0000000000043064
AUTHOR
Lutz Vollrath
Strain differences in the ratio of synaptic body types in photoreceptors of the rat retina.
In the retinal outer plexiform layer of seven different rat strains, synaptic bodies (SB) were counted and, according to their morphology, characterized as synaptic ribbons (SR), synaptic spheres (SS) or intermediate structures. It was found that absolute SB numbers showed relatively small variations while SR/SS ratios differed considerably between the strains investigated. These results are discussed with respect to retinal pigmentation and to formation and degradation, respectively, of synaptic ribbons.
Effects of melatonin on spontaneous electrical activity of neurons in rat suprachiasmatic nuclei: an in vitro iontophoretic study.
Circadian rhythms, endogenously generated in suprachiasmatic nuclei (SCN), seem to be under the direct influence of melatonin. Therefore, the effect of iontophoretically applied melatonin on electrical activity of SCN neurons was investigated in vitro. Usually, melatonin had an inhibitory effect. In the 3-h periods before (2.00-5.00 p.m.) or after (5.00-8.00 p.m.) the light-dark transition the percentage of SCN neurons sensitive to melatonin was very high (80% and 100%, respectively). However, efficacy of melatonin was low in the periods preceeding (20%) and following (33%) this 6-h time interval.
Quantitative analysis of "synaptic" ribbon profiles in the pineal complex of male and female Pirbright-White guinea pigs.
Previous studies have pointed in the direction of sex differences as well as regional differences in the pineal gland of guinea pigs. In the present investigation these aspects were studied at the electron-microscopic level by quantitating different types of “synaptic” bodies, intrinsic to pinealocytes. The two major types of “synaptic” organelles, ribbons and spherules, did not exhibit regional or sex differences. “Synaptic” structures intermediate in appearance to ribbons and spherules were significantly larger in number in males in the distal region of the pineal gland, compared to females. As previous studies have shown that ribbon and spherule numbers undergo characteristic changes dep…
Development of day-night rhythmicity in "synaptic" ribbon numbers in the pinealocytes of posthatch chicks kept under either natural photoperiodic conditions or continuous illumination
: Pineal synaptic ribbons (SR) undergo characteristic changes over a period of 24 hr under natural photoperiodic conditions in various vertebrates, being low in number during daytime and elevated at night. During posthatch development of chicks, the rhythmicity of SR numbers is reported to appear at the age of about 2 weeks. Because the influence of external light during the growth phase of chicks on the development of day-night rhythmicity in SR numbers is unknown, we studied day-night differences in SR numbers in the pinealocytes of chicks at the posthatch ages of 15, 17, and 19 days; chicks had previously been kept under natural photoperiodic conditions or continuous illumination. Under …
Neuropeptide Y (NPY)-like immunoreactivity in the guinea pig pineal organ.
Relatively little is known about mammalian pineal neuropeptides. In the present study neuropeptide Y-like immunoreactivity (NPY-LI) was examined in the guinea pig pineal gland. NPY-LI was restricted to few intrapineal nerve fibers of faint fluorescence intensity. They showed no preferential localization with regard to the different pineal portions. As catecholaminergic fibers are abundant in the guinea pig pineal gland, the scarcity of NPY-LI fibers indicates that in the pineal colocalization of noradrenaline and NPY-LI is not a regular feature, in contrast to other organs. The possibility exists that in the pineal NPY-LI fibers are not of peripheral sympathetic but of central origin.
Day/night serotonin levels in the pineal gland of male BALB/c mice with melatonin deficiency
Abstract. Studies from another laboratory have shown that several strains of laboratory mice have a genetic defect for melatonin synthesis. In non-deficient species, melatonin synthesis undergoes a typical, β-adrenergically regulated day/night rhythm with low melatonin levels during daytime and high levels at night, the precursor serotonin showing an inverse behaviour. This study examines whether a day/night rhythm of pineal serotonin levels exists in melatonin-deficient male BALB/c mice. Mice kept under a lighting schedule of 12 h light (lights on at 07.00 h) and 12 h dark were killed at 13.00 and 01.00 h, respectively. Serotonin amounted to 12-15 ng/pineal and did not show regular day/nig…
Nitric oxide is formed in a subpopulation of rat pineal cells and acts as an intercellular messenger.
In the rat pineal, formation of the second messenger cyclic GMP (cGMP) is under adrenergic control. Two important sequential steps mediate adrenergic signal transduction by cGMP, receptor-stimulated nitric oxide (NO) formation by the enzyme NO synthase I (NOS I), and NO-induced cGMP formation by the cytosolic enzyme guanylyl cyclase. With regard to the first step in cGMP transduction (i.e. NO formation) we found, by means of NOS I immunostaining and NADPH-diaphorase staining, that the presence of NOS I was restricted to a subpopulation of pineal cells, generally surrounded by NOS I-negative cells. Considering the fact that NO is able to permeate the cell membrane, the question arises whethe…
Lack of "Synaptic" Ribbons in the Pineal Gland of BALB/c Mice
In mammalian pinealocytes "synaptic" ribbons (SR) are regularly occurring organelles that are functionally poorly understood. Since in a number of studies on the mouse pineal gland the presence of SR has not been mentioned, it was the aim of this investigation to quantitate SR in mice. BALB/c mice were chosen, which have recently been shown to have a genetic defect for melatonin synthesis. The pineals of 15 mice killed at night, when SR numbers are normally high, were examined electron microscopically, scanning an area of greater than 20,000 micron 2 per gland. In none of these pineals were SR detected. It is concluded that the lack or extreme rarity of SR in laboratory mice may be related …
Protein gene product (PGP) 9.5 immunoreactivity in nerve fibres and pinealocytes of guinea-pig pineal gland: interrelationship with tyrosine- hydroxylase- and neuropeptide-Y-immunoreactive nerve fibres.
This light-microscopic (LM) immunohistochemical study has evaluated the presence and distribution of the pan-neural and neuroendocrine marker protein gene product (PGP) 9.5 in pinealocytes and nerve fibres of guinea-pig pineal gland. The pattern of PGP 9.5-immunoreactive (ir) nerve fibres has been compared with that of fibres staining for tyrosine hydroxylase (TH) or neuropeptide Y (NPY). The vast majority of pinealocytes stained for PGP 9.5, although with variable intensity. PGP 9.5 immunoreactivity was localized in pinealocytic cell bodies and processes. Double-immunofluorescence revealed that PGP 9.5 immunoreactivity was absent from glial cells identified with a monoclonal antibody again…
Structural dissimilarities in different regions of the pineal gland of Pirbright white guinea-pigs.
In the present study, the proximal, intermediate and distal parts of dumbbell-shaped pineal glands of Pirbright White guinea-pigs were investigated with respect to structural dissimilarities. The most conspicuous difference was that while in the proximal part the pinealocytes were arranged in a random fashion, the intermediate and distal regions, except a small ventral area in the latter, exhibited a prominent follicular arrangement of the pinealocytes. Concretions were restricted to the subcapsular region of the intermediate part, whereas cells with hyperchromatic nuclei increased in number in an anterior-posterior direction. In males, pinealocyte nuclear size did not differ significantly …
Developmental Changes and Daily Rhythm in Melatonin-Induced Inhibition of 3′,5′-Cyclic AMP Accumulation in the Rat Pituitary
Melatonin's transduction mechanisms were investigated using in vitro cultured anterior hemipituitaries. Melatonin inhibited cAMP and 3',5'-cyclic GMP accumulation in neonatal rat anterior pituitary stimulated with LHRH. Maximal inhibitory effect was reached within 25 min and persisted for at least 20 min. Inhibition of cAMP accumulation is specific for melatonin because its analogs N-acetylserotonin and 5-methoxytryptamine are 1000 times less potent. Melatonin effect is age- and time-dependent. Marked inhibition was observed in 5-, 10-, and 14-day-old rats but not in 29-day-old ones. Melatonin was significantly more potent when applied at the end of the light period as compared with the fir…
Day/night changes of pineal gland volume and pinealocyte nuclear size assessed over 10 consecutive days
Previous studies aimed at elucidating day/night changes of pineal gland size and pinealocyte nuclear volume have yielded contradictory results. The aim of the present investigation was to examine the above parameters over a period of 10 successive days, at 6-hour intervals under a lighting regimen of LD 12:12 (7 a.m. to 7 p.m.). It was found that changes in pineal gland volume could not be correlated with the light/dark phases; instead, a rhythm with a two-day period was encountered. Pinealocyte nuclear volume, by contrast, showed clear circadian changes, exhibiting troughs during photophase and peaks during scotophase. In this experimental series, the karyometric results conformed to the m…
The “Mainzer EMF-Wachhund”: results from a watchdog project on self-reported health complaints attributed to exposure to electromagnetic fields
The "Mainzer EMF-Wachhund," a watchdog project, offered a system of self-notification of health complaints attributed to exposures to electromagnetic fields (EMFs) to a population of a part of Germany with about 4 million inhabitants. By using a self-administered questionnaire, which was provided online and for download from the Internet, 192 persons reported such health complaints in the period from October 2003 to March 2005. Of these, 56% classified themselves as electromagnetic hypersensitive (EH). Predictors of this self classification were being affected by all kinds of EMF rather than single EMF sources and being female. On average, EH subjects reported a high degree of suffering, 77…
The effects of short pulses of light at night on numbers of pineal "synaptic" ribbons and serotonin N-acetyltransferase activity in male Sprague-Dawley rats.
To characterize further the functionally enigmatic "synaptic" ribbons (SR) of the mammalian pineal gland and to study possible relationships to melatonin synthesis, in the present investigation rats were exposed to short pulses of light at night when both SR numbers and serotonin N-acetyltransferase (NAT) activity are high in comparison to day-time values. Male Sprague-Dawley rats were killed at 13:00 and 01:00 h, respectively, and at 01:10 and 02:00 h after exposure to light for 10 and 60 min, respectively. The pineals were rapidly taken out and cut sagittally in half. One half was processed for electron-microscopic quantitation of SR numbers and the other half for NAT determinations. It w…
Anatomy online: presentation of a detailed WWW atlas of human gross anatomy--reference for medical education.
We present an online anatomy atlas based on the Visible Human Project (VHP) of the US National Library of Medicine. The objective is to provide original unlabeled as well as labeled sections of the human body of high quality and resolution on the Internet, for use in basic and continuing medical education. For a representative overview of the body, 370 axial sections were selected from the male and female data base of the VHP with special regard to regions of clinical interest. Each section is accompanied by its corresponding computer tomography (CT) image and, if available, magnetic resonance images (MRI) for quick and easy comparison of morphologic and radiologic structures. The sections …
Depressive effect of LHRH on the numbers of "synaptic" ribbons and spherules in the pineal gland of diestrous rats.
Previous studies have shown that LHRH or LHRH-like substances are present in the pineal gland. In order to investigate whether exogenous LHRH may affect the pineal gland, in the present study the effects of a single dose of LHRH (1 microgram, i.p.) on pineal "synaptic" ribbons and spherules as well as serum melatonin levels were examined in diestrous Wistar rats. One hour after the injection both ribbons and spherules exhibited a statistically significant decrease in number. Serum melatonin levels were not affected. It is concluded that humoral feedback mechanisms may exist between the hypothalamus and the pineal gland.
An intrinsic neuronal-like network in the rat pineal gland
Recent studies have shown that in rat pineal glands kept in vitro action potential-producing cell clusters are demonstrable. To test whether the clusters interact, multiple-unit recordings were carried out simultaneously from different clusters, with or without electrical stimulation. Clusters with rhythmic burst activity exhibit highly synchronized firing and electrical stimulation of one cluster elicits an immediate response in another one, apparently involving synapses but not gap junctions. It is hypothesized that the interacting clusters form a network. As the firing is affected by norepinephrine, acetylcholine and Ca2+, the network may monitor the interstitial concentrations of these …
Magnetic Sensitive Pineal Cells in Pigeons
The electrical activity of about 30% of a homing pigeon’s pineal cells can be altered by changing the horizontal or vertical component of the natural magnetic field, using two pairs of Helmholtz coils. The cells respond to a rapid and/or gradual change in the magnetic field with either excitation or inhibition. In some of the sensitive cells the responses to magnetic field changes could not be repeated.
Teaching and learning gross anatomy using modern electronic media based on the visible human project
This article reviews online (Internet) anatomy projects and multimedia productions (books and CD-ROMs) based on the Visible Human Project (VHP) of the United States National Library of Medicine. The focus of these projects and productions is on the teaching of 3D anatomy using the digitized sections of the visible human male and female. The article also provides information about the VHP, its goals and what it has achieved thus far.
Pineal ?synaptic? ribbons and spherules during the estrous cycle in rats
In previous studies pineal "synaptic" ribbons have been shown to undergo striking numerical changes under various physiological and experimental conditions and to be regulated by beta-adrenergic mechanisms. The aim of the present investigation was to study the numbers of pineal "synaptic" ribbons and spherules in Wistar rats throughout the estrous cycle and to compare them with those in males. There were no statistically significant differences in the numbers of ribbons and spherules between males and females and in the females at the different stages of the estrous cycle, indicating that the structures in question, in vivo, do not appear to be regulated by naturally occurring changes of se…
Effects of an artificial magnetic field on serotonin N-acetyltransferase activity and melatonin content of the rat pineal gland
In the present study the effects of artificial magnetic fields on pineal serotonin-N-acetyltransferase (NAT) activity and melatonin content in male Sprague-Dawley rats were investigated to study the secretory activity of the pineal gland. Experimental inversion of the horizontal component of the natural magnetic field, performed at night-time, led to a significant decrease of both parameters investigated. During day-time, this effect was less conspicuous. During night-time, inversion of the horizontal component is followed by a reduced pineal secretory activity for about 2 h. After 24 h exposure to the inverted horizontal component, return to the natural condition was followed by a renewed …
Circadian gene expression patterns of melanopsin and pinopsin in the chick pineal gland
The directly light-sensitive chick pineal gland contains at least two photopigments. Pinopsin seems to mediate the acute inhibitory effect of light on melatonin synthesis, whereas melanopsin may act by phase-shifting the intrapineal circadian clock. In the present study we have investigated, by means of quantitative RT-PCR, the daily rhythm of photopigment gene expression as monitored by mRNA levels. Under a 12-h light/12-h dark cycle, the mRNA levels of both pigments were 5-fold higher in the transitional phase from light to dark than at night, both in vivo and in vitro. Under constant darkness in vivo and in vitro, the peak of pinopsin mRNA levels was attenuated, whereas that of melanopsi…
The phototransduction cascade in the isolated chick pineal gland revisited.
It is well established that the isolated chick pineal gland is directly light sensitive and that melatonin synthesis of the gland can be inhibited by exposing the gland to light during scotophase. Since not all the steps of the phototransduction cascade have been clarified to the same extent as in the retina, we have treated isolated chick pineal glands with 90 min of light during scotophase and with drugs that affect key-components of vertebrate phototransduction, i.e., cyclic guanosine monophosphate (cGMP) phosphodiesterase 6 (PDE6), cGMP levels and cGMP-gated calcium channels. The endpoint measured was the activity of the rate-limiting enzyme of melatonin synthesis, arylalkylamine N-acet…
Electrophysiology of the Guinea-pig Pineal Organ: Sympathetic Influence and Different Reactions to Light and Darkness
Publisher Summary Recent electrophysiological studies have shown that the guinea-pig pineal organ comprises two main categories of intrinsic cells, which are (a) pinealocytes which are predominantly influenced by central commissural fibers and (b) pinealocytes which are exclusively influenced by peripheral sympathetic fibers. The first group comprises spontaneously active cells which respond to olfactory, acoustic and short-term optic stimuli respectively. After sympathectomy their spontaneous activity is diminished but not suppressed. The second group comprises spontaneously active cells, which do not respond to short-term sensory stimulation and whose activity depends on the environmental…
The response to isoproterenol of synaptic ribbon numbers in the rat pineal gland changes during postnatal development
Abstract In the mammalian pineal gland synaptic ribbons (SRs) are dynamic organelles of pinealocytes undergoing a day/night rhythm, with small numbers during daytime and significantly higher numbers at night, similar to the formation of the pineal hormone melatonin. Whereas the day/night rhythm of melatonin synthesis is adrenergically regulated, data on the adrenergic regulation of SR numbers in the rat pineal gland are at variance. While some authors have demonstrated that isoproterenol (ISO) stimulates SR numbers, others could not find any effect. To clarify the issue, we carried out identical experiments in two age groups. It was found that in male Sprague-Dawley rats, administration of …
Distribution of dopamine-beta-hydroxylase—like immunoreactivity in the rat pineal organ
The aim of the present investigation was to study the distribution in the rat pineal gland of dopamine-beta-hydroxylase (DBH) which is essential for the formation of the melatonin synthesis-regulating substance noradrenaline (NA). In 5- and 8-month-old male Sprague-Dawley rats DBH-like immunoreactivity (DBH-LI) was studied using polyclonal antibodies against DBH and the indirect immunofluorescent technique. DBH-LI was mainly located in pineal nerve fibres coming from the superior cervical ganglia. The intensity of the staining reaction was considerably lower than in non-pineal noradrenergic nerve fibres and the impression was gained by comparison of DBH-LI specimens with glyoxylic acid-trea…
The role of the hypothalamic paraventricular nuclei for the regulation of pineal melatonin synthesis: New aspects derived from the vasopressin-deficient Brattleboro rat
Abstract There is evidence for an involvement of the hypothalamic paraventricular nuclei (PVN) in the regulation of pineal melatonin synthesis in rats. Since electrical stimulation of the PVN or the systemic administration of arginine-vasopressin (AVP) result in a depression of the nocturnal melatonin surge, this neuropeptide appears to be pivotal for the transduction of PVN-efferent, pinealopetal signals. We therefore used an AVP-deficient animal model, the Brattleboro rat, to further investigate the mechanisms responsible for pineal regulation. Anesthetized adult male animals received 2 min of bilateral electrical stimulation of the PVN either during the day or at night. Thirty min later,…
Inverse behaviour of "synaptic" ribbon and spherule numbers in the pineal gland of male guinea-pigs exposed to continuous illumination.
There is increasing evidence that pineal “synaptic” ribbons are a heterogeneous population of organelles. In addition to “synaptic” ribbons (SR) sensu stricto, which consist of an electron-dense rod surrounded by electronlucent vesicles, “synaptic” spherules (SS) exist, the electrondense core of which is round and much wider than that of the SR. In the guinea-pig SR and SS numbers exhibit an inverse day/night rhythmicity. To gain more insight into the functional significance of SR and SS, guinea-pigs were exposed to continuous illumination for approximately 4 months (LL) and the respective structures in the pineal gland were quantitated under the electron microscope and compared with contro…
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 S…
Effects of LHRH, progesterone, estradiol-17 ? and dexamethasone in vitro on pineal synaptic ribbons and serotonin N-acetyltransferase activity in diestrous rats
Pineal glands of regularly cycling Sprague Dawley rats (180-220 g) killed on the diestrous morning (between 0900-1000 h) were incubated in appropriate media for six hours with LHRH (8.5 microM), progesterone (3.2 microM), estradiol-17 beta (370 nM) or dexamethasone (250 nM). Pineals incubated in hormone-free medium and unincubated glands served as controls. Six rats were used in each group. After incubation the glands were divided into two parts. One part was used to estimate serotonin N-acetyltransferase (NAT) activity. The other part was processed for electron microscopy to quantify synaptic ribbons (SR). The SR numbers were computed to 20,000 microns 2 area of pineal tissue. The number a…
In vitro effects of putative neurotransmitters on synaptic ribbon numbers and N-acetyltransferase activity in the rat pineal gland
The pineal contains a large number of classical transmitters and neuropeptides. Some of these neurochemicals are involved in the regulation of serotonin N-acetyltransferase (NAT) activity and hence in melatonin synthesis. Synaptic ribbons present in the pineal gland also exhibit a numerical day/night rhythm parallel to that of NAT activity. There is scarcity of information regarding the regulation of synaptic ribbon (SR) numbers. In the present study, we have investigated in vitro effects of a number of classical neurotransmitters and neuropeptides. NAT activity was used to monitor melatonin synthesis under the experimental conditions used. Norepinephrine (NE), Delta sleep-inducing peptide …
Response of pineal serotonin N-acetyltransferase activity in male guinea pigs exposed to light pulses at night.
Serotonin N-acetyltransferase (NAT), which is crucial for the formation of melatonin, undergoes a typical day/night rhythm in the pineal gland with low levels during daytime and high levels at night. Short pulses of light given at night have been shown to rapidly depress NAT activity in some species, but not in others, the reasons for this difference being unclear. As diurnality and nocturnality of the experimental animals may play a role and since diurnally active animals have been little investigated in this respect, in the present study the diurnally active guinea pig was investigated. Male guinea pigs kept under a lighting regimen of LD 12:12 (lights off at 1700 hrs) were killed between…
Immunohistochemical evidence for the presence of peptides derived from proenkephalin, prodynorphin and proopiomelanocortin in the guinea pig pineal gland
By using a plethora of region-specific antisera, this light microscopic immunohistochemical study revealed that derivatives from the three opioid precursors, i.e. proenkephalin, prodynorphin and proopiomelanocortin are differentially distributed in the pineal gland of guinea pig. Various molecular forms of immunoreactive opioid peptides derived from proenkephalin or prodynorphin were present in a minority of pinealocytes as well as in nerves. In contrast to this dual distribution pattern of opioid-active peptides, the opioid-inactive derivative from proopiomelanocortin, alpha-melanocyte stimulating hormone, was exclusively present in a large proportion of pinealocytes. A multiple and differ…
Chronoendokinologia — Quo vadis?
The present review deals with important new chronobiological results especially in the field of chronoendocrinology, shedding new light on the circadian organisation of mammals including man. In vitro studies have shown that the concept of the existence of a single circadian oscillator located in the suprachiasmatic nucleus has to be extended. Circadian oscillators have also been found to exist in the retina, islets of Langerhans, liver, lung, and fibroblasts. Another major result is the detection of a new photopigment, melanopsin, present in a subpopulation of retinal ganglion cells which are lightsensitive and project to the suprachiasmatic nucleus, acting as zeitgeber for the photic entr…
Plasticity of synaptic ribbons of the rat pineal gland in vitro — Minor effects of electrical stimulation
Synaptic ribbons (SRs) of mammalian pinealocytes exhibit day/night changes in number and size, changes that are apparently regulated by the suprachiasmatic nucleus via postganglionic sympathetic nerve fibres. Since the neural control of SR changes is far from clear and as pinealocytes produce action potentials, we undertook to investigate whether electrical stimulation affects SR changes. Isolated rat pineal glands removed during the daytime were kept in vitro for 0, 30, 60, 90 or 120 min, with or without continuous electrical stimulation (1 mA, 1 Hz), followed by the quantification of SR profiles (SRPs) by transmission electron microscopy. SRs were categorised as to whether they lay less t…
Nitric oxide inhibits electrically active units in the rat pineal gland.
Extracellular multiple unit recordings were performed in isolated rat pineal glands to determine a possible effect of nitric oxide (NO) on the spontaneous electrical activity of pinealocytes. Spontaneously active cells forming clusters of 3-5 cells fell into two categories: more or less regularly firing clusters (REG, 64%) and irregularly discharging clusters with periodically repeated bursts (RHY, 36%). The NO-donor sodium nitroprusside (SNP) reduced the discharge rate of the great majority of REG clusters and of all the RHY clusters examined. Moreover, the burst activity of RHY clusters was abolished. These results could be completely reproduced by using another NO-donor, S-nitroso-N-acet…
Anomalous occurrence of immunoreactive calcitonin cells in the thymus of the rat.
In a study of the effect of pinealectomy on thyroid C-cell number, 8 animals out of 66 were found to have thymic tissue in close association with the thyroid. Cells containing immunoreactive calcitonin were found in all of the thyroids but in only one of the 8 pieces of thymus. These cells found in a piece of thymic tissue associated with the right thyroid lobe were located immediately under the capsule and did not form or associate with follicles. Unlike the other animals the rat with thymic calcitonin cells had an unequal distribution of C-cells between the left and right thyroid lobes, but the total number of thyroidal C-cells was the same as that of the other rats. Since the thymus prop…
Atypical 24-hour rhythms of serotonin N-acetyltransferase activity in the rat pineal gland.
Previous long-term studies have shown that in the pineal gland of rats melatonin synthesis is subject to infradian rhythms with periods between 4 and 7 days. Since in these studies melatonin-related parameters were measured at one timepoint of a 24-hr cycle only, the aim of the present investigation was to extend these experiments by more frequent sampling, to characterize the infradian rhythmicity in more detail. Male Sprague-Dawley rats kept under a light schedule of LD 12:12 (lights on at 0700) were killed at 6-hr intervals on 8 consecutive days. After decapitation the pineal gland was rapidly dissected out, followed by measurements of one of the melatonin-forming enzymes, serotonin N-ac…
Mammalian Pinealocytes: Ultrastructural Aspects and Innervation
In the mammalian pineal gland it is notoriously difficult to relate structure to function. The pineal-specific cells, the pinealocytes, contain only inconspicuous numbers of secretory granules, and the variable amounts of smooth and rough endoplasmic reticulum also do not point to a particular function. In addition to these widely known cellular components, pinealocytes contain organelles, the so-called 'synaptic' ribbons, histophysiological studies of which provide important insights into the structural and functional complexity of the organ. As synaptic ribbons may be involved in neuronal functions of pinealocytes it is notable that these organelles are structurally heterogeneous. Ribbons…
Neuronal markers in the rodent pineal gland ? an immunohistochemical investigation
Although some embryological and morphological features speak in favour of a neuronal character of rodent pinealocytes, histochemistry and ultrastructure let this issue appear controversial. Using antibodies to different neurofilaments, the neural adhesion molecule L1, synaptophysin and tubulin as neuronal markers, the pineal glands of rat and guinea-pig were studied by means of immunofluorescence. Neurofilament-immunoreactivity was present in some rat pineal nerve fibers and in the majority of guinea-pig pinealocytes, L1 decorated rat intrapineal nerve fibers, synaptophysin was almost ubiquitously distributed in the pineal of both species, while tubulin-immunofluorescence was seen in nerve …
Melatonin modulates diacylglycerol and arachidonic acid metabolism in the anterior pituitary of immature rats.
In pituitary glands of immature rats prelabeled in vitro with [3H]arachidonic acid, melatonin diminished the luteinizing hormone-releasing hormone (LHRH)-induced increase in [3H]diacylglycerol accumulation as well as [3H]arachidonic acid release from the tissue. Melatonin reduced also LHRH-stimulated incorporation of [3H]glycerol into pituitary [3H]diacylglycerol. The effect was day-time dependent: in the evening experiment melatonin was effective at 0.1 nM concentration while in the morning it had no effect even at 10 nM concentration. The effect of melatonin was also abolished by pretreatment with pertussis toxin. Diacylglycerol and/or arachidonic acid might serve as 2nd messengers transd…
Rat pineal arylalkylamine-N-acetyltransferase: cyclic AMP inducibility of its gene depends on prior entrained photoperiod.
The nocturnal biosynthesis of melatonin in the rat pineal depends on strongly enhanced expression of the enzyme N-acetyltransferase [arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87]. AA-NAT transcription is stimulated during darkness by adrenergic inputs to the pineal from the suprachiasmatic nucleus (SCN). Nocturnal activation of the AA-NAT promotor following stimulation of pinealocyte adrenoceptors involves cAMP-dependent stimulation of protein kinase A (PKA). The nocturnal rise in AA-NAT depends on the lighting conditions. As compared with light/dark (LD) 12:12, the delay between dark onset and the nocturnal rise in AA-NAT is shortened under long photoperiods and prolonged under…
Correlation of the number of pineal ?synaptic? ribbons and spherules with the level of serum melatonin over a 24-hour period in male rabbits
Previous studies have shown that pineal "synaptic" ribbons and spherules may respond differently under normal and experimental conditions. It has been suggested that the increase in the number of ribbons may be a prerequisite for enhanced melatonin formation. In the present study, the number of ribbons and spherules as well as the level of serum melatonin were monitored over a 24-h period in the male rabbit, the pineal gland of which is known to contain many spherules. It was found that both the number of ribbons and the levels of serum melatonin show the typical nocturnal increase, exhibiting peaks at 02:00 and 06:00 h, respectively. There is a good correlation (R = 0.8) of the two paramet…
24-hour-variation of pineal gland volume, pinealocyte nuclear volume and mitotic activity in male Sprague-Dawley rats
In two experiments carried out on two alternate days, the 24-h-rhythmicity of pineal gland volume, pinealocyte nuclear size in cortex and medulla and mitotic activity were studied in male Sprague-Dawley rats, to assess to what extent morphological parameters reflect the pronounced day/night differences in pineal melatonin formation. Pineal volume exhibited statistically significant changes in the second experiment only, with a distinct trough at 6 p.m. Karyometry revealed highly variable patterns. In the first experiment, pinealocyte nuclear changes lacked parallelism in cortex and medulla. The cortex exhibited a bimodal curve with peaks at noon of the first day and at 6 a.m. of the second …
Melatonin formation in different parts of the guinea-pig pineal complex as assessed over 24 hours.
There is morphological evidence that the pineal gland is not a uniformly built organ but rather a complex. In the guinea-pig the gland is 6--7 mm in length and dumbbell-shaped, the proximal part coming into intimate contact with central commissural fibres. The aim of the present 24-hour study was to examine in male guinea-pigs whether the proximal, intermediate and distal areas of the gland are involved in melatonin formation and to compare their rhythmicities. Levels of melatonin in serum and the whole pineal gland as assessed by RIA show day/night ratios of 1 : 1.25 and 1 : 3.3, respectively. Serotonin N-acetyltransferase activity was found to exhibit ratios of 1 : 1.75 (Experiment I) and…
Effects of electrical stimulation of the superior cervical ganglia on the number of ?synaptic? ribbons and the activity of melatonin-forming enzymes in the rat pineal gland
Melatonin metabolism in the mammalian pineal gland is under the clear influence of sympathetic fibers originating in the superior cervical ganglia (SCG). Previous studies suggested that pineal "synaptic" ribbons (SR) as well are regulated by the gland's sympathetic innervation. To gain more insight into the mechanisms involved, we examined the effects of sympathetic stimulation on SR number and on the activity of melatonin forming enzymes, serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT). The SCG in adult male rats were stimulated electrically during daytime for either 15 or 120 min. Immediately following stimulation, the glands were removed and processed fo…
No short‐term effects of high‐frequency electromagnetic fields on the mammalian pineal gland
There is ample experimental evidence that changes of earth-strength static magnetic fields, pulsed magnetic fields, or alternating electric fields (60 Hz) depress the nocturnally enhanced melatonin synthesis of the pineal gland of certain mammals. No data on the effects of high-frequency electromagnetic fields on melatonin synthesis is available. In the present study, exposure to 900 MHz electromagnetic fields [0.1 to 0.6 mW/cm2, approximately 0.06 to 0.36 W/kg specific absorption rate (SAR) in rats and 0.04 W/kg in Djungarian hamsters; both continuous and/or pulsed at 217 Hz, for 15 min to 6 h] at day or night had no notable short-term effect on pineal melatonin synthesis in male and femal…
Electrophysiological investigations on the central innervation of the rat and guinea-pig pineal gland
The possible influence of central nervous structures on the electrical activity of single pineal cells was investigated in rat and guinea-pig. In the rat electrical stimulation of the hippocampal formation elicited both single cell responses with different latencies and mostly long-term excitations in single pineal cells, while stimulation of the habenular nuclei caused clear orthodromical responses with different latencies, alterations in the rate of spontaneous electrical activity and evoked discharges of "silent" units. In the guinea-pig electrical stimulation of the paraventricular nucleus influenced predominantly cells in the deeper layers of the posterior part of the pineal gland. Ele…
Mouse photoreceptor synaptic ribbons lose and regain material in response to illumination changes
Abstract Chemical synapses equipped with ribbons are tonically active, high-output synapses. The ribbons may play a role in the trafficking of synaptic vesicles. Recent findings in retinal rod cells of BALB/c mice indicate that ribbons are large and smooth in the dark phase, and, due to the formation and release of protrusions, small during the light phase. As a consequence of these changes, ribbons may traffick fewer vesicles in the light than in the dark phases. The aim of the present study was to find out whether the above ribbon changes in this mouse strain are strictly illumination-dependent and which signalling processes may be involved. Here, we show that ribbons form protrusions and…
Adaptation of the disector method to rare small organelles in TEM sections exemplified by counting synaptic bodies in the rat pineal gland
The disector is the only objective method for quantifying particles of variable size in a given volume. With this method, cell organelles are identified on adjacent sections, but only those present in one section are counted. When counting extremely rare structures in transmission electron microscope sections (physical disector), the usual procedure of counting on electron micrographs is limited for economic reasons (e.g. micrographs highly outnumbering the investigated structures). Hence, to apply this unbiased stereological method, a modification of the physical disector concerning 3 aspects has been developed. (1) The prerequisite of screening large corresponding tissue areas (here appro…
Fos-related antigen 2 (Fra-2) memorizes photoperiod in the rat pineal gland
As the physiological role of fos-related antigen-2 (Fra-2) is largely unknown and since the pineal plays an important role in the photoperiodic control of the body, we have tested the hypothesis that Fra-2 expression is photoperiod-dependent and may be involved in imprinting photoperiod on the pineal gland and the body as a whole. To this end, we have investigated Fra-2 mRNA expression and Fra-2 protein expression under various light/dark (LD) cycles. A clear nocturnal increase occurs for both monitored parameters under all photoperiodic conditions studied. The level of Fra-2 protein expression clearly depends on photoperiod, because the amount of protein at dark onset and during the night …
Involvement of cyclic guanosine monophosphosphate (cGMP) and cytosolic guanylate cyclase in the regulation of synaptic ribbon numbers in rat pineal gland
In the rat pineal gland N-acetyltransferase (NAT) activity and synaptic ribbon (SR) numbers display a circadian rhythm. It is well-known that NAT activity is regulated by adrenergic mechanisms involving cyclic adenosine monophosphate (cAMP) as a second messenger. However, the mechanism involved in the regulation of SR numbers has not been established so far. In the present in vitro study, we have investigated the effects of 8-bromo-cyclic guanosine monophosphate (8-bromo-cGMP), a cyclic guanosine monophosphate (cGMP) analog, and stimulation of guanylate cyclase on SR numbers. Incubation with 8-bromo-cGMP increased SR numbers in a dose- and time-dependent manner. Further, stimulation of the …
Circadian variations of ?synaptic? bodies in the pineal glands of Brattleboro rats
The function of the mammalian pineal gland is regulated primarily by the sympathetic system. Arginine-vasopressin (AVP) may also be involved in the regulation of pineal melatonin synthesis under experimental conditions. The present study was conducted in the AVP-deficient rat strain, the Brattleboro rat, to investigate whether the numbers and rhythms of pineal "synaptic" bodies in this strain are different from those found in intact rats. AVP or its non-vasoconstrictive analog, deamino-D-AVP, was also injected intra-arterially in Brattleboro or Sprague-Dawley rats to test whether this procedure influences "synaptic" body numbers. Brattleboro rats were killed at different time-points through…
The Use of Electron Microscopy and Stereology in the Study of the Mammalian Pineal Gland
Now that transmission electron microscopy (TEM) has been used for many years to study the pineal gland it is timely to evaluate what progress has been achieved by using this technique and to speculate which aspects of pineal research may benefit most by its application in the future.
Comparative Morphology of the Vertebrate Pineal Complex
Publisher Summary This chapter explores whether there is evidence for the assumption that the mammalian pineal organ is a complex rather than a single organ with a uniform function. It is pointed out that the pineal organ is structurally very complex, especially in rodents. The uneven development of the pineal organ in rodents and the topographical relationships of the pineal organs in other mammalian orders have prompted a classification which is based on the shape, the size, and localization of the pineal organ. A tentative classification of the pineal organs of thoroughly studied species shows that closely related species have similar types of pineals. Based on the different localization…
The shape of synaptic ribbons in the rat pineal gland
Under the transmission electron microscope, synaptic ribbons (SRs) of the mammalian pineal gland appear as rod-like organelles. Their three-dimensional structure is not precisely known. In the present study, pineal SRs were investigated using serial sections obtained from rats killed at noon and midnight. The shape of the SRs was reconstructed based on SR profile length and the number of sections in which the profiles were contained. The results obtained show that SRs are basically flat plate-like structures with polymorphic lateral edges. Reconstructions of SRs revealed that they had average dimensions of 300x150x35 nm and were 19.3% larger at night than at day; the difference in SR size p…
Alterations in the spontaneous activity of cells in the guinea pig pineal gland and visual system produced by pineal indoles
The indoles serotonin (SER), melatonin (MEL), 5-methoxytryptophol (5-MTL) and 5-hydroxytryptophol (5-HTL) were administered during daytime microelectrophoretically to 240 cells in the pineal gland of the guniea-pig. The action of SER and 5-HTL was predominantly depressant on the electrical activity, MEL and 5-MTL caused an excitation in most of the units. Although MEL and 5-MTL caused fairly similar reactions on average, they appear to act on different cells. The effects of microelectrophoretically applied MEL and 5-MTL on the spontaneous or evoked activity in the visual system (retinal ganglion cells, optic tract, lateral lateral geniculate body, superior colliculus) of the guinea-pig were…
Investigations on day-night differences of vesicle densities in synapses of the rat suprachiasmatic nucleus
The present study was conducted to test whether the well-known circadian alterations in physiological and metabolical parameters of the hypothalamic suprachiasmatic nucleus (SCN) are accompanied by day-night differences in the number of vesicles in intrinsic synapses of the nucleus. Two groups of 5 adult male rats each were killed at mid-light or mid-dark, respectively, by perfusion with Karnovsky's fluid. The SCN were removed and processed for routine electron microscopy. In medial parts of the nucleus, synapses were characterized as being of Gray type I (asymmetrical), Gray type II (symmetrical) or of intermediate form, and the vesicles per synaptic profile (VPSP) were counted over a defi…
Quantitative electron microscopic observations on Paneth cells of germfree and ex-germfree Wistar rats.
Ultrastructural changes of Paneth cells of germfree (Gf) rats which had been inoculated with bacteria-containing feces from conventionally-reared (SPF) rats were quantitatively examined. 12 and 24 h after inoculation, the Paneth cells showed a striking decrease in the number of secretory granules and the occurrence of large vacuoles. Phagosomes containing bacteria were not seen. After 4 days, the secretory granules reaccumulated and smooth-surfaced apical vesicles increased in number. It is discussed that the large vacuoles may be related to membrane-retrieval events following the massive extrusion of secretory granules whereas the apical vesicles appear to serve this function when exocytos…
Substance P- and calcitonin gene-related peptide-like immunoreactive neurons in the rat trigeminal ganglion — with special reference to meningeal and pineal innervation
Summary The distribution of perikarya showing substance P- ( SP ) or calcitonin gene-related peptide-like inununoreactivity ( CGRP-LI ) in the rat trigeminal ganglion ( TG ) were investigated by means of immunohistochemical methods. Approximately 50% of the perikarya contain CGRP while SP-Ll was observed in 1/3 of the cells. IR fibres were seen to leave the ganglion via the ophtalmic, maxillary, and mandibular nerves. The combination of peptide histochemistry and retrograde labelling of cells in the ganglion following injection of a fluorescent tracer into the pineal gland reveals that few SP- or CGRP-LI trigeminal neurons innervate the pineal gland. In contrast, the vast majority of perika…
In vitro effects of estradiol, testosterone, and progesterone on 5-methoxyindole content, cyclic adenosine 3',5'-monophosphate synthesis, and norepinephrine release in different parts of the female guinea pig pineal complex.
To examine the effects of estradiol, testosterone, or progesterone on cyclic adenosine 3',5'-monophosphate (AMP) accumulation, 5-methoxyindole levels, and norepinephrine (NE) release by the female guinea pig pineal complex, samples of the deep, intermediate, or superficial portions of the complex were incubated in vitro with varied concentrations of either hormone. Exposure for 10 minutes to physiological amounts of estradiol (10 nM) or to 100 microM NE increased significantly cyclic AMP levels to the same extent in the three pineal regions. A maximal effect on cyclic AMP accumulation was observed at 100-nM concentrations of estradiol, with a tendency to return to basal levels at 1-10 micro…
Biphasic 24-Hour Variations in Cyclic GMP Accumulation in the Rat Pineal Gland Are Due to Corresponding Changes in the Activity of Cytosolic and Particulate Guanylate Cyclase
Various parameters of the rat pineal gland display a 24-h rhythm. However, nothing is known about possible 24-h variations in cyclic GMP (cGMP) metabolism. In the present study, 24-h variations in pineal gland cGMP accumulation were investigated by determining the increase in cGMP level with and without inhibitors of phosphodiesterase at different time points over a light/dark cycle (12/12 h). Furthermore, the activity of guanylate cyclase (GC) was determined under substrate-saturated conditions regarding the cytosolic and particulate forms of the enzyme. It has been found that cGMP accumulation and GC activity display biphasic 24-h variations with two peaks--one approximately 7 h after lig…
The role of arginine-vasopressin for pineal melatonin synthesis in the rat: involvement of vasopressinergic receptors.
Abstract The endogenously synthesized nonapeptide arginine vasopressin (AVP) is thought to be involved in transduction of photic information to the pineal gland. The enhancement of circulating AVP leads to a suppression of the nocturnal melatonin surge the mechanisms of which are unknown so far. We therefore studied the effect of dDAVP, an AVP analog with antidiuretic but without vasopressor activity, on pineal melatonin synthesis in Sprague-Dawley and AVP-deficient Brattleboro rats. The nocturnal intra-arterial application of dDAVP mimicked the inhibitory effect of AVP on the activity of the rate-limiting enzyme for pineal melatonin synthesis, N-acetyltransferase (NAT), in both rat strains…
p-Chlorophenylalanine treatment depresses the number of synaptic ribbon profiles in the rat pineal gland, but does not abolish their day-night rhythm
It is largely unknown as to how the day/night rhythm of pineal synaptic ribbon number is regulated. Transcriptional events related to the nocturnal formation of new synaptic ribbons occur early in the morning, when pineal serotonin levels begin to increase. The present study was carried out in order to elucidate as to how altered serotonin levels affect the day/night changes in the number of synaptic ribbon profiles. To this end, male Sprague-Dawley rats received a single dose of p-chlorophenylalanine (pCPA, 300 mg/kg body weight, 72 hours before sacrifice), which depresses tryptophan hydroxylase activity and hence pineal serotonin levels. Control animals received saline injections. Experim…
Synaptic ribbons of the rat pineal gland: responses to in-vivo and in-vitro treatment with inhibitors of protein synthesis.
To elucidate the role of protein synthesis in the nocturnal increase of synaptic ribbons in the rat pineal gland, actinomycin-D, which inhibits transcription, and cycloheximide, an inhibitor of translation, were used. To assure that the drugs were effective and to relate morphological changes to pineal biosynthetic phenomena, the activity of N-acetyltransferase and levels of pineal indoleamine were measured. Results of in-vivo, short-term and long-term treatment with either drug suggest that transcription of proteins related to synaptic ribbon formation occurs during the first half of the light phase, whereas translation takes place during the first few hours of the dark phase. In contrast,…
Ultrastructural observations on the central innervation of the guinea-pig pineal gland.
In the present study the central innervation of the guinea-pig pineal gland was investigated. The habenulae and the pineal stalk contain myelinated and non-myelinated nerve fibres with few dense-cored and electron-lucent vesicles. Some myelinated fibres leave the main nerve fibre bundles, lose their myelin-sheaths and terminate in the pineal gland. Although direct proof is lacking, the non-myelinated fibres appear to end near the site where the bulk of the myelinated fibres are located. Here a neuropil area exists where synapses between non-myelinated fibre elements are abundant. Neurosecretory fibres were also seen. The results support the concept of functional interrelationships between h…
Tentative immunohistochemical demonstration of melatonin in the rat pineal gland
In the present study an attempt was made to demonstrate melatonin in the rat pineal gland by means of immunohistochemistry. The anti-body used was raised against 5-methoxy-N-acetyltryptophan which is chemically similar to melatonin. Specific fluorescence was demonstrable only in pineals from rats killed during the night, when melatonin formation is high. It was restricted to parenchymal cells lying in a marginal zone of the organ. These results are discussed in relation to a subdivision of the pineal parenchyma into cortical and medullary areas.
No Correlation of Pineal "Synaptic" Ribbon Numbers and Melatonin Formation in Individual Rat Pineal Glands
As previous circadian studies of pineal "synaptic" ribbon numbers and melatonin formation suggested that a positive correlation of the two variables exists, in the present investigation this problem was examined in individual pineal glands of rats killed at 1200 h and 2400 h, respectively. For this purpose, one half of the gland was processed for electron microscopy and the ribbons were counted in an area of 20,000 micron2 tissue; in the other half serotonin N-acetyltransferase (NAT) activity and melatonin content were determined. No correlation was found to exist between ribbon numbers and pineal NAT activity, pineal melatonin levels and serum melatonin levels, either at day- or at nightti…
Down-regulation of the nocturnally elevated guanylyl cyclase activity in the rat pineal gland
Abstract Previous studies have shown that in the rat pineal, the cytosolic and the particulate forms of guanylyl cyclase (GC) activity undergo a biphasic 24-h rhythm with two prominent peaks, one in the middle of the light phase and the other in the middle of the dark phase. In this study we investigated whether the well established photo-neural adrenergic regulatory processes identified for pineal melatonin synthesis also apply to the nocturnal elevation of GC activity. A 10-min light pulse given in the middle of the dark phase decreases the cytosolic and the particulate forms of GC. Administration of the β-receptor blocker propranolol did not depress the nocturnally elevated GC activity. …
A possible role for cyclic guanosine monophosphate in the rat pineal gland.
Abstract Adrenergic stimulation of pinealocytes induces an increase of both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). However, for cGMP no biological effects have been demonstrated so far. Therefore we tested the effects of the analog 8-bromo-cGMP on synaptic ribbon numbers and on melatonin synthesis as reflected by N -acetyltransferase (NAT) activity in the rat pineal gland in vitro. Incubation for 6 h with 8-bromo-cGMP did not change the activity of serotonin NAT but in increased the number of synaptic ribbons. These results indicate that cGMP is involved as a second messenger in the regulation of synaptic ribbon numbers in the rat pineal gland.
Lack of effect of oxytocin on the numbers of ?synaptic? ribbons, cyclic guanosine monophosphate and serotonin N-acetyltransferase activity in organ-cultured pineals of three strains of rats
In addition to the stimulating influence of the sympathetic system on the function of the mammalian pineal gland, neuropeptides such as neuropeptide Y, vasoactive intestinal polypeptide and arginine-vasopressin (AVP) are thought to function as modulators. Since AVP has been shown to influence pineal melatonin synthesis, the aim of the present study was to investigate the possible effects of the second hypothalamic nonapeptide oxytocin (OT), which likewise has been detected in the pineal gland. We therefore studied "synaptic" ribbon (SR) numbers, N-acetyltransferase (NAT) activity and the intracellular concentration of cyclic guanosine monophosphate (cGMP) following in vitro incubation of ra…
Day-night differences in the sensitivity of adrenoceptors in the Syrian hamster pineal gland: an in vivo iontophoretic study.
Abstract Investigations on the regulation of pineal melatonin synthesis in the Syrian hamster revealed distinct differences compared to this well-understood mechanism in rat. E.g., a circadian profile of pineal norepinephrine (NE) is absent, there is no β-adrenoceptor sensitivity during daytime and adrenergic receptor supersensitivity is not easily achieved. To elucidate the action of NE on pineal receptor sites, the effects of iontophoretic application of adrenergic compounds on spontaneous electrical discharge rates of pinealocytes were investigated during day- and nighttime. Following application of either NE, isoproterenol or clonidine, cells were activated, inhibited or not affected. W…
Ribbon synapses of the mammalian retina contain two types of synaptic bodies--ribbons and spheres.
The present paper reports that the synaptic bodies of the retinal ribbon synapses in rat, guinea pig, golden hamster and mouse are a heterogeneous population of organelles. In addition to the well-known synaptic ribbons sensu stricto which consist of a platelike electron-dense central structure surrounded by electron-lucent synaptic vesicles, there are what is termed synaptic spheres, in which the core is not platelike, but round to oval. In rat retinae procured at day, ribbons outnumbered spheres by a factor of 4. At night spheres were not seen in photoreceptor cells. Spheres, like ribbons, may lie some distance from the synaptic site, perhaps indicating transit from their site of origin t…
Electrophysiological properties of rat pinealocytes: Evidence for circadian and ultradian rhythms
Extracellular single-unit recordings were made during day- and night-time in the pineal gland of urethane-anesthetized adult male Sprague-Dawley rats. All cells exhibiting spontaneous electrical activity had firing frequencies from less than 1 Hz to about 100 Hz, and their discharge patterns were characterized as regular, irregular or bursting. While most of the spontaneously active cells (n = 163) showed a uniform activity level throughout the recording period (30-120 min), a group of 9 cells exhibited oscillatory rhythms with periods of 4-8 min. In addition, long-term recordings across day- and night-time from five cells revealed increasing activity during night-time in three cells, while…
A differential role of CREB phosphorylation in cAMP-inducible gene expression in the rat pineal
In the rat pineal gland cAMP mediates nocturnal induction of the enzyme arylalkylamine N-acetyltransferase (AA-NAT) as well as of transcription factors such as inducible cAMP early repressor (ICER), Fos-related antigen-2 (Fra-2) and JunB. Cyclic AMP stimulates the phosphorylation of the DNA binding protein cAMP response element binding protein (CREB). While cAMP-induced CREB phosphorylation appears to be a prerequisite for AA-NAT and ICER gene expression, it is not known whether CREB phosphorylation accounts for the full cAMP response of the two genes. Furthermore, the significance of CREB phosphorylation in cAMP-activated Fra-2 and JunB transcription is unknown. In the present in vitro stu…
Serine/Threonine Phosphatase Inhibitors Decrease Adrenergic Arylalkylamine N -Acetyltransferase Induction in the Rat Pineal Gland
Adrenergic regulation of the pineal enzyme serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87] accounts for the circadian rhythm in melatonin formation. In the present study, the role of protein phosphatases in the adrenergic regulation of rat pineal AA-NAT was investigated using specific inhibitors. In cultured pineals, the serine/threonine phosphatase type 1 and type 2A inhibitors okadaic acid and calyculin A significantly decreased adrenergically or cAMP-induced AA-NAT activity, whereas the serine/threonine phosphatase type 2B inhibitor cypermethrin and tyrosine phosphatase inhibitor dephostatin were ineffective. Reverse transcriptase-polymerase chain…
Sulfhydryl G Proteins and Phospholipase A2-Associated G Proteins Are Involved in Adrenergic Signal Transduction in the Rat Pineal Gland
The rat pineal gland with its circadian noradrenaline-regulated melatonin rhythm is an excellent model for studying adrenergic signal transduction with respect to cAMP and cGMP formation. The stimulatory G(s) proteins play a well-established role in this process. In contrast, the potential roles of the inhibitory G(i) proteins, the functionally unclear other G(o) proteins, and a number of G protein subtypes are not known. The present study examines the effects on beta(1)- and beta(1)-plus-alpha(1)-stimulated cAMP and cGMP formation of a number of G protein modulators in rat pinealocyte suspension cultures. The effects of the nitric oxide donor sodium nitroprusside on cGMP were also examined…
The pineal complex in Roman high avoidance and Roman low avoidance rats.
Previous studies have shown that the pineal gland of Roman high avoidance (RHA/Verh) rats is larger than that of Roman low avoidance rats (RLA/Verh). In the present study measurement of enzyme activities (serotonin-N-acetyl-transferase, hydroxyindole-O-methyltransferase) revealed that pineals of RHA/Verh rats are twice as active in melatonin production than pineals of RLA/Verh rats. Indoleamine content was also higher in RHA/Verh rats, whereas noradrenaline content was the same in both lines. When values were expressed per mg protein, these differences disappeared except for N-acetyl-serotonin and noradrenaline which were higher or lower in RHA/Verh rats, respectively. Both lines had higher…
Influence of melatonin and serotonin on the number of rat pineal ?synaptic? ribbons and spherules in vitro
Previous studies have shown that the "synaptic" ribbons (SR) and spherules (SS) of the mammalian pineal gland may respond differently under physiological and various experimental conditions. The aim of the present study was to gain insight into the mechanisms that may be responsible for the numerical changes of these organelles during a 24-h cycle. As the possibility exists that the structures are influenced by substances synthesized within the pinealocyte, rat pineal glands were cultured with and without added melatonin or serotonin, using an experimental protocol such that the addition of melatonin and serotonin mimicks the circadian changes of the respective substances within the pineal.…
Evidence for the presence of two 24-h rhythms 180 degrees out of phase in the pineal gland of male Pirbright-White guinea pigs as monitored by counting "synaptic" ribbons and spherules.
Previous studies have shown that the "synaptic" bodies of mammalian pinealocytes are a heterogeneous group of organelles. Whereas "synaptic" ribbons (SR) exhibit a day/night rhythm with small numbers during daytime and high numbers at night, the so-called "synaptic" spherules (SS) show species-specific differences in their rhythmicity. In the present study the numbers of SR and SS were monitored in male guinea pigs over a full 24-h period at 4-hourly intervals (LD 12:12, lights on at 7:00). The results obtained show that the rhythms of SR numbers and SS numbers are out of phase by 180 degrees. SR numbers are small during daytime and high at night, whereas SS numbers are high during daytime …
Adrenergic Stimulation of Cyclic GMP Formation Requires NO-Dependent Activation of Cytosolic Guanylate Cyclase in Rat Pinealocytes
Cyclic GMP (cGMP) formation in rat pinealocytes is regulated through a synergistic dual receptor mechanism involving beta- and alpha 1-adrenergic receptors. The effects of NG-monomethyl-L-arginine (NMMA), which inhibits nitric oxide (NO) synthase and NO-mediated activation of cytosolic guanylate cyclase, and methylene blue (MB), which inhibits cytosolic guanylate cyclase, were investigated in an attempt to understand the role of NO in adrenergic cGMP formation. Both NMMA and MB inhibited beta-adrenergic stimulation of cGMP formation as well as alpha 1-adrenergic potentiation of beta-adrenergic stimulation of cGMP formation, whereas they had no effect in unstimulated pinealocytes. The inhibi…
Day-night rhythm of acetylcholine in the rat pineal gland
Abstract Using high-performance-liquid-chromatography (HPLC) measurement of acetylcholine, choline acetyltransferase (ChAT) enzyme assay and anti-ChAT immunohistochemistry, we have investigated the expression of the cholinergic system in pineal glands of male rats. Glands procured during the day period (1200 h) contained significant amounts of acetylcholine (0.5 pmol/gland). A similar content was found in pineal glands after a 48 h culture period, i.e. when the intrapineal nerve fibres have degenerated. This strongly indicates that the pinealocytes are the cells which contain acetylcholine. To confirm this conclusion we demonstrate substantial ChAT-like immunoreactivity in pinealocytes. ChA…
Electrophysiological characterization of the pineal gland of golden hamsters.
In one of the most-widely used species in pineal gland research, the strongly photoperiodic golden hamster, Mesocricetus auratus, no electrophysiological data on pinealocytes are currently available. To fill this gap, in the present study 185 spontaneously active pinealocytes from male golden hamsters were recorded extracellularly, both during day- and night-time (light: dark cycle 12 ∶ 12, lights on at 07.00 h). As in other species, pinealocytes exhibited action potentials of 1–2 ms duration. An irregular firing pattern was observed in 95% of the pinealocytes, the remainder fired more regularly or showed a phasic discharge pattern. The firing frequencies ranged from 0.2 to 25 Hz and showed…
Morphological and electrophysiological evidence for habenular influence on the guinea-pig pineal gland
The central innervation of the guinea-pig pineal gland was investigated by histological and electrophysiological methods: Staining the pineal gland and the epithalamus, a double route of central innervation could be shown in the anterior part of the organ: (a) Fibres from the habenular nuclei, mainly from the lateral part, penetrate the organ via the pineal stalk. (b) Other fibres join the striae medullares and running in the habenulae reach the organ more dorsally. The fibres end in the intercellular space where they form a dense network. In 15 male guinea-pigs under urethane anesthesia, two series of unit recording experiments were performed: (a) Recordings were made from 128 units in the…
Plasticity of retinal ribbon synapses.
Ribbon synapses differ from conventional chemical synapses in that they contain, within the cloud of synaptic vesicles (SV's), a specialized synaptic body, most often termed synaptic ribbon (SR). This body assumes various forms. Reconstructions reveal that what appear as rod- or ribbon-like profiles in sections are in fact rectangular or horseshoe-shaped plates. Moreover, spherical, T-shaped, table-shaped, and highly pleomorphic bodies may be present. In mammals, ribbon synapses are present in afferent synapses of photoreceptors, bipolar nerve cells, and hair cells of both the organ of Corti and the vestibular organ. Synaptic ribbons (SR's) are also found in the intrinsic cells of the third…
In vitro synthesis of dopamine and noradrenaline in the isolated rat pineal gland: day-night variations and effects of electrical stimulation.
: Isolated rat pineal glands were incubated in vitro in a medium containing [I4C]dopamine or [14C]tyrosine, and the tissue contents of l4C-labelled and total dopamine and noradrenaline were determined by HPLC followed by electrochemical detection and scintillation spectrometry. During incubation with [l4C]dopamine, the labelled amine accumulated in pineal glands and was partially converted into [l4C]noradrenaline. Nomifensine, a neuronal amine uptake blocker, largely inhibited the accumulation of [l4C]dopamine and the formation of [14C]noradrenaline. These experiments demonstrated dopamine β-hydroxylase activity in the sympathetic nerves of the pineal gland. During incubation with [14C]tyro…
The effects of a number of short-term exogenous stimuli on pineal serotonin-N-acetyltransferase activity in rats
The present study deals with the question as to what extent the sympathetically innervated rat pineal gland is affected by a number of short-term exogenous stimuli given during day-time, as assessed by measuring pineal serotonin-N-acetyltransferase activity (NAT) which is directly proportional to melatonin formation. In male Sprague-Dawley rats kept under LD 12:12 pineal NAT was statistically significantly depressed by physical immobilization for 2 hours, swimming for 15 min in water of 10 and 30 degrees C, exposure for 2 hours to cold (5 degrees C) or heat (40 degrees C), noise (90 db) for 2 hours and hunger for 17 hours. An increase in NAT was noted after swimming for 15 min in water of 2…
Synaptic ribbons, spheres and intermediate structures in the developing rat retina
The present study was conducted to investigate the qualitative and quantitative development of synaptic bodies in retinae of Wistar rats during postnatal days 4-28. In addition, the effects of different light regimens and of eye pigmentation on SB numbers were studied. Synaptic bodies were counted and measured in the outer plexiform layer of retinal tissue fixed and processed by routine electron microscopical techniques. At postnatal days 4 and 5, retinae showed only few synaptic bodies. The main numerical development of synaptic bodies occurred between postnatal days 4 and 9, numbers remaining more or less constant thereafter. The intracellular location of synaptic ribbons changed from pre…
Light and drug induced changes of epiphysial synaptic ribbons.
In the present investigation experiments were carried out to determine whether the functionally obscure synaptic rebbons 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 (nor…
No effects of pulsed radio frequency electromagnetic fields on melatonin, cortisol, and selected markers of the immune system in man.
There is growing public concern that radio frequency electromagnetic fields may have adverse biological effects. In the present study eight healthy male students were tested to see whether or not radio frequency electromagnetic fields as used in modern digital wireless telecommunication (GSM standard) have noticeable effects on salivary melatonin, cortisol, neopterin, and immunoglobulin A (sIgA) levels during and several hours after exposure. In a specifically designed, shielded experimental chamber, the circularly polarized electromagnetic field applied was transmitted by an antenna positioned 10 cm behind the head of upright sitting test persons. The carrier frequency of 900 MHz was pulse…
Perspectives of Comparative Anatomy of the Mammalian Pineal Gland
As the precise role of the pineal gland has been established in a few mammalian species only, every effort should be made to elucidate the function of this potentially important neuroendocrine organ in as many species as possible. One way in which the morphologist can contribute to reach this goal is to carry out comparative studies. Comparative anatomical studies with respect to the pineal gland can be, and should be, carried out at different structural levels: (a) At the gross anatomical level; (b) at the tissue level; (c) at the cellular level, and (d) at the level of cell organelles.
Arylalkylamine N-acetyltransferase gene expression in retina and pineal gland of rats under various photoperiods
Abstract The present study examines how the circadian oscillators in the retina and the suprachiasmatic nucleus (SCN) respond to changes in photoperiod. ArylalkylamineN-acetyltransferase (aa-nat) gene expression studied by quantitative RT-PCR revealed that in adult Sprague–Dawley rats kept under different light–dark (LD) cycles for two weeks the temporal pattern of AA-NAT mRNA expression was identical in retina and pineal gland. In both tissues, the time span between the onset of darkness and the nocturnal rise in AA-NAT mRNA expression was 3 h under LD 20:4, 6 h under LD 12:12, and 15 h under LD 4:20. As aa-nat expression in the pineal gland is regulated by the circadian oscillator in SCN,…
Modulatory role of acetylcholine in the rat pineal gland
Abstract The function of acetylcholine (ACh) in the mammalian pineal gland is unknown. To test the hypothesis that ACh exerts a modulatory role in this organ, in the present study electrophysiogical multiunit recordings were carried out in ex-vivo rat pineal glands superfused with different drugs. It was found that ACh (10 −7 M) as well as the cholinergic agonists oxotremorine (10 −7 M) and nicotine (10 −6 M) increased the discharge rates of most of the spontaneously active units and led to burst activity in previously regularly firing cells. It is concluded that ACh may play a modulatory role in the pineal by influencing the firing of a special population of pineal cells with perhaps recep…
Expression of Nitric Oxide Synthase I Determines the Strength of Adrenergic Cyclic GMP Response in the Rat Pineal Gland
In the rat pineal gland the adrenergic cGMP response is not static but changes during postnatal development and depends on environmental lighting conditions. Since the adrenergic cGMP transduction is mediated by stimulation of the enzyme nitric oxide synthase I (NOS I), we investigated whether changes in the strength of adrenergic cGMP response coincide with the expression of NOS I. Using immunoblot analysis, we found that the increase in adrenergic cGMP transduction in the second postnatal week coincides with enhanced NOS I expression. Similarly, we observed that changes in the intensity of adrenergic cGMP transduction under different environmental lighting conditions correspond to NOS I e…
In rat pinealocytes the cyclic GMP response to NO is regulated by Ca2+ and protein kinase C
There is ample evidence that beta-adrenergic stimulation of cyclic GMP formation is potentiated by alpha1-adrenergic mechanisms, the latter leading to elevation of intracellular Ca2+ concentration ([Ca2+]i) and protein kinase C (PKC) activation. Recent studies have shown that nitric oxide synthase (NOS) and nitric oxide (NO) are a component of the adrenoceptor-cyclic GMP signalling pathway. The aim of the present investigation was to study the roles of alpha1-adrenergic mechanisms, Ca2+ and PKC on NO-stimulated cyclic GMP formation. To this end suspension cultures of rat pinealocytes were treated with the NO donor sodium nitroprusside (SNP) in the presence of alpha1-adrenergic agonists, [Ca…
Electrophysiological evidence for circadian rhythmicity in a mammalian pineal organ
Long-term electrophysiological recordings from the guinea-pig pineal organ show that three types of intrinsic cells can be distinguished: (i) Cells showing constant firing rates over periods of up to 24 hours. (ii) Cells which are highly active during the day and show a low firing rate during the night. (iii) Cells which exhibit low activity during the day and enhanced activity during the night; these cells can be strongly inhibited by 1 min of light given during the night. Both the light- and darkness-activated cells show activity patterns which closely follow season-dependent differences in day- and night-lengths. In addition, both cell types show an oscillatory pattern in maintained acti…