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 s...

<ol>
	<li>Nagel, G., 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=Channelrhodopsin-2,+a+directly+light-gated+cation-selective+membrane+channel.">Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.</a> Proc Natl Acad Sci U S A. 100 (24), 13940-13945 (2003).</li><li>Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond-timescale,+genetically+targeted+optical+control+of+neural+activity.">Millisecond-timescale, genetically targeted optical control of neural activity.</a> Nat Neurosci. 8 (9), 1263-1268 (2005).</li><li>Tye, K. M., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+investigation+of+neural+circuits+underlying+brain+disease+in+animal+models.">Optogenetic investigation of neural circuits underlying brain disease in animal models.</a> Nat Rev Neurosci. 13 (4), 251-266 (2012).</li><li>Yizhar, O., Fenno, L. E., Davidson, T. J., Mogri, M., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetics+in+neural+systems.">Optogenetics in neural systems.</a> Neuron. 71 (1), 9-34 (2011).</li><li>Petreanu, L., Huber, D., Sobczyk, A., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Channelrhodopsin-2-assisted+circuit+mapping+of+long-range+callosal+projections.">Channelrhodopsin-2-assisted circuit mapping of long-range callosal projections.</a> Nat Neurosci. 10 (5), 663-668 (2007).</li><li>Cetin, A., Komai, S., Eliava, M., Seeburg, P. H., Osten, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotaxic+gene+delivery+in+the+rodent+brain.">Stereotaxic gene delivery in the rodent brain.</a> Nat Protoc. 1 (6), 3166-3173 (2006).</li><li>Huang, Z. J., Zeng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+approaches+to+neural+circuits+in+the+mouse.">Genetic approaches to neural circuits in the mouse.</a> Annu Rev Neurosci. 36, 183-215 (2013).</li><li>LeDoux, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emotion+circuits+in+the+brain.">Emotion circuits in the brain.</a> Annu Rev Neurosci. 23, 155-184 (2000).</li><li>Pape, H. C., Pare, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastic+synaptic+networks+of+the+amygdala+for+the+acquisition,+expression,+and+extinction+of+conditioned+fear.">Plastic synaptic networks of the amygdala for the acquisition, expression, and extinction of conditioned fear.</a> Physiol Rev. 90 (2), 419-463 (2010).</li><li>Myers, K. M., Davis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+fear+extinction.">Mechanisms of fear extinction.</a> Mol Psychiatry. 12 (2), 120-150 (2007).</li><li>Quirk, G. J., Mueller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+mechanisms+of+extinction+learning+and+retrieval.">Neural mechanisms of extinction learning and retrieval.</a> Neuropsychopharmacology. 33 (1), 56-72 (2008).</li><li>Vidal-Gonzalez, I., Vidal-Gonzalez, B., Rauch, S. L., Quirk, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microstimulation+reveals+opposing+influences+of+prelimbic+and+infralimbic+cortex+on+the+expression+of+conditioned+fear.">Microstimulation reveals opposing influences of prelimbic and infralimbic cortex on the expression of conditioned fear.</a> Learn Mem. 13 (6), 728-733 (2006).</li><li>Sierra-Mercado, D., Padilla-Coreano, N., Quirk, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissociable+roles+of+prelimbic+and+infralimbic+cortices,+ventral+hippocampus,+and+basolateral+amygdala+in+the+expression+and+extinction+of+conditioned+fear.">Dissociable roles of prelimbic and infralimbic cortices, ventral hippocampus, and basolateral amygdala in the expression and extinction of conditioned fear.</a> Neuropsychopharmacology. 36 (2), 529-538 (2011).</li><li>Cho, J. H., Deisseroth, K., Bolshakov, V. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+encoding+of+fear+extinction+in+mPFC-amygdala+circuits.">Synaptic encoding of fear extinction in mPFC-amygdala circuits.</a> Neuron. 80 (6), 1491-1507 (2013).</li><li>Arruda-Carvalho, M., Clem, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathway-Selective+Adjustment+of+Prefrontal-Amygdala+Transmission+during+Fear+Encoding.">Pathway-Selective Adjustment of Prefrontal-Amygdala Transmission during Fear Encoding.</a> J Neurosci. 34 (47), 15601-15609 (2014).</li><li>Hubner, C., Bosch, D., Gall, A., Luthi, A., Ehrlich, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+dissection+of+optogenetically+activated+mPFC+and+hippocampal+inputs+to+neurons+in+the+basolateral+amygdala:+implications+for+fear+and+emotional+memory.">Ex vivo dissection of optogenetically activated mPFC and hippocampal inputs to neurons in the basolateral amygdala: implications for fear and emotional memory.</a> Front Behav Neurosci. 8, 64 (2014).</li><li>Strobel, C., Marek, R., Gooch, H. M., Sullivan, R. K., Sah, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prefrontal+and+Auditory+Input+to+Intercalated+Neurons+of+the+Amygdala.">Prefrontal and Auditory Input to Intercalated Neurons of the Amygdala.</a> Cell Rep. 10 (9), 1435-1442 (2015).</li><li>Sigurdsson, T., Doyere, V., Cain, C. K., LeDoux, J. 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+potentiation+in+the+amygdala:+a+cellular+mechanism+of+fear+learning.">Long-term potentiation in the amygdala: a cellular mechanism of fear learning.</a> Neuropharmacology. 52 (1), 215-227 (2007).</li><li>Ehrlich, I., Humeau, Y., Grenier, F., Ciocchi, S., Herry, C., Luthi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amygdala+inhibitory+circuits+and+the+control+of+fear+memory.">Amygdala inhibitory circuits and the control of fear memory.</a> Neuron. 62 (6), 757-771 (2009).</li><li>Millhouse, O. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intercalated+cells+of+the+amygdala.">The intercalated cells of the amygdala.</a> J Comp Neurol. 247 (2), 246-271 (1986).</li><li>Busti, D., 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=Different+fear+states+engage+distinct+networks+within+the+intercalated+cell+clusters+of+the+amygdala.">Different fear states engage distinct networks within the intercalated cell clusters of the amygdala.</a> J Neurosci. 31 (13), 5131-5144 (2011).</li><li>Palomares-Castillo, E., Hernandez-Perez, O. R., Perez-Carrera, D., Crespo-Ramirez, M., Fuxe, K., Perez de la Mora, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intercalated+paracapsular+islands+as+a+module+for+integration+of+signals+regulating+anxiety+in+the+amygdala.">The intercalated paracapsular islands as a module for integration of signals regulating anxiety in the amygdala.</a> Brain Res. 1476, 211-234 (2012).</li><li>Asede, D., Bosch, D., Luthi, A., Ferraguti, F., Ehrlich, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+inputs+to+intercalated+cells+provide+fear-learning+modulated+inhibition+to+the+basolateral+amygdala.">Sensory inputs to intercalated cells provide fear-learning modulated inhibition to the basolateral amygdala.</a> Neuron. 86 (2), 541-554 (2015).</li><li>Tamamaki, N., Yanagawa, Y., Tomioka, R., Miyazaki, J., Obata, K., Kaneko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein+expression+and+colocalization+with+calretinin,+parvalbumin,+and+somatostatin+in+the+GAD67-GFP+knock-in+mouse.">Green fluorescent protein expression and colocalization with calretinin, parvalbumin, and somatostatin in the GAD67-GFP knock-in mouse.</a> J Comp Neurol. 467 (1), 60-79 (2003).</li><li>Mar, L., Yang, F. C., Ma, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+marking+and+characterization+of+Tac2-expressing+neurons+in+the+central+and+peripheral+nervous+system.">Genetic marking and characterization of Tac2-expressing neurons in the central and peripheral nervous system.</a> Mol Brain. 5, (2012).</li><li>Jackman, S. L., Beneduce, B. M., Drew, I. R., Regehr, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Achieving+high-frequency+optical+control+of+synaptic+transmission.">Achieving high-frequency optical control of synaptic transmission.</a> J Neurosci. 34 (22), 7704-7714 (2014).</li><li>Li, H., Penzo, M. A., Taniguchi, H., Kopec, C. D., Huang, Z. J., Li, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-dependent+modification+of+a+central+amygdala+fear+circuit.">Experience-dependent modification of a central amygdala fear circuit.</a> Nat Neurosci. 16 (3), 332-339 (2013).</li><li>Petreanu, L., Mao, T., Sternson, S. M., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+subcellular+organization+of+neocortical+excitatory+connections.">The subcellular organization of neocortical excitatory connections.</a> Nature. 457 (7233), 1142-1145 (2009).</li><li>Felix-Ortiz, A. C., Beyeler, A., Seo, C., Leppla, C. A., Wildes, C. P., Tye, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BLA+to+vHPC+inputs+modulate+anxiety-related+behaviors.">BLA to vHPC inputs modulate anxiety-related behaviors.</a> Neuron. 79 (4), 658-664 (2013).</li><li>Chu, H. Y., Ito, W., Li, J., Morozov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target-specific+suppression+of+GABA+release+from+parvalbumin+interneurons+in+the+basolateral+amygdala+by+dopamine.">Target-specific suppression of GABA release from parvalbumin interneurons in the basolateral amygdala by dopamine.</a> J Neurosci. 32 (42), 14815-14820 (2012).</li><li>Zhang, Y. P., Oertner, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+induction+of+synaptic+plasticity+using+a+light-sensitive+channel.">Optical induction of synaptic plasticity using a light-sensitive channel.</a> Nat Methods. 4 (2), 139-141 (2007).</li><li>Britt, J. P., Benaliouad, F., McDevitt, R. A., Stuber, G. D., Wise, R. A., Bonci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+and+behavioral+profile+of+multiple+glutamatergic+inputs+to+the+nucleus+accumbens.">Synaptic and behavioral profile of multiple glutamatergic inputs to the nucleus accumbens.</a> Neuron. 76 (4), 790-803 (2012).</li><li>Kohl, M. M., Shipton, O. A., Deacon, R. M., Rawlins, J. 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">Surgery</td>
		</tr>
		<tr>
			<td>Stereotactic frame</td>
			<td>Stoelting, USA</td>
			<td>51670</td>
			<td rowspan="2">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>
		</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)&#160;</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)&#160;</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></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Recordings, light stimulation, and analysis</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&#160;</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 &#38; Neumann, Germany</td>
			<td>200-100 500 0145</td>
			<td></td>
		</tr>
		<tr>
			<td>Three axis micromanipulator Mini 25</td>
			<td>Luigs &#38; Neumann, Germany</td>
			<td>210-100 000 0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator controller SM7</td>
			<td>Luigs &#38; 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&#160;</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 rowspan="4">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>
		</tr>
		<tr>
			<td>LED unit, USL 70/470</td>
			<td>Rapp Optoelectronic</td>
			<td>L70-000</td>
		</tr>
		<tr>
			<td>Dual port adapter</td>
			<td>Rapp Optoelectronic</td>
			<td>inquire with company</td>
		</tr>
		<tr>
			<td>Filter set red (excitation)</td>
			<td>AHF, Germany</td>
			<td>F49-560</td>
			<td rowspan="3">Filters can be bought as set F46-008</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (beamsplitter)</td>
			<td>AHF, Germany</td>
			<td>F48-585</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (emission)</td>
			<td>AHF, Germany</td>
			<td>F47-630</td>
		</tr>
		<tr>
			<td>Filter set green (excitation)</td>
			<td>AHF, Germany</td>
			<td>F39-472</td>
			<td rowspan="3">Alternatives: filterset F36-149 or F46-002 (with bandpass emission)</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (beamsplitter)</td>
			<td>AHF, Germany</td>
			<td>F43-495W</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (emission)</td>
			<td>AHF, Germany</td>
			<td>F76-490</td>
		</tr>
		<tr>
			<td>LaserCheck, handheld power meter</td>
			<td>Coherent, USA</td>
			<td><a>1098293</a></td>
			<td></td>
		</tr>
		<tr>
			<td>IgorPro Software, Version 6</td>
			<td>Wavemetrics, USA</td>
			<td></td>
			<td rowspan="2">for electrophysiology data analysis, other alternative software packages can also be used&#160;</td>
		</tr>
		<tr>
			<td>Neuromatic suite of macros for IgorPro</td>
			<td><a href="http://www.neuromatic.thinkrandom.com/">http://www.neuromatic.thinkrandom.com</a></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Post hoc analysis of injections and projections</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 ...

The overall goal of this experiment is to investigate functional and synaptic properties of neural projections and micro-circuits using ex-vivo optogenetics. This includes expression of channel rhodopsin and its activation in specific projections combined with patch-clamp recordings. This method can help answer key questions about synaptic properties of local and long-range connectivity within and between brain areas.This is illustrated here investigating the neural circuits that support fear behavior. The main advantage of this technique is that it allows to study circuits and synapses that are not amenable with conventional electrical stimulation techniques. Generally, people new to this method will have to overcome the challenge of performing all the required steps in an optimal way.This includes the stereotactic targeting of viral injections, identifying the optimal expression time for channel rhodpsin, the preparation of good quality brain slices, and stable light activation and recordings. Before beginning this part of the procedure, a recombinant adeno-associated viral vector engineered to express channel rhodopsin fused to a fluorescent protein was introduced into the medial pre-frontal cortex by stereotactic surgery. Prepare the vibratome by fitting a sapphire blade and setting it to cut at 320 micron thickness.After sacrifice according to approved methods and removal of the brain, use a scalpel to cut off the cerebellum, then isolate the anterior portion of the brain containing the medial pre-frontal cortex. Place the anterior portion of the brain in ice-cold cutting solution until slicing. Fill the cutting chamber with ice-cold cutting solution maintained at 4 degrees Celsius using a cooling unit.Oxygenate the solution. Blot the brain dry with filter paper. Glue the four per cent agar block that that has been cut at a 35 degree angle to the vibratome stage, and glue the posterior part of the brain tissue onto this block.Glue two additional agar blocks in front of and behind the brain block for stability while slicing. Place the stage with tissue attached in the cutting chamber and ensure it is submerged. Begin cutting the acute slices.Cut each slice in half and transfer to an interface chamber supplied with oxygenated ACSF at room temperature. After cutting the acute amygdala slices, place the interface chamber in a water bath at 36 degrees Celsius and allow the slices to recover for 35 to 45 minutes. Fix a subset of the slices containing the injection site for post-hoc analysis by sandwiching them between two pieces of filter paper and submerging them in four per cent paraformaldehyde in PBS overnight.Here, a recombinant adeno-associated viral vector engineered to express channel rhodopsin fused to a fluorescent protein was introduced into the medial division of the medial geniculate nucleus and adjacent posterior intra-laminar nucleus by stereotactic surgery. After obtaining the brain, use a scalpel to remove the cerebellum and anterior part of the brain and then cool the middle portion of the brain in ice-cold cutting solution as before. Blot the brain with filter paper and then glue it to the vibratome stage.Again, fix an additional agar block behind the brain for stability while slicing. After fitting the stage into the cutting chamber filled with four degree Celsius cutting solution, cut 320 micron coronal sections through the amygdala, cut them in half, and collect the sections in the interface chamber as before. Under a stereomicroscope using fluorescent illumination check the injection site.To prepare the patch microscope for optogenetic activation of fibers and cells, center the mounted light-emitting diode or LED onto the light delivery pathway. Use a power meter to measure the LED light intensity at the back focal plane and at the output of each objective at a wavelength of 470 nanometers. Use a spreadsheet to calculate the light intesity in milliwatts per millimeter squared and create a calibration curve for each objective.Next, retrieve an acute amygdala slice from the interface chamber and place it in the slice chamber mounted onto the microscope. Position the slice so that the slice surface facing upward in the interface chamber is also facing upward in the recording chamber. Perfuse the slice with fresh, oxygenated ACSF at a rate of one to two milliliters per minute.The temperature should be approximately 31 degrees Celsius. Turn on the fluorescent lamp and select the appropriate filter set for the specific fluoresent protein expressed. Use the 5x objective to obtain an overview.And the 60x objective for assessment of fiber density within the target area. Next, open or restrict the aperture in the microscope light pathway as necessary for the experiment. To obtain a patch recording, fill a patch pipette with internal solution and mount it in the electrode holder.Apply positive pressure to the patch pipette and slowly lower it into the bath solution. Then, under visual control use the micromanipulator to lower the patch pippette into the slice. Approach the neuron of interest with the patch pipette from the side and top.Release the positive pressure when the pipette reaches the surface of the cell as indicated by a dimple visible on the cell surface. Apply negative pressure to obtain a gigaseal. Apply further suction to rupture the membrane patch and obtain the whole cell recording.Next, stimulate the labeled fibers with a connected LED by activating channel rhodopsin with 470 nanometer wavelength light while recording electrical responses from the cell. For synaptic stimulation, use the digital outputs of the data-acquisition software to trigger the LED. Adjust the LED stimulation intensity manually.Repeat the stimulation with an opened or restricted aperture as necessary for the next recorded cell or in the presence of specific test substances. After recording, fix slices for post-hoc analysis by sandwiching them between two filter papers and submerging them in four percent paraformaldehyde overnight. Fibers from the medial pre-frontal cortex were stimulated using optogenetic paired pulse stimulation while excitatory post-synaptic currents were recorded from basolateral amygdala principal neuron and an interneuron.These representative traces show the paired pulse facilitation and paired pulse depression elicited from the stimulation. The following two images demonstrate feed-forward inhibition elicited by optogenetic activation of fibers from the medial prefrontal cortex. This first image shows a representative excitatory postsynaptic current at 70 millivolts and inhibitory postsynaptic current at zero millivolts in a basolateral amygdala principal neuron.The inhibitory postsynaptic current has a longer synaptic latency compared to the excitatory postsynaptic current indicating disynaptic and monosynaptic input, respectively. This image shows that the light evoked by phasic excitatory and inhibitory postsynaptic current sequence at 50 millivolt is blocked by the AMPA kainate antagonist, CNQX, further supporting the disynaptic nature of the inhibitory postsynaptic current. This image shows the effects of a subsequent block of the excitatory and inhibitory postsynaptic current sequence at 50 millivolts by the chloride channel-blocker, picrotoxin, and picrotoxin plus CNQX.The inhibitory postsynaptic current is blocked by picrotoxin and the remaining excitatory postsynaptic current by CNQX. Once mastered, the preparation of brain slices and the recording of light-evoked responses can be done within several hours to a full day. Prerequisites are good injection sites and sufficient expression of channel rhodopsin in cells and projections to be studied.Following this procedure, other methods like anatomical studies of labeled axons and synapses can be performed at the light and electron microscopic level. This allows to correlate functional with anatomical and molecular properties of activated synapses. After watching this video, you should have good understanding of all the required steps for analysis of light-activated fibers and brain slices.This includes channel rhodopsin expression, evaluation and activation of labeled fibers, as well as recording and analysis of the synaptic responses.

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

Research

JoVE Journal

Neuroscience

15.4K Görüntüleme.  University of Tuebingen. The overall goal of this experiment is to investigate functional and synaptic properties of neural projections and micro-circuits using ex-vivo optogenetics. This includes expression of channel rhodopsin and its activation in specific projections combined with patch-clamp recordings. This method can help answer key questions about synaptic properties of local and long-range connectivity within and between brain areas.This is illustrated here investigating the neural circuits that suppo...