Cells expressing preproenkephalin mRNA in the rat pineal gland are not serotonin-producing pinealocytes: Evidence usingin Situ hybridization combined with immunocytochemistry for serotonin

Summary 1. Preproenkephalin (PPEnk) mRNA expressing cells have been identified in rat pineal gland using radioactivein situ hybridization histochemistry. 2. Approximately 7% of the cells in the pineal gland (7.5±0.86, mean ± 95% CI) express PPEnk mRNA. These cells are distributed throughout the pineal as either scattered single cells or small groups of cells with large round or oval nuclei. 3. Usingin situ hybridization combined with ABC immunocytochemistry for serotonin (5-HT) in the same pineal sections, the PPEnk mRNA labeling cells are found not to be serotonin-immunoreactive cells. These data indicate that the PPEnk mRNA is expressed in a certain discrete subpopulation of cells in the rat pineal gland and these cells are not serotonin-producing pinealocytes. 4. The physiologic role of PPEnk-derived peptides in the pineal remains unknown. It is possible that these peptides either are synthesized and secreted as hormones or act as pineal paracrine signals.     

Building an asymmetric brain: Development of the zebrafish epithalamus

The human brain exhibits notable asymmetries. Little is known about these symmetry deviations; however scientists are beginning to understand them by employing the lateralized zebrafish epithalamus as a model. The zebrafish epithalamus consists of the pineal and parapineal organs and paired habenular nuclei located bilateral to the pineal complex. While zebrafish pineal and parapineal organs arise from a common population of cells, parapineal cells undergo a separate program that allows them to migrate left of the pineal anlage. Studying the processes that lead to brain laterality in zebrafish will allow a better understanding of how human brain laterality is established.     

Intranuclear filament bundles in the intestinal epithelium of the oligochete Dero obtusa

Summary Bundles of filaments present inside the nuclei of columnar intestinal epithelial cells of Dero obtusa are described. The filaments appear beaded, each bead consisting of still smaller subunits. The marked similarity of these filament bundles to those previously described within the nuclei of rat pineal gland cells is discussed.     

Regional distribution and cellular expression of tryptophan hydroxylase messenger RNA in postmortem human brainstem and pineal gland

The characterization and cellular localization of tryptophan hydroxylase mRNA in the human brainstem and pineal gland were investigated by using northern blot analysis and in situ hybridization histochemistry. Northern analysis of human pineal gland revealed the presence of two mRNA species that were absent in RNA isolated from human raphe. In situ hybridization experiments revealed very dense hybridization signal corresponding to tryptophan hydroxylase mRNA in cells throughout the pineal gland. In contrast, tryptophan hydroxylase mRNA was heterogeneously distributed in neurons in the dorsal and median raphe nuclei. Within the dorsal raphe, the ventrolateral and interfascicular subnuclei contained the greatest number of tryptophan hydroxylase mRNA-positive neurons. Also, the cellular concentration of tryptophan hydroxylase mRNA varied widely within the dorsal and median raphe. Comparison of the cellular concentration of tryptophan hydroxylase mRNA between the pineal gland and the raphe nuclei revealed an 11- and 46-fold greater average grain density of tryptophan hydroxylase mRNA positive cells in the pineal gland compared with the dorsal and median raphe, respectively. These findings are the first to demonstrate the cellular localization of tryptophan hydroxylase mRNA in the human brain and pineal gland as well as heterogeneity in the cellular concentration within and between these tissues.     

Histomorphogenesis and immunohistochemical study of the bovine pineal gland (Bos taurus) during prenatal development (160 days of gestation to birth)

The ontogenesis of the pineal gland of 20 bovine embryos (Bos taurus) has been analysed from 160 days of gestation to birth by means of optical microscopy and immunohistochemical techniques. For this study, the specimens were grouped into two stage in accordance with the most relevant histological characteristics: Stage 1 (160 to 200 days of prenatal development) and Stage 2 (220 days of prenatal development to birth). At 160 days of gestation some rounded structures with a central lumen, which we refer to as glandular rosettes, begin differentiation from the epithelium of the pineal recess, experiencing an extraordinary increase in number and size at 200 days of intrauterine life. In the interior of the pineal parenchyma we observed some morphologically rounded cells with oval euchromatic nuclei and a well-differentiated nucleolus that we refer to as the pinealoblasts. We also observed other cells characterised by the presence of low cytoplasm and rounded and highly stained nuclei that we refer to as the interstitial cells. The glandular stroma is formed from the capsular, trabecular, and perivascular connective tissue as well as from the reticular network that comes from the cellular processes of the interstitial cells. The blood vessels, at 240 of gestation, show well-formed walls where the endothelial cells stand out. At 160 days of gestation we witnessed some cells with small, dense, oval nuclei, positive to the glial fibrillary acidic protein (GFAP). At this age NPY positive fibres were detected, distributed around the blood vessels and among the pinealoblasts. We conclude by clarifying that the changes detected in the morphology as well as in the number and size of glandular rosettes appear to be related to the functional activity of the pineal gland during embryonic development.     

Occurrence and regional distribution of striated muscle fibers in the rat pineal gland

Summary In a total of 96 rat pineals studied 31 were found to contain striated muscle fibers or their precursors. The muscle fibers were most frequently present in the stalk region and more frequently found in the left than in the right hemisphere. Size measurements revealed that the lengths of pineal muscle cell nuclei differ only slightly from those of the sphincter muscle of the iris. However, the yellowish appearance of pineal muscle cell nuclei under darkfield investigation, a phenomenon observed in all muscular tissues of mesenchymal origin and connective tissue cells, may support the hypothesis that pineal musculature is of mesenchymal rather than ectodermal origin.Supported by a grant (Vo 135/4) of the Deutsche Forschungsgemeinschaft within the Schwerpunktprogramm Neuroendokrinologie     

Complex relationships between the pineal organ and the medial habenular nucleus-pretectal region of the mouse as revealed by S-antigen immunocytochemistry

Summary S-antigen-immunoreactive pinealocytes located in the deep portion of the pineal organ of inbred and wild pigmented mice give rise to long, beaded processes penetrating into the habenular and pretectal regions. In addition, the medial habenular nuclei and the pretectal area contain S-antigen-immunoreactive perikarya, which resemble pinealocytes in size, shape and immunoreactivity and are considered as pinealocyte-like epithalamic cells. Immunoblotting techniques reveal that a single protein band of approximately 48 kDa molecular weight accounts for this immunoreactivity. As shown with the use of the electron microscope, the majority of the S-antigen-immunoreactive processes is closely apposed to immunonegative neuronal profiles and perikarya of the habenular and pretectal regions. S-antigen-immunoreactive processes and perikarya of both pinealocytes of the deep pineal organ and pinealocyte-like epithalamic cells may form the postsynaptic element in conventional synapses involving axons provided with clear synaptic vesicles. Thus, certain mammalian pinealocytes may receive and transmit signals via point-to-point connections resembling neuro-neuronal contacts. These results challenge the concept that the mammalian pineal organ exerts its influence exclusively via the release of melatonin into the general circulation. Furthermore, they provide evidence (i) that neuronal circuits not involving the sympathetic system participate in the regulation of pineal functions in mammals, and (ii) that intimate histogenetic and functional relationships exist between the pineal organ and the habenular-pretectal nuclei in mammals.     

Radioautographic study of the rat brain and pituitary after injection of 3H dexamethasone

The central nervous system and the pituitary of adrenalectomized male rats injected with 3H-dexamethasone were examined by radioautography. At 1 hr after the injection, radioactivity concentration was high in the medial basal hypothalamus, the pituitary and the pineal gland. In the hypothalamus, radioactive material was found to be selectively concentrated in neurons in the ventral part of nucleus arcuatus and in the infundibular region. In the anterior pituitary, a large proportion of cells showed silver grains both in the cytoplasm and over the cell nuclei. However, in a small number of cells, the radioactive material was associated with the cell nuclei. Less radioactivity was present in the intermediate and posterior lobes. The pineal gland contained more silver grains than did other regions of the brain. The results obtained in the present study suggest essentially an action of dexamethasone in the medial basal hypothalamus and at the level of the pituitary.     

Effect of photoperiod on pineal gland volume and pinealocyte size in the Chinese hamster, Cricetulus griseus

Male adult (200-day-old) Chinese hamsters (Cricetulus griseus) raised from weaning under either LD 16:8 or LD 8:16 were used. The pineal gland of the Chinese hamster consists of superficial (major) and deep (minor) components and a continuous, or interrupted, narrow parenchymal stalk interposed between them. The volume of the superficial pineal including the parenchymal stalk is greater under LD 16:8 than under LD 8:16. Under both photoperiods, pinealocytes in the superficial pineal have larger nuclei and more abundant cytoplasm than those in the deep pineal. Nuclei in the superficial pineal appear pale and usually have irregular profiles, whereas those in the deep pineal appear dark and have round profiles. In the superficial pineal, pinealocyte nuclei are larger, paler, and more irregular; and, in addition, nuclear density is lower under LD 16:8 than under LD 8:16. Similar, but less prominent, photoperiod-induced changes occur in the volume of the deep pineal, the size of pinealocytes, and pinealocyte nuclear morphology in the deep pineal. The results indicate that the development and differentiation of pinealocytes in both pineal portions may be advanced under long photoperiods and delayed under short photoperiods, although pinealocytes in the deep pineal may remain not fully differentiated even in adults. Since testicular weights and body weights are similar under both photoperiods, the photoperiod may exert marked influences on the development of the pineal gland without affecting reproductive activity and growth rates of animals.     

Morphologic changes in the pineal gland of rats exposed to continuous darkness

We examined the pineal structure of rats exposed to constant darkness (DD) at light microscopic level. Two groups of rats were exposed to 12:12 light/dark cycle (LD) or DD from their prenatal ontogenesis and then for 3 months after birth. The gland structure of DD rats was observed to have an active appearance. Some of the observed pinealocytes with light nuclei from DD rats were determined to contain double nucleoli. Nuclear area and perimeter of both dark and light types were greater in rats kept in DD than in LD. Rats exposed to DD had more cells with light nuclei and lesser cells with dark ones than rats kept in LD. No significant differences in nuclear characteristics of intermediate type were found between rats kept in LD and those kept in DD. The activity of mammalian pineal can be altered by light conditions to which the animal is exposed.     

Immunocytochemical localization of vitamin A in the retina and pineal organ of the frog, Rana esculenta

Vitamin A immunoreactive sites were studied in the retina and pineal organ of the frog, Rana esculenta, by the peroxidase antiperoxidase, avidin-biotinperoxidase and immunogold methods. In dark-adapted material, strong immunoreaction was found in the outer and inner segments of the photoreceptor cells of both retina and pineal organ, as well as in the pigment epithelium, retinal Müller cells and pineal ependymal cells. In light-adapted retina, cones and green (blue-sensitive) rods were immunopositive. At the electron microscopic level, immunogold particles were found on the membranes of the photoreceptor outer segments as well as on the membranes of the endoplasmic reticulum and mitochondria. Individual retinal photorecptor cells exhibited strong immunoreaction in the distal portion of the inner segment, the ciliary connecting piece and the electron-dense material covering the outer segment. In the pigment epithelium, the immunolabeling varied in intensity in the basal and apical cytoplasm and phagocytosed outer segments. The immunocytochemical results indicate that retinoids (retinal, retinol and possibly retinoic acid) are present not only in the photoreceptor cells of the retina but also in those of the pineal organ. The light-dependent differences in the immunoreactivity of vitamin A underlines its essential role in the visual cycle of the photopigments. Our results suggest that the pineal ependyma plays a role comparable to that of the Müller cells and pigment epithelium of the retina with regard to the transport and storage of vitamin A. The presence of a retinoid in nuclei, mitochondria and cytoplasmic membranes suggests an additional role of vitamin A in other metabolic processes.     

Physiology of avian circadian pacemakers.

The pineal gland plays a cental role in the circadian organization of birds, although it is clearly only one component in a system with other components that have not yet been positively identified. The relative importance of the pineal and other components may vary from one group of birds to another. In the most thoroughly studied species, the house sparrow, pineal removal abolishes circadian rhythmicity; rhythmicity is restored by transplantation of a donor bird's pineal and the restored rhythm has the phase of the donor. This, and other evidence, argues convincingly that the pineal is a pacemaker in the sparrow circadian system. The pineal of the chicken has circadian rhythms in several biochemical parameters that result in the rhythmic synthesis of melatonin. The activity of one enzyme in this pathway is rhythmic for at least two cycles in organ culture. In view of this result it is interesting that pineal removal does not abolish circadian rhythmicity in chickens. The fact that lesions of the suprachiasmatic nuclei abolish circadian rhythms in sparrows, several mammalian species, and perhaps Japanese quail and reptiles, suggests that vertebrate circadian organization may be based on differentially weighted interactions between the pineal, the suprachiasmatic nuclei, and perhaps other brain regions.     

SUBCELLULAR LOCALIZATION OF TRYPTOPHAN-5-MONO-OXYGENASE IN BOVINE PINEAL GLANDS AND RAPHE NUCLEI

Abstract— Tryptophan-5-mono-oxygenase from both bovine raphe nuclei and pineal glands was activated by preincubation with dithiothreitol and ferrous ion at pH 8.5. The optimum pH for the enzyme activity lies between pH 6.4 and 7.3. Preincubation increased the activity of the enzyme from raphe nuclei by about 20 times in both the homogenate and 105,000 g precipitate prepared from it. Activity in the 105,000 g supernatant fraction was about trebled. Corresponding increases in pineal gland enzyme activity were noted: 100 times in homogenate and 105,000 g precipitate and 15 times in 105,000 g supernatant fluid. Total recoveries of activated enzyme from the homogenate prepared in hypo-osmotic medium, in the 105,000 g supernatant and precipitate, were 87.1% and 79.0% for raphe nuclei and pineal glands respectively. Of this, 89.5-91.3% in the case of the raphe nuclei and 76.0-82.0% in the case of the pineal glands, was found in the precipitate. In contrast, 85-90% of the lactate dehydrogenase activity was found in the supernatant fraction. The results of subcellular fractionation revealed that the raphe nuclear enzyme was located in both 'mitochondrial' and 'microsomal' fraction while the pineal gland enzyme was effectively restricted to the 'mitochondrial' fraction. The structural characteristics of the fraction were confirmed by electron microscopy.     

Comparative ultrastructural investigations of the pineal organ of the blind cave fish,Anoptichthys jordani,and its ancestor,the eyed river fish,Astyanax mexicanus

A comparative ultrastructural study has been made of the pineal organ in specimens of two closely related populations of the characid fish, Astyanaz mexicanus. The specimens of one population are living in the river, under natural light conditions. The specimens of the other population, originally described as Anoptichthys jordani, are living in a completely dark cave. In specimens of both populations the pineal organ consists of a spindle shaped end-vesicle, connected to the diencephalic roof by a slender stalk. The pineal tissue is compact and consists predominantly of glia-like supporting cells and sensory cells resembling the photoreceptor cells of the lateral vertebrate eye. Phagocytotic microglia-like cells can be found in close contact with the outer segments of the sensory cells. Nerve cells are located in the neighbourhood of neuropil formations, in which synaptic contacts are established between sensory cells and nerve cells. From these nerve cells fibers are emerging, forming the pineal tract that runs down the pineal stalk towards the diencephalon. On the basis of the ultrastructure described by other authors it is concluded that the pineal organ in specimens of the river population of Astyanax mexicanus resembles the pineal organ of other fish species. In specimens of the river population, reared under normal light-dark conditions for 3, 9 or 18 months, conspicuous morphological changes have not been detected in the presumably light-sensitive outer segments of the sensory cells or in other parts of the pineal tissue. In specimens of the cave populations, reared under identical conditions, an age-dependent, gradual regression of the regular outer segment organization of the pineal sensory cells takes place. In other parts of the pineal tissue, only small morphological changes can be observed. In specimens of the cave population, reared in constant darkness, the regression of the pineal outer segment organization begins earlier and is obvious. It is postulated that the gradual age-dependent regression of the regular organization of the outer segments in the pineal organ of cave specimens of Astyanax mexicanus is genetically determined and indicates a regressive evolution of the pineal light sensitivity. The expression of the regressive traits is dependent on the environmental light conditions.     

Ultrastructure of the pineal body of the common vampire bat, Desmodus rotundus

The type AB pineal body of the common vampire bat, Desmodus rotundus, was recessed and lobulated, was extensively vascularized and intimately related to great veins, and was unassociated with the epithalamic region. The habenular and the posterior commissures coursed anteriorly and were unassociated with the pineal. The saccular suprapineal recess of the third ventricle extended dorsally juxtaposed to the pineal body. These anatomical features are likely to make pinealectomies in the vampire more difficult to manage. The pineal parenchyma consisted of light pinealocytes surrounded by canaliculi of various sizes, often transmitting unmyelinated nerve fibers and glial processes. Desmosomes were common. The pinealocyte nuclei were large and highly infolded; characteristic cytoplasmic constituents included abundant dilated Golgi complexes associated with clear vesicles, numerous polyribosomes, few single cisternae of ribosome-studded rough endoplasmic reticulum, mitochondria, and occasional multivesicular bodies and lysosomes. Almost all pinealocytes exhibited centrioles and some, in addition, displayed basal bodies but rarely ciliary shafts. A conspicuous feature of the pinealocyte cytoplasm was the presence of branched bundles of intermediate filaments, especially in the perinuclear zone. Siderotic macrophages, lipofuscin-pigment-containing phagocytic cells, mast cells, myelin bodies, and both fenestrated and continuous capillaries were present. The perivascular compartment was densely packed with unmyelinated nerve bundles containing small to large fibers exhibiting axoaxonic densities. Other constituents of the perivascular compartment were club-shaped pinealocyte processes filled with clear vesicles, microtubules, an occasional mitochondrion, glial processes, and collagen fibers. "Synapselike" contacts were observed between the axons and pinealocyte processes. Abundant pinocytotic vesicles in the capillary endothelium indicated active pinocytosis. Myelinated nerve fibers were lacking. The pineal ultrastructure of Desmodus is in part unlike that reported for other mammals, including bats.     

Electrical and pharmacological properties of the suprachiasmatic nuclei

The suprachiasmatic nuclei (SCN) of the mammalian hypothalamus are in important circadian pacemaker. The electrical activity of these nuclei exhibits an intrinsic circadian rhythm. The rhythmicity of the SCN is also reflected in cyclic glucose consumption and serotonin metabolism. These rhythms are entrained to the light-dark cycle via the retinohypothalamic projection. This pathway, possibly together with a visual projection via the ventral lateral geniculate nuclei, innervates light-responsive SCN cells, which exhibit the functional properties of luminance detectors. The SCN contain various peptides, acetylcholine, and serotonin either intrinsically or in terminals of afferent projections. For acetylcholine it has been demonstrated that the SCN mediate the process of photic entrainment and light suppression of pineal synthetic activity. In the case of serotonin and vasopressin it seems certain that the SCN do not depend on their presence for generating circadian rhythms or for entrainment. Both substances may modulate the intrinsic pacemaker frequency through mechanisms that remain to be established.     

Presence and distribution of LHRH- and alpha-MSH-like immunoreactive nerve fibers in the epithalamus of the sheep

The presence of luteinizing-hormone-releasing hormone (LHRH)-like and alpha-melanocyte-stimulating hormone (alpha-MSH)-like immunoreactive fibers, nerve terminals or cellular elements in the pineal gland of the sheep has been investigated by immunohistochemistry. No LHRH-or alpha-MSH-like immunoreactive fiber, nerve terminals or cellular elements have been demonstrated in the pineal organ of the sheep. However, LHRH- and alpha-MSH-like immunoreactive fibers are present in the posterior commissure. Immunoreactive LHRH and alpha-MSH nerve endings are evident in the medial and lateral habenular nuclei. Discrepancies with the results obtained in other mammals are indicative of species differences in the distribution of LHRH- and alpha-MSH-like immunoreactive material in the pineal region.