Optogenetic modulation of dopaminergic neurons
Matthias Prigge, Cristian González-Cabrera
Abstract
This protocol describes an optogenetic activation of neuromelanine-laden dopaminergic neurons in the SNc-VTA. We induce neuromelanin (NM) in the SNc-VTA through viral expression of a humane tyrosinase virus. Furhtermore we express a red-absorbing opsin in one hemispherend and blue absorbing in the other hemisphere. We detail how we optognetic stimulateanimals in over four weeks.
THis optogenetic deep brain stimulation induces a reduction in neuromelanine levels in dopaminergic neurons, and rescues behavioral phenotypes as evaluate in the grip strength test.
Steps
Viral Injection
Stereotactic injection of viruses into DAT-Cre (JAX# 006660) animals
Viral Constructs:
1.- pAAV-hSyn1-dlox-ChrimsonR_tdTomato(rev)-dlox-WPRE
2.- pAAV-hSyn1-dlox-hCHR2(H134R)_mcherry(rev)-dlox-WPRE
3.- pAAV_Ef1a_DIO_hTyrHA_minBack
Injections:
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Bilateral injections were performed.
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Right hemisphere: viruses 1 and 3.
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Left hemisphere: viruses 2 and 3.
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Viruses were combined 50:50 and injected a total Volume of 600ul per hemisphere
Steroetactic coordinates:
- Subtantia nigra
ML: 1.4mm AP: 3.25mm DV: 4.0 (blunt needle / NF34BL-2)
Injection set-up:
- Hamiliton syringe couple t oa WPI injector (UMP3T-1) (link)
- Injection speed 100nl/min
Experimental Timeline
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Animals were left to accumulate neuromelanin for 6 or 10 weeks.
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Animals were stimulated for 30 minutes for 4 weeks. Stimulation during 5 days and two days rest.
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The stimulation was 635nm, 5ms pulse, 10 Hz at at 10mW. 3 seconds ON 10 seconds OFF.
Optical stimulation
fibers are attached bilaterally to the animals* the animal is placed in open field arena
- stimulation is controlled via PulsePal (OpenEphys link) on a MRL_III-635L laser
