Effects of 5-hydroxytryptophan on serotonin in nerve endings

—Preparations of synaptosomes (P₂) from the telencephalon and from the diencephalon plus optic lobes of the pigeon and from the telencephalon of the rat were used to study the effects of 5-hydroxytryptophan (5-HTP) on (a) the levels of serotonin (5-HT) in nerve endings and (b) the release of 5-HT from nerve endings. The levels of 5-HT were significantly higher (3.21 × 0.35 nmol/g original tissue weight) in the P₂ fraction isolated from the telencephalon of pigeons given intramuscular injections of 50mg/kg of d ,l -5-HTP in comparison to control values (1.42 ± 0.07). A similar twofold increase was observed with the P₂ fraction isolated from the diencephalon plus optic lobes. In addition, the levels of 5-HTP and 5-hydroxyindoleacetic acid also increased significantly in these P₂ fractions isolated from pigeons given d ,l -5-HTP injections in comparison to values obtained for pigeons given saline injections. In vitro studies using preparations of synaptosomes (from both pigeon and rat) labelled with [³H]5-HT indicated that 0.10 mil l -5-HTP increased the release of [³H]5-HT twofold over control values. A concentration as low as 0.001 mm l -5-HTP was tested on the P₂ fraction from the telencephalon of the pigeon and was found to significantly increase the release of [³H]5-HT over control values. This effect by l -5-HTP was blocked if a decarboxylase inhibitor was added to the medium. l -5-HTP at a concentration of 1.5 mm had no apparent effect on the release of [³H]norepinephrine or [³H]dopamine from synaptosomes prepared from the telencephalon of the rat or pigeon. The results are discussed in terms of the role of serotonin in producing certain types of behavioral depressions exhibited by pigeons and rats given injections of 5-HTP.     

Increased Extracellular Serotonin in Rat Brain After Systemic or Intraraphe Administration of Morphine

Abstract: The effect of morphine on serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in the CNS of unanesthetized rats was investigated by microdialysis. Morphine was administered either subcutaneously, by local perfusion into the diencephalon, or by intraraphe microinjection. Systemic administration of morphine resulted in a significant increase in both extracellular 5-HT and 5-HIAA in the diencephalon. The effect of morphine on 5-HT was dose dependent during local perfusion of the diencephalon with inhibitors of uptake or monoamine oxidase. Systemic morphine also produced significant increases in extracellular 5-HT in the striatum and hippocampus during uptake inhibition. The site of opioid effects on 5-HT was tested by locally perfusing morphine into the diencephalon. This had no effect on 5-HT or 5-HIAA. In contrast, intraraphe injection of morphine caused a dose-dependent increase in extracellular 5-HT and 5-HIAA in the diencephalon. These results suggest that systemic morphine induces an increase in 5-HT release in widespread areas of the forebrain. This appears to be due to an effect on 5-HT cell bodies and not on 5-HT nerve endings in projection sites.     

Localization of serotonin in the hypothalamus and the mesencephalon of the guinea-pig

Summary The distribution of serotonin in the hypothalamus and the mesencephalon of guinea-pigs pretreated with both pargyline and L-tryptophan was investigated immunohistochemically using monoclonal antibodies to 5-HT. 5-HT-positive fibers and varicosities appeared distributed throughout the hypothalamus. Some areas showed a greater density of immunoreactivity: the suprachiasmatic nucleus, the region of the supraoptic crest, the area of the medial forebrain bundle, the ventral part of the nucleus ventromedialis, the median eminence and the ventral part of the mammillary bodies. 5-HT nerve fibers were also scattered in the posterior lobe of the pituitary. An extensive supraependymal plexus of immunoreactive axons was observed in most ventricular regions. No 5-HT positive cell bodies were present in the hypothalamus of the guinea-pig under our experimental conditions, whereas an intense serotonin immunoreactivity was detected in perikarya of the brain stem. 5-HT cell bodies were found predominantly in the raphe region including the nucleus raphe dorsalis and raphe medianus, nucleus interpeduncularis, reticular formation and dorsal area of the medial lemniscus.     

Effects of tryptophan on serotonin in nerve endings.

—Preparations of crude synaptosome fractions (P₂) from the telencephalon and from the diencephalon plus optic lobes of the pigeon and from the telencephalon of the rat were used to study the effects of l -tryptophan on (a) the levels of serotonin (5-HT), norepinephrinc (NE) and dopamine in nerve endings and (b) the release of radioactive 5-HT, NE and dopamine from nerve endings. The level of 5-HT was significantly higher (P < 0–05) in the P₂ fraction isolated from the telencephalon of pigeons given intramuscular injections of 300mg/kg of l -tryptophan in comparison to control values (1.11 ± 0.09 vs 0.74 ± 0.13 nmol/g original tissue wt). A smaller but not statistically significant increase in 5-HT was noted in the P₂ fractions isolated from the diencephalon plus optic lobes of pigeons given injections of l -tryptophan. In vitro studies using preparations of synaptosomes (from both pigeon and rat) labelled with [³H]5–HT demonstrated that 1.0 mm -l -tryptophan caused a 30% increase (P < 0.05) in the release of [³H]5-HT over control values. This effect by l -tryptophan was blocked when a decarboxylase inhibitor was added to the medium. Tryptophan had no effect on the levels of NE or dopamine in these nerve endings nor did it have any effect on the release of these two amines from these preparations of synaptosomes. The results are discussed in terms of the role of serotonin in producing depression in pigeons working on a certain learned behavioural task.     

Intracisternal neurotoxins and monoamine neurons innervating the spinal cord: Acute and chronic effects on cell and axon counts and nerve terminal densities

Summary The total number of catecholamine (CA) and 5-hydroxy-trydroxy-tryptamine (5-HT) containing nerve cell bodies in groups A1-7 and B1-3 were counted using Falck-Hillarp fluorescence histochemistry and found to be about 5000 and about 6300 respectively. The total number of CA axons in the white matter of the spinal cord was found to be about 4300 at the cervical level and about 3300 at the lumbar level as revealed in cryostat sections. Together with previous studies our quantitative results suggest that individual cell bodies in locus coeruleus send axon collaterals to the spinal cord, cortex cerebri, cortex cerebelli and probably also other areas of the brain.The decrease in number of CA axons in spinal cord occurred approximately at the level where the CA nerve terminal innervation of the sympathetic lateral column ends. Single CA axons were observed to give off multiple branches at right angles, suggesting that single CA axons innervate several different levels of the grey matter.A transient loss of visible CA cell bodies in groups A1-7 was observed after intracisternal injection of 25–50 g 6-hydroxydopamine (6-OH-DA). A permanent loss of visible 5-HT cell bodies in groups B1-3 was observed after 25 g 5,6-dihydroxy-tryptamine (5,6-HT) given intracisternally, a transient decrease was obtained when the same dose of 5,6-HT was given intraventricularly.CA nerve terminals regenerated in a cranio-caudal direction along the motoneuron columns after lesioning with 6-OH-DA given intracisternally. The reappearing CA nerve terminals seemed to outline the longitudinally oriented dendritic bundles of the motoneurons and was exclusively found in these areas of the ventral horn. Thus, no substantial CA nerve terminal reinnervation of zona intermedia or the dorsal horn was observed. About 10% of the CA axons running in white matter were left after intracisternal 6-OH-DA and found at all levels of the cord. The number of remaining CA axons did not increase with time.After 10–50 g 5,6-HT given intracisternally a return of 5-HT nerve terminals was only observed in cervical segments. There was a 60–90% decrease in number of axons that accumulated 5-HT after an acute transverse lesion and no recovery of the number of such accumulations was observed during the following six months.It was concluded that regeneration of CA nerve terminals in the spinal cord can occur after lesioning with 6-OH-DA given intracisternally. Regeneration of 5-HT nerve terminals following intracisternal 5,6-HT is very restricted probably because of the permanent loss of 5-HT cell bodies obtained by the intracisternal route of administration as opposed to the intraventricular route. It was suggested that the reappearing CA nerve terminals were derived from locus coeruleus and that they were specifically guided by the motoneuron columns.     

Fetal and postnatal development of cyclic GMP phosphodiesterase activity in the rat brain

Cyclic GMP concentration and cyclic GMP phosphodiesterase activity were studied in rat mothers and fetuses at 17, 19 and 21 days of intrauterine life and 0, 1, 4, 10, 15,20, 30 and 45 days after birth. During this developmental period, the increase in cyclic GMP concentration was discrete and the value in 15-day-old rats was already similar to the adult level. Cyclic GMP phosphodiesterase activity increased from 17- to 19-day fetuses and was significantly reduced in 21-day fetuses, neonates, and 1-day-old rats. This reduction may be a result of fetal endocrine preparation for parturition. During postnatal development, cyclic GMP phosphodiesterase activity increased in a parallel way in the limbic system, corpora striata, cerebral hemispheres, and diencephalon, reaching maximal level between 20 and 30 days after birth, and then decreasing to the adult value. The highest activity was found in corpora striata and the lowest in diencephalon. Cerebellar cyclic GMP phosphodiesterase activity was very high in the 4-day-old rat (257% of adult value) and diminished significantly in the 10-day-old rat with no subsequent changes. Kinetic analysis of the enzyme during postnatal forebrain development showed an increase in both the V_max and the apparent K_m. A decrease in the enzyme's V_max was observed only in the cerebellum.The importance of cyclic GMP phosphodiesterase regulation of cyclic GMP concentrations in the brain during development is discussed.     

Distribution of atrial natriuretic factor-like immunoreactivity in the brain of the frog Rana ridibunda

The localization of atrial natriuretic factor (ANF)-like immunoreactivity in the central nervous system of the frog Rana ridibunda was examined by the indirect immunofluorescence technique, using an antiserum against synthetic ANF (Arg101-Tyr126). Immunoreactive cell bodies were principally found in the dorsal and medial pallium, the medial septal nucleus, the ventrolateral and anteroventral areas of the thalamus, the lateral forebrain bundle, the posterolateral thalamic nuclei, the preoptic nucleus, the dorsal infundibular nucleus, and the anteroventral tegmentum nucleus of the mesencephalon. Numerous cell bodies and a very dense fiber bundle were visualized in the interpeduncular nucleus. All the areas mentioned above contained a high density of immunoreactive fibers. In addition, the amygdala, the infundibular nucleus, the median eminence, and most of the areas of the mesencephalon contained a moderate number of ANF-positive nerve processes. In the frog pituitary, fibers and nerve terminals were found in the peripheral zone of the neural lobe. The intermediate and anterior lobes of the frog pituitary were totally devoid of ANF immunoreactivity. These results indicate that ANF-like material is widely distributed in the frog brain and that ANF may be involved in various brain functions including neuroendocrine regulations.     

Male and female mice living in differently-sized groups

Summary The influence of crowding in different group sizes on the metabolism of serotonin (5-HT) in six different brain areas and on some peripheral data in male and female mice was investigated. In the cortex, the diencephalon and in the basal ganglia, the 5-HT turnover of male mice increases with group size. The females react differently (Figs. 2, 3, 4). The densityinduced changes in the 5-HT metabolism of the mesencephalon correlated individually with the locomotor reactivity of the animals in an open-field (Figs. 8, 9). The septal area is the only part of the brain in male and female mice in which the 5-HT turnover increase depends on group size (Fig. 5).We wish to thank the Deutsche Abbott GmbH which supplied us with pargyline.     

Amino Acid Content of Nerve Endings (Synaptosomes) in Different Regions of Brain: Effects of Gabaculine and Isonicotinic Acid Hydrazide

Abstract: The amino acid content of synaptosomes was determined in six regions of rat brain, and in all regions the five predominant amino acids were glutamate, glutamine, aspartate, taurine, and GABA (γ-aminobutyrate). However, the proportions of the individual amino acids varied considerably from one region to another, the GABA content being particularly high and the taurine content low in synaptosomes from the diencephalon and mesencephalon. Administration of isonicotinic acid hydrazide to rats lowered the synaptosomal GABA level by similar amounts in all brain regions, but the administration of gabaculine resulted in a particularly long-acting elevation in GABA levels in the nerve endings of the diencephalon and mesencephalon. The possibility is raised that the high GABA levels in the nerve terminals of the diencephalon may be involved in the gabaculine-induced lowering of the body temperature of the rats. A constancy in the amount of the synaptosomal pool of "aspartate + glutamate + glutamine + GABA" was observed despite large changes in the relative amounts of the four amino acids brought about by gabaculine.     

Distribution of immunoreactive thyrotropin-releasing hormone in the forebrain and hypophysis of the bullfrog,Rana catesbeiana

The distribution of immunoreactive thyrotropin-releasing hormone (TRH) in the forebrain and hypophysis of Rana catesbeiana was studied by means of specific radioimmunoassay and immunohistochemistry based on peroxidase-antiperoxidase (PAP) techniques. A relatively high concentration of immunoassayable TRH is present in the hypothalamus. Immunoreactive TRH cell bodies are found in the anterior part of the preoptic nucleus, the dorsal infundibular nucleus, the nucleus of diagonal band of Broca, and the medial part of the amygdala. Immunoreactive nerve terminals are observed in the neurohypophysis and the external layer of the median eminence, where the terminals are in close contact with the capillary loops of the hypophyseal portal vessels. The possible role of TRH in the frog brain is discussed.     

Serotonin immunoreactivity in the intermediate lobe of the rat pituitary.

Immunocytochemical staining for serotonin (5-HT) in paraffin-embedded sections of rat pituitary resulted in the localization of reactive nerve fibres and cell bodies in the intermediate lobe. Immunostaining was also found in the anterior and posterior lobes. Labelled nerve fibres appear to enter the intermediate lobe from the neural lobe through the interlobular spaces. These fibres are relatively scarce and lightly stained. Neuroglandular contacts were identified between varicose nerve endings containing serotonin and immunoreactive perykarion. It is not clear whether intermediate lobe cells produced 5-HT themselves or, alternatively, these cells take in 5-HT from serotoninergic nerve terminals.     

Glucose increases synaptic transmission from vagal afferent central nerve terminals via modulation of 5-HT3 receptors

Acute hyperglycemia has profound effects on vagally mediated gastrointestinal functions. We have reported recently that the release of glutamate from the central terminals of vagal afferent neurons is correlated directly with the extracellular glucose concentration. The present study was designed to test the hypothesis that 5-HT(3) receptors present on vagal afferent nerve terminals are involved in this glucose-dependent modulation of glutamatergic synaptic transmission. Whole-cell patch-clamp recordings were made from neurons of the nucleus tractus solitarius (NTS) in thin rat brainstem slices. Spontaneous and evoked glutamate release was decreased in a concentration-dependent manner by the 5-HT(3) receptor selective antagonist, ondansetron. Alterations in the extracellular glucose concentration induced parallel shifts in the ondansetron-mediated inhibition of glutamate release. The changes in excitatory synaptic transmission induced by extracellular glucose concentration were mimicked by the serotonin uptake inhibitor, fenfluramine. These data suggest that glucose alters excitatory synaptic transmission within the rat brainstem via actions on tonically active 5-HT(3) receptors, and the number of 5-HT(3) receptors on vagal afferent nerve terminals is positively correlated with the extracellular glucose concentration. These data indicate that the 5-HT(3) receptors present on synaptic connections between vagal afferent nerve terminals and NTS neurons are a strong candidate for consideration as one of the sites where glucose acts to modulate vagovagal reflexes.     

Localization of atrial natriuretic factor (ANF)-related peptides in the central nervous system of the elasmobranch fish Scyliorhinus canicula

We have investigated the localization of atrial natriuretic factor (ANF)-like immunoreactivity in the central nervous system of the cartilaginous fish, Scyliorhinus canicula, using the indirect immunofluorescence technique. Immunoreactive perikarya and fibers were observed in two regions of the telencephalon, the area superficialis basalis and the area periventricularis ventrolateralis. In the diencephalon, the hypothalamus exhibited a moderate number of ANF-containing neurons and fibers located in the preoptic and periventricular nuclei and in the nucleus lateralis tuberis. The most important group of ANF-immunoreactive cells was observed in the nucleus tuberculi posterioris of the diencephalon. In contrast, the mesencephalon showed only a few ANF-positive nerve processes located in the tegmentum mesencephali. Numerous fine fibers and nerve terminals were found in the dorsal area of the neurointermediate lobe of the pituitary. These results provide the first evidence for the presence of ANF-related peptides in the brain of a cartilaginous fish. The widespread distribution of ANF-positive cells and fibers in the brain and pituitary suggests that this peptide may act both as a neurotransmitter and (or) a neurohormone in fish.     

Retrograde axonal transport after radioactive serotonin injections into the olfactory bulb: a biochemical analysis of transported radioactive material

A biochemical analysis of radioactive compounds was performed in the olfactory bulb (OB) and raphe dorsalis (RD) after injection of radioactive [³H] or [¹⁴C]serotonin (5-HT ranging from 10^?2 M to 10^?7 M) into the OB of rats treated or not with a monoamine-oxidase inhibitor (MAOI).In the OB of untreated rats, radioactivity was associated with precipitated protein and soluble perchloric acid (PCA) fractions. High performance liquid chromatography (HPLC) analysis of the PCA-supernatant gave 4 radioactive peaks: one associated with endogenous 5-HT, another with endogenous 5-hydroxyindole acetic acid (5-HIAA) and two without any relationship with endogenous hydroxyindoles: a ‘5-HT derivative A’ and a ‘5-HT derivative B’. The presence of these ‘5-HT derivatives’ was significantly reduced after treatment with 5,6-dihydroxytryptamine.In the RD, radioactivity was associated with the protein fraction and with ‘5-HT derivative A’. The kinetic analysis (from 30 min to 46 h) of the ‘5-HT derivative A’ was characterized by a disappearance in the OB and an accumulation in the RD corresponding to a rate of migration in a range of 0.7 to 2 mm/h. This compound was absent or negligible in other non-serotoninergic neurons (such as the Locus Coeruleus, Amygdala and Cortex piriformis). No clear evidence for retrograde transport of radioactive 5-hydroxytryptophan (5-HTP) or 5-HIAA was found.At lower concentration of 5-HT injected into the OB, the half lives and the times of maximal accumulation for 5-HIAA, ‘5-HT derivative A’ and ‘5-HT derivative B’ were increased. The specific activity of 5-HT and 5-HIAA was also increased.The selective radioactive accumulation in the cell bodies of RD neurons after injection of radioactive 5-HT into the OB is discussed as resulting from a selectivity in (a) the uptake by 5-HT nerve terminals; (b) the metabolism of 5-HT into ‘5-HT derivative A’ in the OB; (c) the retrograde axonal transport of ‘5-HT derivative A’. This ‘5-HT derivative A’ could represent a messenger between nerve terminals and cell bodies and could be involved in homeostatic mechanisms that maintain cellular dynamics.When a MAOI was used, ‘5-HT-derivative A’ and [³H]5-HT were found in the OB and also in the RD cell bodies, and to a lesser extent, in the non-serotoninergic cell bodies. These results indicate that MAO inhibition produces a relative non-selectivity in the ‘uptake-metabolism and retrograde axonal transport’ systems.     

Modulation by Monoamines of Somatostatin-Sensitive Adenylate Cyclase on Neuronal and Glial Cells from the Mouse Brain in Primary Cultures

Primary cultures of mouse embryonic neuronal or glial cells from the cerebral cortex, striatum, and mesencephalon were used to identify and determine the cellular localization of somatostatin receptors coupled to an adenylate cyclase. Somatostatin inhibited basal adenylate cyclase activity on neuronal but not on glial crude membranes in the three structures examined. The somatostatin-inhibitory effect on neuronal crude membranes was still observed in the presence of (-)-isoproterenol, 3,4-dihydroxyphenylethylamine (dopamine, DA), or 5-hydroxytryptamine (5-HT, serotonin) used at a concentration (10(-5) M) inducing maximal adenylate cyclase activation. In addition, in most cases biogenic amines modified the pattern of the somatostatin-inhibitory effect, triggering either an increase in the peptide apparent affinity for its receptors or an increase in the maximal reduction of adenylate cyclase activity or both. However, 5-HT did not modify the somatostatin-inhibitory response on striatal and cortical neuronal crude membranes. The changes in somatostatin-inhibitory responses were interpreted as a colocalization of the amine and the peptide receptors on subtypes of neuronal cell populations. Finally, somatostatin was shown to inhibit adenylate cyclase activity following its activation by (-)-isoproterenol on glial crude membranes of the striatum and the mesencephalon but not on those of the cerebral cortex.     

NEUROCHEMICAL CORRELATES OF BEHAVIOR

Abstract— Pigeons working on a multiple fixed ratio 50, fixed interval 10 schedule for food reinforcement were killed at 0, 50, 90, 150 and 240 min after an i.m. injection of 300mg/kg l -tryptophan. The levels of tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, 5-hydroxyindole acetic acid, tyrosine, dopamine and norepinephrine were concurrently measured in crude nerve ending fractions (P₂) isolated from the telencephalon, diencephalon plus mesencephalon and pons plus medulla-oblongata of each pigeon. Increases in 5-hydroxytryptamine levels in the nerve ending fraction from the telencephalon were correlated with the onset of the decreased response rates, whereas a return to baseline responding was correlated with a return to normal serotonin levels in this fraction. Changes in dopamine or norepinephrine were not related to the onset of or recovery from the decreased response rate. One group of pigeons were found which did not display any behavioral disruption even though each had received an injection of l -tryptophan; the content of 5-hydroxytryptophan, 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in the nerve ending fraction isolated from the telencephalon of these birds did not differ from control values.     

Regional distribution of 5-hydroxytryptamine in cat brain.

The regional distribution of serotonin (5-HT) in the brain of the cat is poorly understood. In this work, serotonin was analyzed fluorometrically along the brain stem and prosencephalon of the cat. The hypothalamus had the highest concentration of serotonin. Serotonin decreased gradually at the mesencephalon, preoptic area, medulla oblongata, hippocampus, pons, visual cortex, spinal cord and frontal cortex. Significant differences were found between the raphe (3 mm thick) and the lateral blocks of the brain stem. The concentration of serotonin is higher in the raphe blocks, though it decreases caudally. There is no significant difference between the raphe (4 mm thick) and the lateral block of the brain stem. The results demonstrate the regional concentration of serotonin in the CNS of a normal cat, the relationships between serotoninergic neuron groups and serotonin concentration,and the probable significance of nerve terminals and varicosities in storing serotonin.     

Localization of atrial natriuretic factor (ANF)-related peptides in the central nervous system of the elasmobranch fish Scyliorhinus canicula

We have investigated the localization of atrial natriuretic factor (ANF)-like immunoreactivity in the central nervous system of the cartilaginous fish, Scyliorhinus canicula, using the indirect immunofluorescence technique. Immunoreactive perikarya and fibers were observed in two regions of the telencephalon, the area superficialis basalis and the area periventricularis ventrolateralis. In the diencephalon, the hypothalamus exhibited a moderate number of ANF-containing neurons and fibers located in the preoptic and periventricular nuclei and in the nucleus lateralis tuberis. The most important group of ANF-immunoreactive cells was observed in the nucleus tuberculi posterioris of the diencephalon. In contrast, the mesencephalon showed only a few ANF-positive nerve processes located in the tegmentum mesencephali. Numerous fine fibers and nerve terminals were found in the dorsal area of the neurointermediate lobe of the pituitary. These results provide the first evidence for the presence of ANF-related peptides in the brain of a cartilaginous fish. The widespread distribution of ANF-positive cells and fibers in the brain and pituitary suggests that this peptide may act both as a neurotransmitter and (or) a neurohormone in fish.     

Selective inhibition of MAO-A in serotonergic synaptosomes by two amphetamine metabolites, p-hydroxyamphetamine and p-hydroxynorephedrine

The present study was carried out mainly to clarify whether the two amphetamine metabolites, p-hydroxyamphetamine (P-OHA) and p-hydroxynorephedrine (p-OHN) are taken up by mouse brain 5-hydroxytryptamine (5-HT) nerve terminals to inhibit type A monoamine oxidase (MAO-A) and then potentiate the abnormal behavior, head-twitch. Of the two metabolites, only intracerebroventricular p-OHA, at 80 μg/mouse, sufficient to cause a head-twitch response (HTR), appreciably inhibited MAO-A activity without affecting MAO-B activity in homogenates of the mouse striatum, hypothalamus and the rest of the forebrain; and p-OHN did not inhibit either type of MAO at the dose tested. Estimation of intra- and extrasynaptosomal MAO-A activity showed that both metabolites significantly inhibited only the intrasynaptosomal deamination of 5-HT by MAO-A with p-OHA being more potent. Taken together with our previous findings, these present results clearly indicate that p-OHA may accumulate in the 5-HT nerve terminals through the uptake system, and concomitantly inhibit MAO-A activity. These actions of p-OHA may increase intraneuronal 5-HT levels and then potentiate 5-HT release to cause interaction with the post-synaptic 5-HT receptors.     

Regulation of synaptic vesicles pools within motor nerve terminals during short-term facilitation and neuromodulation.

The reserve pool (RP) and readily releasable pool (RRP) of synaptic vesicles within presynaptic nerve terminals were physiologically differentiated into distinctly separate functional groups. This was accomplished in glutamatergic nerve terminals by blocking the glutamate transporter with dl-threo-beta-benzyloxyaspartate (TBOA; 10 microM) during electrical stimulation with either 40 Hz of 10 pulses within a train or 20- or 50-Hz continuous stimulation. The 50-Hz continuous stimulation decreased the excitatory postsynaptic potential amplitude 60 min faster than for the 20-Hz continuous stimulation in the presence of TBOA (P < 0.05). There was no significant difference between the train stimulation and 20-Hz continuous stimulation in the run-down time in the presence of TBOA. After TBOA-induced synaptic depression, the excitatory postsynaptic potentials were rapidly (<1 min) revitalized by exposure to serotonin (5-HT, 1 microM) in every preparation tested (P < 0.05). At this glutamatergic nerve terminal, 5-HT promotes an increase probability of vesicular docking and fusion. Quantal recordings made directly at nerve terminals revealed smaller quantal sizes with TBOA exposure with a marked increase in quantal size as well as a continual appearance of smaller quanta upon 5-HT treatment after TBOA-induced depression. Thus 5-HT was able to recruit vesicles from the RP that were not rapidly depleted by acute TBOA treatment and electrical stimulation. The results support the notion that the RRP is selectively activated during rapid electrical stimulation sparing the RP; however, the RP can be recruited by the neuromodulator 5-HT. This suggests at least two separate kinetic and distinct regulatory paths for vesicle recycling within the presynaptic nerve terminal.