Aug 14, 2023

Public workspaceCNO Preparation and Consumption Monitoring

DOI
dx.doi.org/10.17504/protocols.io.n2bvj33oblk5/v1
  • Victoria Vance1,
  • Katerina Rademacher1,
  • Ken Nakamura1
  • 1Gladstone Institute of Neurological Disease
Kelsey Barcomb
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DOI: dx.doi.org/10.17504/protocols.io.n2bvj33oblk5/v1
Protocol Citation: Victoria Vance, Katerina Rademacher, Ken Nakamura 2023. CNO Preparation and Consumption Monitoring. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj33oblk5/v1
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: In development
We are still developing and optimizing this protocol
Created: August 12, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 86415
Keywords: ASAPCRN, DREADD, Mouse
Funders Acknowledgements:
ASAP-CRN
Grant ID: 020529
Abstract
Chemogenetic models utilize the consumption of clozapine N-oxide (CNO) to activate Designer Receptors Activated Only by Designer Drugs (DREADDs) in order to modulate neuronal activity. This protocol describes the preparation of a CNO solution for consumption by mice, as well as the daily monitoring of water intake.
Protocol materials
ReagentClozapine N-oxideTocrisCatalog #4936
ReagentSucroseMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9378
ReagentMouse Water BottleBioanalytical SystemsCatalog #CS-5010
Materials and Solutions
Materials and Solutions
Materials:
  • Clozapine N-oxide ReagentClozapine N-oxideBioanalytical SystemsCatalog #4936
  • Sucrose ReagentSucroseBioanalytical SystemsCatalog #S9378
  • Autoclaved water

Making CNO and vehicle water stock stolutions

Note
  • Store CNO and vehicle water in 4C fridge.
  • Make fresh weekly.

CNO Water (2% sucrose, 200 mg/L CNO):
20g sucrose and 300mg CNO in 1L autoclaved water
  • Carefully weigh CNO to the nearest 0.05mg, then protect from light
  • Slowly add CNO in small amounts to autoclaved water while stirring, then add sucrose
  • Wrap bottle in foil to protect from light
Vehicle water (2% sucrose):
20g sucrose in 1L autoclaved water
Monitoring water intake
Monitoring water intake
On day 0, discard the autoclaved water, and give 10mL of medicated water (e.g. CNO or vehicle water) in a water bottle per mouse. Record the time when mice were given medicated water.

ReagentMouse Water BottleBioanalytical SystemsCatalog #CS-5010
Guardian Hamster Water Bottle (Amazon cat: B0713XMP98)


Monitor water intake at the same time every day of the experiment.

For each cage, perform the following substeps:
Remove from rack and check that there is sufficient food available on cage floor. Add more if needed.
Use a 10 or 25 mL serological pipette to measure how much water is still left in the bottle. Record this value.
For single-housed cages (e.g. mice with activity wheels), if water level is below 8.0 mL, use a clean pipette to add 5 mL more from the stock. If water level is below 4.0 mL, add 10 mL more. Record how much water is added.
For group-housed cages, top off with enough water to ensure at least 5mL per mouse plus overage (e.g. 15mL for two mice, 35mL for 5 mice).
Return cage to rack and repeat for remaining cages.
Enter recorded water levels into an Excel spreadsheet to track water intake per mouse over time.