In the pineal gland of the mouse the distribution of serotonin (5-HT) and its eventual relationships to a protein secretion were examined by means of fluorescence histochemical (Falck-Hillarp) and ultracytochemical (chromaffin and argentaffin) methods. (1) Yellow formaldehyde-induced fluorophores, characteristic of high concentrations of 5-HT, were found in pinealocytes, interstitial cells and sympathetic adrenergic nerve endings. The 5-HT content was studied according to the circadian variations and different drug treatments. (2) By use of ultracytochemical methods in untreated control mice precipitates indicative of 5-HT were found over dense-core vesicles (DCV; mean diameter: 100 nm) of the pinealocytes and the vesicular compartment of the sympathetic nerve fibers. After reserpine and p-CPA treatments, the reactivity of the DCV disappeared, but the protein secretion accumulated within the DCV was still present. After nialamide treatment the precipitates in the DCV increased and, similar to control mice, masked the protein secretion. Taking into account the specificity and sensitivity of these three complementary methods, previous biochemical data in mammals and ultracytochemical data in submammalian vertebrates, synthesis, storage, catabolism and release of 5-HT in the mouse and hamster pineals are discussed at the cellular level. Different pools of 5-HT are present: agranular and granular in pinealocytes and sympathetic nerve endings; agranular in interstitial cells. In the mouse, only a small portion of the total 5-HT content appears to bae secondarily taken up by the DCV. Within the DCV, 5-HT is possibly bound to a protein secretion of unknown significance (peptidergic neurohormone?). In the pinealocytes, which are sensitive to a large variety of inputs, processes of indole and protein secretion are found. These cells apparently are the recepto-secretory elements of the mammalian pineal gland. They can thus be classified as a member of the diffuse neuroendocrine system of the paraneuron group.

The uptake of 5-hydroxytryptophan (3H-5-HTP) and serotonin (3H-5-HT) and their distribution have been demonstrated, at the ultrastructural level, in the secretory rudimentary photoreceptor cells (SRP cells), in the pineal organ of the parakeet and the duck. In vivo experiments. When the parakeets were injected with 3H-5-HTP alone or pretreated with drugs interfering with monoamine metabolism (e.g. R04-4602, nialamide) prior to injection of the label, the SRP cells always exhibited specific radioautographic reactions, in the regions of the dense secretory granules of the cell body and asynaptic pedicles. The labeling concentration avried quantitatively with time (interval between injection of the label and sacrifice) and drugs used. In vitro experiments. The uptake of 3H-5-HTP and 3H-5-HT in the pineal organ of the duck has also been studied, using an incubation procedure. The results with the two precursors confirmed those obtained "in vivo" with the parakeet. From these results it is suggested that the uptake of precursors in some compartments of SRP cells is qualitatively independent of: a) the experimental period (experiments performed in winter, spring, and summer; uptake of precursors between 11.00 a.m. and 12.00) b) the precursor (3H-5-HTP or 3H-5-HT) c) the observation times. It is concluded that the SRP cell, an important step in the cytological and functional evolution of the pineal organ (Collin, 1969, 1971), is responsible for the synthesis of 5-HT. The metabolism of 5-HT seems also possible in such cells.

Summary. The pineal organ of the pike (Esox lucius L.). IV. Endogenous serotonin and monoamine oxidase activity : An histochemical, ultracytochemical and pharmacological studyThe pineal organ of the pike was studied in natural conditions in autumn and winter (short days). Using fluorescence histochemistry and drug treatments, the agranular pool of 5-HTP/5-HT (5-hydroxytryptophan and/or 5-hydroxytryptamine), the melatonin precursor, was found in different receptor line cells (Falcon, 1979b). This result confirmed the possible existence of photoneuroendocrine cells. The 5-HTP/5-HT pool was also present, but less abundant, in ependymal (supportive = interstitial) cells. 5-HTP/5-HT fluorophores varied quantitatively, depending on the pineal region and the light-dark cycle. The distal part was poor in indoles as compared to the middle and proximal regions. 5-HTP/5-HT appeared to be maximum during the light phase and minimum during the dark phase ; it was absent after p-CPA and reserpine treatments and very abundant only in the middle and proximal regions after nialamide injection.Rare nerve fibers with green fluorescence (indicating a catecholamine neurotransmitter) were found in the meningeal tissue outside the pineal organ ; they probably corresponded to those containing clear and dense-cored vesicles which were observed previously with electron microscopy (Falcon, 1979a).Monoamine oxidase (MAO) activity was studied using light and electron microscopy. The oxidative deamination sites in the pineal epithelium were revealed in receptor line cells and interstitial cells, i.e. in the outer membrane of mitochondria, the membranes of the rough and smooth endoplasmic reticulum, the membranes of the nuclear envelope and the plasma membrane. The reaction product, indicative of MAO activity, was completely absent in nialamide-treated fish. Endogenous 5-HT may be the main substrate for MAO in the pike pineal organ.Introduction.

Using two immunocytochemical procedures (i.e., immunofluorescence and the unlabeled peroxidase-antiperoxidase method), the localization of a serotonin(HT)-like and of a N-acetylserotonin (aHT)-like immunoreactivity in the pineal organ of the pike was studied during winter. It was shown that immunostaining was exclusively restricted to the cells of the receptor line (CRL = typical and modified photoreceptors). The intensity of the reactions varied through the light-dark cycle, HT-like immunoreactivity being high during the photophase and low during the scotophase. In contrast, aHT-like immunoreactivity was highest at the beginning of the scotophase. HT and aHT-like immunoreactivities were detected in all cell types of the pineal epithelium after administration of a monoamine oxidase inhibitor. Up to now, only HT immunoreactivity could be localized at the ultrastructural level. In a number of typical and modified photoreceptors, a HT-positive staining seemed to be confined within the hyaloplasm of the inner segment, particularly with that of the perikaryon and basal pedicle. Our previous and present results strongly suggest that indole compounds, which are involved in the regulation of various neuroendocrine processes in fish, are synthetized within the CRL. Taking into account that the CRL of the pike are also photosensitive, it appears more and more likely that they are photoneuroendocrine cells involved in mediating the effects of the photoperiod on various physiological and behavioral processes.