The authors thank Bryan Krause for technical support and guidance on this project.
This work was supported by the International Anesthesia Research Society (IMRA to AR), National Institutes of Health (R01 GM109086 to MIB), and the Department of Anesthesiology, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA.

All procedures involving animals described in this protocol were approved by the University of Wisconsin-Madison School of Medicine and Public Health Animal Care and Use Committee.
1. Breeding mice to express fluorescent reporter protein in interneuron subpopulations
<ol>
	<li>Pair homozyogous, Cre-dependent tdTomato male mouse with either homozygous SOM-Cre female or homozygous PV-Cre female mouse. 
	NOTE: Other specific neuronal populations may be targeted by using the appropriate Cre lines.</li>
	<li>Allow heterozygous offspring to mature to at least 3 weeks of age before proceeding. For experiments described here, genotyping is not necessary, as homozygous parents produce offspring that are all heterozygous for both cell type-specific Cre recombinase and Cre-dependent reporter alleles.</li>
</ol>
2. Performing unilateral stereotaxic injection of viral construct
<ol>
	<li>Adjust settings of the micropipette puller for injection pipettes as indicated in the instrument user manual (see Table 1 for recommended settings). Pull the glass micropipette.</li>
	<li>Break the tip of the sharp end of the pipette such that the tip diameter is approximately 30 &#181;m with minimal taper over several millimeters.</li>
	<li>Using previously documented procedures, decide on the appropriate titer and volume of virus to be injected. In the experiments described here, 1.0 &#956;L (titer: 3.1-5.7 TU/mL) injected unilaterally produced good results.</li>
	<li>Backfill the full volume of the pipette with mineral oil. Load the pipette onto the microsyringe and flow a small amount of mineral oil through the tip to ensure the tip is not clogged.</li>
	<li>Frontfill at least 1.0 &#956;L of viral construct. The recombinant adeno-associated viral vector used in these experiments was AAV2-hSyn-hChR2(H134R)-EYFP.</li>
	<li>Arrange sterile drape in a surgical area. Sterilize tools for stereotaxic procedure and place it on the drape.</li>
	<li>Anesthetize SOM-tdTomato or PV-tdTomato heterozygous animal using isoflurane (3% for induction, 1.5-2% for maintenance) and oxygen mixture. Periodically confirm surgical level of anesthesia with toe pinch throughout surgery. Ensure animal does not move beyond the surgical plan of anesthesia by monitoring respirations every 10-15 min.</li>
	<li>Shave the top of the animal&#8217;s head. Apply 70% isopropyl alcohol and iodine-based solution liberally to surgical area and ophthalmic ointment to eye sockets to prevent drying of the membrane. Administer bupivacaine/lidocaine (1:1 ratio, 1.0 mg/kg) subcutaneously to surgical site for local anesthetic.</li>
	<li>Fit the animal into stereotaxic frame.</li>
	<li>Use scalpel to make incision along sagittal axis of skin overlying the dorsal surface of the skull. Retract skin using forceps. Hydrate skull with 0.9% saline as necessary.</li>
	<li>On the surface of the skull, lightly mark the intersection of anterior and lateral coordinates with a cross in pencil. Drill a hole at the appropriate coordinates in the transverse plane (in mm relative to Bregma, for cingulate cortex (Cg) injection: anterior 0.2, lateral 0.3; for posterior thalamus (Po) injection: posterior 2.25, lateral 3.4). 
	NOTE: Markings should extend beyond the boundaries of the burr hole to provide guidance for accurate placement of pipette.</li>
	<li>Turn on and balance the air table.</li>
	<li>Reposition electrode manipulator of the stereotaxic frame at 0&#176; for injections into Cg, or 45&#176; in the coronal plane for injections into Po.</li>
	<li>Attach the syringe pump to the electrode manipulator. Attach the microsyringe pump controller to the syringe pump.</li>
	<li>Navigate pipette tip near (but not touching) the surface of the brain, at the intersection of markings created in Step 2.11. Advance the pipette at approximately 1 mm/s along its longitudinal axis into the brain either 0.9 mm (injection in Cg) or 3.1 mm (injection in Po). Wait for 10 min before proceeding.</li>
	<li>Inject 1.0 &#181;L of viral construct over a period of 10 min (100 nL/min). If welling of virus from pipette insertion site is observed, slow the injection rate to 50 nL/min.</li>
	<li>After injection, wait for 10 min before slowly retracting the injection pipette.</li>
	<li>Suture to close the scalp incision and administer 2-5 mg/kg meloxicam subcutaneously.</li>
	<li>Discontinue isoflurane and monitor animal during emergence from anesthesia. Allow to recover according to procedures described by Institution&#8217;s Animal Care and Use Committee, including further administration of analgesics.</li>
</ol>
3. Preparation of acute brain slices
<ol>
	<li>Allow at least 3 weeks for the expression of viral construct before harvesting tissue.</li>
	<li>Prepare 1 L of artificial cerebral spinal fluid for slicing procedure (slicing artificial cerebral spinal fluid, sACSF). See Table 2 for ingredients.</li>
	<li>Throughout the slicing procedure, supply sACSF with dissolved 95% O2/5% CO2 mixture, delivered via gas dispersion tube.</li>
	<li>Prepare ice cold bath for vibrating blade microtome. Mount ice-cold specimen stage onto microtome and fix sapphire blade in place for tissue sectioning.</li>
	<li>Anesthetize mouse with 3% isoflurane and oxygen until loss of righting reflex.</li>
	<li>Decapitate the mouse using guillotine and immediately submerge head in 4&#176;C sACSF. To preserve the health of the tissue, complete the following steps as swiftly as possible.</li>
	<li>Open skull cavity by making a small incision at the base of the skull and gently removing each skull plate. Gently remove underlying dura mater.</li>
	<li>While the brain is still in the skull cavity, use the razor blade to remove the cerebellum. Make a second vertical cut along the sagittal plane in the left hemisphere, just lateral to the midline.</li>
	<li>Prepare tissue block for sectioning.
	<ol>
		<li>Gently lift the brain from the skull cavity. Place the brain on the filter paper with the flat, sagittal plane down. Guide the filter paper over the blocking template and align the brain to the underlying template outline (Supplementary Figure 1).</li>
		<li>Make two parallel cuts in the coronal plane as indicated by the lines on the template. Add a small drop of sACSF to keep filter paper wet, if necessary.</li>
		<li>Place the tissue block in 4&#176;C sACSF briefly while step 3.8.4 is conducted.</li>
		<li>Apply a small amount of super glue to the ice-cold specimen stage.</li>
		<li>Lift the tissue block from cold sACSF. Use the corner of an absorbent towel to wick away excess sACSF. Glue the posterior coronal plane of the tissue block to the specimen stage, with the dorsal surface of the brain facing the sapphire blade.</li>
	</ol>
	</li>
	<li>Collect 500 &#181;m thick coronal brain slices. Place slices of interest on nylon mesh (Supplementary Figure 2) in 34 &#176;C sACSF and allow the container to reach room temperature. 
	NOTE: For experiments described here, electrophysiological recordings were collected from a coronal section centered approximately 2.25 mm posterior to bregma to study a non-primary sensory area, medial secondary visual cortex (V2MM).</li>
</ol>
4. Preparation of experimental artificial cerebral spinal fluid (eACSF) bags containing dissolved volatile anesthetic isoflurane
<ol>
	<li>Prepare 300 mL of a stock mixture of 3.0% isoflurane.
	<ol>
		<li>In a sealed polytetrafluoroethylene gas bag, add ~100 mL of 95% O2/5% CO2 gas mixture to 20-30 mL of liquid isoflurane and a small amount of 0.9% saline. Wait at least for 30 min to allow equilibration of isoflurane between liquid and gas phases.</li>
		<li>Determine the amount of saturated isoflurane gas, Vsat, to add to the stock bag using the following equation: 
		<img alt="Equation 1" src="/files/ftp_upload/61333/61333equ01.jpg" /> 
		where P%stock is the target composition of the stock gas (3% in this case), Vstock is the final volume of the stock gas bag, Pisoflurane is the partial pressure of isoflurane at room temperature (~240 mmHg), and Ptotal is the atmospheric pressure (~760 mmHg).</li>
		<li>Add the calculated amount of saturated gas to an empty gas bag and fill the bag with a volume of 95% O2/5% CO2 gas mixture to bring the total volume of stock bag to 300 mL.</li>
	</ol>
	</li>
	<li>Prepare 2 L of artificial cerebral spinal fluid for perfusion of the slice during the experiment (experimental ACSF, eACSF). See Table 2 for ingredients. Dissolve 95% O2/5% CO2 gas mixture into solution.</li>
	<li>Prepare two separate bags of Control and Isoflurane solutions.
	<ol>
		<li>To an empty polytetrafluoroethylene gas bag, add 600 mL eACSF and 600 mL of 95% O2/5% CO2 gas mixture. Label this bag as Control.</li>
		<li>To another empty polytetrafluoroethylene gas bag, add 300 mL of eACSF. Label this bag as Isoflurane.</li>
		<li>Choose a physiologically relevant equilibrated gas phase concentration of isoflurane. Experiments were conducted using gas concentrations equivalent to 1.3% isoflurane. Mice lose righting reflex, and presumably consciousness, at 0.9% inhaled isoflurane.</li>
		<li>Use the following equation to calculate the equivalent gas phase concentration at room temperature, P%(Troom)31: 
		<img alt="Equation 2" src="/files/ftp_upload/61333/61333equ02.jpg" /> 
		where P%(Tbody) is the physiologically relevant gas phase concentration chosen in Step 4.3.3, Troom is 25 &#176;C, and Tbody is 37 &#176;C.</li>
		<li>Use the following equation to determine volume of gas from stock gas bag, Vstock, to add to the Isoflurane solution. 
		<img alt="Equation 3" src="/files/ftp_upload/61333/61333equ03.jpg" /> 
		where Vsolution is the volume of eACSF in ISOFLURANE bag (300 mL), P%(Troom) is entered from equation (2), &#955; is the saline/gas Ostwald partition coefficient of isoflurane (&#955; = 1.232), and P%stock is the gas phase concentration of the stock gas bag (P%stock = 3.0%).</li>
		<li>To the Isoflurane solution bag, add the volume of gas from the stock gas bag, Vstock, calculated in Step 4.3.5.</li>
		<li>To the Isoflurane solution bag, add a volume of 95% O2/5% CO2 gas mixture to bring the total volume of gas in the Isoflurane solution bag to 300 mL.</li>
	</ol>
	</li>
	<li>Shake both Control and Isoflurane bags on shaker for at least 1 h to allow isoflurane phase equilibration.</li>
	<li>After all data has been collected, the correct concentration may be verified by using an anesthetic gas monitor to measure equilibrated gas concentration of isoflurane above the remaining solution in bag.</li>
	<li>Report experimental concentrations of volatile gases in aqueous units, as millimolar concentrations are more robust to changes in temperature. Use the following equation to convert room temperature gas phase concentration, P%(Troom), to equivalent aqueous concentration (Caqueous, in mM)31: 
	<img alt="Equation 4" src="/files/ftp_upload/61333/61333equ04.jpg" /> 
	where &#945; is the saline/gas Bunsen partition coefficient for isoflurane at 25&#176;C32.</li>
</ol>
5. Preparation of hardware and software for multi-channel recordings
<ol>
	<li>Set up 16-channel data acquisition system according to manufacturer instructions. 
	NOTE: Several commercially available amplifiers and data acquisition systems can be used to collect multi-channel recordings. In the experiments described here, analog signals are delivered via an electrode reference panel to two amplifiers, where they are amplified (2000x) and filtered (0.1-10kHz). Analog inputs to the data acquisition system are digitized at 40kHz.</li>
	<li>Fasten the appropriate 16-channel headstage adaptor to a microscope micromanipulator. Orient the adaptor such that the female connector ports are facing downward.</li>
	<li>Adjust the angle of operation of this micromanipulator such that it is oriented downward toward the recording chamber, at an angle approximately 70&#176; relative to horizontal.</li>
	<li>Connect the headstage input to a 16 x 1 probe for in vitro electrophysiology via the headstage adaptor anchored to the micromanipulator.</li>
	<li>Connect the headstage output connector to the data acquisition system.</li>
	<li>Install appropriate software for data acquisition. Configure 15 input channels to correspond to input signals from the first 15 multi-channel probe contacts. Configure the remaining channel to receive input from the intracellular electrode. 
	NOTE: Take care to consider electrode and adaptor maps when collecting and analyzing data, to ensure the appropriate signal corresponds to the electrode contact from which it was collected.</li>
</ol>
6. Configuration of light stimulation protocols
<ol>
	<li>Set up light delivery system and install the accompanying software.</li>
	<li>Open the software. Choose hardware wiring configuration in which a Trigger Source (Digital/TTL Out) provides Trigger In signal to the light delivery system, and the light delivery system provides Trigger Out signal to a 470 nm LED.</li>
	<li>Mount high-power objective lens. Using digital camera, calibrate high-power objective for use with light delivery system.</li>
	<li>Create new profile sequence of light stimulation profiles.
	<ol>
		<li>Create a pattern of choice. In the experiments described here, a circle of diameter 150 &#181;m is used to allow layer-specific activation of axon terminals.</li>
		<li>To construct a profile sequence, copy and paste this profile for each of any number of trials.</li>
		<li>Create a waveform list that contains waveforms of any light intensity, pulse duration, or pulse number.</li>
		<li>Randomly assign waveforms to each profile. Each profile with its assigned waveform corresponds to one trigger pulse from a Digital TTL input, or one trial.</li>
		<li>Save the profile sequence.</li>
	</ol>
	</li>
	<li>In the data acquisition software, create a new protocol.
	<ol>
		<li>Set the number of trials to equal the number of profiles in the profile sequence just created.</li>
		<li>Choose signal inputs to match those configured in Step 5.6. Configure a protocol that provides a single digital TTL output, recording from these 16 input channels for an appropriate amount of time before and after the digital trigger.</li>
	</ol>
	</li>
</ol>
7. Placing multi-channel probe in ex vivo brain tissue slice
<ol>
	<li>Perfuse bubbled eACSF (not in sealed bags) at 3-6 mL/min.</li>
	<li>Transfer the brain slice containing area of interest onto mesh grid in microscope perfusion chamber. Anchor with platinum harp (see Supplementary Figure 3).</li>
	<li>Rotate mesh grid such that the line of electrode contacts on the distal end of the multi-channel probe is approximately perpendicular to the pial surface.</li>
	<li>Under broadfield illumination and under fine control of the micromanipulator, lower the multi-channel probe toward the surface of the slice.</li>
	<li>Rotate the filter cube turret to engage the appropriate filter cube for visualization of the fluorescent reporter protein expressed in axon terminals of cortical afferents. If necessary, rotate the slice to more precisely align the probe with the pial surface.</li>
	<li>Position the probe just above the plane of the slice, ~200 &#181;m short of the final target position along the x-axis, leaving at least one channel outside the boundary of the area of tissue being recorded</li>
	<li>Slowly insert the probe into the slice by moving the manipulator along its longitudinal axis. To minimize damage to the tissue, only advance the probe to the extent that the sharp tips are just visible below the tissue surface. This will minimize damage to the tissue while still ensuring the electrode contacts are in contact with the tissue.</li>
</ol>
8. Patch clamping targeted neurons and obtaining whole-cell configuration
<ol>
	<li>Switch eACSF source to bagged Control solution.</li>
	<li>Identify fluorescently labeled cell for targeted patch clamp recording.
	<ol>
		<li>Restrict the aperture iris diaphragm to the smallest diameter. Engage a low-power objective lens and bring the tissue into focus.</li>
		<li>Center the light over an area of tissue adjacent to (but not overlapping) the multi-channel probe.</li>
		<li>Engage the high-power (40x or 60x) water immersion objective, using caution to avoid contact between the multi-channel probe and objective lens.</li>
		<li>Rotate the filter cube turret to engage the appropriate filter set to allow imaging of cells expressing Cre-dependent fluorescent marker.</li>
		<li>Identify a fluorescently labeled cell as a target for patch clamp recording. Raise the objective lens to create ample space to lower a patch pipette.</li>
	</ol>
	</li>
	<li>Load a patch pipette (see Table 1) with internal solution (Table 2) and mount pipette into electrode holder. Using 1 mL syringe, apply positive pressure corresponding to ~0.1mL air.</li>
	<li>Lower patch pipette into the solution. Bring the pipette tip into focus under visual guidance.</li>
	<li>Obtain whole-cell recording from the targeted cell using the steps previously demonstrated33.</li>
	<li>If planning to assess changes to intrinsic properties of the cell (e.g., input resistance, action potential firing rate in response to current steps), conduct these recordings. Otherwise, move to axon stimulation protocol in below.</li>
</ol>
9. Layer-specific optogenetic activation of axon terminals
<ol>
	<li>Manipulate field of view in the x-y plane to align light stimulation profile with desired location on slice.</li>
	<li>Load light stimulus protocol and prepare the light delivery system to receive a digital TTL pulse.</li>
	<li>Optogenetically activate axon terminals while simultaneously recording extracellular field potentials and intracellular membrane fluctuations.</li>
	<li>Switch eACSF source to Isoflurane solution and wash drug in for 15 min. If necessary, collect spontaneous recordings during the wash-in.</li>
	<li>Repeat step 9.2-9.3.</li>
	<li>Switch eACSF source to Control solution and wash drug out for 20 min. If necessary, collect spontaneous recordings during wash-out.</li>
	<li>Repeat step 9.2-9.3.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+inhibition+of+frontal-to-parietal+feedback+connectivity+is+a+neurophysiologic+correlate+of+general+anesthesia+in+surgical+patients.">Preferential inhibition of frontal-to-parietal feedback connectivity is a neurophysiologic correlate of general anesthesia in surgical patients.</a> PLoS.One. 6 (10), 25155 (2011).</li><li>Lee, M., 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=Network+Properties+in+Transitions+of+Consciousness+during+Propofol-induced+Sedation.">Network Properties in Transitions of Consciousness during Propofol-induced Sedation.</a> Scientific Reports. 7 (1), 16791 (2017).</li><li>Murphy, M., 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=Propofol+anesthesia+and+sleep:+a+high-density+EEG+study.">Propofol anesthesia and sleep: a high-density EEG study.</a> Sleep. 34 (3), 283-291 (2011).</li><li>Murphy, C., Krause, B., Banks, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+effects+of+isoflurane+on+cortico-cortical+feedback+afferent+responses+in+murine+non-primary+neocortex.">Selective effects of isoflurane on cortico-cortical feedback afferent responses in murine non-primary neocortex.</a> British Journal of Anaesthesiology. 123 (4), 488-496 (2019).</li><li>Raz, 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=Preferential+effect+of+isoflurane+on+top-down+versus+bottom-up+pathways+in+sensory+cortex.">Preferential effect of isoflurane on top-down versus bottom-up pathways in sensory cortex.</a> Frontiers in System Neurosciences. 8, 191 (2014).</li><li>Schrouff, J., 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=Brain+functional+integration+decreases+during+propofol-induced+loss+of+consciousness.">Brain functional integration decreases during propofol-induced loss of consciousness.</a> Neuroimage. 57 (1), 198-205 (2011).</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>Cruikshank, S. J., Urabe, H., Nurmikko, A. V., Connors, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathway-Specific+Feedforward+Circuits+between+Thalamus+and+Neocortex+Revealed+by+Selective+Optical+Stimulation+of+Axons.">Pathway-Specific Feedforward Circuits between Thalamus and Neocortex Revealed by Selective Optical Stimulation of Axons.</a> Neuron. 65 (2), 230-245 (2010).</li><li>Gredell, J. A., Turnquist, P. A., MacIver, M. B., Pearce, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+diffusion+and+partition+coefficients+of+propofol+in+rat+brain+tissue:+implications+for+studies+of+drug+action+in+vitro.">Determination of diffusion and partition coefficients of propofol in rat brain tissue: implications for studies of drug action in vitro.</a> BJA: British Journal of Anaesthesia. 93 (6), 810-817 (2004).</li><li>Benkwitz, 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=Determination+of+the+EC50+amnesic+concentration+of+etomidate+and+its+diffusion+profile+in+brain+tissue:+implications+for+in+vitro+studies.">Determination of the EC50 amnesic concentration of etomidate and its diffusion profile in brain tissue: implications for in vitro studies.</a> Anesthesiology. 106 (1), 114-123 (2007).</li><li>Franks, N. P., Lieb, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+actions+of+volatile+general+anaesthetics+at+molecular+and+cellular+levels.">Selective actions of volatile general anaesthetics at molecular and cellular levels.</a> British Journal of Anaesthesia. 71 (1), 65-76 (1993).</li><li>Honemann, C. W., Washington, J., Honemann, M. C., Nietgen, G. W., Durieux, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partition+coefficients+of+volatile+anesthetics+in+aqueous+electrolyte+solutions+at+various+temperatures.">Partition coefficients of volatile anesthetics in aqueous electrolyte solutions at various temperatures.</a> Anesthesiology. 89 (4), 1032-1035 (1998).</li><li>Au - Segev, A., Au - Garcia-Oscos, F., Au - Kourrich, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-cell+Patch-clamp+Recordings+in+Brain+Slices.">Whole-cell Patch-clamp Recordings in Brain Slices.</a> Journal of Visualized Experiments. (112), e54024 (2016).</li><li>Lin, J. Y., Lin, M. Z., Steinbach, P., 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=Characterization+of+engineered+channelrhodopsin+variants+with+improved+properties+and+kinetics.">Characterization of engineered channelrhodopsin variants with improved properties and kinetics.</a> Biophysical Journal. 96 (5), 1803-1814 (2009).</li><li>Lin, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+user's+guide+to+channelrhodopsin+variants:+features,+limitations+and+future+developments.">A user's guide to channelrhodopsin variants: features, limitations and future developments.</a> Experimental Physiology. 96 (1), 19-25 (2011).</li><li>Banks, M. I., Pearce, R. 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+actions+of+volatile+anesthetics+on+GABAA+IPSCs:+dissociation+of+blocking+and+prolonging+effects.">Dual actions of volatile anesthetics on GABAA IPSCs: dissociation of blocking and prolonging effects.</a> Anesthesiology. 90 (1), 120-134 (1999).</li><li>Hagan, C. E., Pearce, R. A., Trudell, J. R., MacIver, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+measures+of+volatile+anesthetics+in+the+aqueous+phase+using+calcium+sensitive+electrodes.">Concentration measures of volatile anesthetics in the aqueous phase using calcium sensitive electrodes.</a> Journal of Neuroscience Methods. 81, 177-184 (1998).</li></ol>

The authors have nothing to disclose.

In this manuscript, a protocol for evaluating intra- and extracellular responses to selectively activated afferent pathways in ex vivo brain slices is described.
The use of optogenetic tools and parallel recording schemes allows investigators to probe responses of local populations to afferent inputs from distant brain regions, while recording simultaneously from targeted populations of interneurons. The use of optogenetic technology allows for axon terminals of afferent projections to be preserved and activated even though their cell bodies are no longer attached. This relieves geometric restrictions previously imposed upon ex vivo slices, as preservation of long-range electrical connections is no longer paramount. Still, care should be taken to prepare slices in a geometrical plane that preserves any remaining connections of interest. For example, pyramidal cells are oriented vertically along the cortical column, and evoked network activity measured by the multichannel probes in these experiments requires such local connections to be preserved as much as possible. Thus, coronal slices were prepared to keep local connectivity intact.
When choosing optogenetic constructs and relevant fluorescent reporter proteins, properties of their excitation/emission spectra and microscopic optics must be considered. Persistent light stimulation may result in partial inactivation of many channelrhodopsin variants34, which can be avoided by choosing reporter proteins whose excitation spectra do not overlap with that of the opsin. Alternative variants with different kinetics or light sensitivities may also be chosen depending on the experimental paradigm35, including manipulations using alternative excitatory or inhibitory opsins. Filter cubes must also be appropriately aligned with the chosen fluorescent reporters, such that afferent axon terminals or interneurons may be imaged independently and without activating expressed opsins. To account for the variability in virus expression, it may also be pertinent for investigators to normalize any optogenetically-induced activity to the expression level of the viral construct, measured by the fluorescent output of the reporter protein.
Delivery of pre-calculated concentrations of volatile anesthetics to slice tissue is also possible using the methods outlined here. When choosing appropriate physiologically relevant gas equilibrium percentages, investigators should account for 10-15% loss of dissolved isoflurane gas between the perfusion line and tissue36. The methods applicable to isoflurane have been presented, but other drugs such as halothane, sevoflurane, or desflurane can be handled similarly using the appropriate Ostwald and Bunsen coefficients (Supplementary Table 1). The partitioning properties of volatile anesthetics assure that they will predictably dissolve into ACSF. However, because partial pressures are more sensitive to changes in temperature than aqueous EC50 concentrations37, gas equilibrium volume percentages of volatile anesthetics must be converted to predicted room temperature millimolar concentrations to compare observed effects to physiologically relevant doses in vivo. If opting to study intravenous anesthetics such as etomidate or propofol in brain slices, investigators must consider diffusion profiles of the drugs under study, as equilibration times and physiologically relevant concentrations may vary greatly30.
In this manuscript, a protocol is described for testing the effects of volatile anesthetics on distinct components of thalamocortical circuits in ex vivo brain slices. Many of the variables and parameters in the methods described may be manipulated for further investigations. For example, different brain areas, afferent pathways, cell targets, or volatile anesthetics may be studied by adapting the outlined methods to answer novel questions. Combined with other theoretical and experimental methods, study of unique cellular and network components using ex vivo brain slices will advance our understanding of the dynamic brain, and the changes it undergoes during pharmacological and pathophysiological changes in consciousness.

Volatile anesthetics have been used ubiquitously in a variety of clinical and academic settings for more than a century. Distinct classes of anesthetics have unique, often non-overlapping molecular targets1,2,3, yet nearly all of them produce unconsciousness. While their behavioral effects are quite predictable, the mechanisms by which anesthetics induce loss of consciousness are largely unknown. Anesthetics may ultimately influence both the level and contents of consciousness via actions on corticothalamic circuits, disrupting integration of information throughout the cortical hierarchy4,5,6,7,8,9. More broadly, modulation of corticothalamic circuits may play a role in experimentally10 or pharmacologically11 altered states of consciousness, and may also be implicated in sleep12 and in pathophysiological disorders of consciousness13,14.
The elusiveness of the mechanisms underlying loss and return of consciousness during anesthesia may be attributed partially to non-linear, synergistic actions of anesthetics at the cellular, network, and systems levels15. Isoflurane, for example, suppresses activity within the selected brain regions16,17,18, impairs connectivity between distant brain regions19,20,21,22,23, and diminishes synaptic responses in a pathway-specific manner24,25. Which effects of anesthetics, from the molecular to the systems level, are necessary or sufficient to effect loss of consciousness remains unclear. In addition to substantive clinical investigations of consciousness using non-invasive techniques19,20,26, it is important that experimentalists seek to disentangle the distinct cellular and network interactions that subserve the conscious experience.
By simplifying the complex interactions found in the intact brain, ex vivo brain slices allow the study of isolated components of the brain&#8217;s dynamic systems9. A reduced slice preparation combines the benefits of relatively intact anatomical structures of local neural circuits with the versatility of in vitro manipulations. However, until recently, methodological constraints have precluded the study of synaptic and circuit properties of long-range inputs in brain slices27,28; the tortuous path of corticothalamic fiber tracts made activation of independent afferent pathways all but impossible by electrical stimulation.
Investigating the effects of anesthetic agents on the brain slice preparations presents additional challenges. Absent an intact respiratory and circulatory system, anesthetic agents must be bath-applied, and concentrations carefully matched to estimated effect site concentrations. For many intravenous anesthetic agents, the slow rate of equilibration in the tissue renders traditional pharmacological investigations laborious29,30. Investigating the effects volatile gas anesthetics in ex vivo preparations is more tractable, but also presents challenges. These include converting inhaled partial pressure doses to aqueous concentrations, and the need for a modified delivery system of the drug to the tissue via artificial cerebral spinal fluid31.
Here, methods are described by which investigators may capitalize on the well-documented physicochemical properties of the volatile anesthetic isoflurane for drug delivery to ex vivo brain slices, activate pathway- and layer-specific inputs to a cortical area of interest with high spatiotemporal resolution, and conduct simultaneous laminar recordings and targeted patch clamp recordings from select populations of neurons. Combined, these procedures allow investigators to measure volatile anesthetic-induced changes in several observable electrophysiological response properties, from the synaptic to local network level.

<table>
	<tbody>
		<tr>
			<td>2.5x broadfield objective lens</td>
			<td>Olympus</td>
			<td>MPLFLN2.5X</td>
			<td></td>
		</tr>
		<tr>
			<td>40x water immersion objective lens</td>
			<td>Olympus</td>
			<td>LUMPLFLN40XW</td>
			<td></td>
		</tr>
		<tr>
			<td>95% O2/5% CO2 mixture</td>
			<td>Airgas</td>
			<td>Z02OX95R2003045</td>
			<td></td>
		</tr>
		<tr>
			<td>A16 probe</td>
			<td>NeuroNexus</td>
			<td>A16x1-2mm-100-177-A16</td>
			<td>16-channel probe</td>
		</tr>
		<tr>
			<td>AAV2-hSyn-hChR2(H134R)-EYFP</td>
			<td>Karl Deisseroth Lab, UNC Vector Core</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthetic gas monitor (POET II)</td>
			<td>Criticare</td>
			<td>602-3A</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP, Magnesium Salt</td>
			<td>Sigma Aldrich</td>
			<td>A9187</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J</td>
			<td>The Jackson Laboratory</td>
			<td>007914</td>
			<td>Cre-dependent tdTomato mouse</td>
		</tr>
		<tr>
			<td>B6;129P2-Pvalbtm1(cre)Arbr/J</td>
			<td>The Jackson Laboratory</td>
			<td>008069</td>
			<td>PV-Cre mouse</td>
		</tr>
		<tr>
			<td>Belly Dancer Shaker</td>
			<td>Thomas Scientific</td>
			<td>1210H86-TS</td>
			<td>for equilibration of sealed gas bags</td>
		</tr>
		<tr>
			<td>Betadine solution</td>
			<td>Generic brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Generic brand</td>
			<td></td>
			<td>for silver chloriding patch clamp electrode</td>
		</tr>
		<tr>
			<td>Bupivicaine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2)</td>
			<td>Dot Scientific</td>
			<td>DSC20010</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>Capillary glass (patch clamp recordings)</td>
			<td>King Precision Glass, Inc.</td>
			<td>KG-33</td>
			<td>Borosilicate, ID: 1.1mm, OD: 1.7mm, Length: 90.0mm</td>
		</tr>
		<tr>
			<td>Capillary glass (viral injections)</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-203-G/X</td>
			<td>3.5&#34;</td>
		</tr>
		<tr>
			<td>Control of junior micromanipulator</td>
			<td>Luigs and Neumann</td>
			<td>SM8</td>
			<td>for control of junior micromanipulator</td>
		</tr>
		<tr>
			<td>Control of manipulators and shifting table</td>
			<td>Luigs and Neumann</td>
			<td>SM7</td>
			<td>for control of multichannel electrode and shifting table</td>
		</tr>
		<tr>
			<td>Digidata 1440A + Clampex 10</td>
			<td>Molecular Devices</td>
			<td>1440A</td>
			<td>Digitizer and software</td>
		</tr>
		<tr>
			<td>E-3603 tubing</td>
			<td>Fisher Scientific</td>
			<td>14171208</td>
			<td>for delivery of 95% O2/5% CO2 gas mixture to incubation chamber + application of pressure during patch clamping</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Dot Scientific</td>
			<td>DSE57060</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>ERP-27 EEG Reference/Patch Panel</td>
			<td>Neuralynx</td>
			<td>Retired</td>
			<td></td>
		</tr>
		<tr>
			<td>Filling needle</td>
			<td>World Precision Instruments</td>
			<td>50821912</td>
			<td>for filling patch clamp pipettes</td>
		</tr>
		<tr>
			<td>Filter cube for imaging EYFP</td>
			<td>Olympus</td>
			<td>U-MRFPHQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Fisher Scientific</td>
			<td>09801E</td>
			<td>lay over slice template during preparation of tissue block</td>
		</tr>
		<tr>
			<td>Flaming/Brown micropipette puller</td>
			<td>Sutter Instrument</td>
			<td>P-1000</td>
			<td>2.5x2.5 Box filament</td>
		</tr>
		<tr>
			<td>Gas dispersion tube</td>
			<td>Sigma Aldrich</td>
			<td>CLS3953312C</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass syringe (100 mL)</td>
			<td>Sigma Aldrich</td>
			<td>Z314390</td>
			<td>for filling gas-sealed bags</td>
		</tr>
		<tr>
			<td>Gluconic Acid, Potassium Salt (K-gluconate)</td>
			<td>Dot Scientific</td>
			<td>DSG37020</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Dot Scientific</td>
			<td>DSG32040</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>GTP, Sodium Salt</td>
			<td>Sigma Aldrich</td>
			<td>G8877</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>Headstage-probe adaptor</td>
			<td>NeuroNexus</td>
			<td>A16-OM16</td>
			<td>adaptor to connect 16-channel probe to headstage input</td>
		</tr>
		<tr>
			<td>Hemostatic Forceps</td>
			<td>VWR International</td>
			<td>76192-096</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Dot Scientific</td>
			<td>DSH75030</td>
			<td>ACSF,intracellular solution</td>
		</tr>
		<tr>
			<td>HS-16 Headstage</td>
			<td>Neuralynx</td>
			<td>Retired</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>07-893-1389</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol (70%)</td>
			<td>VWR International</td>
			<td>101223-746</td>
			<td></td>
		</tr>
		<tr>
			<td>Junior micromanipulator</td>
			<td>Luigs and Neumann</td>
			<td>210-100 000 0090-R</td>
			<td>for manipulation of patch clamp electrode</td>
		</tr>
		<tr>
			<td>LED Light Source Control Module</td>
			<td>Mightex</td>
			<td>BLS-PL02_US</td>
			<td>optogenetic light source control</td>
		</tr>
		<tr>
			<td>Lidocaine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lynx-8 Amplifier</td>
			<td>Neuralynx</td>
			<td>Retired</td>
			<td></td>
		</tr>
		<tr>
			<td>Lynx-8 Power Supply</td>
			<td>Neuralynx</td>
			<td>Retired</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate (MgSO4)</td>
			<td>Dot Scientific</td>
			<td>DSM24300</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>mCherry, Texas Red filter cube</td>
			<td>Chroma</td>
			<td>49008</td>
			<td>for imaging tdTomato fluorescent reporter</td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette holder</td>
			<td>Fisher Scientific</td>
			<td>NC9044962</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsyringe pump</td>
			<td>World Precision Instruments</td>
			<td>UMP3-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Generic brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MultiClamp 700A</td>
			<td>Molecular Devices/Axon Instruments</td>
			<td>700A</td>
			<td>Amplifier</td>
		</tr>
		<tr>
			<td>Nitrogen (for air table)</td>
			<td>Airgas</td>
			<td>NI200</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon mesh</td>
			<td>Fisher Scientific</td>
			<td>501460083</td>
			<td>stretched over horseshoe of flattened platinum wire, slice rest on top of this during recordings</td>
		</tr>
		<tr>
			<td>Nylon, cut from pantyhose</td>
			<td>Generic brand</td>
			<td></td>
			<td>small piece to create slice platform in incubation chamber, single fibers to create platinum harp</td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Fisher Scientific</td>
			<td>NC1697520</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Dot Scientific</td>
			<td>307</td>
			<td>For transferring tissue to rig</td>
		</tr>
		<tr>
			<td>Platinum wire</td>
			<td>VWR International</td>
			<td>BT124000</td>
			<td>2 cm, flattened, to make platinum harp</td>
		</tr>
		<tr>
			<td>Polygon400</td>
			<td>Mightex</td>
			<td>DSI-E-0470-0617-000</td>
			<td>optogenetic light delivery system, comes with PolyScan2 software</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Dot Scientific</td>
			<td>DSP41000</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>Potassium Phosphate (KH2PO4)</td>
			<td>Dot Scientific</td>
			<td>DSP41200</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr>
			<td>Sapphire blade (for vibratome)</td>
			<td>VWR International</td>
			<td>100492-502</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>Santa Cruz Biotechnology, Inc.</td>
			<td>sc-361445</td>
			<td></td>
		</tr>
		<tr>
			<td>Sealed gas bag</td>
			<td>Fisher Scientific</td>
			<td>109236</td>
			<td></td>
		</tr>
		<tr>
			<td>Shifting table for microscope</td>
			<td>Luigs and Neumann</td>
			<td>380FMU</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (HCO3-)</td>
			<td>Dot Scientific</td>
			<td>DSS22060</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Dot Scientific</td>
			<td>DSS23020</td>
			<td>ACSF, intracellular solution</td>
		</tr>
		<tr>
			<td>Ssttm2.1(cre)Zjh/J (SOM-IRES-Cre)</td>
			<td>The Jackson Laboratory</td>
			<td>013044</td>
			<td>SOM-Cre mouse</td>
		</tr>
		<tr>
			<td>Stereotaxic instrument</td>
			<td>Kopf</td>
			<td>Model 902</td>
			<td>Dual Small Animal</td>
		</tr>
		<tr>
			<td>Super glue</td>
			<td>Staples</td>
			<td>886833</td>
			<td>to fix tissue block to specimen stage during slice preparation</td>
		</tr>
		<tr>
			<td>Surgical drill</td>
			<td>RAM Products Inc.</td>
			<td>DIGITALMICROTORQUE</td>
			<td>Microtorque II</td>
		</tr>
		<tr>
			<td>Syringe (1 mL) with LuerLock tip</td>
			<td>Fisher Scientific</td>
			<td>309628</td>
			<td>for application of pressure during patch clamping</td>
		</tr>
		<tr>
			<td>Syringe (1 mL) with slip tip</td>
			<td>WW Grainger, Inc.</td>
			<td>19G384</td>
			<td>for filling patch clamp pipettes</td>
		</tr>
		<tr>
			<td>Syringe Filters</td>
			<td>VWR International</td>
			<td>66064-414</td>
			<td></td>
		</tr>
		<tr>
			<td>Upright microscope</td>
			<td>Olympus</td>
			<td>BX51</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrating microtome</td>
			<td>Leica Biosystems</td>
			<td>VT1000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Wypall towels</td>
			<td>Fisher Scientific</td>
			<td>19-042-427</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

A timeline of steps described in the protocol is shown in Figure 1. Cortical inputs arriving from higher order cortical areas or from non-primary thalamic nuclei have partially overlapping terminal fields in layer 1 of non-primary visual cortex24. To isolate independent thalamocortical or corticocortical afferent pathways, a viral vector containing ChR2 and an eYFP fluorescent reporter into either Po or Cg was injected. Cells within the injection radius take up the viral vector and, after 2-4 weeks, express the non-specific cation channel ChR2 and the reporter in both the soma and projecting axons (Figure 2A). Coronal slices were collected. With the appropriate filter cube engaged, axons expressing the viral construct were imaged (Figure 2B). The use of ChR2 to activate axon terminals allows for activation of afferents without the prerequisite for an attached soma.
The animals used in the experiments described here were SOM-tdTomato or PV-tdTomato hybrid animals, which express the fluorescent reporter protein tdTomato in either somatostatin- (SOM+) or parvalbumin-positive (PV+) interneurons, respectively. SOM+ or PV+ interneurons in layer 2/3 were targeted for patch clamping under visual guidance with the appropriate filter cube engaged (Layer 1C). These interneurons have dendrites in layer 1 and are targets of corticocortical inputs (Figure 3A).
Addition of 125 mL of 3.0% isoflurane gas and 175 mL of 95% O2/5% CO2 to a sealed bag resulted in a pre-equilibrium concentration of gas of 1.3%. Gas dissolved into eACSF according to its partition coefficient; the predicted gas phase equilibrium concentration of isoflurane at room temperature was 0.6% (Figure 2D). This was confirmed via gas monitor.
The tissue slice was transferred to the recording chamber and the 16x1 multi-channel recording probe was placed orthogonally to the cortical laminae (Figure 2E). A 150 &#956;m circle of 470 nm light centered over cortical layer 1 was delivered via the objective light path, while extracellular field potentials were collected using the 16 x 1 multi-channel probe and targeted whole-cell patch clamp recordings were conducted in interneurons. A schematic of the recording set-up is shown in Figure 2F.
Post-synaptic potentials (PSPs) were observed in interneurons in response to a train of four 2 ms pulses of light (10 Hz; Figure 3A). Local field potentials were also recorded (Figure 3B). Current source density (CSD; Figure 3C) and multi-unit activity (MUA; Figure 3D) were extracted from local field potentials. Ten trials at several different light intensities were used to conduct post hoc analyses. The amplitude of current sinks extracted from the CSD increased as a function of light intensity (Figure 4A). A three-parameter nonlinear logistic equation was fit to the data for comparisons across pathways. PSP amplitude also increased with current sink amplitude (Figure 4B).
Synaptic responses to thalamocortical and corticocortical inputs were measured during control, isoflurane (0.28 mM), and recovery conditions. Post-synaptic responses of somatostatin- (Figure 5A) to corticocortical stimuli were suppressed during isoflurane, as were evoked current sinks (Figure 5B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61333/61333fig01.jpg" /> 
Figure 1: A schematic outlining timeline of important steps in protocol. 
Top: Describes timeline of steps necessary for breeding of transgenic animals and expression of viral vector. Bottom: Depicts steps and timeline for preparing materials and conducting experiment on the day of slice preparation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61333/61333fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61333/61333fig02.jpg" /> 
Figure 2: Injection of viral vector and preparation ex vivo coronal brain slices. 
(A) Schematic representation of injection of viral vector into SOM-tdTomato or PV-tdTomato hybrid mice. (B) Coronal slices of the medial parietal association area (mPtA) were harvested, and thalamocortical (top) or corticocortical (bottom) afferent fibers were identified by their eYFP reporter in layer 1. This figure is modified with permission from24. (C) Overlay of eYFP-labeled axon terminals in layer 1 (green) and tdTomato-labelled SOM+ interneurons (red) in superficial layer 2/3. (D) Sealed bags were prepared with a 50:50 solution-to-gas mixture. (E) Placement of a 16 x 1 probe into mPtA (black outline). (F) Schematic of the recording set-up in the cortical slice. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61333/61333fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61333/61333fig03v2.jpg" /> 
Figure 3: Simultaneous intracellular and multi-channel extracellular recordings in cortical slice. 
(A) Whole-cell current clamp patch recording from the soma of a layer 2/3 PV+ interneuron. Four pulses (2 ms each, blue arrows) of blue light (2.2 mW) at 10 Hz were delivered to corticocortical axon terminals in L1. Average (red trace) of ten trials (grey traces) are shown. (B) Raw data from 16 channels of extracellular 16 x 1 probe. Channels placed in cortical tissue are shown in black, and those lying outside of cortex in grey. (C) A current source density diagram, extracted from the local field potential signal, shows synaptic current sinks (blue) in layer 1. (D) Multi-unit activity, generated by applying a high-pass filter to the local field potential signal, isolates spiking activity evoked in lower layers. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61333/61333fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61333/61333fig04.jpg" /> 
Figure 4: Comparison of responses from recordings in two different slices. 
Multiple light intensities were used to evoke synaptic responses in cortical layer 1. For each trial, the peak amplitude of the evoked response was extracted from the layer 1 extracellular current sink and EPSPs in layer 2/3 PV+ interneurons. (A) Extracellular response profiles of thalamocortical and corticocortical afferents are compared as a function of light intensity. (B) The relationship between current sink amplitude and EPSP amplitude is pathway dependent. Within each stimulus pathway, data from (A) and (B) were collected simultaneously. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61333/61333fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61333/61333fig05.jpg" /> 
Figure 5: Bath application of isoflurane dissolved in eACSF during simultaneous recordings. 
(A) Intracellular whole-cell current clamp recording from layer 2/3 SOM+ interneuron upon activation of corticocortical afferents during control, isoflurane, and wash conditions. Vertical blue lines indicate light stimuli (2 ms; 1.65 mW). (B) Current source density trace extracted from electrode in layer 1. Data were collected simultaneously with those collected in (A). Recovery of responses upon wash demonstrates depression of synaptic responses by isoflurane. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61333/61333fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="5">Micropipette for virus injection</td>
		</tr>
		<tr>
			<td>Glass</td>
			<td>ID: 0.05 mm, OD: 0.11 mm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Loops</td>
			<td>1</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="5"></td>
		</tr>
		<tr>
			<td>Heat</td>
			<td>Pull</td>
			<td>Vel</td>
			<td>Time</td>
			<td>Pressure</td>
		</tr>
		<tr>
			<td>Ramp + 10</td>
			<td>20</td>
			<td>40</td>
			<td>200</td>
			<td>300</td>
		</tr>
		<tr>
			<td colspan="5"></td>
		</tr>
		<tr>
			<td colspan="5">Micropipette for whole-cell patch clamp recordings</td>
		</tr>
		<tr>
			<td>Glass</td>
			<td>ID: 1.1 mm, OD: 1.7 mm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Loops</td>
			<td>4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="5"></td>
		</tr>
		<tr>
			<td>Heat</td>
			<td>Pull</td>
			<td>Vel</td>
			<td>Time</td>
			<td>Pressure</td>
		</tr>
		<tr>
			<td>Ramp</td>
			<td>0</td>
			<td>25</td>
			<td>250</td>
			<td>500</td>
		</tr>
	</tbody>
</table>
Table 1: Recommended glass and parameters for pulling micropipettes for viral injections and whole-cell patch clamp recordings. Glass used for viral injections and whole-cell patch clamp recordings is described, as well as the parameters for pulling micropipettes using the micropipette puller. Consult instruction manuals for micropipette puller for further recommendations or fine-tuning of settings.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Slicing ACSF, sACSF (in mM)</td>
			<td>Experiment ACSF, eACSF (in mM)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>111</td>
			<td>111</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>35</td>
			<td>35</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>20</td>
			<td>20</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>1.8</td>
			<td>1.8</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>1.05</td>
			<td>2.1</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>2.8</td>
			<td>1.4</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>1.2</td>
			<td>1.2</td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>10</td>
			<td>10</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Internal Solution</td>
			<td></td>
		</tr>
		<tr>
			<td>K-gluconate</td>
			<td>140</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>10</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>10</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>0.1</td>
			<td></td>
		</tr>
		<tr>
			<td>MgATP</td>
			<td>4</td>
			<td></td>
		</tr>
		<tr>
			<td>NaGTP</td>
			<td>0.3</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>pH = 7.2</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Composition of artificial cerebral spinal fluid and intracellular solution. Reagents and concentrations for sACSF, eACSF, and intracellular pipette solution for patch clamp recordings are listed.
Supplementary Figure 1: Template for preparing block of tissue to collect brain slices. The template is adjusted to the appropriate size, printed, and glued to a microscope slide. A cover slip is glued over the template to prolong its use. The tissue block is placed on a piece of filter paper with the sagittal plane down, aligned to the pink background, and a vertical cut is made in the coronal plane along the black line. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61333/61333supfig01.jpg" target="_blank">Please click here to download this figure.</a>
Supplementary Figure 2: Incubation chamber for harvested brain slices. The chamber is filled with sACSF and bubbled with 95% O2/5% CO2 gas mixture via a bent needle attached to tubing. Incubation platform is made of nylon stretched over a plastic circular fitting. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61333/61333supfig02.jpg" target="_blank">Please click here to download this figure.</a>
Supplementary Figure 3: Platinum structures for slice in recording chamber. Brain slice is transferred to recording chamber via pipette and placed on top of nylon mesh, which is stretched over a horseshoe-shaped piece of flattened platinum wire and super glued in place. Platinum harp is placed over brain slice to anchor it in place during recording. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61333/61333supfig03.jpg" target="_blank">Please click here to download this figure.</a>
Supplementary Table 1: Ostwald (&#955;) and Bunsen (&#945;) coefficients for other volatile anesthetics. Adapt this protocol for study of other volatile gas anesthetics, such as halothane, sevoflurane, or desflurane. Substitute the equations described in the protocol with the appropriate coefficients as listed in this table. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61333/Supplementary_Table_1.xlsx" target="_blank">Please click here to download this table.</a>

Watch this Scientific Journal Video about Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics at JoVE.com

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

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 ...

optogenetic-activation-afferent-pathways-brain-slices-modulation

Nanyang Technological University

Research

JoVE Journal

Neuroscience

2.1K Views.  University of Wisconsin. 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.

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

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

Larval zebrafish are emerging as a model for describing the development and function of simple neural circuits. Due to their external fertilization, rapid development, and translucency, zebrafish are particularly well suited for optogenetic approaches to investigate neural circuit function. In this approach, light-sensitive ion channels are expressed in specific neurons, enabling the experimenter to activate or inhibit them at will and thus assess their contribution to specific behaviors. Applying these methods in larval zebrafish is conceptually simple but requires the optimization of technical details. Here we demonstrate a procedure for expressing a channelrhodopsin variant in larval zebrafish somatosensory neurons, photo-activating single cells, and recording the resulting behaviors. By introducing a few modifications to previously established methods, this approach could be used to elicit behavioral responses from single neurons activated up to at least 4 days post-fertilization (dpf). Specifically, we created a transgene using a somatosensory neuron enhancer, CREST3, to drive the expression of the tagged channelrhodopsin variant, ChEF-tdTomato. Injecting this transgene into 1-cell stage embryos results in mosaic expression in somatosensory neurons, which can be imaged with confocal microscopy. Illuminating identified cells in these animals with light from a 473 nm DPSS laser, guided through a fiber optic cable, elicits behaviors that can be recorded with a high-speed video camera and analyzed quantitatively. This technique could be adapted to study behaviors elicited by activating any zebrafish neuron. Combining this approach with genetic or pharmacological perturbations will be a powerful way to investigate circuit formation and function.

Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera.

Larval  zebrafish are emerging as a model for describing the development and function  of simple neural circuits. Due to their  external fertilization, ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

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.

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.

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

Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug application during electrophysiological recording is the puff technique, which can be used to study the effect of pharmacological reagents on neuronal activities in brain slices. The greatest advantage of puff application is that the drug concentration around the recording site increases rapidly, thus preventing desensitization of membrane receptors. Successful use of puff application involves careful attention to the following elements: the concentration of the drug, the parameters of the puff micropipette, the distance between the tip of a puff micropipette and the neuron recorded, and the duration and pressure driving the puff (pounds per square inch, psi). This article describes a step-by-step procedure for recording whole-cell currents induced by puffing gamma-aminobutyric acid (GABA) onto a neuron of a prefrontal cortical slice. Notably, the same procedure can be applied with minor modifications to other brain areas such as the hippocampus and the striatum, and to different preparations, such as cell cultures.

We describe the puff technique, by which pharmacological reagents can be administered during whole-cell patch-clamp recording, and highlight various aspects of the features that are crucial for its success.

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition follows a combinatorial coding scheme, where one OR can be activated by a set of odorants and one odorant can activate a combination of ORs. Through such combinatorial coding, organisms can detect and discriminate between a myriad of volatile odor molecules. Thus, an odor at a given concentration can be described by an activation pattern of ORs, which is specific to each odor. In that sense, cracking the mechanisms that the brain uses to perceive odor requires the understanding odorant-OR interactions. This is why the olfaction community is committed to &#34;de-orphanize&#34;&#160;these receptors. Conventional in vitro systems used to identify odorant-OR interactions have utilized incubating cell media with odorant, which is distinct from the natural detection of odors via vapor odorants dissolution into nasal mucosa before interacting with ORs. Here, we describe a new method that allows for real-time monitoring of OR activation via vapor-phase odorants. Our method relies on measuring cAMP release by luminescence using the Glosensor assay. It bridges current gaps between in vivo and in vitro approaches and provides a basis for a biomimetic volatile chemical sensor.

Physiologically, odorant receptors are activated by odorant molecules inhaled in the vapor phase. However, most in vitro systems utilize liquid phase odorant stimulation. Here, we present a method that allows real-time in vitro monitoring of odorant receptor activation upon odorant stimulation in vapor phase.

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition ...

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.

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

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In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetic Stimulation of the Auditory Nerve

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase

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