Feb 19, 2025
Optogenetic Silencing of Projection Terminals
- Richard H. Roth1,
- Jun B. Ding1
- 1Stanford University
Protocol Citation: Richard H. Roth, Jun B. Ding 2025. Optogenetic Silencing of Projection Terminals. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqdp7yvk5/v1
Manuscript citation:
Thalamic integration of basal ganglia and cerebellar circuits during motor learning
Richard H. Roth, Michael A. Muniak, Charles J. Huang, Fuu-Jiun Hwang, Yue Sun, Cierra Min, Tianyi Mao, Jun B. Ding
bioRxiv 2024.10.31.621388; doi: https://doi.org/10.1101/2024.10.31.621388
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: February 10, 2025
Last Modified: February 19, 2025
Protocol Integer ID: 119918
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-020551
Abstract
This protocol describes the combination of protocols and techniques that are employed to optogenetically silence projections from various areas in the brain.
Safety warnings
Wear appropriate PPE as required by your institution.
Ethics statement
Prior ethics approval (e.g. IACUC) should be obtained before performing these experiments. Approval was obtained by the Stanford University IACUC before any procedures were performed.
Before start
Read all protocols and linked protocols beforehand to ensure understanding when combining protocols together.
Intracranial Virus Injection & Optic Fiber Implantation
Intracranial Virus Injection & Optic Fiber Implantation
Follow steps 1-11 in protocol for viral injection of desired virus before implanting optic fiber: dx.doi.org/10.17504/protocols.io.8epv5r514g1b/v1
Follow protocol starting on step 7 for optic fiber implant: dx.doi.org/10.17504/protocols.io.n2bvj9yrwlk5/v1
NOTE-viral injection and optic fiber implantation occur in the same surgery.
A head plate is be placed if a mouse is planned to be used in a head-fixed behavior (lever-pushing task).
Wait 2 weeks for recovery before any behavior and recording experiments.
These experiments are to test behaviors when SNr or DCN projections are silenced in different behavior paradigms. A mouse will only perform one behavior (e.g. either lever-pushing task or rotarod task).
Optogenetic Silencing & Lever-Pushing Task6 steps
Follow procedure in protocol dx.doi.org/10.17504/protocols.io.eq2ly63pwgx9/v1 for task habituation and training process.
Mice have two optogenetic silencing sessions.
At the start of each new session/day: a baseline is collected for 2-3 minutes without inhibition.
Please refer to protocol dx.doi.org/10.17504/protocols.io.eq2ly63pwgx9/v1 for details on trial structure.
To initiate the silencing using the inhibitory opsin eOPN3, a laser is turned on for 3 minutes with 500 ms pulses at 0.1 Hz and a power of 10mW from the fiber tip.
The silencing period is quantified starting at 1 minute after optogenetic laser onset as 'inhibition period'.
The 2 minute window starting 1 minute after optogenetic laser was turned off is the 'post-inhibition washout period'.
Mice are trained for another 9-10 minutes following inhibition.