Homeostatic regulation of serotonergic function by the serotonin transporter as revealed by nonviral gene transfer.

With the aim of exploring the relationship between the serotonin transporter (5-HTT or SERT) and the activity level of serotonin (5-HT) neurotransmission, in vivo expression of this protein was specifically altered using a nonviral DNA transfer method. Plasmids containing the entire coding sequence or a partial antisense sequence of the 5-HTT gene were complexed with the cationic polymer polyethylenimine and injected into the dorsal raphe nucleus of adult male rats. Significant increase or decrease in both [(3)H]citalopram binding and [(3)H]5-HT synaptosomal uptake were observed in various brain areas up to 2 weeks after a single administration of the sense plasmid or 7 d after injection of the short antisense plasmid, respectively. Such changes in 5-HTT expression were associated with functional alterations in 5-HT neurotransmission, as shown by the increased capacity of 5-HT(1A) receptor stimulation to enhance [(35)S]GTP-gamma-S binding onto the dorsal raphe nucleus in sections from rats injected with the sense plasmid. Conversely, both a decrease in 5-HT(1A)-mediated [(35)S]GTP-gamma-S binding and a reduced potency of the 5-HT(1A) receptor agonist ipsapirone to inhibit neuronal firing were observed in the dorsal raphe nucleus of antisense plasmid-injected rats. Furthermore, changes in brain 5-HT and/or 5-HIAA levels, and sleep wakefulness circadian rhythm in the latter animals demonstrated that altered expression of 5-HTT by recombinant plasmids has important functional consequences on central 5-HT neurotransmission in adult rats.

Opposite changes in dopamine metabolites and met-enkephalin levels in the ventricular CSF of patients subjected to thalamic electrical stimulation.

High-frequency electrical stimulations of thalamic nuclei are currently used for the suppression of parkinsonian or essential tremor and for the relief of some types of intractable pain in man. However, the mechanisms by which such stimulations exert their therapeutic effects are essentially unknown. Attempts were made to provide some insight into these mechanisms by measuring the levels of the dopamine metabolites homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC), the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) and met-enkephalin-like immunoreactivity in ventricular cerebrospinal fluid (CSF) of patients with Parkinson's disease (PD) or multiple sclerosis (MS) after a 30-minute therapeutic electrical stimulation of the ventralis intermedius nucleus of the thalamus. In nonstimulated control patients, the levels of these compounds did not significantly differ in two CSF samples taken 30 minutes apart. In stimulated patients, a decrease in dopamine metabolite levels associated with a relative increase in met-enkephalin-like immunoreactivity were observed in the CSF sample taken after the 30-minute stimulation as compared to the sample taken immediately before the stimulation. In contrast, the levels of 5-HIAA remained unaffected by the stimulation. These data confirmed the existence of negative interactions between dopaminergic and enkephalinergic systems in man similar to those previously demonstrated in rats. In addition, they suggest that alterations in dopaminergic or enkephalinergic neurotransmission might be involved in the therapeutic action of thalamic electrical stimulation in patients with parkinsonian symptoms and other patients.

Chronic alnespirone-induced desensitization of somatodendritic 5-HT1A autoreceptors in the rat dorsal raphe nucleus.

The effects of long-term (7, 14 or 21 days) administration of the 5-HT1A receptor agonist alnespirone [5 mg/(kg day), i.p.] on the binding characteristics of 5-HT1A, 5-HT2A and 5-HT3 receptors, and the functional status of 5-HT1A autoreceptors were assessed using biochemical and electrophysiological approaches in rats. Whatever the treatment duration, the specific binding of [3H]8 hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT), [3H]trans,4-[(3Z)3-(2-dimethylaminoethyl) oxyimino-3(2-fluorophenyl) propen-1-yl] phenol hemifumarate ([3H]SR 46349B), and [3H]S-zacopride to 5-HT1A, 5-HT2A and 5-HT3 receptors, respectively, were unaltered in all the brain areas examined. In contrast, in vitro electrophysiological recordings performed 24 h after the last injection of alnespirone showed that the potency of the 5-HT1A receptor agonist, 8-OH-DPAT, to depress the firing of serotoninergic neurons in the dorsal raphe nucleus, was significantly reduced after a 21-day treatment with alnespirone. However, no changes were noted after a 7-day or 14-day treatment. These data indicate that desensitization of somatodendritic 5-HT1A autoreceptors is a selective but slowly developing adaptive phenomenon in response to their chronic stimulation in rats.

Differential membrane targeting and pharmacological characterization of chimeras of rat serotonin 5-HT1A and 5-HT1B receptors expressed in epithelial LLC-PK1 cells.

The serotonin 5-HT1A and 5-HT1B receptors are two structurally related but pharmacologically distinguishable 5-HT receptor types. In brain, the 5-HT1A receptor is localized on the soma and dendrites of neurons, whereas the 5-HT1B receptor is targeted to the axon terminals. We previously showed that these two receptors are targeted in different membrane compartments when stably expressed in the epithelial LLC-PK1 cell line. Further investigations on the mechanisms responsible for their differential targeting were done by constructing chimeras of 5-HT1A and 5-HT1B receptors still able to bind specifically [3H]lysergic acid diethylamide and selective agonists and antagonists. Their cellular localization examined by confocal microscopy suggests that the third intracellular domain of the 5-HT1B receptor was responsible for its Golgi-like localization in transfected LLC-PK1 cells. In contrast, the third intracellular domain of the 5-HT1A receptor apparently allowed the sorting of the chimeras to the plasma membrane. Further inclusion of the C-terminal domain of the 5-HT1A receptor in their sequence led to a basolateral localization, whereas that of the 5-HT1B receptor allowed an apical targeting, suggesting the existence of a targeting signal in this portion of the receptor(s).

Is the potent 5-HT1A receptor agonist, alnespirone (S-20499), affecting dopaminergic systems in the rat brain?

The effects of the new methoxy-chroman 5-HT1A receptor agonist, alnespirone (S-20499), on the dopamine systems in the rat brain were assessed in vivo by means of electrophysiological and neurochemical techniques. Cumulative doses of alnespirone (0.032-4.1 mg kg(-1), i.v.) did not modify the spontaneous firing rate of dopamine neurons in the substantia nigra as well as in the ventral tegmental area. The local application of alnespirone (0.1-10 microM) by reverse microdialysis into the dorsal striatum did not affect the dopamine output but induced a moderate, although dose-independent, increase of 5-HT (5-hydroxytryptamine, serotonin) concentrations in the dialysate. As expected of a 5-HT1A receptor agonist, intraperitoneal (i.p.) administration of alnespirone at 2-32 mg kg(-1) markedly decreased 5-HT turnover in the striatum. Parallel measurements of dopamine turnover showed that alnespirone exerted no effect except at the highest dose (32 mg kg(-1), i.p.) for which a significant increase was observed. Interestingly, both alnespirone-induced reduction in 5-HT turnover and increase in dopamine turnover could be prevented by pretreatment with the selective 5-HT1A receptor antagonist WAY-100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexa ne carboxamide). Altogether, these data indicate that alnespirone does not exert any direct influence on central dopamine systems. The enhanced dopamine turnover due to alnespirone at high dose appeared to result from 5-HT1A receptor stimulation, further supporting the idea that this receptor type may play a key role in 5-HT-dopamine interactions in brain.

[Central serotonin receptors and chronic treatment with selective serotonin reuptake inhibitors in the rat: comparative effects of fluoxetine and paroxetine]. / Récepteurs centraux de la sérotonine et traitements chroniques avec un inhibiteur sélectif de la recapture de la sérotonine chez le rat: effets comparés de la fluoxétine et de la paroxétine.

The hypothesis that a dysfunction of serotonergic neurotransmission is implicated in depression is supported by the clinical efficiency of selective serotonin (5-hydroxytryptamine, 5-HT) reuptake inhibitors (SSRIs) in the treatment of depressive disorders. These drugs, such as fluoxetine and paroxetine, exert their antidepressant activity by increasing 5-HT concentration in the synaptic cleft and thus enhancing serotonergic neurotransmission. However, two to three weeks of treatment are necessary to see the first signs of clinical efficiency. Several hypothetical mechanisms have been put forward to account for this delay, taking into account pharmacokinetic considerations, neurotransmitter metabolism, and/or adaptive regulation of pre and/or post-synaptic receptors. The aim of this study was to look for such adaptive changes in the course of a 3-week treatment with fluoxetine (5 mg/kg/day, i.p.) or paroxetine (5 mg/kg/day, i.p.) in adult rats. In vitro binding and quantitative autoradiographic studies showed that neither 5-HT1A, 5-HT1B, 5-HT2A, nor 5-HT3 receptor binding sites in various brain areas were affected by these treatments. Furthermore, comparison of the specific binding of [3H]8-OH-DPAT to 5-HT1A receptors functionally coupled to G proteins with that of [3H]WAY 100635 to all 5-HT1A receptor binding sites (i.e. coupled and uncoupled with regard to G proteins) revealed no significant change in rats treated with either SSRI. Accordingly, the proportion of functional 5-HT1A receptors (i.e. those physically coupled to G proteins) appeared to remain unaltered all along a 3-week treatment with either fluoxetine or paroxetine. Nevertheless, in vitro electrophysiological recordings of serotonergic neurons in the dorsal raphe nucleus allowed the demonstration of a clearcut functional desensitization of somatodendritic 5-HT1A autoreceptors. Thus, the potency of the 5-HT1A autoreceptor agonist, 8-OH-DPAT, to depress the firing of serotonergic neurons in brain stem slices was significantly reduced as soon as after a 3-day treatment with either SSRI. The proportion of recorded neurons showing desensitization of somatodendritic 5-HT1A autoreceptors then increased along the treatment, and was generally larger with fluoxetine than with paroxetine. As 5-HT1A autoreceptor desensitization can contribute to facilitate serotoninergic neurotransmission, the remarkable efficiency of fluoxetine to trigger this adaptive regulatory mechanism might account, at least partly, for its potent antidepressant activity.

Effects of dorsal rhizotomy and selective lesion of serotonergic and noradrenergic systems on 5-HT1A, 5-HT1B, and 5-HT3 receptors in the rat spinal cord.

Autoradiographic studies were performed in combination with dorsal rhizotomy or selective lesion of descending serotonergic or noradrenergic systems in an attempt to identify the neuronal cell types endowed with the serotonin 5-HT1A, 5-HT1B and 5-HT3 receptors in the rat spinal cord. Unilateral sectioning of seven dorsal roots (C4-T2) at the cervical level produced a marked decrease (approximately-75%, 10 days after the surgery) in the binding of [125I]iodozacopride to 5-HT3 receptors in the superficial layers of the ipsilateral dorsal horn, further confirming the preferential location of these receptors on primary afferent fibres. In addition, a significant decrease (approximately 20%) in the binding of [3H]8-OH-DPAT to 5-HT1A receptors and of [125I]GTI to 5-HT1B receptors was also observed in the same spinal area in rhizotomized rats, suggesting that a small proportion of these receptors are also located on primary afferent fibres. The labelling of 5-HT1B receptors was significantly decreased (-12%) in the dorsal horn at the cervical (but not the lumbar) level, and that of 5-HT3 receptors was unchanged in the whole spinal cord in rats whose descending serotonergic projections had been destroyed by 5,7-dihydroxytryptamine. Conversely, the labelling of 5-HT1A receptors was significantly increased in the cervical (+13%) and lumbar (+42%) dorsal horn in 5,7-dihydroxytryptamine-lesioned rats. Similarly, [3H]8-OH-DPAT binding to 5-HT1A receptors significantly increased (+26%) in the lumbar (but not the cervical) dorsal horn in rats whose noradrenergic systems had been lesioned by DSP-4. The labelling of 5-HT1B receptors was also increased (+31% at the cervical level; +17% at the lumbar level), whereas that of 5-HT3 receptors remained unchanged in these animals. These data indicate that complex adaptive changes in the expression of 5-HT1A and 5-HT1B receptors occurred in the rat spinal cord following the lesion of descending monoaminergic systems.

In situ hybridization evidence for the synthesis of 5-HT1B receptor in serotoninergic neurons of anterior raphe nuclei in the rat brain.

The regional distribution of the mRNA encoding the serotonin 5-HT1B receptor was studied in the central nervous system of the rat by in situ hybridization histochemistry and Northern blot analysis. A 180 base pair probe, corresponding to a highly selective portion of the third intracellular loop of the rat 5-HT1B receptor, was used. In most regions, a single 5 kb message was found by Northern blot analysis. However, two additional bands (2.5 and 4 kb) were detected in the striatum. The rank order of 5-HT1B mRNA abundance was striatum >> septum = ventral tegmentum > or = colliculi = hypothalamus = hippocampus > brain stem > or = cerebellum > or = dorsal horn of the spinal cord > cerebral cortex > or = ventral horn of the spinal cord > olfactory tubercle. This distribution was confirmed by in situ hybridization, which further revealed that the 5-HT1B mRNA was present in dorsal root ganglia, the layer IV of the cerebral cortex, the Purkinje cell layer of the cerebellum, the pyramidal neurons in the CA1 area of the hippocampus, and the dorsal and median raphe nuclei. In situ hybridization was also performed in nomifensine (10 mg/kg/i.p.)-pretreated rats whose serotoninergic neurons were extensively and selectively lesioned by microinjection of 5,7-dihydroxytryptamine (8 micrograms/1 microliter) directly into the anteroventral vicinity of anterior raphe nuclei 3 weeks before sacrifice. In lesioned rats, 5-HT1B mRNA was present in the same areas and at the same levels as in control rats, except in the dorsal and median raphe nuclei, where a marked decrease (-75%) in its local concentration was observed. These data provide the first demonstration of the synthesis of 5-HT1B receptor within serotoninergic neurons, as expected of their presynaptic autoreceptor function at the level of serotoninergic terminals.

Central pre- and postsynaptic 5-HT1A receptors in rats treated chronically with a novel antidepressant, cericlamine.

Biochemical and electrophysiological approaches were used to assess the possible changes in 5-hydroxytryptamine (serotonin) 5-HT1A receptors in the rat brain after a long-term treatment with cericlamine [2-(3,4-dichlorobenzyl)-2-dimethylamino-1-propanol], a novel serotonin reuptake inhibitor with antidepressant properties. Possible changes in other serotonin receptor binding sites (5-HT2A, 5-HT2C and 5-HT3) were also investigated after this treatment. Cericlamine was injected for 2 weeks at a dose (16 mg/kg i.p., twice daily) that ensured complete prevention of 4-methyl-alpha-ethyl-meta-tyramine-induced depletion of brain serotonin. In vitro binding and quantitative autoradiographic studies showed that neither 5-HT1A, 5-HT2A, 5-HT2C nor 5-HT3 receptor binding sites in various brain areas were affected by the 14-day treatment with cericlamine. Although forskolin-stimulated adenylate cyclase activity was significantly increased in hippocampal homogenates from cericlamine-treated rats, the reduction in this enzymatic activity due to 5-HT1A receptor stimulation by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) was unchanged in these animals as compared with controls. In contrast, in vitro and in vivo electrophysiological recordings of serotoninergic neurons in the dorsal raphe nucleus revealed a clearcut functional desensitization of somatodendritic 5-HT1A autoreceptors. Thus the potency of 8-OH-DPAT and ipsapirone to depress the firing rate of these neurons in brain stem slices was significantly reduced after the 2-week treatment with cericlamine. In vivo, the potency of an injection of cericlamine to inhibit the discharge of serotoninergic neurons was also markedly less in rats that had been pretreated for 2 weeks with this drug as compared with controls. However, the inhibitory effects of systemically injected 8-OH-DPAT and ipsapirone on the electrical activity of serotoninergic neurons were as pronounced in cericlamine-treated rats as in controls. In addition, the reduction in serotonin synthesis due to an acute treatment with 8-OH-DPAT (0.1 or 0.3 mg/kg s.c.) was not significantly different in both groups of rats. These data support the idea that postsynaptic (in the hippocampus) and somatodendritic (in the dorsal raphe nucleus) 5-HT1A receptors are differently regulated in the rat brain, because only the latter receptors desensitized after a long-term blockade of serotonin reuptake by cericlamine. They also suggest that the inhibitory influence of systemically administered direct 5-HT1A agonists such as 8-OH-DPAT and ipsapirone on the electrical and metabolic activity of serotoninergic neurons does not result solely from the stimulation of somatodendritic 5-HT1A autoreceptors.

Characteristics of [14C]guanidinium accumulation in NG 108-15 cells exposed to serotonin 5-HT3 receptor ligands and substance P.

In the presence of substance P (SP; 10 microM), serotonin (5-HT; 1 microM) triggered a cation permeability in cells of the hybridoma (mouse neuroblastoma x rat glioma) clone NG 108-15 that could be assessed by measuring the cell capacity to accumulate [14C]guanidinium for 10-15 min at 37 degrees C. In addition to 5-HT (EC50 0.33 microM), the potent 5-HT3 receptor agonists 2-methyl-serotonin, phenylbiguanide, and m-chlorophenylbiguanide, and quipazine, markedly increased [14C]guanidinium uptake in NG 108-15 cells exposed to 10 microM SP. In contrast, 5-HT3 receptor antagonists prevented the effect of 5-HT. The correlation (r = 0.97) between the potencies of 16 different ligands to mimic or prevent the effects of 5-HT on [14C]guanidinium uptake, on the one hand, and to displace [3H]zacopride specifically bound to 5-HT3 receptors on NG 108-15 cells, on the other hand, clearly demonstrated that [14C]guanidinium uptake was directly controlled by 5-HT3 receptors. Various compounds such as inorganic cations (La3+, Mn2+, Ba2+, Ni2+, and Zn2+), D-tubocurarine, and memantine inhibited [14C]guanidinium uptake in NG 108-15 cells exposed to 5-HT and SP, as expected from their noncompetitive antagonistic properties at 5-HT3 receptors. However, ethanol (100 nM), which has been reported to potentiate the electrophysiological response to 5-HT3 receptor stimulation, prevented the effects of 5-HT plus SP on [14C]guanidinium uptake. The cooperative effect of SP on this 5-HT3-evoked response resulted neither from an interaction of the peptide with the 5-HT3 receptor binding site nor from a possible direct activation of G proteins in NG 108-15 cells. Among SP derivatives, [D-Pro9]SP, a compound inactive at the various neurokinin receptor classes, was the most potent to mimic the stimulatory effect of SP on [14C]guanidinium uptake in NG 108-15 cells exposed to 5-HT. Although the cellular mechanisms involved deserve further investigations, the 5-HT-evoked [14C]guanidinium uptake appears to be a rapid and reliable response for assessing the functional state of 5-HT3 receptors in NG 108-15 cells.

Possible involvement of K(ATP) channels in the control of 5-HT neurons.

The possible involvement of ATP-sensitive potassium channels in the control of the electrical activity of central serotoninergic neurons was investigated by recording their firing rate in the dorsal raphe nucleus of rat brain stem slices exposed to various blockers and openers of these channels. Whereas the channel openers lemakalim and aprikalim produced no change in the firing rate of these neurons, the channel blockers glibenclamide and gliquidone were strongly inhibitory. As expected from an effect through ATP-sensitive potassium channels, the inhibition by glibenclamide could be prevented in a competitive manner by lemakalim and aprikalim. In contrast, the inactive isomer of the latter drug, RP 61499, did not alter the glibenclamide effect. In addition to the channel openers, the GABA receptor antagonists, bicuculline and phaclofen, but not the antagonist of somato-dendritic 5-HT1A autoreceptors, (-)tertatolol, prevented the negative influence of glibenclamide on the firing rate of serotoninergic neurons. This suggests that GABA acting at both GABAA and GABAB receptors (but not serotonin through the possible stimulation of autoreceptors) was responsible for the effect of glibenclamide. Accordingly, the blockade by the latter drug of ATP-sensitive potassium channels on GABAergic interneurons probably triggered the release of GABA, which in turn, inhibited serotoninergic neurons. In agreement with this hypothetical mechanism, autoradiographic studies demonstrated that ATP-sensitive potassium channels are not located on serotoninergic neurons (but probably on GABAergic interneurons) as the extensive lesion of these neurons by 5,7-dihydroxytryptamine did not reduce the specific labelling of the dorsal raphe nucleus by [3H]glibenclamide.

5-HT3 receptors in the rat central nervous system are mainly located on nerve fibres and terminals.

Autoradiographic and membrane binding studies with [3H](R,S)- or [3H](S)-zacopride were performed in combination with lesions using various neurotoxins in an attempt to identify which neuronal cell types are endowed with 5-HT3 receptors in the rat central nervous system. Lesions of noradrenergic (by DSP-4), dopaminergic (by 6-hydroxydopamine) and serotonergic (by 5,7-dihydroxytryptamine) systems had little effect generally on the density of 5-HT3 receptors labelled with [3H](R,S)- or [3H](S)-zacopride in various regions of the brain and the spinal cord. The only exception was the amygdala where a significant loss (approximately -20%) of 5-HT3 receptors labelled by [3H](R,S)-zacopride was associated with the selective lesion of serotonergic fibres by 5,7-dihydroxytryptamine. Microinjection of kainic or ibotenic acid into the dorsal and ventral hippocampus reduced the density of 5-HT1A receptors labelled with [3H]8-OH-DPAT (approximately -45%) as expected from their known location on intrinsic neuronal cell bodies and/or dendrites. In contrast, the same lesion did not affect 5-HT3 receptors, suggesting their location on fibres 'en passage'. At the spinal level, 5-HT3 receptors were found to exist on primary afferent fibres terminating within the superficial layers of the dorsal horn, as shown by the marked reduction in the local autoradiographic labelling by [3H](S)-zacopride after either dorsal rhizotomy (-81%) or neonatal capsaicin treatment (-72%). These data suggest that 5-HT3 receptors in the central nervous system are generally located presynaptically on nerve terminals or fibres of non-monoaminergic neurones.

(-)Tertatolol is a potent antagonist at pre- and postsynaptic serotonin 5-HT1A receptors in the rat brain.

The potential 5-HT1A antagonist properties of the beta-antagonist tertatolol were assessed using biochemical and electrophysiological assays in the rat. (+/-) Tertatolol bound with high affinity (Ki = 38 nM) to 5-HT1A sites labelled by [3H]8-OH-DPAT in hippocampal membranes. The (-)stereoisomer (Ki = 18 nM) was about 50-fold more potent than the (+)stereoisomer (Ki = 864 nM) to inhibit the specific binding of [3H]-8-OH-DPAT. As expected of a 5-HT1A antagonist, (-)tertatolol prevented in a concentration-dependent manner (Ki = 24 nM) the inhibitory effect of 8-OH-DPAT on forskolin-stimulated adenylate cyclase activity in rat hippocampal homogenates. Furthermore in vivo pretreatment with (-)tertatolol (5 mg/kg s.c.) significantly reduced the inhibitory influence of 8-OH-DPAT (0.3 mg/kg s.c.) on the accumulation of 5-hydroxytryptophan in various brain areas after the blockade of aromatic L-amino acid decarboxylase by NSD-1015 (100 mg/kg i.p.). In vitro (in brainstem slices; Ki approximately 50 nM) and in vivo (in chloral hydrate anaesthetized rats; ID50 approximately 0.40 mg/kg i.v.), (-)tertatolol prevented the inhibitory effects of the 5-HT1A receptor agonists 8-OH-DPAT, ipsapirone and lesopitron on the firing rate of serotoninergic neurones within the dorsal raphe nucleus. In about 25% of these neurones, the basal firing rate was significantly increased by (-)tertatolol (up to +47% in vitro, and +30% in vivo). These data indicate that (-)tertatolol is a potent competitive antagonist at both pre (in the dorsal raphe nucleus)-and post (in the hippocampus)-synaptic 5-HT1A receptors in the rat brain.

New methoxy-chroman derivatives, 4[N-(5-methoxy-chroman-3-yl)N- propylamino]butyl-8-azaspiro-(4,5)-decane-7,9-dione [(+/-)-S 20244] and its enantiomers, (+)-S 20499 and (-)-S 20500, with potent agonist properties at central 5-hydroxytryptamine1A receptors.

The potential interaction of the new methoxy-chroman derivatives: (+/-)-S 20244 (4-[N-(5-methoxy-chroman-3-yl)N-propylamino]butyl-8-azaspiro- (4,5)-decane-7,9-dione) and its enantiomers (+)-S 20499 and (-)-S 20500 with central 5-hydroxytryptamine1A (5-HT1A) receptors was assessed using biochemical and electrophysiological tests in the rat. In vitro binding assays revealed that these drugs bound with high affinity to 5-HT1A sites in hippocampal membranes (Ki: 0.19 nM for (+)-S 20499, 0.95 nM for (-)-S 20500 and 0.35 nM for the racemate (+/-) S 20244). As seen with the prototypical 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino) tetralin, (+/-)-S 20244, (+)-S 20499 and (-)-S 20500 inhibited forskolin-activated adenylate cyclase in hippocampal homogenates with potencies corresponding to their respective affinities for 5-HT1A sites. The maximal inhibitory effect of the chroman derivatives was not additive with that of 8-hydroxy-2-(di-n- propylamino)tetralin and could be competitively reduced by 5-HT1A antagonists such as (-)-propranolol and (+/-)-tertatolol. Electrophysiological recordings within the dorsal raphe nucleus both in vitro (in brain-stem slices) and in vivo (in chloral hydrate anesthetized rats) showed that (+)-S 20499, (+/-)-S 20244 and (-)-S 20500 induced, in that order of (decreasing) potency, a dose-dependent reduction in the spontaneous firing of serotoninergic neurons. In vitro, as well as in vivo, the inhibitory influence of the chroman derivatives on the discharge frequency of serotoninergic neurons could be competitively antagonized by (+/-)-tertatolol. Finally, oral administration of increasing doses of the most potent enantiomer, (+)-S 20499, induced a marked reduction in the rate of 5-HT turnover, without affecting that of dopamine, in various brain areas. All these biochemical and electrophysiological data indicate that (+)-S 20499 is a highly potent agonist at both presynaptic (i.e., somatodendritic) and postsynaptic 5-HT1A receptors in the rat brain.

Cardiovascular effects of the local injection of 5,7-dihydroxytryptamine into the nodose ganglia and nucleus tractus solitarius in awake freely moving rats.

The role of the nucleus tractus solitarius (NTS) serotonergic afferents in cardiovascular (CV) regulation is yet to be established. However, several findings suggest that in this nucleus the serotonergic endings coming from the nodose ganglia (NG) are involved in the control of blood pressure (BP). The purpose of the present study was to identify the CV effects of the destruction of this NG-NTS serotonergic pathway. For that, the BP, BP variability (BPV) and heart rate (HR) effects of the local microinjection of 5,7-dihydroxytryptamine (5,7-DHT), into the NG and NTS were investigated in awake freely moving rats. The local degeneration of serotonergic elements was associated with a significant decrease in the 5-HT and 5-hydroxyindole acetic acid levels within the NG and NTS in 5,7-DHT treated rats. In addition, the microinjection of the neurotoxin in both structures produced a transient and significant increase in BP. This effect was of greater amplitude and associated with an increase in BPV in NG lesioned rats. These results may indicate that the NG-NTS serotonergic pathway participates in the transfer of the messages arising from the aortic baroreceptors. However, the vagal component of the baroreflex assessed with the phenylephrine test was not significantly modified in NG lesioned animals as compared to controls. Consequently, if the present data suggest that the NG-NTS serotonergic pathway plays a depressor role in BP regulation, its involvement in the reflex CV responses triggered by the stimulation of the aortic baroreceptors has yet to be established.

Differential effects of d-fenfluramine and p-chloroamphetamine on H75/12-induced depletion of 5-hydroxytryptamine and dopamine in the rat brain.

The effects of the two 5-HT-releasing drugs, p-chloroamphetamine and d-fenfluramine, on central serotoninergic and dopaminergic systems were compared in adult rats. Both drugs (0.5-5.0 mg/kg i.p., 2 hr before death) produced a dose-dependent reduction in levels of 5-HT, but only p-chloroamphetamine decreased the levels of 5-hydroxyindoleacetic acid (5-HIAA) in the hippocampus, striatum and cerebral cortex. Within the dose range tested, d-fenfluramine did not affect the levels of DA and of its metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in brain. By contrast, p-chloroamphetamine significantly increased the levels of DA and HVA and decreased the levels of DOPAC, notably in the striatum. As expected of a 5-HT uptake inhibitor, d-fenfluramine at small doses (0.2-0.5 mg/kg) prevented the depletion of 5-HT due to 4-methyl-alpha-ethyl-meta-tyramine (H75/12, 40 mg/kg i.p.), whereas at large doses (1.0-5.0 mg/kg) d-fenfluramine, like p-chloroamphetamine (0.2-1.0 mg/kg), slightly enhanced the effect of H75/12. Neither d-fenfluramine (0.5 mg/kg) nor p-chloroamphetamine (0.5 mg/kg) affected the depletion of DA due to H75/12. These data indicate that p-chloroamphetamine is a 5-HT-releasing drug, at any dose between 0.2 and 5.0 mg/kg, whereas d-fenfluramine acts as a 5-HT uptake inhibitor at 0.2-0.5 mg/kg and as a 5-HT releasing drug at larger doses. On account of the potential neurotoxicity of 5-HT-releasing drugs but not 5-HT uptake inhibitors, it can be inferred that d-fenfluramine is very probably devoid of any neurotoxic action in the dose range (less than 1.0 mg/kg) required for its anorectic action.

Alterations of central serotoninergic and dopaminergic neurotransmission in rats chronically treated with ipsapirone: biochemical and electrophysiological studies.

Inasmuch as sustained treatment with the 5-hydroxytryptamine1A (5-HT1A) agonist, ipsapirone, is necessary for inducing anxiolytic and antidepressant effects in the clinic, investigations were performed for assessing the possible changes in serotoninergic and dopaminergic neurotransmission in the brain of rats 24 hr after a 2-week treatment with this drug. Receptor binding assays with membranes and quantiative autoradiography indicated that the twice-daily administration of ipsapirone (5 mg/kg i.p.) for 14 days did not alter the characteristics of 5-HT1A sites in the hippocampus, septum and dorsal raphe nucleus. In contrast, significant decreases in the Bmax values for 5-HT2 sites (-24%) and 5-HT3 sites (-19%) were found in the frontal and posterior cortex, respectively. As expected from unchanged postsynaptic 5-HT1A receptors, inhibition of forskolin-stimulated adenylate cyclase by 5-HT1A agonists (8-hydroxy-2-(di-n-propylamino)tetralin, ipsapirone) exhibited the same characteristics in hippocampal homogenates from both control and ipsapirone-treated animals. An acute administration of 8-hydroxy-2-(di-n-propylamino)tetralin (0.5 mg/kg i.p.) or ipsapirone (1 or 5 mg/kg i.p.) 24 hr after the last injection for the chronic treatment produced a similar decrease in the rate of 5-HT turnover in various brain areas in rats treated for 2 weeks with saline or ipsapirone. At the highest dose (5 mg/kg i.p.), acute ipsapirone also increased the rate of dopamine turnover in the striatum and cerebral cortex approximately to the same extent in both treatment groups. In vitro recording of the firing of serotoninergic neurons in brain stem slices revealed a desensitization of the somatodendritic 5-HT1A receptors, which might be responsible for the increased 5-HT turnover in the brain stem and striatum of rats chronically treated with ipsapirone as compared with controls. These data demonstrated that chronically administered ipsapirone produces adaptive changes in central serotoninergic neurotransmission which might account for the anxiolytic and antidepressant properties of this drug after sustained treatment.

Aging associated changes in serotoninergic and dopaminergic pre- and postsynaptic neurochemical markers in the rat brain.

Measurements of endogenous levels of serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA), dopamine (DA) and dihydroxyphenyl acetic acid (DOPAC), and biochemical and autoradiographic investigations on 5-HT and DA receptors were made in various brain regions in male rats at three different ages: 3 months, 10 months and 22 months. Age-dependent decreases in 5-HT levels associated with parallel increases in 5-HIAA/5-HT ratio were observed in the hypothalamus, striatum, hippocampus and cerebral cortex, suggesting an accelerated 5-HT turnover in aged rats. Similarly, DA levels were lower, and DOPAC/DA ratio was higher in the striatum of 22-month-old compared to 3-month-old or 10-month-old rats. Of the three different classes of 5-HT receptors which were examined, 5-HT1B sites exhibited the largest age-dependent decrease in density, followed by 5-HT2 sites, while 5-HT1A sites remained practically unchanged during aging. By comparison, the loss of striatal D2 receptors in 22-month-old rats compared to young adults was much greater than that of any 5-HT receptor subtype. Such differential age-dependent alterations of the various classes of 5-HT receptors and of dopaminergic versus serotoninergic synaptic markers might be responsible for at least some of the functional deficits in aged animals.

Tianeptine stimulates uptake of 5-hydroxytryptamine in vivo in the rat brain.

The neurochemical effects of the atypical tricyclic antidepressant, tianeptine, were further assessed on central serotoninergic and dopaminergic systems in the rat. Acute treatment with tianeptine (10 mg/kg i.p.) significantly enhanced the levels of metabolites of 5-HT and DA, 5-hydroxyindole acetic acid and dihydroxyphenylacetic acid respectively, in the brain stem, striatum and cerebral cortex. These effects could be prevented by the administration of drugs acting selectively (or preferentially) on serotoninergic systems such as d,l-fenfluramine and 4-methyl-alpha-ethyl-metatyramine (H75/12), suggesting that the increased metabolism of DA was secondary to a modification of serotoninergic systems in tianeptine-treated rats. In contrast to that found with inhibitors of the uptake of 5-HT, treatment with tianeptine markedly enhanced depletion of 5-HT due to administration of H75/12. However, depletion of DA induced by H75/12, was not altered by tianeptine. In vitro measurement of the uptake of [3H]5-HT also confirmed that tianeptine exerted opposite effects to those of classical tricyclic antidepressants, since the in vivo administration of tianeptine (2 x 10 mg/kg i.p.) induced a significant increase in the uptake of [3H]5-HT in cortical synaptosomes. The fact that both inhibitors of the uptake of 5-HT and tianeptine which, in contrast, enhanced the in vivo uptake of 5-HT, are potent antidepressants, challenges the current hypothesis on the central mechanisms of action of these drugs.

Biochemical and electrophysiological evidence for an agonist action of CM 57493 at pre- and postsynaptic 5-hydroxytryptamine1A receptors in brain.

The potential interaction of CM 57493 [4-(3-trifluoromethyl-phenyl)-1-(2-cyanoethyl)-1,2,3,6-tetrahydropyri din e] with central 5-hydroxytryptamine (5-HT) receptors was assessed using biochemical and electrophysiological tests in the rat and in the cat. In vitro binding assays with rat brain membranes revealed that CM 57493 bound to 5-HT1A sites in a concentration range (pIC50 = 7.1) at least two orders of magnitude lower than that required for its interaction with 5-HT1B/5-HT1D, 5-HT2, 5-HT3 and 5-HTPre sites. The affinity of CM 57493 for 5-HT1A sites labeled by [3H]-8-OH-DPAT in hippocampal membranes was enhanced by Mn++ and reduced by GTP, as expected for an agonist. Like 8-OH-DPAT, CM 57493 inhibited forskolin-activated adenylate cyclase activity in hippocampal homogenates. The inhibitory effects of these two compounds were not additive and were prevented by 5-HT1A antagonists such as spiperone and dl-propranolol. In vivo treatment with CM 57493 decreased the levels of 5-hydroxyindole acetic acid in various brain areas, as observed with other 5-HT1A agonists such as 8-OH-DPAT and ipsapirone. Electrophysiological recording within the dorsal raphe nucleus in chloral hydrate anesthetized rats showed that CM 57493 administration induced a dose-dependent reduction of the spontaneous firing of serotoninergic neurons. In vitro, CM 57493 (5-20 microM) also reduced neuronal firing in the nucleus raphe dorsalis within brainstem slice, and this effect could be prevented by dl-propranolol. Finally, in cats pretreated with reserpine, CM 57493 induced a decrease in ponto-geniculo-occipital activity, which could be antagonized by methiothepin.(ABSTRACT TRUNCATED AT 250 WORDS)