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In Vitro Autoradiographic Localization of 5-HT1A Receptor-Activated G-Proteins in the Rat Brain
Brain Research Bulletin, Vol. 44, No. 1, pp. 39–45, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/97 $17.00 / .00
PII S0361-9230(97)00061-0
In Vitro Autoradiographic Localization of 5-HT1A Receptor-Activated G-Proteins in the Rat Brain LAURA J. SIM, RUOYU XIAO AND STEVEN R. CHILDERS 1 Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Wake Forest University, Medical Center Blvd., Winston-Salem, NC 27157, USA [Received 20 June 1996; Revised 9 November 1996; Accepted 16 December 1996] ABSTRACT: Serotonin 5-HT1A receptors belong to the superfamily of G-protein-coupled receptors. Receptor activation of Gproteins can be determined by agonist-stimulated [ 35S]GTPgS binding in the presence of excess GDP, and in vitro autoradiographic adaptation of this technique allows visualization of receptor-activated G-proteins in tissue sections. The present study was performed to examine 5-HT1A receptor activation of G-proteins using 8-OH-DPAT–stimulated [ 35S]GTPgS binding in membranes and brain sections. In hippocampal membranes, 8OH-DPAT stimulated [ 35S]GTPgS binding by twofold, with an ED50 value of 25 nM. 5-HT1 antagonists, but not 5-HT2 antagonists, increased the ED50 of 8-OH-DPAT in a manner consistent with competitive antagonists. Scatchard analysis of [ 35S]GTPgS binding showed that 8-OH-DPAT induced the formation of high affinity [ 35S]GTPgS binding sites with a KD for GTPgS of 3.2 nM. [ 35S]GTPgS autoradiography, performed in brain sections with the 5-HT1A agonist 8-OH-DPAT, revealed high levels of 5-HT1A stimulated [ 35S]GTPgS binding in the hippocampus, lateral septum, prelimbic cortex, entorhinal cortex, and dorsal raphe nucleus. 5-HT1A –stimulated [ 35S]GTPgS binding in sections was blocked by the addition of the 5-HT1 antagonist methiothepin. These results show that the use of agonist-stimulated [ 35S]GTPgS autoradiography for the 5-HT1A receptor system should provide new information regarding signal transduction in specific brain regions. Q 1997 Elsevier Science Inc.
The relevance of 5-HT 1A receptors to psychiatric disorders makes it especially important to examine functional measures of 5-HT 1A 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 a 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]GTPgS binding in the presence of excess GDP, which reduces basal binding and allows detection of agonist-stimulated binding [14,32]. The [ 35S]GTPgS membrane binding assay has been applied to the study of several G-protein–coupled receptors, including the cloned human 5-HT 1A receptor [30]. Recently, this methodology was adapted to in vitro autoradiography of agoniststimulated [ 35S]GTPgS binding in brain sections [40]. When sections are incubated with appropriate agonists with [ 35S]GTPgS 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 Gproteins in brain membranes and sections by the 5-HT 1A agonist 8-OH-DPAT, and report on the neuroanatomical distribution of 5-HT 1A receptor-activated G-proteins in rat brain.
KEY WORDS: [ 35S]GTPgS autoradiography, G-protein–coupled receptors, 5-HT, 5-HT1A receptors.
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-HT 1A receptor, which has been implicated in depression, anxiety, panic disorder, and alcohol abuse [3,7,9,17,21,28,37,38]. The 5-HT 1A receptor acts via pertussis toxin sensitive G-proteins [5,8,12] to inhibit adenylyl cyclase [6] and increase potassium channel conductance [43,49]. The 5-HT 1A 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]. 1
MATERIALS AND METHODS Male Sprague–Dawley rats (200 g) were purchased from Zivic–Miller (Zelienople, PA). [ 35S]GTPgS (1000 Ci/mmol) was purchased from New England Nuclear Corp. (Boston, MA). R( / )-8-hydroxy-2-(di-n-propylamino)tetralin HBr (8-OHDPAT), pindobind-5-HT 1A , methiothepin mesylate, ketanserin tartrate, ritanserin, and spiperone-HCl were purchased from Research Biochemicals International (Natick, MA). GTPgS and GDP were purchased from Boehringer Mannheim (New York, NY). Reflectionst autoradiography film was purchased from New England Nuclear Corp. (Boston, MA). All other reagent
To whom requests for reprints should be addressed.
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grade chemicals were obtained from Sigma Chemical Co. or Fisher. Agonist-Stimulated [ 35 S]GTPgS Binding in Membranes [ 35S]GTPgS binding was performed as previously described [40]. Rat hippocampus was dissected and homogenized in 20 vol cold Tris-Mg 2/ buffer (50 mM Tris-HCl, 3 mM MgCl2 , 1 mM EGTA, pH 7.4) with a Polytron. The homogenate was centrifuged at 48,000 1 g at 47C for 10 min, resuspended in buffer, centrifuged, and resuspended in assay buffer (50 mM Tris-HCl, 3 mM MgCl2 , 0.2 mM EGTA, 100 mM NaCl, pH 7.4). Membranes (10 mg protein) were incubated at 307C for 1 h in assay buffer with various concentrations of 8-OH-DPAT, with or without antagonists, in the presence of 20 mM GDP and 0.05 nM [ 35S]GTPgS in a 1 ml total volume. Basal binding was measured in the absence of agonist and antagonist, and nonspecific binding was assessed with 10 mM GTPgS. Experiments were also performed using 1 mM 8-OH-DPAT with varying concentrations of 1) GDP (0.5–50 mM) and 2) GTPgS (0.02–200 nM). The reaction was terminated by rapid filtration under vacuum through Whatman GF/B glass fiber filters, followed by three washes with cold Tris buffer (50 mM Tris-HCl, pH 7.4). Bound radioactivity was determined by liquid scintillation spectrophotometry, at 95% efficiency for 35S, after overnight extraction of the filters in 5 ml Ecolite scintillation fluid. Data are reported as mean { SE values of at least three experiments that were each performed in triplicate. Nonlinear iterative regression analyses of agonist concentration–effect curves were performed with JMP (SAS Institute, Cary, NC). The resulting ED50 values were used to determine Ke values for antagonism of the agonist-stimulated response, using the relationship (Ke Å [Ant]/(DR 0 1), where [Ant] is the concentration of antagonist, and DR is the ratio of ED50 values in the presence and absence of antagonist. Scatchard analysis of [ 35S]GTPgS binding was fit to one- or two-site models by LIGAND.
[14,40,44]. To determine the optimal concentration of GDP for 8-OH-DPAT–stimulated [ 35 S]GTPgS binding in membranes, hippocampal membranes were incubated with 1 mM 8-OHDPAT in the presence of 0.5 to 50 mM GDP (Fig. 1). GDP inhibited basal [ 35S]GTPgS binding in a concentration-dependent manner (Fig. 1A), with less than 10% of total [ 35S]GTPgS binding remaining with 50 mM GDP. Addition of 8-OH-DPAT had little effect on [ 35S]GTPgS binding at concentrations of GDP below 1 mM, but significantly stimulated binding at GDP concentrations higher than 1 mM. The relative percent stimulation of [ 35S]GTPgS binding by 8-OH-DPAT was increased with increasing concentrations of GDP, so that the agonist stimulation was greater than twofold at GDP concentrations greater than 10 mM (Fig. 1B). From these data, an optimal GDP concentration in producing significant percent stimulation by 8-OH-DPAT, while maintaining reasonable levels of basal [ 35S]GTPgS binding, was determined to be 20 mM.
Agonist-Stimulated [ 35 S]GTPgS Autoradiography [ 35S]GTPgS autoradiography was performed as previously described [40]. Animals were sacrificed and brains were removed and frozen in isopentane at 0357C. Twenty micron horizontal or coronal brain sections were cut on a cryostat, thaw mounted onto gelatin coated slides, dried under a vacuum and stored desiccated at 0807C until use. Slides were incubated in assay buffer for 10 min at 257C, then in 2 mM GDP in assay buffer at 257C for 15 min. Slides were then incubated with 0.3 mM 8-OH-DPAT with 2 mM GDP and 0.04 nM [ 35S]GTPgS in assay buffer at 257C for 2 h, with and without 0.2 mM methiothepin. Basal binding was assessed in the absence of agonist and antagonist. Slides were rinsed twice for 2 min each in cold 50 mM Tris buffer and once (30 s) in deionized H2O. Slides were dried well and exposed to film for 48–72 h. Films were digitized with a Sony XC-77 video camera and analyzed with the NIH IMAGE program for Macintosh computers. RESULTS 5HT1A Agonist-Stimulated [ 35 S]GTPgS Binding in Membranes [ 35S]GTPgS binding assays were performed in isolated rat hippocampal membranes to pharmacologically characterize 5HT 1A –stimulated [ 35S]GTPgS binding. For other G-protein– coupled receptors, the percent stimulation of [ 35S]GTPgS binding by agonists depends upon the concentration of GDP: increased GDP concentration decreases basal [ 35S]GTPgS binding and increases percent stimulation of binding by agonist
FIG. 1. Effect of GDP on basal and 8-OH-DPAT–stimulated [ 35S]GTPgS binding. Hippocampal membranes were incubated with 0.05 nM [ 35S]GTPgS, 1 mM 8-OH-DPAT, and 0.5–50 mM GDP. Values are expressed as (A) percent total [ 35S]GTPgS binding (as determined in the absence of GDP), and (B) percent stimulation of [ 35S]GTPgS binding. In the absence of GDP, basal [ 35S]GTPgS binding was 2174 { 75 fmol/mg protein.
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41 TABLE 1 EFFECT OF 5-HT ANTAGONISTS ON STIMULATION OF [35S]GTPgS BINDING BY 8OH-DPAT IN RAT HIPPOCAMPAL MEMBRANES Antagonist (mM)
None Ritanserin (0.2 mM) Ketanserin (0.2 mM) Spiperone (0.2 mM) Methiothepin (0.2 mM) Pindobind-5-HT1A (0.2 mM)
FIG. 2. Concentration–effect curves of 8-OH-DPAT–stimulated [ 35S]GTPgS binding in the presence and absence of 5-HT 1 / 1A antagonists. Hippocampal membranes were incubated with 20 mM GDP, 0.05 nM [ 35S]GTPgS, and various concentrations of 8-OH-DPAT, with and without 0.2 mM methiothepin, spiperone, or pindobind-5-HT 1A . Values are expressed as percent stimulation of [ 35S]GTPgS binding { SE.
Under these conditions, concentration–effect curves showed that stimulation of [ 35S]GTPgS binding by 8-OH-DPAT was concentration dependent and saturable (Fig. 2). Maximal stimulation (Emax ) of [ 35S]GTPgS binding by 8-OH-DPAT was 136 { 3% compared to basal, with an ED50 value of 25 { 5 nM. From these data, a concentration of 0.3 mM 8-OH-DPAT was chosen for use in autoradiography experiments in brain sections (see below). The pharmacological specificity of 5-HT 1A –stimulated [ 25S]GTPgS binding was determined by incubating hippocampal membranes with 0.3 mM of 8-OH-DPAT in the presence of 0.2 mM serotonergic antagonists (Table 1). In the absence of antagonist, 8-OH-DPAT stimulated [ 35S]GTPgS binding by approximately twofold (104%). Neither of the 5-HT 2 -selective antagonists, ritanserin or ketanserin, had any significant effect on 8-OH-DPAT–stimulated [ 35S]GTPgS binding. In contrast, addition of antagonists with selectivity at 5-HT 1 / 1A receptors, including spiperone, methiothepin, and pindobind-5-HT 1A , significantly reduced 8-OH-DPAT–stimulated [ 35S]GTPgS binding, with methiothepin (0.2 mM) inhibiting the agonist effect by greater than 90%. Potencies of these antagonists were determined by Ke analysis, using single concentrations of antagonist to increase agonist ED50 values. Results (Fig. 2) showed that all three 5-HT 1 / 1A antagonists acted in a manner consistent with competitive antagonists by increasing agonist ED50 values with no apparent effect on the Emax . From these data, the estimated Ke values were 12.3 { 0.9 nM for spiperone, 14.3 { 0.1 nM for pindobind-5-HT 1A , and 2.4 { 0.2 nM for methiothepin. Previous opioid-stimulated [ 35S]GTPgS binding studies showed that addition of agonist induced high-affinity [ 35S]GTPgS binding sites that could be separated from the lowaffinity sites of basal [ 35S]GTPgS binding by net Scatchard analysis [41,44]. Similar effects were observed in the 5-HT 1 / 1A receptor system (Fig. 3). Scatchard analysis was performed using increasing concentrations of GTPgS in the presence and absence of 1 mM 8-OH-DPAT, as previously described for opioid receptors [39,41]. Both basal and 8-OH-DPAT–stimulated [ 35S]GTPgS binding exhibited multiphasic Scatchard plots that were best fit to two-site models. For both basal and agonist-stimulated [ 35S]GTPgS binding, the low-affinity binding compo-
% Stimulation
104 103 101 26 6 36
{ { { { { {
2 2 3 8 3 2
Rat hippocampal membranes were incubated with 0.05 nM [35S]GTPgS, 20 mM GDP and 0.1 mM 8-OH-DPAT in the presence and absence of 0.2 mM of various 5-HT receptor antagonists. Results are expressed as mean values of percent stimulation by 8-OH-DPAT { SE.
nents were essentially identical, with KD values for GTPgS ranging between 350–500 nM. The basal high affinity site exhibited a KD of 10.6 { 3 nM, with a Bmax of 3.5 { 0.3 pmol/mg protein. Addition of agonist decreased the KD of the high affinity site, with a KD of 3.2 { 0.8 nM and a Bmax of 3.9 { 1.0 pmol/mg. The inset of Fig. 3 shows net Scatchard analysis, in which net agonist-stimulated [ 35S]GTPgS binding was calculated by subtracting basal from 8-OH-DPAT–stimulated [ 35S]GTPgS binding at each concentration of GTPgS. The net Scatchard plot was monophasic, with a KD of 2.3 { 0.3 nM and a Bmax of 3.0 { 0.5 pmol/mg, similar to the corresponding values for the high affinity site of the biphasic plot for 8-OH-DPAT–stimulated binding. 5HT1A Agonist-Stimulated [ 35 S]GTPgS Autoradiography Recent studies have shown that receptor activation of G-proteins can be localized in brain sections by in vitro autoradiogra-
FIG. 3. Scatchard analysis of [ 35S]GTPgS binding in hippocampal membranes. Membranes were incubated with 0.05 nM [ 35S]GTPgS in the presence of 0–200 nM GTPgS with and without 1 mM 8-OH-DPAT. Data are from a typical experiment that was performed in triplicate and replicated three times. Inset: Scatchard analysis of net 8-OH-DPAT– stimulated [ 35S]GTPgS binding (agonist-stimulated binding minus basal binding at each concentration of GTPgS).
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phy of agonist-stimulated [ 35S]GTPgS binding using opioid, cannabinoid and GABAB agonists [40]. To determine whether 5-HT 1 / 1A receptors can be localized using the same technique, rat brain sections were incubated with 0.3 mM 8-OH-DPAT, in the presence and absence of 0.2 mM of the 5-HT 1 / 1A antagonist methiothepin. These concentrations of agonist and antagonist were optimized in membrane experiments using 8-OH-DPAT–stimulated [ 35S]GTPgS binding. Representative sections are shown in Fig. 4. In the sections shown in Fig. 4A, the highest levels of 5-HT 1 / 1A -stimulated [ 35S]GTPgS binding were found in the lateral septum. A low level of 8-OH-DPAT–stimulated [ 35S]GTPgS binding was also found in the cortex. No significant stimulation of [ 35S]GTPgS binding was found in the caudateputamen. The addition of methiothepin inhibited 8-OH-DPAT stimulation of [ 35S]GTPgS binding, so that these sections were comparable to basal [ 35S]GTPgS binding. High levels of 8-OHDPAT–stimulated [ 35S]GTPgS binding were also found throughout the rostral–caudal extent of the hippocampus. This is shown in Fig. 4B, where particularly high 5-HT 1A –stimulated [ 35S]GTPgS binding was observed in the dentate gyrus. In the mesencephalon, 5-HT 1A –stimulated [ 35S]GTPgS binding was found in the dorsal raphe nucleus (Fig. 4C). 8-OH-DPAT–stimulated [ 35S]GTPgS binding was also observed in the entorhinal cortex and subiculum in these sections. The addition of methiothepin again inhibited agonist-stimulated [ 35S]GTPgS binding, verifying the receptor specificity of the effect. Another view of the anatomical distribution of 5-HT 1A –stimulated [ 35S]GTPgS binding is seen the horizontal sections in Fig. 4D, where agoniststimulated [ 35S]GTPgS binding is seen in the lateral septum, prelimbic cortex, hippocampus, subiculum, entorhinal cortex, and dorsal raphe nucleus. DISCUSSION Although 5-HT 1A receptor-stimulated [ 35S]GTPgS binding has been recently reported in membranes from cells transfected with 5-HT 1A 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-HT 1A –stimulated [ 35S]GTPgS binding is significant, with approximately twofold stimulation in the presence of 20 mM GDP. As shown in Fig. 1, this level of stimulation can be altered by varying GDP concentrations, with higher GDP levels producing lower net binding values but higher percent stimulation. The level of stimulation by 8-OH-DPAT is somewhat lower than the threefold stimulation reported for 5-HT 1A receptor-transfected CHO cells [30], but this difference is explained by the heterogeneous nature of brain membranes compared to membranes from transfected cells. This level of stimulation by 8-OH-DPAT was maintained at concentrations of GTPgS at least to 10 nM, so that this level of stimulation is not an artifact of a low concentration of radioligand compared to the number of G-protein a subunits. The potency of 8-OH-DPAT in stimulating [ 35S]GTPgS binding in hippocampal membranes, 25 nM, is similar to that reported for this agonist in transfected cells [30]; moreover, the Ke values of the antagonists spiperone, pindobind 5-HT 1A and methiothepin, are similar to the potencies of these antagonists in binding to 5-HT 1A receptors [18,23,26]. The [ 35S]GTPgS Scatchard analysis (Fig. 3) shows the typical biphasic nature that has been reported for other receptor systems [44]; the fact that the KD and Bmax for the net Scatchard analysis are the same as corresponding values for the high affinity site of agonist-stimulated [ 35S]GTPgS binding, shows that net Scatchard analysis is a valid method for quantifying these parameters without the complexities of multisite analysis. The Bmax
value for 8-OH-DAPT–stimulated [ 35S]GTPgS binding in hippocampal membranes was 3 pmol/mg. Interestingly, measurements of G-protein a subunit content in brain are within the same order of magnitude. For example, the level of Gia2 in hippocampal membranes has been estimated as 13 pmol/mg [2]. Because 5-HT 1A receptors are likely to be coupled to more than one Gprotein subtype, and because a number of other G-protein-coupled receptors exist in hippocampus, these numbers may be valid comparisons. In many cell culture systems, basal [ 35S]GTPgS binding exhibits a single low affinity site with KD ú 100 nM [44]; however, the present study in rat hippocampal membranes confirms previous results in rat striatal membranes [41] that basal [ 35S]GTPgS binding contains a moderate-affinity site of 10–20 nM. The significance of this site in brain membranes is not clear; it could represent precoupled (i.e., spontaneously-active receptors) or may not in fact be relevant to receptor-transducer coupling. In any case, regardless of the tissue or receptor, addition of agonist produces high affinity [ 35S]GTPgS sites with KD values between 1–3 nM. 5-HT 1A receptors have been localized in the brain by traditional receptor binding autoradiography [24,34], while 5-HT 1A mediated second messenger effects, including inhibition of adenylyl cyclase [6] and activation of potassium channels [43,49], have been identified in cells and isolated membranes. The use of in vitro autoradiography of agonist-stimulated [ 35S]GTPgS binding represents a combination of these two approaches: it combines the neuroanatomical resolution of autoradiography with the functional information of a signal transduction assay. The functional aspects of this methodology can be seen in the present study by the fundamentally different effects of agonists and antagonists of the 5-HT 1A receptor system. The agonist 8OH-DPAT produced significant stimulation of [ 35S]GTPgS binding that was eliminated by the addition of 5-HT 1 / 1A , but not 5-HT 2 , antagonists. Moreover, the antagonists had no effect on their own. These results demonstrate that [ 35S]GTPgS autoradiography in brain sections can be used to localize an intracellular signal transduction mechanism (in this case, activation of G-protein a subunits) when performed in the presence of appropriate concentrations of agonist and GDP. Although opioid, cannabinoid, GABAB , and ORL-1 receptors have previously been localized by agonist-stimulated [ 35S]GTPgS autoradiography [40,42], the present study represents the first monoaminergic agonist to be used successfully in the autoradiographic assay. Attempts in our laboratory to adapt this technique to other monoaminergic receptors, particularly dopamine receptors, have been less successful (L. Sim and S. Childers, unpublished observations). It is clear that the [ 35S]GTPgS binding signal depends upon both the number of receptors and the efficiency of coupling between receptors and G-proteins [40,41]; therefore, G-protein activity stimulated by monoaminergic receptors with low receptor number and/or low catalytic amplification (i.e., the number of G-proteins activated per receptor) may be difficult to detect. It appears that the combination of receptor number and catalytic amplification is sufficiently high in the 5-HT 1A receptor system in the brain to detect a considerable signal. As demonstrated in Fig. 1, the magnitude of this signal depends upon the concentration of GDP, and in membranes 20 mM GDP was optimal for agonist stimulation of [ 35S]GTPgS binding. However, as we have reported for other receptor systems [40], the concentration of GDP necessary in the autoradiographic assay is approximately a hundred times higher (1–2 mM GDP) than that required for the [ 35S]GTPgS assay in membranes. The reason for this concentration difference between membranes and sections is not clear, but may be due to either the high level of protein in brain sections or differences
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FIG. 4. Representative sections of rat brain showing the distribution of 5-HT 1A -stimulated [ 35S]GTPgS binding, in the presence and absence of antagonist, compared to basal [ 35S]GTPgS binding. Sections were incubated with 2 mM GDP, then with 0.04 nM [ 35S]GTPgS, 2 mM GDP, and 0.3 mM 8-OH-DPAT, with and without 0.2 mM methiothepin. Basal binding was assessed in the absence of agonist. Coronal sections are shown at the level of (A) septum, (B) hippocampus, and (C) dorsal raphe nucleus. 8-OH-DPAT-stimulated [ 35S]GTPgS binding in these regions, as well as the entorhinal cortex and prelimbic cortex, is also shown in the autoradiogram of a horizontal brain section in Fig. 4D.
between the kinetics of G-protein binding in situ compared to isolated membranes. The receptor specificity of 8-OH-DPAT–stimulated [ 35S]GTPgS binding was verified by several criteria. Agoniststimulated [ 35S]GTPgS binding was blocked by the addition of the 5-HT 1 antagonist methiothepin. The anatomical distribution of 8-OH-DPAT–stimulated [ 35S]GTPgS binding also correlated with that of 5-HT 1A receptors [24,34] with high levels of both
receptors and receptor-activated G-proteins in the hippocampus, septum, cortex, and dorsal raphe nucleus. This is in contrast to other serotonergic receptors. For example, 5-HT 1B receptors are highest in the basal ganglia and substantia nigra [34], and 5-HT 2 receptors are found in the cortex, nucleus accumbens, claustrum, caudate-putamen, and choroid plexus [27,33]. The areas in which high levels of 5-HT 1A -stimulated [ 35S]GTPgS binding were identified (i.e., hippocampus, septum,
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cortex, and dorsal raphe nucleus) are components of the limbic system. The serotonergic system, and particularly the 5-HT 1A receptor, has been implicated in depression, because 5-HT 1A receptor agonists alleviate depression in animal models [3,7,17]. However, the effects of antidepressant drugs on 5-HT 1A receptors [13,15,36,47,48] and 5-HT 1A –mediated inhibition of adenylyl cyclase [29,31,45] are equivocal. There is also evidence that antidepressant treatment alters the levels of G-protein a subunit protein and mRNA in the brain [19,20], indicating that antidepressants may act in part via alterations in signal transduction pathways. However, changes in G-protein levels may not necessarily accompany changes in function [22], and membrane proteins such as tubulin may be important modulators in regulating G-protein a subunits during antidepressant treatment [4]. For this reason, it is crucial to have a direct functional measure of those G-proteins that are coupled to specific receptors. Thus, the [ 35S]GTPgS binding technique may be particularly important in the analysis of the underlying neurochemical disturbances that lead to depression, as well as elucidation of the mechanisms by which antidepressant drugs act. 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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guanylyl 5 *-[g-[35S]thio]-triphosphate binding. Proc. Natl. Acad. Sci. USA 92:7242–7246; 1995. Sim, L. J.; Selley, D. E.; Xiao, R.; Childers, S. R. Differences in Gprotein activation by mu and delta opioid, and cannabinoid, receptors in rat striatum. Eur. J. Pharmacol. 307:95–107; 1996. Sim, L. J.; Xiao, R.; Childers, S. R. Identification of opioid receptorlike (ORL1) peptide-stimulated [35S]GTPgS binding in rat brain. Neuroreport 7:729–733; 1996. Sprouse, J. S.; Aghajanian, G. K. Electrophysiological responses of serotonergic dorsal raphe neurons to 5-HT 1A and 5-HT 1B agonists. Synapse 1:3–9; 1987. Traynor, J. R.; Nahorski, S. R. Modulation by m-opioid agonists of guanosine-5 *-O-(3-[35S]thio)triphosphate binding to membranes from human neuroblastoma SH-SY5Y cells. Mol. Pharmacol. 47:848–854; 1995. Varrault, X.; Leviel, V.; Bockaert, J. 5-HT 1A -sensitive adenylyl cyclase of rodent hippocampal neurons: Effects of antidepressant treatments and chronic stimulation with agonists. J. Pharmacol. Exp. Ther. 257:433–438; 1991. Verge, D.; Daval G.; Marcinkiewicz, M.; Patey, A.; El Mestikawy, S.; Gozlan, H.; Hamon, M. Quantitative autoradiography of multiple 5-HT 1 -receptor subtypes in the brain of control or 5,7-dihydroxytryptamine-treated rats. J. Neurosci. 6:3474–3482; 1986. Watanbe, Y.; Sakai, R. R.; McEwen, B. S.; Mendelson, S. Stress and antidepressant effects on hippocampal and cortical 5-HT 1A and 5-HT 2 receptors and transport sites for serotonin. Brain Res. 615:87– 94; 1993. Welner, S. A.; DeMontigny, C.; Desroches, J.; Desjardins, P.; Suranyi-Cadotte, B. E. Autoradiographic quantification of serotonin1A receptors in rat brain following antidepressant drug treatment. Synapse 4:347–352; 1989. Williams, J. T.; Colmers, W. F.; Pan, Z. Z. Voltage- and ligandactivated inwardly rectifying currents in dorsal raphe neurons in vitro. J. Neurosci. 8:3499–3506; 1988.
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In Vitro Autoradiographic Localization of 5-HT1A Receptor-Activated G-Proteins in the Rat Brain LAURA J. SIM, RUOYU XIAO AND STEVEN R. CHILDERS 1 Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Wake Forest University, Medical Center Blvd., Winston-Salem, NC 27157, USA [Received 20 June 1996; Revised 9 November 1996; Accepted 16 December 1996] ABSTRACT: Serotonin 5-HT1A receptors belong to the superfamily of G-protein-coupled receptors. Receptor activation of Gproteins can be determined by agonist-stimulated [ 35S]GTPgS binding in the presence of excess GDP, and in vitro autoradiographic adaptation of this technique allows visualization of receptor-activated G-proteins in tissue sections. The present study was performed to examine 5-HT1A receptor activation of G-proteins using 8-OH-DPAT–stimulated [ 35S]GTPgS binding in membranes and brain sections. In hippocampal membranes, 8OH-DPAT stimulated [ 35S]GTPgS binding by twofold, with an ED50 value of 25 nM. 5-HT1 antagonists, but not 5-HT2 antagonists, increased the ED50 of 8-OH-DPAT in a manner consistent with competitive antagonists. Scatchard analysis of [ 35S]GTPgS binding showed that 8-OH-DPAT induced the formation of high affinity [ 35S]GTPgS binding sites with a KD for GTPgS of 3.2 nM. [ 35S]GTPgS autoradiography, performed in brain sections with the 5-HT1A agonist 8-OH-DPAT, revealed high levels of 5-HT1A stimulated [ 35S]GTPgS binding in the hippocampus, lateral septum, prelimbic cortex, entorhinal cortex, and dorsal raphe nucleus. 5-HT1A –stimulated [ 35S]GTPgS binding in sections was blocked by the addition of the 5-HT1 antagonist methiothepin. These results show that the use of agonist-stimulated [ 35S]GTPgS autoradiography for the 5-HT1A receptor system should provide new information regarding signal transduction in specific brain regions. Q 1997 Elsevier Science Inc.
The relevance of 5-HT 1A receptors to psychiatric disorders makes it especially important to examine functional measures of 5-HT 1A 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 a 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]GTPgS binding in the presence of excess GDP, which reduces basal binding and allows detection of agonist-stimulated binding [14,32]. The [ 35S]GTPgS membrane binding assay has been applied to the study of several G-protein–coupled receptors, including the cloned human 5-HT 1A receptor [30]. Recently, this methodology was adapted to in vitro autoradiography of agoniststimulated [ 35S]GTPgS binding in brain sections [40]. When sections are incubated with appropriate agonists with [ 35S]GTPgS 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 Gproteins in brain membranes and sections by the 5-HT 1A agonist 8-OH-DPAT, and report on the neuroanatomical distribution of 5-HT 1A receptor-activated G-proteins in rat brain.
KEY WORDS: [ 35S]GTPgS autoradiography, G-protein–coupled receptors, 5-HT, 5-HT1A receptors.
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-HT 1A receptor, which has been implicated in depression, anxiety, panic disorder, and alcohol abuse [3,7,9,17,21,28,37,38]. The 5-HT 1A receptor acts via pertussis toxin sensitive G-proteins [5,8,12] to inhibit adenylyl cyclase [6] and increase potassium channel conductance [43,49]. The 5-HT 1A 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]. 1
MATERIALS AND METHODS Male Sprague–Dawley rats (200 g) were purchased from Zivic–Miller (Zelienople, PA). [ 35S]GTPgS (1000 Ci/mmol) was purchased from New England Nuclear Corp. (Boston, MA). R( / )-8-hydroxy-2-(di-n-propylamino)tetralin HBr (8-OHDPAT), pindobind-5-HT 1A , methiothepin mesylate, ketanserin tartrate, ritanserin, and spiperone-HCl were purchased from Research Biochemicals International (Natick, MA). GTPgS and GDP were purchased from Boehringer Mannheim (New York, NY). Reflectionst autoradiography film was purchased from New England Nuclear Corp. (Boston, MA). All other reagent
To whom requests for reprints should be addressed.
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grade chemicals were obtained from Sigma Chemical Co. or Fisher. Agonist-Stimulated [ 35 S]GTPgS Binding in Membranes [ 35S]GTPgS binding was performed as previously described [40]. Rat hippocampus was dissected and homogenized in 20 vol cold Tris-Mg 2/ buffer (50 mM Tris-HCl, 3 mM MgCl2 , 1 mM EGTA, pH 7.4) with a Polytron. The homogenate was centrifuged at 48,000 1 g at 47C for 10 min, resuspended in buffer, centrifuged, and resuspended in assay buffer (50 mM Tris-HCl, 3 mM MgCl2 , 0.2 mM EGTA, 100 mM NaCl, pH 7.4). Membranes (10 mg protein) were incubated at 307C for 1 h in assay buffer with various concentrations of 8-OH-DPAT, with or without antagonists, in the presence of 20 mM GDP and 0.05 nM [ 35S]GTPgS in a 1 ml total volume. Basal binding was measured in the absence of agonist and antagonist, and nonspecific binding was assessed with 10 mM GTPgS. Experiments were also performed using 1 mM 8-OH-DPAT with varying concentrations of 1) GDP (0.5–50 mM) and 2) GTPgS (0.02–200 nM). The reaction was terminated by rapid filtration under vacuum through Whatman GF/B glass fiber filters, followed by three washes with cold Tris buffer (50 mM Tris-HCl, pH 7.4). Bound radioactivity was determined by liquid scintillation spectrophotometry, at 95% efficiency for 35S, after overnight extraction of the filters in 5 ml Ecolite scintillation fluid. Data are reported as mean { SE values of at least three experiments that were each performed in triplicate. Nonlinear iterative regression analyses of agonist concentration–effect curves were performed with JMP (SAS Institute, Cary, NC). The resulting ED50 values were used to determine Ke values for antagonism of the agonist-stimulated response, using the relationship (Ke Å [Ant]/(DR 0 1), where [Ant] is the concentration of antagonist, and DR is the ratio of ED50 values in the presence and absence of antagonist. Scatchard analysis of [ 35S]GTPgS binding was fit to one- or two-site models by LIGAND.
[14,40,44]. To determine the optimal concentration of GDP for 8-OH-DPAT–stimulated [ 35 S]GTPgS binding in membranes, hippocampal membranes were incubated with 1 mM 8-OHDPAT in the presence of 0.5 to 50 mM GDP (Fig. 1). GDP inhibited basal [ 35S]GTPgS binding in a concentration-dependent manner (Fig. 1A), with less than 10% of total [ 35S]GTPgS binding remaining with 50 mM GDP. Addition of 8-OH-DPAT had little effect on [ 35S]GTPgS binding at concentrations of GDP below 1 mM, but significantly stimulated binding at GDP concentrations higher than 1 mM. The relative percent stimulation of [ 35S]GTPgS binding by 8-OH-DPAT was increased with increasing concentrations of GDP, so that the agonist stimulation was greater than twofold at GDP concentrations greater than 10 mM (Fig. 1B). From these data, an optimal GDP concentration in producing significant percent stimulation by 8-OH-DPAT, while maintaining reasonable levels of basal [ 35S]GTPgS binding, was determined to be 20 mM.
Agonist-Stimulated [ 35 S]GTPgS Autoradiography [ 35S]GTPgS autoradiography was performed as previously described [40]. Animals were sacrificed and brains were removed and frozen in isopentane at 0357C. Twenty micron horizontal or coronal brain sections were cut on a cryostat, thaw mounted onto gelatin coated slides, dried under a vacuum and stored desiccated at 0807C until use. Slides were incubated in assay buffer for 10 min at 257C, then in 2 mM GDP in assay buffer at 257C for 15 min. Slides were then incubated with 0.3 mM 8-OH-DPAT with 2 mM GDP and 0.04 nM [ 35S]GTPgS in assay buffer at 257C for 2 h, with and without 0.2 mM methiothepin. Basal binding was assessed in the absence of agonist and antagonist. Slides were rinsed twice for 2 min each in cold 50 mM Tris buffer and once (30 s) in deionized H2O. Slides were dried well and exposed to film for 48–72 h. Films were digitized with a Sony XC-77 video camera and analyzed with the NIH IMAGE program for Macintosh computers. RESULTS 5HT1A Agonist-Stimulated [ 35 S]GTPgS Binding in Membranes [ 35S]GTPgS binding assays were performed in isolated rat hippocampal membranes to pharmacologically characterize 5HT 1A –stimulated [ 35S]GTPgS binding. For other G-protein– coupled receptors, the percent stimulation of [ 35S]GTPgS binding by agonists depends upon the concentration of GDP: increased GDP concentration decreases basal [ 35S]GTPgS binding and increases percent stimulation of binding by agonist
FIG. 1. Effect of GDP on basal and 8-OH-DPAT–stimulated [ 35S]GTPgS binding. Hippocampal membranes were incubated with 0.05 nM [ 35S]GTPgS, 1 mM 8-OH-DPAT, and 0.5–50 mM GDP. Values are expressed as (A) percent total [ 35S]GTPgS binding (as determined in the absence of GDP), and (B) percent stimulation of [ 35S]GTPgS binding. In the absence of GDP, basal [ 35S]GTPgS binding was 2174 { 75 fmol/mg protein.
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41 TABLE 1 EFFECT OF 5-HT ANTAGONISTS ON STIMULATION OF [35S]GTPgS BINDING BY 8OH-DPAT IN RAT HIPPOCAMPAL MEMBRANES Antagonist (mM)
None Ritanserin (0.2 mM) Ketanserin (0.2 mM) Spiperone (0.2 mM) Methiothepin (0.2 mM) Pindobind-5-HT1A (0.2 mM)
FIG. 2. Concentration–effect curves of 8-OH-DPAT–stimulated [ 35S]GTPgS binding in the presence and absence of 5-HT 1 / 1A antagonists. Hippocampal membranes were incubated with 20 mM GDP, 0.05 nM [ 35S]GTPgS, and various concentrations of 8-OH-DPAT, with and without 0.2 mM methiothepin, spiperone, or pindobind-5-HT 1A . Values are expressed as percent stimulation of [ 35S]GTPgS binding { SE.
Under these conditions, concentration–effect curves showed that stimulation of [ 35S]GTPgS binding by 8-OH-DPAT was concentration dependent and saturable (Fig. 2). Maximal stimulation (Emax ) of [ 35S]GTPgS binding by 8-OH-DPAT was 136 { 3% compared to basal, with an ED50 value of 25 { 5 nM. From these data, a concentration of 0.3 mM 8-OH-DPAT was chosen for use in autoradiography experiments in brain sections (see below). The pharmacological specificity of 5-HT 1A –stimulated [ 25S]GTPgS binding was determined by incubating hippocampal membranes with 0.3 mM of 8-OH-DPAT in the presence of 0.2 mM serotonergic antagonists (Table 1). In the absence of antagonist, 8-OH-DPAT stimulated [ 35S]GTPgS binding by approximately twofold (104%). Neither of the 5-HT 2 -selective antagonists, ritanserin or ketanserin, had any significant effect on 8-OH-DPAT–stimulated [ 35S]GTPgS binding. In contrast, addition of antagonists with selectivity at 5-HT 1 / 1A receptors, including spiperone, methiothepin, and pindobind-5-HT 1A , significantly reduced 8-OH-DPAT–stimulated [ 35S]GTPgS binding, with methiothepin (0.2 mM) inhibiting the agonist effect by greater than 90%. Potencies of these antagonists were determined by Ke analysis, using single concentrations of antagonist to increase agonist ED50 values. Results (Fig. 2) showed that all three 5-HT 1 / 1A antagonists acted in a manner consistent with competitive antagonists by increasing agonist ED50 values with no apparent effect on the Emax . From these data, the estimated Ke values were 12.3 { 0.9 nM for spiperone, 14.3 { 0.1 nM for pindobind-5-HT 1A , and 2.4 { 0.2 nM for methiothepin. Previous opioid-stimulated [ 35S]GTPgS binding studies showed that addition of agonist induced high-affinity [ 35S]GTPgS binding sites that could be separated from the lowaffinity sites of basal [ 35S]GTPgS binding by net Scatchard analysis [41,44]. Similar effects were observed in the 5-HT 1 / 1A receptor system (Fig. 3). Scatchard analysis was performed using increasing concentrations of GTPgS in the presence and absence of 1 mM 8-OH-DPAT, as previously described for opioid receptors [39,41]. Both basal and 8-OH-DPAT–stimulated [ 35S]GTPgS binding exhibited multiphasic Scatchard plots that were best fit to two-site models. For both basal and agonist-stimulated [ 35S]GTPgS binding, the low-affinity binding compo-
% Stimulation
104 103 101 26 6 36
{ { { { { {
2 2 3 8 3 2
Rat hippocampal membranes were incubated with 0.05 nM [35S]GTPgS, 20 mM GDP and 0.1 mM 8-OH-DPAT in the presence and absence of 0.2 mM of various 5-HT receptor antagonists. Results are expressed as mean values of percent stimulation by 8-OH-DPAT { SE.
nents were essentially identical, with KD values for GTPgS ranging between 350–500 nM. The basal high affinity site exhibited a KD of 10.6 { 3 nM, with a Bmax of 3.5 { 0.3 pmol/mg protein. Addition of agonist decreased the KD of the high affinity site, with a KD of 3.2 { 0.8 nM and a Bmax of 3.9 { 1.0 pmol/mg. The inset of Fig. 3 shows net Scatchard analysis, in which net agonist-stimulated [ 35S]GTPgS binding was calculated by subtracting basal from 8-OH-DPAT–stimulated [ 35S]GTPgS binding at each concentration of GTPgS. The net Scatchard plot was monophasic, with a KD of 2.3 { 0.3 nM and a Bmax of 3.0 { 0.5 pmol/mg, similar to the corresponding values for the high affinity site of the biphasic plot for 8-OH-DPAT–stimulated binding. 5HT1A Agonist-Stimulated [ 35 S]GTPgS Autoradiography Recent studies have shown that receptor activation of G-proteins can be localized in brain sections by in vitro autoradiogra-
FIG. 3. Scatchard analysis of [ 35S]GTPgS binding in hippocampal membranes. Membranes were incubated with 0.05 nM [ 35S]GTPgS in the presence of 0–200 nM GTPgS with and without 1 mM 8-OH-DPAT. Data are from a typical experiment that was performed in triplicate and replicated three times. Inset: Scatchard analysis of net 8-OH-DPAT– stimulated [ 35S]GTPgS binding (agonist-stimulated binding minus basal binding at each concentration of GTPgS).
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phy of agonist-stimulated [ 35S]GTPgS binding using opioid, cannabinoid and GABAB agonists [40]. To determine whether 5-HT 1 / 1A receptors can be localized using the same technique, rat brain sections were incubated with 0.3 mM 8-OH-DPAT, in the presence and absence of 0.2 mM of the 5-HT 1 / 1A antagonist methiothepin. These concentrations of agonist and antagonist were optimized in membrane experiments using 8-OH-DPAT–stimulated [ 35S]GTPgS binding. Representative sections are shown in Fig. 4. In the sections shown in Fig. 4A, the highest levels of 5-HT 1 / 1A -stimulated [ 35S]GTPgS binding were found in the lateral septum. A low level of 8-OH-DPAT–stimulated [ 35S]GTPgS binding was also found in the cortex. No significant stimulation of [ 35S]GTPgS binding was found in the caudateputamen. The addition of methiothepin inhibited 8-OH-DPAT stimulation of [ 35S]GTPgS binding, so that these sections were comparable to basal [ 35S]GTPgS binding. High levels of 8-OHDPAT–stimulated [ 35S]GTPgS binding were also found throughout the rostral–caudal extent of the hippocampus. This is shown in Fig. 4B, where particularly high 5-HT 1A –stimulated [ 35S]GTPgS binding was observed in the dentate gyrus. In the mesencephalon, 5-HT 1A –stimulated [ 35S]GTPgS binding was found in the dorsal raphe nucleus (Fig. 4C). 8-OH-DPAT–stimulated [ 35S]GTPgS binding was also observed in the entorhinal cortex and subiculum in these sections. The addition of methiothepin again inhibited agonist-stimulated [ 35S]GTPgS binding, verifying the receptor specificity of the effect. Another view of the anatomical distribution of 5-HT 1A –stimulated [ 35S]GTPgS binding is seen the horizontal sections in Fig. 4D, where agoniststimulated [ 35S]GTPgS binding is seen in the lateral septum, prelimbic cortex, hippocampus, subiculum, entorhinal cortex, and dorsal raphe nucleus. DISCUSSION Although 5-HT 1A receptor-stimulated [ 35S]GTPgS binding has been recently reported in membranes from cells transfected with 5-HT 1A 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-HT 1A –stimulated [ 35S]GTPgS binding is significant, with approximately twofold stimulation in the presence of 20 mM GDP. As shown in Fig. 1, this level of stimulation can be altered by varying GDP concentrations, with higher GDP levels producing lower net binding values but higher percent stimulation. The level of stimulation by 8-OH-DPAT is somewhat lower than the threefold stimulation reported for 5-HT 1A receptor-transfected CHO cells [30], but this difference is explained by the heterogeneous nature of brain membranes compared to membranes from transfected cells. This level of stimulation by 8-OH-DPAT was maintained at concentrations of GTPgS at least to 10 nM, so that this level of stimulation is not an artifact of a low concentration of radioligand compared to the number of G-protein a subunits. The potency of 8-OH-DPAT in stimulating [ 35S]GTPgS binding in hippocampal membranes, 25 nM, is similar to that reported for this agonist in transfected cells [30]; moreover, the Ke values of the antagonists spiperone, pindobind 5-HT 1A and methiothepin, are similar to the potencies of these antagonists in binding to 5-HT 1A receptors [18,23,26]. The [ 35S]GTPgS Scatchard analysis (Fig. 3) shows the typical biphasic nature that has been reported for other receptor systems [44]; the fact that the KD and Bmax for the net Scatchard analysis are the same as corresponding values for the high affinity site of agonist-stimulated [ 35S]GTPgS binding, shows that net Scatchard analysis is a valid method for quantifying these parameters without the complexities of multisite analysis. The Bmax
value for 8-OH-DAPT–stimulated [ 35S]GTPgS binding in hippocampal membranes was 3 pmol/mg. Interestingly, measurements of G-protein a subunit content in brain are within the same order of magnitude. For example, the level of Gia2 in hippocampal membranes has been estimated as 13 pmol/mg [2]. Because 5-HT 1A receptors are likely to be coupled to more than one Gprotein subtype, and because a number of other G-protein-coupled receptors exist in hippocampus, these numbers may be valid comparisons. In many cell culture systems, basal [ 35S]GTPgS binding exhibits a single low affinity site with KD ú 100 nM [44]; however, the present study in rat hippocampal membranes confirms previous results in rat striatal membranes [41] that basal [ 35S]GTPgS binding contains a moderate-affinity site of 10–20 nM. The significance of this site in brain membranes is not clear; it could represent precoupled (i.e., spontaneously-active receptors) or may not in fact be relevant to receptor-transducer coupling. In any case, regardless of the tissue or receptor, addition of agonist produces high affinity [ 35S]GTPgS sites with KD values between 1–3 nM. 5-HT 1A receptors have been localized in the brain by traditional receptor binding autoradiography [24,34], while 5-HT 1A mediated second messenger effects, including inhibition of adenylyl cyclase [6] and activation of potassium channels [43,49], have been identified in cells and isolated membranes. The use of in vitro autoradiography of agonist-stimulated [ 35S]GTPgS binding represents a combination of these two approaches: it combines the neuroanatomical resolution of autoradiography with the functional information of a signal transduction assay. The functional aspects of this methodology can be seen in the present study by the fundamentally different effects of agonists and antagonists of the 5-HT 1A receptor system. The agonist 8OH-DPAT produced significant stimulation of [ 35S]GTPgS binding that was eliminated by the addition of 5-HT 1 / 1A , but not 5-HT 2 , antagonists. Moreover, the antagonists had no effect on their own. These results demonstrate that [ 35S]GTPgS autoradiography in brain sections can be used to localize an intracellular signal transduction mechanism (in this case, activation of G-protein a subunits) when performed in the presence of appropriate concentrations of agonist and GDP. Although opioid, cannabinoid, GABAB , and ORL-1 receptors have previously been localized by agonist-stimulated [ 35S]GTPgS autoradiography [40,42], the present study represents the first monoaminergic agonist to be used successfully in the autoradiographic assay. Attempts in our laboratory to adapt this technique to other monoaminergic receptors, particularly dopamine receptors, have been less successful (L. Sim and S. Childers, unpublished observations). It is clear that the [ 35S]GTPgS binding signal depends upon both the number of receptors and the efficiency of coupling between receptors and G-proteins [40,41]; therefore, G-protein activity stimulated by monoaminergic receptors with low receptor number and/or low catalytic amplification (i.e., the number of G-proteins activated per receptor) may be difficult to detect. It appears that the combination of receptor number and catalytic amplification is sufficiently high in the 5-HT 1A receptor system in the brain to detect a considerable signal. As demonstrated in Fig. 1, the magnitude of this signal depends upon the concentration of GDP, and in membranes 20 mM GDP was optimal for agonist stimulation of [ 35S]GTPgS binding. However, as we have reported for other receptor systems [40], the concentration of GDP necessary in the autoradiographic assay is approximately a hundred times higher (1–2 mM GDP) than that required for the [ 35S]GTPgS assay in membranes. The reason for this concentration difference between membranes and sections is not clear, but may be due to either the high level of protein in brain sections or differences
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FIG. 4. Representative sections of rat brain showing the distribution of 5-HT 1A -stimulated [ 35S]GTPgS binding, in the presence and absence of antagonist, compared to basal [ 35S]GTPgS binding. Sections were incubated with 2 mM GDP, then with 0.04 nM [ 35S]GTPgS, 2 mM GDP, and 0.3 mM 8-OH-DPAT, with and without 0.2 mM methiothepin. Basal binding was assessed in the absence of agonist. Coronal sections are shown at the level of (A) septum, (B) hippocampus, and (C) dorsal raphe nucleus. 8-OH-DPAT-stimulated [ 35S]GTPgS binding in these regions, as well as the entorhinal cortex and prelimbic cortex, is also shown in the autoradiogram of a horizontal brain section in Fig. 4D.
between the kinetics of G-protein binding in situ compared to isolated membranes. The receptor specificity of 8-OH-DPAT–stimulated [ 35S]GTPgS binding was verified by several criteria. Agoniststimulated [ 35S]GTPgS binding was blocked by the addition of the 5-HT 1 antagonist methiothepin. The anatomical distribution of 8-OH-DPAT–stimulated [ 35S]GTPgS binding also correlated with that of 5-HT 1A receptors [24,34] with high levels of both
receptors and receptor-activated G-proteins in the hippocampus, septum, cortex, and dorsal raphe nucleus. This is in contrast to other serotonergic receptors. For example, 5-HT 1B receptors are highest in the basal ganglia and substantia nigra [34], and 5-HT 2 receptors are found in the cortex, nucleus accumbens, claustrum, caudate-putamen, and choroid plexus [27,33]. The areas in which high levels of 5-HT 1A -stimulated [ 35S]GTPgS binding were identified (i.e., hippocampus, septum,
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cortex, and dorsal raphe nucleus) are components of the limbic system. The serotonergic system, and particularly the 5-HT 1A receptor, has been implicated in depression, because 5-HT 1A receptor agonists alleviate depression in animal models [3,7,17]. However, the effects of antidepressant drugs on 5-HT 1A receptors [13,15,36,47,48] and 5-HT 1A –mediated inhibition of adenylyl cyclase [29,31,45] are equivocal. There is also evidence that antidepressant treatment alters the levels of G-protein a subunit protein and mRNA in the brain [19,20], indicating that antidepressants may act in part via alterations in signal transduction pathways. However, changes in G-protein levels may not necessarily accompany changes in function [22], and membrane proteins such as tubulin may be important modulators in regulating G-protein a subunits during antidepressant treatment [4]. For this reason, it is crucial to have a direct functional measure of those G-proteins that are coupled to specific receptors. Thus, the [ 35S]GTPgS binding technique may be particularly important in the analysis of the underlying neurochemical disturbances that lead to depression, as well as elucidation of the mechanisms by which antidepressant drugs act. 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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