Elsevier

Brain Research

Volume 1022, Issues 1–2, 1 October 2004, Pages 214-220
Brain Research

Research report
Regulators of G-protein signaling 4: modulation of 5-HT1A-mediated neurotransmitter release in vivo

https://doi.org/10.1016/j.brainres.2004.06.073Get rights and content

Abstract

Regulators of G-protein signaling (RGS) play a key role in the signal transduction of G-protein-coupled receptors (GPCRs). Specifically, RGS proteins function as GTPase accelerating proteins (GAPs) to dampen or “negatively regulate” GPCR-mediated signaling. Our group recently showed that RGS4 effectively GAPs Gαi-mediated signaling in CHO cells expressing the serotonin-1A (5-HT1A) receptor. However, whether a similar relationship exists in vivo has yet to be identified. In present studies, a replication-deficient herpes simplex virus (HSV) was used to elevate RGS4 mRNA in the rat dorsal raphe nuclei (DRN) while extracellular levels of 5-HT in the striatum were monitored by in vivo microdialysis. Initial experiments conducted with noninfected rats showed that acute administration of 8-OH-DPAT (0.01–0.3 mg/kg, subcutaneous [s.c.]) dose dependently decreased striatal levels of 5-HT, an effect postulated to result from activation of somatodendritic 5-HT1A autoreceptors in the DRN. In control rats receiving a single intra-DRN infusion of HSV-LacZ, 8-OH-DPAT (0.03 mg/kg, s.c.) decreased 5-HT levels to an extent similar to that observed in noninfected animals. Conversely, rats infected with HSV-RGS4 in the DRN showed a blunted neurochemical response to 8-OH-DPAT (0.03 mg/kg, s.c.); however, increasing the dose to 0.3 mg/kg reversed this effect. Together, these findings represent the first in vivo evidence demonstrating that RGS4 functions to GAP Gαi-coupled receptors and suggest that drug discovery efforts targeting RGS proteins may represent a novel mechanism to manipulate 5-HT1A-mediated neurotransmitter release.

Introduction

Regulators of G-protein signaling (RGS) proteins comprise a diverse family of proteins capable of modulating G-protein-coupled receptors (GPCRs)-mediated signaling [4]. A highly conserved 120-amino acid region or “RGS-box” interacts with specific regions of the Gα protein [26] to provide conformational stability and acceleration of endogenous Gα hydrolysis [27]. Therefore, RGS proteins primarily function as GTPase accelerating proteins (GAPs) reducing the activated GTP-bound state of the α subunit and dampening intracellular signaling cascades mediated by GPCRs [14], [15], [18], [28]. The initial description of RGS proteins to “negatively regulate” receptor signaling has recently expanded to show that RGS proteins may also act directly on adenylyl cyclase and function as effector molecules themselves [8], [9]. These characteristics, in addition to their enriched CNS distribution [10], promote the hypothesis that RGS proteins represent a novel and intriguing class of drug targets for neuroscience discovery research.

While the purported GAP activity and biochemical roles of RGS proteins in vitro are well documented, their functional consequence in intact neuronal (or in vivo) systems remains poorly characterized. Recently, RGS-insensitive (RGS-i) rats have been developed, which renders the Gα subunit insensitive to interacting with RGS proteins. Interesting behavioral phenotypes including decreased body mass, shaking or tremors and lethality induced by the administration of serotonin and cholinergic receptor agonists at doses that are benign to normal rats have been reported in these rats [11], [12], [23]. More recently, Rahman et al. [21] used viral-mediated techniques to demonstrate that overexpression of RGS9 in the rat ventral striatum is capable of diminishing the behavioral effects of dopamine D2 but not D1 receptor agonists. These later findings represent the first series of studies revealing a functional interaction in vivo between RGS9 and dopaminergic receptor systems.

While the work from Rahman et al. is encouraging, very little information is known concerning the function of other RGS proteins in vivo. Recently, our research group showed that RGS4 effectively GAPs Gαi-mediated signaling in CHOK1 cells stably expressing serotonin-1A (5-HT1A) receptors [9]. The goal, therefore, of the present study was to investigate whether RGS4 proteins could also GAP a Gαi-coupled receptor in vivo. To address this question, RGS4 was virally expressed in the rat dorsal raphe nucleus (DRN), an area of the brain predominately expressing Gαi-coupled 5-HT1A receptors [3]. In these infected animals, microdialysis techniques were employed to monitor the effects of RGS4 expression in the DRN on basal and 5-HT1A agonist-stimulated levels of extracellular 5-HT in the rat striatum. The striatum was chosen for these neurochemical studies because it receives dense serotonergic projections originating from the DRN [2] and because regulation of 5-HT release in this region is linked to 5-HT1A autoreceptors in the DRN [16]. To our knowledge, these experiments are the first to evaluate whether RGS proteins modulate neurotransmitter release in an in vivo model.

Section snippets

Animals

All experimental procedures followed specifications of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and Wyeth's Institutional Animal Care and Use Committee. Male Sprague–Dawley rats (Charles River, Wilmington, MA), weighing 280 and 350 g, were group-housed before surgery in an AAALC-accredited facility and maintained on a 12-h light/dark cycle. Experiments were conducted during the light period (lights on at 06:00 h) and animals had access to food and water

RGS4 expression in the DRN

Coronal sections of the rat DRN were subjected to in situ hybridization techniques. A single intra-DRN HSV-RGS4 infusion produced marked elevations in local RGS4 mRNA at 2, 3, 4 and 5 days but noticeably less at 7 days postinfection (data not shown). Based on these preliminary findings, all microdialysis studies were performed 4 days postinoculation. A line drawing of the intended path and destination of the virus infusion needle is shown in Fig. 1A along with representative photomicrographs of

Discussion

RGS proteins comprise a diverse family of proteins capable of modulating GPCR-mediated signaling [4], [18]. A recent work shows that RGS4 effectively GAPs Gαi-mediated signaling in CHOK1 cells stably expressing the 5-HT1A receptor [9]. To examine whether a similar relationship could be identified in vivo, molecular biology and microdialysis techniques were combined in freely moving rats. The results of the present study demonstrate that a single injection of HSV-RGS4 in the DRN blocked

Acknowledgements

The authors would like to thank Drs. Karen L. Marquis, Paul McGonigle and Zia Rahman for their valuable insight, suggestions and dialogue and Dr. Eric Nestler for the HSV viral reagents used in this study. We also are grateful to Juan Mercado, Sue Asbury and Brian Strassle for their exceptional technical assistance.

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  • Cited by (0)

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    Neurological Disorders, J&J Pharmaceutical Research and Development, Raritan, NJ 08869, USA.

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    Neuropharmacology, Psychiatry CEDD, GlaxoSmithKline, Harlow, Essex, UK.

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