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Method Article

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

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DOI:

10.3791/61333

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July 23rd, 2020

 ,  ,  , 
1Department of Anesthesiology, University of Wisconsin, 2Department of Anesthesiology, Rambam Health Care Campus, the Ruth and Bruce Rappaport Faculty of Medicine, Technion - Israel Institute of Technology

In This Article

Summary

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.

Abstract

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.

Introduction

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’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.

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Protocol

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

  1. 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.
  2. 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.

2. Performing unilateral stereotaxic injection of viral construct

  1. 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.
  2. Break the tip of the sharp end of the pipette such that the tip diameter is approximately 30 µm with minimal taper over several millimeters.
  3. Using previously documented procedures, decide on the appropriate titer and volume of virus to be injected. In the experiments described here, 1.0 μL (titer: 3.1-5.7 TU/mL) injected unilaterally produced good results.
  4. 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.
  5. Frontfill at least 1.0 μL of viral construct. The recombinant adeno-associated viral vector used in these experiments was AAV2-hSyn-hChR2(H134R)-EYFP.
  6. Arrange sterile drape in a surgical area. Sterilize tools for stereotaxic procedure and place it on the drape.
  7. 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.
  8. Shave the top of the animal’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.
  9. Fit the animal into stereotaxic frame.
  10. 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.
  11. 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.
  12. Turn on and balance the air table.
  13. Reposition electrode manipulator of the stereotaxic frame at 0° for injections into Cg, or 45° in the coronal plane for injections into Po.
  14. Attach the syringe pump to the electrode manipulator. Attach the microsyringe pump controller to the syringe pump.
  15. 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.
  16. Inject 1.0 µL 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.
  17. After injection, wait for 10 min before slowly retracting the injection pipette.
  18. Suture to close the scalp incision and administer 2-5 mg/kg meloxicam subcutaneously.
  19. Discontinue isoflurane and monitor animal during emergence from anesthesia. Allow to recover according to procedures described by Institution’s Animal Care and Use Committee, including further administration of analgesics.

3. Preparation of acute brain slices

  1. Allow at least 3 weeks for the expression of viral construct before harvesting tissue.
  2. Prepare 1 L of artificial cerebral spinal fluid for slicing procedure (slicing artificial cerebral spinal fluid, sACSF). See Table 2 for ingredients.
  3. Throughout the slicing procedure, supply sACSF with dissolved 95% O2/5% CO2 mixture, delivered via gas dispersion tube.
  4. Prepare ice cold bath for vibrating blade microtome. Mount ice-cold specimen stage onto microtome and fix sapphire blade in place for tissue sectioning.
  5. Anesthetize mouse with 3% isoflurane and oxygen until loss of righting reflex.
  6. Decapitate the mouse using guillotine and immediately submerge head in 4°C sACSF. To preserve the health of the tissue, complete the following steps as swiftly as possible.
  7. 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.
  8. 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.
  9. Prepare tissue block for sectioning.
    1. 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).
    2. 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.
    3. Place the tissue block in 4°C sACSF briefly while step 3.8.4 is conducted.
    4. Apply a small amount of super glue to the ice-cold specimen stage.
    5. 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.
  10. Collect 500 µm thick coronal brain slices. Place slices of interest on nylon mesh (Supplementary Figure 2) in 34 °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).

4. Preparation of experimental artificial cerebral spinal fluid (eACSF) bags containing dissolved volatile anesthetic isoflurane

  1. Prepare 300 mL of a stock mixture of 3.0% isoflurane.
    1. 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.
    2. Determine the amount of saturated isoflurane gas, Vsat, to add to the stock bag using the following equation:
      Equation for isoflurane concentration in solution, V_sat calculation formula, chemistry.
      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).
    3. 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.
  2. 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.
  3. Prepare two separate bags of Control and Isoflurane solutions.
    1. 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.
    2. To another empty polytetrafluoroethylene gas bag, add 300 mL of eACSF. Label this bag as Isoflurane.
    3. 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.
    4. Use the following equation to calculate the equivalent gas phase concentration at room temperature, P%(Troom)31:
      Humidity correction formula equation related to temperature differences.
      where P%(Tbody) is the physiologically relevant gas phase concentration chosen in Step 4.3.3, Troom is 25 °C, and Tbody is 37 °C.
    5. Use the following equation to determine volume of gas from stock gas bag, Vstock, to add to the Isoflurane solution.
      Static equilibrium formula, Vstock calculation, displaying variables like Vsolution and P% used in science.
      where Vsolution is the volume of eACSF in ISOFLURANE bag (300 mL), P%(Troom) is entered from equation (2), λ is the saline/gas Ostwald partition coefficient of isoflurane (λ = 1.232), and P%stock is the gas phase concentration of the stock gas bag (P%stock = 3.0%).
    6. To the Isoflurane solution bag, add the volume of gas from the stock gas bag, Vstock, calculated in Step 4.3.5.
    7. 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.
  4. Shake both Control and Isoflurane bags on shaker for at least 1 h to allow isoflurane phase equilibration.
  5. 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.
  6. 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:
    Chemical equilibrium equation: C<sub>aqueous</sub>=0.44614*α*P°%(T<sub>room</sub>) in scientific research.
    where α is the saline/gas Bunsen partition coefficient for isoflurane at 25°C32.

5. Preparation of hardware and software for multi-channel recordings

  1. 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.
  2. Fasten the appropriate 16-channel headstage adaptor to a microscope micromanipulator. Orient the adaptor such that the female connector ports are facing downward.
  3. Adjust the angle of operation of this micromanipulator such that it is oriented downward toward the recording chamber, at an angle approximately 70° relative to horizontal.
  4. Connect the headstage input to a 16 x 1 probe for in vitro electrophysiology via the headstage adaptor anchored to the micromanipulator.
  5. Connect the headstage output connector to the data acquisition system.
  6. 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.

6. Configuration of light stimulation protocols

  1. Set up light delivery system and install the accompanying software.
  2. 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.
  3. Mount high-power objective lens. Using digital camera, calibrate high-power objective for use with light delivery system.
  4. Create new profile sequence of light stimulation profiles.
    1. Create a pattern of choice. In the experiments described here, a circle of diameter 150 µm is used to allow layer-specific activation of axon terminals.
    2. To construct a profile sequence, copy and paste this profile for each of any number of trials.
    3. Create a waveform list that contains waveforms of any light intensity, pulse duration, or pulse number.
    4. 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.
    5. Save the profile sequence.
  5. In the data acquisition software, create a new protocol.
    1. Set the number of trials to equal the number of profiles in the profile sequence just created.
    2. 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.

7. Placing multi-channel probe in ex vivo brain tissue slice

  1. Perfuse bubbled eACSF (not in sealed bags) at 3-6 mL/min.
  2. Transfer the brain slice containing area of interest onto mesh grid in microscope perfusion chamber. Anchor with platinum harp (see Supplementary Figure 3).
  3. 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.
  4. Under broadfield illumination and under fine control of the micromanipulator, lower the multi-channel probe toward the surface of the slice.
  5. 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.
  6. Position the probe just above the plane of the slice, ~200 µm 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
  7. 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.

8. Patch clamping targeted neurons and obtaining whole-cell configuration

  1. Switch eACSF source to bagged Control solution.
  2. Identify fluorescently labeled cell for targeted patch clamp recording.
    1. Restrict the aperture iris diaphragm to the smallest diameter. Engage a low-power objective lens and bring the tissue into focus.
    2. Center the light over an area of tissue adjacent to (but not overlapping) the multi-channel probe.
    3. Engage the high-power (40x or 60x) water immersion objective, using caution to avoid contact between the multi-channel probe and objective lens.
    4. Rotate the filter cube turret to engage the appropriate filter set to allow imaging of cells expressing Cre-dependent fluorescent marker.
    5. 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.
  3. 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.
  4. Lower patch pipette into the solution. Bring the pipette tip into focus under visual guidance.
  5. Obtain whole-cell recording from the targeted cell using the steps previously demonstrated33.
  6. 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.

9. Layer-specific optogenetic activation of axon terminals

  1. Manipulate field of view in the x-y plane to align light stimulation profile with desired location on slice.
  2. Load light stimulus protocol and prepare the light delivery system to receive a digital TTL pulse.
  3. Optogenetically activate axon terminals while simultaneously recording extracellular field potentials and intracellular membrane fluctuations.
  4. Switch eACSF source to Isoflurane solution and wash drug in for 15 min. If necessary, collect spontaneous recordings during the wash-in.
  5. Repeat step 9.2-9.3.
  6. Switch eACSF source to Control solution and wash drug out for 20 min. If necessary, collect spontaneous recordings during wash-out.
  7. Repeat step 9.2-9.3.

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Results

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...

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Discussion

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...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
2.5x broadfield objective lensOlympusMPLFLN2.5X
40x water immersion objective lensOlympusLUMPLFLN40XW
95% O2/5% CO2 mixtureAirgasZ02OX95R2003045
A16 probeNeuroNexusA16x1-2mm-100-177-A1616-channel probe
AAV2-hSyn-hChR2(H134R)-EYFPKarl Deisseroth Lab, UNC Vector Core
Anesthetic gas monitor (POET II)Criticare602-3A
ATP, Magnesium SaltSigma AldrichA9187intracellular solution
B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/JThe Jackson Laboratory007914Cre-dependent tdTomato mouse
B6;129P2-Pvalbtm1(cre)Arbr/JThe Jackson Laboratory008069PV-Cre mouse
Belly Dancer ShakerThomas Scientific1210H86-TSfor equilibration of sealed gas bags
Betadine solutionGeneric brand
BleachGeneric brandfor silver chloriding patch clamp electrode
Bupivicaine
Calcium Chloride (CaCl2)Dot ScientificDSC20010ACSF
Capillary glass (patch clamp recordings)King Precision Glass, Inc.KG-33Borosilicate, ID: 1.1mm, OD: 1.7mm, Length: 90.0mm
Capillary glass (viral injections)Drummond Scientific Company3-000-203-G/X3.5"
Control of junior micromanipulatorLuigs and NeumannSM8for control of junior micromanipulator
Control of manipulators and shifting tableLuigs and NeumannSM7for control of multichannel electrode and shifting table
Digidata 1440A + Clampex 10Molecular Devices1440ADigitizer and software
E-3603 tubingFisher Scientific14171208for delivery of 95% O2/5% CO2 gas mixture to incubation chamber + application of pressure during patch clamping
EGTADot ScientificDSE57060intracellular solution
ERP-27 EEG Reference/Patch PanelNeuralynxRetired
Filling needleWorld Precision Instruments50821912for filling patch clamp pipettes
Filter cube for imaging EYFPOlympusU-MRFPHQ
Filter paperFisher Scientific09801Elay over slice template during preparation of tissue block
Flaming/Brown micropipette pullerSutter InstrumentP-10002.5x2.5 Box filament
Gas dispersion tubeSigma AldrichCLS3953312C
Glass syringe (100 mL)Sigma AldrichZ314390for filling gas-sealed bags
Gluconic Acid, Potassium Salt (K-gluconate)Dot ScientificDSG37020intracellular solution
GlucoseDot ScientificDSG32040ACSF
GTP, Sodium SaltSigma AldrichG8877intracellular solution
Headstage-probe adaptorNeuroNexusA16-OM16adaptor to connect 16-channel probe to headstage input
Hemostatic ForcepsVWR International76192-096
HEPESDot ScientificDSH75030ACSF,intracellular solution
HS-16 HeadstageNeuralynxRetired
IsofluranePatterson Veterinary07-893-1389
Isopropyl alcohol (70%)VWR International101223-746
Junior micromanipulatorLuigs and Neumann210-100 000 0090-Rfor manipulation of patch clamp electrode
LED Light Source Control ModuleMightexBLS-PL02_USoptogenetic light source control
Lidocaine
Lynx-8 AmplifierNeuralynxRetired
Lynx-8 Power SupplyNeuralynxRetired
Magnesium Sulfate (MgSO4)Dot ScientificDSM24300ACSF
mCherry, Texas Red filter cubeChroma49008for imaging tdTomato fluorescent reporter
Meloxicam
Micropipette holderFisher ScientificNC9044962
Microsyringe pumpWorld Precision InstrumentsUMP3-4
Mineral oilGeneric brand
MultiClamp 700AMolecular Devices/Axon Instruments700AAmplifier
Nitrogen (for air table)AirgasNI200
Nylon meshFisher Scientific501460083stretched over horseshoe of flattened platinum wire, slice rest on top of this during recordings
Nylon, cut from pantyhoseGeneric brandsmall piece to create slice platform in incubation chamber, single fibers to create platinum harp
Ophthalmic ointmentFisher ScientificNC1697520
PipetteDot Scientific307For transferring tissue to rig
Platinum wireVWR InternationalBT1240002 cm, flattened, to make platinum harp
Polygon400MightexDSI-E-0470-0617-000optogenetic light delivery system, comes with PolyScan2 software
Potassium Chloride (KCl)Dot ScientificDSP41000ACSF
Potassium Phosphate (KH2PO4)Dot ScientificDSP41200ACSF
Razor bladeFisher Scientific12-640
Sapphire blade (for vibratome)VWR International100492-502
Scalpel bladeSanta Cruz Biotechnology, Inc.sc-361445
Sealed gas bagFisher Scientific109236
Shifting table for microscopeLuigs and Neumann380FMU
Sodium Bicarbonate (HCO3-)Dot ScientificDSS22060ACSF
Sodium Chloride (NaCl)Dot ScientificDSS23020ACSF, intracellular solution
Ssttm2.1(cre)Zjh/J (SOM-IRES-Cre)The Jackson Laboratory013044SOM-Cre mouse
Stereotaxic instrumentKopfModel 902Dual Small Animal
Super glueStaples886833to fix tissue block to specimen stage during slice preparation
Surgical drillRAM Products Inc.DIGITALMICROTORQUEMicrotorque II
Syringe (1 mL) with LuerLock tipFisher Scientific309628for application of pressure during patch clamping
Syringe (1 mL) with slip tipWW Grainger, Inc.19G384for filling patch clamp pipettes
Syringe FiltersVWR International66064-414
Upright microscopeOlympusBX51
Vibrating microtomeLeica BiosystemsVT1000S
Wypall towelsFisher Scientific19-042-427

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Extracellular RecordingsWhole-Cell Patch ClampIsoflurane DeliverySomatostatin InterneuronsParvalbumin InterneuronsCurrent Source DensityMulti-Unit Activity
Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics
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