Mar 29, 2024

Public workspaceValidating Responsiveness of AAV-DIO-hM3Dq-DREADD

eLife
DOI
https://dx.doi.org/10.17504/protocols.io.6qpvr3yrpvmk/v1
  • Katerina Rademacher1,
  • Ken Nakamura1
  • 1Gladstone Institute of Neurological Disease
Haru Yamamoto
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DOI: https://dx.doi.org/10.17504/protocols.io.6qpvr3yrpvmk/v1
External link: https://doi.org/10.7554/eLife.98775
Protocol Citation: Katerina Rademacher, Ken Nakamura 2024. Validating Responsiveness of AAV-DIO-hM3Dq-DREADD. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr3yrpvmk/v1
Manuscript citation:
Rademacher K, Doric Z, Haddad D, Mamaligas A, Liao S, Creed R, Kano K, Chatterton Z, Fu Y, Garcia JH, Vance VM, Sei YJ, Kreitzer A, Halliday G, Nelson AB, Margolis E, Nakamura K Chronic hyperactivation of midbrain dopamine neurons causes preferential dopamine neuron degeneration. eLife 13(). doi: 10.7554/eLife.98775
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 07, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 93034
Keywords: ASAPCRN, dopamine neurons with cno, activation of dreadd receptor, dreadd receptor, dopamine neuron, dreadd in datirescre mice, datirescre mice, responsiveness of aav, neuron, hm3dq, robust increase in locomotion, locomotion, dreadd, mice
Funders Acknowledgements:
ASAP
Grant ID: 020529
Abstract
To confirm responsiveness of AAV-DIO-hM3Dq-DREADD in DATIRESCre mice. Activation of DREADD receptors in dopamine neurons with CNO should induce a robust increase in locomotion.
Materials
  • DATIRESCre mice bilaterally injected in the midbrain with AAV-DIO-hM3Dq-DREADD
  • Med Associates wireless activity wheels (ENV-047)
  • Med Associates Hub (DIG-807)
  • Med Associates software (SOF-860)
  • Clozapine-n-oxide (CNO; Tocris 4936-MTO)
  • Sterile saline
Troubleshooting
Preparation
Mice should be at least two-weeks post-surgery.
Habituate mice to wireless running wheels in individually-housed cages.
See Victoria Vance, Katerina Rademacher, Ken Nakamura 2023. Behavior Tracking with Running Wheels. protocols.io https://protocols.io/view/behavior-tracking-with-running-wheels-cymrxu56
Prepare CNO for IP injection.
0.5mg/kg CNO dissolved in sterile saline, 500µL volume per mouse.
Prepared fresh the day of injection.
Procedure
Note: All injections should be performed at the same time of day, at least 2–3 hours before the dark cycle.
Day 1: IP inject mice with 500µL of sterile saline.
Day 2: IP inject mice with 500µL CNO.
Day 3 or 4: IP inject mice again with 500µL CNO.
Responders increase their running wheel activity in the 10-60min following IP injection of CNO. Exclude mice that do not respond to IP CNO from further analysis.