Jan 31, 2024
Optogenetic modulation of dopaminergic neurons
- 1Neuromodulatory Network Group, Leibniz Institute for Neurobiology, Magdeburg.
- TeamPrigge
Protocol Citation: Cristian González-Cabrera, Matthias Prigge 2024. Optogenetic modulation of dopaminergic neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp3kppvzp/v1
Manuscript citation:
Optical deep brain stimulation can reactivate NM-laden neurons and rescue behavioral phenotypes.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 30, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94413
Keywords: Optogenetics, optical deep brain stimulation, Neuromelanin, ASAPCRN
Funders Acknowledgements:
ASAP
Grant ID: 020505
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.
Viral Injection
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:
- Bilateral injections were performed.
- Right hemisphere: viruses 1 and 3.
- Left hemisphere: viruses 2 and 3.
- 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:
- Injection speed 100nl/min
Experimental Timeline
Experimental Timeline
- Animals were left to accumulate neuromelanin for 6 or 10 weeks.
- Animals were stimulated for 30 minutes for 4 weeks. Stimulation during 5 days and two days rest.
- The stimulation was 635nm, 5ms pulse, 10 Hz at at 10mW. 3 seconds ON 10 seconds OFF.
Optical stimulation
Optical stimulation
- fibers are attached bilaterally to the animals
- the animal is placed in open field arena