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The regeneration of the arm of the crinoid Antedon mediterranea has been studied by means of LM, TEM, and SEM techniques as regards the development of both the external form and the internal anatomical organization. According to the timing and the modalities of development of the different structures and their reciprocal interactions, three main stages can be recognized in the morphogenesis of the whole arm: 1) a coelom-dependent stage, 2) a nervous system-dependent stage and 3) a skeleton-dependent stage. Moreover, using histofluorescence and immunohistochemical methods to visualize catecholamines and indolamines, it has been possible to reveal the presence of dopamine and serotonin and follow the fluctuations and the different involvements of these neuro-trasmitters during the regeneration process.

The endogenous compounds, serotonin (5-HT) and tryptamine (TA), have been found to alter function in the CNS, supporting the hypothesis that they serve as neurotransmitters. It is reported here that intrathecal injections of either 5-HT or tryptamine elicited similar behavioral syndromes consisting of caudally-directed biting, or licking and scratching. Serotonin was found to be considerably more potent than tryptamine. However, administration of both indoleamines produced synergistic interactions with respect to this behavioral syndrome. Administration of fluoxetine with either 5-HT or tryptamine potentiated the scratching behavior produced by either indoleamine, but failed to enhance the effect of an injection of 5-HT plus tryptamine. Administration of methysergide blocked the effects of both indolamines. The results are discussed in terms of a possible mechanism of the synergism between 5-HT and tryptamine.

Publisher Summary Although “the profession of the pineal is to regulate reproduction whereas its avocation is to influence the functioning of other endocrine organs,” yet some other target organs can be envisaged, and some new insights may be obtained. The different responses may be tentatively attributed either to the secretion by the epithalamo-pineal complex of several indolederivatives, acting altogether differently than MLT alone, and/or to an activation of eventually melatonergic nerves to the bone marrow. The long latency of the blood platelet increase suggests that MLT or the epithalamic indolamines induce an acceleration of the development from stem- to adult cells, and/or from megakaryocytoblast to mature megakaryocyte. This chapter also presents various other perspectives in pineal functions and in conclusion outlines the results obtained.

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.

The present paper presents the evaluation of a mixed mode column (reversed phase and cation exchange) performance for the separation of 12 catecholamines, indolamines and their precursors and metabolites. The influence of different parameters, like organic modifier nature and percentage, salts nature and percentage, on the compounds separation was investigated. Good separation of the 12 selected compounds was obtained in the isocratic mode using a mobile phase composed of 90 % methanol and 10% ammonium acetate 20 mM, however the analysis time was excessively long (80 minutes). Thus an elution gradient was optimized and it reduced the analysis time to less than half (35 minutes).

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.