Manipulating and probing nerve cells by light. PhD Arto Lipponen Depart. of psychology
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1 Manipulating and probing nerve cells by light PhD Arto Lipponen Depart. of psychology
2 Presentation PART 1 (theory) - Manipulating and probing techniques Optogenetics - manipulating and probing nerve cells by light Optogenetics - methodology Optogenetics summary PART 2 (examples) - Neural substrates of awakening probed with optogenetic control of hypocretin neurons. Adamantidis et al. Nature Functional imaging of hippocampal place cells at cellular resolution during virtual navigation. Dombeck et al. Nature Neuroscience 2010
3 Manipulating and probing techniques Methodology Manipulation Pharmacology Lesioning Electrical stimulation Imaging EEG MEG MRI Temporal scale (how fast?) Spatial scale (how accurate?)
4 Optogenetics - manipulating and probing nerve cells by light - Light could provide a rapid way to to control and readout the activity of nerve cells (or any other cell), if A) Cells would be able to respond to light B) Cells would be able to send light if activated
5 Optogenetics - manipulating and probing nerve cells by light - Our vision is based on rod and cone cells capability to react to light/photons (opsins) Algea express specific ion pumps able to respond to light/photons (channel-, haloand bacteriorhodopsin) Yizhar et al., Neuron, 2011
6 Optogenetics - manipulating and probing nerve cells by light Photon causes modifications in retinal molecule -> change in the opsin protein conformation -> activation of the channel - > activation of the second messenger system -> increase the probability of the cell be activated or inhibited 20Materials/Physiology%20101/Chapter% 20Notes/Fall%202007/chapter_10% 20Fall% htm Boyden 2011
7 Optogenetics - manipulating and probing nerve cells by light - The Aequorea Victoira can produce flash of lights by releasing calcium to distract predators (bioluminescence)
8 Optogenetics - manipulating and probing nerve cells by light - Binding of calcium ions to aequorin leads to the oxidation of coelenterazine to colenteramide. Colenteramide relaxes to the ground state while emitting a photon of 470 nm - Grienberger 2012 After binding of calcium to GCaMP conformational intramolecular changes lead to an increase in the emitted fluorescence of 515 nm
9 Optogenetics -definition genetic targeting of specific neurons or proteins with optical technology for imaging or control of the targets within intact, living neural circuits (Deisseroth et al., 2006).
10 Optogenetics - methodology Isolate corresponding gene for the protein Transfer and merge the gene in the DNA of nerve cells Stimulate the tissue with light Read out the light signal
11 Optogenetics - methodology transfer Transfer by 1. viruses 2. Electroporation 3. Transgenic lines Allows transfer only to certain cells Grienberger 2012 Pama 2012
12 Optogenetics - methodology illumination of the brain tissue Pama 2012
13 Optogenetics - methodology probing the brain Wide field view of a mouse head after the embedment of a cover glass to the thinned bone. Fluorescence images of a yellow red VSFP variant captured through the glass window. Scale bars: 2mm. Knöpfel 2012
14 Optogenetics - methodology probing the brain
15 Optogenetics - summary Optogenetical methods allow temporally and spatially specific method to manipulate and probe neurons
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17 Neural substrates of awakening probed with optogenetic control of hypocretin neurons - - The neural underpinnings of sleep involve interactions between sleeppromoting areas and arousal systems Hypocretin-producing neurons are important for arousal stability and loss of Hcrt function has been linked to narcolepsy However, it is unknown whether electrical activity arising from Hcrt neurons is sufficient to drive awakening from sleep states or is simply correlated with it
18 Neural substrates of awakening probed with optogenetic control of hypocretin neurons Channelrhodopsin was isolated and transferred by viral vectors to hypocretin cells The neurons in the hypothalamus with channelrhodopsin were illuminated by laser
19 Neural substrates of awakening probed with optogenetic control of hypocretin neurons Light stimulation activated labeled hypocretin neurons
20 Neural substrates of awakening probed with optogenetic control of hypocretin neurons Light stimulation of labeled hypocretin neurons reduced latency to wakefulness.
21 Functional imaging of hippocampal place cells at cellular resolution during virtual navigation
22 Functional imaging of hippocampal place cells at cellular resolution during virtual navigation A place cell is a type of pyramidal neuron within the hippocampus that becomes active when an animal enters a particular place in its environment; this place is known as the place field. - Before tetrode EEG recording of individual spiking activity Attempt to screen multiple neurons simultaneously
23 Functional imaging of hippocampal place cells at cellular resolution during virtual navigation Calcium indicator was transferred by viral vector AAV2/1-synapsin-1-GCaMP3
24 Functional imaging of hippocampal place cells at cellular resolution during virtual navigation A glass window was implanted onto the skull and animal was attached to a two photon microscope
25 Functional imaging of hippocampal place cells at cellular resolution during virtual navigation Animal was allowed to study virtual environment
26 Functional imaging of hippocampal place cells at cellular resolution during virtual navigation
27 Functional imaging of hippocampal place cells at cellular resolution during virtual navigation
28 Functional imaging of hippocampal place cells at cellular resolution during virtual navigation
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