Regulation Of Serotonin Release Research Articles

Ligand-Induced 5-HT3 Receptor Internalization in Enteric Neurons in Rat Ileum

Release of 5-hydroxytryptamine (5-HT) from mucosal enterochromaffin cells and activation of 5-HT(3) receptors (5-HT(3)Rs) on neurons in the gut wall is important in the response of the gut to the luminal environment. Intestinal inflammation is associated with increased levels of mucosal 5-HT. The aims of the study were to determine the following: (1) if 5-HT(3)R undergoes ligand-induced internalization in myenteric neurons, and (2) the effect of long-term increase of mucosal 5-HT on 5-HT(3)Rs. Acute effects of exogenous 5-HT or endogenous release of 5-HT by luminal glucose on cellular localization of 5-HT(3)Rs was determined by immunohistochemistry and confocal microscopy. Treatment with the serotonin re-uptake inhibitor, fluoxetine, for 6 days (20 mg/kg daily orally) was used to increase mucosal 5-HT chronically in rats. Net ileal fluid movement was measured in anesthetized rats by the weight change of a 2.5% agarose cylinder. Acute increases in 5-HT induced by exogenous or endogenous 5-HT decreased 5-HT(3)R immunoreactivity at the neuronal cell membrane by 70% and 60%, respectively. Chronic fluoxetine treatment increased mucosal levels of 5-HT and decreased membrane 5-HT(3)R immunoreactivity by 27%. Net fluid absorption was decreased by a 5-HT(3)R agonist or by luminal glucose; this was attenuated 88% and 99%, respectively, by fluoxetine treatment. Long-term increase in 5-HT in the intestinal mucosa results in increased 5-HT(3)R internalization in myenteric neurons. Chronic changes in mucosal 5-HT may alter gastrointestinal secretory and motor function via ongoing loss of receptor from neuronal membrane, causing a mismatch between luminal content and absorption.

Regulation of serotonin release in the Syrian hamster intergeniculate leaflet region.

In mammals, the thalamic intergeniculate leaflet (IGL) conveys behavioral (non-photic) phase-resetting information to the circadian clock of the suprachiasmatic nucleus. Here we report a 24 h fluctuation in in vivo serotonin release in the hamster IGL region, peaking at night. Novel wheel exposure at midday, a stimulus that can reset circadian phase, activates the release of serotonin in the IGL region. In addition, electrical stimulation of the dorsal raphe nucleus acutely increases serotonin release the IGL region, confirming a functional serotonergic projection from this nucleus to the IGL. Collectively, these findings suggest that behavioral and/or circadian changes in dorsal raphe nuclear activity could modulate serotonin-mediated activities of the IGL.

Regulation of Serotonin Release in the Lateral Septum and Striatum by Corticotropin-Releasing Factor

The serotonergic dorsal raphé nucleus (DRN) is innervated by corticotropin-releasing factor (CRF)-immunoreactive fibers and contains CRF receptor-binding sites, suggesting that endogenous CRF regulates this system. The present study examined the possibility that CRF in the DRN regulates the release of serotonin (5-HT) in forebrain terminal regions. Intracerebroventricular administration of CRF produced a bimodal effect on extracellular levels of 5-HT in the lateral septum. Doses of 0.3 and 1.0 microg decreased extracellular 5-HT levels, whereas both a higher (3.0 microg) and a lower (0.1 microg) dose had no effect. The reduction of extracellular 5-HT in the lateral septum by CRF (0.3 microg, i.c.v.) was blocked by pretreatment with the CRF receptor antagonist d-PheCRF(12-41) (3.0 microg, i.c.v.). Direct administration of CRF (30 ng) into the DRN reduced extracellular 5-HT levels in the lateral septum and the striatum. Furthermore, injection of d-PheCRF(12-41) (10 ng) into the DRN before ventricular administration of CRF (0.3 microg, i.c.v.) blocked the decrease in extracellular 5-HT in both the lateral septum and striatum. Taken together, these data support the hypothesis that CRF may modulate 5-HT release in terminal regions via its effects at the level of the DRN. This modulation supports a potential interaction between CRF and 5-HT in stress-related psychiatric disorders in which both systems have been implicated.

Regulation of serotonin release by GABA and excitatory amino acids.

Regulation of serotonin release by gamma-aminobutyric acid (GABA) and glutamate was examined by microdialysis in unanaesthetized rats. The GABA(A) receptor agonist muscimol, or the glutamate receptor agonists kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolaproprionate or N-methyl-D-aspartate were infused into the dorsal raphe nucleus (DRN) while extracellular serotonin was measured in the DRN and nucleus accumbens. Muscimol produced decreases, and the glutamate receptor agonists produced increases in serotonin. To determine if these receptors have a tonic influence on serotonergic neurons, glutamate or GABA(A) receptor antagonists were infused into the DRN. Kynurenate, a nonselective glutamate receptor blocker, produced a small, 30% decrease in serotonin. A similar decrease was obtained with combined infusion of AP-5 and DNQX into the DRN. The GABAA receptor blocker bicuculline produced an approximately three-fold increase in DRN serotonin. In conclusion, glutamate neurotransmitters have a weak tonic excitatory influence on serotonergic neurons in the rat DRN. However, the predominate influence is mediated by GABA(A) receptors.

Regulation of serotonin release in the frontal cortex and ventral hippocampus of homozygous mice lacking 5-HT1B receptors: in vivo microdialysis studies.

To assess the involvement of the serotonin receptor subtype 5-HT1B as terminal autoreceptor regulating 5-HT release in mice, we compared basal values and potassium-evoked changes of extracellular 5-HT levels obtained by in vivo microdialysis in two serotoninergic terminal projection areas of conscious wild-type mice with those measured in homozygous mutant mice lacking the gene encoding the 5-HT1B receptor. In the frontal cortex and ventral hippocampus, basal and K+-evoked 5-HT release did not differ between the two strains of mice studied. The infusion via reverse microdialysis of the selective 5-HT1B receptor agonist CP-93,129 (500 nM) decreased significantly K+-evoked 5-HT release in the frontal cortex (by -44%) and ventral hippocampus (by -32%) of wild-type mice but had no effect in mutants. In a similar manner, the mixed 5-HT1B-5-HT1D receptor agonist sumatriptan (800 nM) decreased significantly K+-evoked 5-HT release in the frontal cortex (by -46%) of wild-type mice but had no effect in mutants. These results demonstrated that 5-HT1B knockout mice are not as sensitive to full (CP-93,129) and mixed (sumatriptan) 5-HT1B receptor agonists as are wild-type mice. These data provide in vivo evidence that, in mice, 5-HT1B, but not 5-HT1D, autoreceptors inhibit 5-HT release at nerve terminals located in the frontal cortex and ventral hippocampus.

Protein kinase C: regulation of serotonin release from rat brain cortical slices

The effects of phorbol esters on serotonin release were examined in an attempt to investigate the role of protein kinase C in the regulation of serotonin release. Rat brain parietal cortical slices were incubated with [ 3H]5-HT in the presence of pargyline in order to label the serotonin stores. Potassium stimulated (30 s) release and spontaneous [ 3H]5-HT efflux were examined in slices during superfusion with Krebs-Ringer solution containing chlorimipramine. Repeated K + stimulations elicited reproducible responses with release ratios of approximately 1.0. Introduction of phorbol 12-myristate,13-acetate (PMA) or phorbol 12,13-dibutyrate (PDBu) 20 min prior to S 2, or S 3 resulted in dose-related increases in [ 3H]5-HT or [ 3H]NE release. PMA was slightly more potent (93% increase) than PDBu in potentiating K +-stimulated [ 3H]5-HT release. Phorbol and 4α-phorbol 12,13-didecanoate (4αPDD) which do not activate protein kinase C did not alter serotonin release. In contrast, basal [ 3H]5-HT and [ 3H]NE release were altered to a far lesser extent which was not always dose related. The response to the phorbol esters was reversible, Ca 2+-dependent and reached maximal effect after 20 min of superfusion. The putative protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) inhibited K +-induced [ 3H]5-HT release significantly (11%) but did not alter basal efflux. The PMA facilitation of serotonin release was, however, markedly prevented by the enzyme inhibitor. The effect of PMA on release was found not to be directly mediated through the prejunctional serotonin autoreceptors. This is suggested by (1) the similarity in PMA-induced release potentiation observed in the presence or absence of the serotonin agonist, 5-methoxytryptamine, and (2) by the inability of the kinase inhibitor, H-7, to alter K +-stimulated release by the serotonin receptor antagonist, methiothepin. These results on serotonin and norepinephrine release and those of previous workers dealing with other transmitters point to an important role of protein kinase C in the modulation of neurotransmitter release in the central nervous system.

Regulation of serotonin release from the in vitro rat hippocampus: effects of alterations in levels of depolarization and in rates of serotonin metabolism.

We analyze the time course of 5-hydroxytryptamine (5-HT, serotonin) release from K+-depolarized hippocampal slices using a two-compartment kinetic model. The model is based on the assumptions that the rate of release is dependent on the amount of 5-HT in a releasable pool and that this pool may be resupplied during depolarization by newly synthesized 5-HT. Comparisons were made between predictions of the model and observed changes in 5-HT metabolism and in 5-HT release studied under a variety of experimental conditions. In accordance with predictions of the model, experimental manipulation of 5-HT synthesis and breakdown rates did not affect release immediately after depolarization but did affect the release rate during prolonged depolarization. Increasing bath tryptophan from 0 to 10 microM approximately doubled both 5-HT synthesis and the release rate after 40 min of K+-induced depolarization while having a smaller effect on release during the first 2 min. Inhibition of 5-HT breakdown did not significantly affect release during the first 2 min of depolarization but increased it over threefold after 40 min. In contrast, altering the concentrations of K+ or Ca2+ in the incubation medium affected mainly the early phase of 5-HT release and not the late phase. Reducing Ca2+ from 2.4 to 0.4 mM reduced 5-HT release by about 30% during the first 9 min of depolarization but did not affect release during the subsequent 30 min. Increasing the concentration of K+ from 18 to 60 mM stimulated release by sixfold during the first 2 min but only twofold after a subsequent 30 min. These results support our kinetic model and suggest that regulation of 5-HT metabolism at the site of the nerve terminal could be a mechanism for modulation of 5-HT release during prolonged discharge of serotonergic neurons.

Synthesis and release of serotonin by brain slices: Effect of ionic manipulations and cationic ionophores

The effect of various ionic manipulations and cationic ionophores on the rates of release and synthesis of serotonin were investigated in brain slices prepared from adult male rats. Two depolarizing, univalent cationic ionophores, gramacidin (10 μg/ml) and valinomycin (10 μ/ml), and the non-depolarizing, calcium-specific ionophore, A23187 (190 μM), stimulated both the release and synthesis of serotonin in this tissue preparation. Electrical field depolarization of the brain slices also stimulated both the release and synthesis of serotonin, effects which were completely blocked by a calcium-free media and 1 mM EGTA or by 10 mM Mg 2+. Lithium partially blocked the release of serotonin by the stimulated brain slices, but markedly augmented the rates of serotonin biosynthesis. Although electrical stimulation significantly increased the rate of [ 3H]tryptophan uptake by the slices, the increased rates of release and synthesis of serotonin following treatment of the tissues with the ionophores and lithium were not associated with increased rates of tryptophan uptake. Furthermore, despite the inhibition of the release and synthesis of serotonin by stimulated slices incubated in the presence of magnesium or in the absence of calcium, rates of tryptophan uptake by the stimulated brain slice remained increased. These results taken together with the results of other studies suggest that release and synthesis of serotonin by the serotonergic neuron is tightly coupled to the ionic events attending depolarization and further argue than transcellular calcium fluxes may be of special importance in this regulation. These results do not support the postulate that regulation of serotonin release and synthesis is dependent upon substrate availability as a major variable.