Optogenetic Manipulation of Neural Circuits During Monitoring Sleep/wakefulness States in Mice

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 diseases^1,2,3. 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.......

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 GABA^BNST neurons. Our data showed that optogenetic excitation of GABA^BNST 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 .......

Materials

Name	Company	Catalog Number	Comments
1x1 Fiber-optic Rotary Joints	Doric	FRJ 1x1 FC-FC	for optogenetics
6-pin header	KEL corporation	DSP02-006-431G
6-pin socket	Hirose	21602X3GSE
A/D converter	Nippon koden	N/A	Analog to digital converter
AAV10-EF1a-DIO-ChR2-EYFP			3.70×1013(genomic copies/ml)
AAV10-EF1a-DIO-EYFP			5.82×1013(genomic copies/ml)
Ampicillin	Fuji film	014-23302
Amplifier	Nippon koden	N/A	for EEG/EMG recording
Anesthetic vaporizer	Muromachi	MK-AT-210D
Automatic injecter	KD scientific	780311
Carbide cutter	Minitor	B1055	φ0.7 mm. Reffered as dental drill, used with high speed rotary micromotor
Cyanoacrylate adhesion  (Aron alpha A) and acceleration	Konishi	#30533
Dental curing light	3M	Elipar S10
Epoxy adhesive	Konishi	#04888	insulation around the solder of 6-pin and shielded cable
Fiber optic patch cord (branching)	Doric	BFP(#)_50/125/900-0.22
Gad67-Cre mice	provided by Dr. Kenji Sakimura		Cre recombinase gene is knocked-in in the Gad67 allele
Hamilton syringe	Hamilton	65461-01
High speed rotary micromotor kit	FOREDOM	K.1070	Used with carbide cutter
Interconnecting sleeve	Thorlab	ADAF1	φ2.5 mm Ceramic
Isoflurane	Pfizer	871119
Laser	Rapp OptoElectronic	N/A	473nm wave length
Laser intesity checker	COHERENT	1098293
Laser stimulator	Bio research center	STO2	reffered as pulse generator in text
Optic fiber with ferrule	Thorlab	FP200URT-CANNULA-SP-JP
pAAV2-rh10	provided by PennVector Core
pAAV-EF1a-DIO-EYFP-WPRE-HGHpA	Addgene	plasimid # 20296
pAAV-EF1a-DIO-hChR2(H134R)-EYFP-WPRE-HGHpA	provided by Dr. Karl Deisseroth
Patch cord	Doric	D202-9089-0.4	0.4m length, laser conductor between laser and rotary joint
pHelper	Stratagene
Photocurable dental cement	3M	56846
Serafin clamp	Stoelting	52120-43P
Shielded cable	mogami	W2780	Soldering to 6-pin socket for EEG/EMG recording
Sleep recording chamber	N/A	N/A	Custum-made (21cm× 29cm × 19cm) with water tank holder
Sleep sign software	KISSEI COMTEC	N/A	for EEG/EMG analysis
Slip ring	neuroscience,inc	N/A	for EEG/EMG analysis
Stainless screw	Yamazaki	N/A	φ1.0 x 2.0
Stainless wire	Cooner wire	AS633	0.0130 inch diameter
Stereotaxic frame with digital console	Koph	N/A	Model 940
Syringe needle	Hamilton	7803-05
Vital recorder software	KISSEI COMTEC	N/A	for EEG/EMG recording