Ultrastructural observations on synaptic ribbons in the pineal organ of the goldfish

Summary Synaptic ribbons in the pineal organ of the goldfish were examined electron microscopically with particular attention to their topography. These structures were formed of parallel membranes, which were poorly preserved with OsO₄ fixation and could be extracted from thin sections with pronase indicating their proteinaceous nature. Synaptic ribbons were closely apposed to the plasma membrane bordering dendrites of ganglion cells, but were also related to processes of both photoreceptor and supportive cells. Their close proximity to invaginations of the plasma membrane and portions of the endoplasmic reticulum suggest that they are involved in the turnover of cytoplasmic membranes. Tubular and spherical organelles of unknown function are also described.     

Ultracytochemistry of the synaptic ribbons in the rat pineal organ

Summary The synaptic complexes of the rat pinealocytes are neither cholinergic nor adrenergic. In the synaptic vesicles, a neurotransmitter carrier substance of lipid nature reacting with OsO₄-Zn I₂ mixture (similar to that present in both cholinergic and adrenergic vesicles) was not found.In addition, there were no indications of glucose-6-phosphatase or thiamine-pyrophosphatase activity in the synaptic vesicles. Thus, it appears that the synaptic vesicles do not originate from the rough or smooth endoplasmic reticulum.The synaptic ribbons do not contain carbohydrates, are of protein nature and possess some chemical resemblance to microtubules and microtubular bouquets.Appropriate ultracytochemical reactions have not shown detectable quantities of sodium and calcium ions in pinealocyte synaptic complexes.Grateful acknowledgment is made to Mr. P.-A. Milliquet for technical assistance and to Dr. T. Jalanti (C.M.E., Lausanne) for his help in the use of the X-ray microanalyser.Dedicated to Professor Dr. med. G. Töndury on the occasion of his 70th birthday.     

Circadian changes in synaptic ribbons and spherules in pinealocytes of the Syrian hamster (Mesocricetus auratus)

Summary In the present study, synaptic ribbons were studied morphologically and quantitatively in hamster pineal gland. The number of ribbons and spherules of hamster pinealocytes was counted over a 24-h period. The 24-h variations in the quantity of synaptic ribbons were found to parallel fluctuations in pineal melatonin concentrations. No significant circadian changes were observed for synaptic spherules, indicating different roles for these two structures.     

Synaptic ribbons of a mammalian pineal gland circadian changes

Summary Synaptic ribbons (SR) of the mammalian pineal gland are functionally enigmatic. In the present study it is shown that in male guinea-pigs kept under a lighting regimen of 12 hrs illumination (7.00–19.00) and 12 hrs darkness (19.00–7.00) the SR of pinealocytes vary about 25-fold in number over a period of 24 hrs. An increase is found between 15.00 and 6.00 and a decrease between 6.00 and 15.00. Analysis of the intracellular localization and the topographical relationships indicate that SR lie near the cell membrane of pinealocytes throughout the 24 hr period and that they are related to adjacent pinealocytes. A working hypothesis put forward implies that SR represent cell organelles involved in intercellular communication and that it is their function to either enhance the secretory activity of the pineal gland or to establish circuits within the gland between adjacent pinealocytes, similar to neuronal circuits.The skilled technical assistance of Mrs. C. Howe is gratefully acknowledged.     

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     

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     

Acetylcholinesterase-containing nerve cells and their distribution in the pineal organ of the goldfish,Carassius auratus

Summary Histochemically, an intense acetylcholinesterase (AChE) reaction has been observed in the perikarya of the nerve cells and in the neuropil formations of the pineal organ in the goldfish, Carassius auratus. A group of AChE-rich nerve cells has also been observed between the caudal end of the pineal stalk and the habenular ganglion. No component of the complex revealed butyrylcholinesterase (BuChE) activity.Two different types of nerve cells were recognized on the basis of their size, AChE activity and distribution. Type I cells are characterized by large perikarya possessing a moderate AChE activity and by the presence of an extensive AChE-rich neuropil formation in their vicinity; they are restricted to the rostro-lateral regions of the pineal vesicle. Type II cells are situated in the medio-rostral area of the pineal vesicle and along the entire length of the stalk, and are smaller than Type I cells; they show an intense AChE activity in their perikarya.The neuropil formations in the medio-rostral area of the pineal vesicle are almost as large as those in the vicinity of the Type I cells; they exhibit a strong AChE activity. In the rostral half of the vesicle several sensory cells are associated with each nerve cell, while in the caudal portion only a few cells are apposed to each nerve cell. Thus, the ratio of the number of sensory cells to that of AChE-containing nerve cells in the anterior half of the pineal vesicle is high when compared with the remaining area. In the anterior half of the vesicle the outer segments of the sensory cells are more distinct and their inner segments possess a higher AChE activity than those in the posterior region and the stalk. A gradation in the degree of development of neuropil formations along the pineal axis is remarkable; their size and AChE activity gradually diminish in a caudal direction. In view of the structural specialization of the rostral region of the pineal organ, it has been argued that its terminal portion is more photosensitive.This work was supported by a fellowship from the Alexander von Humboldt Foundation, Federal Republic of Germany.     

Feeding rhythms in constant light and constant darkness: the role of the eyes and the effect of melatonin infusion

Exposure to constant light abolishes circadian behavioral rhythms of locomotion and feeding as well as circulating melatonin rhythms in pigeons (Columba livia). To determine if feeding rhythmicity could be maintained in pigeons exposed to constant light, periodic infusions (10h/day) of melatonin were administered to pinealectomized and bilaterally retinectomized/pinealectomized pigeons under conditions of both constant darkness and constant light. The infusions were sufficient to entrain rhythmicity in pinealectomized pigeons in constant darkness and to restore and maintain rhythmicity in bilaterally retinectomized/pinealectomized pigeons in constant darkness. On subsequent exposure to constant light, rhythmicity remained phase locked to the melatonin infusions in bilaterally retinectomized/pinealectomized pigeons but was abolished in sighted pinealectomized birds. These results suggest that while endogenous melatonin rhythms are both necessary and sufficient to maintain behavioral rhythms in DD, their effect can be overridden by constant light but only if perceived by the eyes. Thus, constant light may abolish behavioral rhythmicity in intact pigeons (and perhaps in other species) by a mechanism other than suppression of endogenous melatonin rhythmicity. Such a mechanism might involve direct stimulation of locomotor or feeding activity by retinally perceived (but not by extra-retinally perceived) light, or alternatively by suppression of a hypothalamic oscillator that receives its major light input from the retinae.Abbreviations PX pinealectomized - EX bilaterally enucleated - LD light:dark cycle - LL constant light - DD constant darkness - DD_b constant darkness before exposure to constant light - DD_a constant darkness after exposure to constant light     

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     

Melatonin in the regulation of annual testicular events in carp Catla catla: evidence from the studies on the effects of exogenous melatonin, continuous light, and continuous darkness

The physiological significance of melatonin in the regulation of annual testicular events in a major carp Catla catla was evaluated through studies on the effects of graded dose (25, 50, or 100 µg/100 g body wt.) of melatonin exogenously administered for different durations (1, 15, or 30 days) and manipulation of the endogenous melatonin system by exposing the fish to constant darkness (DD) or constant light (LL) for 30 days. An identical experimental schedule was followed during the preparatory (February-March), pre-spawning (April-May), spawning (July-August), and post-spawning (September-October) phases of the annual cycle. Irrespective of the reproductive status of the carp, LL suppressed while DD increased the mid-day and mid-night values of melatonin compared to respective controls. Influences of exogenous melatonin varied in relation to the dose and duration of treatment and the reproductive status of the carp. However, testicular response to exogenous melatonin (at 100 µg, for 30 days) and DD in each reproductive phase was almost identical. Notably, precocious testicular maturation occurred in both DD and melatonin-injected fish during the preparatory phase and in LL carps during the pre-spawning phase. In contrast, testicular functions in both the melatonin-treated and DD fish were inhibited during the pre-spawning and spawning phases, while the testes did not respond to any treatment during the post-spawning phase. In conclusion, this study provided the first experimental evidence that melatonin plays a significant role in the regulation of annual testicular events in a sub-tropical surface-dwelling carp Catla catla, but the influence of this pineal hormone on the seasonal activity of testis varies in relation to the reproductive status of the concerned fish.     

Long-term effects of constant light or darkness on chicken pineal hydroxyindole-O-methyltransferase expression: biochemical and cellular aspects

1. Chickens kept in constant light, as opposed to constant darkness, display a twofold increase in the activity of pineal hydroxyindole-O-methyltransferase (HIOMT), the last acting enzyme in the melatonin pathway. 2. Using an immunological approach, we presently show that this regulation of HIOMT activity reflects changes in the concentration of a single molecular form of the enzyme protein (a 38 kDa polypeptide). Immunohistofluorescence indicates that these concentration changes concurrently affect modified photoreceptors and pinealocyte-like cells in the chicken pineal organ. 3. Together, the present data support the hypothesis that environmental lighting might regulate the expression of the HIOMT gene.     

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     

Dissociation between the circadian rhythm of locomotor activity and the pineal clock in the Japanese newt

The circadian locomotor activity rhythm of the Japanese newt has been thought to be driven by a putative brain oscillator(s) subordinate to the pineal clock. The existence of mutual coupling between the pineal clock and the brain oscillator(s) in vivo was examined. We covered the newt's skull with aluminum foil and simultaneously reversed the light-dark cycle, thereby allowing the pineal organ to be exposed to constant darkness while the rest of the animal was exposed to the reversed light-dark cycle. In control animals, whose heads were covered with transparent plastic, the rhythm of synaptic ribbon number in the pineal photoreceptor cells was entrained to the reversed light-dark cycle. Rhythms from newts whose heads were shielded, however, were similar to those observed in the unoperated newts kept under constant darkness. The locomotor activity rhythms of both head-covered animals and control animals were entrained to the reversed light-dark cycle. These data suggest that extrapineal photoreception can entrain the putative brain oscillator(s), but not the pineal clock. Thus, at least in an aspect of photic entrainment, there seems to be little or no mutual coupling between the pineal clock and the putative brain oscillator(s) in the circadian system of the Japanese newt.Abbreviations LD light-dark - DD constant darkness - SCN suprachiasmatic nucleus - SR synaptic ribbon     

Expression of circadian rhythmicity in Djungarian hamsters under constant light: Effects of light intensity and the circadian system's state

Summary Djungarian hamsters (Phodopus sungorus), were exposed to constant light with increasing intensities (20, 60, 350 lux), and wheel running activity was recorded. With increasing light intensity the percentage of hamsters showing a split in their daily activity pattern increased and the free running period was lengthened for both the unsplit and the split state. The fact that the free running period of both states depended on the light intensity together with the observation that the highest incidence of acircadian activity occurred under 350 lux, provoked the idea that the emergence of splitting or acircadian rhythmicity is a direct consequence of the light induced lengthening of the free running period. However, analysis of the data failed to support the idea that emergence of a split or acircadian activity is a threshold phenomenon with respect to the free running period.Due to differences in circadian function some Djungarian hamsters do not exhibit photoinduction following short day exposure. In these individuals splitting also occurred but required exposure to a higher light intensity than in photo-responsive hamsters. This observation is in accordance with the idea that the two phenotypes differ in the interaction of the two component oscillators underlying circadian rhythmicity.Abbreviations LD long day photoperiod - LL constant light - SD short day photoperiod - free running period     

Retinally perceived light is not essential for photic regulation of pineal melatonin rhythms in the pigeon: studies with microdialysis

Using in vivo microdialysis, effects of retinally perceived light on pineal melatonin release and its rhythmicity was examined in the pigeon. In the first experiment, light-induced suppression of pineal melatonin release was studied. Although light given to the whole body during the dark strongly suppressed pineal melatonin release to a daytime level, light exclusively delivered to the eyes did not remarkably inhibit melatonin release. In the second experiment, in order to determine whether retinally perceived light has phase-shifting effects on pineal melatonin rhythms, pigeons were given a single light pulse of 2 h at circadian time (CT) 18 and the phases of the second cycle after the light pulse were compared with those of control pigeons without the light pulse. In this experiment, phase advances of pineal melatonin rhythms were observed when the light was given to the whole body but not when only the eyes were illuminated. In a third experiment, after entrainment to light-dark 12:12 (LD 12:12) cycles, birds whose heads were covered with black tapes were transferred into constant light (LL) conditions and only the eyes were exposed to new LD cycles for 7 days (the phase was advanced by 6 h from the previous cycles) using a patching protocol. This procedure, however, could not entrain pineal melatonin rhythms to the retinal LD cycles. These results indicate that the eyes are not essential for photic regulation of pineal melatonin release and its rhythmicity in the pigeon.Abbreviations CT circadian time - LD light-dark - LL constant light - SCN suprachiasmatic nucleus - LLdim constant dim light - NE norepinephrine - SCG superior cervical ganglia - WB whole body - E eye - EX extraretina - C control     

The synaptic ribbons of the guinea-pig pineal gland under normal and experimental conditions

Summary Synaptic ribbons (SR), functionally enigmatic structures of mammalian pinealocytes, were studied electron microscopically with regard to number, intracellular localization and topographical relationships, both under normal and experimental conditions. Pineal glands of guinea-pigs serving as controls contained 1.75 ribbon fields/unit area in the males and 2.58 in the females. In animals subjected to continuous illumination for 64 days the number of ribbon fields increased 20-fold in the males and 9-fold in the females. Continuous darkness (26 to 70 days) had varying effects; in some animals SR increased either strongly or moderately, in others they appeared unchanged. Under continuous illumination a higher percentage of ribbon fields bordered the cell membrane than in the controls. Moreover, paired ribbon fields occurred. The topographical analysis revealed that 98 % of the ribbon fields bordering the cell membrane lay opposite another pinealocyte and the remainder opposite nerve fibres, blood vessels and collagenous fibres. It is suggested that SR of mammalian pinealocytes do not represent non-functioning phylogenetic relics but true organelles possibly involved in coupling adjacent pinealocytes functionally.This study was supported by a grant from the Deutsche Forschungsgemeinschaft, Bonn.     

Pineal-dependent locomotor activity of lamprey,Lampetra japonica,measured in relation to LD cycle and circadian rhythmicity

Summary Locomotor activity of the river lamprey, Lampetra japonica, was investigated under a light-dark (LD 1212) cycle and under continuous dark conditions. Intact lampreys were entrained to the light:dark cycle. They were active mainly in the early half of the dark period and inactive in light period. The light:dark entrainment continued in 72.7% of lampreys after the removal of bilateral eyes, but additional pinealectomy made the entrainment disappear in all lampreys. When lampreys were pinealectomized with their eyes intact, light: dark entrainment was abolished in most cases. The results indicate that the pineal organ of the lamprey is a photoreceptive organ responsible for synchronizing locomotor activity to LD cycle. Under continuous dark conditions, the locomotor activity began to free-run with a period of 21.3 ± 0.9 h (mean ± SD, n = 53). This circadian rhythmicity was not affected by the removal of lateral eyes but was abolished by pinealectomy. The pineal organ appears to function as an oscillator, or as one of the oscillators, for the circadian locomotor rhythm of lampreys.Abbreviations DD continuous dark - LD light:dark     

Electron microscopy of the rabbit pineal organ in vitro

The results presented in this study show that the rabbit pineal organ cultured in vitro retained its in vivo fine structure for at least eight days. However, the Golgi complex in the light pinealocytes stopped forming dense core vesicles while vesicle-crowned ribbons increased in number. After addition of norepinephrine to the culture medium, the Golgi complex once more began the production of dense core vesicles. Terminals of light pinealocytic processes then often contained Golgi dense core vesicles in close contact with the cell membrane, suggesting the release of the vesicular content into the intercellular and perivascular spaces. A close topographical relationship between Golgi dense core vesicles and vesicle-crowned ribbons was observed.