In Vitro Autoradiographic Localization of 5-HT1A Receptor-Activated G-Proteins in the Rat Brain
Introduction
Molecular cloning has established that several subtypes of serotonin receptors are members of the superfamily of G-protein coupled receptors 1, 10, 25, 35. Of particular interest is the 5-HT1A receptor, which has been implicated in depression, anxiety, panic disorder, and alcohol abuse 3, 7, 9, 17, 21, 28, 37, 38. The 5-HT1A receptor acts via pertussis toxin sensitive G-proteins 5, 8, 12to inhibit adenylyl cyclase [6]and increase potassium channel conductance 43, 49. The 5-HT1A receptor has been localized by in vitro autoradiography, with the highest levels in the hippocampus, cortex, septum, and dorsal raphe nucleus 24, 34. These receptors have been characterized as both presynaptic autoreceptors in the dorsal raphe nucleus and postsynaptic receptors in other regions 13, 46.
The relevance of 5-HT1A receptors to psychiatric disorders makes it especially important to examine functional measures of 5-HT1A receptor activity. Unfortunately, although traditional receptor binding autoradiography provides neuroanatomical resolution, it does not provide information regarding receptor-coupled intracellular signal transduction. For G-protein-coupled receptors, the initial step in mediating agonist efficacy is the activation of G-proteins [16], which occurs when a receptor agonist changes the conformation of the G-protein α subunit from a GDP-preferring state into a state with high affinity for GTP [11]. This activation process can be measured in vitro with the use of [35S]GTPγS binding in the presence of excess GDP, which reduces basal binding and allows detection of agonist-stimulated binding 14, 32. The [35S]GTPγS membrane binding assay has been applied to the study of several G-protein-coupled receptors, including the cloned human 5-HT1A receptor [30]. Recently, this methodology was adapted to in vitro autoradiography of agonist-stimulated [35S]GTPγS binding in brain sections [40]. When sections are incubated with appropriate agonists with [35S]GTPγS and a high concentration of GDP, the resulting activation of intracellular G-proteins can be observed autoradiographically. This technique allows visualization of receptor-activated G-proteins with high anatomical resolution, and has been used with other G-protein-coupled receptors including opioid, cannabinoid, GABAB, and opioid receptor-like (ORL1) receptors 40, 42. In this study, we characterize the activation of G-proteins in brain membranes and sections by the 5-HT1A agonist 8-OH-DPAT, and report on the neuroanatomical distribution of 5-HT1A receptor-activated G-proteins in rat brain.
Section snippets
Materials and Methods
Male Sprague-Dawley rats (200 g) were purchased from Zivic-Miller (Zelienople, PA). [35S]GTPγS (1000 Ci/mmol) was purchased from New England Nuclear Corp. (Boston, MA). R(+)-8-hydroxy-2-(di-n-propylamino)tetralin HBr (8-OH-DPAT), pindobind-5-HT1A, methiothepin mesylate, ketanserin tartrate, ritanserin, and spiperone-HCl were purchased from Research Biochemicals International (Natick, MA). GTPγS and GDP were purchased from Boehringer Mannheim (New York, NY). Reflections® autoradiography film was
5HT1a agonist-Stimulated [35S]GtpγS Binding in Membranes
[35S]GTPγS binding assays were performed in isolated rat hippocampal membranes to pharmacologically characterize 5-HT1A-stimulated [35S]GTPγS binding. For other G-protein-coupled receptors, the percent stimulation of [35S]GTPγS binding by agonists depends upon the concentration of GDP: increased GDP concentration decreases basal [35S]GTPγS binding and increases percent stimulation of binding by agonist 14, 40, 44. To determine the optimal concentration of GDP for 8-OH-DPAT-stimulated [35S]GTPγS
Discussion
Although 5-HT1A receptor-stimulated [35S]GTPγS binding has been recently reported in membranes from cells transfected with 5-HT1A receptors [30], the present study is the first to report this effect in brain membranes and in brain sections by autoradiography. In hippocampal membranes, 5-HT1A-stimulated [35S]GTPγS binding is significant, with approximately twofold stimulation in the presence of 20 μM GDP. As shown in Fig. 1, this level of stimulation can be altered by varying GDP concentrations,
Acknowledgements
The authors thank Dr. Dana E. Selley for providing helpful discussions. This research was supported by USPHS grant DA06634 (to S.R.C.) and DA-00287 (to L.J.S.) from the National Institute on Drug Abuse.
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