Showing papers in "Journal of Pineal Research in 1984"

Effects of pinealectomy and aging on the serum corticosterone circadian rhythm in rats.

Abstract: Male Sprague-Dawley rats were housed in alternate light/dark conditions (light on, 7:00 AM, light off, 7:00 PM). Corticosterone was determined by radioimmunoassay from blood samples that were obtained by tail clip at 4-h intervals. Pinealectomized animals have shown significant increase of corticosterone levels at 7:00 AM, 11:00 AM and 7:00 PM in comparison with 2-month-old intact rats. There were no differences in serum corticosterone rhythm between 24-month-old and pinealectomized animals. Twelve-month-old rats have shown significant increase of corticosterone levels at 7:00 and 11:00 AM in comparison with 2-month-old animals. The age-associated increase of serum corticosterone and the similarity between serum corticosterone circadian rhythm in aged and pinealectomized animals suggest that an age-related decrease in melatonin production [Reiter et al., 1981] may contribute to age related changes of hypothalamic-pituitary adrenal axis regulation.

The Influence of Various Irradiances of Artificial Light, Twilight, and Moonlight on the Suppression of Pineal Melatonin Content in the Syrian Hamster

Abstract: The purpose of the present studies using artificial light was to determine how the timing and duration of exposure influence the light-induced suppression of pineal melatonin levels in hamsters. An 8-min exposure to 0.186 microW/cm2 of cool white fluorescent light caused a continued depression of pineal melatonin even when animals were returned to darkness. In addition, the pineal gland does not appear to change its sensitivity to light throughout the night. A 20-min exposure to 0.019 microW/cm2 of cool white fluorescent light did not significantly suppress pineal melatonin during any time of the melatonin peak, whereas a 20-min exposure to 0.186 microW/cm2 was capable of always suppressing melatonin. Furthermore, increasing the duration of 0.019-microW/cm2 exposure to 30, 60, 120, or 180 min does not increase the capacity of this irradiance to depress melatonin. Similar to artifical light, natural light has a variable capacity for suppressing nocturnal levels of pineal melatonin. Twilight irradiances of 0.138 microW/cm2 or less did not suppress nocturnal melatonin whereas twilight irradiances of 3.0 microW/cm2 or greater did suppress pineal melatonin. A few animals did have lower melatonin after a 40-min exposure to full moonlight during July (0.045 microW/cm2) or January (0.240 microW/cm2). However, pineal melatonin levels remained high in the majority of animals exposed to full moonlight.

Distribution of substance P neurons in the epithalamus of the rat: an immunohistochemical investigation.

Abstract: Substance P (SP) immunoreactivity was studied in the epithalamus of intact and superior cervical ganglionectomized female rats by using the peroxidase-antiperoxidase immunocytochemical technique. Cells containing immunoreactive SP were located in the medial habenula, and SP fibers were observed in the lateral habenula, habenular commissure, pineal stalk, and the pineal organ. Bilateral removal of the superior cervical ganglia did not affect the immunocytochemical staining of SP-containing cells and fibers 15-30 days later. It is suggested that habenular neurons containing SP send projections into the pineal organ of the rat.

Pineal Neural Connections With the Brain in Two Teleosts, the Crucian Carp and the European Eel

Abstract: The neural connections between the pineal organ and the brain were investigated in two teleost species: the crucian carp and the European eel. Horseradish peroxidase (HRP) was administered into the pineal parenchyma in vivo or in vitro. After 12-96 hours, the brains were fixed, and HRP was reacted in frozen sections with either diaminobenzidine (DAB) or Hanker-Yate's reagent. The HRP had entered the pineal neurons, and had labeled their axonal processes (the pineal tract) in a Golgi-like manner. In both species, HRP-labeled axons were observed in the habenular nuclei, the pretectal area, the dorsal and ventral thalamus, the dorsal periventricular tegmentum, and in the posterior periventricular hypothalamus. In the eel, single axons were also observed in the anterior hypothalamus and in the brain stem. Thin, varicose (preterminal) axons were observed in great numbers in the pretectal area and dorsal thalamus in both species, and in small numbers in the habenular nuclei and posterior periventricular hypothalamus. For comparison, the retinofugal projections were visualized after intraocular HRP-injection. A close association of retinal and pineal terminal fields were noted in the pretectal area and the dorsal thalamus, whereas other diencephalic retinal and pineal recipient areas seem more segregated.

Comparison of the Effects of β‐Adrenergic Agents on Pineal Serotonin N‐Acetyltransferase Activity and Melatonin Content in Two Species of Hamsters

Abstract: The nighttime rise in pineal melatonin levels can be blocked by administration of the beta-adrenergic receptor antagonist, propranolol, in both Syrian hamsters and rats. Although the administration of beta-adrenergic receptor agonists such as norepinephrine or isoproterenol stimulates pineal melatonin production in the rat, these drugs are without apparent effect on indole production in the Syrian hamster. To determine whether this lack of stimulatory effect in the Syrian hamster is characteristic of this species, a comparison of the effects of norepinephrine and isoproterenol on pineal serotonin N-acetyltransferase activity and melatonin content was conducted. In contrast to their lack of effect in the Syrian hamster, norepinephrine and isoproterenol stimulated pineal serotonin N-acetyltransferase activity and melatonin content in the Djungarian hamster. Hourly injection of norepinephrine during a continuation of light into the normal dark period stimulated increases in the activity of serotonin N-acetyltransferase and melatonin content in the Djungarian hamster but was without effect on these pineal parameters in the Syrian hamster.

Characterization of dopaminergic receptor sites in bovine pineal gland.

Abstract: In addition to beta-adrenergic receptor agonists, L-dopa and dopamine have been also shown to activate the production of melatonin and its synthesizing enzyme, serotonin N-acetyltransferase. In an attempt to characterize dopaminergic receptor sites, bovine pineal synaptosomes were prepared by differential centrifugation techniques. Washed disrupted synaptic membranes were used to study 3H-spiroperidol binding, using standard membrane-binding techniques. Association of 3H-spiroperidol to pineal membranes was very rapid, reaching equilibrium within 2 min and remaining stable for 20 min. Dissociation was also rapid with a t 1/2 of 3 min. Analysis of saturation studies (0.035 to 20 nM spiroperidol, 16 concentrations) using the LIGAND program indicated the presence of two binding sites with KDS (dissociation equilibrium constant) of 0.18 nM and 2.1 nM. The Bmax's (receptor density) of the sites were 37 and 630 fmoles/mg protein respectively. The IC50S of haloperidol, cis-flupenthixol, and chlorpromazine were 8, 12, and 80 nM, respectively, while those of pipamperone, cyproheptadine, and cinanserin were 60, 400, and 1500 nM. These and other data indicate that the most abundant site is a dopamine D2 receptor while the less abundant site may be a serotonin receptor. The function of dopamine and dopaminergic neurons in bovine pineal gland is not known and has not been established.

Influence of the pineal gland and circadian rhythms in circulating levels of thyroid hormones of male hamsters

Abstract: Thyroxin (T4) and triiodothyronine (T3) were measured by radioimmunoassay in serum of hamsters sacrificed at 4-hr intervals throughout the daily light-dark cycle (14L/10D). Both T4 and T3 concentrations increased significantly during the L period of the daily cycle and decreased during the D period of the cycle; A.M. versus P.M. differences in free thyroxin indices (FTI) were also studied using the T4 and T3 uptake assays of Nuclear Medical Laboratories (Dallas, Texas). The free thyroxin index was significantly greater in serum samples of hamsters sacrificed at 7 P.M. than at 7 A.M. (lights on at 6:30 A.M.). Serum taken at 7 P.M. had less unsaturated binding sites than serum taken at 7 A.M. No significant A.M. versus P.M. differences in free thyroxin index were found in blind hamsters, although blind hamsters had significantly lower T4 and FTI than controls. Placing melatonin in the drinking water at a dose of 80 micrograms/ml did not significantly influence hormone levels. The greatest difference in hormone concentrations between control and blinded hamsters was found in P.M. samples. Blind hamsters had FTIs that were 48% of P.M. controls. Pinealectomy prevented the effects of blinding on T4 levels and FTIs.

Increases in volume, fluid content, and lens weight of eyes following systemic administration of melatonin.

Abstract: Melatonin's effects were studied in male golden hamsters (Mesocricetus auratus) distributed among five surgical groups (nonoperated, sham-pinealectomized, sham-pinealectomized plus black plastic shielding of the pineal region, pinealectomized, and pinealectomized plus black plastic shielding of the pineal region) and three injection groups (vehicle only, 25 micrograms melatonin, and 2,500 micrograms melatonin). Injections (s.c.) were daily for 28 d at L11 to L11.75 in a (light:dark) L:D 14:10 artificial photoperiod. Animals (N = 112) were killed and dissected on the day after the last injection (at 55-65 d of age). None of the surgical procedures affected weights of eyes or their parts, nor did they influence the effects of administered melatonin on the eyes. Melatonin caused an increase in absolute and relative eye weight and an increase in fluid content of intraocular space. The magnitudes of these effects were positively related to melatonin dose. These same eyes had a progressively lower weight of nonlenticular tissues with low to high doses of melatonin, probably in relation to greater fluid content, and suspected increase in intraocular pressure. Lens wet and dry weights were significantly greater in animals receiving melatonin, but only at the high dose. These actions of melatonin are likely to be direct and are shown to not require the presence of the pineal. Experiments of other designs are suggested in order to determine whether the effects of the low, near physiological, dose of melatonin represent physiological actions of endogenous melatonin, synthesized and released within the eye. However, effects of large doses of melatonin on the eye are still noteworthy in relation to interpretation of experiments employing such dosages, and of disease states involving changes in intraocular pressure.

Structure and ultrastructure of the aging rat pineal gland.

Abstract: The pineal gland of rats 12-28 months old was studied with light and electron microscopes. All pineal components exhibited regressive changes of different intensity with age. In type I pinealocytes, there was a marked increase in dense bodies as well as the occasional appearance of wide cell profiles full of vesicles. Type II pinealocytes showed nuclear infoldings and cytoplasmic deposits of lipofuscin. Pineal stroma displayed an increase in connective tissue fibers, both collagen and oxytalan, as well as remains of basement membranes and other materials of unknown nature. Calcareous concretions were also found, mostly in the pineal capsule. All regressive changes were more intense with increasing age.

Studies on the kinetics of melatonin and N-acetylserotonin in the rat at mid-light and mid-dark.

Abstract: The distribution and disappearance of melatonin and N-acetylserotonin after intravenous injection were studied at mid-light and mid-dark. Male rats were adapted under a photoperiod of 12 h light and 12 h dark cycle for a minimum of 1 week. After the intravenous injection of melatonin (10 micrograms), N-acetylserotonin (10 micrograms), or 3H-melatonin (76 ng), blood samples were collected at 1, 3, 5, 10, 20, 30, 45, 60, 75, 90, and 120 min. The concentrations of indoles in the plasma samples were determined by radioimmunoassay, while the radioactivity of 3H-melatonin was determined by scintillation counting. The distribution of melatonin and N-acetylserotonin followed a two-compartment model, showing an initial distribution phase followed by an elimination phase. The biological half-life of melatonin at mid-light and mid-dark were similar but the volume of distribution (V beta and Vc) and metabolic clearance rate (C1s) were greater at mid-dark than at mid-light. The estimated melatonin secretory rate at mid-dark was fivefold greater than that at mid-light. When 3H-melatonin was used, the half-life was significantly longer. A diurnal difference in the distribution was also evident in the N-acetylserotonin studies. The volume of distribution (V beta and Vc) was greater at mid-dark while the C1s was not significantly faster at mid-dark compared with that at mid-light. However, the estimated N-acetylserotonin secretory rate at mid-dark was eightfold greater than at mid-light.

Effect of short photoperiod on hypothalamic methionine-enkephalin and LHRH content and serum beta-endorphin-like immunoreactivity (beta-end LI) levels in golden hamsters.

Abstract: Adult female golden hamsters were used to study the effect of short photoperiod on the endogenous opioid system and the effect of pinealectomy on the serum beta-endorphin-like immunoreactivity (beta-end LI) levels. Hamsters were housed under either long photoperiod (14L:10D) or short photoperiod (2L:22D) and the regularity of the estrous cycles was determined by daily vaginal exfoliative cytology. Hamsters under short photoperiod became acyclic after about 7 wk. At the end of 8 wk, all the hamsters were decapitated and medial basal hypothalamic (MBH) content of LHRH and methionine-enkephalin (met-enkephalin) were measured by specific radioimmunoassays (RIA). Both LHRH and met-enkephalin levels of the MBH were significantly elevated in the short-photoperiod hamsters as compared to the normally cycling control animals under long photoperiod. In a second experiment, the effect of pinealectomy (PNX) on the serum levels of beta-end LI in the short-photoperiod hamsters was determined. The serum beta-end LI levels were increased approximately threefold in the noncyclic hamsters housed under 8 wk of short-photoperiod conditions. Pinealectomized hamsters kept under 8 wk of short-photoperiod exhibited lower serum beta-end LI levels similar to those of normally cycling hamsters kept under long photoperiod. These results indicate a possible functional relationship between increased pineal activity (as a result of short photoperiod) and increased MBH met-enkephalin, LHRH, and serum beta-end LI levels.

Cytochemical analysis of calcium distribution in the superficial pineal gland of the Mongolian gerbil.

Abstract: The potassium pyroantimonate-osmium method was employed to determine the distribution of cations in the superficial pineal gland of the Mongolian gerbil. The reaction product in the glands was presumed to be calcium following the use of both the chelator EGTA and x-ray microanalysis. A gradient of reaction product was found in the pineal with the highest concentrations occurring in and around the pineal concretions (PC). The periphery of mature PC demonstrated a heavy precipitate as did the surrounding pinealocytes. PC-associated pinealoeytes contained reaction product in many organelles, including small vesicles that appeared to be opening in the direction of the concretions. The vacuoles of the pinealocytes contained various concentrations of precipitate. The results obtained in the present investigation have led to a hypothesis on the formation of pineal concretions: (1) a pinealocyte vacuole is formed, (2) calcium is transported to the vacuole, (3) the cation content of the vacuole increases, and (4) the cations precipitate into an immature concretion. Fibrillar material and an amorphous substance located within the pinealocyte vacuole appear to form cation containing vesicles that contribute to the periphery of the developing PC, resulting in the continued growth of the concretion.

Melatonin inhibits prostaglandin E release from the medial basal hypothalamus of pinealectomized rats.

Abstract: The effects of pinealectomy and of melatonin administration on prostaglandin E synthesis in the medial basal hypothalamus were studied in male rats. Melatonin treatment significantly decreased prostaglandin E release from the medial basal hypothalamus in pinealectomized rats. The results of the present study suggest that melatonin modulates hypothalamo-hypophyseal function, at least in part, via inhibition of hypothalamic prostaglandin synthesis.

Quantitative cytological analysis of functional changes in adrenomedullary chromaffin cells in normal, sham-operated, and pinealectomized rats in relation to time of day: I. Nucleolar size.

Abstract: Adrenaline (A)- and noradrenaline (N)-cells in juxtacortical and central areas of adrenal medullas of nonoperated, sham-operated, and pinealectomized male rats (n = 125) were investigated by quantitative electron and light microscopy. Animals were killed at eight time points during a standardized 24-h, light-dark (12:12) cycle 14 d after surgery. Diameters of nucleoli (n = 9,600) and the nucleolar margination rate were studied chiefly by light microscopy. Major findings include the following: Twenty-four-hour changes occurred in nucleolar diameters in adrenomedullary chromaffin cells in nonoperated animals, with the peak in the late dark phase (X +/- SD, A-cells: 1.29 +/- 0.06 micron; N-cells; 1.23 +/- 0.05 micron) and the minimum in the mid-light (A-cells: 1.16 +/- 0.04 micron) or early dark (N-cells: 1.14 +/- 0.03 micron) phase. These changes were markedly dampened (diminished) in sham-operated animals. Since this dampening of time-of-day changes after sham-operations was not seen in A-cells after pinealectomy, mediation by the gland of this dampening is suggested. Pinealectomized animals showed 24-h changes in nucleolar diameters, but these changes differed from those of nonoperated controls in their temporal and phase relations. For example, there was phase advance in A-cells in the juxtacortical area and desynchronization in N-cells. Furthermore, rhythm amplitude was slightly increased in A-cells and slightly decreased in N-cells after pinealectomy. A- and N-cells differed from each other consistently and significantly in the quantitative morphology of their nuleoli and nuclei, and slightly in the patterns of their time-of-day changes in nucleolar size. Nucleolar size usually showed a tendency for a regional gradient, decreasing slightly from periphery toward the center. These observations contribute to our understanding of the chronobiology and functional relations of adrenomedullary chromaffin cells, and of the effects of pinealectomy and sham operations upon this system.

No correlation of pineal "synaptic" ribbon numbers and melatonin formation in individual rat pineal glands.

Abstract: 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 nighttime. It is concluded that the ribbons may perhaps be more closely related to the innervation of the pineal gland than to melatonin formation.

Electroconvulsive shock: effect on pineal and hypothalamic indoles.

Abstract: Chronic electroconvulsive shock (ECS) did not significantly alter pineal concentrations of melatonin. Statistically significant decreases of serotonin, however, were found in both the pineal and hypothalamus. These results support the previous reports of serotoninergic receptor supersensitivity following repeated ECS, and suggest serotoninergic involvement but do not necessarily exclude adrenergic involvement in the antidepressant effect of electroconvulsive therapy.

Synaptic junctions between the adrenergic axon varicosity and the pinealocyte in the rat.

Abstract: Intercellular relationships of sympathetic nerve fibers and cells in pineal glands of Long-Evans rats and yellow-bellied country rats (Rattus losea Swinhoe) were studied with conventional electron microscopy. Typical synapses were found between adrenergic axon varicosities and pinealocytes. The synaptic cleft was 20-30 nm thick and contained granular or filamentous material of moderate electron density. Some small granulated vesicles attached to, or fused with, the presynaptic axolemma, a feature suggesting an exocytotic release of the vesicle content. The occurrence of membranous cisternae or tubules in the synaptic nerve ending may connote the phenomenon of synaptic vesicle recycling. Beneath the postsynaptic pinealocyte plasmalemma there was a dense plaque corresponding to the postsynaptic density of neurons. Possible origins and functional significance of the synapse-making nerve fibers are also discussed.

The ultrastructure of the nerve fibers and pinealocytes in the rat pineal stalk.

Abstract: In view of the increasing interest in the central innervation of the mammalian pineal gland, this aspect was studied in depth in the rat. This species is especially suited since the nerve fibers in question form a distinct bundle running from the deep to the superficial pineal gland through the pineal stalk. The axons were counted and analysed ultrastructurally in the pineal stalks cut transversely at three levels (proximal, intermediate, and distal) relative to the neural axis and in longitudinal sections. The number of nerve fibers was highly variable, ranging from 551 to 1, 132 proximally and from 110 to 448 distally, indicating that many fibers terminate in the stalk or leave the stalk after forming a loop. Large myelinated axons, which are abundant proximally, appear to lose their sheaths along their course through the stalk. Most of the axons were small and unmyelinated. A few of these had the appearance of sympathetic fibers and disappeared after sympathectomy. Others contained abundant neurosecretory granules, and, according to the literature, may originate in the hypothalamic paraventricular nuclei. The majority of the small axons which are apparently devoid of granules and dense-cored vesicles may come from the habenular nuclei and the stria medullaris. In addition to axons, the stalk contains astrocytes, a few oligodendrocytes and Schwann cells, as well as pinealocytes identical to those of the superficial pineal gland.

Pineal Arginine Vasotocin Activity Increases 200-Fold During August in Adult Rats and Hamsters

Abstract: In a previous study, measurements of arginine vasotocin-immunoactivity (iAVT) in immature rats over a period of 14 months, led to the discovery of a significant yearly variation, with peak levels of iAVT in August. In the present study, iAVT was measured in pineals obtained from mature male and female rats and hamsters once or twice weekly from July until early September 1982. For all groups, mean pineal AVT-immunoactivity was less that 7 pg/gland in early July, but then increased significantly by August 11-13. For hamsters, maximum values of 1,272 +/- 49 (mean +/- S.E.: n = 3) and 1,065 +/- 62 pg/gland were recorded for males and females, respectively. For rats, peak values measured were 940 +/- 12 pg/gland for males and 1,040 +/- 34 for females. The AVT-activity levels then decreased to less than 100 pg/gland by early September. Thus, a dramatic August elevation of pineal iAVT is characteristic of hamsters as well as rats, and of mature as well as immature animals.

Effect of acute light exposure upon melatonin content, NAT activity, and nuclear volume in the gerbil pineal complex.

Abstract: This report describes the response of both the superficial and deep components of the gerbil pineal gland to a 30-min pulse of fluorescent light at the time of the nocturnal peak in pineal activity. When gerbils were maintained in a 14 h light:10 h dark photoperiod, mean melatonin concentrations in the superficial pineal gland ranged between 72 and 108 pg/gland during the day and first 4 h of darkness, rose to a peak of 399 pg/gland 7 h after lights out, then returned to basal levels at the expected time of light onset. Pineal melatonin contents were reduced to daytime values within 30 min after exposure of gerbils to fluorescent light at the time of the nocturnal peak. These changes in superficial pineal melatonin content reflected the circadian pattern in serotonin N-acetyltransferase (NAT) activity, as would be expected if NAT controls melatonin synthesis. In addition to the reductions in NAT and melatonin content induced by nocturnal photic stimulation, there was a 30% reduction in mean pinealocyte nuclear volume. A nocturnal elevation in melatonin content but not in NAT activity occurred in the deep pineal gland. Unlike the superficial pineal gland, the deep pineal gland did not demonstrate a response in melatonin content, NAT activity, or pinealocyte nuclear volumes upon exposure of gerbils to light at the time of the nocturnal peak in superficial pineal activity.

Daily Profiles of N‐Acetyltransferase Measured at a Single Time in Rat Pineal Glands, Retinas, and Harderian Glands

Abstract: Serotonin N-acetyltransferase activity (NAT) exhibited a daily cycle in light:dark (LD) 14:10 when it was measured in pineal glands taken from rats killed at a sequence of time points. The ratio of peak subjective night NAT to minimum subjective day NAT was 10.9/0.3 nmol per pineal gland per hour. When the rats were placed in constant dark the rhythm persisted (8.2/0.02). When the rats were placed in constant light the rhythm persisted with markedly attenuated amplitude (0.6/0.02). We also measured NAT profiles in rat pineal glands, Harderian glands, and retinas with alternative methods. We kept rats on six LD 14:10 light-dark cycles with lights-out beginning at midnight, 2 AM, 4 AM, 6 AM, 8 AM, or 10 AM and killing all the rats at one time point, 10 AM. We examined the NAT time profiles 4, 8, and 11 days following placement of the rats in the phase-shifted cycles. In addition, we measured the NAT profile in LD 2:22 and LD 22:2 by keeping the rats on twelve cycles for 11 days and killing all the rats at one time. Pineal NAT exhibited a rhythm in all the cycles: peak-dark/nadir-light values (nmol product per gland per hour) were 15.6/0.1 in LD 14:10, controls killed at successive time points. The ratios for the profiles obtained using the one time point procedure were 16.7/0.1 in LD 14:10 8.5/0.2 in LD 22.2, and 12.9/0.2 in LD 2.22. Increasing the photoperiod reduced the time to the NAT peak. In LD 14:10, Harderian NAT was 31–39 nmol per gland per hour but the peak/ nadir radio was only 1.2; retinal NAT was low (0.2–0.7 nmol per retina per hour) and had only a 3.5-fold peak/nadir ratio.

Quantitation of ultrastructural twenty-four-hour changes in pineal nuclear dimensions.

Abstract: An ultrastructural stereological analysis was made of the nucleus in pinealocytes of 28 male Fischer rats sampled at seven times in a light:dark 12:12 photoperiod cycle. Comparisons of the data from the seven times, in relation to daily means, showed variations in mean nuclear cross-sectional surface area of +/- 16%, and in mean nuclear perimeter of +/- 10%. Peaks in both nuclear dimensions occurred at the middle of the light period and were coincident with the presence of elaborate nuclear configurations and deep indentations. At 2330 and 0430 (1 and 6 h after lights off) nuclear folding was generally less complex. Mitochondria were found in close association with the nuclear folds. Forty percent of nucleoli were marginated at all of the sampling times, except at 1130 (1 h after lights on) and at 2330 (1 h after lights off), when 50% and 35%, respectively, were marginated.

An Immunocytochemical Study of the Luteinizing Hormone‐Releasing Hormone (LHRH) System in the White‐Footed Mouse: Effect of Blinding and Melatonin

Abstract: Previous studies have provided evidence that pineal-mediated gonadal atrophy involves the luteinizing hormone-releasing hormone (LHRH) system in some rodents. The purpose of the present study was to visualize the LHRH system in the brain of the white-footed mouse by immunocytochemical methods and to assess the influence of blinding or melatonin on the LHRH immunostaining characteristics of the median eminence. Six intact adult female mice and six adult males (two intact, two blinded, and two with subcutaneous melatonin-beeswax implants) treated for 6 wk were sacrificed by perfusion of fixative, and their brains were removed, cut into 60-micron sections and stained for LHRH by the peroxidase-antiperoxidase (PAP) method. Since the tissues were batch-processed in separate wells in a common chamber, semiquantitive comparisons may be made between similar sections from animals subjected to different treatments. The testes were also collected and prepared for microscopic examination. The distribution of LHRH-stained cell bodies and fibers was found to be essentially the same as has been described in other rodents, i.e., predominantly confined to the preoptic area and hypothalamus. The perikarya appear as unipolar and bipolar fusiform cells with "beaded fiber" type processes. In sections that included the median eminence of the blinded and melatonin-treated males, the degree of immunopositive staining appears to be greater than in the median eminence of untreated mice. The histological appearance of the testes from the treated animals revealed a profound inhibition of gametogenesis. These observations support the suggestion that pineal-mediated gonadal atrophy is the result of an inhibition of LHRH release from the median eminence.

A stereological study of the effects of the pineal gland on the ventral prostate of the rat.

Abstract: Several procedures known to influence pineal gland secretion were studied for their effects on the ventral prostate of rats. Stereological measurements were made of volume fractions of acini and glandular epithelium, surface fractions of glandular epithelium, and length fractions of acini. From these figures and the weights of the glands, the volumes, surface areas, and lengths of these features were calculated, as well as the mean epithelial heights, mean acinar diameters, and mean distances between glandular acini. None of these measurements differed in sham-operated controls, pinealectomized, or blinded rats. In blind-anosmic rats, however, there were significant decreases in the weights of the prostates and the volumes of the acini, reflecting significant decreases in the mean diameters of acini. None of the other parameters differed from those of controls, except the length fraction of acini. These results differ from those described by others following castration in rats, indicating that the action of the pineal gland on the ventral prostate may be different from that of androgen deprivation, in this species. Caution should therefore be exercised in the interpretation of the results of experiments in which prostate weights are used as indications of the actions of pineal hormones on the neuroendocrine-gonadal axis.

Relative Efficacy of Melatonin and 5‐Methoxytryptamine in Terms of Their Antigonadotrophic and Counterantigonadotrophic Actions in Male Syrian Hamsters

Abstract: Antigonadotrophic and counterantigonadotrophic activities of melatonin and 5-methoxytryptamine (5-MT) were quantitatively compared in male Syrian hamsters. In long day conditions, the daily afternoon administration of either 5, 15, 25, 50, 100 or 200 micrograms melatonin induced testicular regression within 10 wk; under the same circumstances, only the 200-microgram dosage of 5-MT was able to suppress testicular weights. Thus, 5-MT appears to have about one-tenth the antigonadotrophic action of melatonin in the male Syrian hamster. In short days, the subcutaneous implantation of either 50 or 100 micrograms melatonin (every 2 wk in beeswax) prevented testicular regression whereas it required much larger doses of 5-MT (1 mg every 2 wk in beeswax) to achieve the same counterantigonadotrophic action. In terms of both their antigonadotrophic and counterantigonadotrophic effects, hamsters seem to be more sensitive to melatonin than to 5-MT.

Seasonal Changes in Glycogen Level and Size of Pinealocytes of the White‐Footed Mouse, Peromyscus leucopus: A Semiquantitative Histochemical Study

Abstract: Glycogen level in and size of pinealocytes of the feral, white-footed mouse Peromyscus leucopus, were studied by a semiquantitative histochemical method to determine whether seasonal changes exist in them under natural conditions, what temporal pattern they exhibit, and whether 24-hour changes in these parameters exist in different seasons, as shown in the laboratory dd-mice. Marked seasonal changes were seen in both glycogen levels and nuclear densities (ANOVA p less than 0.005). The size of pinealocytes at 09:00 to 10:00 showed one peak (and nadir) seasonal change, with the smallest size in winter (December and February) and a larger size in warmer seasons, with the maximum value in July. Glycogen level in pinealocytes at 09:00 to 10:00 showed bimodal seasonal changes, with lower levels in fall and spring and higher levels in winter and summer. In fall, a circadian trend in glycogen level in pinealocytes was seen, with a higher level at the end of the light period. In winter, the glycogen levels were very high at 09:00, 13:00, 17:00, and 21:00 examined and showed dampening of time-of-day differences. On the other hand, the size of pinealocytes followed a time-of-day change (P less than 0.005), being largest at 13:00 and smallest at 21:00. Thus, marked changes in quantitative structure and chemical activities, suggesting changes in functional activity, in pinealocytes were noted especially in severe, cold winter.

The Effect of Pinealectomy on Plasma Levels of Gonadotrophins and Growth Hormone in the Pigeon (Columba livid)

Abstract: The effects of pinealectomy on plasma follicle-stimulating hormone (FSH), luteinizing hormone (LH), and growth hormone (GH) concentrations were studied for a period of 9 weeks in pigeons reared under naturally increasing day length. After pinealectomy, plasma LH and GH concentrations decreased in both sexes when compared with sham-operated birds. In pinealectomized female pigeons, the plasma level of FSH was elevated, while in male birds the effect on FSH was negligible. A trend toward increased weight of testis, ovary, and oviduct was also seen after pinealectomy, but the increase was not statistically significant. The present results suggest that pinealectomy is able to modify the secretion of gonadotrophins and GH in the pigeon and indicate a stimulatory role for the pineal gland in LH and GH secretion and, in female pigeons, an inhibitory role in FSH secretion.

Responses of Synaptic Ribbons in Pineal Photoreceptors Under Normal and Experimental Lighting Conditions

Abstract: The size of synaptic ribbons (SR) in photoreceptor cells of the goldfish pineal organ was quantified over 24-h light:dark cycles of long (16:8) and short (10:14) photoperiods during summer and winter months, respectively. The amplitude of both rhythms was similar with peak values occurring toward the latter part of the photophase or early dark. When fish were entrained to the long photoperiod and exposed to continual light, SR size continued to increase during the expected dark time. The effect of extending the photoperiod into the expected dark time was diminished when fish were entrained to a short photoperiod and presented with 6 h of darkness at the end of the 24-h period. The size increase in response to environmental lighting is believed to reflect a greater demand for either vesicle attachment sites or neurotransmitter storage sites since vesicles (neurotransmitter) have been hypothesized to accumulate in the synaptic pedicles during inhibition by light. From a comparative standpoint it is noteworthy that synaptic ribbons (vesicle-crowned rods) in mammals react in a similar manner to both normal and experimental lighting conditions.

Nuclear and Cytoplasmic Inclusion Bodies in Pinealocytes of the Cotton Rat, Sigmodon hispidus: An Electron Microscopic Study

Abstract: Electron microscopic observations on pinealocytes of cotton rats (Sigmodon bispidus) killed in October revealed the presence of cytoplasmic inclusion bodies (CIBs) and two kinds of nuclear inclusions--coiled bodies and granular inclusion bodies. These inclusions were usually not bounded by a membrane. CIBs showed round or irregular profiles and consisted of granular and filamentous materials of moderate electron opacity intermingled with electron-lucent areas. Nuclear granular inclusions appeared as homogeneous or heterogeneous granules of variable electron opacity, which usually exhibited round profiles of variable diameters and consisted of granular and filamentous elements. Electron-opaque granules were observed in perinuclear spaces, dilated cisternae of rough endoplasmic reticulum, as well as in the central portion of CIBs. In CIBs containing granules, the peripheral region was composed of the moderately electron-opaque substance seen in usual CIBs, while the central core was occupied by a granular and filamentous substance and an intervening electron-lucent matrix. Since an electron-opaque granular substance in nuclei, perinuclear spaces, cisternae of rough endoplasmic reticulum, and the central portion of CIBs may occur concomitantly, the granules seen in these sites may be related. Coiled bodies were round in profile and were composed of electron-opaque strands consisting of granular and filamentous elements and intervening material of low electron opacity. This structure, frequently located in close proximity to the nuclear granular inclusions, may play a role in the formation of the intranuclear granules in cotton rat pinealocytes.

Effects of Melatonin and Control Injections on Pineal Serotonin and Norepinephrine: Afternoon Injections Lower Serotonin Levels Thirty‐Six Percent at Light‐Dark Transition

Abstract: Pineal weight and serotonin (5-HT) and norepinephrine (NE) contents were studied in male Sprague-Dawley rats that were maintained under controlled light:dark conditions (LD 14:10; lights on 0700-2100) and that received daily subcutaneous injections of either melatonin (20 micrograms in 0.1 ml per animal) or the same volume (0.1 ml) of vehicle alone, at one of two times (0800-0900 or 1800-1900). Animals were sacrificied at four times (1000, 1400, 2000, or 2300) on the day after the last of the 7 consecutive d of injection. Pineal glands were quickly weighed and then frozen for 5-HT and NE assay by the Maickel and Miller extraction and fluorescence methods. Pineal NE content showed differences related to time of day, in confirmation of early work. But no effects attributable specifically to melatonin were found. Melatonin also failed to affect pineal 5-HT content significantly. But injection of either melatonin or vehicle at 1800-1900 led to a reduction in 5-HT content averaging 36% when sampled at either 2000 or 2300, and in comparison with animals injected at 0800-0900. It is suggested that a stresslike or zeitgeberlike effect of injections within a critical period at the end of the daily light phase can cause an earlier-than-normal daily fall in pineal 5-HT content.