8 day social groups did not differ from one another behaviorally and were therefore combined for statistical purposes. The two mother reared groups and the two artificially reared groups did not differ from one another and were therefore combined to compare just the main reanng conditions, AR versus MR. Social behaviors were analyzed using a one-way ANOVA. The main finding was that anificial rearing result in an increase in agonistic behaviors towards social conspecifics. There were significant effects for charging (F( 1, 34) = , pc ), climbing (F ( 1.34) = 4.984, p<o.os), hairpull (F (1,34) = , pco.00 1), pinning playmate (F ( 1,34) = 4.606, p<0.05), pouncing (F (1,34) = 6 1.O62, pco.00 l), mounting, (F ( 1,34) = , pco.00 1). running (F ( 1.34) = p<o.oo 1 ), twitching (F ( 1,34) = , p<o.oo 1 ) and jumping (F ( 1,34) = 3.169, p<o.oj), with AR anirnals showing higher durations in al1 of these behaviors than MR animals (see Figure 7). There was also a significant main effect for grooming self (F ( 1.34) = , p<0.02) and a marginal effect for huddling in the corner with the playmate (F (1,34) = , p=0.096), with MR animals showing higher durations for these behaviors than AR animals (see Figure 7). The frequencies of social behaviors followed the same pattern of results as the duration of social behaviors and there fore are not reported as a separate set of analyses.... Insen Figure 7 about here * Plus-Maze Test of Emotionality

31 Artificial Rearing and Juvenile Matemal Behavior 30 The number of entries into open arms of the plus-maze and the percent of time spent in the open arms were analyzed using a one-way ANOVA comparing AR and MR animals. As can be seen in Figure 8, AR animals spent a higher proportion of time in the open arms of the elevated plus-maze (F (1,94) = 34.40, p<0.001) but they also showed a significant number to entries into the arms (F (1.91) = 94.41, p<o.oo 1). If a three group comparison is performcd, followed by a post hoc comparison it is evident that both the AR-MA\( and AR-MIN groups are significantly different frorn the MR groups but that the MAX group Falls behveen the two other groups for both percent of time spent in open arms (F (2.94) = 48.3 IO,, p<0.001) and number of entries into opcn arms (F (2.94) = pc ). See Figure 9. Insen Figures 8 & 9 about here *----- Behaviors Exhibited During Reexposure Jwenile Maternal Behavior There were no significant differences between AR and MR animals during the 2- hour exposure phase prior to sacnficing in total time for each behavior. However, when comparing the 3 rearing groups there were significant main effects for hovering over pups, (F (1, 39) = 4.362, pc0.05). pup body licking (F (1-39) = 3-277, p=0.05), jerking, (F ( 1, 39) = , p<o.oo 1 ), and mnning (F( 1,39) = 6.602, pco.02); consistent with earlicr differences. As can be seen in Figure 10, MAX animals did less body licking in the PUP4 group but more in the PUP8 group than the MIN animais. MN animals exhibited less hovering during the exposure phase than both the MR and MAX groups;

32 Artificial Rearing and Juvenile Materna1 Behavior 3 1 post hoc tests reveal a significant difference only when compared to the MAX animals. There were significant juvenile condition X rearing condition interactions found for running (F (2,31) = , p~0.00 1) and jerking (F (2,34) = , pco.00 1). For boih running and jerking behavior, in the PUPl group MIN animals exhibited the highest levels of these behaviors, but in the PUP8 day group MAX animals had higher levels than the MM and MR groups (see Figure 10). There were no significant differences between conditions for percent reinevals. There was also no significant difference bchveen groups for percent of animals spending time in contact with pups during the 15-minute spot-checks over a 2-hour penod. *-----* * Insert Figure 10 about here There was a main effect for playing behavior between the PUP4 and PUP8 groups. with animals in the four day playing in the presence of pups significantly more than animals in the eight day group (F ( 1, 39) = 6.629, p<0.02). There were no other significant differences found between juvcnile conditions during the IO-minute reexposure test. Jrtvenile Social Behavior During Reexposrtre Cornpanson of reanng conditions (AR vs. MR) for social behaviors on the day of sacrifice revealed significant main effects for hair pulling (F (1.34) = 6.062, pco.05). pouncing (F( 1,34) = 9.158, p<o.oz). and a marginal effect for running around the cage (F (1,34) = 3.537, p=0.076). As can be seen in Figure 1 1, AR animals engaged in more of these behaviors than MR animals. There were also significant main effects between


34 Artificial Rcaring and Juvenile Matemal Behavior 33 When cornparing the 3 reanng conditions (MAX vs. MIN vs. MR), there were main effects for the MPOA (F (2,94) = 2.870, p=0.06) and the piriform cortex (F (2,941 = 3.550, p<o.os), with MR animals within the pup groups having the highest ce11 densities and the MIN having the lowest (see Figure 13). There were no other significant di fferences. Jzivenile Condition Effecrs Insert Figures 13 a-c about here... The arcas with significant main effects for juvenile condition included. the MPOA (F(1,91) = , p~0.00 l), vbnst (F(1,94) = 7.982, p<o.ooi), BLA (F (1,94) = , p<0.05), lateral habenula (F ( 1.94) = 3.935, p<o.oz), PAG (F ( 1,94) = 2.85, pe0.05, and the VMH (F (1,94) = 4.755, p4.02). In all cases the two pup groups had the highest ce11 densities than the social and isolated control groups. Post hoc cornparisons show that for the MPOA both pup groups were significantly different from the social and isolated controls. The PUP8 group was significantly different from the control group for the BLA. For the BNST, the PUP group was significantly different from the al1 of the other groups. For the VMH the PUP4 group was significantly different from the SOC group, and was significantly different from both the SOC and the CONTROL group for the latenl habenula. There were no other significant differences between juveniie conditions, and there were no significant interactions (see Figures 12 a-h).

35 Artificial Rearing and Juvenile Matemal Behavior 34 A series of 3 (MAX vs. MM vs. MR) X 4 (PUP4 vs. PUP8 vs. SOC vs. CON) ANOVAs were analyzed for the various neural sites, the results replicate the above 2 X 4 analyses and are therefore not described or illustrated. To assess whether retrieval differences may be contributing to the observeci differences in Fos-lir expression in the above analyses the two pup groups were collapsed and divided into animals that retrieved (n= 28)or did not retrieve pups (n= 12). 2 (AR vs. MR) X condition (retrieve vs. no retrieve vs. social vs. control) ANOVAs were computed on each of the relevant neural sites. These fmdings replicate the results descnbed above. Correfarions Beriteen Behavior and Demity of Fos Labelirig To determine whether there was a relation between density of Fos reactive cells at the different brain sites and different behavior, Pearson r correlations were computed across juvenile conditions for each cornparison. Most of the correlations were low and not significant, pco.0 1, this level was established given the large number of correlations. Brain scheniarics and phoiomicrograplis Figure 14 shows the MPOA for AR and MR animals within each of the pup conditions and a representative animal (either AR or imr) from each of the social and isolated control groups. Figures 15 and 16 show the pinfom and parietal cortex respectively, with one AR and one MR animal represented for each of the four juvenile conditions. Each figure shows, a) a schematic of each neural site, b) a representative distribution of Fos-labeled cells, where each cell represents five labeled cells and c) a photomicrograph at 1OX magnification using an inverse light microscope. Figures show schematics of the vbnst, BLA, LH, VMH, and PAG for each of the four juvenile conditions, a representative animal (either AR or MR) was chosen for each figure. Each

36 Artificial Rearing and Juvenile Materna1 Behavior 35 figure shows, a) a schematic of each neural site, b) a representative distribution of Foslabeled cells, where each cell represents five labeled cells and c) a photomicrograph at 1 OX magnification using an inverse light microscope Insen Figures about here _-_ Discussion The purpose of the present study was to investigate the effects of 24-hour matemal deprivation and replacement "licking-like" stimulation, on juvenilc matemal and social behaviors and the pattem of neural activation in the brain circuitry that underlies these behavion. The results indicate that manipulations of the earl y preweaning experience of pups result in both a change in their pup-directed behavior matemal behaviors during the juvenile period and a change in the activation pattern ofcfos in the brain. In comparison to mother-reared (MR) animals, anirnals that were arti ficially-reared (AR) showed decreased pup body licking and an increase in play behaviors in the presence ofpups, which persisted even arer the animals staned to retrieve pups and show the full-fledged pattern of matemal behaviors. In cornpanson to MR animals, AR animals also had less pup-stimulated c-fos expression in the media1 preoptic area (MPOA), the parietal cortex, and the pirifom cortex- al1 sites that are normally activated durkg the expression of matemal behavior. The MPOA Functions as the 'last-common pathway' for the expression of the behavior; the pirifom (olfactory) and parietal (somatosensory) conices process the primary sensory modalities that are activated during interactions with pups. The addition of early simulated "licking-like"

37 Artificial Reuing and JuveniIe Materna1 Behavior 36 stimulation provided to the artificially-reared (AR-MAX) animals arneliorated some of the deficits in behavior. For example, for pup licking, AR-MAX animals showed levels that fell between levels exhibited by AR-MM animals and MR animals. A similar trend was evident in c-fos expression in the MPOA. AR-MAX animals had higher numben of Fos labelcd celis in the MPOA than did AR-MIN animals, but these numbers did not reach the same levels found in the MPOA of MR animals. The extra tactile stimulation provided to the AR-MA>( animals. while having a clear effect on brain activation, was not sufficient to bring them to the level of functioning similar to that displayed by MR animals. A second purpose of the study was to investigate the effects of early reanng on the development of juvenile social behavior and the activation pattem of its underlying circuitry. The results show that early reanng expenencc (AR) had effects on many social behaviors; however, in this case thcre were no associated changes in the pattern of activation in neural sites beiieved to be involved in social behavior. While AR animals engaged in more rough-and-tumble play behaviors, such as charging, mounting and hairpulling, than did MR animals. there were no group differences in the density of c-fos expression in any of the relevant sites, including the VMH. globus pallidus, nucleus accumbens or the PVN. areas that have previously been implicated in social behavior (Gordon, Kollack-Walker, Panskepp, & Bingham, 1999; Panksepp, Siviy, & Normansell, 1984; Vandershcuren, Stein, Wiegant, & VanRee, 1995). These results address a number of issues. The first revolves around the nature of the behavion exhibited by juvenile animals towards rat pups, whether it should be charactenzed as materna1 or social. (or both) and the underlying neural bais of these

38 Arti ficial Rearing and Juvenile Materna1 Behavior 37 behaviors. Of issue is the extent to which behavior and brain are functionally and neuroanatomically similar in the juvenile as in the adult. The second issue to be addressed in the forgoing is the effect of early experiences with mother and in the nest on the development of this affiliative system. The issue at question is whether the elimination of mother and nest experiences alters the pattern of developrnent of juvenile affiliative behaviors and whether the juvenile brain exhibits plasticity and is able to be altered by these early deprivations experiences. First, the juvenile expenence or stimulus situation was manipulated to optimize the likelihood that juvenile animals would become maternal with prolonged exposure to pups. Previous studies have investigated the issue of maternal behavior in juveniles using only a five-day exposure period to pups (Brunelli, et al., L985; Oxley & Fleming, 2000: Drmic. Oxley, O'Day, & Fleming. submitted); in the present study a distinction was made between the two pup-exposed groups, one exposed for four days (hence, on the threshold of retrieving and becoming 'matemal') and a second group exposed for eight days (most ofwhorn retrieve pups). In terms of maternal behavior, a higher percentage of animals in the PUP8 than PUP4 groups retrieved pups. with 100% of MR animals in PUP8 group were retrieving by the day of sacrifice compared to only 30% within the PUP4 group. The fact that there were no other behavionl differences between the two groups is not surprising as other researchers have also found that the juvenile rat lacks the initial avenive reactions to pups that characterize the virgin adult animal and hence, the juvenile shows many of the proximal licking and crouching responses that are normally shown by materna1 rats well before they retrieve pups. By cornparison, adult virgin animals presented with foster pups, have altogether longer latencies and tend to crouch

39 Artiticial Rearing and Juvenile Matemal Behavior 38 over and lick pups for the first time only afier retrieval (Gray & Chelsey, 1984; Mayer & Rosenblatt, 1979; Stern, 1987; Stem & Rogers, 1988). Consistent with other studies of juvenile animals, the present study found increases in Fos-ir in the vbnst (Drmic, et al.. submitted) and the lateral habenula (Kalinchev, et al., 2000) in matemal juvenile animals. These results are also consistent with studies done in adult matemal (postpartum) animals, where, in cornparison to non-pup stimulated animals there is a similar increase in Fos-lir in the vbnst and lateral habenula (Kalinchev, et al., 2000; Lonstein et al., 1998; Nurnan & Numan, 1995; Numan et al., 1998). Interestingly, the group with the highest increase in Fos-ir in the lateral habenula was the PUP4 group who does not show the Full pattern of matemal behaviors. Previous research bas found that the lateral habenula is necessary for the onset, but not the maintenance of adult matemal behavior (Corodimas, Rosenblatt, Canfield. & MorrelI, 1993; Corodimas, Rosenblatt, & Monell. 1992; Matthews-Felton. Corodimas, Rosenblatt, & Morrell, 1995). It may well have a similar function in the juvenile. Contrary to other studies in the juvenile (Drmic, et al., submitted; Kalinchev. et al., 2000). but consistent with the pattern of results reported for adults, the present study found significant differences in the MPOA, BLA. and the PAG between pup-exposed and social/isolate control animais, with animals exposed to pups showing increases in Fos-ir in these areas (Calamandrei & Keveme, 1994; Fleming et al., 1994; Fleming & Korsmit, 1996; Numan & Numan, 1994; Numan et al., 1998). The MPOA results compliment previous lesion studies in the juvenile animal (Kalinchev, et al., 2000; Oxley & Fleming, 2000) as well as in the adult (Jacoboson, Terkel, Bridges, & Sawyer, 1979; Numan, 1974; Numan, 1990; Numan & Numan, 1996); in both the MPOA is necessary for the final

40 Artificial Rearing and Juvenile Materna1 Behavior 39 expression of maternal behavior. The BLA is known to be involved in the formation of associations (Everitt & Robbins, 1992) and is suspected to be activated when matemal animals become familiar with pups and become conditioned to cues associated with them (Fleming & Korsmit, 1996). Given this information, it is not surpnsing that the group with the highest numbers of Fos-lir was the PUP8 group, which had the most exposure to the pups, thereby allowing the formation of an association for pup cues. One possible explanation for discrepancy found between the current study and that of previous juvenile work in the pattern of fos expression (Drmic, et al., subrnitted; Kalinchev et al ) is that the paradigms used in the various studies are different. The two previous studies assigned a temporal definition of maternal criterion in order for the juveniles to be considered matemal. Kalinchev et ai. (2000) stated that matemal behavior was considered to be established when the juvenile fernale retrieved and grouped pups in the nest within the fint 15 minutes of testing on two consecutive days. Given that retrieval did not occur in most of the animals in the Drmic et al., (submitted) study, they adopted a different definition of materna1 criterion that did not include reineval but included, hovenng and genital licking of pups for two consecutive days. In both studies the juveniles had only two days during which they exhibited materna1 behavior before sacrifice. In the present study animals were exposed for a predetermined set amount of time to pups; for either 4 or 8 consecutive days regardless of the behavior they exhibited. Although there were no differences between groups in the latency to become matemal the range was from 2-5 days. Hence, the juvenile animals in this study had prolonged exposure to pups and had the opportunity to act matemally towards the pups for more

41 Artificial Rearing and Juvenile Materna1 Behavior 40 than two consecutive days. Perhaps, for the juvenile animal, prolonged exposure is the necessary component for Fos-ir activation within key materna1 sites. The results for the VMH from the current study conflict with previous results reported in the juvenile animal (Drmic, et al., submitted). It has been previously reported that animals exposed to pups or social stimuli show decreases in Fos-lir within the VMH (Drmic, et al., submitted), which is consistent with research that has been reported in adult female virgins (Numan & Sheehan, 1999; Sheehan, et al., 2000). Within the MR animals there is no difference between juvenile conditions, it is within the AR group that a difference between the juvenile conditions is evident. Specifically, the PUP4 group has the highest amount of Fos-ir expression in the VMH compared to that of the control animals. This increase expression in Fos may reflect the fact that AR animals engage in more prosocial behaviors in the presence of pups. The VMH is known to be involved in play behaviors; lesions to the VMH cause a decrease in play behaviors (Panskepp et al ) and increased activation during social, rough-and-tumble play behaviors (Gordon, et al., 1999). Given that AR animals engage in many play behaviors in the presence of pups this rnay be one explanation for the differences between studies. It is evident that these c-fos results suggest that early experiences during the preweaning penod affect the bnin. What is not clear is what the mechanism is that causes this change in Fos expression as a funciion of early rearing. We suspect that these changes may reflect the absence of stimulatory input from the mother, such as olfactory and somatosensory stimulation, that must alter the pattern of activation in Fos expression in the juvenile rat brain. This lack of stimulation normally provided by the mother and nest environment may directly affect the brain, or indirectly affect the MPOA through

42 Artificiai Rearing and Juvenile Materna1 Behavior 3 1 somatosensory areas, such as the panetal and piriform cortices. The precise nature of the brain changes is not known. For instance, whether the elevations in c-fos reflect the direct activation by experience- induced elevations in behavior; or, whether the elevations in c-fos reflect direct effects on brain structure/fùnction that in turn affect later behavioral expression. This is relevant to the present data in two noteworthy ways. In some cases the behavior and c-fos are clearly related and in others there is dissociation. For example, although early deprivation altered both matemal behavior and c-fos expression in the same direction. the two were not correlated. With respect to social behavior there wcre clear behavioral effects of early experience, but no c-fos expression. Elevations in c-fos expression as a function of earlier experiences could reflect changes in reactivi ty of nrurons within the affected regions due to al terations in electrochemical properties of the neurons (changes in neurotransmitter function. enzymes, ions, etc) (Hall. Wilkinson, Hurnby, & Robbins, 1998; Kehoe, Triaoo, Suresh, Shoemaker & Arons, 1998; Matthews, Wilkinson, & Robbins, 1996) or supporting cells (Featherstone, Fleming, & Ivy, 2000); altematively, they it could reflect experienceinduced synaptogenesis (Lui, Diorio, Day, Francis. & Meaney, 2000) or even neurogenesis (Tanapat. Galea. & Gould, 1998; Young, Lawlor, Leone, Dragunow. & During, 1999); or it could produce alterations in the rate of ce11 death (Zhang, Xing, Levine, Post, & Smith, 1997; Zhang, Dent, Levine, Post, & Smith, 19%). In the literature there are a number of rnodels for early experience effects on the brain. For instance. one line of siudies has focused on early olfactory learning and the plasticity within the olfactory system. In the normal nest environment, the young leam about odors that can influence later behavior towards sexual partners (Fillion & Blass,

43 Artificial Rearing and Juvenile Materna1 Behavior ; Wilson & Sullivan, 1994) and towards pups (Lovic, et al., in prep). These early experiences of olfactory-somatosensory associations are encoded by the brain and produce long-lasting changes in brain; such as alterations in the catecholamine and gabagerenic properties of ceils within the olfactory bulb (Najbauer & Leon, 1995, Wilson & Sullivan, 1994). Depnvation of matemal behavior is known to induce a vanety of other physiological, behavioral, rnetabolic, and neurochemical changes that are differentially regulated by environmental and social stimuli (Hall, 1998). These could be independent effects ofearly experience and CO-occur with the changes in neural activation or thcy could induce the neural effects. There is a large body of researc h that shows negative effects of materna1 deprivation on the hypothalamic-pituitary-adrenal (HPA) axis. Pups that are matemally depnved have enhanced corticosterone response to various stressors. increased sensitivity to ACTH, increased c-fos expression in the PVN (which synthesizes CRF), altered metabolisrn, down regulation of rninenlocorticoid and glucocorticoid mrna receptors in the CA 1 region of the hippocampus and serum growth decreases (Kuhn& Schanberg, 1998; van Oers, de Kloet, Whelan & Levine, 1998; Vasquez. van Oers, Levine & Akil, 1996; Suchecki, Rosenfeld & Levine, 1993). Matemal separation during infancy produces an animal with an altered hippocarnpal corticoid receptor system, with, enhanced HPA responsiveness to stress and a stress response that is prolonged (Liu, Diono, Tannenbaum, Caldji, Francis, Freedman, Sharma, Plotsky & Meaney, 1997; Vasquez. van Oers, Levine & Akil, 1996; Suscheki. Nelson, van Oers & Levine, 1995). Stroking has been found to suppress the elevated ACTH stress response and charactenstics of deprivcd rats and to normalize corticosterone and glucocorticoid

44 Artificial Rearing and Juvenile Materna1 Behavior 43 receptor mrna levels (van Oers, de Kloet, Whelan & Levine, 1998), growth hormone secretion and ornithine decarboxlase activity (Suchecki, Rosen feld & Levine, 1993; Evoniuk, Kuhn, & Schanberg, 1979). Hence, by reinstating critical components of the dams' nurturing behavior some of the negative physiological responses due to maternal deprivation can be reversed. Alterations seen in HPA activity have also been found with handling and with normal variations in materna1 care (Francis, Caldji, Champagne, PIotsky & Meaney, 1999; Francis. et al, 1999; Francis & Meaney, 1999; Caldji, et al, 1998; Liu et al, 1997). Differences in the mechanisms that regulate HPA axis and nerve growth properties have also been produced in pups naturally occumng variations in maternal licking and grooming of rat pups and in pups that receive added licking from mothen following 'handling' regime (Caldji, et al, 1998; Liu, et al, 1997; Liu, et al., 2000; Meaney, Diorio, Francis. Weaver. Yau et al., 2000). Taken together these studies show plasticity of various systems that mediate stress and fearfulness in the adult rat that is affected by early matemal care. Although, there has been no direct link established between HPA activity and maternal behavior, an indirect link through the effects of HPA activation and emotional responsivity may be important. Finally, in a recent study by Francis, Champagne, and Meaney (2000) found that naturally occurring variations in matemal care are associated with oxytocin receptor levels in the brain that are involved in the expression of matemal behavior, such as the MPOA and BNST. Dunng the lactation period, offspnng from mothers who were high Iickinghrched-back nuning had higher oxytocin binding in the MPOA and BNST than offspring from low licking/arched back nursing mothers. These findings are interesting

45 ArtificiaI Rexing and Juvenile Matemal Behavior 44 given that oxytocin has been implicated in materna1 behavior, social affiliation and stress (Insel, 1997; Noonan, Caldwell, Li, Walker, Pedenen & Mason, 1994; Panksepp, Nelson & Siviy, 1994). Taken together these studies illustrate how early reanng expenence can affiect matemal affiliative behavion and the underlying neural and neurochemical systems that contributes to these behaviors. As these studies demonstrate the early rearing experience, in particular, the matemal care one receives as a neonate is critical. The mother is not only a provider of nutrients, warmth and protection but also is the source that provides offspring with their expenences that shape their behavioral and physiological development. Materna1 behavior is a crucial fom olearly experience. It is increasingly evident early experience impinges upon very labile systems and circuitries within the brain that cause changes in the expression of later behavior in various stimulus situations.

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50 Artificial Rearing and Juvenile Matemal Behavior 49 Figure Captions Figure 1 Effects of reanng condition, artificial reanng (AR) vs. mother reanng (MR) and juvenile condition, four days of exposure to pups (PUP4) vs. eight days of exposure to pups (PUPI) on percent of juvenile fernale rats exhibiting pup retrieval behavior. Main effect of juvenile condition, Chisquare (p<o.o 1 ). Figures 2 a- Effects of reanng condition (AR vs. MR) and juvenile condition (PUP4 vs. PUPI) on juvenile materna1 behaviors, pup body licking, pup genital licking, hovenng over pups, nest building, mouthing pups and pup body sni ffing. Figure 3 Effects of rearing condition (AR vs. MR) and j uvenile condition ( PUP4vs. PUPI) on frequency of retrievals. Significant differcnce between juvenile conditions (~~0.05) Figures 4 a-c Effects of rearing condition (AR vs. MR) and juvenile condition (PUPJ vs. PUPI) on play behaviors in the presence of pups. hopping, jerking, and running around the cage. Figure 5 Effects of stimulation replacement (AR-MAX or AR-MIN) compared to MR juvenile female rats and juveniie condition (PUP4 vs. PUPI) on percent of pup retrievals. Significant main effect of juvenile condition for the AR-MIN and MR groups, both showing a higher percent of rats retrieving in the PUPI group; whereas, the AR-MAX animals were the same in the two juvenile conditions. Figures 6 a-i Effects of stimulation replacement (AR-MAX or AR-MIN) compared to MR juvenile female rats and juvenile condition (PUP4 vs. PUP8) on juvenile maternai behaviors, pup body lic king, pup genital licking, hovenng over pups, nest building, pup body sniffing and play behaviors in the presence of pups. Bars with symbols above them indicate a significant main effect of rearing condition (pi0.05). For pup body licking and hopping, there was a significant difference between AR-MIN and MR rats. For running around the cage both the AR-MM and AR-MAX groups were significantly different From the MR group. Figure 7 Figure 8 Effects of reanng condition (AR vs. MR) on juvenile social behaviors, charge, hair-pull, mount, pounce, pin, thvitch, and self-grooming. There were significant differences in al1 behaviors except for wrestling behveen the two groups. Effects on reanng condition (AR vs. MR) on ernotionality as measured in the elevated-plus maze on PND 21. The proportion of time spent in the

51 Artificial Rearing and Juvenile Materna1 Behavior 50 open arms (a) and the number of entries into open arms (b) both showed a significant effect of rearing condition. Figure 9 Figure 10 Figure 11 Effects of stimulation condition (AR-MAX vs. AR-MM) compared to MR rats on emotionality as measured in the elevated-plus mare on PND 21. The proportion of time spent in the open arms (a) and the number of entries into open arms (b) both showed a significant effect of stimulation condition. Ident ical symbols indicate signi ficant di fference between groups. Effects of stimulation replacement (AR-MAX or AR-MIN) compared to MR juvenile fernale rats within each juvenile condition (PUP4 vs. PUPI) on pup body licking, hovering, niming. hopping and jerking on the day of reexposure/sacrifice. There was a significant main effect of stimulation condition (p<0.001) for body licking, hovering, jerking and running and a significant interaction between stimulation and juvenile conditions (p<o.oj) for ninning and jerking. Identical symbols indicatc significant difference between groups. Effects of rearing condition (AR vs. MR) on juvenile social behaviors on the day of reexposure/sacrifice. There was a significant main effect for hair-pull, pounce, and running around the cage. with AR rats exhibiting more of this behavior than MR rats. There was also a significant effect for darting away lrom playmate, being pinned by playmate and wrestling, with MR animals having higher durations of thcse behaviors than AR anima!^. Figures 12 a-h Number of cells showing fos-like immunoreactivity in the (a) MPOA. (b) parietal cortex. (c) piriform cortex, (d) bed nucleus of stria terminalis. (e) basolateral amygdala, (0 ventromedial hypothalamus, (g) lateral habenula. and (h) periaqueductal grey area in the artificially reared rats and mother reared rats within each juvenile condition, PUP4, PUP8, SOCIAL and CONTROL. Figures 13 a-c Number of cells showing fos-like immunoreactivity in the (a) MPOA, (b) parietal cortex, (c) pinform cortex in AR-MM, AR-MAX and MR rats within each juvenile condition PUPJ, PW8, SOCIAL and CONTROL. Figures (A) Schematic of eacli of brain regions, MPOA, parietal and pinform cortex. (B) Representative distribution of Fos-labeled cells, where each dot represcnts ftve labeled cells, except within the MPOA where each dot represents ten labeled cells for each rearing condition (AR vs. MR) and each juvenile condition (PUP4, PUPB, SOC, and CONTROL).