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,...

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 ...

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 ...

ex-vivo-optogenetic-dissection-of-fear-circuits-in-brain-slices

Research

JoVE Journal

Neuroscience

15.9K Views.  University of Tuebingen. Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that 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.

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 ...

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 ...

Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural circuits. Due to the fractional change in the light signal, it has been difficult to use this method as a quantitative assay. This article describes special optics and slice handling systems, which render this technique stable and reliable. The present article demonstrates the slice handling, staining, and recording of the VSD-stained hippocampal slices in detail. The system maintains physiological conditions for a long time, with good staining, and prevents mechanical movements of the slice during the recordings. Moreover, it enables staining of slices with a small amount of the dye. The optics achieve high numerical aperture at low magnification, which allows recording of the VSD signal at the maximum frame rate of 10 kHz, with 100 pixel x 100-pixel spatial resolution. Due to the high frame rate and spatial resolution, this technique allows application of the post-recording filters that provide sufficient signal-to-noise ratio to assess the changes in neural circuits.

We introduce a reproducible and stable optical recording method for brain slices using voltage-sensitive dye. The article describes voltage-sensitive dye staining and recording of optical signals using conventional hippocampal slice preparations.

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural ...

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/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/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

Simultaneous Optical and Electrophysiological Monitoring of Neuronal Cells in Brain Slices

Source:&#160; Murphy, C. A., et al. <a href="https://app.jove.com/v/61333">Optogenetic activation of afferent pathways in brain slices and modulation of responses by volatile anesthetics.</a> J. Vis. Exp. (2020)
This video outlines a method for simultaneously imaging and recording electrical activity from transgenic brain slices that contain neuron-specific fluorescent proteins. The procedure involves maintaining the brain slice in a perfusion chamber with aerated artificial cerebrospinal fluid, using a light source to excite fluorescent-labeled cells, and recording the electrical signals with a multichannel probe and patch clamp.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.&#160;&#160;&#160;&#160;&#160;
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Generation of Tilted Amygdala Slices from a Mouse Brain

Source: Bosch, D., et al. <a href="https://app.jove.com/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

Summary

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