N-Methyl-d-Aspartic Acid/Glycine Interactions on the Control of 5-Hydroxytryptamine Release in Raphe Primary Cultures

Abstract: 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 μM) significantly stimulated (by 69%) release of newly synthesized 5-[³H]hydroxytryptamine ([³H]5-HT). d -2-Amino-5-phosphonopentanoate (AP-5; 50 μM) 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 μM) inhibited [³H]5-HT release in the absence of Mg²⁺. Strychnine (50 μM) 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 μM). 7-Chlorokynuren-ate 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).     

Effect of vasoactive intestinal peptide on serotonin release in the suprachiasmatic area of the rat. Modulation by oestradiol

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 E₂ on the interaction VIP-5-HT, the experiments were conducted on male, ovariectomized (OVX) and ovariectomized oestradiol implanted rats (OVX-E₂). 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 ³H-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-E₂ 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 E₂. This interaction between E₂, VIP and 5-HT at the SCA level may be involved in the regulation of phasic LH and prolactin surge.     

Effects of pinealectomy and melatonin treatments on serotonin uptake and release from synaptosomes of rat hypothalamic regions

This study examined the effects induced by long-term pinealectomy, daily melatonin treatment to pinealectomized and intact rats, and a single melatonin injection on [¹⁴C]-serotonin (5-HT) uptake and release from synaptosomes obtained of hypothalamic regions. Pinealectomy inhibited the accumulation of labeled 5-HT by synaptosomes of the preoptic area-anterior hypothalamus (POA-AH), but it failed to alter the [K⁺]-evoked 5-HT release. Melatonin treatment for 10 consecutive days to pinealectomized rats restored 5-HT uptake in POA-AH, and also increased 5-HT release in medial and posterior hypothalamus. These results suggest that pineal melatonin plays a stimulatory role on the serotoninergic terminals of the hypothalamus. Moreover, when daily melatonin treatment was administered to intact rats a significant increase in 5-HT uptake activity by synaptosomes of all the hypothalamic regions was observed, but 5-HT release was unaffected. In contrast, a single melatonin injection induced a significant decrease in 5-HT release from synaptosomes of the POA-AH was observed. The results suggest the existence of a differential sensitivity in the mechanisms mediating melatonin actions on 5-HT uptake/release, which depends on the presence of the pineal gland in the animals and on the frequency of the treatments with the pineal hormone.     

Microtubule inhibitors alter the secretion of beta-glucuronidase by human blood platelets: involvement of microtubules in release reaction II

The effects of the antimicrotubular drugs colchicine and vinblastine on the blood platelet release reaction were studied by measuring release of ¹⁴C-5-hydroxytryptamine (¹⁴C-5-HT, release I) and β-glucuronidase (release II) from gel-filtered human platelets. β-glucuronidase release induced by thrombin was significantly inhibited by colchicine (0.01-1 mM) or vinblastine (0.05–0.1 mM). Release of ¹⁴C-5-HT, however, was unaffected at low concentrations of colchicine and only slightly inhibited at higher concentrations. Inhibition of β-glucuronidase release depended on colchicine or vinblastine concentrations and decreased with longer time intervals (1′, 5′, 20′) after thrombin stimulation. Levels of the cytoplasmic enzyme, lactic acid dehydrogenase, in supernatants of colchicine treated platelets were not significantly different from controls. Colchicine also inhibited β-glucuronidse release, but not ¹⁴C-5-HT release, induced by trypsin and sodium arachidonate. Binding of ¹⁴C-colchicine by platelets was measured and it was found that platelet aggregation and release of 5-HT induced by adenosine diphosphate, epinephrine and collagen proceeded without any alteration in colchicine binding. However, significant increases in the rate and degree of colchicine binding were observed when platelets were stimulated by thrombin, trypsin and arachidonic acid which induced aggregation, release of both 5-HT and β-glucuronidase. The results suggest that an alteration in platelet microtubules is correlated with the physiologic response resulting in release II and that the cellular mechanisms effecting release I and II by platelets differ qualitatively in that the microtubules may facilitate release II.     

[3H]Acetylcholine and [3H]5-hydroxytryptamine release from rat midbrain slices and the effects of calcium and phenobarbital

The K-stimulated release of [³H]ACh from rat midbrain slices prelabeled by incubation with [³H]choline was dependent on extracellular Ca. Phenobarbital inhibited the K-stimulated [³H]ACh release and the IC₅₀ was equal to that found for K-stimulated endogenous ACh release. These results support the suggestion that barbiturates primarily inhibit the Ca-dependent stimulated release of ACh and affect ACh synthesis only indirectly. K-Stimulated release of [³H]5-HT was also inhibited by removing Ca from the medium or by adding phenobarbital which further supports the effects of barbiturates on the depolarization-induced release process. Fluoxetine, an inhibitor of 5-HT uptake, increased the amount of [³H]5-HT found in the medium but did not fully block the uptake of [³H]5-HT in this slice preparation.     

Antagonism of 5-Hydroxytryptamine by Methiothepin shown in Micro-electrophoretic Studies of Neurones in the Lateral Geniculate Nucleus

THERE has been little success in the search for a specific antagonist of the actions of 5-hydroxytryptamine (5-HT) on central neurones, although several compounds reduce the effects of both tryptamine derivatives and catecholamines in the central nervous system^1,2. The recent report that lysergic acid diethylamide blocked the excitant action of 5-HT, but not that of noradrenaline, on medullary reticular neurones³ has not been confirmed⁴. Moreover, an earlier investigation of olfactory bulb neurones indicated that lysergic acid diethylamide blocked the action of noradrenaline more readily than that of 5-HT⁵.     

Presynaptic Control of the Synthesis and Release of Dopamine from Striatal Synaptosomes: A Comparison Between the Effects of 5-Hydroxytryptamine, Acetylcholine, and Glutamate

The effects of 5-HT and glutamate on dopamine synthesis and release by striatal synaptosomes were investigated and compared with the action of acetylcholine, which acts presynaptically on this system. 5-HT inhibited (28%) synthesis of [¹⁴C]dopamine from L-[U-¹⁴C]tyrosine, at 10-⁵M and above. This contrasts with the action of acetylcholine, which stimulated [¹⁴C]-dopamine synthesis by 24% at 10-⁴ M. Tissue levels of GABA were unaffected by either 5-HT or acetylcholine up to concentrations of 10-⁴ M. The inhibitory action of 5-HT (5 × 10^?5 M and 2 × 10^?4 M) on [¹⁹C]dopamine synthesis was completely abolished by methysergide (2 × 10^?6 M). Higher concentrations of methysergide (10^?4 M) or cyproheptadine (10^?5 M) inhibited [¹⁴C]dopamine synthesis by 28% and 25%, respectively, when added alone to synaptosomes. However, only methysergide prevented the further inhibition of synthesis caused by 5-HT. At concentrations of 2 × 10^?5 M and above, 5-HT stimulated [¹⁴C]dopamine release. This releasing action differed from that of acetylcholine, which occurred at lower concentrations (e.g., 10^?6 M). Methysergide (up to 10^?4 M) or cyproheptadine (2 × 10^?4 M) did not reduce the 5-HT (5 × 10^?5 M)-induced release of [¹⁴C]dopamine, but methysergide (10^?4 M) showed a potentiation (49%) of this increased release. The stimulatory effects of 5-HT (2 × 10^?5 M) and K⁺ (56 mM) on [¹⁴C]dopamine release were additive, indicating that two separate mechanisms were involved. However, when both agents were present the stimulatory effect of K⁺ (56 mM) on [¹⁴C]dopamine synthesis was not seen above the inhibitory effect of 5-HT. Glutamate (0.1-5 mM) did not affect [⁴C]dopamine release or its synthesis from L-[U-¹⁴C]tyrosine. It is concluded that 5-HT modulates the synthesis of dopamine in striatal nerve terminals through a presynaptic receptor mechanism, an action antagonised by methysergide. The releasing action of 5-HT apparently occurs through a separate mechanism which is also distinct from that involved in the response to K⁺ depolarisation.     

Heterogeneity,position, and functional capability of the macrophages in Peyer's patches

Summary Radioenzymatic assays and light microscope radioautographic studies performed on photophores of Porichthys notatus demonstrated (1) significant amounts of catecholamines (dopamine, noradrenaline, adrenaline) and 5-hydroxytryptamine (serotonin) in these organs; (2) selective uptake and storage of [³H]noradrenaline ([³H]NA) by axon terminals innervating the photocytes, and (3) strong accumulation of [³H]5-hydroxytryptamine ([³H]5-HT) within the photocytes. Uptake and storage of [³H]NA in the nerve fibers were seemingly unaffected by the addition of ten-fold molar concentrations of unlabelled serotonin. Accumulation of [³H]5-HT by the photocytes was dose-dependent and diminished markedly in the presence of ten-fold molar concentrations of non-radioactive noradrenaline. Neither neuronal uptake of [³H]5-HT or [³H]A, nor photocytic accumulation of [³H]A were detectable under the conditions of the present experiments. This information should provide a framework for further investigations of the regulation of photophore luminescence by the biogenic amines.Supported by grants from the National Research Council and Medical Research Council of CanadaJacques de Champlain and Lise Farley provided facilities and expertise with the radioenzymatic techniques. The technical assistance of Sylvia Garcia and Marie-Hélène Parizeau was also appreciated     

Electrochemical measurement of release of dopamine and 5-hydroxytryptamine from synaptosomes.

Liquid chromatography with electrochemical detection has been used to determine endogenous dopamine (DA) and serotonin (5-HT) in a striatal crude synaptosomal fraction isolated by centrifugal methods. The electrochemical determinations are accomplished with a new type of electrode material, pressure annealed pyrolytic graphite. The analysis scheme permits direct quantitation of DA and 5-HT with minimal sample pretreatment. The distribution of endogenous 5-HT in each subfraction throughout the centrifugation procedure is found to be approximately parallel to that of endogenous DA. The release of DA and 5-HT from the crude synaptosome preparation exhibits the properties expected for stimulus-coupled, depolarization-induced release. The release of both neurotransmitters is Ca²⁺ dependent and is induced by high K⁺ or veratridine in the external medium. The latter is blocked by tetrodotoxin. Release is independent of ascorbic acid, but is dependent on the temperature of incubation.     

The stimulation of GABAB receptors increases serotonin release in the rat suprachiasmatic area

The action of γ-aminobutyric acid (GABA) and related compounds on the spontaneous release of newly synthesized [³H]5-hydroxytryptamine ([³H]5-HT) was studied in the suprachiasmatic area (SCA) using a superfusion system. GABA (10 μM) increased [³H]5-HT release from SCA by up to 190%. Bicuculline or picrotoxin (10 μM) failed to inhibit the stimulatory effect of GABA. Muscimol (10 μM), a GABA_A agonist, was ineffective, however β-p-chlorophenyl GABA, R(−)baclofen, enhanced over 200% the release of the indoleamine; this latter effect was stereospecific. RS baclofen was twice less potent than the R(−)isomer in increasing the [³H]5-HT release. S(+)baclofen failed to affect the release of the indoleamine, whereas it attenuated the effect of its enantiomer. The effect of R(−)baclofen was Ca²⁺ dependent and was abolished by tetrodotoxin (TTX).Taken together these results suggest that in the SCA, [³H]5-HT release is facilitated by the stimulation of GABA_B receptors. The possible localization of these receptors is discussed in the light of morphological data recently reported by Bosler et al. (1985) and results obtained after TTX application.     

Release of [3H]serotonin from brain slices

1. Labelled serotonin ([³H]5-HT) accumulated by slices of rat brain either in vivo or in vitro is released by depolarizing procedures such as electrical stimulation or high external potassium concentrations. Electrical stimulation predominantly affects the liberation of the unchanged amine, rather than of its principal metabolite, 5-HIAA. 2. Release of [³H]5-HT does not appear to be calcium-dependent. 3. Amount of release parallels the density of serotonin-containing nerve terminals in each of several cerebral regions tested. Release from several extracerebral tissues was similar to that obtained from cerebral tissues having relatively little endogenous 5-HT. 4. Electrically induced release of [³H]5-HT is markedly inhibited by desipramine, chlorpromazine, LSD, lithium and ouabain.     

Studies on the Role of α2-Adrenoceptors in the Control of Synaptosomal [3H]5-Hydroxytryptamine Release: Effects of Antidepressant Drugs

The sensitivity of alpha 2-adrenoceptors on 5-hydroxytryptamine (5-HT) nerve endings obtained from rat cerebral cortex was investigated following treatment with the antidepressant drugs desipramine (10 mg/kg/day for 21-28 days) or clorgyline (1 mg/kg/day for 21-28 days). [3H]5-HT (100 nM) was used to load cortical synaptosomes (P2) after experiments with uptake inhibitors confirmed that this concentration of amine ensured exclusive uptake into 5-HT nerve terminals. The sensitivity of K+-stimulated release of [3H]5-HT to alpha 2-adrenoceptor occupancy was assessed in a superfusion system by means of the dose-dependent inhibition of [3H]5-HT release by clonidine. This is blocked by yohimbine (1 microM), which, when administered alone, enhances release, suggesting that endogenous catecholamines released from other synaptosomes act on these alpha 2-heteroreceptors. The effect of addition of citalopram (1 microM) to superfusates suggests that some reuptake of [3H]5-HT occurs during superfusion. Of the tritium released into superfusates during "background" and K+-stimulated release, 17 and 90%, respectively is [3H]5-HT. The attenuation of K+-stimulated release by clonidine is apparently diminished by the chronic clorgyline regimen but not by desipramine. However, clorgyline elevates catecholamine levels, and this might increase endogenous noradrenaline (NA) efflux, which by competition with clonidine could appear to alter alpha 2-adrenoceptor sensitivity. This possibility was investigated by depleting NA with the neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4). These studies showed that the apparent effect of chronic clorgyline on alpha 2-adrenoceptor sensitivity to clonidine was due to competition with increased levels of endogenous NA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoreceptor-mediated inhibition of 3H-5-hydroxytryptamine release from rat brain cortex slices by analogues of 5-hydroxytryptamine

Rat brain cortex slices preincubated with ³H-5-hydroxytryptamine (³H-5-HT) were superfused with physiological salt solution containing paroxetine, an inhibitor of 5-hydroxytryptamine (5-HT) uptake. The effects of various indolethylamines on the electrically evoked tritium overflow (containing 66.3% unmetabolized ³H-5-HT) were investigated (the percentage of unmetabolized ³H-5-HT was not altered by the indolethylamines or metitepin). 6,7-Dihydroxytryptamine (6,7-DHT) did not affect the stimulation-evoked tritium overflow, whereas the latter was inhibited by the other tryptamine derivatives investigated; when the compounds were compared to each other on the basis of their inhibitory potencies the following rank order was obtained: unlabelled 5-HT > 5-methoxytryptamine > 4-HT > 6-HT > 5,6-DHT > tryptamine > 7-HT > 5,7-DHT. The inhibitory effects of these compounds were antagonized by metitepin. It is concluded that the indolethylamines inhibit the stimulation-evoked ³H-5-HT release by activating the presynaptic 5-HT autoreceptors on the 5-HT neurones of the rat brain cortex. Similarities may exist between these receptors and the postsynaptic 5-HT_l binding sites of this brain area.     

Effects of Methiothepin and Lysergic Acid Diethylamide on Serotonin Release In Vitro and Serotonin Synthesis In Vivo: Possible Relation to Serotonin Autoreceptor Function

Abstract: An in vitro system characterizing the presyn- aptic serotonin (5-HT) autoreceptor which controls the release of 5-HT from rat brain slices is described. Using this system, methiothepin (1–10 μ M) demonstrated 5-HT autoreceptor antagonist activity -by enhancing 5-HT release, while several recognized postsynaptic 5-HT receptor antagonists were inactive: mianserin, cinanserin, cyproheptadine, methysergide. The activity of methiothepin was highest in hypothalamic slices and lowest in striatal slices and was inhibited by the autoreceptor agonists lysergic acid diethylamide (LSD) and 5-methoxy- tryptamine (5-MT). The reversal of the methiothepin-enhanced 5-HT release from hypothalamic slices by LSD was not influenced by 0.3 μ M tetrodotoxin. The peripheral administration of LSD to rats has been shown to reduce 5-HT synthesis and release by a mechanism thought to involve, in part, an autoreceptor-mediated reduction in impulse flow of 5-HT neurons. In the present experiments, intraperitoneal injection of methiothepin antagonized the LSD-induced reduction in hypothalamic 5-HT synthesis (5-hydroxytryptophan accumulation) while exerting no influence by itself. Conversely, compounds which were not active as 5-HT autoreceptor antagonists in vitro (i.e., cyproheptadine, methysergide, cinanserin) did not influence the effect of LSD on 5-HT synthesis. Further, the reduction in 5-hydroxytryptophan (5-HTP) accumulation by LSD showed regional differences in inhibition by methiothepin (hypothalamus > cortex > striatum) which paralleled the autoreceptor antagonist activity of methiothepin in vitro. These data suggest that similar autoreceptor mechanisms control 5-HT release and synthesis in terminal 5-HT projection areas and that the reduction in 5-HT accumulation by LSD and the antagonism by methiothepin may represent a useful biochemical measure of 5-HT autoreceptor activity in vivo.     

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.     

Effects ofp-chlorophenylalanine on cortical monoamines and on the activity of noradrenergic neurons

The catecholamines noradrenaline, dopamine, adrenaline, the indoleamine 5-hydroxy-tryptamine (5-HT; serotonin), and some of their major metabolites were assayed, using high performance liquid chromatography (HPLC), in the neocortex of normal rats as well as in animals in which 5-HT synthesis had been inhibited withp-chlorophenylalanine. Besides important depletions in serotonin and in 5-hydroxyindole-3-acetic acid, noradrenaline levels were significantly reduced, but the content in 3-methoxy-4-hydroxyphenylglycol was increased, indicating an augmented utilization of this amine. The levels of dopamine and 3-methoxytyramine were also reduced, although homovanillic acid and 3,4-dihydroxyphenylacetic acid levels remained constant. The spontaneous unitary activity of identified noradrenergic neurons in the Locus coeruleus was increased, indicating an hyperactivity of this system. These results can be interpreted in relation to functional interactions between the catecholamines and serotonin; i.e.: a decrease in endogenous serotonin results in the loss of a negative feedback control of noradrenaline release.Special Issue dedicated to Prof. Eduardo De Robertis.     

The effects of injections of D,L-5-hydroxytryptophan on the efflux of endogenous serotonin and 5-hydroxyindoleacetic acid from a synaptosomal fraction.

Abstract— The effects of i.p. injections of SO mg/kg d,l-5-hydroxytryptophan (5-HTP) and saline alone on the in uitro release of endogenous serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were studied using preparations of axon terminals (P₂ isolated from the telencephalon of rats. The level of 5-HT was 2-fold greater and the level of 5-HIAA was 5-fold greater in the P₂ fraction isolated from rats given the d,l-5-HTP injection than from rats given saline injections. At 37°C the in vitro efflux of 5-HT and 5-HIAA from the P₂ fractions of animals injected with 5-HTP 30min before killing was approx 3 times higher than the saline control group. The amount of 5-HT and 5-HIAA released at 37°C was 3–5 times higher than the amount released at 0°C for both the 5-HTP and saline injected rats. Increasing the concentration of potassium ions in the media to 55 mm significantly increased the release of 5-HT but not 5-HIAA in both groups of animals. The amount of 5-HT released by 55mm-K⁺ was about 2-fold higher from the P₂ fraction isolated from rats given 5-HTP injections with respect to those given saline injections. The potassium stimulated release of 5-HT was calcium dependent. The data thus indicate that injection of 50 mg/kg d,l-5-HTP in rats can cause an increase in the level of 5-HT and 5-HIAA in a crude synaptosomal fraction and that as a result of this increase, there is a temperature dependent increased release of 5-HT and 5-HIAA under normal resting membrane conditions. There is also an increased release of 5-HT as a result of membrane depolarizing conditions induced by elevated potassium levels which is calcium dependent.     

Inhibition of tryptophan hydroxylase: Neurochemical action of a catecholamide seizure-inducing agent

H13/04, an audiogenic seizure-inducing catecholamide, has previously been demonstrated to decrease the accumulation of 5-hydroxytryptophan (5-HTP), while increasing the accumulation of dihydroxyphenylalanine (DOPA) after aromatic acid decarboxylase inhibition in vivo. The present study examined the effect of H13/04 on intracellular storage, release, and metabolism of serotonin (5-HT) and noradrenaline (NA) in vitro in order to differentiate between the primary effects of the drug and possible secondary effects due to neurotransmitter interaction. H13/04 had no effect on NA synthesis by brain minces from C57BL/6 mice, but did have a marked effect on [³H]5HT synthesis from [³H]tryptophan in mouse brain minces. H13/04 was subsequently shown to competitively inhibit tryptophan hydroxylase. The data presented in this study indicate that the primary action of H13/04 on biogenic amines is to decrease the synthesis rate of 5-HT by competitive inhibition of tryptophan hydroxylase. The lack of any effect on NA in vitro is consistent with the hypothesis that the primary biochemical action of the drug is on the 5-HT system and that the action on NA in vivo is an indirect effect possibly secondary to the inhibition of 5-HT synthesis.     

Nonexocytotic serotonin release tonically suppresses serotonergic neuron activity

The firing activity of serotonergic neurons in raphe nuclei is regulated by negative feedback exerted by extracellular serotonin (5-HT)_o acting through somatodendritic 5-HT1A autoreceptors. The steady-state [5-HT]_o, sensed by 5-HT1A autoreceptors, is determined by the balance between the rates of 5-HT release and reuptake. Although it is well established that reuptake of 5-HT_o is mediated by 5-HT transporters (SERT), the release mechanism has remained unclear. It is also unclear how selective 5-HT reuptake inhibitor (SSRI) antidepressants increase the [5-HT]_o in raphe nuclei and suppress serotonergic neuron activity, thereby potentially diminishing their own therapeutic effect. Using an electrophysiological approach in a slice preparation, we show that, in the dorsal raphe nucleus (DRN), continuous nonexocytotic 5-HT release is responsible for suppression of phenylephrine-facilitated serotonergic neuron firing under basal conditions as well as for autoinhibition induced by SSRI application. By using 5-HT1A autoreceptor-activated G protein–gated inwardly rectifying potassium channels of patched serotonergic neurons as 5-HT_o sensors, we show substantial nonexocytotic 5-HT release under conditions of abolished firing activity, Ca²⁺ influx, vesicular monoamine transporter 2–mediated vesicular accumulation of 5-HT, and SERT-mediated 5-HT transport. Our results reveal a cytosolic origin of 5-HT_o in the DRN and suggest that 5-HT_o may be supplied by simple diffusion across the plasma membrane, primarily from the dense network of neurites of serotonergic neurons surrounding the cell bodies. These findings indicate that the serotonergic system does not function as a sum of independently acting neurons but as a highly interdependent neuronal network, characterized by a shared neurotransmitter pool and the regulation of firing activity by an interneuronal, yet activity-independent, nonexocytotic mechanism.     

Evaluation of monoaminergic neurotransmitters in the acute focal ischemic human brain model by intracerebral in vivo microdialysis

The release of neurotransmitters principally glutamate during cerebral ischemia has been extensively studied. It is well recognized that ischemia induced release of glutamate plays a key role in “excitotoxic” neuronal death. The role of monoaminergic neurotransmitters is however unclear. The purpose of this study was to evaluate the extracellular norepinephrine, 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA) and serotonin (5-HT) under varied degrees of ischemia in the acute focal ischemic model of the human brain by in-vivo microdialysis. The ischemic response of these amines was correlated with the glutamate levels. Our study concludes that these amines and metabolites can be detected in the human “stroke” model. No marked fluctuations were noted in the levels of norepinephrine and DOPAC. However, significant changes to partial and total ischemia were noted in the extracellular levels of 5-HIAA and 5-HT. These compounds showed a dramatic increase with the onset of ischemia with higher detectable levels in the partial ischemic state in comparison to the total ischemic dialysate levels. The exact role played by the differential increase in the levels of 5-HT to the other catecholamines in the pathogenesis of ischemic neuronal damage remains unclear and warrants further study.