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DREADD‐Induced cFos Expression in the Basal Forebrain of Male Rats
Author(s) -
Marciante Alexandria Beverly,
Cunningham J. Thomas
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.866.8
Subject(s) - receptor , forebrain , basal forebrain , microinjection , chemistry , microbiology and biotechnology , medicine , ectopic expression , biology , endocrinology , biochemistry , central nervous system , gene
Designer Receptors Exclusively Activated by Designer Drugs, or DREADDs, are genetically modified G‐protein coupled receptors (GPCR) that are sensitive to an exogenous pharmacological agent, clozapine‐ N ‐oxide (CNO). DREADDs can be packaged in viral vectors with specific promoters or combined with CRE dependent platforms to express these receptors in specific neuronal phenotypes. This chemogenetic approach can be used to activate (G q ), inhibit (G i ), or stimulate cAMP (G s ) in neurons. In the present study, we tested the effects of a CRE independent G q DREADD, using a CaM Kinase (CaMKIIa) promoter, and a mCherry reporter (rAAV5‐CaMKIIa‐hM3D(G q )‐mCherry) on Fos staining in the basal forebrain. Adult male Sprague‐Dawley rats (250–300 g bw, Charles River) were anesthetized with isoflurane and stereotaxically injected with an AAV containing the hM3D(G q ) or a control virus (rAAV5‐CaMKIIa‐mCherry) in either the diagonal band of broca (DBB) or the median preoptic nucleus (MnPO). Animals were allowed two weeks of recovery prior to administered CNO or vehicle. CNO was dissolved into dimethylsulfoxide (DMSO) and saline (ratio 20% to 80%) and given via intraperitoneal injection (IP) at a concentration of 10mg/kg. Rats were food and water deprived for 90 minutes following the injections. Each rat was anesthetized with inactin (100 mg/kg IP) and prepared for immunohistochemistry. Forebrains were sliced into 40 um coronal sections using cryostat. The sections were processed for Fos immunohistochemistry using DAB‐peroxidase and mCherry immunofluorescence to verify cells transfected with either the DREADD or control virus. Overall, rats transfected with the G q DREADD virus and treated with CNO showed significantly elevated Fos staining in the DBB or MnPO than groups transfected with either G q DREADD and treated with vehicle or the control virus and treated with CNO or vehicle. Those transfected with the G q DREADD virus in the DBB and treated with CNO, showed significantly more Fos staining than those transfected with either G q DREADD and treated with vehicle (P<0.001) or the control virus and treated with CNO (P<0.001) or vehicle (P=0.002). The CNO did not appear to have a significant effect on Fos staining in rats injected with the control vector. Injection of G q DREADD virus into the DBB also appears to have less of an effect on downstream targets, specifically the paraventricular nucleus (PVN) and the supraoptic nucleus (SON), than did injection of G q DREADD virus into the MnPO. These results indicate that G q DREADD can be used to differentially activate neurons in either the DBB or the MnPO to influence activity in downstream regions that control autonomic and neuroendocrine function. Support or Funding Information Supported by P01 HL088052