Changes of Ganglioside Pattern During Cerebellar Development of Normal and Staggerer Mice

Saturable and specific binding sites for 5-[3H]hydroxytryptamine (5-HT, serotonin) characterized by a KD of 3.5-4.5 nM were detected in the chick embryo brain and were shown to develop linearly as a function of age, weight, and protein content. Saturation and displacement studies using unlabeled 5-HT as the displacing ligand suggested a single population of binding sites. However, displacement studies using 5-methoxytryptamine, lysergic acid diethylamide (LSD), 2-bromo-lysergic acid diethylamide (BOL), methysergide, and spiperone as competing ligands suggested the existence of subclasses of [3H]5-HT binding sites because the Hill coefficients were less than unity. When compared with the reported [3H]5-HT binding sites (5-HT1) in the rat forebrain, the IC50 values of the competing ligands were similar. However, the Hill coefficients for LSD and methysergide were less than unity which suggested that the [3H]5-HT binding sites in the chick embryo brain may be more similar to those found in rat spinal cord than rat forebrain. To study [3H]5-HT binding site regulation and development, various serotonergic compounds were injected into the chorioallantoic fluid of the eggs at different times during embryonic development. Multiple pretreatments with d,l-5-hydroxytryptophan, 5-HT, or BOL were found to have no significant effects on either the affinity (KD) or number (Bmax) of specific [3H]5-HT binding sites. Multiple pretreatments with p-chlorophenylalanine were found to increase the Bmax of specific [3H]5-HT binding by 23% (p less than 0.01) whereas multiple pretreatments with LSD were found to decrease the Bmax of specific binding by 45% (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Demonstration of aromatic l-amino acid decarboxylase activity in human brain with l-dopa and l-5-hydroxytryptophan as substrates by high-performance liquid chromatography with electrochemical detection

The enzymatic decarboxylations of l-DOPA and l-5-hydroxytryptophan (l-5-HTP) by aromatic l-amino acid decarboxylase (AADC) were measured with homogenates from human brain regions, caduate nucleus and hypothalamus, using our new and highly sensitive methods for l-DOPA decarboxylase and l-5-HTP decarboxylase by high-performance liquid chromatography with electrochemical detection (HPLC-ED). Dopamine formed from l-DOPA as substrate was measured for DOPA decarboxylase activity using d-DOPA for the blank. For 5-HTP decarboxylase activity, serotonin (5-HT) formed from l-5-HTP was measured, and the blank value in presence of NSD-1055 was subtracted. NSD-1055 inhibited 5-HTP decarboxylase activity completely at a concentration of 0.2 mM. In this study, the properties of l-5-HTP decarboxylase activity in human caudate nucleus were first examined. AADC activities in human brains were found to be widely variable for both l-DOPA and l-5-HTP as substrates. The ratio of the activities for l-DOPA and l-5-HTP were found to be significantly higher in hypothalamus than in caudate nucleus. AADC activity for l-DOPA in the brain was found to be linear up to 40 min of incubation, while that for l-5-HTP was found to be linear up to 240 min of incubation. The optimum pyridoxal phosphate concentration was found to be similar for both substrates and was between 0.01 and 0.1 mM. The optimum pH values were found to be 7.2 and 8.2 for l-DOPA decarboxylase and l-5-HTP decarboxylase, respectively. K_m and V_max values for a human caudate nucleus l-DOPA decarboxylase were found to be 414 μM and 482 pmol/min/g wet weight, respectively, while those for l-5-HTP decarboxylase were found to be 90 μM and 71 pmol/min/g wet weight, respectively.     

New and highly sensitive assay for l-5-hydroxytryptophan decarboxylase activity by high-performance liquid chromatography—voltammetry

This paper describes a new, inexpensive and highly sensitive assay for aromatic l-amino acid decarboxylase (AADC) activity, using l-5-hydroxytryptophan (l-5-HTP) as substrate, in rat and human brains and serum by high-performance liquid chromatography (HPLC) with voltammetric detection. l-5-HTP was used as substrate and d-5-HTP for the blank. After isolating serotonin (5-HT) formed enzymatically from l-5-HTP on a small Amberlite CG-50 column, the 5-HT was eluted with hydrochloric acid and assayed by HPLC with a voltammetric detector. N-Methyldopamine was added to each incubation mixture as an internal standard. This method is sensitive enough to measure 5-HT, formed by the enzyme, 100 fmol to 140 pmol or more. An advantage of this method is that one can incubate the enzyme for longer time (up to 150 min), as compared with AADC assay using l-DOPA as substrate, resulting in a very high sensitivity. By using this new method, AADC activity was discovered in rat serum.     

Suppressive effect of nantenine,isolated from Nandina domestica Thunberg,on the 5-hydroxy-L-tryptophan plus clorgyline-induced head-twitch response in mice

We investigated the effects of nantenine (9,10-Methylenedioxy-1,2 dimethoxyaporphine), a major alkaloid isolated from the fruit of Nandina domestica Thunb (Berberidaceae), on the 5-HT2A receptor-mediated head-twitch response (HTR) in mice. Intraperitoneal (i.p.) injection of nantenine (13.3, 20 and 30 mg/kg) as well as the 5-HT2A receptor antagonist ketanserin (0.0625, 0.25 and 1 mg/kg) inhibited the 5-hydroxy-L-tryptophan (l-5-HTP; 75 mg/kg, i.p.) plus monoamine oxidase inhibitor, clorgyline (1 mg/kg, i.p.)-induced HTR in a dose-dependent manner. In contrast, neither l-5-HTP plus clorgyline nor 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT; 5 microg/mouse, i.c.v.)-induced head weaving was affected by nantenine or ketanserin. Furthermore, neither nantenine (up to 30 mg/kg) nor ketanserin (up to 1 mg/kg) affect on the locomotor activity. In the receptor binding studies, nantenine showed affinity to the 5-HT2A receptors (Ki = 0.4 microM), while it had less affinity toward alpha1-adrenergic (Ki = 2.1 microM) and D2-dopaminergic (Ki = 1.7 microM) receptors of the mouse brain. These results suggest that nantenine inhibits l-5-HTP plus clorgyline-induced head- twitch response by blocking 5-HT2A receptors in the central nervous system.     

Enhancement in extracellular serotonin levels by 5-hydroxytryptophan loading after administration of WAY 100635 and fluoxetine

It has been demonstrated that synthesis of serotonin (5-HT) is dependent on the availability of precursor, as well as the activity of 5-HT neurons. In the present series of experiments, we examined the effects of precursor (5-HTP) loading on extracellular hypothalamic 5-HT after administration of fluoxetine alone or in combination with WAY 100635, a selective 5-HT1A antagonist. In the first experiment, fluoxetine alone (10 mg/kg i.p.) caused 5-HT levels to significantly increase to 150% of basal levels. Subsequent administration of 5-HTP at 10, 20, and 40 mg/kg i.p. caused 5-HT levels to further increase to a maximum value of 254%, 405%, and 618%, respectively. In the second experiment, either vehicle or WAY 100635 (1 mg/kg/hour s.c.) was infused, then fluoxetine (10 mg/kg i.p.) and 5-HTP (10 mg/kg i.p.) were administered. By itself, WAY 100635 led to a slight but significant increase in hypothalamic 5-HT levels one hour after the start of administration (130% of basal levels). In the WAY 100635-treated group, fluoxetine caused an increase to 240% of basal levels after one hour, which rose to 290% of basal levels after two hours. Subsequent administration of 5-HTP further increased 5-HT levels to 580% of basal levels after one hour. In the vehicle-treated group, fluoxetine caused an increase of 160% of basal levels which was stable over two hours, and subsequent administration of 5-HTP led to a slight increase in 5-HT levels of 220% after one hour. These results suggest that combining blockade of 5-HT1A autoreceptors with 5-HT uptake inhibition results in a synergistic increase in synthesis and release of 5-HT when precursor is administered.     

5-Hydroxytryptamine affects rat migrating myoelectric complexes through different receptor subtypes: evidence from 5-hydroxytryptophan administration

The effect of the serotonin precursor 5-hydroxytryptophan (5-HTP) on jejunal migrating myoelectric complexes (MMCs) was investigated in conscious rats. Subcutaneous administration of low doses of 5-HTP (1-2 mg/kg) shortened the period between migrating complexes, whereas high doses of the compound (4-8 mg/kg) disrupted the MMC pattern. The serotonin (5-HT2) antagonist methysergide (8 mg/kg s.c.) did not alter basal MMC, neither did it prevent the effect of a low dose of 5-HTP; conversely, it antagonized the disruption due to the high dose. The 5-HT3 antagonist ICS 205-930 (30 micrograms/kg s.c.) decreased MMC frequency; administration of 2 mg/kg 5-HTP following ICS 205-930 brought the frequency of myoelectric complexes back to basal values. Both effects of 5-HTP were prevented by the decarboxylase inhibitor benserazide (85 mg/kg i.p.), which per se caused a transient inhibition of spiking activity. The results suggest that rat MMCs can be influenced in a composite fashion by progressively increasing concentrations of 5-HT, which in turn activate different receptor subtypes. A peripheral neuronal receptor, probably belonging to the 5-HT3 subclass, mediates the increase in MMC frequency observed after low doses of 5-HTP; higher levels of serotonin activate 5-HT2 receptors, causing disruption of cycling activity. Additionally, 5-HT3 receptors, but not 5-HT2, appear to be relevant for the regulation of the MMC pattern by the endogenous amine.     

Comparison of [125I]Iodolysergic Acid Diethylamide Binding in Human Frontal Cortex and Platelet Tissue

The human platelet contains a functional 5-hydroxytryptamine (5-HT) receptor that appears to resemble the 5-HT2 subtype. In this study, we have used the iodinated derivative [125I]iodolysergic acid diethylamide ([125I]iodoLSD) in an attempt to label 5-HT receptors in human platelet and frontal cortex membranes under identical assay conditions to compare the sites labelled in these two tissues. In human frontal cortex, [125I]iodoLSD labelled a single high-affinity site (KD = 0.35 +/- 0.02 nM). Displacement of specific [125I]iodoLSD binding indicated a typical 5-HT2 receptor inhibition profile, which demonstrated a significant linear correlation (r = 0.97, p less than 0.001, n = 17) with that observed using [3H]ketanserin. However, [125I]iodoLSD (Bmax = 136 +/- 7 fmol/mg of protein) labelled significantly fewer sites than [3H]ketanserin (Bmax = 258 +/- 19 fmol/mg of protein) (p less than 0.001, n = 6). In human platelet membranes, [125I]iodoLSD labelled a single site with affinity (KD = 0.37 +/- 0.03 nM) similar to that in frontal cortex. The inhibition profile in the platelet showed significant correlation with that in frontal cortex (r = 0.96, p less than 0.001, n = 16). We conclude that the site labelled by [125I]iodoLSD in human platelet membranes is biochemically similar to that in frontal cortex and most closely resembles the 5-HT2 receptor subtype, although the discrepancy in binding capacities of [125I]iodoLSD and [3H]ketanserin raises a question about the absolute nature of this receptor.     

Chronic ACTH treatment: influence on 5-HT2 receptors and behavioral supersensitivity induced by 5,7-dihydroxytryptamine lesions

The capacity of the serotonin (5-HT) precursor 5-HIP to induce the ACTH-responsive myoclonic-convulsive disorder infantile spasms in patients with Down's syndrome has been cited as evidence for altered serotonergic neurotransmission in infantile spasms. Since there is no animal model of infantile spasms, the suitability of behavioral supersensitivity (myoclonus) evoked by 5-HTP in rats with 5,7-dihydroxytryptamine (DHT) lesions as a model was tested by determining the effect of chronic treatment with ACTH (40 IU/kg) on 5-HTP-evoked myoclonus. In rats treated with DHT as adults, ACTH administration did not alter the "serotonergic behaviors," such as myoclonus, induced by 30 mg/kg 5-hydroxytryptophan (5-HTP), but induced a small significant increase in Bmax of neocortical 5-HT2 sites of the DHT group, with no change in rats without lesions. In rats treated with DHT as neonates, there was also no significant difference in behaviors evoked by several doses of 5-HTP. These data suggest that ACTH minimally modifies the effects on 5-HT receptors of DHT lesions, but the intracisternal DHT model is not a suitable model for infantile spasms because chronic ACTH was not antimyoclonic.     

Assessment of binding indices and physiological responsiveness of the 5-HT2 receptor on human platelets

This is the first report of parallel studies of binding indices and physiological responsiveness of the "Serotonin-two" (5-HT2) receptor on the human platelet membrane. Binding indices were measured by a microassay employing [125I]ILSD as radioligand and ketanserin to define specific binding. A single receptor population was found, characterized by a KD of 1.69 +/- 0.45 nM and Bmax of 14.5 +/- 6.0 pmol/g protein in healthy subjects. Functional responsiveness of the platelet 5-HT2 receptor complex was assessed by measurement of the extent to which serotonin (10uM) augmented platelet aggregation induced by threshold concentrations of adenosine diphosphate (ADP). A statistically significant positive correlation was found between the number of platelet 5-HT2 receptor sites (Bmax) and the magnitude of the serotonin-amplified aggregation response (r = .70, n = 38, p less than 0.001). Assessment of binding indices and physiological responsiveness of the platelet 5-HT2 receptor complex should facilitate study of age, hormonal, disease, and drug effects on 5-HT2 receptor function in human subjects.     

Characterization of [3H]Quipazine Binding to 5-Hydroxytryptamine3 Receptors in Rat Brain Membranes

[3H]Quipazine was used to label binding sites in rat brain membranes that display characteristics of a 5-hydroxytryptamine3 (5-HT3) receptor. The radioligand binds with high affinity (KD, 1.2 +/- 0.1 nM) to a saturable population of sites (Bmax, 3.0 +/- 0.4 pmol/g of tissue) that are differentially located in the brain. Specific [3H]quipazine binding is not affected by guanine or adenine nucleotides. ICS 205-930, BRL 43964, Lilly 278584, and zacopride display less than nanomolar affinity for these sites whereas MDL 72222 is approximately one order of magnitude less potent. The pharmacological profile of the binding site is in excellent agreement with that of 5-HT3 receptors characterized in peripheral physiological models. We conclude that [3H]quipazine labels a 5-HT3 receptor in the rat CNS.     

Hyperleptinemia elicited by the 5-HT precursor, 5-hydroxytryptophan in mice: involvement of insulin

Mechanisms for hyperleptinemia elicited by a serotonin (5-hydroxytryptamine, 5-HT) precursor, 5-hydroxytryptophan (5-HTP), were investigated. 5-HTP elicited apparent increases in serum leptin levels of mice. Administration of 5-HTP did not alter expression of leptin mRNA in white adipose tissues. Furthermore, neither 5-HTP nor 5-HT increased leptin secretion from isolated fat pads of mice. Since insulin is known to enhance leptin release, involvement of insulin in 5-HTP-induced hyperleptinemia was examined. 5-HTP significantly elevated serum insulin levels. In mice treated with streptozotocin, which depletes insulin, 5-HTP did not increase serum leptin levels. These results suggest that hyperinsulinemia participates the elevation of serum leptin levels elicited by 5-HTP.     

New and highly sensitive assay for l-5-hydroxytryptophan decarboxylase activity by high-performance liquid chromatography—voltammetry

This paper describes a new, inexpensive and highly sensitive assay for aromatic l-amino acid decarboxylase (AADC) activity, using l-5-hydroxytryptophan (l-5-HTP) as substrate, in rat and human brains and serum by high-performance liquid chromatography (HPLC) with voltammetric detection. l-5-HTP was used as substrate and d-5-HTP for the blank. After isolating serotonin (5-HT) formed enzymatically from l-5-HTP on a small Amberlite CG-50 column, the 5-HT was eluted with hydrochloric acid and assayed by HPLC with a voltammetric detector. N-Methyldopamine was added to each incubation mixture as an internal standard. This method is sensitive enough to measure 5-HT, formed by the enzyme, 100 fmol to 140 pmol or more. An advantage of this method is that one can incubate the enzyme for longer time (up to 150 min), as compared with AADC assay using l-DOPA as substrate, resulting in a very high sensitivity. By using this new method, AADC activity was discovered in rat serum.     

Artifactual High-Affinity and Saturable Binding of [3H]5-Hydroxytryptamine Induced by Radioligand Oxidation

The binding of [3H]5-hydroxytryptamine (5-HT, serotonin) to cerebellar membranes was examined after preincubation of [3H]5-HT in the presence or absence of ascorbate. The tissue preparation was identical in all experiments and consisted of rat cerebellar homogenates in Tris-HCl buffer with 0.1% ascorbate. Cerebellar membranes were used because of their low density of 5-HT1 binding sites. In the presence of ascorbate during a 4-h preincubation period, minimal specific binding of 2 nM [3H]5-HT is detected. Similar results are obtained with equimolar concentrations of other antioxidants (butylated hydroxytoluene, sodium dithionite, and sodium metabisulfite). Apparent specific binding increases 14-fold following a 4-h preincubation of [3H]5-HT in the absence of ascorbate. The increase in apparent specific [3H]5-HT binding is time-dependent and plateaus after 4-6 h of preincubation. When ascorbate is present during the 4-h preincubation, Scatchard analysis of [3H]5-HT binding reveals a KD value of 3.0 +/- 0.3 nM and a Bmax value of 1.9 +/- 0.2 pmol/g tissue. When ascorbate is absent during the preincubation, the KD is essentially unchanged at 3.6 +/- 0.1 nM but the Bmax is significantly increased to 36.5 +/- 7 pmol/g tissue. Drug competition studies reveal that the apparent specific "[3H]5-HT binding" in the absence of ascorbate appears to be displaced by nanomolar concentrations of hydroxylated tryptamines (5-HT, bufotenine) but not by nonhydroxylated tryptamines (5-methoxytryptamine, tryptamine). HPLC analysis demonstrates that [3H]5-HT is essentially destroyed by a 4-h incubation at 22 degrees C in the absence of ascorbate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for Distinct 5-Hydroxytryptamine2 Binding Site Subtypes in Cortical Membrane Preparations

5-Hydroxytryptamine (5-HT) displays a sixfold higher affinity for 5-HT2 binding sites labeled by [3H]ketanserin in rat (IC50 = 200 +/- 40 nM) and human (IC50 = 190 +/- 50 nM) cortex than for 5-HT2 sites in bovine cortex (IC50 = 1,200 +/- 130 nM). The Hill slopes of the 5-HT competition curves are 0.67 +/- 0.04 in rat, 0.69 +/- 0.08 in human, and 0.96 +/- 0.02 in bovine cortex. Scatchard analysis of (+/-)-[3H]4-bromo-2,5-dimethoxyamphetamine ([3H]DOB) binding in the rat indicates a population of binding sites with a KD of 0.38 +/- 0.04 nM and a Bmax of 1.5 +/- 0.05 pmol/g tissue. In contrast, specific [3H]DOB binding cannot be detected in bovine cortical membranes. These data indicate that species variations exist in 5-HT2 binding site subtypes and that [3H]ketanserin appears to label a homogeneous population of 5-HT2 binding site subtypes in bovine cortex.     

Fluorescence microscopy of the 5-HTP turnover in the exocrine pancreas of mice and rats

Summary The turnover ofl-5-HTP,d-5-HTP and 5-HT in the exocrine pancreas have been studied by means of the fluorescence method ofFalck andHillarp. l- andd-5-HTP are easily taken up by the acinar cells, whereas 5-HT seems to pass into the cells only to a minor extent. After the administration ofl-5-HTP (and in some cases after 5-HT administration), specific fluorescence is seen in the form of apically located granules (probably identical with the zymogen granules) for a short period, which is prolonged, if the animals are pretreated with a MAO inhibitor. Decarboxylase inhibition prevents the appearance of these fluorescent granules. Administration ofd-5-HTP does not give rise to this granular fluorescence but to a diffuse fluorescence throughout the cells. Thus, there are reasons to assume that the granular fluorescence derives from 5-HT. The results obtained in this work correspond well with those from a similar study withl-DOPA and some of its analogues.abbreviations DOPA 3,4-dihydroxyphenylalanine - DA dopamine - NA noradrenaline - A adrenaline - 5-HTP 5-hydroxytryptophan - 5-HT 5-hydroxytryptamine - MAO monoamine oxidase This work was supported by grants from the Swedish Medical Research Council (B68-12X-712-03B and B68-14X-56-04B), the United States Public Health Service (06701-02) and the Faculty of Medicine, University of Lund, Lund, Sweden.     

Effect of serotonin on murine macrophages: suppression of Ia expression by serotonin and its reversal by 5-HT2 serotonergic receptor antagonists

Serotonin (5-HT), a mediator released from platelets at sites of inflammation, suppressed IFN-gamma-induced Ia expression in mouse bone marrow macrophages maintained in vitro. (Mean percent suppression = 63.9% +/- 9.2, n = 40.) This suppression was not toxic or endotoxin-related, was concentration-dependent, and occurred at the physiologic concentrations of 5-HT present at inflammatory sites. The concentration of 5-HT producing the half-maximal effect was 2.5 to 5.5 X 10(-8) M. Related compounds, dopamine, histamine, and tryptamine, were much less potent in suppressing IFN-gamma-induced Ia, with maximally suppressing concentrations more than 100-fold higher than the maximally suppressing 5-HT concentration. L-5-hydroxytryptophan (5-HTP), the most potent analog tested, was 10-fold less potent than 5-HT in suppressing Ia expression. The concentration of 5-HTP producing the half-maximal effect = 4 X 10(-7) M. 5-HT suppression of IFN-gamma-induced Ia expression was antagonized by the 5-HT2 type receptor antagonists spiperone, ketanserin, and LY53857. Concentrations of these agents resulting in 50% inhibition of the serotonin effect were 1.5 X 10(-8) M, 7.5 X 10(-8) M, and 4.5 X 10(-12) M, respectively. 5-HT was most effective in suppressing IFN-gamma-induced Ia when added early in culture simultaneously with IFN-gamma. These data provide functional evidence that 5-HT suppression of IFN-gamma-induced Ia expression is mediated through a 5-HT receptor with some characteristics of the 5-HT2 type. 5-HT may play a physiologic role at sites of inflammation as a modulator of the effects of IFN-gamma on macrophage function.     

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.     

5-Hydroxytryptamine Measurements in Molluscan Ganglia and Neurons Using a Modified Radioenzymatic Assay

A radioenzymatic procedure for the determination of sub-picomole amounts of 5-hydroxytryptamine (5-HT) is described. It is a modification of the method originally described by Saavedra et al. (1973), in which 5-HT was measured as the radiolabelled product [3H]5-methoxy-N- acetyltryptamine , after incubation with [3H]S-adenosylmethionine, acetyl-CoA, and the enzymes hydroxyindole-O-methyltransferase (EC 2.1.1.4) and N-acetyltransferase (EC 2.3.1.5). Ganglia from various gastropod molluscs (Aplysia californica, Tritonia diomedia , Lymnaea stagnalis, and Helisoma trivolvis ), as well as individual neuronal somata isolated from these ganglia, were assayed for 5-HT. Among the homologous giant cerebral cells in these animals, the 5-HT concentrations were similar. Statistical analysis of the 5-HT values in paired 5-HT-containing neurons demonstrated that the variability was considerably greater in samples obtained from different animals than in those obtained from the same animal. This suggests that experiments aimed at manipulating amine levels in individual neurons may benefit by using a paired-cell paradigm. The effects of incubating Aplysia ganglia with 5-HT or with the 5-HT precursors tryptophan and 5-hydroxytryptophan (5-HTP) were studied. High concentrations of 5-HTP and 5-HT (100 microM) increased the levels of 5-HT in ganglia, but only incubation in high concentrations of 5-HTP resulted in an increase of 5-HT in the isolated somata of 5-HT-containing neurons C1 and P5.     

The effects of 5-hydroxytryptophan and 5-hydroxytryptamine on dopamine synthesis and release in rat brain striatal synaptosomes.

The effects of 5-hydroxytryptophan (5-HTP) and serotonin (5-HT) on dopamine synthesis and release in rat brain striatal synaptosomes have been examined and compared to the effects of tyramine and dopamine. Serotonin inhibited dopamine synthesis from tyrosine, with 25% inhibition occurring at 3 μM-5-HT and 60% inhibition at 200 μM. Dopamine synthesis from DOPA was also inhibited by 5-HT, with 30% inhibition occurring at 200 μ. At 200 μM-5-HTP, dopamine synthesis from both tyrosine and DOPA was inhibited about 70%. When just the tyrosine hydroxylation step was measured in the intact synaptosome, 5-HT, 5-HTP, tyramine and dopamine all caused significant inhibition, but only dopamine inhibited soluble tyrosine hydroxylase [L-tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2] prepared from lysed synaptosomes. Particulate tyrosine hydroxylase was not inhibited by 10 μM-5-HT, but was about 20% inhibited by 200 μM-5-HT and 5-HTP. At 200 μM both 5-HT and 5-HTP stimulated endogenous dopamine release. These experiments suggest that exposure of dopaminergic neurons to 5-HT or 5-HTP leads to an inhibition of dopamine synthesis, mediated in part by an intraneuronal displacement of dopamine from vesicle storage sites, leading to an increase in dopamine-induced feedback inhibition of tyrosine hydroxylase, and in part by a direct inhibition of DOPA decarboxylation.