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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.

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.

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.

Publisher Summary The results presented in this chapter show that it is possible to differentiate between catechol and indolamines in synaptic vesicles. Two methods have been successfully assayed in the pineal nerves of the rat, one of them based on the use of current fixation methods combined with pharmacological treatment and the other using a histochemical technique. After both fixation methods, the typical population of clear and granulated vesicles could be observed with minor differences such as a greater density of the intermediate type of vesicles after glutaraldehyde-osmium tetroxide and darker and better defined cores of the small vesicles after osmium tetroxide alone. However, the heterogeneity of monoamine-containing vesicles as revealed by current and special fixation methods indicates that these results cannot be extrapolated to other granulated vesicles without demonstration. The chapter also shows that different fixatives may preserve different components in synaptic vesicles even if they give a similar picture at the electron microscopic level. Not only employed the fixative but also the used precise schedule may involve substantial differences in the preservation of monoamines. Differences in binding may be an important parameter in the localization of monoamines in synaptic vesicles.

Neural control over pituitary gonadotropin secretion implies distinct regulation levels. Integrative centres, located mainly in the anterior hypothalamus, process sensory or somesthetic information coming from higher parts of the CNS. These integrative centres then forward their information to the neurosecretory neurons responsible for the elaboration of “releasing factors”, which are located within the “Halasz zone”. Finally releasing factors are released into the portal circulation at the level of the median eminence.

A new methodological approach to the fluorescence microscopical visualization of biogenic monoamines, based on condensation with gaseous glyoxylic acid, is presented. Glyoxylic acid treatment seems both to be more sensitive for the well-known biogenic catecholamines and indolamines, and to allow the detection of other biogenic amines—such as melatonin—which are not demonstrable with available techniques.

Pineal glands of normal adult mice, 7 to 42 days after bilateral superior cervical gangliectomy and 5 and 16 hours after one dose of reserpine (10 mg/kg) were studied under the electron microscope. The architecture of the gland is basically similar to that of other mammalian pineal glands previously studied. Mouse pinealocytes are polymorphic cells with perivascular and intercellular processes. Its most prominent feature is the presence of abundant granulated vesicles with a mean diameter of 1100 A and a dense core of about 800 A intermingled with clear vesicles of similar size distributed throughout the cytoplasm and more concentrated in perivascular and intercellular processes. These processes were seen in continuity with the perykaryon and remained after bilateral superior cervical gangliectomy. Processes containing the plurivesicular component of adrenergic nerves situated in perivascular and intercellular spaces disappeared after bilateral superior cervical gangliectomy. Reserpine depleted small granulated vesicles of nerves but the larger ones of perikaryon and polar processes remained almost unchanged. The histochemical technique of Wood was positive for catechol- and indolamines in the nerves. The reaction was negative in the perikaryon and polar processes. The significance of these findings is discussed.

The effects of reserpine after mono-aminoxidase (MAO) inhibition has been studied by observing and recording spontaneous activity in mice. The injection of reserpine following a MAO inhibitor produced a biphasic pattern of excitation, the first period lasting for 2–3 hr and the second until 5–6 hr after the injection. The administration of DOPA to mice pretreated with a MAO inhibitor caused stimulation similar to the first phase of reserpine action. Under the same conditions, an excitatory effect analogous to that of the second phase was produced by 5-HTP. When animals were “depleted” with an injection of reserpine, 5-HTP and DOPA given after a MAO inhibitor were both able to reverse the depression caused by reserpine and even produce their characteristic stimulation. Under the same conditions, the stimulating effect of a second dose of reserpine completely disappeared 24 and 48 hr after the “depleting” dose of the alkaloid, gradually returning 72 hr thereafter. Accepting that reserpine, 5-HTP and DOPA act through the biogenic amines in the CNS, we have suggested, on the basis of our results, that both catechol-amines and serotonin would have definite and distinct stimulant actions in the brain.

To determine the effect of biogenic amines on LH release in rats, blood samples were drawn by cardiac puncture in etheranesthetized animals bearing chronic cannulae in the third ventricle (3rd V) immediately before and 15 min after intraventricular injection of various drugs. In the interim between removal of the 2 blood samples the rats recovered from anesthesia. Plasma LH was measured by radioimmunoassay. Serotonin (5-HT) did not alter plasma LH levels significantly at any stage of 4-day estrous cycles. Norepinephrine (NE) also failed to produce over-all significant changes in plasma LH although there were some positive responses. Dopamine (DA) raised LH to levels as high as 8- or 10-fold above controls (p

The capacity of biogenic amines to form fluorophores in histochemical gas phase reactions has been tested with 20 compounds having a carbonyl group (> C = O) as a common characteristic. Significant visible fluorescence was induced from catecholamines and indolamines with aldehydes, ketones, α-keto acids and carboxylic acids, suggesting that all of these compounds can enter fluorophore-forming cyclization reactions under the histochemical gas phase conditions. The most "reactive" reagents are found among the low molecular aldehydes, and formaldehyde and glyoxylic acid seem to be the most suitable reagents, combining high reactivity with good selectivity. Fluorescence, interesting for the histochemical demonstration of N-acetylated indolamines, was obtained from melatonin and N-acetyl-5-hydroxytryptamine with some acid reagents (glyoxylic acid, formic acid, acetic acid and pyruvic acid). A direct cyclodehydration according to the classical Bischler-Napieralski reaction is the most likely mechanism underlying this fluorophore formation. The usefulness of the various carbonyl reagents for the fluorescence histochemical demonstration of other biologically interesting amines, such as histamine, p-tyramine and octopamine, has been specially investigated.

IT is well known that the neonatal rat brain contains relatively little serotonin1,2 and that the level of this amine increases slowly with age. Karki et al.3 have shown that the enzymes mediating the synthesis and oxidation of serotonin are also low in the immature rat brain, and increase thereafter with growth of the animal. The aforementioned authors suggest that the low levels of indolamines in the rat brain at birth can be attributed to a deficiency of the binding or storage system, and are not a reflexion of the insufficiency of the enzyme systems. Schanberg and Giarman4,5 have shown that endogenous brain serotonin distributes itself between the particulate matter of brain homogenates and the supernatants, and that this relationship is susceptible to alteration by drugs. It was, therefore, of interest to investigate the sub-cellular distribution of serotonin in the developing rat brain and to determine whether it will parallel other maturational changes.

This chapter investigates the origin of the nerve fibers innervating the pineal gland, particularly the nature of the granular vesicles present in the nerve endings. For this purpose, two principal approaches are used: some rats are submitted to the bilateral extirpation of the superior cervical ganglia and some are submitted to the action of drugs that may alter the metabolism of catechol and indolamines. In both cases, the glands are fixed and studied under the electron microscope, and the changes occurring in the nerve fibers and endings are recorded. Preliminary determinations of 5-hydroxytryptamine (5-HT) content are also carried out in normal and denervated glands and in some of the rats treated with agents that may change the content in this indolamine. The chapter presents the results in an extended form. The chapter discusses the actions of other drugs, such as dopamine (DOPA) 5-hydroxytryptophan (5-HTP), α-methylmetatyrosine, aramine, and nialamide on the ultrastructure of nerve endings and on the 5-HT content of the gland.

Granulated vesicles in rat pineal nerve endings consist of 2 compartments; the core and the matrix. The core can be stained by a variety of techniques including osmium tetroxide alone and a mixture of cosmium tetroxide-zinc iodide (ZIO), and gives histochemical reactions for cathecol and indolamines. The matrix can be stained with ZIO. To determine whether the matrix ZIO-reactive material represents a store of noradrenaline, different amounts of tyramine were adminisered i.p. in 50 mg/kg doses at 15-min intervals. The pineal glands were then studied under the electron microscope using the above techniques. Noradrenaline was determined fluorimetrically. It was observed that after the first dose of tyramine, no significant change in the noradrenaline content occurred although the matrix ZIO-reactive material decreased by 80%; this strongly suggests that the matrix ZIO-reactive material is not a noradrenaline store but represents some other component of the vesicle which is also depleted by tyramine. In contrast, the core components revealed by the techniques used disappeared gradually.

Publisher Summary This chapter describes the occurrence of indole amines in the pituitary proper of mammals, excluding the median eminence region. The localization of the serotonin in pituitary cells is not established yet, but a new fluorigenic substance, which was not identical with either catecholamines or serotonin, was recently found. This compound has some similarities to the fluorescence characteristics of peptides with tryptophan in the NH,-terminal position. The possible functional significance of local monoamines in the control of pituitary secretion is also discussed. Investigations undertaken to clarify this question showed that peptides with tryptophan in the NH,-terminal position react in the formaldehyde-ozone procedure to give an intense fluorescence spectrally similar to that of tryptamine. It remains to be determined whether N-terminal tryptophyl peptides actually exist in the pituitary and whether these peptides are responsible for the pituitary fluorescence. At present there are no experimental data relating the piuitary stores of serotonin, tryptamine, or histamine to the secretion of pituitary hormones.

p-Chloropherrylalaninc, a drug that selectively inhibits the synthesis of serotooin, has been used in order to invcstigate the localization of this amine in nerves of the pineal gland of rats. Glands of normal and treated rats were studied under the electron microscope, after fixing with osmium tetroxide and glutaraldehyde, and applying specific histochemistry for catechol and indolamines. Fluorometric determinations of scrotonin, noradrenalinc and dopaminc were also made in normal and treated glands. In normal glands about 35 % of granulated vesicles were observed in nerve endings fixed with osmium tetroxide or glutaraldehyde. After treatment with p-chlorophenylalanine the dense core of the granulated vesicles disappeared in glands fixed with glutaraldehyde but remained unchanged or increased in number in those fixed in osmium tetroxide. p-Chlorophcnylalanine produced an 87% decrease in serotonin content of the pineal gland while noradrenalinc and dopamine content did not diminish, The correlation between morphological and biochemical data suggests that both catechol and indolamines are stored in granulated vesicles in the pineal nerves of the rat. This assumption was confirmed with the specific histochemical technique of wood to differentiate catechol and indolamines.

Summary Drugs that may change the metabolism of catecholamines and indolamines have been used in order to investigate the nature of the granulated vesicles found in adrenergic nerve endings min the pineal gland of the rat. In normal endings, 30–40 per cent of the vesicles are granulated and the rest are homogeneous. The proportion of granulated vesicles diminish considerably between 2–48 hr after a single injection of reserpine and this is slowly restored to normal levels in a period of 8 days. A single injection or chronic treatment with iproniazid (a MAO inhibitor) increases the proportion of granulated vesicles, and also the size of the vesicles and granules. Iproniazid prior to reserpine protects the nerve endings from the depletion of granulated vesicles. Pyrogallol (a COMT inhibitor) does not change the concentration of granulated vesicles and does not protect the nerve ending from the action of reserpine. These results are interpreted as supporting the concept that the granulated vesicles contain the adrenergic transmitter. The possible mechanism of action of these drugs on the submicroscopic structure of adrenergic nerve endings is discussed.