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...

<ol>
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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 pres...

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 vi...

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 ...

This protocol can be used to investigate whether volatile anesthetics disrupts some synopsis more than others, to determine if their effects are cell type or as pathway specific, and to determine what effects occur at the population level. The main advantage of this technique is the specificity with which we can assess the effects of volatile anesthetics. We can target specific pathways and cell types in simultaneously assay population activity.Begin by preparing the tissue block for sectioning. Gently lift the brain from the skull cavity and place it on the filter paper. Make a cut along the sagittal plane of the brain parallel to the midline and place the tissue block on this sagittal plane.Guide the filter paper over the blocking template, and align the brain with the underlying template outline. Make a parallel cut in the coronal plane as indicated by the lines on the template, adding a small drop of slicing aCSF to keep the filter paper wet if necessary. Place the tissue block in four degrees Celsius slicing aCSF and apply a small amount of glue to the ice-cold specimen stage.Lift the tissue block from the slicing aCSF and wick away excess solution with a corner of an absorbent towel. Glue the posterior coronal plane of the tissue block to the specimen stage with the dorsal surface of the brain facing the sapphire blade. Collect 500 micrometer thick coronal brain slices and place them on a nylon mesh in 34 degrees Celsius slicing aCSF.Allow the container to reach room temperature. To prepare experimental aCSF bags with dissolved isoflurane, add 600 milliliters of experimental aCSF and 600 milliliters of 95%oxygen and 5%carbon dioxide gas mixture to an empty polytetrafluoroethylene gas bag. Label this bag control.Add 300 milliliters of experimental aCSF to another polytetrafluoroethylene gas bag, and label this bag isoflurane. Proceed with preparing the isoflurane bag according to the manuscript directions. Configure the light simulation protocols by randomly assigning wave forms to each profile.Each profile with its assigned wave form corresponds to one trigger pulse from a digital TTL input or one trial. When finished, save the profile sequence. To place the multi-channel probe in the ex vivo brain tissue slide, perfuse bubbled experimental aCSF at three to six milliliters per minute, and transfer the brain slice containing the area of interest onto the mesh grid in the microscope perfusion chamber.Anchor the brain slice with a platinum harp. Rotate the mesh grid so that the line of electrode contacts on the distal end of the multi-channel probe is approximately perpendicular to the peel surface. Under broad field illumination and fine control of the micromanipulator, lower the multichannel probe toward the surface of the slice.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 peel surface. Position the probe just above the plane of the slice 200 micrometers short of the final target position along the x-axis, leaving at least one channel outside of the area of tissue being recorded.Slowly insert the probe into the slice 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. Switch experimental aCSF source to bagged control solution and identify the fluorescently-labeled cell for targeted patch clamp recording.Restrict the aperture to the smallest diameter and engage the high power water immersion objective, taking care to avoid contact between the multichannel probe and objective lens. Bring the tissue into focus. Center the light over an area of tissue adjacent to but not overlapping the multi-channel probe.Engage the appropriate filter set to allow imaging of cells expressing the Cre-dependent fluorescent marker. Raise the objective lens to create ample space for lowering a patch pipette. Load a patch pipette with internal solution and mount it on the electrode holder.Use a one milliliter syringe to apply positive pressure corresponding to approximately 0.1 milliliter air. Lower the patch pipette into the solution bringing the tip into focus under visual guidance, and obtain whole cell recording from the targeted cell. The animals used in this protocol expressed fluorescent reporter protein tdTomato in either somatostatin or parvalbumin-positive interneurons.These interneurons were targeted for patch clamping under visual guidance with the appropriate filter cube engaged. Postsynaptic potentials were observed in interneurons in response to a train of four two millisecond pulses of light. Local field potentials were also recorded.Current source density and multi-unit activity were extracted from local field potentials. The amplitude of current sinks extracted from the current source density increased as a function of light intensity. A three parameter non-linear logistic equation was fit to the data for comparisons across pathways.Postsynaptic potential amplitude also increased with current sink amplitude. Synaptic responses to the thalamocortical and cortico-cortical inputs were measured during control, isoflurane, and recovery conditions. Post synaptic responses of somatostatin and cortico-cortical stimuli and the evoked current sinks were suppressed in isoflurane conditions.When preparing the brains slices, maintaining the health of the tissue is paramount. It's important to keep the tissue cool throughout the slicing procedure, working quickly without damaging the tissue. Multichannel extracellular recordings may be post-processed in a variety of ways.For example, high pass filtering of the wideband signal isolates population spiking activity while current source density calculations highlight synaptic activity. Together, these techniques have allowed us to explore anesthetic effects on specific isolated components of cortical circuits, providing insight into the mechanisms underlying loss of consciousness in vivo.

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JoVE Journal

Neuroscience

2.1K visualizações.  University of Wisconsin. This protocol can be used to investigate whether volatile anesthetics disrupts some synopsis more than others, to determine if their effects are cell type or as pathway specific, and to determine what effects occur at the population level. The main advantage of this technique is the specificity with which we can assess the effects of volatile anesthetics. We can target specific pathways and cell types in simultaneously assay population activity.Begin by preparing the tissue block for s...