Neuropeptide Y localization in telencephalic and diencephalic structures of the ground squirrel brain.

The distribution of neuropeptide Y-immunoreactive (NPY-IR) perikarya, fibers, and terminals was investigated in the brain of two species of hibernatory ground squirrels, Spermophilus tridecemlineatus and S. richardsonii, by means of immunohistochemistry. In the telencephalic and diencephalic structures studied, distinct patterns of NPY-IR were observed which were essentially identical in male and female animals of both species. No differences in amount or distribution of NPY-IR structures were observed between animals which had been in induced hibernation for several months before sacrifice in March/April and those sacrificed one week after their capture in May. In some brain structures (e.g., the hypothalamic arcuate nucleus), IR cell bodies were observed only after pretreatment with colchicine. NPY-IR perikarya and fibers were found in the cerebral cortex, caudate nucleus-putamen, and dorsal part of the lateral septal nucleus. Dense fiber plexuses were seen in the lateral and medial parts of the bed nucleus of the stria terminalis. The numbers of IR perikarya observed in the medial part of the nucleus increased following intraventricular colchicine injections. The accumbens nucleus exhibited few IR cells and many fibers. Claustrum and endopiriform nuclei showed a considerable number of stained cells and fibers that increased in number and staining intensity in colchicine-treated ground squirrels. The induseum griseum showed a small band of IR cell bodies and varicose fibers. Bipolar of multipolar IR cells and varicose fibers were found in the basal nucleus of the amygdala. Dense fiber plexuses as well as IR terminals were seen in the median, medial, and lateral preoptic areas of the hypothalamus. Terminals and relatively few fibers were located in the periventricular, paraventricular, and supraoptic nuclei. The anterior, lateral, dorsomedial, and ventromedial hypothalamic nuclei contained relatively large numbers of terminals and fibers. In the suprachiasmatic nuclei, dense terminals were distributed mainly in the ventromedial subdivision. In the median eminence, immunoreactive terminals were concentrated in the external layer, with fibers predominant in the internal layer. NPY-IR perikarya were observed only in the arcuate nucleus of the hypothalamus and only following colchicine treatment. In the epithalamus (superficial part of the pineal gland and habenular nuclei), varicose fibers appeared mainly in perivascular locations (pineal) or as a dense plexus (habenular nuclei). These results from ground squirrels are discussed in comparison to those obtained in other species and with regard to considerations of the physiological role of NPY.

Immunohistochemical evidence for the presence of neuropeptides in the hypothalamic suprachiasmatic nucleus of ground squirrels.

The cytoarchitecture and immunocytochemical distribution of neuropeptides (corticotropin-releasing factor, CRF; neuropeptide Y, NPY; oxytocin, OXY; vasopressin, VP; and vasoactive intestinal polypeptide, VIP) were studied in the hypothalamic suprachiasmatic nuclei (SCN) in male and female ground squirrels of two species (Spermophilus tridecemlineatus and S. richardsonii). Immunoreactive (IR) perikarya were found in sections incubated with VP or VIP antisera. VP-IR cell bodies were seen in the dorsal and medial parts of the nucleus in colchicine-treated animals. IR fibers were distributed throughout the SCN. In the ventral part of the nucleus, VIP-IR cells were seen in untreated animals and were more pronounced in colchicine-treated animals. VIP-IR fibers and terminals form a dense plexus throughout the nucleus. Furthermore, NPY-IR terminals and fibers with multiple varicosities, but no IR perikarya, were present in the suprachiasmatic nuclei. Within the borders of the SCN, no cell bodies or fibers were stained with CRF or OXY antisera in any animal.

Pineal sensitivity to nighttime swimming stress changes during the active season in Richardson's ground squirrels (Spermophilus richardsonii).

Melatonin synthesis in the pineal gland, which is primarily regulated by the environmental lighting regime, can also be influenced by other factors that elicit modifications in sympathetic tone. The objectives of this study were to determine if forced swimming alters the normal pattern of melatonin production in the pineal gland of the Richardson's ground squirrel (Spermophilus richardsonii). In early June, the squirrels were forced to swim for 10 min during the photophase or during the scotophase. In mid-July squirrels swam only during the scotophase. Animals were sacrificed 15, 30, or 60 min after the onset of swimming. Activities of pineal N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) were assessed by radioenzyme assay, and pineal melatonin content was measured by radioimmunoassay. Daytime swimming elicited no major changes in enzyme activity or pineal melatonin. In June, swimming at night prevented the normal rises in NAT activity and pineal melatonin seen in nonswimming controls. In contrast, the pineals of squirrels that were tested 6 weeks later in mid-July did not appear to be as sensitive to nighttime swimming, as there were only minor differences in both NAT activity and melatonin content compared to controls. These results demonstrate that forced nighttime swimming, unlike several other aversive stimuli, can evoke changes in the normal pattern of pineal melatonin production in this species. Furthermore, the pineal's response to such stimuli may not be stable over the course of the active season.

Thyroxine 5'-deiodination in brown adipose tissue and pineal gland: implications for thermogenic regulation and role of melatonin.

T4 type II 5'-deiodinase (5'-D II) activity was studied in wild-captured Richardson's ground squirrels. As previously reported for other species, 5'-D II activity was detected in frontal cortex, cerebellum, pineal gland, and brown adipose tissue (BAT); in the median eminence the levels of 5'-D II activity were undetectable with our methodology. When pineal gland, frontal cortex, and cerebellum nyctohemeral profiles were studied, none of them showed variations. Cold exposure for 4 h led to an increase in the enzymatic activity 10-fold above the basal values for BAT, while in the pineal gland the values were doubled; cold exposure failed to change the 5'-D II activity in the frontal cortex. Acute melatonin treatment caused a 7-fold increase in 5'-D II activity in BAT, but did not affect enzyme activity in either the pineal gland or frontal cortex. The data indicate that 5'-D II in Richardson's ground squirrel shows classical localizations. Additionally, two new regulatory factors of 5'-D II are reported, i.e. melatonin for BAT and cold for the pineal gland.

Melatonin synthesis in the pineal gland of the Richardson's ground squirrel (Spermophilus richardsonii): influence of age and insulin-induced hypoglycemia.

The nocturnal rises in pineal N-acetyltransferase (NAT) activity and melatonin levels were compared in young (25-35 days old) and adult (at least 1 year old) Richardson's ground squirrels. When expressed as NAT activity per pineal gland, the nighttime rise in the activity of this enzyme was less in young than in the adult animals; conversely, the melatonin content of the pineal glands of young animals was higher at one point (4 a.m., 8 hours after darkness onset) when compared to that in adult squirrels. When data were expressed relative to total protein, the NAT and melatonin rhythms in the pineals of young and adult animals were very similar. The effect of insulin-induced hypoglycemia on both daytime and nighttime NAT and melatonin levels in the pineal gland of the Richardson's ground squirrel was also assessed. Low daytime levels of these constituents were not influenced by the administration of 10 units insulin, a treatment which caused a marked drop in circulating glucose levels. At night, when pineal NAT and melatonin levels were high insulin injection had a very modest stimulatory effect on NAT activity (one point was elevated above saline injected controls) while melatonin levels remained unchanged by the treatment. These findings in the ground squirrel in reference to insulin-induced hypoglycemia, and stressors in general, appear to differ from those in the rat where stress can have a substantial influence or both low daytime and high nighttime levels of pineal NAT and melatonin.

Changes in serotonin levels, N-acetyltransferase activity, hydroxyindole-O-methyltransferase activity, and melatonin levels in the pineal gland of the Richardson's ground squirrel in relation to the light-dark cycle.

Pineal serotonin and melatonin levels and the activities of hydroxyindole-O-methyltransferase (HIOMT) and N-acetyltransferase (NAT) were studied over a 24-hour period in the pineal gland of the diurnally active Richardson's ground squirrel (Spermophilus richardsonii). Under alternating light-dark conditions (light:dark hours 14:10), pineal serotonin and melatonin levels exhibited a rhythm with high values occurring either during the day (serotonin) or during the night (melatonin). NAT activity was also markedly increased during darkness. HIOMT activity exhibited no 24-hour variation. Exposure of squirrels to constant light for 7 days exaggerated the serotonin rhythm, but obliterated the cycles of NAT and melatonin. Under constant darkness (for 7 days), the rhythms in serotonin, melatonin and NAT persisted, each having a period of about 24 h. In the second study, ground squirrels were exposed to light-dark cycles of either 8:16, 10:14 or 14:10. Under each of these photoperiodic environments, rhythms in pineal NAT and melatonin were apparent. Increasing the daily dark period from 10 to 14 h caused a prolongation of the elevated NAT and melatonin levels. However, a further prolongation of the daily dark period (to 16 h) did not further increase the duration of the rise in NAT and melatonin. The results show that continual light exposure (irradiance of 200 microW/cm2) for 7 days suppresses the pineal rhythms in both NAT activity and melatonin level in the Richardson's ground squirrel. Conversely, light exposure, rather than depressing the serotonin rhythm, actually exaggerates it. Constant darkness for 7 days has little influence on the 24-hour rhythms of either NAT or melatonin.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of various irradiances of artificial light, twilight, and moonlight on the suppression of pineal melatonin content in the Syrian hamster.

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.

Influence of light irradiance on hydroxyindole-O-methyltransferase activity, serotonin-N-acetyltransferase activity, and radioimmunoassayable melatonin levels in the pineal gland of the diurnally active Richardson's ground squirrel.

When Richardson's ground squirrels were kept under light:dark cycles of 14:10 h there was no nocturnal rise in pineal hydroxyindole-O-methyltransferase (HIOMT) activity. Conversely, the 10 h dark period was associated with large nocturnal rises in both pineal serotonin-N-acetyltransferase (NAT) activity and radioimmunoassayable melatonin levels. The nighttime rises in pineal NAT and melatonin were not suppressed by the exposure of the animals to a light irradiance of 925 mu W/cm2 during the normal dark period. On the other hand, when the light irradiance was increased to 1850 mu W/cm2 the rise in pineal NAT activity was eliminated while the melatonin rise was greatly reduced. When ground squirrels were acutely exposed to a light irradiance of 1850 mu W/cm2 for 30 min beginning at 5.5 h after lights out, pineal NAT activity and melatonin levels were reduced to daytime values within 30 min. The half-time (t 1/2) for each constituent was less than 10 min. Exposure to a light irradiance of either 5 s or 5 min (beginning at 5.5 h into dark period) was equally as effective as 30 min light exposure in inhibiting pineal NAT activity and melatonin levels. When animals were returned to darkness after a 30 min exposure to a light irradiance of 1850 mu W/cm2 at night, both pineal NAT activity and melatonin levels were restored to high nighttime levels within 2 h of their return to darkness. The results indicate that the pineal gland of the wild-captured, diurnal Richardson's ground squirrel is 9000 X less sensitive to light at night than is the pineal gland of the laboratory raised, nocturnal Syrian hamster.

Ultrastructure of pinealocytes of the kangaroo rat (Dipodomys ordi).

The ultrastructure of the pinealocytes of the wild-captured ord kangaroo rat (Dipodomys ordi) was examined. A homogeneous population of pinealocytes was present in the pineal gland of the kangaroo rat. The Golgi apparatus, granular endoplasmic reticulum, mitochondria, lysosomes, dense-core vesicles, vacuoles containing a flocculent material and lipid droplets were consistent components of the pinealocyte cytoplasm, whereas infrequently-observed organelles included centrioles, multivesicular bodies, subsurface cisternae, "synaptic" ribbons and cilia. The number of dense-core vesicles was relatively high and dense-core vesicles and vacuoles containing a flocculent material were present in the same cell. Although it has been recently suggested that two different secretory processes, i.e., neurosecretory-like (Golgi apparatus - dense-core vesicles) and ependymal-like (granular endoplasmic reticulum - vacuoles containing a flocculent material) may be involved in different regulatory mechanisms in the pinealocytes, the definitive answer to this is still far from clear. Therefore, the pineal gland of the kangaroo rat appears to be a good model for the study of the potential relationship between these two secretory processes, especially in respect to seasonal changes.

Studies on the regulation of pineal melatonin production in the Richardson's ground squirrel (Spermophilus richardsonii).

The acute exposure of wild-captured Richardson's ground squirrels to fluorescent light (intensity = 370-400 ftc) at 2400h, 4 hours after the onset of darkness, was followed by a slight depression in the activity of pineal N-acetyltransferase (NAT); during the same 60 min period, pineal melatonin levels were not inhibited. Conversely, when laboratory-raised squirrels were either exposed to light at night or kept in their normal period of darkness, pineal NAT activity and melatonin levels differed greatly between the two groups. In darkness both NAT and melatonin rose sharply and remained elevated during most of the night. When animals were exposed to light at night the NAT rhythm was completely suppressed and the rise in melatonin was severely dampened. Finally, the administration of isoproterenol (6 mg/kg) increased otherwise low daytime levels of both NAT activity and melatonin levels in the pineal gland of the ground squirrel.

The effects of the light:dark cycle and sympathetically-active drugs on pineal N-acetyltransferase activity and melatonin content in the Richardson's ground squirrel, Spermophilus richardsonii.

When adult Richardson's ground squirrels, born in the wild, are kept in the laboratory under light:dark cycles of 14:10 (lights out at 2000 h), pineal N-acetyltransferase (NAT) activity and melatonin levels begin to rise shortly after lights out; both reach peak levels by 4 h into the dark phase. The rise in pineal NAT activity is partially blocked by phentolamine, an alpha-receptor blocker, and totally blocked by propranolol, a beta-receptor blocker. Phentolamine had no inhibitory influence on the nocturnal rise in pineal melatonin while propranolol partially prevented the increase in melatonin. Isoproterenol, a beta-receptor agonist, seemed to stimulate both NAT activity and the melatonin content in the pineal gland of the Richardson's ground squirrel. In the final study adult squirrels were kept in either 10 h of darkness at night or they were exposed to light (intensity = 370-400 ftc) during the normal period of darkness. Groups of squirrels were killed at 2000, 2400, 0200, 0400 and 0800 h. Darkness was associated with a marked rise in both pineal NAT activity and melatonin content. However, even when the squirrels were kept in light during the normal dark period, the activity of the acetylating enzyme and melatonin levels rose greatly and reached peak values at 0400 h.

Studies on pineal melatonin levels in a diurnal species, the eastern chipmunk (Tamias striatus): effects of light at night, propranolol administration or superior cervical ganglionectomy.

Five experiments were carried out on the control of melatonin levels in the pineal gland of a diurnal species, the Eastern chipmunk (Tamias striatus). We confirmed that the exposure of chipmunks of fluorescent white light of 3,981-4,304 lux during the normal dark period does not prevent the rise in pineal melatonin levels normally associated with darkness. Also, the administration of propranolol (20 mg/kg) at 8 p.m. did not block the rise in pineal melatonin in animals exposed to either dark or light at night. Similarly, if chipmunks received propranolol 4 hours into the dark phase, pineal melatonin levels were not depressed 2 hours later. When animals were superior cervical ganglionectomized, however, the pineal content of melatonin remained low regardless of whether animals were exposed to darkness or light at night. The exposure of chipmunks acutely to light at midnight (4 hours after darkness onset) had only a slight depressive effect on pineal melatonin 30 min later; by comparison, when chipmunks were acutely exposed to light at 3 a.m. (7 hours after darkness onset) daytime pineal melatonin levels were reached within 15 min after light onset. These findings in a diurnal species, The Eastern chipmunk, differ markedly when compared to previously reported observations on nocturnal laboratory rodents.

Day-night differences in the number of pineal "synaptic" ribbons in two diurnal rodents, the chipmunk (Tamias striatus) and the ground squirrel (Spermophilus richardsonii).

Daytime numbers of pineal "synaptic" ribbons higher than reported in the pineal gland of any other mammalian species were observed in two diurnal rodents, the eastern chipmunk and Richardson's ground squirrel. The number of "synaptic" ribbons was lower during the daytime and higher at night in both of these species.