Jan 30, 2024

Public workspaceChemogenetic modulation of catecholaminergic neurons

DOI
dx.doi.org/10.17504/protocols.io.rm7vzxdz5gx1/v1
  • 1Neuromodulatory Network Group, Leibniz Institute for Neurobiology, Mageburg
Prigge Lab
Open access
DOI: dx.doi.org/10.17504/protocols.io.rm7vzxdz5gx1/v1
Protocol Citation: Cristian González-Cabrera, Csilla Novák, Maria P. Contreras, Ernesto Durán, Matthias Prigge 2024. Chemogenetic modulation of catecholaminergic neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzxdz5gx1/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: Working
We use this protocol and it's working
Created: January 30, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94402
Keywords: Chemogenetics, hM4Di, hM3D, DREADDs, long-term modulation, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: 020505
Abstract
This protocol outlines a method for modulating noradrenergic and dopaminergic neuronal activity in the LC and SNc-VTA, respectively, using Cre-dependent hM3D and hM4D expression. It details stereotactic injection techniques for these chemogenetic tools and the preparation of the J60 agonist for intraperitoneal delivery. Developed for longitudinal studies spanning up to four weeks, the protocol aims to assess neuromelanin's activity-dependent accumulation through daily agonist administration.
Materials
JHU37160 aka J60
Stereotactic injection
Stereotactic injection
Stereotactic injection of viruses into DbH-Cre (JAX# 033953) or DAT-Cre (JAX# 006660) animals

Viral Construct:
pAAV-hSyn-DIO-hM4D(Gi)-mCherry (Addgene link)
Volume: 400ul
pAAV-hSyn-DIO-hM3D(Gq)-mCherry (Addgene link)
Volume: 400ul

(in our experiment we mix our DIO-hTyr Virus 1:1 with DREADDs)

Steroetactic coordinates:
  • Subtantia nigra

ML: 1.4mm AP: 3.25mm DV: 4.0mm (blunt needle / NF34BL-2)

  • Locus coeruleus

ML: 1.2mm AP: 5.2mm DV: 3.5mm (blunt needle / WPI NF34BL-2)
ML: 1.5mm AP: 5.2mm DV: 4.2mm (beveled needle / WPI NF34BV-2)

Injection set-up:
  • Hamiliton syringe couple t oa WPI injector (UMP3T-1) (link)
  • Injection speed 100nl/min

Expression
Expression
Assess accepted pain management protocol for injected animals, Start experiment as earlyas 3 weeks post-injections
Preparation agonist solution
Preparation agonist solution
Preparation of agonist solution

Several agonists are available for chemogenetic modulation with advantages and disadvantages, i.e., clozapine N-oxid (CNO), clozapine, or JP60. We are using J60 (JHU37160)
Please look at the reference for more detail (Bonaventura et al., 2019)

chemical structure JHU37160 aka J60 (Bonaventura et al., 2019)

  • Solve entire vial (1mg) in 10ml saline (0.9% NaCl)
(Compound can be ordered from Torcris (link))

The solubility is very good, so by vortexing for a couple of seconds it should be already dissolved.

  • Aliquot in a volume of 0.3ml and freeze at -20degree

DO NOT USE IT FOR MORE THAN 1 MONTH!


Modulation of Neuronal Activity
Modulation of Neuronal Activity
  • Animals are injected at a dosage of 1mg/kg body weight. Therefore, each aliquot is the maximum for one animal. The injections are intraperitoneal, as shown in the picture. When administering repeated injections, use different locations in the belly to avoid the same injection site.


Intraperitoneal injection of mice. Please consult a local vet for more details.


Protocol references
Bonaventura J, Eldridge MAG, Hu F, Gomez JL, Sanchez-Soto M, Abramyan AM, Lam S, Boehm MA, Ruiz C, Farrell MR, Moreno A, Galal Faress IM, Andersen N, Lin JY, Moaddel R, Morris PJ, Shi L, Sibley DR, Mahler SV, Nabavi S, Pomper MG, Bonci A, Horti AG, Richmond BJ, Michaelides M. High-potency ligands for DREADD imaging and activation in rodents and monkeys. Nat Commun. 2019 Oct 11;10(1):4627. doi: 10.1038/s41467-019-12236-z. PMID: 31604917; PMCID: PMC6788984.