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The study was conducted to investigate diurnal changes in pinealocyte ultrastructure, pineal serotonin content and plasma melatonin concentration in the domestic pig. The immature pigs (n=24) were kept under a cycle of 12 h light : 12 h dark, with a photophase between 0800 and 2000. During the photophase the animals were exposed to direct sunlight. After four weeks the gilts were slaughtered at 0900, 1400, 2100 and 0200. The pineals were removed and divided into two parts - one for quantitative ultrastructural study (by a point count method) and one for serotonin assay. Simultaneously, blood samples were taken for melatonin assay. The relative volume of mitochondria in pinealocyte perikarya was significantly higher at 1400 than at 0200 and 0900 as well as at 2100 than at 0200. The relative volume of Golgi apparatus was higher at 0900 and 1400 than at 0200. The relative volume of dense bodies of the MBB-1 type in pinealocyte perikarya was significantly lower at 1400 and 2100 than at 0900. In contrast, the relative volume of MBB-2 was higher at 1400 than at 0900 and 0200. The numerical density of DCV in perikarya was significantly higher at 0200 than at 1400. No significant differences were found in rough endoplasmic reticulum, lysosomes and multivesicular bodies. The pineal serotonin content showed a prominent rhythm with the maximum at 1400. The plasma melatonin concentration was significantly higher at 0200 than at 0900, 1400 and 2100. The obtained results demonstrate that both pinealocyte ultrastructure and pineal biochemistry in the pig undergo significant changes in the course of the diurnal rhythm.

The pineal functions are modulated by some neuropeptides including PHI and VIP. The presence of PHI-immunoreactive and VIP-immunoreactive nerve fibers in the pineal gland has been shown in several mammalian species. Both peptides influence the pineal serotonin N-acetyltransferase activity and melatonin synthesis. The aim of the present study was to examine the localization of PHI- and VIP-immunoreactive nerve fibers in the pig pineal gland. Four three-month old female pigs housed in natural light conditions, with free access to food and water, were used in the study. The pineals were fixed by perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer. An immunohistochemical ABC streptavidin-biotin-complex method was used for the demonstration of PHI and VIP. PHI- and VIP-immunopositive nerve fibers were found in the pineal gland as well as in the habenular and posterior commissural areas. In the pineal gland, the density of PHI-immunoreactive nerve fibers was considerably higher than that of the fibers containing VIP. PHI- and VIP-immunopositive nerve fibers were more abundant in the cortical than in the medullary part of the gland. The nerve fibers formed bundles in the pineal capsule, from where they penetrated to the connective tissue septa and formed a dense meshwork surrounding blood vessels. In the parenchyma, PHI- and VIP-immunoreactive nerve terminals created baskets around clusters of pinealocytes. No PHI- or VIP-immunopositive cells were found in the pig pineal gland.

The sympathetic nerve fibers originating from the superior cervical ganglia and supplying the pineal gland play the most important role in the control of the pineal activity in mammals. NPY and CPON are also present in the majority of the pinealopetal sympathetic neurons. In this study, immunohistochemical techniques were used to demonstrate the existence and coexistence of tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH) as well as NPY and CPON in the nerve fibers supplying the chinchilla pineal gland. Ten two-year-old female chinchillas housed in natural light conditions were used in the study. The pineals were fixed by perfusion. ABC immunohistochemical technique and immunofluorescence labelling method were employed. TH-immunoreactive (TH-IR) varicose nerve fibers were observed in the pineal gland as well as in the posterior commissural area. Within the chinchilla pineal gland, TH-IR nerve fibers were located in the capsule and connective tissue septa. Numerous varicose TH-IR branches penetrated into the parenchyma and formed a network showing the highest density in the proximal region of the gland. In the central and distal parts of the pineal parenchyma, a subtle network, composed of thin varicose nerve branches, was observed. Double immunostaining revealed that the majority of TH-IR nerve fibers was positive for DbetaH or NPY. TH- and DbetaH-positive neuron-like cells were observed in the proximal region of the gland. The pattern of pineal innervation immunoreactive to CPON was similar to the innervation containing NPY, TH and DbetaH. The chinchilla intrapineal innervation containing TH, DbetaH, NPY and CPON is characterized by the higher density in the proximal part of the gland than in the middle and distal ones. The specific feature of the chinchilla pineal is also the presence of single TH/DbetaH-immunoreactive neuron-like cells in the proximal part of the gland.

Vasoactive intestinal peptide (VIP) is one of neuropeptides involved in the regulation of the pineal gland function. The acute treatment of rat pinealocytes with VIP caused changes in their biochemical parameters. The present study concerns the effects of the chronic treatment with VIP on ultrastructure and function of the rat pinealocytes in organ culture. The pineals of adult male rats were assigned to one of three groups and placed in organ culture for four consecutive days. The pineals of the first group were incubated in the control medium, the pineals of the second group--12 hrs in control medium and 12 hrs in medium with 1 microM VIP (between 20.00 and 8.00) during each day, the pineals of the third group--24 hrs per day in medium with 1 microM VIP. The melatonin concentration was measured using RIA and activity of enzymes using radiochemical methods. Point count method was used in quantitative ultrastructural analysis. Both modes of chronic treatment with VIP increased significantly the level of melatonin secretion during four days of the culture and the content of this hormone in the pineal explants at the end of the experiment. Treatment with the neuropeptide for 12 hrs and 24 hrs per day elevated also the activity of arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase. On the other hand, VIP had no effect on the activity of arylamine-N-acetyltransferase. VIP increased the relative volume of rough endoplasmic reticulum, Golgi apparatus and mitochondria and did not influence the relative volume of lysosomes and lipid droplets as well as the numerical density of dense core vesicles in the examined rat pinealocytes. The obtained results indicate stimulatory effect of chronic treatment with VIP on the synthesis and secretion of melatonin in the rat pinealocytes in vitro. The results of morphological study are in agreement with the obtained biochemical data and point to the increase in secretory and metabolic activity of the rat pinealocytes in response to VIP.

An immunohistochemical study of the pineal gland of the domestic pig was carried out using the antisera raised against vasopressin (VP). The pineal glands were taken from the newborn, 21-day- and 7-month old female pigs. The pig pineal gland is moderately innervated by VP-immunoreactive nerve fibers. They run from the habenular commissure into the connective tissue septa and further into the pineal parenchyma. In the subependymal tissue as well as in the connective tissue septa, the fibers are smooth or with small varicosities and in the parenchyma with large ones. The obtained results point to extrapineal and extraepithalamic source of the fibers. The density of VP-immunoreactive fibers in the pineal gland of 7-month old pigs is higher than in the younger animals.