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Alterations of the hypothalamic-pituitary-adrenal (HPA) axis function characterized by a decreased negative feedback capacity are often associated with affective disorders and are corrected by treatment with antidepressant drugs. To gain a better understanding of the effects of the antidepressant drug fluoxetine, a specific serotonin (5-HT) reuptake inhibitor, on central corticosteroid receptors, the effects of short-term activation of serotonin transmission on central corticosteroid receptor expression were analysed in adrenalectomized (ADX) rats either supplemented or not with corticosterone. Serotonin transmission was stimulated either by a single injection of the 5-HT precursor, 5-hydroxy-L-tryptophan (5-HTP), or by a 2-day treatment with fluoxetine. In ADX rats, administration of 5-HTP decreased hippocampal mineralocorticoid (MR) and glucocorticoid (GR) receptor numbers 24 h later, while their respective mRNAs were unchanged and these effects of 5-HTP were mediated by 5-HT2 receptors. In the hypothalamus, GR mRNAs and binding sites decreased 3 h and 24 h after 5-HTP, respectively. By contrast, fluoxetine treatment increased hippocampal MR and GR mRNAs and MR binding sites while GR number remained unchanged. In ADX rats supplemented with corticosterone, 5-HTP and fluoxetine treatment had the same effects on corticosteroid receptors compared to those observed in non supplemented ADX rats: 5-HTP decreased hippocampal MR and GR and hypothalamic GR while fluoxetine treatment increased hippocampal MR. These results show that short-term stimulation of 5-HT transmission by 5-HTP decreases hippocampal and hypothalamic corticosteroid receptor numbers through a corticosterone-independent mechanism. It is hypothesized that the delayed maximal increase in extracellular 5-HT contents after fluoxetine treatment, due to negative feedback regulations induced by the activation of 5-HT1A and 5-HT1B autoreceptors, is not the primary cause for the delayed normalization of corticosteroid receptor numbers that regulates the HPA axis functioning.

Transgenic mice bearing a transgene coding for a glucocorticoid receptor antisense mRNA, which partially blocks glucocorticoid receptor expression, were used in order to clarify the role of glucocorticoid receptors in the regulation of 5-HT(1A), 5-HT(1nonA) and 5-HT(2) binding sites labelled by quantitative autoradiography in the frontal and prefrontal cortex, striatum, hypothalamus, amygdala and raphe nuclei. We found that 1 nM [3H]8-hydroxy-2-[di-N-propylamino]tetralin ([3H]8-OH-DPAT) binding to 5-HT(1A) sites was decreased in strata oriens (-15.1+/-3.5%) and radiatum-lacunosum-moleculare (-13.3+/-4.3%) of the hippocampal CA(3) area, and 2 nM [3H]5-hydroxytryptamine binding to 5-HT(1nonA) sites in the presence of 100 nM 8-OH-DPAT and mesulergine was decreased in the dorsal subiculum (-17.8+/-6.9%). By contrast, 5-HT(2) sites labelled by 0.5 nM of (+/-)-1-(2, 5-dimethoxy-4-[125I]iodophenyl)-2-aminopropane was increased in the dorsal subiculum (+35.2+/-11.5%) and CA(2) area (+29.2+/-11.3%). The observed differences in binding to 5-HT(1) and 5-HT(2) sites were all located in areas of the hippocampus that contain both gluco- and mineralo-corticoid receptors, and no difference was observed in anatomical structures which contain only glucocorticoid receptors. Therefore, it seems that the important factor for the regulation of these 5-HT receptors is the interaction between gluco- and mineralo-corticoid receptors rather than the absolute density of glucocorticoid receptors. These results suggest that some of the alterations of the serotonergic neurotransmission observed in depressed patients might be secondary to an altered glucocorticoid receptor function.

The purpose of the present study was to investigate the possible cellular location of 5-HT(1B) receptors on retinal and geniculate afferents in the rat suprachiasmatic nucleus (SCN). Biocular enucleation significantly decreased 5-HT(1B) binding site labeling (35%), specifically in the ventral part of the SCN, while monocular enucleation produced a decrease of smaller magnitude (12%), limited to the ventral part of the contralateral SCN, these results being consistent with the known distribution of retinal afferents in the nucleus. By contrast, bilateral geniculate lesion did not induce any significant variation of 5-HT(1B) binding site labeling in the SCN. Previously, we reported that serotonin (5-HT) synthesis inhibition by parachlorophenylalanine increases 5-HT(1B) binding site labeling in the SCN. Using saturation studies, we have now demonstrated that this upregulation reflected an increase in the total number of 5-HT(1B) binding sites (+41% in the dorsal and +67% in the ventral part of the SCN). Furthermore, we evaluated the effects of bilateral geniculate lesion after 5-HT stores depletion in order to overcome problems of technical resolution limits. The magnitude of upregulation was significantly decreased (27%) after bilateral geniculate lesion, suggesting that part of the 5-HT(1B) receptor population was located on geniculate axon terminals within the SCN. The possible involvement of 5-HT(1B) receptors, according to their cellular locations evidenced in the present study, in photic and nonphotic entrainment of the circadian clock is discussed.

The bilateral olfactory bulbectomy resulted in significantly higher plasma concentration of corticosterone, but not of ACTH in basal conditions and much higher plasma ACTH and corticosterone concentrations after 15 min of immobilization stress than were observed in sham-operated animals. Daily treatment with fluoxetine-a specific serotonin reuptake inhibitor-(15 mg/kg/day) had no effect on basal ACTH and corticosterone concentrations in OB rats. Fluoxetine treatment caused lower levels of ACTH, but not of corticosterone secretion, in response to immobilization stress. Bulbectomy significantly reducing 5-HT concentration in the amygdala. Stress increased serotonergic activity in the hypothalamus but not in the amygdala of OB rats. Chronic fluoxetine treatment of both unstressed and stressed OB rats resulted in a lower turnover rate in the two structures. Our results suggest that the hypercorticosteronemia observed after bulbectomy in unstressed OB rats is independent of the serotonergic system in both hypothalamus and amygdala. In contrast, they also demonstrate hypothalamic 5-HT changes in the HPA hyperactivity of OB rats in response to stress. Chronic fluoxetine treatment may normalize pituitary ACTH secretion in response to stress, possibly desensitization of the 5-HT receptors in the hypothalamus due to 5-HT being move available at the synapses.

Removal of the olfactory bulbs results in numerous physiological and behavioral changes in rats. The most frequent and characteristic change is an abnormally high level of corticosterone in the blood, possibly due to changes in the activity of the hypothalamic neurons which synthesize corticotrophin-releasing hormone (CRH). Some of these neurons also synthesize vasopressin (AVP). They are located in the parvocellular part of the paraventricular nucleus of the hypothalamus, which projects into the external layer of the median eminence. We investigated whether there was such a change in activity by studying the synthesis and storage activity of CRH neurons in bulbectomized rats. CRH and AVP axon terminals in frozen sections of the external layer of the median eminence were labeled by immunofluorescence techniques and the degree of labeling was analyzed semi quantitatively. There was no difference in the area or intensity of CRH-labeling in control and bulbectomized rats. However, a significantly larger area was stained for AVP in the bulbectomized than in control rats. We also used in situ hybridization, with single- and double-labeling, to study the effects of bulbectomy on expression of the genes encoding CRH and AVP. No significant difference was found in the levels of mRNA for CRH and the number of CRH+/AVP+ cell bodies was similar in the parvocellular part of the paraventricular nucleus in bulbectomized and normal rats. Our results suggest that the hypothalamo-pituitary-adrenal (HPA) axis changes observed after olfactory bulbectomy may be due to plastic changes in hypothalamic CRH neurons, resulting in greater storage of increased AVP in CRH neurosecretory nerve terminals in the external layer of the median eminence.

Transgenic (TG) mice deficient in glucocorticoid receptors (GR) were used in order to study the effects of a reduced GR function on adrenocorticotropin hormone and corticosterone plasma levels and on serotonin metabolism in different brain areas under basal resting conditions, after a 30-min restraint stress and 60 min after the end of the restraint stress. There was no difference in basal or stress-induced levels of either adrenocorticotropin hormone or corticosterone in control and TG mice, but the return of adrenocorticotropin hormone to basal values after the end of the stress was delayed in TG mice. Under basal conditions, the ratio 5-hydroxyindoleacetic acid/5-hydroxytryptamine was decreased only in the hippocampus of TG mice compared to controls. In the brain stem, the ratio 5-hydroxyindoleacetic acid/5-hydroxytryptamine increased compared to basal values after a 30-min restraint stress and values were still high 60 min after the end of the restraint stress in both control and TG mice. In the hippocampus, the ratio 5-hydroxyindoleacetic acid/5-hydroxytryptamine increased at the end of the stress and returned to basal levels 60 min later in control mice, whereas there was no change at the end of the stress but an increase 60 min later in TG mice. Finally there was no change in serotonin metabolism in the cortex, striatum or hypothalamus in either group or situation. Our results support the hypothesis of a tonic activation of serotonin turnover by corticosterone through GR in the mouse hippocampus. Moreover, stress-induced stimulation of serotonin metabolism in the brain stem and hippocampus appears to be delayed in TG mice compared to control mice. These results are particularly relevant for mood disorders such as depression where alterations of serotoninergic transmission might be secondary to an impairment of GR functions.

Luteinizing hormone-releasing hormone (LHRH release, which serves as the primary drive to the hypothalamic-pituitary gonaldal axis, is controlled by many neuro-mediators. Serotonin has been implicated in this regulation. However, it is unclear whether the central effect of serotonin on LHRH secretion is exerted directly on LHRH neurosecretory neurons or indirectly via multisynaptic pathways.

Bilateral olfactory bulbectomy (BOX) has major biochemical and behavioral effects, and is one of the most widely investigated of animal models of depression. We studied the consequences of BOX in male rats, on the organization of endogenous circadian rhythms for ACTH, corticosterone (Cort), motor activity (MA) and body temperature (BT). Mean levels were increased for Cort and MA, whereas no significant changes were observed for ACTH and BT. Significantly higher plasma Cort morning values were evidenced in BOX than sham-operated animals. In addition, compared with the single prominent power spectrum for the 24 hours period of control rats, the BOX animals displayed substantially lower 24 hours spectral power for the MA and BT circadian rhythms. These alterations suggest that olfactory bulbectomy, by disruption of the afferences and efferences, induced drastic changes in the function of the endogenous clock or of its regulating systems. From this point of view, bulbectomized rats may therefore be a valuable model to studying the etiology of psychiatric disorders with rhythm disturbance.

1. 1. We examined the binding of [3H]-8-hydroxy-2.(DI-n-propilamino)tetralin ([3H]-8OH-DPAT) to 5-hydroxytriptamine-1A (5-HT1A) receptors in rat hippocampal membranes. 2. 2. Computer analysis of [3H]-8OH-DPAT displacement curves in the absence and in the presence of 100 μM guanosine-5'-O-(3-thiotriphosphate) (GTPγS) were best fitted with a three-site model with apparent dissociation constants (Kd) of 0.45, 2.8 and 30 nM; the corresponding binding capacity (Bmax) existing in the three affinity states were 4, 2 and 12 pmol/g of tissue (wet weight), respectively. 3. 3. These results suggest that [3H]-8OH-DPAT binding can be resolved as complex isotherms and we provided evidence that [3H]-8OH-DPAT labels 2 high-affinity GTPγS-sensitive and one low-affinity GTPγS-insensitive state of 5-HT1A receptors.

Previous works have suggested an interactive stimulatory effect of progesterone (P) and serotonin (5-HT) on luteinizing hormone release. The purpose of the present study was to determine whether 5-HT via 5-HT1A receptors interacts with P in the process of luteinizing hormone-releasing hormone (LHRH) release. Using fetal hypothalamic neurons in primary cell cultures the first goal of this study was to determine the effects of 5-HT1A receptor agonists on LHRH secretion. 8-Hydroxy-2 (di-n-propylamino) tetralin (8-OH-DPAT) or ipsapirone (10–5M) significantly stimulated LHRH release. Pharmacological studies have allowed to rule out the possible involvement of α2-or β-adrenore-ceptors, or 5-HT uptake sites, in the stimulatory effect of 8-OH-DPAT on LHRH release, thus demonstrating the specific involvement of 5-HT1A receptors in the stimulation of LHRH release. The second goal was to test the ability of P to stimulate LHRH release from fetal hypothalamic neurons. P (10–6M) applied for 30 or 120 min significantly stimulated LHRH secretion. The maintenance of the stimulation of LHRH release by P after a cycloheximide treatment or by an impermeable analog of P, P-3-BSA, has suggested a nonge-nomic effect of P on LHRH release. The effects of a pretreatment of cells by P on 8-OH-DPAT-induced LHRH release were tested. While 10–7M P alone did not stimulate LHRH release, this concentration of steroid potentiated the LHRH response to 10–5M 8-OH-DPAT. These findings led to the conclusion that P acting at the level of the plasma membrane potentiates the stimulatory effect of 5-HT1A receptor agonists on LHRH release.

Previous studies have shown that melanotrope cells of the pars intermedia of Rana ridibunda are inhibited by dopaminergic D2 agonists and stimulated by β-adrenergic agonists. In the present study, we have examined the possible involvement of α-adrenoreceptors in the regulation of frog melanotrope cells. Reversed-phase HPLC analysis combined with electrochemical detection revealed the presence of both dopamine and noradrenaline in pars intermedia extracts (74.1 and 3.2 ng/mg protein, respectively), while adrenaline was undetectable. Administration of graded doses of noradrenaline and adrenaline (from 0.1 to 10 µM) to perifused frog neurointermediate lobes induced a dose-dependent inhibition of α-MSH release. The inhibitory effect of adrenaline was partially blocked by the D2-dopaminergic antagonist sulpiride and totally suppressed by concomitant administration of sulpiride and yohimbine (an α2-adrenergic antagonist). Conversely, in the presence of sulpiride, noradrenaline provoked a strong stimulation of α-MSH secretion which was totally blocked by the β-adrenergic antagonist propranolol. Taken together, our results indicate that endogenous catecholamines may exert a complex regulatory action on frog melanotrope cells through activation of dopaminergic D2, α2- and β-adrenergic receptors.

Control of serotonin release and synthesis by amino acid neurotransmitters was investigated in rat rostral and caudal rhombencephalic raphe cells in primary cultures respectively. Endogenous amounts of taurine, glycine, GABA and glutamate were measured in both types of cultures. These amino acids were spontaneously released to the incubating medium. Exogenous taurine (10(-4) M) inhibited release and synthesis of newly formed [3H]serotonin [3H]5-HT from [3H]-tryptophan only in rostral raphe cells. Glycine (10(-3) M) decreased [3H]5-HT release in both types of cells, synthesis being diminished only in rostral raphe cells. Glycine inhibitory effect was totally blocked by strychnine (5 x 10(-5) M). GABA (10(-4) M) reduced [3H]5-HT metabolism in rostral as well as caudal raphe cells. This effect was totally antagonized in caudal and partially in rostral raphe cells by bicuculline (5 x 10(-5) M) a GABAA receptor antagonist. Baclofen (5 x 10(-5) M), a GABAB receptor agonist, induced a decrease of 5-HT release in rostral raphe cells. These observations suggest that monoamine release was entirely mediated by GABAA receptors in caudal raphe cells although GABAA and GABAB receptors were involved in control of 5-HT metabolism in rostral raphe cells. L-glutamate (10(-4) M) stimulated 5-HT metabolism in both types of cells, effect totally blocked by PK26124 (10(-6) M). N-methyl-D-aspartate (10(-4) M) enhanced 5-HT metabolism and the induced-effect was antagonized by the selective N-methyl-D-aspartate receptor antagonist D,L-2 amino-5-phosphonovaleric acid. Quisqualate (10(-5) M) stimulated [3H]5-HT release only in caudal raphe cells. This effect was mimicked by (RS)-a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, a quisqualate "ionotropic" receptor agonist, this increase being blocked by 6,7-dinitroquinoxaline 2,3-dione. These observations suggest that the glutamate stimulating-induced effect on serotonin metabolism is entirely mediated by N-methyl-D-aspartate receptor-type in rostral raphe cells and that quisqualate "ionotropic" receptors are also involved in caudal raphe cells. Taken together these results show that [3H]5-HT metabolism is controlled by taurine, glycine, GABA and glutamate in rhombencephalic raphe cells in primary cultures. However, some difference in amino acid receptor-types involved in the control of serotonin metabolism are observed according to the rostral or caudal origin of raphe cells.

The purpose of this study was to test the capacity of oestradiol to modulate the stimulating effect of a-aminobutyric acid (GABA) on serotonin (5-HT) metabolism, previously described in the Suprachiasmatic area of the male rat. After an in vivo stimulation of GABA transmission by systemic administration of a GABA-transaminase inhibitor (amino-oxyacetic acid) or a GABA(B) agonist (RS-baclofen), the 5-HT metabolism was studied in the Suprachiasmatic area of ovariectomized, and ovariectomized oestradiol-treated rats. Amino-oxyacetic acid or RS-baclofen treatment increased the endogenous content of 5-HT in the Suprachiasmatic area of males and ovariectomized rats. These two treatments were without effect in ovariectomized oestradiol-treated rats. GABA transmission stimulation induced by amino-oxyacetic acid treatment failed to affect the release and synthesis of 5-HT in ovariectomized oestradiol-treated rats while it increased these two parameters of 5-HT metabolism in the Suprachiasmatic area of male and ovariectomized rats. To investigate the main target of oestradiol effect, comparative studies of the serotoninergic and GABAergic metabolism in the Suprachiasmatic area were performed in the three experimental groups. Under our experimental conditions the endogenous 5-HT metabolism was similar between ovariectomized and ovariectomized oestradiol-treated rats. Nevertheless, 5-HT metabolism was higher in the two female groups than in the male group. Neither GABA metabolism nor GABAergic response to GABA-related drug treatment differed between ovariectomized, and ovariectomized oestradiol-treated rats. However, the turnover of GABA was higher when compared to the two female groups. It is concluded that the lack of 5-HT responsiveness to GABA transmission stimulation in ovariectomized oestradiol-treated rats was not related to an effect of oestradiol on 5-HT metabolism or to an effect of the steroid on GABA turnover. Furthermore, our results suggest a sex difference in the activity of serotoninergic and GABAergic systems in the Suprachiasmatic area.

The hypothalamus of both adult and fetal rats contains a population of cells which can exhibit some features of serotoninergic (5-HT) neurons under certain circumstances. However, their neuronal serotoninergic nature is still controversial. In fact the presence of tryptophan hydroxylase activity has not yet been clearly established. This study attempted to verify whether [3H]5-HT can be synthesized from [3H]tryptophan ([3H]TRP) in hypothalamic cell cultures from 16-day-old fetuses. Data showed that [3H]5-HT was synthesized from [3H]TRP and the amounts of [3H]5-HT increased linearly as a function of time for 60 min. Pargyline markedly increased the quantities of [3H]5-HT and decreased those of [3H]5-hydroxyindole acetic acid, [3H]5-HT synthesis was inhibited by p-chlorophenylalanine, while α-methyl-p-tyrosine had no effect. The present biochemical study shows the presence of an intrinsic 5-HT neuronal system in the hypothalamus of the fetal rat.

Bilateral olfactory bulbectomy (OB) has drastic biochemical and behavioral effects and is often associated with an increase in plasma corticosterone concentrations. This experiment examined the effects of OB on adrenocorticotropin (ACTH) and corticosterone release under basal and stress conditions and on proopiomelanocortin (POMC) gene expression. Bulbectomy potentiated hypophysal ACTH and adrenal corticosterone release induced by ether stress but had no effect on ACTH release under basal conditions, despite a significant increase of circulating corticosterone. POMC gene expression was stronger (+60%) in OB rats than in sham-operated rats. These results suggest that olfactory bulbectomy substantially altered the negative feed-back exerted by glucocorticoids on anterior pituitary corticotropic cells in the male rat.

Many studies have shown the existence of functional interactions between central neurotransmitter systems and the hypothalamo-pituitary adrenal axis. Mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) are regulated by multiple factors including glucocorticoids themselves. Neurotransmitters such as serotonin (5-hydroxytryptamine: 5-HT) can regulate brain corticosteroid receptors in a complex way. The present study examined the short-term (48 h) effects of parachlorophenylalanine (PCPA), a drug which specifically inhibits 5-HT synthesis, on corticosteroid receptor levels and on the expression of their respective messenger ribonucleic acids (mRNA) in the rat hippocampus, hypothalamus and brain stem. The study was performed in bilaterally adrenalectomized animals, in order to avoid potential drug-induced changes in plasma corticosterone levels, which could secondarily regulate MR and GR. Short-term inhibition of 5-HT synthesis by PCPA significantly increased the number of hippocampal MR-binding sites. PCPA treatment did not alter the number of GR-binding sites in the hippocampus, hypothalamus and brain stem. We observed no change in the affinities of GR and MR sites in all the structures studied. In PCPA-treated rats, restoration of control 5-HT levels by injection of its immediate precursor, 5-hydroxytryptophan (5-HTP) brings the number of hippocampal MR-binding sites back to control levels. It can therefore be concluded that the increase in number of MR-binding sites induced by acute PCPA treatment is dependent on the decrease in 5-HT levels. The increase in hippocampal MR binding sites was correlated with an induction of their messengers, suggesting that 5-HT modulates the synthesis of MR protein. Although PCPA did not modify the number of hippocampal GR-binding sites, a decrease in hippocampal GR mRNA expression was observed. This study shows that 5-HT inhibits hippocampal mineralocorticoid receptor synthesis and that this effect is not mediated by changes in corticosterone hormone secretion, and illustrates the existence of complex mechanisms for corticosteroid receptor regulation in the hippocampus.

International audience; We have previously reported that selective axotomy of serotoninergic neurons produced by an intraventricular injection of 5, 7‐dihydroxytryptamine is followed by an increase in 5‐HT1B binding sites in the suprachiasmatic nucleus of the hypothalamus. This post‐lesion up‐regulation is shown here to be spontaneously reversed after long‐term survival in spite of an incomplete reinnervation of the nucleus. Recovery may be accelerated by fetal raphe transplants that produce more rapid reinnervation.

Bilateral olfactory bulb lesions in rats simulate aspects of chronic major depression in humans (1,2). Among the animal models of depression that have been presented in the literature, numerous studies have demonstrated that some or all of endocrine and behavioral effects of olfactory bulbectomy can be reversed by subchronic administration of antidepressant drugs, including tricyclic agents such as imipramine and atypical agents such as mianserin (3, 4, 5). Broekkamp et al(6) have also reported that acute administration of single doses of several drugs believed to facilitate serotoninergic activity (fluoxetine, fen-fluramine, quipazine) reversed the passive avoidence deficit shown by bulbectomized rats.

Serotonin1B (5-HT1B) receptor binding in the suprachiasmatic nucleus (SCN) following impairment of serotoninergic transmission was studied by quantitative autoradiography. Serotonin (5-HT) denervation with 5,7-dihydroxytryptamine (5,7-DHT) caused a significant increase in the density of 5-HT1B receptors in both the ventral (62%) and dorsal (53%) parts of the SCN as early as 3 days after axotomy. The magnitude of this increase did not differ 3, 15 or 21 days post-lesion. An up-regulation of 5-HT1B receptors with similar magnitude was obtained in the two parts of the SCN after inhibition of 5-HT synthesis by chronic parachlorophenylalanine treatment. In this case, up-regulation was shown to be reversible after restoration of 5-HT synthesis with l-5-hydroxytryptophan. These results indicate that 5-HT1B receptor density in the SCN was inversely correlated with 5-HT levels. These plastic properties exhibited by 5-HT1B receptors in the SSN are discussed in relation to the mode of 5-HT transmission and possible localization of the receptors onto the main chemically defined cell populations of the nucleus.

The action of serotonin on growth hormone (GH) secretion is controversial because of interspecies differences and lack of specificity of serotoninergic drugs. Serotonin (5-HT) appears to inhibit GH release in the sheep and in man. We have investigated the site of action of tianeptine, a 5-HT uptake enhancer, in sheep since it is possible to collect hypophysial portal blood for the simultaneous determination of growth hormone-releasing hormone (GHRH) and somatostatin in this species under conscious, unstressed conditions. Tianeptine injection (10 mg/kg i.v.) resulted in a significant, immediate and short-lasting (30 min) increase in peripheral GH (+750%; P < 0.01) and hypophysial portal GHRH (+180%; P < 0.01). No change in the secretion of somatostatin was recorded during the same time. These data suggest that serotoninergic inputs are inhibitory to GH secretion. Tianeptine acts centrally to stimulate GH secretion in the sheep and its effect is mediated through changes in GHRH but not somatostatin release into hypophysial portal blood.

International audience; Glutamic acid and glycine were quantified in cells and medium of cultured rostral rhombencephalic neurons derived from fetal rats. In the presence of 1 mM Mg2+, NMDA (50 microM) significantly stimulated (by 69%) release of newly synthesized 5‐[3H]hydroxytryptamine ([3H]5‐HT). D‐2‐Amino‐5‐phosphonopentanoate (AP‐5; 50 microM) blocked the stimulatory effect of NMDA. AP‐5 by itself inhibited [3H]5‐HT release (by 25%), suggesting a tonic control of 5‐HT by glutamate. In the absence of Mg2+, basal [3H]5‐HT release was 60% higher as compared with release with Mg2+. AP‐5 blocked the increased [3H]5‐HT release observed without Mg2+, suggesting that this effect was due to the stimulation of NMDA receptors by endogenous glutamate. Glycine (100 microM) inhibited [3H]5‐HT release in the absence of Mg2+. Strychnine (50 microM) blocked the inhibitory effect of glycine, indicating an action through strychnine‐sensitive inhibitory glycine receptors. The [3H]5‐HT release stimulated by NMDA was unaffected by glycine. In contrast, when tested in the presence of strychnine, glycine increased NMDA‐evoked [3H]5‐HT release (by 22%), and this effect was prevented by a selective antagonist of the NMDA‐associated glycine receptor, 7‐chlorokynurenate (100 microM). 7‐Chlorokynurenate by itself induced a drastic decrease in [3H]5‐HT release, indicating that under basal conditions these sites were stimulated by endogenous glycine. These results indicate that NMDA stimulated [3H]5‐HT release in both the presence or absence of Mg2+. Use of selective antagonists allowed differentiation of a strychnine‐sensitive glycine response (inhibition of [3H]5‐HT release) from a 7‐chlorokynurenate‐sensitive response (potentiation of NMDA‐evoked [3H]5‐HT release).

The occurrence of serotonin in the human adrenal gland was demonstrated both by immuno-histochemical and biochemical approaches. Using specific polyclonal antibodies to serotonin, the presence of numerous immunoreactive cells was revealed by means of the peroxidase-antiperoxidase technique. These cells exhibited the morphological characteristics of mast cells. Combination of high performance liquid chromatography and electrochemical detection showed the presence of substantial amounts of both serotonin and its metabolite 5-hydroxyindolacetic acid in adrenocortical extracts. The role of serotonin in the regulation of steroidogenesis from human adrenocortical slices was studied in vitro using a perifusion system technique coupled to a specific radioimmunoassay for cortisol. Graded doses of serotonin (from 10(-8) M to 3 x 10(-7) M) increased cortisol production in a dose-dependent manner. Prolonged exposure of adrenal fragments to serotonin (10(-7) M) induced a biphasic response, i.e. a rapid and transient increase in cortisol secretion followed by a plateau phase, suggesting the existence of a desensitization phenomenon. The stimulatory effect of serotonin (10(-7) M) was not altered during infusion of the serotonin1 and/or serotonin2 receptor antagonists methysergide (10(-6) M) and ketanserin (10(-6) M), respectively. In contrast, ICS 205 930 (10(-6) M), a non-selective serotonin3/serotonin4 antagonist, totally abolished the response of adrenal slices to serotonin (10(-7) M). The benzamide derivative zacopride, considered as a serotonin4 agonist, induced a robust stimulation of cortisol secretion. In addition, the corticotropic effects of serotonin (10(-7) M) and zacopride (10(-6) M) were not additive. Incubation of adrenocortical fragments with zacopride (10(-6) M) or serotonin (10(-6) M) caused a significant increase in cAMP formation. Taken together, these data suggest that serotonin, locally released by intra-adrenal mast-like cells, may act as a paracrine factor to stimulate cortisol secretion in man. Our results also indicate that serotonin-induced corticosteroid production is mediated through activation of a serotonin4 receptor subtype positively coupled to adenylate cyclase.

The inter-renal (adrenal) gland of amphibians is composed of chromaffin and steroidogenic cells which can interact through a paracrine mode of communication. We have previously shown that serotonin is present in secretory granules of frog adrenochromaffin cells; concurrently, we have demonstrated that serotonin is a potent stimulator of corticosterone and aldosterone secretion by adrenocortical cells. The aim of the present study was to determine the origin of the amine contained in frog chromaffin cells. Using 3 H-labelled tryptophan as a precursor, we observed the formation of substantial amounts of serotonin and its metabolite 5-hydroxyindoleacetic acid by frog inter-renal slices. Newly synthesized serotonin was secreted into the incubation medium and the release process was enhanced by depolarizing concentrations of KCl. Fluoxetine, an inhibitor of serotonin uptake, caused an increase of 3 H-labelled serotonin in the incubation medium, suggesting that the indoleamine was taken up again by adrenal chromaffin cells. The capacity of the frog inter-renal gland to synthesize serotonin was also demonstrated by incubating inter-renal slices with non-labelled tryptophan or 5-hydroxytryptophan. In these conditions, we observed that the rate of synthesis was higher when 5-hydroxytryptophan was used as a precursor, rather than tryptophan. Taken together, these results indicate that chromaffin cells, which have the capacity for synthesizing and releasing serotonin, behave like authentic serotonergic paraneurons. As far as is known, these data provide the first evidence for the occurrence of tryptophan-5-hydroxylase activity within the adrenal gland.

This study aimed at analyzing the regulation of in vitro serotonin expression by neurons taken from different regions of the embryonic rat rhombenceph-alon. We studied the influence of co-culture with alar-plate tissue using immunocytochemical and biochemical methods. Computer-assisted densitometry was used to estimate the co-culture effects on the serotonin content of the cell bodies. The more dynamic aspects of serotonin expression, such as synthesis and release, were studied by measuring (3H)serotonin newly synthe-sized from (3H) tryptophan. The density of the immunostaining was significantly decreased in B1, B2 cells by co-culture with both caudal and rostral alar-plate tissue. For B4–B9 cells, only co-culture with rostral alar-plate tissue produced a significant decrease. The de novo synthesis of serotonin was significantly decreased in B1, B2 neurons co-cultured with caudal alar-plate tissue only. Once again, the B4–B9 cells proved to be less influenced by the experimental conditions, as co-culture with both types of alar-plate tissue produced no significant effect. We concluded that the in vitro expression of serotonin can be modulated by environmental factors, but the relative influence of these factors is very different in rostral versus caudal serotonin expressing cell populations.

Autoregulatory mechanisms affecting serotonin [5-hydroxytryptamine (5-HT)] release and synthesis during the early period of development were investigated in dissociated cell cultures raised from embryonic rostral rat rhombencephalon. The presence of 5-HT1A and 5-HT1B receptors in serotoninergic neurons was assessed using binding assays. The involvement of 5-HT1A and 5-HT1B receptors in the control of the synthesis and release of [3H]5-HT was studied using biochemical approaches with several serotoninergic receptor ligands. A mean decrease of 30% in [3H]5-HT synthesis and release was observed in the presence of 5-HT (10−8M), the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), the 5HT1B/1A agonist 5-methoxy-3-(1,2,5,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969), the 5-HT1B agonist 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP-93, 129), and the 5-HT1D/1B agonist sumatriptan. Inhibition of 5-HT synthesis and release induced by 8-OH-DPAT was blocked by chiral N-tert-butyl-3-[1-[1-(2-methoxy)phenyl]piperazinyl]-1-phenylpropionamide dihydrochloride quaternary-hydrate (WAY 100135) (10−7M) or methyl 4-[4-[4-(1,1,3-trioxo-2H-1,2-benzoisothiazol-2-yl)butyl]-1-piperazinyl]-1H-indole-2-carboxylate (SDZ 216–525) (10−7M), and that of CP-93, 129 was blocked by methiothepin (10−7M). Paradoxically, extracellular levels of [3H]5-HT increased in the presence of 8-OH-DPAT and RU 24969 at 10−6M. 5-HT uptake experiments showed that these two agonists interacted with the 5-HT transporter. 5-HT1 binding sites (620 fmol/mg of protein) and 5-HT1A (482 fmol/mg of protein) and 5-HT1B (127 fmol/mg of protein) receptors were detected in 12-day in vitro cell cultures. Experiments carried out with tetrodotoxin suggested that 5-HT1A receptors are located on nerve cell bodies, whereas 5-HT1B receptors are located on the nerve terminals. We concluded that autoregulatory mechanisms involving 5-HT1A and 5-HT1B autoreceptors are functionally mature in cells from rostral raphe nuclei during the early period of development.

The local effect of L-glutamic acid (5 x 10(-5) M) on the release of [3H]serotonin continuously synthesized from [3H]tryptophan was examined in the caudate nucleus of 'encephale isole' unanaesthetized cats implanted with push-pull cannula. L-Glutamic acid (5 x 10(-5) M) decreased [3H]serotonin release from nerve terminals of the dorsalis raphe-striatal serotonergic neurons. The effect was antagonized by 2-amino-6-trifluoromethoxybenzothiazole (PK 26124) (10(-6) M), an antagonist of glutamatergic transmission. This effect was mimicked by N-methoxy-D-aspartic acid NMDA (5 x 10(-5) M) and prevented by DL-2-phosphono-valeric acid (APV) (5 x 10(-6) M), indicating that L-glutamic acid decreased serotonin release via a N-methoxy-D-aspartate type receptor. The superfusion of serotonergic nerve terminals in the caudate nucleus with tetrodotoxin prevented the inhibitory L-glutamic acid-induced effect on serotonin release. Furthermore, L-glutamic acid-induced inhibition of [3H]serotonin release was antagonized by bicuculline (5 x 10(-5) M). These data suggest that the glutamatergic receptors involved were not located directly on serotonin nerve terminals. The inhibitory control exerted by L-glutamic acid on serotonergic transmission could involve gamma-aminobutyric acid interneurons. Since no reduction of spontaneous [3H]serotonin release was observed in the presence of bicuculline, GABAergic neurons appeared to exert a phasic influence on serotonin release. Indirect inhibitory presynaptic control on serotonin release mediated by corticostriatal glutamatergic fibers is discussed in light of previous findings.

Using a push-pull cannula technique and an isotopic method for estimating [ 3 H]serotonin continuously synthesized from [ 3 H]tryptophan, the effects of changes in the release of serotonin in the dorsalis raphe nucleus on in vivo release of [ 3 H]serotonin in the cat caudate nucleus were investigated. The increase in the release of serotonin in the dorsalis raphe nucleus caused by local application of parachlorophenylethylamine (10 −6 M) reduced striatal [ 3 H]serotonin release. This inhibition in serotonin release in the striatum was blocked by the prior and continuous local superfusion of the dorsal raphe with methiothepin (10 −6 M), a serotonin autoreceptor antagonist. GABA (5 × 10 −5 M) applied to the dorsalis raphe reduced both local and striatal release of [ 3 H]serotonin. However, picrotoxin (10 −5 M), a OABA A receptor antagonist applied locally in the dorsalis raphe nucleus increased [ 3 H]serotonin release while decreasing striatal [ 3 H]serotonin release. This decrease in serotonin release in the striatum was again blocked by continuous superfusion of the raphe with methiothepin. Furthermore, superfusion of serotonergic cell bodies of the dorsalis raphe nucleus with methiothepin alone never altered local release or striatal release of [ 3 H]serotonin. These data strongly suggest that the release of serotonin from the cell body in the dorsalis raphe nucleus phasically controls release of the amine at the axonal nerve ending through serotonergic autoreceptors located on serotonergic nerve cell bodies in the dorsalis raphe nucleus. The origin of the serotonin released in the dorsalis raphe nucleus and the possibility that this type of regulation could be related to changes in nerve impulse conduction of the serotonergic raphe-striatal system are discussed.

The effect of vasoactive intestinal peptide (VIP) on spontaneous and induced release of newly synthesized 5-hydroxytryptamine (5-HT) was studied in the suprachiasmatic area (SCA) using a superfusion system. To test the possible modualtion by E 2 on the interaction VIP-5-HT, the experiments were conducted on male, ovariectomized (OVX) and ovariectomized oestradiol implanted rats (OVX-E 2 ). VIP (10 −7 M) infused for 15 min caused an increase of 5-HT release from SCA of male and OVX. The positive effect of VIP on 5-HT release results partially from an inhibition of the reuptake of 5-HT: in male and OVX SCA, VIP inhibited the 3 H-5-HT uptake by 40 to 50%. The infusion of VIP before a pulse of K + (10-20-30-56 mM) leads to a potentialisation of the evoked release suggesting that VIP sensitized the presynaptic membrane to the process linking depolarization and release. When SCA taken from OVX-E 2 were exposed to VIP, 5-HT uptake and consequently 5-HT release were unchanged. The present results suggest that the metabolism of 5-HT in the SCA is influenced by VIP and that this regulation may be modulated by E 2 . This interaction between E 2 , VIP and 5-HT at the SCA level may be involved in the regulation of phasic LH and prolactin surge.

Various stressful manipulations in rats (cold-water swim, electric foot-shock administration, imparied access to food reward) were found to reduce the convulsant potency of drugs which interfere with GABA or benzodiazepine central processes. The convulsant threshold dosages of picrotoxin (0.4 mg/ml) or pentetrazol (10 mg/ml) administered after the stress by infusion (0.2 ml/min) via a vein of the tail were enhanced. The onset of generalized seizures induced by isoniazid (800 mg/kg) or by thiosemicarbazide (64 mg/kg) i.p. was delayed after cold-water swim. However, convulsant threshold dosages of bemegride or strychnine perfused at 2 and 0.2 mg/ml respectively were not changed by stress. Cold-water swim increased the number of cortical (but not cerebellar) [3H]flunitrazepam binding sites (+ 24%) but failed to alter cortical [3H]muscimol binding. This post-stress enhancement of binding sties, although suppressed by bicuculline (10(-4) M) seems not to be dependent on GABAergic mechanisms. Indeed cold-water stress did not reduce the ability of muscimol (10(-6) and 10(-5) M) and GABA (5 x 10(-6) and 5 x 10(-5) M) to increase flunitrazepam binding. Finally, this post-stress enhancement of benzodiazepine binding was not found to be paralleled by changes in the protective effects of diazepam against picrotoxin- or pentetrazol-induced seizures.

The presence of serotonin (5-HT) in dissected intestinal muscular wall of the caecum was demonstrated by the determination of endogenous level of the amine by both spectrofluorimetric and radioenzymatic assays. Biosynthesis of [ 3 H]5-HT from [ 3 H]tryptophan in in vitro conditions revealed the presence of tryptophan hydroxylase in these muscular layers and therefore strongly suggest the presence of serotoninergic neurons. Following dissection of the mucosa from the muscular layers containing the nerve plexuses, endogenous 5-HT and 5-hydroxyindol acetic acid levels as well as amounts of [ 3 H]5-HT synthesized from [ 3 H]tryptophan were always higher than those found in intact fragments of the caecum. These results are discussed in terms of metabolic processes involved in the regulation between the two 5-HT containing compartments.

Serotonin of the superior cervical ganglion is contained in a distinct and separate population of small intensely fluorescent (SIF) cells. We provide evidence, in in vitro experiments, that newly synthesized serotonin can be released in the cat superior cervical ganglion from the serotonin-containing SIF cells. Resting steady state in the release of [ 3 H]serotonin was observed 30 min after the beginning of the superfusion with l -[ 3 H]tryptophan. A marked increase was seen in the serotonin release either in the presence of fluoxetine, a potent reuptake blocker of serotonin, or during depolarization with potassium chloride or veratridine. Calcium-free medium led to a decrease of spontaneous and potassium-evoked release. The veratridine-stimulating response was abolished by tetrodotoxin. Paradoxically, a slight increase in the spontaneous release of serotonin was observed in the presence of tetrodotoxin. Serotonin released from serotonin-containing SIF cells could be involved in the modulation of ganglionic transmission.

Since metergoline (1-methyl-8-beta-carbobenzyloxy-aminomethyl-10-alpha-ergoline) is a potent 5-HT antagonist in peripheral organs, its possible blocking effects on 5-HT receptors in the rat brain were investigated. In vitro, metergoline inhibited both the specific high affinity binding of 3-H-5-HT onto synaptosomal membranes (IC 50 = 18 nM) and the stimulating effect of 10 micron 5-HT on the adenylate cyclase activity in colliculi homogenates from newborn rats (IC 50 = 12 micron. In vivo, the administration of metergoline (10 mg/kg i.p., 60 min before death) resulted in a significant decrease in the 3-H-5-HT binding capacity of synaptosomal membranes from the forebrain of adult rats. Taken together, these data clearly indicated that metergoline is a potent blocker of some serotoninergic receptors in the rat brain. Surprisingly, the changes in 5-HT turnover occurring in the brainstem and in the forebrain 1 h after metergoline (2-10 mg/kg) treatment were similar to those normally induced by a central 5-HT agonist: both the rate of 5-HT utilisation and that of 5-HT synthesis were significantly decreased. These changes were in contrast to the acceleration of 5-HT turnover induced by the administration of another potent central 5-HT antagonist, methiothepin. These results are discussed in relation to the possible existence of several types of serotoninergic receptors in the rat brain. It is possible that the positive feedback regulation of 5-HT turnover is triggered by the blockade of serotoninergic receptors sensitive to methiothepin, but not to metergoline.

Summary The intraventricular administration of 6-hydroxydopamine (6-OHDA) to kittens between 5 days and 4 months of age induced marked changes in the endogenous levels of DA, NE and 5-HT in various brain areas. In contrast to the well-known selective effect of 6-OHDA against catecholaminergic neurones in the rat, serotoninergic neurones were also markedly affected by 6-OHDA treatment in kittens; particularly in the hippocampus and the colliculi, 5-HT levels were markedly and permanently decreased after the intraventricular administration of 6-OHDA. A significant but less pronounced reduction in 5-HT levels was also noted in other areas such as the piriform cortex, the cerebral neocortex, the cerebellum and the septum. Only very discrete changes were detected in the caudate nucleus, the olfactory tubercle and the raphe area. The administration of chlorimipramine (10 mg/kg, i.p.) 1 h before 6-OHDA treatment completely prevented the effects of the neurotoxic agent on serotoninergic innervation. Marked regional differences were also noted concerning the effects of 6-OHDA treatment on dopaminergic neurones. Whereas DA levels in the raphe area and the hypothalamus were almost unaffected, they were permanently reduced by about 50% in the caudate nucleus and the olfactory tubercle after the intraventricular administration of 6-OHDA. In the caudate nucleus, the reduction was even much more pronounced (−90%) when 6-OHDA was administered during the first 3 postnatal weeks. In most forebrain areas (hippocampus, piriform cortex and cerebral neocortex) and in the cerebellum, NE levels were permanently reduced to about 10% of those of control kittens as soon as the third day following the intraventricular injection of 6-OHDA. In contrast, after a transient drop, NE levels in the lateral brain stem (containing the locus coeruleus) of 6-OHDA-treated kittens returned and even surpassed (+100%) those found in age-paired controls. Analyses of the characteristics of l -[ 3 H]NE uptake in synaptosomes indicated that 6-OHDA treatment resulted in both a striking loss of specific uptake sites in forebrain areas and a significant increase in the V max of the NE uptake process in synaptosomes from the lateral brain stem. However, in contrast to the rapid increase in NE levels, this change in V max occurred much later, since it was first detected at more than one month after 6-OHDA treatment. This delay suggests that the doubling in NE levels occurring for the first month following the intraventricular administration of 6-OHDA simply resulted from an increased accumulation of the catecholamine in noradrenergic terminals in the lateral brain stem. Later on, the change in V max might indicate a sprouting of new noradrenergic terminals in the vicinity of the locus coeruleus area. The intraventricular administration of 6-OHDA resulted in a significant increase in the maximal stimulatory effect of l -isoproterenol on adenylate cyclase activity in homogenates of the cerebral cortex and the lateral brain stem. Therefore, not only the degeneration (in the cerebral cortex) but also the proliferation (in the lateral brain stem) of noradrenergic terminals were associated with an increase in the density of beta-adrenergic receptors. These results are discussed in relation to the possible function of the noradrenergic sprouts in the lateral brain stem.

Serotonin (5-HT) metabolism was studied on isolated mucosa of the rat caecum in order to determine its characteristics in enterochromaffin cells. High levels of 5-HT were found in these cells and weak catabolic processes were demonstrated. Enterochromaffin cells of the mucosa are able to synthesize ((3)H)5-HT from ((3)H)tryptophan in vitro indicating that these cells might contain tryptophan hydroxylase. In addition, a high affinity uptake of exogenous ((3)H)5-HT is demonstrated. These results show that enterochromaffin cells of the mucosa present similar biochemical properties compared to those described for serotoninergic neurons of the central nervous system.

Summary The effects of a 96 h paradoxical sleep deprivation (PS) on the metabolism of serotonin have been studied in the brain stem-mesencephalon, cortex-diencephalon and spinal cord of rats. A marked increased formation of [ 3 H]serotonin was observed in tissues of PS-deprived animals after intracisternal administration of [ 3 H]tryptophan or after incubation with [ 3 H]tryptophan when compared with control animals. No effect could be seen when [ 3 H]5-hydroxytryptophan was used instead of [ 3 H]tryptophan in experiments in vivo as well as in vitro . As indicated by the changes on [ 3 H]tryptophan accumulation in tissues and on serotonin-specific activity in the experiment in vitro , the increased [ 3 H]serotonin synthesis in PS-deprived animals seemed to result from both an increased transport of [ 3 H]tryptophan and an increased rate of conversion of [ 3 H]tryptophan to [ 3 H]5-hydroxytryptophan. The utilization of brain [ 3 H]serotonin endogenously synthesized from [ 3 H]tryptophan was accelerated during PS deprivation. The increased turnover of serotonin in PS-deprived animals appeared to be related to the impossibility of triggering PS.

Unilateral kainic acid lesion of the rat centromedian-parafascicular complex (CM-PF) of the thalamus induced a decrease in the 5-hydroxyindole acetic acid/5-hydroxytryptamine ratio both in ipsi and contralateral striatum and substantia nigra, and an increase in both ipsi and contralateral frontal cerebral cortex. No change in apparent serotonin turnover was detected in anterior raphe nuclei. Serotonin synthesis, estimated by measuring 5-hydroxytryptophan accumulation after injection of a decarboxylase inhibitor, was not affected by the CM-PF lesion. The possible pathways involved in the control of serotonin transmission by CM-PF are discussed.

"In vitro" 3H-5-HT and 3H-5-HIAA, newly synthesized from 3H-TRP, are measured in the brainstem, the anterior raphe nuclei and the locus coeruleus of C57BL and BALBc mice. As found for the caudate nucleus and the hippocampus, higher synthesis and release are determined in C57BL than in BALBc, for the locus coeruleus and more globally, for the brainstem. But these differences disappear when the study is carried on the raphe dorsalis and the raphe centralis nuclei. Therefore the serotonergic activity could be independently regulated at the level of cell bodies and at those of terminals. However, the 5-HT metabolism of NRD of BALBc mice could be submitted to a specific autoinhibition which could explain a lower 5-HT synthesis and release at the corresponding terminal level, compared to C57BL.

Daily changes in tryptophan-5-hydroxylase (TrH) activity have been studied in six different 5-HT-containing cell groups (B 3 , B 4 , B 5 , B 6 , B 7 , B 8 ) in the brain stem of rats maintained in a regular cycle of 12 h of light and 12 h of darkness. Significant circadian alternations of TrH activity were observed in most of the raphe nuclei tested, although the changes were not identical from one structure to another. The striatum showed daily variations in TrH activity in opposite phase to the nucleus raphe dorsalis which projects specific terminals into this area. Monoamine oxidase (MAO) activity was simultaneously estimated in these nuclei but did not exhibit any significant rhythm, suggesting a specific regulation of TrH. Total protein levels were also subject to daily changes.

The release of 3H-serotonin (3H-5-HT) endogenously synthesized from 3H-tryptophan was estimated in both dorsalis and magnus (MRN) raphe nuclei of anaesthetized “encephale isole” cats, by using push-pull cannulae. Resting steady state in the release of 3H-5-HT was observed 30 min after the beginning of superfusion with L-3H-tryptophan. The amounts of 3H-5-HT released in the DRN and the MRN are much greater than those measured simultaneously in the caudate nucleus. A marked increase either in the 5-HT release was seen in the presence of fluoxetine, a potent reuptake blocker of 5-HT, or during local depolarization with potassium chloride. The spontaneous release was diminished by removing Ca++ and by adding cobalt to the medium. Tetrodotoxin (TTX) decreased the 5-HT release in the DRN and, based on previously established, blocked the stimulating effect of batrachotoxin. According to the pharmacological characteristics of the monoamine dendritic release determined for dopamine in the substantia nigra [17], our results suggest that 5-HT release processes in the DRN correspond to a release from nerve endings, not from dendrites. The purpose of this study was to determine if the 5-HT released in the DRN is released from either axon terminals or dendrites. Morphological studies performed on the DRN do not consistently demonstrate the high density of serotoninergic varicosities in the DRN. In addition, two types of 5-HT axonal varicosities, characterized by their synaptic or non-synaptic junctions, are present in the brain. The concept that the quantities of 5-HT released could vary from one type compared to the other is discussed.

The mechanisms involved in parafascicularis nucleus control on serotoninergic neurons projecting into the caudate nucleus were investigated in “ence´phale-isole” cats. The effects of unilateral stimulation of the parafascicularis nucleus on the release of newly synthesized [3H]serotonin were simultaneously determined in the ipsilateral caudate nucleus and the dorsalis raphe nucleus using push-pull cannulae. The actions of various pharmacological treatments performed either in the caudate nucleus or in the dorsalis raphe nucleus were also examined. The electrical or chemical stimulation of the parafascicularis nucleus induced a decrease in striatal [3H]serotonin release and an increase in [3H]serotonin release in the dorsalis raphe nucleus. The blockade of cholinergic (mecamylamine) and glutamatergic (PK 26124) transmissions at the striatal level did not modify the thalamic stimulation induced effect on serotonin release in the caudate nucleus or in the dorsalis raphe nucleus. However, a decrease induced by parafascicularis nucleus stimulation in serotonin release in the caudate nucleus could not be observed when the autoreceptors present on serotoninergic nerve cell bodies localized in the dorsalis raphe nucleus were blocked by a methiothepin perfusion within the nucleus. These results indicate that the parafascicularis nucleus controls striatal serotonin transmission by inducing changes in the nerve activity of serotoninergic neurons in the dorsalis raphe nucleus via somatodendritic serotonin release and autoreceptors.

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Summary Electrolytic raphe lesion was performed in 4–6-day-old rats and the resulting changes of 5-HT metabolism within the central nervous system were analyzed up to 9 months later. As soon as the 2nd day following the selective destruction of B7 and B8 nuclei, forebrain 5-HT levels were decreased by more than 75%. This reduction persisted for at least 9 months with no sign of recovery. The time course of 5-HIAA decrease was parallel to that of the indoleamine so that the ratio of 5-HIAA over 5-HT levels in the forebrain of lesioned rats was similar to that estimated in controls, whatever their age. This result would suggest that the remaining serotoninergic neurons in the lesioned rats did not develop a compensatory hyperactivity. The raphe lesion induced no change in MAO activity and synaptosomal tryptophan uptake but a pronounced decrease in the Vmax of synaptosomal 5-HT uptake process in various forebrain areas occurred. The serotonin sensitive adenylate cyclase activity in colliculi homogenate was not altered by the lesion suggesting that this enzyme was probably located in postsynaptic membranes. In addition, this observation would indicate that 5-HT receptors which are linked to this adenylate cyclase did not become supersensitive following the selective degeneration of serotoninergic neurons. Animals without forebrain serotoninergic innervation might be of great interest to analyse the role of serotoninergic neurons in various functions (sleep, analgesia, thermoregulation).

Vasoactive intestinal peptide (VIP) inhibits serotonin (5-HT) uptake in the suprachiasmatic area (SCA) of the rat. The present study investigates the possibility of a functional relationship between 5-HT uptake mechanisms and 5-HT autoreceptor activity in this effect of VIP in the SCA. The hypothesis of a linkage between these two mechanisms of 5-HT regulation has been recently proposed. We investigated the possibility of the presence of 5-HT autoreceptors in the SCA. Using superfusion system, exogenous 5-HT (500 and 50 nM) increased the release of newly synthesized 3 H-5-HT. In contrast, 5 nM of exogenous 5-HT inhibited this release. This latter effect was antagonized by methiothepin (10 −7 M). Infusion of VIP increased the level of 3 H-5-HT in the effluent fractions; this effect was not modified by methiothepin (10 −7 M). In contrast, the concentration of methiothepin required to inhibit the VIP effect was 10 −6 or 10 −5 M, the same molarity found to decrease the 5-HT uptake. On the other hand, the increase of the 3 H-5-HT in the synaptic cleft, induced by VIP, did not modify the inhibition of 3 H-5-HT release induced by 5 nM of exogenous 5-HT. We conclude that the effect of VIP on 5-HT metabolism in the SCA is linked to the 5-HT uptake mechanism but not to the activity of 5-HT presynaptic autoreceptors. In our experimental conditions, the activity of 5-HT autoreceptors is independent of the 5-HT uptake processes.

Using a push-pull cannula technique and an isotopic method for the estimation of [ 3 H]serotonin continuously synthesized from [ 3 H]tryptophan, the effects of acetylcholine were investigated on the in vivo release of [ 3 H]serotonin in the cat basal ganglia and the dorsal raphe nucleus. The unilateral striatal application of acetylcholine (5 × 10 −5 M) reduced local release of [ 3 H]serotonin. This effect was mimicked by nicotine (5 × 10 −5 M) and prevented by mecamylamine (10 −6 M). Oxotremorine (5 × 10 −5 M) had no effect on the local release of [ 3 H]serotonin. All these treatments failed to modify [ 3 H]serotonin release in the ipsilateral substantia nigra or in the dorsal raphe nucleus. The superfusion of serotonergic nerve terminals of the caudate nucleus with tetrodotoxin prevented the inhibitory acetylcholine-induced effect on serotonin release. Furthermore, bicuculline (5 × 10 −5 M) in the caudate nucleus blocked the effect of nicotine, while γ-aminobutyric acid (10 −4 M) induced a decrease in local release of [ 3 H]serotonin. These data strongly suggest that the inhibitory control exerted by acetylcholine on serotonergic transmission could involve γ-aminobutyric acid interneurons. Acetylcholine-induced changes in [ 3 H]serotonin release were only observed in non-anaesthetized “encephale isole” cats and not in halothane-anaesthetized animals. The possibility that such a regulation could be presynaptic (direct or through other neurotransmitters) or related to a change in the activity of the serotonergic raphe-striatal neuronal system is discussed.

Newly synthesized serotonin (5-HT) can be released in the nodose ganglion from the nerve cell bodies of vago-aortic serotoninergic neurones. Free-calcium led to a decrease of spontaneous and potassium-evoked release. The veratridine-stimulating response was abolished by TTX. The concept that 5-HT released from perikarya in the extracellular space could be involved in the self-regulation of the activity of the vago-aortic pathway is discussed.

Daily changes in 5-HT metabolism have been studied in the cortex, hypothalamus and brain stem of rats maintained in a regular cycle of 12 h of light and 12 h of darkness. The synthesis of [3H]5-HT from [3H]Try was first estimated in vivo, at various times of two successive periods of 6 h of light and darkness, by measuring the accumulation of [3H]5-HT and its metabolite [3H]5-HIAA 7 min after the intracisternal administration of the 3H precursor. Endogenous 5-HT levels were always lower during the dark period in the cortex and the brain stem; a similar observation was made for the initial accumulation of [3H]5-HT in all structures examined. From the total accumulation of [3H]5-HT and [3H]5-HIAA and the specific activity of Try in tissues, it appears that 5-HT synthesis decreased significantly at 21.00 h (dark) as compared with 15.00 h (light) in the brain stem and the hypothalamus. Changes in synthesis were dependent on [3H]Try initial accumulation in tissues and were not related to changes in the rate of conversion of Try into 5-HT. The latter results were confirmed in vitro by measuring the accumulation of [3H]5-HT and [3H]5-HIAA in hypothalamic and brain stem slices and their incubating media. The close correlation found between [3H]Try initial accumulation and total [3H]5-HT plus [3H]5-HIAA formation in studies both in vivo and in vitro indicated that 5-HT synthesis was not dependent on plasma tryptophan levels in normal physiological states. Finally the decreased synthesis of 5-HT that occurred during the night was confirmed in the whole brain with the MAOI method. The changes in the relative accumulation of [3H]5-HT and its metabolite [3H]5-HIAA observed in vivo at various times of the light and dark periods and those detected in vitro at 15.00 and 21.00 h, both in the brain stem and the hypothalamus, showed a marked activation of the utilization of the newly synthetized transmitter during the dark period which started immediately after the extinction of light. The estimation of the relative accumulation of [3H]5-HT and [3H]5-HIAA in slices and incubating medium of hypothalamic slices at 15.00 and 21.00 h suggested that the increased utilization observed at 21.00 h corresponded to an enhanced extraneuronal release of the transmitter. Therefore the processes of synthesis and release of 5-HT in central serotoninergic neurones were not synchronized, synthesis was maximal during the light period and release was activated during the dark period. This study not only provided new information concerning the circadian rhythms in 5-HT synthesis and utilization but also enabled us to discover new aspects of the processes of the regulation of 5-HT metabolism in central serotoninergic neurones.