Synaptic ribbons during postnatal development of the pineal gland in the golden hamster (Mesocricetus auratus)

Summary Synaptic ribbons, functionally enigmatic structures of mammalian pinealocytes, were studied during the postnatal development of the pineal gland in the golden hamster (Mesocricetus auratus). On day 4 post partum, synaptic ribbons appear in cells that have already started to differentiate into pinealocytes. Between days 4 and 9, an increase in the number of synaptic ribbons occurs, concomitant with the continuing differentiation of the pineal tissue. Between days 9 and 16, when differentiation of this tissue is almost completed, the number of synaptic ribbons decreases and approaches that characteristic of the adult pineal gland. During development, the synaptic ribbons increase in length, and dense core vesicles are frequently found in the vicinity of these structures. It is assumed that a functional relationship exists between dense core vesicles and the synaptic ribbons, which are considered to be engaged in a certain form of secretory activity of the mammalian pineal gland.Supported by the Deutsche Forschungsgemeinschaft     

Synaptic ribbons in the pineal organ of the goldfish: Circadian rhythmicity and the effects of constant light and constant darkness

Summary Synaptic ribbons in photoreceptor cells of the goldfish pineal organ undergo significant daily changes in their length, distance from the plasma membrane, and number per unit area of pineal end-vesicle. The rhythms persist in fish exposed to constant darkness. Constant light abolishes the rhythms in length and distance of synaptic ribbons from the plasmalemma, but has little effect on numerical changes over a 24-h cycle. These findings suggest that synaptic ribbons in the pineal organ of lower vertebrates might be useful as indicators of metabolic activity.     

Prenatal development of "synaptic" ribbons in the guinea pig pineal gland

Pineal "synaptic" ribbons are a heterogeneous population of organelles. "Synaptic" ribbons (SR) sensu stricto, "synaptic" spherules (SS), and intermediate forms (IMF) are present. Their function and origin are unknown, and a knowledge of their prenatal development is lacking. Thus the pineal glands of prenatal, neonatal, and adult guinea pigs were prepared for electron microscopy. "Synaptic" ribbons were studied morphologically and quantitatively. The three categories of "synaptic" ribbons reported in adult pineal glands were also present in prenatal pineal glands. Their structural features, distribution, grouping, and composition patterns are similar to those in adults. "Synaptic" ribbons were first detected in pinealocytes of the distal region of a 42-day postcoitus (PC) pineal gland and were comparable with those in adults. They increased in number with age and reached a peak at 63 days PC, followed by a steep decline at 66 and 67 days PC. By day 69 PC, the numbers increased again and showed a dramatic increase after birth. Several true ribbon synapses were seen at day 63 PC between pinealocyte cell processes or between pinealocyte cell process and pinealocyte cell body. Since true ribbon synapses have not been found in adult guinea pig pinealocytes, their synaptic nature could have been lost during development. No precursors for the "synaptic" ribbons were found. The endoplasmic reticulum cisternae may be the origin for the ribbon vesicles because of their close association with the "synaptic" ribbons.     

Ultrastructure of the pineal gland of the eastern chipmunk (Tamias striatus)

The ultrastructure of the pineal gland of the wild-captured eastern chipmunk (Tamias striatus) was examined. A homogenous population of pinealocytes was the characteristic cellular element of the chipmunk pineal gland. Often, pinealocytes showed a folliclelike arrangement. Mitochondria, Golgi apparatus, granular endoplasmic reticulum, lysosomes, centrioles, dense-core vesicles, clear vesicles, glycogen particles, and microtubules were consistent components of the pinealocyte cytoplasm. The extraordinary ultrastructural feature of the chipmunk pinealocyte was the presence of extremely large numbers of “synaptic” ribbons. The number of “synaptic” ribbons in this species exceeded by a factor of five to 30 times that found in any species previously reported. In addition to pinealocytes, the pineal parenchyma contained glial cells (oligodendrocytes and fibrous astrocytes). Capillaries of the pineal gland of the chipmunk consisted of a fenestrated endothelium. Adrenergic nerve terminals were relatively sparse.     

Effects of adrenergic agonists and antagonists on the numbers of synaptic ribbons in the rat pineal gland

In the pineal gland numbers of synaptic ribbons (SR) undergo day/night changes which parallel the rhythm of melatonin synthesis. Since pineal biosynthetic activity is controlled by activation of adrenoreceptors, we investigated the effects of adrenergic agonists and antagonists on pineal synaptic ribbon numbers and N-acetyltransferase (NAT) activity, the key enzyme of melatonin synthesis in rats. In vivo application of the beta-adrenergic antagonist propranolol decreased melatonin synthesis when given during the dark phase but did not affect SR numbers. Treatment during daytime with the beta-adrenergic agonist isoproterenol increased pineal NAT activity whereas SR numbers did not change. Norepinephrine stimulated NAT activity in vitro in a dose-dependent manner, but did not elevate SR numbers. Incubation with an analog of the second messenger cyclic adenosine monophosphate increased both NAT activity and SR numbers. These results suggest that the beta-adrenergic system does not play a decisive role in the regulation of the nocturnal increase in SR numbers observed in the rat pineal gland.     

Ultrastructural and biochemical responses of photoreceptor pinealocytes to light and dark in vivo and in vitro

The teleost pineal organ contains functional photoreceptors that synapse with pinealofugal neurons. This study examined the effects of environmental lighting on protein content and levels of putative amino acid transmitters, as well as structural components associated with protein synthesis and neurotransmission. Goldfish subjected to continual illumination for 3 days tended to have increased pineal levels of free amino acids and protein compared to dark adapted glands. Similar responses to environmental lighting occurred in cultured glands suggesting a functional relationship to photosensory mechanisms. Morphometric ultrastructural analyses of pineal photoreceptors showed an increased size of nucleoli (especially the fibrillar component), Golgi bodies, and synaptic ribbons when glands were subjected to continuous light both in vivo and in vitro. The good agreement between protein levels and nucleolar morphology indicates a general effect of environmental lighting on photoreceptor protein metabolism, which may be related to photoreceptor outer segment renewal. Parallel changes in levels of certain amino acids (e.g., glutamate) and size of synaptic ribbons is consistent with an hypothesized role of amino acids in photoreceptor neurotransmission.     

Circadian variations in pinealocytes of the Chinese hamster,Cricetulus griseus

Summary Circadian morphological variations of pinealocytes in the superficial pineal of the Chinese hamster (Cricetulus griseus) were studied using quantitative electron-microscopic techniques. The volume of the nucleus and cytoplasm of pinealocytes exhibited similar circadian variations, with the maximum around the middle of the light period and the minimum during the first half of the dark period. Synaptic ribbons in pinealocytes were classified into three groups, type-1, –2 and –3 synaptic ribbons, which appeared as rods, round or irregular bodies and ring-shaped structures, respectively; a synaptic ribbon index was determined for the respective types. The synaptic ribbon index was expressed as the number of synaptic ribbons in the pinealocyte profile representing the cell size. The type-1 synaptic ribbon index, which was smallest during the second half of the light period, was increased during the dark period. The length of straight or slightly curved rods showed a 24-h change similar to that of the type-1 synaptic ribbon index; the length of the rods was maximal during the first half of the dark period and minimal at the end of the light period. There was no apparent circadian variation in the type-2 synaptic ribbon index. The type-3 synaptic ribbon index was higher during the light period than during the dark period; the index attained zero 3h after the onset of darkness and, thereafter, increased gradually.     

Postnatal development of "synaptic" ribbons and spherules in the guinea pig pineal gland

Previous studies have shown that the functionally enigmatic pineal "synaptic" ribbons are structurally a heterogeneous group of organelles consisting of rodlike ribbons sensu stricto, spherules, and intermediate forms. As ribbons and spherules react differently under various experimental conditions, these organelles were studied qualitatively and quantitatively during the postnatal period in guinea pigs. It was found that the pinealocytes were highly differentiated at birth and contained all three forms of "synaptic" structures. Ribbons and intermediate forms were more abundant than spherules and exhibited a striking increase in number on postnatal days 1 and 2; this increase was followed by a distinct trough and by a second peak at days 12 and 13, after which their numbers declined to reach adult levels by day 20. The spherules were small in number at birth and did not show the large immediate postnatal increase observed for the ribbons and intermediate forms. Instead there was a steady numerical increase up to day 12 (absolute number) or day 15 (relative numbers), followed by a decrease to adult level by day 20. Whereas during the early postnatal period (days 1 to 3) the majority of pinealocytes were characterized by ribbons and intermediate forms, with increasing age spherule-bearing pinealocytes increased in number. As ribbons and spherules were usually not found in the same pinealocyte, the present findings are interpreted to mean that ribbons and spherules characterize different types of pinealocytes showing an inverse numerical development postnatally. Developmentally intermediate forms behave like ribbons.     

Pineal synaptic ribbons in blinded rats

Summary To study the effects of bilateral ophthalmectomy on the circadian rhythm of the pineal gland, the number of the synaptic ribbons and ribbon fields in the pineal gland of female rats were determined by electron microscopy. In a preliminary experiment, the pineal gland was taken from rats at 15 days, and 1, 2, 3, 4, and 6 months after bilateral ophthalmectomy. It was found that the numbers of both synaptic ribbons and ribbon fields decreased markedly and reached a minimum at 1 month postoperatively, but recovered completely after 4 months. In the main experiment, the number of these intracellular elements was counted in both control and enucleated animals killed at 4 h intervals between 02:00 h and 22:00 h at either 1 month or 6 months after operation. All animals were kept under a lighting regimen of 12 h of illumination (06:00 to 18:00) and 12h of darkness (18:00 to 06:00). After 1 month, a remarkable decrease in the number of both the synaptic ribbons and ribbon fields was again noted but the circadian rhythm still remained. Complete quantitative and qualitative recovery in the circadian rhythm was obtained 6 months later.Supported by a grant-in-aid from the Japanese Ministry of Education, Science and Culture The authors wish to thank Mrs. Kazuko Moriwaki for her technical assistance     

Precise annual changes in the numbers of "synaptic" ribbons and spherules in the rat pineal gland

Although pineal "synaptic" ribbons (SR) are frequently examined by means of quantitative electron microscopy, their functional significance remains unclear. The same is true for related structures--"synaptic" spherules (SPH). In the course of such studies, it has been noted that SR counts may differ from laboratory to laboratory. Because seasonal changes may play a role, a 2-year study was performed on male rats kept under routine laboratory conditions and killed at monthly intervals during daytime or nighttime. Both structures examined showed distinct day-night differences throughout the year, with higher numbers at night than during the day. There were significant annual changes in both SR and SPH during both daytime and nighttime. The comparison of the curves from the 2 years showed that they were virtually identical both during daytime and nighttime. The numbers of SR were the highest in October and the lowest in April; the numbers of SPH had two plateaus, one with lower values from November to April, and the other with higher values from May to October. It appears from the present study that SR and SPH numbers in the rat pineal gland show statistically significant and precisely timed seasonal changes that may well account for the variations of SR numbers in the different publications.     

Ultrastructure and serotonin immunocytochemistry of the parietal-pineal complex in the Japanese grass lizard, Takydromus tachydromoides.

The fine structure and immunocytochemical localization of serotonin in the cells of the receptor line were studied in the parietal eye and pineal organ proper of the Japanese grass lizard, Takydromus tachydromoides. Typical photoreceptor cells (PC) were the predominant cell type in the receptor line of the parietal eye, the outer segments of which had regular stacks of numerous disks similar to those of cones. The pineal organ contained relatively few PCs, which showed less well-developed outer segments than those of the parietal eye. In contrast, secretory rudimentary photoreceptor cells (SRPC) accounted for the majority of receptor cells in the pineal organ. These cells were structurally characterized by whorl-like lamellar outer segments and numerous dense-cored vesicles (80-280 nm in diameter). A small number of SRPC were also found in the parietal retina, which were similar to those in the pineal organ. In the parietal-pineal complex, numerous mitochondria located in the PC were larger and rounder than those in the SRPC. In the PC, basal processes prossessed only synaptic ribbons, whereas in the SRPC some of these processes contained synaptic ribbons and others contained dense-cored vesicles, rarely having both. Serotonin-immunoreactive cells were found not only in the pineal organ but also in the parietal eye, which closely resembled the cells of the receptor line in their size and shape. Furthermore, on immunoelectron microscopy for serotonin using the protein A-gold technique, gold particles indicating serotonin-immunoreactive sites were restricted in the core of dense-cored vesicles in the SRPC of the pineal organ. Regional differences in the distributions of the PC, SRPC and serotonin-immunoreactivity were found in the parietal-pineal complex.     

Light and drug induced changes of epiphysial synaptic ribbons

Summary In the present investigation experiments were carried out to determine whether the functionally obscure synaptic ribbons of mammalian pinealocytes can be affected by acute changes in environmental lighting and which chemical processes may be involved in their regulation. Experiments carried out in male guinea-pigs have shown that the amounts of synaptic ribbons are immediately affected by changes in the lighting pattern. Extension of the light period reduced the normally occurring increase, whereas extension of the dark period inhibited the normally occurring decrease in the amount of synaptic ribbons. Results following injections of a number of drugs known to influence pineal function (noradrenaline, L-DOPA, propranolol, reserpine and p-chlorophenylalanine, respectively) suggest that synaptic ribbons may be directly or indirectly regulated by -adrenergic mechanisms.Dedicated to Professor Wolfgang Bargmann on the occasion of his 70th birthday.     

Pattern of synaptic connections in the pineal organ of the ayu,Plecoglossus altivelis (Teleostei)

Summary Synaptic connections were studied by means of electron microscopy in the sensory pineal organ of the ayu, Plecoglossus altivelis, a highly photosensitive teleost species. Three types of specific contacts were observed in the pineal end-vesicle: 1) symmetrically organized gap junctions between the basal processes of adjacent photoreceptor cells; 2) sensory synapses endowed with synaptic ribbons, formed by basal processes of photoreceptor cells and dendrites of pineal neurons; 3) conventional synapses between pineal neurons, containing both clear and dense-core vesicles at the presynaptic site. Based on these findings, the following interpretations are given: (i) The gap junctions may be involved in an enhancement of electric communication and signal encoding between pineal photoreceptor cells. (ii) The sensory synapses transmit photic signals from the photoreceptor cells to pineal nerve cells. (iii) The conventional synapses are assumed to be involved in a lateral interaction and/or summation of information in the sensory pineal organ. A concept of synaptic relationships among the sensory and neuronal elements in the pineal organ of the ayu is presented.Fellow of the Alexander von Humboldt Foundation, Federal Republic of Germany     

Influence of light/dark, seasonal and lunar cycles on serum melatonin levels and synaptic bodies number of the pineal gland of the rat

Synaptic bodies (SB) are ultrastructural organelles observed in the pinealocytes of mammals. According to its shape, they have been classified into synaptic ribbons (SR), synaptic spherules (SS), and intermediate synaptic bodies (ISB). They have been related to the melatonin regulation and production mechanisms of the pineal gland. Circadian and circannual fluctuations of both melatonin and SB have been reported. The possibility that other external factors, apart from light-dark or seasonal cycles, might influence pineal function has been suggested. We studied the evolution of the number of SB and serum melatonin levels not only during light-dark and seasonal phases but also during lunar cycles. Forty male wistar rats were used. Experiment was first carried out in winter and repeated identically in spring. Each season, one group of animals was killed during the new-moon days and a second group during the full-moon days: half of both groups in the photophase and the other half in the scotophase. The number of SB was measured at electron microscopic level whereas serum melatonin levels were determined by radioimmunoassay techniques. Main results showed that SR number and serum melatonin levels were higher during scotophases, winter and full-moon days. The SS only showed a light predominance during winter, whereas predominance of the ISB was found only during the scotophases. These results support the influence of the photophasic factors on the SR and ISB variations. In the case of the SS the influence of the lunar cycles is always dependent on the other factors. Finally, the serum level of melatonin is clearly influenced by the photophasic rhythms and the seasonal periods but not by the lunar cycles.     

Responses of pineal photoreceptors in the brook and rainbow trout

Summary Electron microscopy of the pineal receptor cells in light- and dark-adapted brook trout, Salvelinus fontinalis and the rainbow trout, Salmo gairdneri, revealed no significant differences in the tubular and filamentous elements of the inner segment, neck and supranuclear regions. However, changes in synaptic relations between the photoreceptor and nerve cell were induced by light and darkness. In the light-adapted state, the synaptic relationship between axon terminals and photoreceptor basal processes predominates, while in darkness the synapses between photoreceptor basal processes and ganglion cell dendrites are more prominent. Further, in darkness, the photoreceptor basal processes show a number of synaptic vesicles and synaptic ribbons. These findings suggest that the sensory function of the fish pineal is enhanced during darkness but inhibited by light, and that the synaptic relationships are involved in the control of sensory activity in the pineal photoreceptor and ganglion cells. These results corroborate those of electrophysiological studies in that the maximal spontaneous discharge frequency of the ganglion cells occurs in the dark, and it also shows a burst when light is removed. The typical chemical synapse between the axon terminal and the photoreceptor basal process in light seems to function as an inhibitor.The authors thank Dr. Mary Ann Klyne for her assistance in several aspects of this work. Financial assistance was provided by the NSERC of Canada and the Ministry of Education of Québec     

The pineal gland of the gerbil,Meriones unguiculatus

Morphometric analytical procedures were employed to study the pineal gland of the Mongolian gerbil following superior cervical ganglionectomy (SCGX). The purpose of this study was to define the effects of sympathetic denervation on the morphology of the gland at two time periods, 0500 and 1900 h (one hour before lights-on and lights-off, respectively). Fluorescence histochemistry was employed to determine catecholamine and indoleamine content in intact and denervated pineal glands. After SCGX, the pinealocytes decrease in size, concretions are prevented from forming, and the yellow fluorescence in the gland is lost. Following denervation a depression in the volume of most of the pinealocyte organelles, i.e., SER, RER/ribosomes, free cytoplasm, mitochondria and presumptive secretory vesicles, was also observed. However, synaptic ribbons increased in volume in the gerbils that had been killed at 1900 h. It appears that the sympathetic innervation to the pineal gland is a requirement for the presumptive secretory activity of the pinealocytes.     

Ultrastructural study of the cellular types in the pineal organ of Gambusia affinis (teleost)

The pineal organ of Gambusia affinis was studied via light and electron microscopy. The cell types studied included photoreceptor cells, supporting cells, and a third cell type. The photoreceptor cells, which appear to form clusters, are divided into four regions: outer segment, inner segment, cell soma, and synaptic pedicle. Synaptic ribbons are commonly observed in the synaptic pedicle. The supporting cells separate the photoreceptor cells from the thick basal lamina that surrounds the entire pineal organ. The supporting cells show highly organized membrane formations, some lipid-like inclusions, and a diplosome. One of the centrioles gives rise to an invaginated cilium. The third cell type is observed infrequently and appears to be located mainly in the vicinity of the outer segments. The morphological characteristics of this cell type are similar to those of phagocytic cells. The ultrastructural features of the pineal organ of G. affinis are compared with those of other teleosts.     

The Pineal Gland of the Mink, Mustela vison: Light-, Fluorescence- and Electron Microscopical Studies

The pineal gland of normal and experimental female mink has been studied by light-, fluorescence- and electron microscopy. The general structure of the mink pineal is described. Two main cell types are recognized. One, termed pinealocyte, predominates in number. Though slight morphological differences (e.g. electron density of the cytoplasm and content of organelles) were observed, this study indicates that the pineal of mink only contains one single population of pinealocytes. The other, termed glial cell, inserted between the pinealocytes, is characterized by the presence of elongated processes, containing microfilaments. Different treatments (ovariectomy and LH—RH administration) and different endocrine states during the year induced morphological changes in the pinealocytes. A rich network of nerve fibres containing electron-dense granules (40–50 nm) is observed. Microspectrofluorometrically these fibres exhibit the spectral characteristics of cateholamines. All the pinealocytes show a yellow fluorescence. This cellular fluorophor shows the same microspectrofluorometric characteristics as does the fluorophor of serotonin. Occasionally, synaptic ribbons are observed in the perikaryon and the processes of the pinealocytes. A large number of cellular junctions between pinealocytes and endothelial cells is present. Their presumed function(s) are discussed. There is evidence of a blood-brain barrier within the mink pineal gland.     

A morphological study of the circadian cycle of the pineal gland of the rat

Summary To seek a morphological expression of circadian rhythm, we investigated cytologically pineal glands taken from rats every 2 to 4 h under a lighting regime of 12 h of illumination (6:00 to 18:00) and 12 h of darkness. The changes in the number of synaptic ribbons and ribbon fields was observed by electron microscopy. The number of these intracellular elements was greatest at 2:00 and the lowest at 14:00, the difference being statistically significant. There were no significant local differences in numbers with respect to the part of the pineal gland examined. The data are similar to those of Vollrath from the pineal gland of a guinea pig, and seem to confirm a circadian function in the pineal gland in mammals.Supported by grants-in-aid from the Ministry of Education, Science and Culture, and the Ministry of Health and Welfare