Abstract Title

DREADD-Induced cFos Expression in the Basal Forebrain of Male Rats

RAD Assignment Number

1500

Presenter Name

Alexandria Marciante

Abstract

Purpose: 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 (Gq), inhibit (Gi), or stimulate cAMP (Gs) in neurons expressing DREADD receptors. In the present study, we tested the effects of a CRE independent Gq DREADD, using a CaM Kinase (CaMKIIa) promoter, and a mCherry reporter (rAAV5-CaMKIIa-hM3D(Gq)-mCherry) on Fos staining in the basal forebrain.

Methods: Adult male Sprague-Dawely rats (250-300 g bw, Charles River) were anesthetized with isoflurane and stereotaxically injected with an AAV containing the hM3D(Gq) or a control virus (rAAV5-CaMKIIa-mCherry) in either the diagonal band of bregma (DBB) or the median preoptic nucleus (MnPO). Animals were monitored for recovery for two weeks and then 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 administration of CNO or vehicle and then perfused transcardially using 4% paraformaldehyde. Brains were harvested and placed into 30% sucrose until proper dehydration of the brain. Forebrains were then sliced into 40 micron segments using cryostat. Immunohistological techniques were performed as previously described using peroxidase staining for Fos to determine activation of the DREADD virus and fluorescent staining for mCherry to verify cells transfected with either the DREADD or control virus.

Results: Overall, rats transfected with the Gq DREADD virus and treated with CNO showed significantly elevated Fos staining in the DBB or MnPO than groups transfected with either Gq DREADD and treated with vehicle or the control virus and treated with CNO or vehicle. Those transfected with the Gq DREADD virus in the DBB and treated with CNO, showed significantly more Fos staining than those transfected with either Gq 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 Gq 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 Gq DREADD virus into the MnPO.

Conclusions: These results indicate that Gq 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.

Research Area

Integrative Physiology

Presentation Type

Poster

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DREADD-Induced cFos Expression in the Basal Forebrain of Male Rats

Purpose: 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 (Gq), inhibit (Gi), or stimulate cAMP (Gs) in neurons expressing DREADD receptors. In the present study, we tested the effects of a CRE independent Gq DREADD, using a CaM Kinase (CaMKIIa) promoter, and a mCherry reporter (rAAV5-CaMKIIa-hM3D(Gq)-mCherry) on Fos staining in the basal forebrain.

Methods: Adult male Sprague-Dawely rats (250-300 g bw, Charles River) were anesthetized with isoflurane and stereotaxically injected with an AAV containing the hM3D(Gq) or a control virus (rAAV5-CaMKIIa-mCherry) in either the diagonal band of bregma (DBB) or the median preoptic nucleus (MnPO). Animals were monitored for recovery for two weeks and then 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 administration of CNO or vehicle and then perfused transcardially using 4% paraformaldehyde. Brains were harvested and placed into 30% sucrose until proper dehydration of the brain. Forebrains were then sliced into 40 micron segments using cryostat. Immunohistological techniques were performed as previously described using peroxidase staining for Fos to determine activation of the DREADD virus and fluorescent staining for mCherry to verify cells transfected with either the DREADD or control virus.

Results: Overall, rats transfected with the Gq DREADD virus and treated with CNO showed significantly elevated Fos staining in the DBB or MnPO than groups transfected with either Gq DREADD and treated with vehicle or the control virus and treated with CNO or vehicle. Those transfected with the Gq DREADD virus in the DBB and treated with CNO, showed significantly more Fos staining than those transfected with either Gq 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 Gq 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 Gq DREADD virus into the MnPO.

Conclusions: These results indicate that Gq 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.