Name: ex vivo optogenetic dissection of fear circuits in brain slices
Uploaded: 2016-04-05T17:00:00.000+00:00
Duration: 11 min 13 s
Description: Watch this Scientific Journal Video about Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices at JoVE.com

We thank Cora H&#252;bner and Andrea Gall for help in acquiring some of the representative results. This work was supported by the Werner Reichardt Centre for Integrative Neuroscience (CIN) at the University of Tuebingen, an Excellence Initiative funded by the Deutsche Forschungsgemeinschaft (DFG) within the framework of the Excellence Initiative (EXC 307), and by funds from the Charitable Hertie Foundation.

Ethics statement: All experimental procedures were in accordance with the EU directive on use of animals in research and were approved by the local Animal Care and Use Committee (Regierungspr&#228;sidium Tuebingen, state of Baden-W&#252;rttemberg, Germany) responsible for the University of T&#252;bingen.&#160;
1. Stereotactic Injection Procedure
<ol>
	<li>Prepare sterile tools (scissors, scalpel, clamps, drill, needles, suture material) using a sterilizer. Arrange sterile tools and other required solutions and surgery supplies such as sterile cotton swaps, disinfectant, sterile phosphate buffered saline (PBS, pH 7.4), and H2O2 on a sterile surgical drape.</li>
	<li>Pull glass micropipettes for injections (Figure 1A, inset) using a 3 mm wide box filament on a horizontal microelectrode puller according to the manufacturer&#39;s instructions. 
	Note: For deep injections, the electrode taper should be sufficiently long to reach the ventral most coordinates (approx. 5 mm; for coordinates, see 1.10).</li>
	<li>Premix 1 &#181;l of virus solution and&#160;0.2 &#181;l of 0.1% fast green solution in sterile PBS (for better visibility of solution in the glass pipette). Fill glass pipettes with mixture of virus solution and fast green using a microliter pipette with a fine tip (Figure 1A, inset). 
	Note: Work with recombinant Adeno-associated viral vectors (rAAV) is considered biosafety level 1 (BSL 1) work and needs to be performed in a BSL 1 laboratory. rAAVs used in this study (Figure 1C) were: 1)&#160;mPFC: rAAV-hSyn-ChR2(H134R)-eYFP (serotype 2/9) 16; 2) MGm/PIN: rAAV-CAGh-ChR2(H134R)-mCherry (serotype 2/9) 23; 3) mpITC/BLA: rAAV-EF1a-DIOhChR2(H134R)-YFP (serotype 2/1) 23</li>
	<li>Anesthetize a mouse using a small animal anesthesia machine (Isoflurane: 3% in oxygen for induction). 
	Note: Mice used in this study were 4 - 7 weeks old and of the following strains and genotypes: C57Bl6/J wild type (experiment in Figures 2A and 3A-D); GAD67-GFP 24 (experiment in Figures 2B and 3E-H); Tac2-Cre 25 (experiment in Figures 2C and 3I-J).</li>
	<li>Shave head between ears and eyes. Apply disinfectant (providone-iodine based) to shaved head using cotton swabs.</li>
	<li>Apply an eye ointment to prevent drying of eyes during anesthesia. Subcutaneously inject mouse with analgesic (meloxicam-based, 0.1 ml of 5 mg/ml solution).</li>
	<li>Place mouse in stereotactic frame (Figure 1A) and maintain anesthesia via a gas anesthesia mask (Isoflurane: 2% in oxygen for maintenance). Check anesthesia depth using limb withdrawal reflex before continuing. 
	Note: Maintain sterile conditions as well as possible during the entire surgical procedure; wear disposable facemask, surgical gown, and gloves.</li>
	<li>Make skin incision on top of the head using scissors. Gently pull skin to the side using blunt forceps, fix with clamps to expose skull surface, and clean skull with H2O2.</li>
	<li>Mark injection sites on skull using a fine tip permanent marker and drill holes for both hemispheres (Figure 1B). Coordinates for injections in this study are (from Bregma (mm)): mPFC: anterior 1.9, lateral &#177; 0.3, ventral 2.1; MGm/PIN: posterior 3.0, lateral &#177; 1.8, ventral 3.8; mpITC/BLA: posterior 1.45, lateral &#177; 3.35, and ventral 4.75.</li>
	<li>Mount filled glass pipette onto stereotactic frame connected to a pressure injection device and bring the pipette to Bregma position.</li>
	<li>Break off the tip of the glass pipette using fine straight-tip forceps. Do this gently to prevent aerosol generation of virus solution. Make sure the pipette tip is open by applying a few pressure pulses and observing extrusion of drops of virus solution.</li>
	<li>Go to the desired injection coordinates and inject half of the pipette content (~0.5 &#181;l) using the following settings on the pressure injection device: Pressure: 20 psi, average pulse length: 30 ms, average number of pulses: 50.</li>
	<li>Leave pipette in place for ~1 min before slowly (1 mm/min) retracting it. 
	Note: Make sure pipette is not blocked before repeating procedure with same pipette on other hemisphere. In case of blocked tip, clean or break off tip and zero pipette position at Bregma again.</li>
	<li>Clean skull with PBS (pH 7.4), remove clamps, gently pull the skin together, and suture the incision with individual button suture (3 - 4 knots). Apply disinfectant (providone-iodine based) around the wound.</li>
	<li>Stop the anesthesia and do not leave mouse unattended until it is fully awake. Keep single housed or return to company of other animals only when fully recovered. Postoperatively, continue to monitor the health status, and administer analgesic if necessary. Follow procedures according to rules put forward by the local Animal Care and Use Committee.</li>
</ol>
2. Preparation of Acute Slices
<ol>
	<li>Prepare ACSF, cutting solution, tools (scissors, scalpel, forceps, spatula, Pasteur pipette), and agar blocks. 
	Note: 1 L&#160;of artificial cerebro-spinal fluid (ACSF) is required per experiment, and is prepared by dissolving chemicals in double-distilled H2O as previously published 16,23. Cutting solution is prepared by supplementing 200 ml of ACSF with 0.87 ml of 2 M MgSO4 stock solution.</li>
	<li>Oxygenate ACSF and cutting solution throughout the experiment with 95% O2 and 5% CO2.</li>
	<li>Deeply anesthetize mouse using a small animal anesthesia machine using isoflurane (3% in oxygen). Check anesthesia depth using limb withdrawal reflex before continuing.</li>
	<li>Decapitate mouse using large scissors and immediately chill head in ice-cold cutting solution.</li>
	<li>Open skull by a single midline incision from caudal to rostral and gently push pieces of skull to the sides using forceps. Rapidly remove brain by gently lifting it out of the skull using a small rounded spatula. Cut off cerebellum with scalpel. Place brain in ice-cold cutting solution.</li>
	<li>For mPFC injection sites: cut off anterior part of the brain (containing mPFC) using a scalpel and put in ice-cold cutting solution until slicing.</li>
	<li>Remove excess cutting solution with a filter paper and glue the posterior part of the brain onto the vibratome stage.</li>
	<li>For tilted amygdala slices, glue the brain on an agar block (4%) cut at a 35&#176; angle (Figure 1D, middle). For coronal amygdala slices, MGm/PIN injection sites and mPFC injection sites, glue the brain directly on stage (Figure 1D, left and right). Place an additional agar block behind brain for stability while slicing.</li>
	<li>Place stage in cutting chamber with ice-cold oxygenated cutting solution that is maintained at 4 &#176;C using a cooling unit. Prepare acute slices of the amygdala (320 &#181;m) using a sapphire blade. Place slices in an interface chamber supplied with oxygenated ACSF at RT.</li>
	<li>After preparation of acute amygdala slices, place the interface chamber in a waterbath at 36 &#176;C to recover slices for 35 - 45 min. Subsequently, return the interface chamber to RT. For recording from these slices, proceed with step 3.2.</li>
	<li>During commencement of step 2.10, cut slices of the injection sites as described before for acute amygdala slices (steps 2.8 - 2.9). Recovery of slices in the waterbath is not required here. Optional: To quickly estimate injection site location, observe slices on a stereoscope equipped with a fluorescent lamp and appropriate filter sets.</li>
	<li>Fix slices containing injection sites for post-hoc analysis by sandwiching them between two filter papers and submerging them in 4% paraformaldehyde (PFA) in PBS solution O/N. For analysis of injection sites proceed with step 4.1.</li>
</ol>
3. Visualization and Stimulation of Presynaptic Fibers
<ol>
	<li>Prepare patch microscope for optogenetic activation of fibers and cells:
	<ol>
		<li>Center the mounted light emitting diode (LED) onto the light delivery pathway.</li>
		<li>Measure the LED light intensity at the back focal plane and at the output of each objective with a power meter choosing the appropriate wavelength of 470 nm.</li>
		<li>Calculate the light intensity in mW/mm2 and create a calibration curve (LED intensity (%) versus light output (mW/mm2)) for each objective for values measured for 470 nm wavelength.</li>
	</ol>
	</li>
	<li>Retrieve an acute amygdala slice from the interface chamber and place in the slice chamber mounted onto the upright microscope equipped with a fluorescent lamp. Take care to position the slice such that the slice surface facing upward in the interface chamber is also facing upward in the recording chamber. Perfuse slice with fresh, oxygenated ASCF at a rate of 1 - 2 ml/min at a temperature of ~31 &#176;C.</li>
	<li>Observe presynaptic fibers in the slice using the fluorescent lamp in combination with appropriate filter sets for the specific fluorescent protein expressed. Use 5x objective to obtain an overview (Figure 1E), and 60x objective for assessment of fiber density within the target area. 
	Note: For GFP and YFP,&#160;use Filter set &#34;green&#34; (Excitation 472/20, Beamsplitter 495, Emission 490 LP) for mCherry use filter set &#34;red&#34; (Excitation 560/40, Beamsplitter 585, Emission 630/70) as specified in the materials/equipment table.</li>
	<li>Open or restrict the aperture in the microscope light pathway as desired for the experiment (Figure 2D).</li>
	<li>To obtain a patch recording, fill a patch pipette with internal solution and mount in electrode holder. Apply positive pressure to the patch pipette and slowly lower it first into the bath solution and then under visual control into the slice using the micromanipulator.
	<ol>
		<li>Approach the neuron of interest with the patch pipette from the side and top. Release positive pressure when the pipette is on the surface of the cell (dimple visible on cell surface) and obtain a &#34;gigaseal&#34; by applying negative pressure.</li>
		<li>Apply further suction to rupture the membrane patch to obtain whole-cell recording. Subsequently, stimulate labeled fibers with the connected LED using the appropriate wavelength for activating ChR (470 nm) while recording electrical responses from the cell.</li>
		<li>For synaptic stimulation start with a low LED intensity and increase until the desired synaptic current amplitude is reached. Trigger the LED by configuring digital outputs in the data acquisition software to control the timing and pulse length (examples in Figure 3). 
		Note: other software and/or TTL-generating devices can be used to trigger LED.</li>
	</ol>
	</li>
	<li>Repeat stimulation with opened or restricted aperture (step 3.4) in the microscope light pathway as desired for the next recorded cell and/or in the presence of specific drugs.</li>
	<li>After recording, fix slices for post-hoc analysis by sandwiching them between two filter papers and submerging them in 4% PFA O/N.</li>
	<li>Analyze electrophysiological data. 
	Note: Use appropriate software to visualize recorded sweep data for each individual stimulation offline. When analyzing effects of drugs, use peak detection routines to obtain time course of drug effect on synaptic current amplitudes for all individual sweeps. Determine when the drug effect reaches steady state. To prepare representative average responses for figures, use software to average &#8805;10 individual sweeps per experimental condition (c.f. Figure 3B-D, F-H, J right panel). 
	Note: several alternative software packages can be used for data analysis.</li>
</ol>
4. Post-hoc Analysis of Injection Sites
<ol>
	<li>Wash slices of injection sites (from step 2.12) and recording sites (if re-imaging is desired, from step 3.7) three times in PBS. This is done by repeatedly replacing solution with fresh PBS on a rotating shaker three times for 10 min.</li>
	<li>Prepare 2% agar-agar solution in PBS, and let it cool down to ~65 &#176;C. Place slices flat on the bottom of a small 30 mm diameter petri dish, embed slices in agar-agar and let it cool down until solid.</li>
	<li>Glue an agar block with embedded slice or slices to the stage of a vibratome and place stage in cutting chamber with PBS.</li>
	<li>Resection embedded slices to 70 &#181;m thickness. Optional: After resectioning, slices can be stained, i.e., with Neurotrace to reveal cytoarchitecture (Figure 3A, C), and/or by immunofluorescence staining for YFP or mCherry to boost the signal from the ChR fusion protein.</li>
	<li>Mount slices on slides and coverslip with mounting media. Image injection sites and, if desired, also image slices containing fibers in the projection areas using a fluorescent microscope or a confocal laser-scanning microscope (Figure 2A-C).</li>
</ol>

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N., Deisseroth, K., Paulsen, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemisphere-specific+optogenetic+stimulation+reveals+left-right+asymmetry+of+hippocampal+plasticity.">Hemisphere-specific optogenetic stimulation reveals left-right asymmetry of hippocampal plasticity.</a> Nat Neurosci. 14 (11), 1413-1415 (2011).</li><li>Morozov, A., Sukato, D., Ito, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+suppression+of+plasticity+in+amygdala+inputs+from+temporal+association+cortex+by+the+external+capsule.">Selective suppression of plasticity in amygdala inputs from temporal association cortex by the external capsule.</a> J Neurosci. 31 (1), 339-345 (2011).</li><li>Davidson, B. L., Breakefield, X. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+vectors+for+gene+delivery+to+the+nervous+system.">Viral vectors for gene delivery to the nervous system.</a> Nat Rev Neurosci. 4 (5), 353-364 (2003).</li><li>Aschauer, D. F., Kreuz, S., Rumpel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+transduction+efficiency,+tropism+and+axonal+transport+of+AAV+serotypes+1,+2,+5,+6,+8,+and+9+in+the+mouse+brain.">Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8, and 9 in the mouse brain.</a> PLoS One. 8 (9), (2013).</li><li>Salegio, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport+of+adeno-associated+viral+vectors+is+serotype-dependent.">Axonal transport of adeno-associated viral vectors is serotype-dependent.</a> Gene Ther. 20 (3), 348-352 (2013).</li><li>Holehonnur, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+viral+serotypes+produce+differing+titers+and+differentially+transduce+neurons+within+the+rat+basal+and+lateral+amygdala.">Adeno-associated viral serotypes produce differing titers and differentially transduce neurons within the rat basal and lateral amygdala.</a> BMC Neurosci. 15, (2014).</li><li>McFarland, N. R., Lee, J. S., Hyman, B. T., McLean, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+transduction+efficiency+of+recombinant+AAV+serotypes+1,+2,+5,+and+8+in+the+rat+nigrostriatal+system.">Comparison of transduction efficiency of recombinant AAV serotypes 1, 2, 5, and 8 in the rat nigrostriatal system.</a> J Neurochem. 109 (3), 838-845 (2009).</li><li>Miyashita, T., Shao, Y. R., Chung, J., Pourzia, O., Feldman, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+channelrhodopsin-2+(ChR2)+expression+can+induce+abnormal+axonal+morphology+and+targeting+in+cerebral+cortex.">Long-term channelrhodopsin-2 (ChR2) expression can induce abnormal axonal morphology and targeting in cerebral cortex.</a> Front Neural Circuits. 7, (2013).</li></ol>

The authors declare that they have no competing financial interests.

This protocol describes a method for ex&#160;vivo optogenetic investigation of neural circuits and local connectivity that can be easily implemented on most, if not all, upright slice patch-clamp recording setups by equipping them with a ~470 nm LED at the epifluorescence light port. A major advantage of optogenetic stimulation of axonal projections in slices is that it allows for specific activation and investigation of properties of connections that were not accessible with conventional electrical stimulation, because the corresponding fiber tracts were not known, not clearly defined, or not preserved in a single brain slice. As a key example relevant for fear circuits, it is illustrated how this can be applied to dissect properties of mPFC inputs to the amygdala.
Even in cases where electrical fiber tract stimulation has been extensively used in past studies, these tracts often carry fibers from different regions of origin. For example, in circuits related to fear learning, projections from different sensory thalamic nuclei or cortical regions run intermingled through the internal and external capsules, respectively. The advantage of optogenetic stimulation is that it enables selective activation of a subset of fibers from a defined region. This is illustrated for specific thalamic inputs from PIN and MGm to intercalated cells and lateral amygdala principal cells. Furthermore, problems arising with electrical stimulation, i.e., activation of other fibers of passage or cells in the vicinity of the stimulating electrode that may make local connections in the target area can be overcome. However, while optogenetic stimulation is equally rapid as electrical stimulation, there may be limitations regarding the stimulation frequency that can be reliably applied. This depends on inactivation and deinactivation kinetics and of the variant of ChR that is being used,&#160;4 as well as expression levels and methods, and light stimulation methods26.
When using an LED mounted to the fluorescence port for light stimulation, typically the whole field of view of a given objective is illuminated, resulting in activation of all fibers or cells in the field and within a certain depth. This can only partially be restricted to a smaller region or set of labeled cells (in this example, the mpITC) using an aperture in the fluorescent light path 23,27. With LEDs, light power is not an issue, as high power blue LEDs are now available from many manufacturers. However, the emission spectra have full-width half maximum values of several tens of nanometers. Therefore, LEDs pose limitations when either spatially precise stimulation for subcellular mapping of connectivity and/or monochromatic stimulation are desired. Both can be improved by using blue lasers as light sources, typically in combination with more sophisticated setups that enable targeting and/or scanning of the beam in small areas and defined tissue depths (e.g., see&#160;28).
With the ex&#160;vivo optogenetic approach described here, several properties of synaptic transmission can be studied. To assess monosynapticity of the early synaptic response component, thorough analysis of latencies and their jitter, as well as response amplitude variance should be used on a representative sample of recorded neurons (e.g., see&#160;16,17,23). In cases where activation of network activity may play a role, the method of choice is to isolate the direct monosynaptic component by applying a combination of tetrodotoxin (TTX) to block action potentials in combination with the K+&#160;channel blocker 4-aminopyridine (4-AP) to prevent repolarization 14,28,29. The receptors mediating mono- and disynaptic components of inputs can be identified using pharmacological blockers. Furthermore, it is possible to study aspects of presynaptic modulation of optogenetically-activated inputs in the amygdala 23,30. A potential caveat is that ChR2 has some calcium permeability 1,4 and its activation in presynaptic fibers and terminals has been suggested to alter release probability by various mechanisms 26,31. Nevertheless, several studies including examples shown here successfully employed ChR2 expression to identify input- and target cell-specific differences in paired-pulse ratios in the amygdala and other brain areas 16,23,26,32 and furthermore demonstrated modulation of release probability by activation of specific signalling pathways in presynaptic terminals that was not precluded by ChR activation 23,30. Further support comes from data in hippocampus and cerebellum, showing that with the appropriate ChR expression method and light stimulation technique, physiological aspects including efficacy of release and fidelity of synaptic transmission can be reliably studied 26. Lastly, optogenetic fiber activation has also been successfully used to apply stimulation protocols that induce synaptic plasticity 31,33,34.
Several aspects are critical for the success of this method. One is the precision of stereotactic targeting of viral vectors. Therefore, it is useful to rapidly assess injection site location before obtaining recordings and then perform a more detailed post-hoc analysis. In addition to spatial precision, the viability of neurons at the injection site is a major determinant for a successful experiment and depends on injection technique. The presented option, using long and thin-tapered glass electrodes, serves this purpose well for both superficial and deeper brain areas, but may have the disadvantage that the taper is very flexible. An alternative is to use microliter syringes especially developed for stereotaxic delivery in neuroscience (neuro-syringes) with more rigid needles that can dispense small volumes. The specificity of targeting ChR-expression to defined cell types and regions can be further improved using conditional expression 7, for example with the Cre-system as illustrated for amygdala intercalated cells. This also allows the use of strong promoters in Cre-dependent viral vectors.
Another critical aspect is the choice of the viral vector. To express ChR, recombinant Adeno-associated viruses (rAAVs) were used which have the advantage of being a biosafety level 1 vectors compared to other popular viral systems. rAAVs have been used for some time and are generally safe and reliable vectors with good neural tropism and little or no immunogenic potential, but their packaging volume is limited (approx. 4-5 kb)35. In these specific experiments, serotypes 2/9 and 2/1 were used for ubiquitous and conditional expression, respectively. In accordance with the literature, these serotypes transduce cells in the target region, but appear not to be taken up by axons to retrogradely label cells, unlike serotypes 2/5 and 2/6 36,37. However, initial experiments should exclude that there are ChR-labeled cell bodies in areas projecting to the injection site, which is especially important when studying reciprocally connected brain regions such as mPFC and amygdala. Several recent studies compared efficacy of rAAV serotypes in different brain areas in rats and mice 36,38,39. An emerging theme is that newer serotypes (e.g., 2/1, 2/8 and 2/9) are generally more efficient than the original 2/2 serotype. Also of note, rAAV-mediated expression of ChR, in contrast to other methods, did not cause detrimental effects on expressing cells or labeled axons 40. The required expression time to achieve effective fiber activation ex vivo appears to depend on the distance of the projection studied. Here and consistent with the literature, for labeling and activation of long-range connections in the mouse (mPFC and sensory inputs), an expression time of 4 - 8 weeks is needed, whereas for studying local connectivity in the amygdala, shorter expression times of 2 - 3 weeks are sufficient 14-17,23,30,34.
For recording of optogenetically-elicited responses in the target area, two factors are critical: 1) the acute brain slice needs to be of good quality and 2) a substantial amount of viable labeled fibers needs to be preserved. The quality of slices can be improved by using sapphire blades for slicing. Preservation of axons and thus, size and stability of light responses can be improved by cutting slices at a certain angle as illustrated for mPFC-amygdala projections (Figure 1D and 2A) 16,34. However, this strongly depends on the orientation of afferents in the target region and will have to be determined for each combination of projection area and target region. In this respect, post-hoc analysis of projections in the target region can be useful. To enhance fiber detection and circumvent bleaching that may have occurred during slice stimulation, immunostaining against the fluorescent tag on the ChR fusion protein can be employed.
Overall, this method of optogenetic circuit mapping has recently not only been applied to fear circuits, but many other systems in the brain. In the future, targeting of subnuclei and specific cell types by combining an increasing array of genetically modified mouse lines and conditional viral vectors will provide an even more detailed understanding of the diversity of functional synaptic properties of specific local and long-range circuits. Furthermore, post-hoc immunolabeling of fluorescently-tagged ChR in investigated functional connections could be combined with ultrastructural analysis (i.e., using electron microscopic methods) to provide insights into precise morphological properties of previously activated synapses.

Precise tools for visualization and concurrent activation of specific connections between brain areas and specific types of neurons are becoming more important in understanding the functional connectivity underlying healthy brain function and disease states. Ideally, this entails physiological investigation of precise synaptic properties with which identified neurons communicate. This is particularly true for connections between brain areas that cannot be preserved in a single acute brain slice. In the past, this has been largely achieved in separate experiments. On the one hand, neural tracers injected in vivo have been employed combined with subsequent light or electron microscopic analysis of pre- and postsynaptic partners. On the other hand, when fiber tracts from the region of origin are preserved and accessible in the slice preparation, electrical stimulation has been used to assess synaptic communication mechanisms with cells in the target region.
With the advent of optogenetics, the targeted expression of light-gated cation-channels, such as Channelrhodopsins (ChRs) fused to fluorescent proteins, now enables activation of neurons and their axonal trajectories while allowing for their visualization and post-hoc anatomical analysis 1-4. Because ChR-expressing axons can be stimulated even when severed from parent somata 5, it is possible in brain slices to:&#160;1) assess inputs from brain regions that were not accessible with conventional electrical stimulation, because fiber tracts are not separable or the specific trajectory is not known;&#160;2) unequivocally identify the region of origin for specific inputs that were postulated but incompletely understood; and 3) investigate the functional connectivity between defined cell types, both locally and in long-range projections. Because of a number of advantages, this optogenetic mapping of circuits in brain slices has become widely used in the last years, and a variety of viral vectors for expression of fluorescently-tagged ChRs are readily available from commercial suppliers. Some key advantages of optogenetic activation over conventional electrical stimulation are no damage to the tissue due to placement of stimulation electrodes, specificity of fiber stimulation because electrical stimulation may also recruit fibers of passage or other nearby cells, and an equally rapid and temporally precise stimulation. In addition, stereotactic injection of viral vectors can easily be targeted to specific brain areas 6 and conditional or cell-type specific expression can be achieved using Cre-dependent expression and/or specific promoters 7. Here, this technique is applied for mapping of long-range and local circuits in the fear system.
The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories 8,9. Apart from the amygdala, the medial prefrontal cortex (mPFC) and hippocampus (HC), structures that are reciprocally connected to the amygdala, are implicated in aspects of acquisition, consolidation and retrieval of fear and extinction memories 10,11. Activity in subdivisions of the mPFC appears to play a double role in controlling both&#160;high and low fear states 12,13. This could in part be mediated by direct connections from mPFC to the amygdala that would control amygdala activity and output. Therefore, in the last years, several studies started in ex vivo slice experiments to investigate synaptic interactions between mPFC afferents and specific target cells in the amygdala 14-17.
During fear learning, sensory information about conditioned and unconditioned stimuli reaches the amygdala via projections from specific thalamic and cortical regions. Plasticity of these inputs to neurons in the lateral part (LA) of the basolateral amygdala (BLA) is an important mechanism underlying fear conditioning 9,18. Increasing evidence suggests that parallel plastic processes in the amygdala involve inhibitory elements to control fear memory 19. A group of clustered inhibitory neurons are the GABAergic medial paracapsular intercalated cells (mpITCs), but their precise connectivity and function is incompletely understood 20-22. Here, optogenetic circuit mapping is used to assess afferent and efferent connectivity of these cells and their impact on target neurons in the amygdala, demonstrating that mpITCs receive direct sensory input from thalamic and cortical relay stations&#160;23. Specific expression of ChR in mpITCs or BLA neurons allows&#160;mapping of local interactions, revealing that mpITCs inhibit, but are also mutually activated by, BLA principal neurons, placing them in novel feed-forward and feedback inhibitory circuits that effectively control BLA activity 23.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Surgery&lt;/strong&gt;</td></tr><tr><td>Stereotactic frame</td><td>Stoelting, USA</td><td>51670</td><td>can be replaced by other stereotactic frame for mice</td></tr><tr><td>Steretoxic frame mouse adaptor</td><td>Stoelting, USA</td><td>51625</td><td>can be replaced by other stereotactic frame for mice</td></tr><tr><td>Gas anesthesia mask for mice</td><td>Stoelting, USA</td><td>50264</td><td>no longer available, replaced by item no. 51609M</td></tr><tr><td>Pressure injection device, Toohey Spritzer</td><td>Toohey Company, USA</td><td>T25-2-900</td><td>other pressure injection devices (e.g. Picospritzer) can be used</td></tr><tr><td>Kwik Fill glass capillaries</td><td>World Precision Instruments, Germany</td><td>1B150F-4</td><td></td></tr><tr><td>Anesthesia machine, IsoFlo</td><td>Eickemeyer, Germany</td><td>213261</td><td></td></tr><tr><td>DC Temperature Controler and heating pad</td><td>FHC, USA</td><td>40-90-8D</td><td></td></tr><tr><td>Horizontal Micropipette Puller Model P-1000</td><td>Sutter Instruments, USA</td><td>P-1000</td><td></td></tr><tr><td>Surgical tool sterilizer, Sterilizator 75</td><td>Melag, Germany</td><td>08754200</td><td></td></tr><tr><td>rAAV-hSyn-ChR2(H134R)-eYFP (serotype 2/9)</td><td>Penn Vector Core, USA</td><td>AV-9-26973P</td><td></td></tr><tr><td>rAAV-CAGh-ChR2(H134R)-mCherry (serotype 2/9)&amp;nbsp;</td><td>Penn Vector Core, USA</td><td>AV-9-20938M</td><td></td></tr><tr><td>rAAV-EF1a-DIOhChR2(H134R)-YFP (serotype 2/1)&amp;nbsp;</td><td>Penn Vector Core, USA</td><td>AV-1-20298P</td><td></td></tr><tr><td>fast green</td><td>Roth, Germany</td><td>0301.1</td><td></td></tr><tr><td>Isoflurane Anesthetic, Isofuran CP (1ml/ml)</td><td>CP Pharma, Germany</td><td></td><td></td></tr><tr><td>Antiseptic, Betadine (providone-iodine)</td><td>Purdure Products, USA</td><td>BSOL32</td><td>can be replaced by other disinfectant</td></tr><tr><td>Analgesic, Metacam Solution (5mg/ml meloxicam)</td><td>Boehringer Ingelheim, Germany</td><td></td><td>can be replaced by other analgesics</td></tr><tr><td>Bepanthen eye ointment</td><td>Bayer, Germany</td><td>0191</td><td>can be replaced by other eye ointment</td></tr><tr><td>Drill NM3000 (SNKG1341 and SNIH1681)</td><td>Nouvag, Switzerland</td><td></td><td></td></tr><tr><td>Sutranox Suture Needle</td><td>Fine Science Tools, Germany</td><td>12050-01</td><td></td></tr><tr><td>Braided Silk Suture</td><td>Fine Science Tools, Germany</td><td>18020-60</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Recordings, light stimulation, and analysis&lt;/strong&gt;</td></tr><tr><td>artificial cerebrospinal fluid (ACSF)</td><td>for composition see references #16 and #23</td><td></td><td></td></tr><tr><td>internal patch solutions</td><td>for composition see references #16 and #23</td><td></td><td></td></tr><tr><td>MagnesiumSulfate Heptahydrate</td><td>Roth, Germany</td><td>P027.1</td><td>prepare 2M stock solution in purified water</td></tr><tr><td>Slicer, Microm HM650V</td><td>Fisher Scientific, Germany</td><td>920120</td><td></td></tr><tr><td>Cooling unit for tissue slicer, CU65</td><td>Fisher Scientific, Germany</td><td>770180</td><td></td></tr><tr><td>Sapphire blade</td><td>Delaware Diamond Knives</td><td></td><td>custom order, inquire with company</td></tr><tr><td>Stereoscope, SZX2-RFA16</td><td>Olympus, Japan</td><td></td><td></td></tr><tr><td>Xcite fluorescent lamp (XI120Q-1492)</td><td>Lumen Dynamics Group, Canada</td><td>2012-12699</td><td></td></tr><tr><td>Patch microscope, BX51WI</td><td>Olympus, Japan</td><td></td><td></td></tr><tr><td>Multiclamp 700B patch amplifier&amp;nbsp;</td><td>Molecular Devices, USA</td><td></td><td></td></tr><tr><td>Digitdata 1440A</td><td>Molecular Devices, USA</td><td></td><td></td></tr><tr><td>PClamp software, Version 10</td><td>Molecular Devices, USA</td><td></td><td>used to control data acquisition and stimulation</td></tr><tr><td>Bath temperature controler, TC05</td><td>Luigs &amp;amp; Neumann, Germany</td><td>200-100 500 0145</td><td></td></tr><tr><td>Three axis micromanipulator Mini 25</td><td>Luigs &amp;amp; Neumann, Germany</td><td>210-100 000 0010</td><td></td></tr><tr><td>Micromanipulator controller SM7</td><td>Luigs &amp;amp; Neumann, Germany</td><td>200-100 900 7311</td><td></td></tr><tr><td>glass capillaries for patch pipettes</td><td>World Precision Instruments, Germany</td><td>GB150F-8P</td><td></td></tr><tr><td>Cellulose nitrate filterpaper for interface chamber&amp;nbsp;</td><td>Satorius Stedim Biotech, Germany</td><td>13006--50----ACN</td><td></td></tr><tr><td>LED unit, CoolLED pE</td><td>CoolLED, UK</td><td>244-1400</td><td>CoolLED or USL 70/470 and appropriate adapters are two alternative choices for LED stimulation</td></tr><tr><td>CoolLED 100 Dual Adapt</td><td>CoolLED, UK</td><td>pE-ADAPTOR-50E</td><td>CoolLED or USL 70/470 and appropriate adapters are two alternative choices for LED stimulation</td></tr><tr><td>LED unit, USL 70/470</td><td>Rapp Optoelectronic</td><td>L70-000</td><td>CoolLED or USL 70/470 and appropriate adapters are two alternative choices for LED stimulation</td></tr><tr><td>Dual port adapter</td><td>Rapp Optoelectronic</td><td>inquire with company</td><td>CoolLED or USL 70/470 and appropriate adapters are two alternative choices for LED stimulation</td></tr><tr><td>Filter set red (excitation)</td><td>AHF, Germany</td><td>F49-560</td><td>Filters can be bought as set F46-008</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (beamsplitter)</td><td>AHF, Germany</td><td>F48-585</td><td>Filters can be bought as set F46-008</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (emission)</td><td>AHF, Germany</td><td>F47-630</td><td>Filters can be bought as set F46-008</td></tr><tr><td>Filter set green (excitation)</td><td>AHF, Germany</td><td>F39-472</td><td>Alternatives: filterset F36-149 or F46-002 (with bandpass emission)</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (beamsplitter)</td><td>AHF, Germany</td><td>F43-495W</td><td>Alternatives: filterset F36-149 or F46-002 (with bandpass emission)</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (emission)</td><td>AHF, Germany</td><td>F76-490</td><td>Alternatives: filterset F36-149 or F46-002 (with bandpass emission)</td></tr><tr><td>LaserCheck, handheld power meter</td><td>Coherent, USA</td><td>1098293</td><td></td></tr><tr><td>IgorPro Software, Version 6</td><td>Wavemetrics, USA</td><td></td><td>for electrophysiology data analysis, other alternative software packages can also be used&amp;nbsp;</td></tr><tr><td>Neuromatic suite of macros for IgorPro</td><td>http://www.neuromatic.thinkrandom.com</td><td></td><td>for electrophysiology data analysis, other alternative software packages can also be used&amp;nbsp;</td></tr><tr><td colspan="4">&lt;strong&gt;Post hoc analysis of injections and projections&lt;/strong&gt;</td></tr><tr><td>Paraformaldehyde powder (PFA)</td><td>Roth, Germany</td><td>0335.2</td><td></td></tr><tr><td>Neurotrace 435/455 blue fluorescent Nissl stain</td><td>Invitrogen</td><td>N-21479</td><td></td></tr><tr><td>agar-agar for embedding and resectioning</td><td>Roth, Germany</td><td>5210.3</td><td></td></tr><tr><td>30 x 10 mm petri dishes for embedding</td><td>SPL Life Sciences</td><td></td><td>alternatives can be used</td></tr><tr><td>Slides, Super Frost</td><td>R. Langenbrinck, Germany</td><td>61303802</td><td>alternatives can be used</td></tr><tr><td>cover slips</td><td>R. Langenbrinck, Germany</td><td>3000302</td><td>alternatives can be used</td></tr><tr><td>Vecta Shield mounting medium</td><td>Vector Laboratories, USA</td><td>H-1000</td><td>alternative mounting media can be used</td></tr><tr><td>cellulose nitrate filter for flattening slices for fixation</td><td>Satorius Stedim Biotech, Germany</td><td>11406--25------N</td><td></td></tr><tr><td>Confocal Laser Scanning Microscope LSM 710</td><td>Zeiss, Germany</td><td></td><td></td></tr></tbody></table>

ex vivo optogenetic dissection of fear circuits in brain slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

This section shows the workflow of an ex&#160;vivo optogenetic approach and representative results from different experimental strategies to investigate the physiological properties of sensory and modulatory long-range projections to BLA and mpITC neurons as well as properties of local connectivity between mpITC and BLA.
After stereotactic injection of the selected viral vector at the desired coordinates into the mouse brain (Figure 1A-C, viral expression time 2 - 6 weeks, depending on experiment), acute brain slices of the injection sites and projection areas in the amygdala are prepared at the appropriate angle for patch-clamp experiments and from the injected brain region for post-hoc analysis of injection sites (Figure 1D). Before starting recordings and the optogenetic stimulation, fluorescently labeled cells or axonal projections in the target area (amygdala) should be checked on the upright microscope for patch-clamp recordings by using the attached fluorescent lamp&#160;(Figure 1E). Upon obtaining a patch-clamp recording from a putative target cell in the projection region of the slice, light stimulation is initiated while time point, interval, and pulse length are controlled via the patch software that triggers the light emitting diode (LED). Depending on the experimental strategy (Figure 2A-C, right) the aperture in the fluorescent light path is either fully opened or restricted (Figure 2D). While keeping the pulse length constant and as short as possible (ideally &#8804;1 msec), LED output intensity is slowly increased to assess which intensity is required to achieve the desired amplitude of the synaptic response in a particular experiment (Figure 2E). After recording, amygdala slices are post-fixed. Upon resectioning and optional staining, injection and recording sites are imaged on a confocal microscope to verify injection location and to exclude data from misplaced injections. Images of injection sites and the associated axonal projections in the amygdala for the three experimental strategies (mPFC inputs to BLA, thalamic inputs to mpITCs, and local mpITC activation) are shown in Figure 2A-C.
Figure 3 shows representative recording results obtained from BLA principal neurons, local BLA interneurons and mpITC neurons illustrating the properties of light-evoked responses for the different experimental strategies used (Figure 3A, E, I). To target GABAergic neurons (local interneurons and mpITCs) for recording, GAD67-GFP reporter mice were used 24. For mPFC and sensory thalamic projections, several aspects of synaptic transmission can be studied. The temporal precision of activation and the kinetic properties of the ChRs used in this study enable reliable stimulation with paired pulses at an inter-stimulus-interval of 50 msec. This allows for analysis of paired-pulse ratios (PPR) of postsynaptic currents (PSCs), which can either be facilitating or depressing depending on projection and target cell type, and serve as indicators of presynaptic release probability (Figure 3B, F, H). Additionally, analysis of synaptic latencies allows for dissection of different postsynaptic response components.
In this example, longer latencies of the inhibitory PSC (IPSC) versus excitatory PSC (EPSC) component indicate a disynaptic and monosynaptic input, respectively (Figure 3C). In other instances, a more thorough analysis of additional EPSC properties such as response jitter or the coefficient of variance of response size may be needed to draw conclusions on its mono- or disynaptic nature 17,23. Furthermore, application of pharmacological blockers for specific receptors can identify the nature of PSCs (i.e., glutamatergic EPSC and GABAergic IPSC, Figure 3C-D). As expected, the early component of the mPFC input was glutamatergic, whereas the late component was GABAergic. The complete block of both EPSC and IPSC with the glutamate receptor blocker CNQX further supports that the IPSC is disynaptic. To investigate modulation of synaptic transmission at optogenetically activated fibers, the effects of agonists and antagonists for metabotropic receptors (here GABAB receptors) on amplitude and PPR can be assessed to evaluate influences on pre- and postsynaptic sites. In this example, the concomitant decrease of amplitude with an increase in PPR is indicative of a presynaptic modulation of light-activated fibers (3G, H). Finally, local interactions of cells in the amygdala can be assessed, for example, when mice that express Cre under control of the Tac2 promoter 25 are injected with a double-floxed ChR expressing viral vector (Figure 1C). Because Tac2 and thus, ChR is expressed in mpITC and central amygdala (Figure 2C), light activation was restricted to the mpITC by closing the fluorescent light path aperture (Figure 2D). Under these conditions, light-evoked action potentials can be elicited in infected mpITCs. Short latency inhibitory synaptic responses in the BLA indicate the presence of a functional inhibitory connection between mpITC and BLA principal neurons.
<img alt="Figure 1" src="/files/ftp_upload/53628/53628fig1.jpg" /> 
Figure 1.&#160;Stereotactic Injections, Preparation of Acute Brain Slices, and Visualization of Presynaptic Fibers.&#160;(A, B) Stereotactic virus injection. A) Picture of anesthetized mouse placed in a stereotactic frame with skull exposed and the injection pipette. Inset: Zoom in picture of injection pipette filled with virus solution mixed with fast green. Scale bar: 3 mm. (B) Schematic of a mouse skull with marked positions of drill holes for different injection areas. (C) Scheme showing different viral constructs used in this study. Dark grey: promoter sequence; blue: Channelrhodopsin2 (ChR2 (H134R)); green/red: fluorescent protein. Expression time was 2 weeks for local amygdala projections and 4 - 6 weeks for projections from mPFC and MGm/PIN. (D) Preparation of acute brain slices: Scheme showing placement of mouse brain on slicer stage for obtaining slices from different injection and projection areas. (E) Visualization of fibers in acute brain slices from a GAD67-GFP mouse injected with ChR2-mCherry virus in MGm/PIN. Pictures are taken on the upright patch microscope with different filter sets: GAD67-GFP expression, middle; MGm/PIN fibers labeled with mCherry, right. Scale bar: 200 &#181;m. mPFC, medial prefrontal cortex; mpITC, medial paracapsular intercalated cells; BLA, basolateral amygdala; MGm, medial geniculate nucleus, medial part; PIN, posterior intralaminar thalamic nucleus; LA, lateral amygdala; BA, basal amygdala; CeA, central nucleus of the amygdala. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53628/53628fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<img alt="Figure 2" src="/files/ftp_upload/53628/53628fig2.jpg" /> 
Figure 2.&#160;Injection Sites, Projection Areas, and Optogenetic Stimulation of Axonal Projections.&#160;(A-C) Confocal images of representative injection sites and projection areas in the BLA, and schemes of the experimental strategies. (A) Left: mPFC injection site in a C57Bl/6 mouse stained with Neurotrace. Scale bar: 500 &#181;m. Middle: corresponding projections in the BLA in a tilted amygdala slice. Scale bar: 200 &#181;m. Right: Schematic of whole field illumination of mPFC projections and recording of neuron in the BLA. (B) Left: MGm/PIN injection site in a GAD67-GFP mouse. Scale bar: 500 &#181;m. Middle: Corresponding projections in mpITC and LA of a coronal amygdala slice. Scale bar: 200 &#181;m. Right: Schematic of whole field illumination of MGm/PIN projections and recording of an mpITC neuron. (C) Left: Local expression of ChR2-YFP in a Neurotrace stained amygdala slice of a Tac2-Cre mouse. Scale bar: 200 &#181;m. Inset: Zoom in of mpITCs expressing ChR2-YFP. Scale bar: 20 &#181;m. Right: Schematic of restricted illumination of the mpITC cluster and recording of a BLA neuron. (D) Pictures of recording chamber during light delivery and image of neurons in acute brain slices (mpITC cluster) in a GAD67-GFP mouse with open and restricted aperture. Scale bar: 30 &#181;m. (E) Excitatory postsynaptic currents (EPSCs) evoked by different LED intensities (top) and plot of light power versus evoked EPSC amplitude (bottom) from a representative mpITC neuron upon optogenetic activation of fibers from MGm/PIN. Scale bar: 100 pA/10 msec. 
Note: Figure 2A is modified from reference #16 and Figure 2C from reference #23. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53628/53628fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53628/53628fig3.jpg" /> 
Figure 3.&#160;Exemplary Results from Optogenetic Activation of Long Range and Local Projections.&#160;(A) Scheme of experimental strategy for (B-D). (B) Representative EPSCs recorded from a BLA principal neuron and BLA interneuron upon optogenetic paired-pulse stimulation (50 msec inter-stimulus-interval) of fibers from mPFC eliciting paired pulse facilitation and paired pulse depression, respectively. Scale bar: 100 pA/25 msec. (C) Optogenetic activation of fibers from mPFC elicits feed-forward inhibition. Left: Representative EPSC (at -70 mV) and inhibitory postsynaptic current (IPSC, at 0 mV) in a BLA principal neuron. The IPSC has a longer synaptic latency compared to the EPSC. Scale bars: 200 pA/10 msec and 200 pA/2 msec. Right: Light evoked biphasic EPSC/IPSC sequence (at -50 mV) is blocked by the AMPA/Kainate antagonist CNQX (10 &#181;m), further supporting the disynaptic nature of the IPSC. Scale bar: 50 pA/5 msec. (D) Effects of subsequent block of EPSC/IPSC sequence (at -50 mV) by the Cl- channel blocker Picrotoxin (PTX, 100 &#181;M) and PTX + CNQX. The IPSC is blocked by PTX and the remaining EPSC by CNQX. Scale bars: 50 pA/5 msec. (E) Scheme of experimental strategy for (F-H). F) Representative EPSCs recorded from an mpITC and a BLA principal neuron upon optogenetic paired-pulse stimulation of fibers from MGm/PIN. Scale bar: 50 pA/20 msec. (G) EPSCs in another mpITC neuron are blocked by the sodium channel blocker Tetrodotoxin (TTX, 0.5 &#181;M), indicating that they are dependent on sodium channel activity. Scale bar: 50 pA/20 msec. (H) Thalamic inputs to mpITC neurons are modulated by presynaptic GABAB receptors. EPSCs under control condition, reduction of EPSC amplitude and concomitant increase in the paired pulse ratio during application of the GABAB agonist Baclofen (2 &#181;M), and recovery of both amplitude and initial paired pulse ration upon co-application of the GABAB antagonist CGP55845 (10 &#181;M). These changes are indicative of a presynaptic modulation by GABAB receptors. Scale bar: 50 pA/20 msec. (I) Scheme of experimental strategy for (J). (J) Light-evoked action potentials recorded from a ChR2-YFP expressing mpITC neuron. Light-evoked IPSCs recorded from a BLA principal neuron at different holding potentials (-90, -70, -50, -20, and 0 mV) reverse around the calculated equilibrium potential for Cl-. Scale bars: 20 mV/100 msec and 10 pA/10 msec. Note: Figure 3B-D is modified from Reference #16, and Figure 3H and J from reference #23. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53628/53628fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices at JoVE.com

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

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Nanyang Technological University

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15.9K Views.  University of Tuebingen.  Optogenetische benaderingen worden wijd gebruikt om neurale activiteit te manipuleren en de gevolgen voor hersenfunctie beoordelen. Hier, wordt een techniek beschreven die bij in vivo expressie van het optische activator Channelrhodopsin, maakt de ex vivo analyse van synaptische eigenschappen specifieke lange afstand en lokale neurale verbindingen in angst gerelateerde circuits. 

Video: Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits&#160;in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations&#160;in vitro.

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the ...

Assessing Rodent Empathy: Observational Fear and Vicarious Freezing Method

Observational Fear as a Model of Affective Empathy in Mice

Empathy, characterized by the ability to recognize and share the emotions of others, plays a fundamental role in shaping social interactions. It allows individuals to respond to the emotional states of others, promoting prosocial behaviors and social bonding. Observational fear is a fundamental aspect of affective empathy, where an observer witnesses a demonstrator undergoing aversive experiences and subsequently exhibits fear behaviors. This socially induced vicarious freezing response in observers, known as emotional contagion or affect sharing, is regarded as an indicator of empathy-like traits in rodents. Here, we present a protocol that delineates the assessment of vicarious freezing responses in observers exposed to conspecific demonstrators receiving aversive foot shocks. The utilization of observational fear assays in rodents has become a widely adopted method for studying the neural mechanisms underlying affective empathy. Given the universality of observational fear across mammals, this methodology further contributes to advancing our understanding of the neural substrates of empathy in humans.

The observational fear paradigm assesses vicarious freezing in rodents as a model for affective empathy. This procedure entails exposing observer rodents to conspecific demonstrators receiving aversive foot shocks to elicit empathic freezing responses. By employing observational fear assays, researchers can investigate the neural mechanisms underlying affective empathy.

Empathy, characterized by the ability to recognize and share the emotions of others, plays a fundamental role in shaping social interactions. It allows ...

Behavior

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional ...

Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect distinct cellular and network components of these circuits remains unclear. Ex vivo brain slices provide a means by which investigators may probe discrete components of complex networks and disentangle potential mechanisms underlying the effects of volatile anesthetics on evoked responses. To isolate potential cell type- and pathway-specific drug effects in brain slices, investigators must be able to independently activate afferent fiber pathways, identify non-overlapping populations of cells, and apply volatile anesthetics to the tissue in aqueous solution. In this protocol, methods to measure optogenetically-evoked responses to two independent afferent pathways to neocortex in ex vivo brain slices are described. Extracellular responses are recorded to assay network activity and targeted whole-cell patch clamp recordings are conducted in somatostatin- and parvalbumin-positive interneurons. Delivery of physiologically relevant concentrations of isoflurane via artificial cerebral spinal fluid to modulate cellular and network responses is described.

Ex vivo brain slices can be used to study the effects of volatile anesthetics on evoked responses to afferent inputs. Optogenetics are employed to independently activate thalamocortical and corticocortical afferents to non-primary neocortex, and synaptic and network responses are modulated with isoflurane.

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. Traditional in vitro electrophysiological techniques use electrical stimulation to evoke synaptic transmission. A major drawback of this method is its nonspecific nature; all axons in the region of the stimulating electrode will be activated, making it difficult to attribute an effect to a particular afferent connection. This issue can be overcome by replacing electrical stimulation with optogenetic-based stimulation. We describe a method for combining optogenetics with in vitro patch-clamp recordings. This is a powerful tool for the study of both basal synaptic transmission and synaptic plasticity of precise anatomically defined synaptic connections and is applicable to almost any pathway in the brain. Here, we describe the preparation and handling of a viral vector encoding channelrhodopsin protein for surgical injection into a pre-synaptic region of interest (medial prefrontal cortex) in the rodent brain and making of acute slices of downstream target regions (lateral entorhinal cortex). A detailed procedure for combining patch-clamp recordings with synaptic activation by light stimulation to study short- and long-term synaptic plasticity is also presented. We discuss examples of experiments that achieve pathway- and cell-specificity by combining optogenetics and Cre-dependent cell labeling. Finally, histological confirmation of the pre-synaptic region of interest is described along with biocytin labeling of the post-synaptic cell, to allow further identification of the precise location and cell type.

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Here we present a protocol describing viral transduction of discrete brain regions with optogenetic constructs to permit synapse-specific electrophysiological characterization in acute rodent brain slices.

Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. ...

Visualization and Mapping of Neuronal Pathways in an Amygdala Brain Slice

Source: Bosch, D., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53628">Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices.</a> J. Vis. Exp. (2016).
This video demonstrates a procedure for analyzing synaptic connectivity between medial prefrontal cortex (mPFC) and amygdala neurons using acute amygdala brain slices from mice. The mPFC neurons in these mice express channelrhodopsin fused to a fluorescent protein. The channelrhodopsins facilitate ion influx and action potential generation in the mPFC neurons upon light activation. Consequently, mPFC neurons release neurotransmitters that bind to postsynaptic amygdala neurons, triggering postsynaptic currents that are recorded to visualize mPFC-amygdala connectivity.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Visualization and ...

JoVE Encyclopedia of Experiments

Neurophysiology

Imaging and Optical Techniques

An Electrophysiological Study of Retinogeniculate and Corticogeniculate Synapses in Mouse Brain Slices

Source: Chen, X., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59680">Electrophysiological Investigations of Retinogeniculate and Corticogeniculate Synapse Function.</a> J. Vis. Exp. (2019).
The video describes the electrophysiological recording of retinogeniculate and corticogeniculate synapses in mouse brain slices. The slices containing the dorsal lateral geniculate nucleus (dLGN) are placed in a recording chamber. A stimulating pipette is positioned on either the optic tract to stimulate retinal ganglion cells (RGCs) and investigate retinogeniculate synapses or the nucleus reticularis thalami to stimulate cortical neurons and investigate corticogeniculate synapses. Upon stimulation, the RGCs and cortical neurons release excitatory neurotransmitters, which produce synaptic currents in the LGN relay neurons. These currents are recorded using a patch-clamp recording pipette.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Solutions

	Dissection solution
	
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Electrophysiology Techniques

Generation of Tilted Amygdala Slices from a Mouse Brain

Source: Bosch, D., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53628">Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices.</a> J. Vis. Exp. (2016)
The video demonstrates the generation of tilted amygdala slices from a mouse brain. The brain is positioned at an angle on the vibratome specimen stage to obtain slices containing the amygdala and axons from the medial prefrontal cortex that form synaptic connections with the amygdala. The brain slices are maintained at the physiological temperature to preserve the tissue for further experiments.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Acute Slices

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Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

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