Neonatal development of circadian rhythm in “synaptic” ribbon numbers in the rat pinealocyte

Populations of “synaptic” ribbons (SR) in pinealocytes of the rat pineal gland at various ages from the neonatal period through early adulthood were analyzed morphometrically. Diurnal and nocturnal numbers of SR were small on day one but had increased dramatically by the end of the first week and beginning of the second week. By day 10, numbers of SR had peaked. Thereafter nocturnal numbers of SR demonstrated no significant increases or decreases. However, between days 10 and 19, diurnal numbers of SR decreased precipitously, appearing to have leveled off at the onset of puberty to levels observed in adults. SR were never scarce or absent in any age group studied. The neonatal appearance of large numbers of SR coincides developmentally with the initial sympathetic innervation of pineal parenchyma. Circadian rhythm in numbers of SR begins shortly thereafter. It is suggested (1) that circadian rhythm in numbers of SR is initiated and regulated or modified by sympathetic innervation of pinealocytes containing SR, and (2) that SR formation may be related to the intracellular flow of membrane relative to the transport and release of specific pineal products.     

Electron microscopical studies on axonal degeneration in the rat iris following ganglionectomy

Superior cervical ganglionectomy causes a very rapid and complete distintegration of adrenergic axons in the rat iris commencing less than 24 hours after operation. At 48 hours post-operatively the electronmicroscopical appearance of the iris is normal with osmium tetroxide fixation because the debris has vanished and the abundant surviving cholinergic axons in the autonomic ground plexus give the impression that the total innervation is still intact. However, with permanganate fixation the denervated iris examined 48 hours to one year after operation lacks granular vesicles in its axons and appears in this single respect different from normal. After ciliary ganglionectomy the same kind of degeneration of individual axons begins within 24 hours, but continues for several days, involving a greater number of axons than the previous operation. Removal of both sets of ganglia intensifies the loss of axons and causes some atrophy of the iris tissues. A residue of apparently intact axons persists, however, for as long as ten months. The previously discordant results of ganglionectomy on iris innervation obtained by light microscopy are shown to be dependent on technical inadequacies, combined with the fact that in the earlier literature it was not known that there is a close intermingling of adrenergic and cholinergic axons in all parts of the iris plexuses.     

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

Summary 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 hydoxyindole-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 for electron microscopy and for the determination of NAT and HIOMT activities. No differences in pineal SR number, size or location were found in rats stimulated with either parameters when compared with sham-stimulated or control animals. While the activity of HIOMT remained unchanged, the activity of NAT was also unaltered following 15 min of stimulation, but was augmented approximately three-fold in animals stimulated for 120 min. It is concluded that if SR in the rat pineal gland are under sympathetic control, the regulation is different from that involved in melatonin formation.     

Inverse behaviour of "synaptic" ribbon and spherule numbers in the pineal gland of male guinea-pigs exposed to continuous illumination

Summary 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 control animals kept under a lighting regiment of 12 h light and 12 h dark. It was found that SR numbers increase following LL whereas SS numbers decrease. The proximal, intermediate and distal parts of the dumbbell-shaped organ respond differently. The increase in SR numbers is significant in the distal and intermediate regions only, whereas the decrease in SS numbers is significant in the proximal and the intermediate regions only. Within each pineal region analyses of parenchymal subareas measuring 65 m by 65 m exhibit an inverse correlation of SR and SS numbers indicating that there are parenchymal domains in which either SR or SS predominate. Morphometric analyses of a number of pinealocytic parameters reveal minor differences between different pineal regions and that exposure to LL does not strikingly affect the pinealocyte perikarya. By contrast, the numbers of pinealocyte processes increase significantly after LL in the distal and intermediate, but not the proximal region of the pineal gland. These observations suggest structural and functional differences between different parts of the guinea-pig pineal gland.     

Adrenalectomy but not gonadectomy affects rat pineal beta-adrenergic receptor density

Bilateral adrenalectomy, like chronic pineal denervation by means of superior cervical ganglionectomy, induced an increase in the morning levels of [125I]iodopindolol (IPIN) binding to rat pineal membranes; this change may be related to the reduced input of catecholamines to the pinealocytes as suggested by the decrease in nocturnal pineal melatonin synthesis previously reported in adrenalectomized rats. Castrated rats did not show changes in IPIN binding to pineal membranes when measured either at middark or in chronically superior cervical ganglionectomized rats, suggesting that the regulation of pineal beta-adrenergic receptors is independent from gonadal control.     

Pup circadian rhythm entrainment--effect of maternal ganglionectomy or pinealectomy

In rodents, during late embryonic and early neonatal development, circadian rhythms develop in synchrony with those of their mothers, which in turn are synchronized with the environmental photoperiod.This paper examines the effect of maternal ganglionectomy (pineal gland sympathetic denervation) or extirpation of the pineal gland on pups' drinking rhythms, a behavior that is continuously monitored in individual animals starting after weaning and studied up to 3 weeks later. Maternal ganglionectomy or pinealectomy performed on the 7th day of gestation significantly disrupts rat pups' drinking behavior, within and among litters. In both treatments, circadian rhythm characteristics of the free-running period (tau), phase, amplitude and alpha were significantly altered compared to those of the control pups born from sham-operated mothers. With the exception of the alpha component, both maternal treatments have similar effects. When melatonin was given to the mothers instead of the endogenous pineal secretory activity for 5 days during the late period of gestation, this treatment reversed the effects of maternal ganglionectomy and pinealectomy. These observations, together with previous studies of our group, indicate that the maternal superior cervical ganglia and pineal gland are necessary components of the mechanism for maternal synchronization, and that maternal melatonin may, directly or indirectly, affect the performance of the pups' central oscillator during early pup rat development.     

Neural control of glycogen content and its diurnal rhythm in mouse pineal cell.

In adult male dd mice, possible mechanisms regulating the glycogen content in the pineal cell were investigated by a semiquantitative histochemical method, with particular reference to the role of the sympathetic innervation. Reserpine, superior cervical ganglionectomy (SCGX), or decentralization of the ganglia (DC), as well as continuous light, prevented the nocturnal decrease in the glycogen content, causing a marked increase, and caused a gradual decrease in the size of the pineal cell. In the SCGX or DC group, the glycogen content reached a peak at 2 days and then decreased gradually. The nocturnal decrease was also prevented by propranolol. Noradrenaline caused a marked decrease in the glycogen content. These findings support the hypothesis that the glycogen metabolism and its diurnal rhythm in the pineal cell are regulated by the sympathetic nerve terminals innervating the pineal gland, presumably by the release of noradrenaline. In addition, the nature of the internal mechanism in the organism generating the pineal glycogen rhythm was examined. Light was considered to induce a phase shift in such a mechanism, but reserpine was not.     

Innervation and functional changes in mesenteric perivascular calcitonin gene-related peptide- and neuropeptide Y-containing nerves following topical phenol treatment

We have previously shown that age-related reduction of innervation and function in mesenteric perivascular calcitonin gene-related peptide-containing vasodilator nerves takes place in spontaneously hypertensive rats. The present study was performed to investigate innervation and functional changes in perivascular calcitonin gene-related peptide- and adrenergic neuropeptide Y-containing nerves after topical treatment with phenol, which damages nerve fibers, around the rat superior mesenteric artery. Under pentobarbital-Na anesthesia, 8-week-old Wistar rats underwent in vivo topical application of phenol (10% phenol in 90% ethanol) or saline (sham rats) to the superior mesenteric artery proximal to the bifurcation of the abdominal aorta. After the treatment, the animals were subjected to immunohistochemistry of the 3rd branch of small arteries proximal to the intestine and to vascular responsiveness testing on day 3 through day 14. The innervation levels of calcitonin gene-related peptide-like immunoreactivity containing fibers and neuropeptide Y-like immunoreactivity containing fibers were markedly reduced on day 3 to day 14 and on day 5 to day 14 after the treatment, compared with those in sham-operated rats, respectively. In perfused mesenteric vascular beds isolated from phenol-treated rats, adrenergic nerve-mediated vasoconstriction and calcitonin gene-related peptide nerve-mediated vasodilation in response to periarterial nerve stimulation (2-12 Hz) were significantly decreased on day 3 and day 7. Neurogenic release of norepinephrine in phenol-treated rats on day 7 was significantly smaller that that in sham-operated rats. Nerve growth factor content in the mesenteric arteries of phenol-treated rats was significantly lower than that in sham-operated rats. Administration of nerve growth factor using osmotic mini-pumps for 7 days after the phenol treatment resulted in greater density of calcitonin gene-related peptide- and neuropeptide Y-like immunoreactivity fibers than in phenol-treated rats and restored decreased vascular responses to periarterial nerve stimulation. These results suggest that topical phenol-treatment of the mesenteric artery effectively induces functional denervation of perivascular nerves, which can be prevented or reversed by nerve growth factor treatment.     

Focal axonal injury: the early axonal response to stretch

Summary The development of a model for axonal injury in the optic nerve of the guinea pig has allowed analysis of early morphological changes within damaged axons. We provide evidence that the initial site of damage after stretch is the nodes of Ranvier, some of which develop nodal blebs. The development of nodal blebs is correlated with the loss of subaxolemmal density, disruption of the neurofilament cytoskeleton and aggregation of membranous profiles of smooth endoplasmic reticulum. Nodal blebs are numerous 15 min after injury but less so at later survivals. The glial-axonal junction is intact at early survivals in damaged nodes. Marked accumulation of membranous organelles occurs in the paranodal and internodal regions adjacent to damaged nodes between two and six hours and is correlated with disruption of the myelin sheath. Axotomy and the formation of degeneration bulbs occurs between 24 and 72 h. The area of axonal injury is invaded by phagocytic cells by 72 h and large numbers of myelin figures occur within the neuropil until 14 days.The results are compared with those of other studies of diffuse axonal injury and other neuropathies. The time course of axonal changes is more rapid than during Wallerian degeneration. Our data from longer surviving animals is exactly comparable with published data. We are confident that the principal site of axonal injury is the node of Ranvier. We suggest that damage at the node results in disruption of axonal transport, which in turn leads to a cascade of events, culminating in axotomy between 24 and 72 h after the initial insult.     

Age-related changes in pineal “synaptic” ribbon populations in rats exposed to continuous light or darkness

“Synaptic” ribbon (SR) populations in the rat pineal gland were analyzed morphometrically at various ages from birth to early adulthood. The rats were born and raised in continuous light or continuous darkness. SR numbers were high irrespective of the presence or absence of light during the early neonatal period. However, SR numbers decreased substantially during the second neonatal week of continuous light, remaining low at subsequent ages, including early adulthood. In contrast, SR numbers apparently remained elevated during continuous darkness at various ages from the early neonatal period to early adulthood. Stimulation of pineal adrenergic receptors with L-isoproterenol during continuous light resulted in an acute, reversible increase in SR numbers; blockade of these receptors with L-propranolol during continuous darkness resulted in an acute decrease in SR numbers. These results indicate that light-mediated changes in SR populations were (1) age related and (2) related to some component of the pineal beta-adrenergic receptor mechanism. A hypothesis that SR formation may be related to beta-adrenergic receptor-associated membrane turnover is presented.