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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we describe methods of optogenetic manipulation of particular types of neurons during monitoring of sleep/wakefulness states in mice, presenting our recent work on the bed nucleus of the stria terminalis as an example.

Abstract

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 (ChR2), is expressed by a virus vector in a particular type of neuronal cells in various Cre-driver mice. Activation of these opsins is triggered by application of light pulses which are delivered by laser or LED through optic cables, and the effect of activation is observed with very high time resolution. Experimenters are able to acutely stimulate neurons while monitoring behavior or another physiological outcome in mice. Optogenetics can enable useful strategies to evaluate function of neuronal circuits in the regulation of sleep/wakefulness states in mice. Here we describe a technique for examining the effect of optogenetic manipulation of neurons with a specific chemical identity during electroencephalogram (EEG) and electromyogram (EMG) monitoring to evaluate the sleep stage of mice. As an example, we describe manipulation of GABAergic neurons in the bed nucleus of the stria terminalis (BNST). Acute optogenetic excitation of these neurons triggers a rapid transition to wakefulness when applied during NREM sleep. Optogenetic manipulation along with EEG/EMG recording can be applied to decipher the neuronal circuits that regulate sleep/wakefulness states.

Introduction

Sleep is essential for optimal cognitive function.Recent findings also suggest that disturbances in sleep are associated with a wide range of diseases1,2,3. Although the functions of sleep are as yet largely unresolved, substantial progress has been made recently in understanding the neural circuits and mechanisms that control sleep/wakefulness states4. In mammals, there are three states of vigilance: wakefulness, non-rapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep. Wakefulness is characterized by fast EEG oscillations (5-12 Hz) of ....

Protocol

All experiments here were approved by the Animal Experiment and Use Committee of the University of Tsukuba, complying with NIH guidelines.

1. Animal Surgery, Virus Injection, Electrode for EEG/EMG, and Optical Fiber Implantation

CAUTION: Appropriate protection and handling techniques should be selected based on the biosafety level of the virus to be used. AAV should be used in an isolated P1A graded room for injection, and the tube carrying AA.......

Representative Results

The present study showed the effect of optogenetic excitation of GABABNST neurons on sleep state transition. ChR2-EYFP was focally expressed in GABA neurons in the BNST. An in situ hybridization histochemical study showed that ChR2-EYFP was colocalized in neurons expressing GAD 67 mRNA signals, indicating that these are GABAergic neurons. Immunohistochemical slice samples confirmed the position of the optic fiber, whose tip was just above the BNST25.

.......

Discussion

We here presented a method to evaluate the effect of optogenetic stimulation of neurons with particular chemical identities on state transitions of sleep/wakefulness and gave an example of manipulation of GABABNST neurons. Our data showed that optogenetic excitation of GABABNST neurons results in immediate transition from NREM sleep to wakefulness.

Various experimental designs are available because of the development of numerous types of optogenetic tools. It is possible .......

Acknowledgements

This study was supported by the Merck Investigator Studies Program (#54843), a KAKENHI Grant-in-Aid for Scientific Research on Innovative Areas, "WillDynamics" (16H06401) (T.S.), and a KAKENHI Grant-in-Aid for Exploratory Research on Innovative Areas (T.S.) (18H02595).

....

Materials

NameCompanyCatalog NumberComments
1x1 Fiber-optic Rotary JointsDoricFRJ 1x1 FC-FCfor optogenetics
6-pin headerKEL corporationDSP02-006-431G
6-pin socketHirose21602X3GSE
A/D converterNippon kodenN/AAnalog to digital converter
AAV10-EF1a-DIO-ChR2-EYFP3.70×1013(genomic copies/ml)
AAV10-EF1a-DIO-EYFP5.82×1013(genomic copies/ml)
AmpicillinFuji film014-23302
AmplifierNippon kodenN/Afor EEG/EMG recording
Anesthetic vaporizerMuromachiMK-AT-210D
Automatic injecterKD scientific780311
Carbide cutterMinitorB1055φ0.7 mm. Reffered as dental drill, used with high speed rotary micromotor 
Cyanoacrylate adhesion  (Aron alpha A) and accelerationKonishi#30533
Dental curing light3MElipar S10
Epoxy adhesiveKonishi#04888insulation around the solder of 6-pin and shielded cable
Fiber optic patch cord (branching)DoricBFP(#)_50/125/900-0.22
Gad67-Cre miceprovided by Dr. Kenji SakimuraCre recombinase gene is knocked-in in the Gad67 allele
Hamilton syringeHamilton65461-01
High speed rotary micromotor kitFOREDOMK.1070Used with carbide cutter
Interconnecting sleeveThorlabADAF1φ2.5 mm Ceramic 
IsofluranePfizer871119
Laser  Rapp OptoElectronicN/A473nm wave length
Laser intesity checkerCOHERENT1098293
Laser stimulatorBio research centerSTO2reffered as pulse generator in text
Optic fiber with ferrule ThorlabFP200URT-CANNULA-SP-JP
pAAV2-rh10provided by PennVector Core
pAAV-EF1a-DIO-EYFP-WPRE-HGHpAAddgeneplasimid # 20296
pAAV-EF1a-DIO-hChR2(H134R)-EYFP-WPRE-HGHpAprovided by Dr. Karl Deisseroth
Patch cordDoricD202-9089-0.40.4m length, laser conductor between laser and rotary joint
pHelperStratagene
Photocurable dental cement3M56846
Serafin clampStoelting52120-43P
Shielded cablemogamiW2780Soldering to 6-pin socket for EEG/EMG recording
Sleep recording chamberN/AN/ACustum-made (21cm× 29cm × 19cm) with water tank holder
Sleep sign softwareKISSEI COMTECN/Afor EEG/EMG analysis
Slip ringneuroscience,incN/Afor EEG/EMG analysis
Stainless screwYamazakiN/Aφ1.0 x 2.0
Stainless wireCooner wireAS633 0.0130 inch diameter
Stereotaxic frame with digital consoleKophN/AModel 940
Syringe needleHamilton7803-05
Vital recorder softwareKISSEI COMTECN/Afor EEG/EMG recording

References

  1. Spoormaker, V. I., Montgomery, P. Disturbed sleep in post-traumatic stress disorder: Secondary symptom or core feature?. Sleep Medicine Reviews. 12 (3), 169-184 (2008).
  2. Dworak, M., Wiater, A., Alfer, D., Stephan, E., Hollmann, W., Struder, H. K.

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Optogenetic ManipulationNeural CircuitsSleep wakefulnessMiceNeuro CircuitSleep Wakefulness RegulationSurface EEGAnesthetized MouseStereotactic ApparatusAdeno associated Virus VectorMicroinjectionBed Nucleus Of The Stria Terminalis BNSTElectroencephalogram EEGElectromyogram EMG

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