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1. The effects of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid and N -acetyl-5-hydroxytryptamine on oocytes of Blaberus craniifer , in which vitellogenesis was prevented by imaginal molt decapitation, were investigated. 2. Sites binding anti-egg-protein antibodies were detected in the periphery of basal oocytes of treated females, with individual variability. 3. In this ovoviviparous cockroach, the onset of vitellogenesis may thus not be triggered solely by juvenile hormone, and indolamines may play a role in the uptake of haemolymphatic proteins by oocytes.

The aim of the present investigation was to look for the mechanisms causing disturbances in carbohydrate metabolism during the action of the epileptogenic agent methionine sulfoximine The levels of glucose, glycogen, and indolamines were measured in seven different regions of rat brain. Methionine sulfoximine induced a decrease in serotonin level which was roughly dose-dependent. There were no obvious, changes in tryptophan and 5-hydroxyindoleacetic levels in any area. Methionine sulfoximine induced the known increase in glucose and glycogen levels. The direct precursor of serotonin, 5-hydroxytryptophan, and benserazide (a decarboxylase inhibitor) were then injected into rats in association with methionine sulfoximine. In this case, methionine sulfoximine failed to induce seizures. Moreover, the serotonin level was unchanged and the carbohydrate content did not significantly increase. There was only a rise in 5-hydroxyindoleacetic acid level. This work shows a striking parallelism between serotonin decrease and glycogen increase.

We developed a chromatographic system which separates cate-cholamines and indolamines as well as a large number of their metabolites (acids, aminoacids and glycols). Waters Radial Pak columns compressed radially to avoid formation of cavities in the packing, were used. These columns must be washed with methanol in order to obtain reproducible results when an ion-pairing mobile phase is employed. The conditioning of bhe columns was not affected by washing with triethylamine or variations in radial pressure. Good resolution of 13 samples was obtained in 28 minutes with a mobile phase composed of 0.1 M KH2PO4, 0.1 mM EDTA and 5 mM heptane sulfonic acid in water and Me OH (85:15), pH = 3.6. Products were identified with the use of a Metrohm 64l electrochemical detector. The technique was used for rapid, simultaneous determination of NA, DA, DOPAC, HVA, 5HT and 5HIAA in deproteinated mouse brain samples.

Drosophila melanogaster homogenates incubated with tritiated 5-hydroxytryptophan, 5-hydroxytryptamine, or N-acetylserotonin have the ability of converting them into labelled indolamines, including melatonin. All these compounds were characterized by two-dimensional thin-layer chromatography by comparison with nonradiolabelled standards, and melatonin by mass spectrometry and radioimmunoassay. On the other hand, the injection of pharmacological doses of melatonin into 2-day-old female flies diminishes the mating speed and the oviposition rate.

The pineal complex of the three-spined stickleback (Gasterosteus aculeatus L.) was investigated by light and electron microscopy, as well as fluorescence histochemistry for demonstration of catecholamines and indolamines. The pineal complex of the stickleback consists of a pineal organ and a small parapineal organ situated on the left side of the pineal stalk. The pineal organ, including the entire stalk, is comprised mainly of ependymal-type interstitial cells and photoreceptor cells with well-developed outer segments. Both unmyelinated and myelinated nerve fibres are present in the pineal organ. Nerve tracts from the stalk enter the habenular and posterior commissures. A small bundle of nerve fibres connects the parapineal organ and the left habenular body. The presence of indolamines (5-HTP, 5-HT) was demonstrated in cell bodies of both the pineal body and the pineal stalk, and catecholaminergic nerve fibres surround the pineal complex.

Chlordimeform is a relatively new acaricide/insecticide, whose mode of action we have investigated. It appears to interfere with amine-mediated control of nervous and endocrine systems in a variety of ways. Specifically, chlordimeform causes a build-up of the amines 5-hydroxytryptamine and to a lesser extent norepinephrine in the rat brain in vivo, antagonizes the in vivo action of reserpine in the rat (reserpine depletes amine stores in the CNS), inhibits monoamine oxidase from rat liver in vitro, and causes hypotension in rabbits. In the American cockroach it directly stimulates the heart in situ, acts synergistically with tryptamine in vivo, inhibits amine-N-acetyltransferase from cockroach head in vitro, causes accumulation of indolamines in cockroaches in vivo, and blocks the stimulation of adenylate cyclase by octopamine in the cockroach CNS in situ. It also inhibits tryptamine metabolism in whole mites in vitro. Images FIGURE 3. FIGURE 4. A FIGURE 4. B FIGURE 4. C FIGURE 4. D

Dense bodies containing high amounts of chrome were localized in the perikarya of substantia nigra and dorsal raphe neurons following the cytochemical reaction of endogenous dopamine and serotonin (respectively) with glutaraldehyde-dichromate (GDC). Energy dispersive X-ray analysis of these bodies revealed chrome levels two to four times higher than those recorded from the cytoplasmic background. Pretreatment with paraformaldehyde blocked the GDC reaction within the dense bodies in the substantia nigra (chrome levels similar to background), while the chrome levels in the dense bodies of the raphe neurons remained elevated. This demonstrates that pretreatment with paraformaldehyde allows selective localization of central nervous system serotonin stores by the GDC technique.

The distribution of serotonin-immunoreactive cell bodies and fibers in the ventral nerve cord of the earthworm has been investigated from whole-mount preparations and serial sections. Serotonin-immunoreactive neurons are organized in seven soma groups per ganglion; these are defined by cell number, soma shape, diameter and fiber projections. Positional variations of this pattern have been studied quantitatively. The number of labeled perikarya is constant in midbody ganglia, but increases markedly rostral to the posterior margin of the clitellum. Variability of position and cell number differs between the cell groups. Stained nerve fibers could only be partially traced; their distribution is described in relation to defined neuronal fiber bundles and segmental nerves. The distribution and morphology of serotonin-immunoreactive cells are compared with previous observations, based on pattern analysis of primary catecholamines and indolamines with the use of a formaldehyde-induced fluorescence technique. The possible role of serotonin-immunoreactive nervous elements in different nerve cord compartments is discussed with respect to physiological effects of serotonin in earthworms and other invertebrates.

Temporal changes in monoaminergic activity in the locus coeruleus (LC) in response to hemorrhage of 10 or 20% of blood volume were assessed using normal pulse voltammetry in alpha-chloralose-urethan-anesthetized cats. Oxidation current was measured with a carbon microelectrode, and changes at 230 and 450 mV were used as estimates of catecholaminergic and indolaminergic activity, respectively. Plasma adrenocorticotropin (ACTH) was measured by radioimmunoassay. Hemorrhage of 20% blood volume caused a transient increase in the catecholaminergic activity in a compact area in the ventral LC (vLC) that preceded increases in the plasma ACTH. The increase in oxidation current at 450 mV was similar to that at 230 mV, suggesting no significant contribution from indolamines. Dorsal rostral pontine sites outside this area exhibited either sustained decreases in oxidation current or no change in response to hemorrhage. The proportion of sites that exhibited transient increases in oxidation current in the vLC after the 10% blood loss was less than that after the 20% blood loss, suggesting that this response was dependent on the magnitude of hemorrhage. Since the LC was implicated previously in the control of ACTH release, we suggest that hemodynamic signals traversing the LC activate catecholaminergic mechanisms that, in turn, participate in the regulation of ACTH release after hemorrhage.

Through a controlled double-blind study in 30 hospitalized patients affected with endogenous depression, the antidepressant action of the combination of nialamide+l-5-HTP has been evaluated and compared with a control group which only received nialamide (+ placebo). The patients treated with nialamide + l-5-HTP achieved a fuller recovery than those who were treated with nialamide alone. The treatment with nialamide + l-5-HTP proved to have a shorter delay of onset. Side effects showed no marked differences except for the orthostatic hypotension which was less apparent in those patients treated with nialamide + l-5-HTP.

Kynuramines occur endogenously in brain and peripheral tissues as metabolites of indoleamino acids and indolamines but little is known regarding their possible physiological and/or pharmacological activity. The present study has investigated the effects of kynuramine and 5-hydroxykynuramine on the cardiovascular system of pithed rats and attempted to correlate effects seen on adrenergic and serotonergic receptors with ligand binding experiments done in vitro using rat brain membranes. Kynuramine was found to release cardiac catecholamines and to act as a weak partial agonist on vascular alpha-adrenoceptors. Hydroxylation in the 5-position (5-hydroxykynuramine) did not alter cardiac potency but increased pressor activity 100-fold. Vasopressor responses to 5-hydroxykynuramine were mediated via a dual agonistic action on both postsynaptic alpha 2-adrenoceptors and vascular serotonin2 receptors. These findings were supported by ligand binding studies, in which both kynuramine and 5-hydroxykynuramine showed affinity for cortical [3H]spiroperidol and [3H]clonidine binding sites. Overall, the results show that kynuramines can exert peripheral (and possibly central) actions which may prove to be of physiological and/or pharmacological significance.

The effect of indolamine derivatives on beta-endorphin (beta-end) release has been studied in vitro using rat anterior pituitary cells. Incubation of primary cultures for 2 h with 100 nmol/l of melatonin, serotonin or 5-methoxytryptamine significantly increased the beta-end release in response to 20 nmol/l of ovine corticotropin-releasing factor (oCRF). Incubation of the cultures with 100 nmol/l of L-tryptophan, 5-hydroxy-L-tryptophan, 5-hydroxytryptophol or 5-methoxytryptophol had no effect on basal or CRF-induced beta-end release. The effect of serotonin and melatonin was further tested in a superfusion system of dispersed rat anterior pituitary cells. Superfusion with oCRF (200 nmol/l) for 4 min elicited an immediate rapid increase in beta-end release which lasted 30-40 min. Simultaneous superfusion with melatonin (1 mumol/l) or serotonin (1 mumol/l) significantly increased the effect of oCRF pulses on beta-end release. We conclude that melatonin and serotonin are able to act directly on anterior pituitary cells to potentiate the effect of oCRF on beta-end release.

The present modification of the formaldehyde condensation method for demonstrating catecholamines and indolamines involves generation of the formaldehyde gas at + 80°C followed by reaction at +25 or 0°C. This procedure results in improved sensitivity for demonstrating noradrenaline and dopamine. Because adrenaline and peptides with NH2-terminal tryptophan give no or very weak fluorescence under these reaction conditions the modification also offers possibilities for improved discrimination between primary catecholamines on one hand and adrenaline and tryptophyl peptides on the other. Under the conditions used, the fluorescence yield of 5-hydroxytryptamine did not seem to be improved by variations in the reaction temperature.

Low MAO activity has been reported in a number of studies to be lower in patients with chronic schizophrenia (see recent review by WYATT and MURPHY,~ cotwins of schizophrenic@ as well as in patients with bipolar (manic depressive) disorders3 .Since MAO is an important enzyme in the degradation of both catecholamines and indolamines, low MAO in schizophrenics would be consistent with two of the leading conceptualizations of biochemical abnormalities in the major psychoses-the transmethylation and dopamine hypotheses. Further, platelet MAO activity is under genetic control as shown by studies of twins.4,5

The hypothalamus exerts an inhibitory influence on prolactin release from the anterior pituitary. Whether or not peptide hypothalamic prolactin-regulating factors, analogous to those for other pituitary hormones, exist, remains to be confirmed. There is evidence that catecholamines and indolamines directly or indirectly affect prolactin release, This concept may explain the pathogenesis of galactorrhœa-amenorrhœa and other endocrine diseases produced by hypothalamic-pituitary disorders.

N-acetylated and tertiary indolamines, some of which are possible neurotransmitter candidates in the CNS, cannot be visualized with the standard Falck-Hillarp histofluorescence method and very little is known about their cellular localization. The present investigation demonstrates that glyoxylic acid (GA), formaldehyde (FA) in combination with aluminum ions (the ALFA method) and trifluoroacetic acid anhydride (TFAA) are capable of forming fluorescent compounds from N-acetylated (e.g. melatonin and N-acetyl-5-hydroxytryptamine) and tertiary (e.g. bufotenin) indolamines in histochemical protein models. With GA and FA-aluminum more vigorous reaction conditions were required for demonstration of these compounds compared to those needed for optimal visualization of primary catecholand indolamines (prolonged reaction time and higher concentration of GA and FA and aluminum ions). The fluorophore formation from N-acetylated and tertiary indolamines, which represents a new reaction principle in amine fluorescence histochemistry, is proposed to proceed as follows. In the first step, the indole reacts in 2-position with the reagent. The intermediate formed is dehydrated in the second step, yielding a strongly fluorescent 2-methylene derivative, which either per se or as the corresponding autoxidized dimer constitutes the main fluorophore. TFAA and related anhydrides represent new and potent reagents for histochemical visualization of N-acetylated indolamines such as melatonin. In contrast to the GA and ALFA reactions the optimal formation of fluorphores with TFAA required only mild reaction conditions (2–10 min at 0–20° C). The main fluorophore formed from melatonin has been identified and the reaction with TFAA is proposed to proceed as follows. An unstable intermediate, the isoimidinium carboxylate, is formed in the first step and this compound is then cyclized to form the fluorophore, 6-methoxy-1-methyl-3,4-dihydro-β-carboline. The GA and ALFA methods are already widely used for visualization of catecholamine systems. The fluorescence microscopical and microspectrofluorometric analysis did not, however, veveal any specific structures containing N-acetylated or tertiary indolamines in the rat CNS. The TFAA reaction was highly specific for N-acetylated indolamines when applied to protein models. However, in tissue a disturbing background fluorescence appeared, which under all reaction conditions tested, developed concomitantly with the specific fluorescence from melatonin. The problem with this background reaction has to be solved before the TFAA reaction can be applied for demonstration of N-acetylated indolamines in tissue.

Les sites d'incorporation du 5-HTP-3H (5-hydroxytryptophane-3H) et de retention de ses derives ont ete recherches dans l'organe pineal de Lacerta vivipara (J.). Les animaux recoivent au cours de l'apres-midi (aux environs de 15h, le 5/VII, T°=21 a 28°C) une injection intraperitoneale de 5-HTP-3H. Ils sont sacrifies apres 2, 10, 15, 21, 30 minutes, 1, 2, 3, 4, 5, 10, 15 heures et 1, 2, 3, 4, 5, 6, 7, jours. De l'etude qualitative et quantitative, il ressort que des reactions radioautographiques specifiques apparaissent dans les photorecepteurs rudimentaires secretoires (=PRS) entre 2 et 10 minutes. La concentration de radioactivite atteint un maximum au niveau des PRS, apres 5 heures. Puis l'intensite du marquage diminue pour disparaitre entre 2 et 3 jours. La radioactivite des PRS est toujours concentree dans les regions de grains denses de secretion proteique (500 a 3400 A) d'origine golgienne. Les autres compartiments cellulaires sont nettement moins radioactifs. Les autres cellules (interstitielles de type ependymaire; nerveuses sensorielles et probablement noradrenergiques) de l'organe pineal et les regions voisines du cerveau ne retiennent pas significativement les composes radioactifs. Ces resultats et ceux anterieurs, cytophysiologiques et biochimiques, montrent le role important des PRS dans la biosynthese et le stockage de la serotonine. D'autres conclusions concernant la biosynthese et le metabolisme des indolamines ne seront exposees que dans une etude ulterieure ou l'incorporation du 5-HTP-3H est envisagee dans les conditions experimentales. Le turnover des indolamines semble plus lent chez Lacerta que chez les Mammiferes.

Catecholamines and indolamines react with formaldehyde to form fluorescent condensation products. This reaction forms the basis for the histochemical method of Falck and Hillarp which permits the demonstration of monoamines at the cellular level. The formation of the fluorophores is believed to require the presence of catalyzing proteins, peptides or amino acids. A similar reaction, however, can be induced also on silica gel thin-layers and this has been utilized for micro-analysis of tissue amines. In the present study formaldehyde-induced fluorophores of noradrenaline and 5-hydroxytryptamine in dry protein films and on silica gel thin-layers were analysed by microspectrofluorometry. The results indicated that the fluorophores formed in protein films and in silica gel were identical. It is suggested that the reaction on thin-layers can be used as a convenient model system permitting a detailed analysis of the various steps in the histochemical technique for the demonstration of catecholamines and 5-hydroxytryptamine.

The rhythmical activity of the liver fluke, Fasciola hepatica, was stimulated by 5-hydroxytryptamine and by lysergic acid diethylamide at very low concentrations. The effect was peripheral and was not mediated through the central ganglion. Other amines also stimulated rhythmical activity, the most potent being the indolamines. Bromolysergic acid diethylamide, and other analogues such as yohimbine, harmine, and dopamine depressed rhythmical movement and antagonized the stimulant action of 5-hydroxytryptamine and lysergic acid diethylamide. Evidence which suggests the presence of tryptamine receptors in the trematode is discussed.

Adult Syrian hamsters were given a subcutaneous injection of reserpine 3 days before an intraperitoneal injection of 3H-3,4 dihydroxyphenylalanine or 3H-5, hydroxytryptophan and the carotid bodies were subsequently prepared for electron microscopic radioautography. Other Syrian hamsters were given a subcutaneous injection of reserpine and the carotid bodies were subjected to a sensitive cytochemical test for the detection of unsubstituted amines. These studies were made to determine whether the labeled amine precursors were incorporated into the cells and to see whether the parenchymal cells were affected by reserpine treatment. Material from hamsters treated first with reserpine and subsequently injected with 3H-3,4 dihydroxyphenylalanine or 3H-5, hydroxytryptophan exhibited reduced grains of silver over the cells which were associated mainly with the dense cores of the cytoplasmic granules. These studies offer evidence that the granules of the carotid body incorporate catecholamine and indolamine precursors. Material from hamsters incubated for the presence of unsubstituted amines gave a positive reaction (opaque cytoplasmic granules) for catecholamines but not for indolamines. The latter substances may not be present in quantities sufficient to register a positive reaction in the cytochemical test. The opaque granules, indicative of the presence of catecholamines, decreased in density after reserpine treatment. 5 days after one reserpine injection the granules had regained opacity and were comparable to those seen in the control cells.

Considering that glutamatergic axons innervate the C1(Ad) medullary nuclei, which are responsible for the excitation of the peripheral adrenal glands, we decided to investigate catecholamines (noradrenaline, adrenaline and dopamine) plus indolamines (plasma serotonin and platelet serotonin) at the blood level, before and after a small oral dose of amantadine, a selective NMDA antagonist. We found that the drug provoked a selective enhancement of noradrenaline plus a minimization of adrenaline, dopamine, plasma serotonin and platelet serotonin circulating levels. Significant enhancement of diastolic blood pressure plus reduction of systolic blood pressure and heart rate paralleled the circulating parameter changes. The above findings allow us to postulate that the drug was able to enhance the peripheral neural sympathetic activity. Minimization of both adrenal sympathetic and parasympathetic activities was also registered after the amantadine challenge. The above findings supported the postulation that this drug should be a powerful therapeutic tool for treating diseases affected by adrenal sympathetic hyperactivity.

1. Neurons from rat superior cervical ganglia were grown in coculture with pineal cells. Action potentials of neurons in cocultures were 25% longer and were 25% greater in amplitude than those recorded from neurons grown in the presence of ganglionic nonneuronal cells alone. 2. Neurons showed an increase in action potential duration with increasing time in culture. This may have been related to the recovery of nonneuronal cell populations after an initial exposure to the antimitotic agent Ara-C. In cultures not initially exposed to Ara-C, a subsequent exposure after 7 days in culture resulted in a shortening of the action-potential duration. 3. Neuronal cultures were exposed to gel slabs containing the pineal indolamines, serotonin,N-acetylserotonin, and melatonin. Serotonin andN-acetylserotonin showed no effect on the neuronal action potentials at the concentrations tested. 4. Melatonin caused an increase in action-potential duration that was associated not with an increase in action-potential amplitude, but with a decrease in actionpotential rise rates. The effects of long-term exposure in melatonin appeared to be reversible in some cells but not in all. 5. Short-term effects of melatonin were observed in older cultures and in younger cultures after the cells were stimulated repeatedly. Older cultures also had higher levels of spontaneous activity. The dependence of the short-term effects of melatonin on electrical activity may suggest a role for melatonin as a neuromodulator.

Desoxyfructo-serotonin oxalate and free base produced a decrease of the blood pressure and heart rate of vagotomized cats (in total doses of 100 mg, i.v.) for 30 and 120 min. respectively, with total blocking of the carotid occlusion pressor response. With desoxyfructo-5-methoxytryptamine the decrease in the blood pressure lasts only 1 minute, followed by a 30 min. moderate increase. With this compound the C.O. pressor response is not blocked. On anaesthetized dogs with opened thorax low doses of the drug (100–200 μg/kg i.v.) produced a similar decrease of the blood pressure and heart rate, however, only for a short time (3 min.). An important increase in the sinusal coronary flow and partial oxygen pressure was also observed. Oral administration of desoxyfructo-serotonin oxalate (50 mg/kg) reduced to 40% the gastric ulcer formation by restraint in rats. With the same compound (100 mg/kg i.p. daily per 4 days) a very significant “anti-stress” activity was observed on mice by increased swimming endurance, Rota-Rod test performance, anti-ulcer effect and by elimination of milk induced leucocytosis.

At concentrations less than 10 −5 Moles, desoxyfructoserotonin oxalate has very little direct effect on human blood platelets, but still interferes with the active transport and release of serotonin.

Sensory stimuli from the external environment (e.g. visual and olfactory stimuli, stress) or internal stimuli cause brain nerve fibres to release neurotransmitters (catecholamines, indolamines and cholinergic agents). These neurotransmitters regulate the secretion of gonadotrophin-releasing hormone (GnRH) from neurosecretory cells of the hypothalamus (Kamberi, 1975). It would seem that under the proper steroid environment, catecholamines (dopamine, norepinephrine or epinephrine) and the cholinergic agent acetylcholine exert a stimulatory influence, whereas indolamines (serotonin or its metabolic product, melatonin) have an opposite effect. In turn, GnRH reaches the anterior pituitary via the hypophyseal portal system, and, through the mediation of cAMP [though may be not as an obligatory intermediate (Naor et al., 1975)], controls gonadotrophin secretion (Labrie et al., 1974).

Uptake and turnover of dopamine (DA) in rat peritoneal mast cells were studied by a cytofluorometric technique. The main advantage of the method is that it permits the study of the distribution of amine content within populations of cells. Catecholamines and indolamines can be differentiated, but subtler structural differences in this group of compounds cannot be distinguished. We, therefore, combined the cytofluorometric measurements with a liquid chromatographic method based on reversed-phase chromatography followed by amperometric detection in a thin layer flow cell. Intraperitoneally injected L-DOPA was rapidly decarboxylated to DA, which was accumulated in mast cell granules. The elimination of DA from the mast cells was much faster than previously published 5-hydroxytryptamine and histamine elimination rates. No evidence of intracellular conversion of DA before its elimination was found and simultaneous heparin quantitations gave no evidence of an elimination pathway due to exocytosis of granules. Electron microscopy disclosed no structural changes that could be related to exocytosis during the elimination phase of DA. The rapid elimination together with absence of inhibition of DA-uptake after storage of exogenous 5-hydroxytryptamine suggest that the mechanism of DA storage differs from the mechanism of storage of endogenous mast cell amines.

Insulin receptors are present in the hypothalamus, but the cell types bearing them are unknown. In order to test the hypothesis that some insulin receptors in the hypothalamus are associated with catecholamine terminals, rats were injected with 50 micrograms or 75 micrograms doses (intracerebroventricular) of 6-hydroxydopamine (6-OHDA). Control rats received vehicle only. The animals were sacrificed 7 days after injection, and catecholamine and indolamine levels in the hypothalamus were measured by high performance liquid chromatography with electrochemical detection. Localization of specific binding sites for [125I]-insulin in the arcuate (ARC), dorsomedial (DMN) and ventromedial (VMN) nuclei were determined by quantitative film autoradiography. Treatment with 6-OHDA resulted in a 70% reduction in hypothalamic norepinephrine content as compared to vehicle-treated controls (P less than 0.01). A slight depletion of epinephrine, dopamine and indolamines was also detected. Computerized image analysis of the autoradiograms was used to determine radioactivity bound (DPM/mm2) in each nucleus. Highest binding was in the ARC and DMN, with much lower binding in the VMN. Insulin binding in the ARC of the 6-OHDA-treated group was decreased by 25% compared to controls (P less than 0.01). No significant change in insulin binding was observed in the DMN or VMN. The 6-OHDA treatment had no significant effect on weight gain or on plasma insulin levels. The reduction of insulin binding in the ARC after 6-OHDA treatment supports the hypothesis that some insulin binding sites are located on catecholamine terminals in the arcuate nucleus.

Ultracytochemical identification of monoamines in the paraventricular organ (PVO) of Xenopus laevis tadpoles was achieved by applying glutaraldehyde-potassium dichromate fixation to the brains. It appears that the amines are concentrated inside elongated or round large granular vesicles within the neurons. The presence of dopamine and, to a lesser extent, of indolamines in neurons of the PVO is discussed.

In p-chlorophenylalanine (PCPA) pretreated insomniac cats, the intraventricular (i.v.t.) injection of artificial cerebrospinal fluid (CSF) did not impair the insomnia. CSF transfer from normal cats was followed in 4 out of 9 cats by the restoration of paradoxical sleep (PS). However, CSF transfer from paradoxical sleep deprived cats did result in 12 out of 13 experiments in a significant increase in slow wave sleep (SWS) and the induction of PS. Biochemical analysis of the CSF from normal or PS deprived cat has shown that the highest quantity of indolamines was at least 1000 times smaller than the threshold dose of 5-HTP (200 microgram) which has been shown to be able to restore sleep by i.v.t. injection in PCPA pretreated insomniac cats. These experiments provide evidence that the transfer of a small quantity of CSF (250 microliter) from a previously paradoxical sleep deprived cat can restore paradoxical sleep in an insomniac PCPA pretreated cat in bypassing the biosynthesis of serotonin (5-HT). These results suggest that a 'paradoxical sleep inducing factor' may be stored in the central nervous system during sleep deprivation.

When injected intraperitoneally (i.p.) into normal cats, methiothepin (1-7.5 mg/kg) or metergoline (0.5-8 mg/kg) induced total insomnia. The duration of suppression of deep slow-wave sleep (SWS2) and paradoxical sleep (PS) was dose-related for methiothepin (10-30 h), but was shorter and not dose-related for metergoline (3-5 h). When injected into the fourth ventricle, methiothepin (20 microgram) induced a selective suppression of PS for 6-7 h). In rho-chlorophenyl-alanine (PCPA)-pretreated, insomniac cats, i.p. injection of 5 mg/kg of DL-5-hydroxytryptophan(5-HTP) was followed by SWS and PS after latencies of 25 and 62 min. When methiothepin (2.5 mg/kg) or metergoline (4 mg/kg) were given before 5-HTP, the latency for the first PS episode increased dramatically to 7 h. Thereafter PS occurred periodically for 10 h but no SWS appeared. These results suggest that a 'PS factor' induced by 5-HTP and different from indolamines is preserved during the antagonistic effect of methiothepin or metergoline until it can act upon the executive mechanisms of PS. Our data also suggest that metergoline and methiothepin suppress but do not delay the mechanism responsible for SWS2 in the PCPA-5-HTP paradigm.

The metabolism of monoamines in the brain of rats in fulminant hepatic failure was studied after administration of branched chain amino acids (BCAA). Male Sprague-Dawley rats were exposed to a total hepatectomy or a sham operation and infused intravenously for 5 hours with a 10% glucose solution alone or 10% glucose + 0.24 mol/l BCAA (0.08 mol/l of each valine, leucine, and isoleucine). Thirty minutes before decapitation, a decarboxylase inhibitor (NSD 1015®), blocking the conversion of 5-hydroxytryphtophan (5-HTP) to 5-hydroxytryptamine (5-HT), and dihydroxyphenylalanine (DOPA) to dopamine (DA), was injected intraperitoneally. The concentrations of 5-HTP, 5-HT, and 5-hydroxyindoleacetic acid (5-HIAA) were analyzed by high-performance liquid chromatography, DOPA, DA, and norepinephrine (NE) by a radio-enzymatic method in 8 different regions of the brain and spinal cord. The concentrations of 5-HTP were increased in the brains of hepatectomized rats, indicating an increased synthesis rate of the indoleamines. After administration of BCAA to hepatectomized rats, the synthesis rate was decreased to normal or, in some brain regions, even to subnormal values. The brain and spinal cord concentrations of DOPA and DA were unchanged in hepatectomized rats compared to sham-operated animals, indicating an unchanged turnover of the catecholamines. The levels of NE in the diencephalon and in the spinal cord were decreased after hepatectomy. The level of consciousness was not influenced by the BCAA infusions.

The development of the concept of chemical transmission at the synapse and its application to the central nervous system has resulted in remarkable advances in our understanding of brain structure, function, and pathology. Among these, it has provided a basis for characterizing neuronal systems other than on morphological criteria. The utility of this characterization is best exemplified by our current knowledge of catecholamine and indolamine neuron systems in the mammalian brain. The presence of an indolamine (5-hydroxytryptamine, or serotonin) and catecholamines (norepinephrine, dopamine) in brain in a nonuniform pattern was first established 20 years ago (Twarog and Page, 1953; Amin et al., 1954; Bogdanski et al., 1957; Bertler and Rosengren, 1959) and led to the proposal that these compounds serve as chemical mediators of synaptic transmission (Amin et al., 1954; Vogt, 1954; Brodie and Shore, 1957). Subsequent studies in which brain amine content was analyzed following placement of localized destructive lesions indicated an association of specific neuron systems, particularly the medial forebrain bundle, with these putative neurotransmitters (Heller et al., 1962; Heller and Moore, 1968). It was not until the development of a specific and sensitive histochemical procedure for the intraneuronal localization of indolamines and catecholamines, the Falck-Hillarp method (Falck et al., 1962; Carlsson et al., 1962; Falck, 1962; Corrodi and Jonsson, 1967; Bjorklund et al., 1972), however, that significant advances were made in analyzing the specific neuronal systems which produce serotonin and catecholamines in brain.

Human Y79 retinoblastoma cells are capable of synthesizing the putative retinal neurotransmitters dopamine and serotonin. Separation of the catecholamines and indolamines by high performance liquid chromatography combined with electrochemical detection showed that the cells readily convert tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and, to a lesser extent, dopamine. When DOPA was added, a large quantity of dopamine was produced, as well as norepinephrine, epinephrine, and 3,4-dihydroxyphenylacetic acid. Exogenous tryptophan added to the cells was partially converted to 5-hydroxytryptophan and serotonin. A larger quantity of serotonin was produced when 5-hydroxytryptophan was added. Y79 cells have a high and low-affinity uptake system for dopamine and serotonin. The K′m and V′max for the high-affinity uptake of dopamine and serotonin are 2.34 ± 0.64 and 3.63 ± 1.15 μM and 4.77 ± 1.12 and 3.20 ± 1.20 pmol min-1 mg protein-1, respectively. These kinetic parameters are similar to those reported for other retinal preparations where dopamine and serotonin have been suggested to function as neurotransmitters. Tyrosine and tryptophan, the physiologic precursors of dopamine and serotonin, respectively, and phenylalanine are also taken up by high and low-affinity transport systems. The kinetic parameters for their high-affinity uptake systems are all very similar, suggesting that they may be taken up by the same transporter. These studies show that a tumor cell line derived from the human retina synthesizes dopamine and serotonin and has high affinity uptake systems for these compounds and their pre cursors. This is consistent with the possibility that dopamine and serotonin may function as neurotransmitters in the human retina and that the Y79 retinoblastoma may be a suitable cellular model system in which to study their transport and metabolic properties.

Test-tube experiments proved that alkaline lead citrate (Reynolds, 1963), which is generally used as an electron-opaque stain, specifically reacts with precipitates formed by glutaraldehyde and biogenic amines (indolamines, primary catecholamines). Glutaraldehyde/osmium tetroxide fixation and staining of thin sections with alkaline lead citrate is recommended as an optimal preparation method of studying the above-mentioned amines at a fine structural level.

This paper describes new procedures for highly sensitive visualization of monoamine stores in peripheral tissues, taking advantage of the recently introduced aluminum-catalysed formaldehyde (ALFA) reaction. The tissues are exposed to an aluminum sulphate solution (with or without formaldehyde fixation) in a perfusion and/or immersion step, followed by formaldehyde vapour treatment. Procedures are described for freeze-dried, paraffin embedded tissue, cryostat sections and whole mount preparations. For all these tissue preparations the ALFA method gives a highly sensitive and precise demonstration of catecholamine-containing neurons and 5-HT-containing cells in a variety of peripheral tissues. For freeze-dried tissue and cryostat sections the ALFA method represents an improvement in comparison with other available methods. This is particularly noticeable for the very delicate adrenergic nerves in such organs as the thyroid, ovary, pancreas and the gastrointestinal tract.

Desoxyribulo-serotonin shows a serotonin-like activity on both D and M type tryptaminergic receptors. Similarly to desoxyfructo-serotonin, its affinity is about 100 times less towards these receptors when compared to serotonin. The intrinsic activities of the two sugar derivatives for D type receptors are nearly equal and very close to the initial value of serotonin. Desoxyfructo-serotonin showed a decreased intrinsic activity for M type receptors with 70 % of the initial value observed for serotonin. The intrinsic activity of desoxyribulo-serotonin for these receptors is even less, showing only 40 % activity. The effect of desoxyribulo-serotonin on platelet functions is revealing : in spite of its decreased intrinsic activity towards M type receptors, the ribose derivative shows unchanged or higher activities in shape-change, aggregation, inhibition of the uptake and the release of serotonin, than desoxyfructo-serotonin, confirming earlier observation, that M type receptors are not involved in platelet functions. It is noteworthy that desoxyribulo-serotonin is a nearly total inhibitor of the release of serotonin from platelets induced by 5,6-dihydroxytryptamine.

Carbon fiber microelectrodes were used with the differential pulse voltammetry method for in vivo determination of indolamines within the extracellular space of the dorsal horn of the spinal cord or chloral hydrate-anesthetized rats. Under these conditions a peak of oxidation current which is characteristic of 5-hydroxyindoles is recorded at 280-300 mV. Stimulation of the nucleus raphe magnus (NRM) with stimulation parameters comparable to those used to elicit analgesia in freely moving animals produced marked alterations in the voltammograms: (1) stimulation of the NRM for 10 min induced an immediate and sustained increase in the peak amplitude; (2) post-effects of variable duration were observed; (3) the increase in the 5-hydroxyindolaminergic signal was significantly reduced during a second series of NRM stimulations indicating some degree of tolerance to central stimulation. The accuracy of these observations is strengthened by the fact that the basal 5 hydroxyindolaminergic signal is strongly depressed after pretreatment of the animal with p-chlorophenylalanine; in addition, under these conditions, NRM stimulation is totally inefficient. We suggest that these results reflect the in vivo modification of 5-HT metabolism. This represents the first evidence for an in vivo release of 5-HT during stimulation of brain stem areas which induces powerful analgesia in freely moving animals.

Melatonin signal transduction was examined in median eminence/pars tuberalis (ME/PT) explants from Djungarian hamsters. High affinity melatonin receptors in hamster ME/PT were first quantified by in vitro autoradiography using the potent melatonin agonist 125I-labeled melatonin ([125I]MEL). Scatchard analysis of [125I]MEL binding in ME/PT revealed high affinity receptors [dissociation constant (Kd) = 2.75 X 10(-11) M]. [125I]MEL binding was markedly reduced by guanine nucleotides; treatment with the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) caused a 10-fold decrease in receptor affinity. Melatonin (10 nM) significantly inhibited forskolin-stimulated cAMP accumulation in ME/PT, but not in pituitary or pineal glands. In ME/PT explants, melatonin and 6-chloromelatonin inhibited forskolin-stimulated cAMP accumulation in a dose-dependent manner with similar potency (significant inhibition for each at concentrations greater than or equal to 100 pM). Serotonin significantly inhibited forskolin-stimulated cAMP levels only at doses greater than or equal to 100 microM. Inhibition of [125I]MEL binding in ME/PT by these three indolamines paralleled that determined for inhibition of forskolin-stimulated cAMP accumulation. Pertussis toxin treatment (1 microgram/ml) blocked the ability of melatonin (10 nM) to inhibit forskolin-stimulated cAMP accumulation and significantly reduced [125I]MEL binding. Pertussis toxin ADP-ribosylated the alpha-subunits of at least two guanine nucleotide-binding proteins in ME/PT explants with molecular weights of approximately 40 K. Melatonin did not increase phosphodiesterase activity in ME/PT explants. The results strongly suggest that a signal transduction pathway for melatonin in mammals involves inhibition of adenylyl cyclase by a pertussis toxin-sensitive guanine nucleotide-binding protein.

Monoaminergic inputs to the caudal neurosecretory complex (CNc) of Poecilia latipinna have been identified using histofluorescence and immunohistochemical techniques. The present study was undertaken to identify specific monoamines and determine the relative contribution of indolamines and catecholamines in supraspinal and intrinsic innervation of the nucleus. The CNc was deafferented by transecting the spinal cord rostral to the CNc. Ten days subsequently, CNc of spinal-transected and control fish were processed for either biochemical or immunohistochemical analysis. Norepinephrine and serotonin were detected in pooled samples of control CNc. Following deafferentation, the content of both monoamines was diminished. Using immunohistochemical labeling for serotonin or for the catecholamine-synthesizing enzymes, tyrosine hydroxylase (TH) or dopamine-β-hydroxylase (DBH), the number of monoamine fibers was decreased in deafferented CNc compared to control. A substantial serotonergic innervation remains after deafferentation, as evidenced by serotonin-positive neurons and heavy, varicose fibers. Occasional TH/DBH-positive cells and fibers remain after deafferentation. These data suggest that both norepinephrine and serotonin are associated with descending supraspinal projections, while serotonin predominates as the intrinsic monoamine.

Neural growth and differentiation is regulated by genetic and epigenetic factors. Although genetic regulation is only recently beginning to be explored, intrinsic epigenetic control of neural growth has been the focus of research for several investigators. The regulatory role of hormones such as thyroid hormones and steroid hormones in neural growth and differentiation has been well documented. More recently, we (Vernadakis and Gibson, 1974) and others (Lauder, 1979) have proposed that neurohumor substances such as acetylcholine, catecholamines and indolamines are involved in cellular processes including neuro-genesis before their involvement in neurotransmitter processes. As it has been abundantly demonstrated with hormones, it is probably the combination of actions and interaction of several neurohumor substances that influence cellular growth. Moreover, neuronalglial interrelationships and the influence of neurohumor substances thereon are important factors in neural growth and differentiation. Lauder and associates will discuss in this symposium their work with serotonin on neurogenesis. We will discuss primarily the role of norepinephrine in neural growth. We have used the chick embryo as an animal model to study maturational processes at the organismic level and neural culture to characterize differential responses of neurons and glial cells to neurohumor substances.

These studies reveal that the postnatal ontogeny of serotonin (5HT) in the sympathetic nuclei of the rat spinal cord is protracted; the adult complement of 5HT-immunoreactive fibers is not achieved until at least 60 days of age. As descending serotonin fibers innervate and demarcate the distribution of preganglionic sympathetic nuclei, rostral-caudal and temporal gradients exist. Additionally, a heterogeneous segmental 5HT ontogenetic pattern is observed in sympathetic nuclei. Most serotonin fibers in laminae VII and X are unorganized at birth except for some sympathetic nuclei in high thoracic regions where the 5HT sympathetic pattern is being initiated. By postnatal day 6 the framework of the 5HT pattern is established in all sympathetic nuclei, and by postnatal day 16 a pattern is formed, which develops into the compact adult state by postnatal day 60. The protracted period of sympathetic 5HT development corresponds with the length of time it takes for the autonomic nervous system to mature. In addition, 5HT intraspinal cell bodies are observed at all time points examined, except for the day of birth, and are found in the same regions as adult 5HT neurons, i.e., dorsal or lateral to the central canal in laminae VII and X and in all spinal segments except cervical levels. Many of the 5HT neurons are pericanalicular and bipolar in appearance. Multipolar 5HT neurons are first observed on postnatal day 45.

Rats maintained on a tryptophan deficient diet had reduced brain and serum tryptophan and brain 5-hydroxy-indolacetic acid levels compared to controls. 5-Hydroxytryptophan and l-tryptophan administered to these deficient rats in a combination (5∶95) produced a greater elevation of indolamines and tryptophan in the brain than either amino acid alone. In rats maintained on a normal diet the urinary output of 3-hydroxykynurenine was considerably reduced by treatment with the combination of amino acids as com pared to tryptophan treatment. 5-Hydroxytryptophan reduced the induction of kynurenine synthesis in the liver produced by tryptophan, implying that it is capable of inhibiting the enzyme tryptophan pyrrolase in vivo. It is suggested that the possession by 5-hydroxytryptophan of tryptophan pyrrolase inhibitory properties may make the administration of the combination a better treatment of depressed patients exhibiting an indolamine deficit than either amino acid alone.

Desoxyfructo-serotonin is a new synthetic sugar derivative of serotonin, however, its formation in physiological conditions can also be expected. Using rat brain monoamine oxidase (MAO) in vitro , desoxyfructo-serotonin is significantly more slowly metabolized than serotonin. “Transcypromine”, a specific inhibitor of MAO, also inhibits the oxidative deamination of the sugar derivative.

Publisher Summary The sex differences that are found in neuronal connectivity suggest that such differences may also be expected with respect to the neurotransmitter content measured within sexually dimorphic areas. This chapter discusses the various aspects of the study of sex differences in neurotransmitter systems, and the vasopressin innervation of the brain is discussed as an example of the use of immunocytochemistry in studying sex differences. Several methods have been developed for studying the anatomy of neurotransmitter systems: (i) the histofluorescent demonstration of the indolamines and catecholamines, (ii) enzymes histochemical procedures for cholinergic neurotransmitter pathways, and (iii) immunocytochemistry, which is potentially the most versatile method, because it does not depend on the detailed knowledge of either the chemical nature or the metabolizing enzymes for a given neurotransmitter. Studies of the vasopressinergic innervation of the brain have demonstrated that immunocytochemistry in conjunction with tracing and lesion studies forms a powerful tool to study the anatomical background of a particular sex difference. However, one major problem of immunocytochemical methods is that it is difficult to raise an antiserum, which is directed specifically against a single antigen, which makes it necessary to extensively test all sera and, if necessary, to purify them. An advantage of the application of immunocytochemistry is that it can readily be combined with other techniques, for example, with steroid receptor labeling. The study of the neurotransmitter content of the various components of a given sexually dimorphic system can also help to elucidate its functional significance, if only because the knowledge about the neurotransmitters involved makes it possible to use pharmacological tools, such as local administration of specific agonists or antagonists.