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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.
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.
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 ....
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.......
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.
.......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 .......
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).
....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 | Â 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 |
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