Name: long-range channelrhodopsin-assisted circuit mapping of inferior colliculus neurons with blue and red-shifted channelrhodopsins
Uploaded: 2020-02-07T13:00:00
Description: Watch this Scientific Journal Video about Long-range Channelrhodopsin-assisted Circuit Mapping of Inferior Colliculus Neurons with Blue and Red-shifted Channelrhodopsins at JoVE.com

This work was supported by a Deutsche Forschungsgemeinschaft Research Fellowship (GO 3060/1-1, project number 401540516, to DG) and National Institutes of Health grant R56 DC016880 (MTR).

Obtain approval from the local Institutional Animal Care and Use Committee (IACUC) and adhere to NIH guidelines for the care and use of laboratory animals. All procedures in this protocol were approved by the University of Michigan IACUC and were in accordance with NIH guidelines for the care and use of laboratory animals.
1. Surgery Preparations
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	<li>Perform surgeries in aseptic conditions. Autoclave/sterilize all surgery tools and materials before surgery. Wear surgery gown and mask f...

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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> Nature Neuroscience. 8, 1263-1268 (2005).</li><li>Klapoetke, N. C., 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=Independent+optical+excitation+of+distinct+neural+populations.">Independent optical excitation of distinct neural populations.</a> Nature Methods. 11, 338-346 (2014).</li><li>Flotte, T. 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K., 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=Simultaneous+fast+measurement+of+circuit+dynamics+at+multiple+sites+across+the+mammalian+brain.">Simultaneous fast measurement of circuit dynamics at multiple sites across the mammalian brain.</a> Nature Methods. 13, 325-328 (2016).</li><li>Lin, J. Y., Knutsen, P. M., Muller, A., Kleinfeld, D., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ReaChR:+a+red-shifted+variant+of+channelrhodopsin+enables+deep+transcranial+optogenetic+excitation.">ReaChR: a red-shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation.</a> Nature Neuroscience. 16, 1499-1508 (2013).</li><li>Mager, T., 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=High+frequency+neural+spiking+and+auditory+signaling+by+ultrafast+red-shifted+optogenetics.">High frequency neural spiking and auditory signaling by ultrafast red-shifted optogenetics.</a> Nature Communications. 9, (2018).</li><li>Oda, K., 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=Crystal+structure+of+the+red+light-activated+channelrhodopsin+Chrimson.">Crystal structure of the red light-activated channelrhodopsin Chrimson.</a> Nature Communications. 9, (2018).</li><li>Hooks, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-Channel+Photostimulation+for+Independent+Excitation+of+Two+Populations.">Dual-Channel Photostimulation for Independent Excitation of Two Populations.</a> Current Protocols in Neuroscience. 85, e52 (2018).</li><li>Schild, L. C., Glauser, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+Color+Neural+Activation+and+Behavior+Control+with+Chrimson+and+CoChR+in+Caenorhabditis+elegans.">Dual Color Neural Activation and Behavior Control with Chrimson and CoChR in Caenorhabditis elegans.</a> Genetics. 200, 1029-1034 (2015).</li><li>Rost, B. R., Schneider-Warme, F., Schmitz, D., Hegemann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+Tools+for+Subcellular+Applications+in+Neuroscience.">Optogenetic Tools for Subcellular Applications in Neuroscience.</a> Neuron. 96, 572-603 (2017).</li><li>Maimon, B. E., Sparks, K., Srinivasan, S., Zorzos, A. N., Herr, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrally+distinct+channelrhodopsins+for+two-colour+optogenetic+peripheral+nerve+stimulation.">Spectrally distinct channelrhodopsins for two-colour optogenetic peripheral nerve stimulation.</a> Nature Biomedical Engineering. 2, 485 (2018).</li><li>Asrican, B., 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=Next-generation+transgenic+mice+for+optogenetic+analysis+of+neural+circuits.">Next-generation transgenic mice for optogenetic analysis of neural circuits.</a> Frontiers in Neural Circuits. 7, (2013).</li><li>Oliver, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+cochlear+nucleus+projections+to+the+inferior+colliculus+in+the+cat:+A+light+and+electron+microscopic+study.">Dorsal cochlear nucleus projections to the inferior colliculus in the cat: A light and electron microscopic study.</a> Journal of Comparative Neurology. 224, 155-172 (1984).</li><li>Oliver, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projections+to+the+inferior+colliculus+from+the+anteroventral+cochlear+nucleus+in+the+cat:+Possible+substrates+for+binaural+interaction.">Projections to the inferior colliculus from the anteroventral cochlear nucleus in the cat: Possible substrates for binaural interaction.</a> Journal of Comparative Neurology. 264, 24-46 (1987).</li><li>Glendenning, K. K., Masterton, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acoustic+chiasm:+efferent+projections+of+the+lateral+superior+olive.">Acoustic chiasm: efferent projections of the lateral superior olive.</a> Journal of Neuroscience. 3, 1521-1537 (1983).</li><li>Adams, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ascending+projections+to+the+inferior+colliculus.">Ascending projections to the inferior colliculus.</a> Journal of Comparative Neurology. 183, (1979).</li><li>Winer, J. A., Larue, D. T., Diehl, J. J., Hefti, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+cortical+projections+to+the+cat+inferior+colliculus.">Auditory cortical projections to the cat inferior colliculus.</a> Journal of Comparative Neurology. 400, 147-174 (1998).</li><li>Salda&#241;a, E., Mercha&#324;, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+and+commissural+connections+of+the+rat+inferior+colliculus.">Intrinsic and commissural connections of the rat inferior colliculus.</a> Journal of Comparative Neurology. 319, 417-437 (1992).</li><li>Sivaramakrishnan, S., Sanchez, J. T., Grimsley, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+concentrations+of+divalent+cations+isolate+monosynaptic+inputs+from+local+circuits+in+the+auditory+midbrain.">High concentrations of divalent cations isolate monosynaptic inputs from local circuits in the auditory midbrain.</a> Frontiers in Neural Circuits. 7, (2013).</li><li>Felix, R. A., Gour&#233;vitch, B., Portfors, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcortical+pathways:+Towards+a+better+understanding+of+auditory+disorders.">Subcortical pathways: Towards a better understanding of auditory disorders.</a> Hearing Research. 362, 48-60 (2018).</li><li>Winer, J. A., Schreiner, C., Winer, J. A., Schreiner, C., 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=.">.</a> The inferior colliculus: with 168 illustrations. , (2005).</li><li>Goyer, 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=A+novel+class+of+inferior+colliculus+principal+neurons+labeled+in+vasoactive+intestinal+peptide-Cre+mice.">A novel class of inferior colliculus principal neurons labeled in vasoactive intestinal peptide-Cre mice.</a> eLife. 8, e43770 (2019).</li></ol>

We have found that CRACM is a powerful technique for identifying and characterizing long range synaptic inputs to neurons in the mouse IC. Following the protocol detailed here, we achieved robust transfection of neurons in the DCN and IC as well as reliable axonal trafficking of Chronos and ChrimsonR to synaptic terminals in the IC. Additionally, we demonstrated that this technique enables the measurement and analysis of postsynaptic events, including PSP amplitude, halfwidth, decay time, and receptor pharmacology. Our e...

Synaptic connections are critical to neural circuit function, but the precise topology and physiology of synapses within neural circuits are often difficult to probe experimentally. This is because electrical stimulation, the traditional tool of cellular electrophysiology, indiscriminately activates axons near the stimulation site, and in most brain regions, axons from different sources (local, ascending, and/or descending) intertwine. However, by using channelrhodopsin assisted circuit mapping (CRACM)1,2, this limitation can now be overcome3. Channelrhodopsin (ChR2) is a light activated, cation-selective ion channel originally found in the green alga Chlamydomonas reinhardtii. ChR2 can be activated by blue light of a wavelength around 450-490 nm, depolarizing the cell through cation influx. ChR2 was first described and expressed in Xenopus oocytes by Nagel and colleagues4. Shortly after that, Boyden and colleagues5 expressed ChR2 in mammalian neurons and showed that they could use light pulses to reliably control spiking on a millisecond timescale, inducing action potentials ~10 ms after activation of ChR2 with blue light. Optogenetic channels with even faster kinetics have been found recently (e.g., Chronos6).
The basic approach to a CRACM experiment is to transfect a population of putative presynaptic neurons with a recombinant adeno-associated virus (rAAV) that carries the genetic information for a channelrhodopsin. Transfection of neurons with rAAV leads to the expression of the encoded channelrhodopsin. Typically, the channelrhodopsin is tagged with a fluorescent protein like GFP (Green Fluorescent Protein) or tdTomato (a red fluorescent protein), so that transfection of neurons in the target region can easily be confirmed with fluorescence imaging. Because rAAVs are non-pathogenic, have a low inflammatory potential and long-lasting gene expression7,8, they have become a standard technique to deliver channelrhodopsins to neurons. If, after transfection of a putative presynaptic population of neurons, activation of a channelrhodopsin through light flashes elicits postsynaptic potentials or currents in the target neurons, this is evidence of an axonal connection from the transfected nucleus to the recorded cell. Because severed axons in brain slice experiments can be driven to release neurotransmitter through channelrhodopsin activation, nuclei that lie outside of the acute slice but send axons into the postsynaptic brain region can be identified with CRACM. The power of this technique is that the connectivity and physiology of identified long range synaptic inputs can be directly investigated.
In addition to channelrhodopsins that are excitable by blue light, investigators have recently identified several red-shifted channelrhodopsins9,10, including Chrimson and its faster analog ChrimsonR, both of which are excited with red light of ~660 nm6. Red-shifted opsins are of interest because red light penetrates tissue better than blue light, and red light may have a lower cytotoxicity than blue light10,11,12. Red-shifted channelrhodopsins also open up the possibility of dual color CRACM experiments, where the convergence of axons from different nuclei on the same neuron can be tested in one experiment6,13,14. However, current red-shifted opsins often exhibit unwanted cross-activation with blue light15,16,17, making two color experiments difficult. In addition, some reports have indicated that ChrimsonR undergoes limited axonal trafficking, which can make it challenging to use ChrimsonR for CRACM experiments16,17.
Nearly all ascending projections from the lower auditory brainstem nuclei converge in the inferior colliculus (IC), the midbrain hub of the central auditory pathway. This includes projections from the cochlear nucleus (CN)18,19, most of the superior olivary complex (SOC)20, and the dorsal (DNLL) and ventral (VNLL) nuclei of the lateral lemniscus21. Additionally, a large descending projection from the auditory cortex terminates in the IC18,19,20,21,22, and IC neurons themselves synapse broadly within the local and contralateral lobes of the IC23. The intermingling of axons from many sources has made it difficult to probe IC circuits using electrical stimulation24. As a result, even though neurons in the IC perform computations important for sound localization and the identification of speech and other communication sounds25,26, the organization of neural circuits in the IC is largely unknown. We recently identified VIP neurons as the first molecularly identifiable neuron class in the IC27. VIP neurons are glutamatergic stellate neurons that project to several long-range targets, including the auditory thalamus and superior colliculus. We are now able to determine the sources and function of local and long-range inputs to VIP neurons and to determine how these circuit connections contribute to sound processing.
The protocol presented here is tailored to investigating synaptic inputs to VIP neurons in the IC of mice, specifically from the contralateral IC and the DCN (Figure 1). The protocol can be easily adapted to different sources of input, a different neuron type or a different brain region altogether. We also show that ChrimsonR is an effective red-shifted channelrhodopsin for long range circuit mapping in the auditory brainstem. However, we demonstrate that ChrimsonR is strongly activated by blue light, even at low intensities, and thus, to combine ChrimsonR with Chronos in two-color CRACM experiments, careful controls must be used to prevent cross-activation of ChrimsonR.

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			<td>AAV1.Syn.ChrimsonR-tdTomato.WPRE.bGH</td>
			<td>Addgene</td>
			<td>59171-AAV1</td>
			<td></td>
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		<tr>
			<td>AAV1.Syn.Chronos-GFP.WPRE.bGH</td>
			<td>Addgene</td>
			<td>59170-AAV1</td>
			<td></td>
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			<td>Ai14 reporter mice (B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J)</td>
			<td>Jackson Laboratory</td>
			<td>stock #007914</td>
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			<td>Amber (590nm) LUXEON Rebel LED</td>
			<td>Luxeon Star LEDs</td>
			<td>SP-01-A8</td>
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			<td>Blue (470nm) LUXEON Rebel LED</td>
			<td>Luxeon Star LEDs</td>
			<td>SP-01-B4</td>
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			<td>Carproject (carprofen)</td>
			<td>Henry Schein Animal Health</td>
			<td>59149</td>
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			<td>Drummond glas capillaries</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-203-G/X</td>
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			<td>Drummond Nanoject 3</td>
			<td>Drummond Scientific Company</td>
			<td>3-300-207</td>
			<td></td>
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			<td>Electrode beveler</td>
			<td>Sutter Instrument</td>
			<td>FG-BV10-D</td>
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			<td>Ethilon 6-0 (0.8 metric) nylon sutures</td>
			<td>Ethicon</td>
			<td>local pharmacy</td>
			<td></td>
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			<td>Fixed stage microscope</td>
			<td>any</td>
			<td>n/a</td>
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			<td>Gas anesthesia head holder</td>
			<td>David Kopf Instruments</td>
			<td>933-B</td>
			<td></td>
		</tr>
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			<td>General surgery tools</td>
			<td>Fine Science Tools</td>
			<td>N/A</td>
			<td></td>
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			<td>Golden A5 pet clipper</td>
			<td>Oster</td>
			<td>078005-010-003</td>
			<td></td>
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			<td>Heating pad</td>
			<td>Custom build</td>
			<td>N/A</td>
			<td></td>
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			<td>Hooded induction chamber w/ vacuum system</td>
			<td>Patterson Scientific</td>
			<td>78917760</td>
			<td></td>
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			<td>Hot bead sterilizer Steri 250</td>
			<td>Inotech</td>
			<td>IS-250</td>
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			<td>Iodine solution 10%</td>
			<td>MedChoice</td>
			<td>local pharmacy</td>
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			<td>Isoflurane vaporizer</td>
			<td>Patterson Scientific</td>
			<td>07-8703592</td>
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			<td>Lidocain topical jelly 2%</td>
			<td>Akorn</td>
			<td>local pharmacy</td>
			<td></td>
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			<td>Micro motor drill 1050</td>
			<td>Henry Schein Animal Health</td>
			<td>7094351</td>
			<td></td>
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			<td>Micro motor drill bits 0.5 mm</td>
			<td>Fine Science Tools</td>
			<td>19007-05</td>
			<td></td>
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			<td>Motorized Micromanipulator</td>
			<td>Sutter Instrument</td>
			<td>MP-285/R</td>
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			<td>Ophthalmic ointment Artificial Tears</td>
			<td>Akorn</td>
			<td>local pharmacy</td>
			<td></td>
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			<td>P-1000 electrode puller</td>
			<td>Sutter Instrument</td>
			<td>P-1000</td>
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			<td>Patch clamp amplifier incl data acquisition software</td>
			<td>any</td>
			<td>n/a</td>
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			<td>Portable anethesia machine</td>
			<td>Patterson Scientific</td>
			<td>07-8914724</td>
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			<td>Small animal steroetaxic frame</td>
			<td>David Kopf Instruments</td>
			<td>930-B</td>
			<td></td>
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			<td>Standard chemicals</td>
			<td>local vendors</td>
			<td>N/A</td>
			<td></td>
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			<td>standard imaging solutions</td>
			<td></td>
			<td></td>
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			<td>Sterile towel drapes</td>
			<td>Dynarex</td>
			<td>4410</td>
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			<td>Surgical marker</td>
			<td>Fine Science Tools</td>
			<td>18000-30</td>
			<td></td>
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		<tr>
			<td>Temperature controller</td>
			<td>Custom build</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>any</td>
			<td>n/a</td>
			<td></td>
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			<td>VIP-IRES-Cre mice (Viptm1(cre)Zjh/J)</td>
			<td>Jackson Laboratory</td>
			<td>stock #010908</td>
			<td></td>
		</tr>
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			<td>Water bath</td>
			<td>any</td>
			<td>n/a</td>
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long-range channelrhodopsin-assisted circuit mapping of inferior colliculus neurons with blue and red-shifted channelrhodopsins

When investigating neural circuits, a standard limitation of the in vitro patch clamp approach is that axons from multiple sources are often intermixed, making it difficult to isolate inputs from individual sources with electrical stimulation. However, by using channelrhodopsin assisted circuit mapping (CRACM), this limitation can now be overcome. Here, we report a method to use CRACM to map ascending inputs from lower auditory brainstem nuclei and commissural inputs to an identified class of neurons in the inferior colliculus (IC), the midbrain nucleus of the auditory system. In the IC, local, commissural, ascending, and descending axons are heavily intertwined and therefore indistinguishable with electrical stimulation. By injecting a viral construct to drive expression of a channelrhodopsin in a presynaptic nucleus, followed by patch clamp recording to characterize the presence and physiology of channelrhodopsin-expressing synaptic inputs, projections from a specific source to a specific population of IC neurons can be mapped with cell type-specific accuracy. We show that this approach works with both Chronos, a blue light-activated channelrhodopsin, and ChrimsonR, a red-shifted channelrhodopsin. In contrast to previous reports from the forebrain, we find that ChrimsonR is robustly trafficked down the axons of dorsal cochlear nucleus principal neurons, indicating that ChrimsonR may be a useful tool for CRACM experiments in the brainstem. The protocol presented here includes detailed descriptions of the intracranial virus injection surgery, including stereotaxic coordinates for targeting injections to the dorsal cochlear nucleus and IC of mice, and how to combine whole cell patch clamp recording with channelrhodopsin activation to investigate long-range projections to IC neurons. Although this protocol is tailored to characterizing auditory inputs to the IC, it can be easily adapted to investigate other long-range projections in the auditory brainstem and beyond.

We crossed VIP-IRES-Cre mice (Viptm1(cre)Zjh/J) and Ai14 Cre-reporter mice (B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J) to generate F1 offspring in which VIP neurons express the fluorescent protein tdTomato. F1 offspring of either sex were used, aged postnatal day (P) 21 to P70. A total of 22 animals were used in this study.
Stereotaxic injection of AAV1.Syn.Chronos-GFP.WPRE.bGH into the r...

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Long-range Channelrhodopsin-assisted Circuit Mapping of Inferior Colliculus Neurons with Blue and Red-shifted Channelrhodopsins

Channelrhodopsin-assisted circuit mapping (CRACM) is a precision technique for functional mapping of long-range neuronal projections between anatomically and/or genetically identified groups of neurons. Here, we describe how to utilize CRACM to map auditory brainstem connections, including the use of a red-shifted opsin, ChrimsonR.

When investigating neural circuits, a standard limitation of the in vitro patch clamp approach is that axons from multiple sources are often intermixed, ...

Channelrhodopsin-assisted circuit mapping is a precision technique for functional mapping of long-range neural projections. Here, we show how to use this approach to investigate circuits in the auditory brain stem. In brain slices, axons from multiple sources are often intermixed, making it difficult to isolate individual inputs using electrical stimulation.By using CRACM, this limitation can be overcome. Finding lambda and consistently defining the stereotaxic coordinate system is challenging as is keeping the duration of the procedure short. Having all steps carefully mapped out will increase your success rate.Begin by spraying the surgery area with 70%ethanol to sanitize and place sterile towel drapes to cover the surgery area. Remove cage bedding to limit risk of asphyxiation from the recovery cage. Next, put a heating pad under the cage and provide a food and water source.Next, transfer the animal to a stereotaxic frame and continue anesthesia. Insert a rectal temperature probe and switch on the homeostatic temperature controller. Then, apply ophthalmic ointment to prevent eyes from drying out.Administer preemptive analgesic. Finally, shave the scalp with electric clippers. Disinfect scalp with three alternating swabs of povidone iodine and 70%ethanol.Begin surgery by making an incision in the scalp along the midline, starting between the ears and continuing rostral to the eyes, exposing the lambda and bregma sutures. Push skin to the side and remove periosteum from the exposed bone if necessary. Next, mark the lambda suture with a surgical marker, position the tip of the nano-injector so that it is just touching lambda and zero the micromanipulator coordinates.Use the nano-injector tip and micromanipulator to measure the difference in elevation between the lambda and bregma sutures. Adjust the palette bar height to bring lambda and bregma to within plus or minus 100 micrometer height difference. Map the injection site using the nano-injector tip and micromanipulator coordinate system and mark the site with a surgical marker.Then, use a micromotor drill with a 0.5 millimeter drill burr to perform a craniotomy over the injection site. To ensure broad transfection of neurons in the target nucleus, use a nano-injector to make injections at various depths in the tissue and in the case of larger brain regions, like the inferior colliculus, make injections over the course of two or more penetrations at different X and Y coordinates. To inject the inferior colliculus, deposit 20 nanoliters of virus in the intervals of 250 micrometers along the Z axis between 2, 250 micrometers and 1, 750 micrometers depth.For injections in the dorsal cochlear nucleus, deposit 20 nanoliters of virus at a depth of 4, 750 micrometers and 4, 550 micrometers respectively. After injecting at each Z coordinate, wait two to three minutes before moving the injector to the next Z coordinate to allow time for the virus to diffuse away from the injection site, reducing the probability that virus will be sucked up the injection tract when the nano-injector is repositioned. Then, after the last injection, wait three to five minutes before retracting nano-injector from the brain.When the nano-injector is removed from the brain between penetrations and between animals, eject a small volume of virus from the tip to check that the tip has not clogged. After injections, use sterile PBS to wet the cut edges of the scalp and then gently move the skin back towards the midline. Close the wound with simple interrupted sutures using 6-0 nylon sutures.Then, apply 0.5 to one milliliter of 2%lidocaine jelly to the wound. Remove the ear bars and temperature probe, turn of isoflurane, and remove the animal from the palette bar and transfer it to the recovery cage. Finally, monitor recovery closely.Once the animal is fully awake, moving around, and showing no signs of pain or distress, transfer it back into its cage and return the cage to the vivarium. Use standard patch clamp methods to make recordings. Begin by placing the slice in a recording chamber under a fixed stage upright microscope and continuously profuse with artificial cerebrospinal fluid at two milliliters per minute.Next, patch neurons under visual control using a suitable patch clamp amplifier. Lastly, during wholesale recordings, activate Chronos by delivering brief pulses of 470 nanometer light or ChrimsonR by brief pulses of 580 nanometer light through commercially available LEDs. Results indicated that ChrimsonR injection led to strong expression in the DCN with tdTomato fluorescence visible in cells and fibers.In the contralateral ICC, fibers strongly labeled with tdTomato were clearly visible after three weeks, demonstrating the long-range trafficking capability of the ChrimsonR-tdTomato construct when injected into auditory brain stem nuclei. Optical activation of ChrimsonR elicted EPSPs in IC VIP neurons, indicating that ChrimsonR is a useful tool for long-range CRACM experiments when the experimental parameters demand the use of red light instead of blue light. However, we found that ChrimsonR was readily activated with blue light, showing the same threshold for blue light activation as Chronos.For this protocol it is most important to make sure that the stereotaxic coordinates are correct and to confirm that the virus was expressed only in your targeted brain region. By exchanging the Chronos ChrimsonR viroconstruct with a different virus, for example, a cell specific Flex virus, you can answer several anatomical or physiological questions without changing the surgery protocol.

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