Detailed pharmacological characterization of 5-HT1A-receptor-mediated [35S]GTP gamma S binding in rat hippocampal membranes

5-HT-stimulated [(35)S]GTPgammaS binding to rat hippocampal membranes was pharmacologically characterized. Signal/noise ratio or percent increase over basal was optimized with 100 microM GDP, 2-10 mM MgCl(2), and 150-200 mM NaCl. However, we preferred the standard condition (20 microM GDP, 5 mM MgCl(2), and 100 mM NaCl: Condition I) to the alternative one (100 microM GDP, 5 mM MgCl(2), and 150 mM NaCl: Condition II) because 1) absolute values of basal and 5-HT-sensitive bindings decreased with higher concentrations of GDP and NaCl; 2) EC(50) values determined under Condition II were 2 - 6 fold higher than those under Condition I; 3) some partial agonists had less intrinsic activities in the presence of higher concentrations of GDP; and 4) Inhibitory effects of WAY100635 were complete under Condition I, while incomplete under Condition II. Pharmacological profile of concentration-dependent stimulation by a series of 5-HT ligands and concentration-dependent inhibition of 5-HT-stimulated binding by several 5-HT-receptor antagonists clearly indicated that this response under Condition I was mediated solely through 5-HT(1A) receptors. Although caution should be paid especially to the apparent intrinsic activities susceptible to the assay conditions, this method appears useful to investigate functional coupling between 5-HT(1A) receptors and their coupled G proteins in native hippocampal membranes.     

Effects of a selective 5-HT reuptake blocker,citalopram, on the sensitivity of 5-HT autoreceptors: Electrophysiological studies in the rat brain

Summary Citalopram (CIT), is a selective serotonin (5-HT) reuptake blocker and a clinically effective antidepressant. The present electrophysiological studies were undertaken to investigate in vivo the acute and long-term effects of CIT administration on 5-HT neurotransmission. In a first series of experiments, a single dose of CIT (0.05–0.5 mg/kg) was administered intravenously to naive rats while recording the activity of a 5-HT-containing neuron in the nucleus raphe dorsalis. A dose-response relationship of the inhibitory effect of CIT on the firing activity of 5-HT neurons was obtained with an ED₅₀ of 0.23±0.03 mg/kg. In a second series of experiments, rats were treated with CIT (20 mg/kg/day, i.p.) for 2, 7 and 14 days. In rats treated for 2 days, there was a marked reduction in the firing activity of 5-HT neurons in the nucleus raphe dorsalis; there was a partial recovery after 7 days and a complete recovery after 14 days of treatment. The response of 5-HT neurons to intravenously administered LSD was decreased in rats treated for 14 days with CIT, indicating a desensitization of the somatodendritic 5-HT autoreceptor. In a third series of experiments, carried out in rats treated with CIT (20 mg/kg/day, i.p.) for 14 days, the suppression of firing activity of CA₃ hippocampal pyramidal neurons produced by microiontophoretically-applied 5-HT and by the electrical activation of the ascending 5-HT pathway was measured. Long-term treatment with CIT did not modify the responsiveness of these neurons to microiontophoretically-applied 5-HT; however, the effect of the electrical activation of the ascending 5-HT pathway on these same neurons was enhanced. To determine if 5-HT reuptake blockade could be responsible for this enhancement, CIT (1 mg/kg) was injected intravenously in naive rats while stimulating the ascending 5-HT pathway; it failed to modify the effectiveness of the stimulation. To assess the involvement of the 5-HT terminal autoreceptor, methiothepin, a 5-HT autoreceptor antagonist, was injected intravenously (1 mg/kg) in naive rats and in rats treated for 14 days with CIT while stimulating the ascending 5-HT pathway. Methiothepin enhanced the effect of the stimulation in naive rats but failed to do so in the CIT-treated rats. It is concluded that long-term CIT treatment enhances 5-HT neurotransmission by desensitizing both the somatodendritic and terminal 5-HT autoreceptors.     

Differential effects of the novel antidepressant agomelatine (S 20098) versus fluoxetine on 5-HT1A receptors in the rat brain

Agomelatine (S 20098) is a novel antidepressant drug with melatonin receptor agonist and 5-HT(2C) receptor antagonist properties, but actual mechanisms underlying its antidepressant action are unknown. Because functional desensitization of 5-HT(1A) autoreceptors in the dorsal raphe nucleus (DRN) occurs after chronic administration of several classes of antidepressants, we investigated whether this adaptive change could also be induced by agomelatine. Neither acute nor chronic treatment with agomelatine (10 mg/kg i.p. for 14 days or 50 mg/kg i.p. for 21 days) changed the density of 5-HT(1A) receptors and their coupling with G proteins in the DRN and the hippocampus in rats. Moreover, these treatments did not affect the basal electrophysiological characteristics and the responses to 5-HT(1A) receptor stimulation of DRN and hippocampal neurons in brain slices. Parallel experiments with melatonin (10 mg/kg i.p. for 14 days) and fluoxetine (5 mg/kg i.p. for 14 days) as reference compounds showed that the former was unable to affect 5-HT(1A) receptors whereas the latter decreased both the 5-HT(1A) receptor-mediated [(35)S]GTP-gamma-S binding and the potency of ipsapirone, a 5-HT(1A) receptor agonist, to inhibit neuronal firing in the DRN. These data indicate that the antidepressant action of agomelatine is not mediated through the same mechanisms as SSRIs or tricyclics.     

5-HT1A receptor agonist properties of antipsychotics determined by [35S]GTPgammaS binding in rat hippocampal membranes

1. 5-Hydroxytryptamine 1A (5-HT1A) receptors have attracted increasing attention as a promising target for antipsychotic therapy. Although many atypical antipsychotic drugs, including the prototype clozapine, have been reported to be partial agonists at 5-HT1A receptors, these results are often fragmental and derived mainly from experiments that used cultured cells. 2. In the present study, [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) binding assay in rat hippocampal membranes was applied to a series of antipsychotic drugs, especially atypical antipsychotics. 3. Most, but not all, of atypical antipsychotic drugs and the classical antipsychotic drug nemonapride behaved as partial agonists at 5-HT1A receptors with varied potencies and relative efficacies. The most potent compound was perospirone with a mean EC50 of 27 nmol/L, followed by aripiprazole (45 nmol/L) > ziprasidone (480 nmol/L) > nemonapride (790 nmol/L) > clozapine (3900 nmol/L) > quetiapine (26,000 nmol/L). The maximal percentage increases over the basal binding (%Emax) for these antipsychotic drugs were 30-50%, with the exception of perospirone (approximately 15%), whereas 5-HT stimulated the binding to a mean %Emax of 105%. 4. Increasing concentrations of the selective and neutral 5-HT1A antagonist WAY100635 shifted the concentration-response curve of nemonapride-stimulated [35S]GTPgammaS binding to the right and in parallel. 5. The relative efficacy or intrinsic activity of a compound was affected differently by the differing concentrations of guanosine diphosphate (GDP) in the assay buffer, which should be taken into consideration when determining the relative efficacies of these antipsychotics as 5-HT1A receptor agonists. 6. These results provide important information concerning the relevance of 5-HT1A receptor partial agonist properties in the treatment for schizophrenic patients with most, if not all, of atypical antipsychotic drugs.     

Agonist-stimulated GTPgamma[35S] binding to 5-HT(1A) receptors in human post-mortem brain

In this study, we have demonstrated that the technique of agonist-stimulated guanosine-5'-O-(3-[35S]thio)-triphosphate (GTPgamma[35S]) binding can be successfully used to study the functional activity of the human 5-HT(1A) receptor in post-mortem tissue. Full agonist and antagonist actions of ligands specific for this site have been shown. Utilising 4-(2'-methoxy-phenyl)-1-[2'-(n-2'-pyridinyl)-p-fluorobenzamido]- ethyl-piperazine ([3H]MPPF), the affinity of several antipsychotics for the 5-HT(1A) receptor was determined; clozapine and quetiapine were found to have K(i) values at this receptor that, relative to their dopamine D(2) receptor affinities, indicated at least partial receptor occupancy at clinical doses. The agonist/antagonist activity of these two antipsychotics was studied using GTPgamma[35S] binding. Both compounds show partial agonism, and in addition, clozapine exhibited a larger degree of antagonism against 5-HT-stimulated binding than did quetiapine.     

Domestication alters 5-HT1A receptor binding in rat brain.

Serotonin-1A receptor binding density was compared in the brains of wild and domesticated adult male Rattus norvegicus using in vitro receptor autoradiography of [3H]8-hydroxy-2-[n-dipropylamino]tetraline (DPAT). While both groups exhibited similar patterns of labeling, [3H]DPAT binding density was significantly (p less than or equal to 0.05) lower in the median raphe nucleus and greater in superficial entorhinal cortex and rostral dentate gyrus of domesticated compared to wild rats. The results suggest that specific serotonergic circuits from the median raphe nucleus to the entorhinal and hippocampal regions might be involved in regulation of the defensive behaviors that differ profoundly between wild and domesticated rats. The relationship of these putative differences to behavioral disorders such as anxiety and depression in humans is discussed.     

Mu and delta opioid-stimulated [35S]GTP gamma S binding in brain and spinal cord of polyarthritic rats

Polyarthritis induced by inoculation with complete Freund's adjuvant alters opioid peptides, but does not affect opioid receptor binding. This study was conducted to measure mu and delta opioid receptor-stimulated G-protein activity in brain and spinal cord of rats 19 days after injection of complete Freund's adjuvant or vehicle. Mu and delta opioid-stimulated [35S]GTPgammaS binding measured autoradiographically in caudate-putamen, medial thalamus and periaqueductal gray was unchanged in polyarthritic rats. Delta opioid-stimulated [35S]GTPgammaS binding was significantly decreased in the spinal cord of polyarthritic rats, whereas mu opioid-stimulated activity was unchanged. These data reveal that the functional activity of delta opioid receptors in the spinal cord is altered in polyarthritis.     

5-HT1A receptor-mediated G protein activation assessed by [35S]GTPgammaS binding in rat cerebral cortex

To date, 5-hydroxytryptamine1A (5-HT1A) receptor-mediated functional assays (adenylyl cyclase inhibition, high-affinity GTPase activity and [35S]guanosine-5'-O-(gamma-thio)-triphosphate ([35S]GTPgammaS) binding) have been performed mainly in hippocampal membranes. In the current study, 5-HT-stimulated G protein activation assays were carried out in rat cerebral cortical membranes. High-affinity GTPase activity was stimulated by 5-HT, but not by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). By contrast, 5-HT- and 8-OH-DPAT-stimulated [35S]GTPgammaS binding displayed sufficient dynamic range enough to warrant further pharmacological analysis. Under standard conditions, which were determined precisely in terms of the concentrations of GDP, MgCl2 and NaCl, the profile of 5-HT-stimulated [35S]GTPgammaS binding investigated using a series of 5-HT receptor agonists and antagonists clearly indicated the involvement of the 5-HT1A receptor subtype. There appeared to be no evidence supporting the presence of regional heterogeneity in coupling efficiency between 5-HT1A and G proteins in the hippocampus or cortex. This method is a useful tool for investigating functional coupling between postsynaptic 5-HT1A receptors and G proteins in cerebral cortical membranes.     

Aging alters in a region-specific manner serotonin transporter sites and 5-HT(1A) receptor-G protein interactions in hamster brain

Key proteins regulating serotonergic activity, specifically the serotonin transporter and 5-HT(1A) receptor, were examined in the midbrain raphe nuclei of young (3-4 months) and old (17-19 months) hamsters (N=7-10/group). An age-related decrease in the maximal density of serotonin transporter sites labelled with [(3)H]paroxetine (fmol/mg protein, Old: 396+/-13; Young: 487+/-27) was observed in the dorsal raphe nucleus (DRN) but not the median raphe nucleus (MRN), without affecting the affinity of [(3)H]paroxetine. In the DRN and MRN, the stimulation of [(35)S]GTP gamma S binding by the 5-HT(1A) receptor agonist 8-OH-DPAT, or the number of 5-HT(1A) receptor sites labeled with [(3)H] MPPF, was not different in old versus young animals. Thus in the DRN, aging decreased serotonin transporter sites without changing 5-HT(1A) receptor activation of G proteins or 5-HT(1A) receptor density. In the CA(1) region of hippocampus, 8-OH-DPAT-stimulated [(35)S]GTP gamma S binding was increased in the older animals (% above basal, Old: 141+/-21; Young: 81+/-17) without changing specific [(3)H] MPPF binding sites, suggesting that the capacity of 5-HT(1A) receptors to activate G proteins is enhanced. Aging also appears to enhance this capacity in the dentate gyrus, because this region exhibited a constant level of 8-OH-DPAT-stimulated [(35)S]GTP gamma S binding in spite of an age-related decrease in the number of [(3)H] MPPF binding sites (fmol/mg protein, Old: 203+/-21; Young: 429+/-51).     

Stimulated [35S]GTPγS binding by 5-HT1A receptor agonists in recombinant cell lines Modulation of apparent efficacy by G-protein activation state

G-protein activation by different 5-HT receptor ligands was investigated in h5-HT_1A receptor-transfected C6-glial and HeLa cells using agonist-stimulated [³⁵S]GTPγS binding to membranes in the presence of excess GDP. 5-HT (10 μM) stimulated [³⁵S]GTPγS binding in the C6-glial membrane preparation to a larger extent than in the HeLa preparation; maximal responses with 30 μM GDP were 490 ± 99 and 68 ± 12%, respectively. With the 5-HT receptor agonists that were being investigated, the two preparations displayed the same rank order of potency for stimulation of [³⁵S]GTPγS binding. In the C6-glial preparation at 0.3 μM GDP, the rank order of maximal effects was: 5-HT (1.00) > 8-OH-DPAT (0.90) = R(+)-8-OH-DPAT (0.87) = 5-CT (0.86) = L694247 (0.84) > S(–)8-OH-DPAT (0.68) = buspirone (0.67) = spiroxatrine (0.67) = flesinoxan (0.64) > ipsapirone (0.53) = (–)-pindolol (0.50) > SDZ216525 (0.25). However, differences in maximal response in the C6-glial preparation were magnified by increasing the GDP concentrations, indicating that the activity state of G-proteins can affect the maximal response. With the exception of 5-CT and L694247, increasing the amount of GDP to 30 μM and higher concentrations resulted in an attenuation of both the ligand’s maximal effect (24 to 56%) and apparent potency (6 to 24-fold). Each of the [³⁵S]GTPγS binding responses was mediated by a 5-HT_1A receptor as indicated by the competitive blockade by WAY100635 and spiperone. Only 5-CT and L694247 in some conditions displayed an efficacy similar to that of 5-HT at the h5-HT_1A receptor; the other agents with intrinsic activity are partial agonists at this receptor. The data also suggest that the activity state of the G-proteins is involved in the maximal effects that can be produced by activating the h5-HT_1A receptor. Received: 5 May 1997 / Accepted: 20 July 1997     

Differential regulation of serotonin-1A receptor-stimulated [35S]GTP gamma S binding in the dorsal raphe nucleus by citalopram and escitalopram

The effect of chronic citalopram or escitalopram administration on 5-HT1A receptor function in the dorsal raphe nucleus was determined by measuring [35S]GTP gamma S binding stimulated by the 5-HT1A receptor agonist (R)-(+)-8-OH-DPAT (1nM-10 microM). Although chronic administration of citalopram or escitalopram has been shown to desensitize somatodendritic 5-HT1A autoreceptors, we found that escitalopram treatment decreased the efficacy of 5-HT1A receptors to activate G proteins, whereas citalopram treatment did not. The binding of [3H]8-OH-DPAT to the coupled, high affinity agonist state of the receptor was not altered by either treatment. Interestingly, escitalopram administration resulted in greater occupancy of serotonin transporter sites as measured by the inhibition of [3H]cyanoimipramine binding. As the binding and action of escitalopram is limited by the inactive enantiomer R-citalopram present in racemic citalopram, we propose that the regulation of 5-HT1A receptor function in the dorsal raphe nucleus at the level of receptor-G protein interaction may be a result of greater inhibition of the serotonin transporter by escitalopram.     

Early desensitization of somato-dendritic 5-HT1A autoreceptors in rats treated with fluoxetine or paroxetine

Electrophysiological and autoradiographic approaches were used to assess possible changes in 5-hydroxytryptamine (serotonin) 5-HT_1A receptors in the rat dorsal raphe nucleus after a subchronic treatment with fluoxetine or paroxetine, two specific serotonin reuptake inhibitors with antidepressant properties. Fluoxetine or paroxetine were injected daily (5 mg/kg, i.p.) for various time periods up to 21 days. Electrophysiological recordings performed 24 h after the last injection showed that the potency of the 5HT_1A receptor agonist, 8-OH-DPAT, to depress the firing of serotoninergic neurons in the dorsal raphe nucleus within brain stem slices was significantly reduced as early as after a 3-day treatment with either drug. The proportion of recorded neurons showing desensitization of somatodendritic 5-HT_1A autoreceptors increased along the treatment from 40% on the 3rd day to 60–80% on the 21st day. At no time during the treatment, was the specific binding of [³H]8-OHDPAT (agonist radioligand) or [³H] WAY-100 635 (antagonist radioligand) to 5-HT_1A receptors modified in the dorsal raphe nucleus or in other brain areas, suggesting that neither the density nor the coupling of these receptors to G-proteins were probably altered in rats injected with fluoxetine or paroxetine for up to 21 days.These results show that adaptive desensitization of somatodendritic 5-HT_1A autoreceptors within the dorsal raphe nucleus can already be detected after a 3-day treatment with selective serotonin reuptake inhibitors. Rather than the desensitization per se, it may be the progressive increase in the number of serotoninergic neurons with desensitized 5-HT_1A autoreceptors which plays a critical role in the (slowly developing) antidepressant action of these drugs.     

Acute or chronic antidepressants do not modify [125I]cyanopindolol binding to 5-HT1B receptors in rat brain

Acute or chronic treatment of young or adult rats with chlorimipramine, tianeptine or iprindole, antidepressants with different effects on 5-HT uptake mechanisms, did not modify the density or the affinity of 5-HT1B receptors of the frontal cortex. No significant receptor change was found after prenatal exposure to these antidepressants. The lack of effect of the antidepressants was not related to the density of 5-HT1B receptors, which was significantly lower in younger animals.     

Autoradiographic characterisation of [35S]GTPgammaS binding stimulation mediated by 5-HT1B receptor in postmortem human brain

G-protein activation mediated by 5-HT1B receptors was studied in human brain by [35S]GTPgammaS autoradiographic methods. 5-HT (10 microM) increased [35S]GTPgammaS binding in caudate-putamen nucleus, globus pallidus, dentate gyrus, CA1, entorhinal cortex and substantia nigra. In basal ganglia and midbrain, this effect was blocked by GR 127935 (5-HT(1B/1D) antagonist). In contrast, WAY 100635 (selective 5-HT1A antagonist) reversed the effect of 5-HT in hippocampus and entorhinal cortex. Therefore, a detailed pharmacological study was carried out in basal ganglia and substantia nigra using 5-HT and the 5-HT(1B/1D) agonists GTI and CP 93129. In these areas, these agonists stimulated [35S]GTPgammaS binding in a concentration-dependent manner, with no significant differences in the potency for a given structure. Furthermore, GTI was more potent in the putamen than in globus pallidus. In caudate-putamen, the three agonists showed the same efficacy, while in globus pallidus and substantia nigra the efficacy of 5-HT was higher than GTI and CP 93129. The selective 5-HT1B antagonist SB-224289 inhibited GTI- and CP 93129-stimulated [35S]GTPgammaS binding in basal ganglia and substantia nigra, while coincubation with BRL 15572 (selective 5-HT1D antagonist) did not result in any significant change. Here we report the anatomical pattern of distribution of 5-HT1B-dependent functionality by using specific pharmacological tools in human brain sections.     

Time course of the effects of adrenalectomy and corticosterone replacement on 5-HT1A receptors and 5-HT uptake sites in the hippocampus and dorsal raphe nucleus of the rat brain: an autoradiographic analysis

Previous studies have shown that adrenalectomy (ADX) increases the binding of³H-DPAT to 5-HT_1A receptors in the hippocampus (HIP) and this effect is partially overcome by corticosterone (CORT) replacement. The present study investigated the time course of the effects of ADX with or without CORT replacement on serotonin (5-HT) pre- and postsynaptic systems in the HIP and dorsal raphe nucleus (DR) by quantitative autoradiography. In the HIP, ADX for 7, 10 or 14 days caused a significant increase in³H-DPAT binding in the CA₁ region (pyramidal layer), CA_2,3 region (molecular and pyramidal layers) and in the dentate gyrus (molecular and granular layers) which returned to control levels when measurements were made 35 days post-ADX. A decrease in³H-DPAT binding was observed 14 days after ADX in the DR but not in the median raphe nucleus (MR). Although replacement with CORT did not lead to a reversal in³H-DPAT binding at early time points, binding was restored to control levels 7–28 days after CORT replacement in all regions of the HIP. In the DR, CORT did not cause a reversal in³H-DPAT binding at any of the time points examined. In contrast to the effects seen on the 5-HT_1A receptor subtype, no significant change was noted on the binding of³H-CN-IMI to uptake sites for 5-HT in the HIP or DR after ADX or CORT replacement. The results of this study indicate that long-term alterations in the HPA axis lead to changes in the 5-HT_1A receptor system that are both region-specific and time-dependent.     

SKF83959 exhibits biochemical agonism by stimulating [(35)S]GTP gamma S binding and phosphoinositide hydrolysis in rat and monkey brain

SKF83959, a benzazepine with high affinity for aminergic receptors, elicits behaviors such as grooming and vacuous chewing that are characteristic of dopamine D(1)-like receptor stimulation in rodents. Unlike classical D(1) agonists, however, SKF83959 does not stimulate adenylyl cyclase. Knowing that some D(1)-like receptors are coupled to phospholipase C-mediated signaling cascades in the brain, the present study aimed to determine whether SKF83959 exhibits an agonistic action at the biochemical level and also whether this benzazepine can modulate phosphoinositide hydrolysis in a manner that would be consistent with the behavioral effects of the drug. Similar to dopamine and the selective D(1)-like agonist SKF38393, SKF83959 competitively displaced the receptor binding of [(3)H]dopamine in an agonist-like manner, significantly stimulated [(35)S]guanosine-5'-O-(3-thio)triphosphate binding, and potently enhanced phospholipase C-mediated phosphoinositide hydrolysis in rat and monkey brain tissues. SKF83959 was generally more potent than SKF38393, whereas SKF38393 consistently exhibited greater pharmacological efficacy. These findings may implicate a role for the phospholipase C signaling cascade in the agonistic behavioral and antiparkinsonian activity of SKF83959. Dopamine-sensitive phospholipase C signaling should probably be considered in subsequent formulations of mechanisms and models of dopaminergic function in the normal or diseased brain.     

Activation of 5-HT(1A) autoreceptors enhances the inhibitory effect of galanin on hippocampal 5-HT release in vivo

The microdialysis technique was used to examine interactions between 5-HT(1A) and galanin receptors in the dorsal raphe nucleus (DRN), by measuring the extracellular levels of 5-HT in the ventral hippocampus of awake rats. The rats were pretreated with the 5-HT(1A) receptor agonist (R,S)-8-OH-DPAT (0.3 mg/kg, s.c.) or saline. 8-OH-DPAT caused a time-dependent reduction of basal 5-HT levels down to 43-48% at 40 min while at 140 min, the hippocampal 5-HT had returned to control values. At that time point, the rats received a second injection of 8-OH-DPAT or galanin (0.15, 0.5 and 1.5 nmol/0.5 microl) infused into the lateral ventricle. The second injection of 8-OH-DPAT caused a significantly smaller reduction of hippocampal 5-HT levels. In contrast, galanin at all three doses in the 8-OH-DPAT-pretreated groups, was significantly more potent in reducing 5-HT levels (maximal reduction to 74%, 52% and 49%, respectively) than it was in saline-pretreated rats (maximal reduction to 96%, 85% and 69%, respectively). The inhibitory effect of galanin (1.5 nmol) on extracellular 5-HT levels in the rat hippocampus was significantly attenuated by co-administration of the 5-HT(1A) receptor antagonists WAY-100635 (0.3 and 0.6 mg/kg s.c.) and, to a lesser extent, with pindolol (20 mg/kg s.c.). These data provide direct in vivo evidence of agonistic 5-HT(1A)-galanin receptor interaction at the presynaptic level. Furthermore, the findings indicate that a down-regulation of the somato-dendritic 5-HT(1A) autoreceptors, following their stimulation with 8-OH-DPAT and possibly also indirectly with 5-HT reuptake inhibitors, may be compensated by a subsequent 'sensitization' of the inhibitory galanin receptors in the DRN. Thus, the enhanced galanin receptor-mediated inhibition of 5-HT neurotransmission may contribute to the pathophysiology of depression or to the reduced and delayed efficacy of antidepressant therapies.     

In vivo control of 5-hydroxytryptamine release by terminal autoreceptors in rat brain areas differentially innervated by the dorsal and median raphe nuclei

Selective serotonin reuptake inhibitors (SSRIs) reduce the 5-HT release in vivo. This effect is due to the activation of somatodendritic 5-HT_1A receptors and it displays a regional pattern comparable to that of selective 5-HT_1A agonists, i.e., preferentially in forebrain areas innervated by the dorsal raphe nucleus (DRN). However, despite a comparatively lower 5-HT_1A-mediated inhibition of 5-HT release and a greater density of serotonergic uptake sites in hippocampus, the net elevation produced by the systemic administration of SSRIs is similar in various forebrain areas, regardless of the origin of serotonergic fibres. As terminal autoreceptors may also limit the SSRI-induced elevations of 5-HT in the extracellular brain space, we reasoned that a differential control of 5-HT release by terminal autoreceptors in DRN- and median raphe-innervated areas might be accountable. To examine this possibility, we have conducted a regional microdialysis study in the DRN, MRN and four forebrain regions preferentially innervated either by the DRN (frontal cortex, striatum) or the median raphe nucleus (MRN; dorsal and ventral hippocampus) using freely moving rats. Dialysis probes were perfused with 1 μM of the SSRI citalopram to augment the endogenous tone on terminal 5-HT autoreceptors. The non-selective 5-HT₁ antagonist methiothepin (10 and 100 μM, dissolved in the dialysis fluid) increased extracellular 5-HT in frontal cortex and dorsal hippocampus in a concentration-dependent manner. The 5-HT_1B/1D antagonist GR 127935 was ineffective at 10 μM and tended to reduce 5-HT in dorsal hippocampus at 100 μM. The local infusion of 100 μM methiothepin significantly elevated the extracellular 5-HT concentration to 142–173% of baseline (mean values of 260 min post-administration) in the DRN, MRN, frontal cortex, striatum and hippocampus (dorsal and ventral). Comparable elevations were noted in the four forebrain regions examined. As observed in frontal cortex and dorsal hippocampus, the perfusion of 10 μM GR 127935 did not elevate 5-HT in DRN, MRN, striatum or ventral hippocampus. Because the stimulated 5-HT release in the DRN has been suggested to be under control of 5-HT_1B/1D receptors, we examined the possible contribution of these receptor subtypes to the effects of methiothepin in the DRN. The perfusion of sumatriptan (0.01–10 μM) or GR 127935 (0.01–10 μM) did not significantly modify the 5-HT concentration in dialysates from the DRN. Thus, the present data suggest that the comparable effects of SSRIs in DRN- and MRN-innervated forebrain regions are not explained by a preferential attenuation of 5-HT release by terminal 5-HT_1B autoreceptors in hippocampus, an area with a low inhibitory influence of somatodendritic 5-HT_1A receptors. Methiothepin-sensitive autoreceptors (possibly 5-HT_1B) appear to play an important role not only in the projection areas but also with respect to the control of 5-HT release in the DRN and MRN. In addition, our findings indicate that GR 127935 is not an effective antagonist of the actions of 5-HT at rat terminal autoreceptors. Received: 27 February 1998 / Accepted: 12 June 1998     

Selective visualization of rat brain 5-HT2A receptors by autoradiography with [3H]MDL 100,907

The recently developed 5-HT_2A receptor selective antagonist [³H]MDL100,907 ((+/–)2,3-dimethoxyphenyl-1-[2-(4-piperidine)-methanol]) has been characterized as a radioligand for the autoradiographic visualization of these receptors. [³H]MDL100,907 binding to rat brain tissue sections was saturable, had sub-nanomolar affinity (Kd=0.2–0.3nM), and presented a pharmacological profile consistent with its binding to 5-HT_2A receptors (rank order of affinity for [³H]MDL100,907-labelled receptors: MDL100,907 > spiperone > ketanserin > mesulergine). The distribution of receptors labelled by [³H]MDL100,907 was compared to the autoradiographical patterns obtained with [³H]Ketanserin, [³H]Mesulergine, and [³H]RP62203 (N-[3-[4-(4-fluorophenyl)-piperazin-1-y1]propyl]-1,8-naphtalenesultam) and to the distribution of 5-HT_2A receptor mRNA as determined by in situ hybridization. As opposed to the other radioligands, [³H]MDL100,907 labelled a single population of sites (5-HT_2A receptors) and presented extremely low levels of non-specific binding. The close similarity of the distributions of [³H]MDL100,907-labelled receptors and 5-HT_2A mRNA further supports the selectivity of this radioligand for 5-HT_2A receptors and suggests a predominant somatodendritic localization of these receptors. The present results point to [³H]MDL100,907 as the ligand of choice for the autoradiographic visualization of 5-HT_2A receptors. Received: 7 April / Accepted: 18 May 1997