Method Article

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

DOI:

10.3791/61333

⸱

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

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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

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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

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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 cortic....

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Protocol

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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....

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Results

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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

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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.......

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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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

References

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  1. Baumgart, J. P., et al. Isoflurane inhibits synaptic vesicle exocytosis through reduced Ca2+ influx, not Ca2+-exocytosis coupling. Proceedings of the National Academy of Sciences U.S.A. 112 (38), 11959-11964 (2015).
  2. Herring, B. E., Xie, Z., Marks, J., Fox, A. P.

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Tags

Optogenetic ActivationBrain SlicesVolatile AnestheticsExtracellular RecordingsWhole Cell Patch ClampIsoflurane DeliverySomatostatin InterneuronsParvalbumin InterneuronsCurrent Source DensityMulti Unit Activity

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