Introduction

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Of the ~15 serotonin receptors that have been identified to date (Boess and Martin, 1994), the 5-hydroxytryptamine (5-HT)_1A receptor has arguably received the most attention, primarily because selective 5-HT_1A receptor agonists (buspirone, ipsapirone, and gepirone) display anxiolytic/antidepressant effects, and because considerable evidence has accumulated that its function is altered after repeated treatment with anxiolytic and antidepressant drugs (Blier and De Montigny, 1994). Very recent studies in 5-HT_1A receptor knockout mice (Heisler et al., 1998; Parks et al., 1998; Ramboz et al., 1998) have directly demonstrated the importance of this receptor in animal models of anxiety and depression: the mutant animals exhibited both increased anxiety and behaviors similar to those observed after antidepressant treatment. The 5-HT_1A receptor is located both presynaptically on 5-HT cell bodies (as somatodendritic autoreceptors) in the dorsal and median raphe nuclei, and postsynaptically primarily in the limbic system (hippocampus, lateral septum, entorhinal and medial prefrontal cortex) (Vergé et al., 1986; Pompeiano et al., 1992; Kia et al., 1996). [Terminal autoreceptors are not 5-HT_1A (Vergé et al., 1985) but 5-HT_1B/D (Sari et al., 1997)]. Somatodendritic autoreceptor stimulation inhibits the firing of 5-HT neurons (Sprouse and Aghajanian, 1988) via membrane hyperpolarization consequent to activation of a pertussis toxin-sensitive G protein-coupled K⁺ conductance (Innis and Aghajanian, 1987); the decrease in impulse flow results in a decrease in 5-HT synthesis (Hjorth and Magnusson, 1988; Meller et al., 1990) and release (Hjorth and Sharp, 1991) in terminal areas innervated by these neurons. Some postsynaptic 5-HT_1A receptors (e.g., in hippocampus) also mediate a hyperpolarizing response by increasing a K⁺ conductance (Beck et al., 1992), whereas others (whose physiological function is unclear) are coupled to inhibition of forskolin-stimulated adenylyl cyclase activity (Bockaert et al., 1987; Yocca et al., 1992).

Studies assessing 5-HT_1A receptor function were complicated by findings that the potency and intrinsic activity of various 5-HT_1A receptor drugs differed when examined in models of pre- and postsynaptic receptor activation; these drugs were more potent and efficacious at somatodendritic autoreceptors in the dorsal raphe than at postsynaptic receptors in, e.g., the hippocampus (for review, see Meller et al., 1990). In a series of studies using a variety of pre- and postsynaptic models of 5-HT_1A receptor function, we showed that this was due to differences in receptor/effector coupling efficiency (Meller et al., 1990, 1992; Yocca et al., 1992; Cox et al., 1993), methodologically defined as differences in receptor reserve (Furchgott and Bursztyn, 1967): somatodendritic autoreceptors displayed a large receptor reserve, whereas postsynaptic receptors did not. From a pharmacological perspective, this served to explain how a particular drug, such as the weak partial agonist BMY 7378, could demonstrate apparent full agonism in the dorsal raphe yet act as an antagonist at postsynaptic receptor sites in the hippocampus (Meller et al., 1990). Although these studies proved valuable in defining the pharmacological activity of 5-HT_1A receptor drugs for eliciting various functional responses, they provided only an overall assessment of the efficiency of the signal transduction cascade between receptor occupation and response at pre- and postsynaptic 5-HT_1A receptor sites.

A direct measure of the initial, activation step of receptor/G protein coupling can be obtained from agonist-stimulated guanosine-5'-O-(3-thio)triphosphate ([³⁵S]GTPS) binding to receptors (Lazareno and Birdsall, 1993; Wieland and Jakobs, 1994). Recently, agonist-stimulated binding of [³⁵S]GTPS to 5-HT_1A receptors has been demonstrated in hippocampal membranes (Sim et al., 1997; Alper and Nelson, 1998), cloned cell membranes (Newman-Tancredi et al., 1997), and brain sections by autoradiography (Sim et al., 1997; Waeber and Moskowitz, 1997; Dupuis et al., 1998b). In the present study, this technique was used to determine whether previously described regional differences in receptor/effector-coupling efficiency are demonstrable at the level of receptor/G protein coupling. Two consequences would be expected if this were the case. First, the potency of a full agonist for stimulating [³⁵S]GTPS binding should be greater (i.e., lower EC₅₀) at somatodendritic autoreceptors in the dorsal raphe than at postsynaptic receptors in the hippocampus. Second, partial irreversible blockade of 5-HT_1A receptors should shift the EC₅₀ for the agonist to the right in the dorsal raphe (Meller et al., 1990) but only reduce the maximum response (without altering the EC₅₀) in the hippocampus (Yocca et al., 1992). The present results demonstrate that neither of these expectations was fulfilled.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Animals. Male Sprague-Dawley rats (200-250 g; Taconic Farms, Germantown, NY) were maintained on a 12-h light/dark cycle and housed four per cage with food and water ad libitum.

Materials. 5-HT hydrochloride, R-(+)-8-hydroxy-dipropylaminotetralin hydrobromide (8-OH-DPAT), (±)-8-OH-DPAT, 5-carboxamidotryptamine (5-CT) maleate, N,N-dipropyl-5-carboxamidotryptamine (N,N-DP-5-CT) maleate, WAY100,635 {N-[2-[4-(2-methoxyphenyl)-1- piperazinyl]ethyl]-N-2-pyridinyl-cyclohexanecarboxamide} maleate, phentolamine mesylate, and phenoxybenzamine hydrochloride (PBZ) were obtained from Research Biochemicals (Natick, MA). EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) was purchased from Aldrich Chemicals (Milwaukee, WI), adenosine deaminase, GDP, and GTPS were purchased from Sigma Chemical Co. (St. Louis, MO), and [³⁵S]GTPS (1250 Ci/mmol) was obtained from NEN (Boston, MA). SuperFrost/Plus slides were purchased from Fisher Scientific Co. (Pittsburgh, PA).

Drug Treatments and Tissue Dissections. In some experiments, rats were injected with vehicle or the irreversible receptor antagonist EEDQ (1 mg/kg s.c.) and sacrificed 24 h later. Whole hippocampus, cerebral cortex, and corpus striatum were grossly dissected as described previously (Meller et al., 1990; Meller and Bohmaker, 1996).

[³⁵S]GTPS Binding in Membranes. The method is essentially identical with that described by Sim et al. (1997). Brain tissues were homogenized in ice-cold homogenization buffer (50 mM Tris-HCl, 3 mM MgCl₂, 1 mM EGTA, pH 7.4), centrifuged at 48,000g for 10 min at 4°C, and pellets were washed once by resuspension. Membrane pellets were resuspended in assay buffer (50 mM Tris-HCl, 3 mM MgCl₂, 0.2 mM EGTA, 100 mM NaCl, pH 7.4) and stored in aliquots at 70°C. Thawed aliquots were diluted with assay buffer and preincubated at 30°C for 10 min with adenosine deaminase (10 mU/ml final concentration) to reduce basal binding (Sim et al., 1998), centrifuged, and resuspended in assay buffer at a protein concentration of 100 µg/ml. In some experiments, membranes were exposed to the receptor alkylating PBZ (0.3 or 1 µM final concentration) or vehicle during the 10-min preincubation period and were washed once by resuspension. Membranes (10 µg of protein) were incubated for 1 h at 30°C with 0.05 nM [³⁵S]GTPS and 20 µM GDP in the presence or absence of various concentrations of drugs (1 ml total volume). Basal binding was measured in the absence of drugs, and nonspecific binding in the presence of 10 µM nonradioactive GTPS. The reaction was stopped by rapid filtration over Whatman GF/B filters, the filters were washed with 3 × 5 ml of ice-cold wash buffer (50 mM Tris-HCl, pH 7.4) and bound radioactivity was quantitated by liquid scintillation counting.

[³⁵S]GTPS Autoradiography. The method is identical with that described by Childers and colleagues (Sim et al., 1997). Harvested brains were frozen by slow immersion in 2-methylbutane maintained at 35°C and stored frozen at 70°C. Coronal 12-µm sections were cut on a cryostat (Reichert-Jung model 2800 Frigocut N) maintained at 17°C, with the atlas of Paxinos and Watson (Paxinos and Watson, 1986) to identify regions of interest. Sections were thaw-mounted on SuperFrost/Plus slides and stored on ice during collection. The slides were dried under vacuum overnight at 4°C, then stored with dessicant at 70°C. Thawed slides were dried in a stream of cool air for 30 min and transferred to five-slide plastic mailers. Sections were equilibrated in assay buffer (50 mM Tris-HCl, 3 mM MgCl₂, 0.2 mM EGTA, 100 mM NaCl, pH 7.4) for 10 min at 25°C; in some experiments PBZ (10 µM) or vehicle was present during this equilibration period. Sections were then preincubated in assay buffer containing 2 mM GDP, adenosine deaminase (10 mU/ml final volume) and, where appropriate, antagonist drugs (WAY100,635 or phentolamine), for 15 min at 25°C, followed by incubation (2 h; 25°C) in fresh assay buffer containing 0.04 nM [³⁵S]GTPS, 2 mM GDP, and adenosine deaminase in the presence or absence of various concentrations of agonist/antagonist drugs. Basal binding was carried out in the absence of any drugs and nonspecific binding in the presence of 10 µM GTPS. Incubations were terminated by two washes (2 min each) in ice-cold wash buffer (50 mM Tris-HCl, pH 7.0) and a brief rinse in ice-cold distilled water. Sections were dried overnight at room temperature and apposed to autoradiographic film (Hyperfilm max; Amersham, Arlington Heights, IL) for 3 to 5 days. Autoradiograms were analyzed with a computerized image analysis system (MCID-M4; Imaging Research, St. Catherines, Ontario, Canada). In a standard assay, various amounts of [³⁵S]GTPS were added to brain pastes, which were frozen and cut on a cryostat. Brain paste sections were exposed to autoradiographic film together with [¹⁴C] microscales (Amersham), which allowed for conversion of optical densities to nCi [³⁵S]/mg tissue equivalent (Sim et al., 1995). However, it was found that radioactivity and optical density were linearly correlated at least up to A_1.5; therefore, autoradiographic films (maximum A, <1.0) were routinely subjected to relative quantitation, and data were plotted as percentage of increase above basal.

Data Analysis. All dose-response curves were fit with the ALLFIT program of De Lean et al. (1978). This iterative program allowed dose-response curves to be simultaneously analyzed for best fit with the four-parameter logistic equation Y = (a  d)/[(1 + X/c)^b] + d, where a is the response at zero dose, b is the slope factor, c is the ED₅₀, and d is the response at "infinite" dose. Y is the response elicited by a particular dose X. Fits were obtained with and without constraints being placed on values for these parameters. The program calculated partial F tests, which were used to determine whether a particular set of constraints significantly worsened the fit obtained relative to a standard set of constraints or no constraints at all. Extensive use of the program has been described previously (Meller et al., 1990, 1992; Yocca et al., 1992; Cox et al., 1993).

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

5-HT_1A Receptor Specificity in Hippocampal Membranes. Dose-response curves for a number of 5-HT agonists were generated in hippocampal membranes (Fig. 1). The mean EC₅₀ and E_max values are shown in Table 1. The rank potency order of the tested agonists was identical with their rank order of affinity (Hibert et al., 1987) for 5-HT_1A receptors (5-CT  N,N-DP-5-CT > R-(+)-8-OH-DPAT > 5-HT > ipsapirone). For R-(+)-8-OH-DPAT, the EC₅₀ (28 nM) and E_max (127% stimulation above basal) were essentially identical with those reported previously (Sim et al., 1997). Because the stimulation elicited by the nonspecific 5-HT₁ agonists 5-HT and 5-CT was ~30% greater than that produced by the selective 5-HT_1A agonists R-(+)-8-OH-DPAT and N,N-DP-5-CT, we investigated the possibility that the former drugs were stimulating additional 5-HT₁ receptor subtypes (5-HT_1B-F) to yield a greater percentage of stimulation. Indeed, the selective 5-HT_1A receptor antagonist WAY100,635 (1 µM) (Forster et al., 1995) completely abolished the stimulation of [³⁵S]GTPS binding produced by 1 µM R-(+)-8-OH-DPAT or 0.1 µM N,N-DP-5-CT, but only eliminated 70% of the increase produced by 0.3 µM 5-CT (Fig. 2), supporting the idea that ~30% of the stimulation produced by 5-CT was via non-5-HT_1A receptor sites. Although WAY100,635 shows some affinity (~100-fold less) for ₁-receptors (Forster et al., 1995), the -adrenergic antagonist phentolamine (1 µM) did not alter R-(+)-8-OH-DPAT-stimulated [³⁵S]GTPS binding (data not shown). As expected (Pauwels et al., 1997; Alper and Nelson, 1998), racemic 8-OH-DPAT displayed lower intrinsic activity, and the known partial agonist ipsapirone displayed ~45% of the intrinsic activity of R-(+)-8-OH-DPAT; WAY100,635 behaved as a neutral antagonist (Fig. 1; Table 1). Based on these results, R-(+)-8-OH-DPAT was used to stimulate [³⁵S]GTPS binding in all subsequent studies.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Dose-response curves for agonist-stimulated binding of [35S]GTPS binding in hippocampal membranes. Each curve is the mean ± S.E. of three independent experiments carried out in quadruplicate.

View this table: [in this window] [in a new window]   TABLE 1 5-HT1A receptor-stimulated [35S]GTPS binding in hippocampal membranes The ALLFIT-derived EC50 and Emax values are for the dose-response curves shown in Fig. 1.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Inhibition by WAY100,635 of hippocampal [35S]GTPS binding stimulated by 1 µM R-(+)-8-OH-DPAT, 0.1 µM N,N-DP-5-CT, or 0.3 µM 5-CT. Each curve is the mean ± S.E. of three or four separate assays, each performed in quadruplicate.

R-(+)-8-OH-DPAT-Stimulated [³⁵S]GTPS Binding in Cortex and Striatum. The specificity of 5-HT_1A-receptor-stimulated [³⁵S]GTPS binding by R-(+)-8-OH-DPAT was further substantiated by detection of agonist-stimulated binding in the cerebral cortex but not in the striatum (Fig. 3), which is known to be devoid of 5-HT_1A receptors (Pompeiano et al., 1992). As in the hippocampus, the stimulation in the cortex was completely blocked in the presence of 1 µM WAY100,635 but not by 1 µM phentolamine (data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   R-(+)-8-OH-DPAT stimulated [35S]GTPS binding in membranes of cerebral cortex (EC50 = 14 nM; Emax = 83%) but not corpus striatum. Each point is the mean of duplicate experiments performed in triplicate.

Receptor/G Protein Coupling Efficiency for Agonist-Stimulated [³⁵S]GTPS Binding in Hippocampus. Dose-response curves were generated for R-(+)-8-OH-DPAT-stimulated binding of [³⁵S]GTPS in hippocampal membranes after partial irreversible receptor blockade either in vitro by treatment with PBZ (Fig. 4A) or in vivo by treatment with EEDQ (Fig. 4B). Either treatment reduced the maximal response without altering the EC₅₀, similar to the results obtained for inhibition of forskolin-stimulated adenylyl cyclase in the hippocampus after partial irreversible receptor blockade (Yocca et al., 1992). Thus, as expected, 5-HT_1A receptor/G protein coupling in the hippocampus exhibited low efficiency (i.e., no receptor reserve).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effects of irreversible receptor blockade in vitro (PBZ) and in vivo (EEDQ) on R-(+)-8-OH-DPAT-stimulated [35S]GTPS binding in hippocampal membranes. Each curve is the mean ± S.E. of three or four separate experiments performed in quadruplicate. A, ALLFIT analysis indicated that all three curves could share the same EC50 (29 nM) and slope factor (0.73) without a significant degradation in the fit; both PBZ Emax values, however, differed significantly from control (control, 166%; PBZ 0.3 µM, 76%; PBZ 1 µM, 51%). B, EEDQ treatment did not alter the EC50 for R-(+)-8-OH-DPAT (shared EC50 = 13 nM), but significantly reduced the maximal response from 178 to 60% stimulation.

Autoradiographic Analysis of R-(+)-8-OH-DPAT-Stimulated [³⁵S]GTPS Binding in Different Brain Regions. Figure 5 shows stimulated binding of [³⁵S]GTPS in the dorsal hippocampus as a function of R-(+)-8-OH-DPAT concentration. Highest stimulated binding was observed in the molecular layer of the dentate gyrus and in strata oriens and radiatum, with lower levels in CA2 and CA3. This laminar and subfield distribution corresponds exactly to that of 5-HT_1A receptor-binding sites (Pompeiano et al., 1992). Incubation with the specific 5-HT_1A receptor antagonist WAY100,635 (1 µM) abolished the increase produced by 1 µM R-(+)-8-OH-DPAT, but the -adrenergic antagonist phentolamine (1 µM) had no effect (data not shown). Stimulated binding also was observed in hypothalamic and amygdaloid nuclei, but did not appear to be completely eliminated by WAY 100,635 and was not characterized further. Figure 6 shows the dose dependence for R-(+)-8-OH-DPAT-stimulated binding in the dorsal raphe, which was likewise abolished by 1 µM WAY100,635 but not by phentolamine (data not shown). Typical sections depicting nonspecific, basal and 1 or 10 µM R-(+)-8-OH-DPAT-stimulated binding in the mid-hippocampal formation, the lateral septum, and the medial prefrontal cortex are shown in Fig. 7. The medial prefrontal cortex exhibited the lowest maximal stimulation (~60%) of the brain regions examined. Consistent with the results in membranes (Fig. 3), there was a low level of stimulated binding in the cortex (Figs. 5-7), but not in the striatum (Fig. 7, middle).

View larger version (44K): [in this window] [in a new window]   Fig. 5.   Representative serial sections at the level of the dorsal hippocampus showing stimulation of [35S]GTPS binding as a function of molar R-(+)-8-OH-DPAT concentration. See text for details. NS, nonspecific binding; DG, dentate gyrus; DPAT, R-(+)-8-OH-DPAT; WAY, WAY100,635.

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Dose-dependent stimulation of [35S]GTPS binding by R-(+)-8-OH-DPAT in serial sections at the level of the dorsal raphe (DR) nucleus.

View larger version (39K): [in this window] [in a new window]   Fig. 7.   Representative sections demonstrating R-(+)-8-OH-DPAT-stimulated [35S]GTPS binding in the mid-hippocampal formation, the lateral septum (LS), and the medial prefrontal cortex (mPFC).

View larger version (26K): [in this window] [in a new window]   Fig. 8.   Dose-response curves for R-(+)-8-OH-DPAT-stimulated [35S]GTPS binding in sections of the mid-hippocampal formation, dorsal hippocampus, lateral septum, and dorsal raphe. Each curve is the mean ± S.E. of four to six experiments, each carried out in two to five replicate sections. For dose-response curves analyzed individually, ALLFIT-derived EC50 (in nanomoles/liter) and Emax (percentage of stimulation above basal) parameters, respectively, were as follows: mid-hippocampal formation, 46 and 110; dorsal hippocampus, 96 and 123; lateral septum, 85 and 111; and dorsal raphe, 67 and 83. Simultaneous analysis of all the data yielded a best-fit (i.e., that which allowed the maximal number of parameters to be shared without a significant degradation in the fit) in which all the curves shared a common EC50 (72 nM) and a common slope factor (0.78) and maximum response (114%) for all except the dorsal raphe curve (slope factor, 0.44; maximum, 84%). Fits were significantly worsened when constrained to share a common slope factor (P = .015) or maximum response (P = .001) for all curves.

View larger version (16K): [in this window] [in a new window]   Fig. 9.   Dose-response curves for R-(+)-8-OH-DPAT-stimulated [35S]GTPS binding in sections of the dorsal hippocampus, mid-hippocampal formation, and dorsal raphe treated with vehicle or PBZ (10 µM; 10 min). Curves represent the mean ± S.E. of three to seven (dorsal hippocampus), four to eight (mid-hippocampal formation), or four or five (dorsal raphe) experiments, each with two to four replicates In each brain region, PBZ significantly decreased the maximum response without altering the EC50. ALLFIT-derived parameters were as follows: dorsal hippocampus, shared EC50, 62 nM; Emax, 120 (control) and 59% (PBZ); mid-hippocampal formation, shared EC50, 41 nM; Emax, 123 (control) and 64% (PBZ); and dorsal raphe, shared EC50, 45 nM; Emax, 79 (control) and 51% (PBZ).

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The results obtained in the present study in regard to the distribution of 5-HT_1A receptor-stimulated [³⁵S]GTPS binding sites in brain, and the intrinsic activity and potency of agonists, are in general agreement with those described previously (Sim et al., 1997; Waeber and Moskowitz, 1997; Alper and Nelson, 1998; Dupuis et al., 1998b). For example, in hippocampal membranes, the intrinsic activity (127%) and EC₅₀ (28 nM) values for R-(+)-8-OH-DPAT-stimulated binding were essentially identical with those reported by Sim et al. (1997). The finding that the nonselective agonists 5-HT and 5-CT stimulated ~30% more binding than the selective agonist R-(+)-8-OH-DPAT (Fig. 1), which was incompletely abolished by the selective antagonist WAY100,635 (Fig. 2), was also similar to that reported previously (Alper and Nelson, 1998). Although the identity of 5-HT₁ receptor subtypes that may be mediating the increased stimulation produced by 5-HT and 5-CT were not investigated in the present study, attempts by others to establish that stimulation of [³⁵S]GTPS binding occurs via other 5-HT receptor subtypes, with purported selective agonists or antagonists, have yielded mixed results. In autoradiographic studies in guinea pig brain, a selective 5-HT_1B/1D antagonist blocked 5-CT-stimulated binding in the substantia nigra (rich in 5-HT_1B/1D receptors) but not in other (5-HT_1A receptor-rich) areas (Waeber and Moskowitz, 1997). Agonists with high affinity, and purported selectivity, for 5-HT_1B/1D receptors stimulated binding not only in the substantia nigra but also in areas enriched in 5-HT_1A receptors (hippocampus, lateral septum); the stimulation in the latter areas (but not in substantia nigra) was abolished by a 5-HT_1A receptor antagonist (Waeber and Moskowitz, 1997). Attempts to visualize stimulated binding via 5-HT_1F receptors in areas enriched in this receptor subtype (e.g., claustrum), with drugs with high affinity for the receptor, were unsuccessful; again, however, 5-HT_1A receptor-stimulated response was easily detected (Waeber and Moskowitz, 1997). Others were unable to demonstrate 5-HT_1B/1D receptor-mediated increased binding in guinea pig brain sections by autoradiography; indeed, no matter what the selectivity profile of the drug used, only 5-HT_1A receptor-stimulated responses appeared to be elicited (Dupuis et al., 1998a,b). Thus, although the demonstration of an increased binding of [³⁵S]GTPS via various 5-HT receptor subtypes continues to be problematic, R-(+)-8-OH-DPAT-stimulated binding via 5-HT_1A receptors was both highly specific and generally robust. The agonist-stimulated binding was completely abolished by the highly selective antagonist WAY100,635, but was unaffected by phentolamine, an antagonist of the only other receptor (₁-adrenergic) for which WAY100,635 has moderate affinity (Forster et al., 1995).

Initial experiments to determine whether differences in 5-HT_1A receptor/G protein coupling efficiency are demonstrable at pre- versus postsynaptic sites focused on assessing the receptor reserve for R-(+)-8-OH-DPAT-stimulated [³⁵S]GTPS binding in membranes. As expected, in hippocampal membranes partial irreversible 5-HT_1A receptor blockade, by treatment with either PBZ in vitro or EEDQ in vivo, reduced the maximum response to the agonist but did not alter its EC₅₀ (Fig. 4), indicative of low receptor/G protein coupling efficiency. This finding is consistent with the low overall receptor/effector coupling efficiency observed at postsynaptic sites in hippocampus with the adenylyl cyclase assay (Yocca et al., 1992). Attempts to evaluate coupling efficiency in membranes of dorsal raphe tissue obtained by micropunch were hampered by the need to pool tissue from many animals, the labor-intensiveness of this task, the relatively low yield of protein recovered, and, not least, by an unexpectedly low percentage of stimulation even in control tissue. This is probably due to the difficulty of obtaining, even by micropunch, dorsal raphe uncontaminated by adjacent tissue. These difficulties rendered the generation of dose-response curves in PBZ-treated membranes highly problematic. Consequently, further experiments were carried out using quantitative autoradiographic analysis of brain sections, where the signal was fairly robust even in the dorsal raphe nucleus and dose-dependent stimulation could be readily ascertained (Figs. 5, 6, and 8).

The EC₅₀ for R-(+)-8-OH-DPAT did not differ significantly between the dorsal raphe and either the dorsal or mid-hippocampus (Fig. 8), in contrast to many previous studies, using behavioral and electrophysiological paradigms, that established that 5-HT_1A agonists such as 8-OH-DPAT are more potent in the former region than the latter (see references cited in Meller et al., 1990). The lack of a regional difference in receptor/G protein coupling efficiency indicated by this finding was clearly corroborated by partial irreversible receptor blockade experiments. With brain sections that visualized the dorsal raphe nucleus and the hippocampus at two different levels, PBZ treatment significantly reduced the maximal response to R-(+)-8-OH-DPAT in each region without significantly affecting its EC₅₀ (Fig. 9). The results in the dorsal raphe using this technique stand in marked contrast to a previous study assessing overall receptor/effector coupling efficiency at somatodendritic autoreceptors in vivo, where partial irreversible 5-HT_1A receptor blockade shifted the dose response for 8-OH-DPAT >8-fold to the right (Meller et al., 1990).

The absence of an observable difference in the efficiency of 5-HT_1A receptor/G protein coupling in the dorsal raphe and the hippocampus may be due to one or more factors. First, and most parsimoniously, the high receptor/effector coupling efficiency observed in vivo at raphe nucleus somatodendritic 5-HT_1A autoreceptors may reflect overall amplification of the individual steps in the signal transduction cascade that is not observed at the level of the activation step of receptor/G protein coupling. Differences in 5-HT_1A receptor density, receptor/G protein ratio (i.e., stoichiometry), G protein subunits available for coupling (and their attendant variables such as kinetics and/or equilibria of receptor/G protein binding), and effectors may all contribute to the overall efficiency of receptor/effector coupling (Newman-Tancredi et al., 1997; Pauwels et al., 1997; Selley et al., 1998). [Receptor densities in the two regions are similar, however (Li et al., 1997)]. Interestingly, results paralleling those obtained herein have been reported for the µ-opiate receptor. Classical in vivo receptor inactivation experiments demonstrated that µ-opiate receptor-mediated antinociception displays high receptor/effector coupling efficiency (receptor reserve) (Zernig et al., 1995), whereas µ-opiate receptor-mediated stimulation of [³⁵S]GTPS binding in thalamic membranes exhibited an absence of receptor reserve (using an indirect method of assessment) (Selley et al., 1998).

The present finding of no difference in the efficiency of 5-HT_1A receptor-stimulated G protein activation between the hippocampus and dorsal raphe supports the indirect findings of a previous study in guinea pig brain sections (Dupuis et al., 1998b). These authors found no significant difference in the relative potency and intrinsic activity of several 5-HT_1A agonists, including racemic 8-OH-DPAT, for stimulating [³⁵S]GTPS binding in hippocampus and dorsal raphe and postulated that the high GDP concentrations used may have masked differences in receptor/G protein coupling efficiency (although a differential receptor reserve in these brain regions in this species has not been established). This postulated masking effect may relate to the necessity of using high concentrations of GDP to promote guanine nucleotide exchange and optimize detection of agonist-stimulated [³⁵S]GTPS binding. It has been repeatedly noted that in membranes and brain sections GDP concentrations of 1 to 50 µM and 1 to 2 mM, respectively, are generally required to maximize the percentage of stimulation of binding by agonists of various receptors (Sim et al., 1995). Moreover, many investigators have noted that intrinsic activity differences among agonists are amplified as the concentration of GDP is increased; concomitantly, agonist potencies are reduced (Pauwels et al., 1997; Alper and Nelson, 1998; Selley et al., 1997, 1998). This is analogous to situations where overall receptor/effector coupling efficiency is low (no receptor reserve), and differences in intrinsic activity (maximal tissue response) are directly related to the efficacy of the agonists. In contrast, when receptor/effector coupling efficiency is high (large receptor reserve), even partial agonists display full intrinsic activity, and the potency of agonists is increased. The suggested mechanism (Selley et al., 1997) posits that high efficacy (i.e., full) agonists are inherently better able to overcome the effect of excess GDP to stabilize the inactive (GDP-bound) form of the G protein, and thus at high GDP concentrations the intrinsic activity differences between full and partial agonists are amplified. This hypothesis is supported by the observation that partial agonists are less able to promote the release of prebound GDP and thus display lower intrinsic activity for stimulating [³⁵S]GTPS binding (Lorenzen et al., 1996). However, there is no basis for expecting that high GDP concentrations would differentially affect the potency of a particular agonist [e.g., R-(+)-8-OH-DPAT] in different brain regions. Thus, although high GDP concentrations may reduce the potency of R-(+)-8-OH-DPAT in both brain regions, a true difference in potency (reflecting a difference in receptor/G protein coupling efficiency) should still be discernable. The analysis of receptor/G protein coupling by this technique may, for other unknown reasons, be inherently limited to the detection of low-efficiency coupling for this activation step in the signal transduction cascade, although it may be well suited for defining differences in intrinsic efficacy among agonists (Selley et al., 1998). Further studies (perhaps with different techniques) are needed to determine the contribution coupling efficiency at individual steps of the signal transduction pathway makes to overall assessments of receptor/effector coupling efficiency.