Effects of hibernation on somatostatin-like immunoreactivity in the brain of the ground squirrel (Spermophilus richardsonii) and European hedgehog (Erinaceus europaeus)

Summary The localization of the somatostatin system in the brains of Richardson's ground squirrels (Spermophilus richardsonii) and European hedgehogs (Erinaceus europaeus) was described by use of immunocytochemical methods. In addition, (i) chemically differing types of somatostatin and (ii) different activity phases of the somatostatin system during the hibernation cycle were investigated in the ground squirrel by means of high pressure liquid chromatography (HPLC) and radioimmunoassay (RIA).In both species, the hypothalamic component of the somatostatin system (periventricular nuclei, fiber projections to the median eminence) is more prominent than the widespread extrahypothalamic representation of the system displaying mainly scattered perikarya and nerve fibers. The reactivity pattern of the somatostatin system varied among hibernating, aroused, and non-hibernating animals; moreover, the interspecific differences were pronounced. The activity of the hypothalamic somatostatin system in the hibernating ground squirrel appeared to be suppressed when compared to non-hibernating controls, whereas in the hibernating hedgehog this system showed signs of increased activity in comparison to non-hibernating controls. In contrast, in the present material the extrahypothalamic components of the somatostatin system did not exhibit significant changes in their activity.     

Immunocytochemical demonstration of somatostatin-containing neurons in the hypothalamus of the domestic mallard

Summary In the hypothalamus of the adult domestic mallard, small to medium-sized perikarya are stained specifically with rabbit antiserum against cyclic somatostatin (PAP technique of Sternberger). The somatostatin-immunoreactive material is located in neurons different from those containing immunoreactive LHRH, vasotocin or mesotocin. Somatostatin-containing perikarya are observed 1) in a chain-like arrangement extending from the area of the median division of the supraoptic nucleus to the caudal end of the paraventricular nucleus; 2) as single cells in the preoptic region; and 3) as a conspicuous formation in the optic tract division of the supraoptic nucleus. In the rostral portion of the median eminence, somatostatin-immunoreactive axons penetrate into the external zone. Fine accessory fiber bundles project to the neural lobe.     

Effect of ionizing radiation on the synthetic activity of blood system cells in ground squirrels in different physiological states of animals

The functional (synthetic) activity of blood lymphocytes and bone marrow haemopoietic cells in ground squirrels during the annual cycle as well as in hibernating and awaken animals in winter have been studied by fluorescent microspectrometry. The effect of ionizing radiation on animals in different functional states of the hibernation-arousal bout was investigated too. It was shown that the synthetic activity (parameter alpha) in blood lymphocytes was minimal in hibernating state in winter and maximal in active euthermic spring animals, then slightly decreased in June and more considerably decreased in the prehibernating autumn period. In awake animals in winter, the values of parameter alpha reached the same values as in summer. The changes of parameter alpha in bone marrow haemopoietic cells were essentially the same: the minimal values were observed in the prehibernation autumn period and in awake animals in winter the alpha values were slightly higher than in active euthermic animals in summer. The maximal synthetic activity in bone marrow haemopoietic cells in active euthermic spring animals is due mainly to cells in G1-G2 phases of the cell cycle. The decrease of the synthetic activity in summer is a result of the cell transition from G2 to mitosis and transition of a part of cells to G0 When investigating the hibernation-arousal bout in ground squirrels in winter, during arousal, we found two stages considerably differing in both the values of parameter alpha in bone marrow haemopoietic cells and the number of blood cells. The synthetic activity and the total number of blood and bone marrow cells in ground squirrels irradiated in the state of deep hibernation did not differ significantly from the state of non-irradiated hibernating animals. The negative effect of radiation appeared upon the arousal of these animals but it was expressed to a lesser degree in comparison with the animals irradiated in the active state. It was found that the acute irradiation of animals during arousal from hibernation in the second stage caused the most pronounced functional inactivation and cell death. The physiological state of ground squirrels subjected to ionizing irradiation at different phases of the hibernation-arousal bout plays a determining role in the changes of the qualitative and quantitative characteristics of blood system cells. Thus, the hypometabolic state of ground squirrels in hibernation is a factor of protection from the action of ionizing radiation on the organism and the immune system.     

The neuroendocrine system in hibernating mammals: present knowledge and open questions

The present review describes the distribution and the function-dependent reactivity pattern of those peptidergic and aminergic components of the neuroendocrine system of hibernating mammals that have been studied by histological, pharmacological and physiological techniques. Particular attention has been paid to the intrinsic connectivity of the peptidergic apparatus and its input systems. Since the reactivity patterns of the neuroendocrine system show remarkable fluctuations in relation to the various stages of hibernation and euthermia, these fluctuations have been analyzed with respect to (1) their causative role in the regulation of hibernation and (2) their secondary response to physiological changes during hibernation. The author's investigations described in this review have mainly been performed in European hedgehogs (Erinaceus europaeus), European and golden hamsters (Cricetus cricetus, Mesocricetus auratus), dormice (Glis glis), and in Richardson's and Columbian ground squirrels (Spermophilus richardsonii, Spermophilus columbianus), by the use of light- and electron-microscopic immunocytochemistry and histochemistry, in situ hybridization, radioimmunoassays and stereotaxically guided application techniques. These experiments were also performed in hypothermic animals. The (partially published) results obtained by the author and his associates are reviewed with reference to the body of evidence found in the recent literature. With respect to their reactivity patterns, several neuropeptide and transmitter systems can be regarded as candidates for control systems of hibernation. Neuronal complexes immunoreactive for endogenous opiates, in particular enkephalin, and also for vaspressin, somatostatin, substance P, corticotropin-releasing factor and serotonin are probably involved in the neuroendocrine control of hibernation.Dedicated to the memory of Dr. Gesa Merker     

A comparison of neuropeptide immunocytochemistry in fluid-fixed and freeze-dried brains

Summary Immunocytochemical staining of luteinizing hormone-releasing hormone (LHRH), somatostatin, and neurophysin was compared in rat brains fixed with 1) formalin, 2) Bouin's solution, 3) freeze-dried (FD), or 4) freeze-dried + paraformaldehyde vapor perfused (FDV). The distribution of LHRH fibers was similar in all preparations; however, beads of granular reaction product often appeared finer and more numerous in the median eminence of FD- and FDV brains. Positively stained LHRH perikarya were not observed in any of the preparations. In contrast, somatostatin-immunoreactive perikarya were present in the fluid-fixed and FD brains, although few were observed in FDV brains. Somatostatin-immunoreactive fibers were present in all preparations, but appeared most numerous in the median eminence of FD brains. Staining of neurophysin-containing perikarya and fibers was similar in all preparations. These observations suggest that the FD brain can provide a suitable tissue substrate for immunocytochemistry, demonstrating staining comparable to or surpassing that of more conventional preparations. However, staining of antigens in FD brain was not uniformly successful and may depend on stereochemical characteristics of each antigen as well as properties of the primary antisera used in the staining procedure.     

Down-regulation in the insulin-like growth factor (IGF) axis during hibernation in the golden-mantled ground squirrel, Spermophilus lateralis: IGF-I and the IGF-binding proteins (IGFBPs)

The golden-mantled ground squirrel, Spermophilus lateralis, undergoes a profound winter hibernation that represents, among other changes, a prolonged period of starvation. In addition to dramatic metabolic and other physiological adaptations during hibernation which serve to reduce fuel energy expenditure, we have hypothesized that there may also be significant changes in the endocrine axis that regulates energetically-expensive somatic growth. As compared with euthermic, non-hibernating controls, hibernating S. lateralis were found to have 75%-reduced serum concentrations of insulin-like growth factor-I (IGF-I; from approximately 625 to approximately 150 ng/ml in both females and males, P < 0.05). While IGFBP-3 was the predominant IGFBP in serum of the euthermic controls, its levels were reduced to a similar degree in serum from the hibernating animals. IGFBP-4 was present at relatively low levels in the euthermic controls, and was reduced to undetectable levels in hibernating animals. Surprisingly, there was no IGFBP detectable in the 30 kDa range in either euthermic or hibernating S. lateralis, suggesting that IGFBP-1 does not play a role in hibernation-related changes in the IGF axis. In accordance with these endocrine changes, when serum from hibernating S. lateralis was added to cartilage explant cultures (at a 5% v/v concentration), it exhibited no ability to alter (35)S-proteoglycan synthetic rate, whereas serum from the euthermic squirrels significantly stimulated synthetic activity by 2-fold. These results suggest that part of hibernation adaptation in S. lateralis includes down-regulation in the growth-regulatory IGF axis. J. Exp. Zool. 289:66-73, 2001.     

Decreases in pineal melatonin content during the hibernation bout in the golden-mantled group squirrel, Spermophilus lateralis

The role of the pineal gland in modulating the rhythmic bouts of hibernation in the golden-mantled ground squirrel (S. lateralis) was explored by comparing pineal melatonin content in hibernating animals with that of euthermic animals at the same time of year. Significant decreases in pineal melatonin content were found in hibernating versus euthermic animals. In addition, significantly lower values for pineal melatonin were observed in hibernating animals that were sacrificed in the late bout period, just prior to expected spontaneous arousal, as compared to hibernating animals that were sacrificed on the first day of their respective bouts. A strong correlation was evident between pineal melatonin content and the duration of the individual hibernation bout. These data suggest that pineal melatonin may be important in determining the duration of individual bouts of hibernation in this species.     

Ultrastructural, morphometrical and immunocytochemical analyses of the exocrine pancreas in a hibernating dormouse

Pancreatic acinar cells of euthermic, hibernating and arousing individuals of the hazel dormouse Muscardinus avellanarius (Gliridae) have been observed at the electron-microscopic level and analysed by means of ultrastructural morphometry and immunocytochemistry in order to investigate possible fine structural changes of cellular components during periods of strikingly different degrees of metabolic activity. During hibernation, the cisternae of the rough endoplasmic reticulum (RER) flatten assuming a parallel pattern, the Golgi apparatus is extremely reduced and the mitochondria contain many electron-dense particles. The cell nuclei appear irregularly shaped, with deep indentations containing small zymogen granules. They also contain abundant coiled bodies and unusual constituents, such as amorphous bodies and dense granular bodies. Large numbers of zymogen granules occur in all animals. However, the acinar lumina are open and filled with zymogen only in euthermic animals, whereas, in hibernating and arousing individuals, they appear to be closed. Morphometrical analyses indicate that, in pancreatic acinar cells, nuclei and zymogen granules significantly decrease in size from euthermia to hibernation, probably reflecting a drastic decrease of metabolic activities, mainly protein synthesis and processing. In all the studied animals, immunocytochemistry with specific antibodies has revealed an increasing gradient in α-amylase content along the RER-Golgi-zymogen granule pathway, reflecting the protein concentration along the secretory pathway. Moreover, during deep hibernation, significantly larger amounts of α-amylase accumulate in RER and zymogen granules in comparison to the other seasonal phases analysed. Upon arousal, all cytoplasmic and nuclear constituents restore their euthermic aspect and all morphometrical and immunocytochemical parameters exhibit the euthermic values, thereby indicating a rapid resumption of metabolic activities.     

Ultrastructural changes in Paneth cells during hibernation in the ground squirrel Spermophilm lateralis

Summary The ultrastructure of Paneth cells from jejuno-ileal segments of the small intestine of the ground squirrel, S. lateralis, was examined under normal euthermic conditions and during the profoundly depressed metabolic conditions of natural hibernation. Paneth cells obtained from hibernating animals gave evidence of markedly reduced activity when compared to Paneth cells from euthermic animals. In hibernating animals, the nuclei were smaller, with less prominent nucleoli and with an increased proportion of heterochromatin. In hibernating animals, the rough endoplasmic reticulum was fragmentary and poorly organized, in contrast to the typical arrangement of concentric lamellae seen in euthermic animals. Although the total number of ribosomes was decreased in hibernating animals, there were proportionally more free ribosomes than in euthermic animals. Paneth cells from hibernating animals also contained a greater number of apical secretory granules which were smaller and more variable in electron density than granules from control animals. These ultrastructural features indicate that during hibernation the Paneth cell is relatively quiescent.Supported by U.S.P.H.S. grants RR 05411 and RR 05583 from the N.I.H.     

Myoglobin content in skeletal muscles of hibernating ground squirrels rises in autumn and winter

The content of myoglobin (Mb) in skeletal muscles of Arctic Yakutian ground squirrel (Citellus undulatus Pallas) was measured in the active euthermic summer and prehibernating autumn animals as well as in hibernating and awake animals in winter. The myoglobin content in winter, irrespective of the state of the animal, was found to be about three times higher than in summer. The content of myoglobin in autumn was also two-fold increased compared to summer, suggesting that high myoglobin level is necessary for hibernation. Analysis of biochemical data available suggests that the increase in myoglobin content in winter is probably related to a high oxygen demand of muscles at the first stage of arousal (non-shivering thermogenesis) when rectal temperature rises from 0 to 10-12 degrees C. At this stage, the oxygen-dependent processes in muscles proceed under the conditions when peripheral blood flow is blocked and anaerobic glycolysis is switched off.     

Beta-adrenergic signal transduction in the hypothalamus of the European hamster: Relation with the seasonal hibernation cycle and the diurnal activity cycle

Mammalian hibernation, an adaptation to survive harsh winter conditions, is one of the most prominent seasonal rhythmic processes exactly regulated on a low metabolic level. Diurnal variations in vegetative physiology are missing during hibernation; however, a precisely working diurnal system is mandatory for both the proper initiation and termination of the annual hibernation phase and the periodical arousal reactions. Biorhythms and the vegetative physiological processes connected with hibernation are, among others, controlled by hypothalamic noradrenaline systems. In this study, the density, binding capacity, and relative proportions of β₁- and β₂-adrenergic receptors (AR) within the hypothalamus of: 1) motorically inactive summer; 2) motorically active summer; 3) aroused, motorically active winter; and 4) deeply hibernating winter European hamsters (Cricetus cricetus) were studied. For further analysis of the β-adrenergic signal transduction cascade, the activity of adenylyl cyclase (AC) was measured by formation of cAMP in controls, after stimulation of G proteins, or after forskolin stimulation without or in presence of manganese ions. While β₁- and β₂-AR subtypes were nearly equally abundant (50% β₁:50% β₂) in active summer, inactive summer, and hibernating hamsters, a significant redistribution in favor of β₂-AR occurred after arousal (40% β₁:60% β₂). The activity of AC was much higher in active summer hamsters than in inactive summer, aroused winter, and hibernating winter hamsters. When AC was stimulated by guanylylimidophosphate [Gpp(NH)p], MnCl₂, forskolin, or by forskolin in presence of MnCl₂ instead of MgCl₂, the potency to stimulate AC was found to show the following rank order: basal < Gpp(NH)_p < MnCl₂ ≤ forskolin + MnCl₂ < forskolin.     

Immunocytochemical localization of neuropeptides in the hypothalamus of the Japanese long-fingered bat,Miniopterus schreibersii fuliginosus

Summary To elucidate the role of hypothalamic neuropeptides in regulation of reproductive phenomena of seasonally breeding feral mammals, we used Japanese long-fingered bats, Miniopterus schreibersii fuliginosus, for immunocytochemical study of distribution of the following neuropeptides in the hypothalamus: arginin vasopressin, oxytocin, luteinizing hormone-releasing hormone, somatostatin, corticotropin-releasing factor, and growth hormone-releasing factor. The size, shape and location of supraoptic, paraventricular, suprachiasmatic, and arcuate nuclei of the bat were determined. Arginin vasopressin-and oxytocin-immunoreactive magnocellular neurons were found in the supraoptic and paraventricular nuclei, where they exhibited separate distribution into two distinct groups. Parvocellular arginin vasopressin neurons occurred only in the suprachiasmatic nucleus. The hibernating bats exhibited slightly increased numbers of vasopressin and oxytocin neurons in the supraoptic and paraventricular nuclei. The pregnant bat displayed further increased numbers of vasopressin and oxytocin neurons in both nuclei. Somatostatin-immunoreactive neurons in the paraventricular nucleus were also immunopositive to anti-oxytocin serum, while those in the ventromedial and arcuate nuclei reacted solely to anti-somatostatin serum. They projected to the anterior median eminence and infundibular stalk. Luteinizing hormone-releasing hormone-immunoreactive perikarya were scattered throughout the basal hypothalamus, being particularly abundant in the arcuate nucleus. They were larger in size in hibernating bats than those in normal (non-pregnant) and pregnant females. They projected fibers mainly to the internal layer of the median eminence and infundibular stalk. A few luteinizing hormone-releasing hormone-reactive fibers were also observed in the organum vasculosum laminae terminalis, lateral habenular nuclei, pineal stalk, retroflexus fasciculus, and olfactory tubercle. Corticotropin releasing factor-immunoreactive perikarya were distributed in the paraventricular nucleus and medial preoptic area and projected into the external layer of the anterior median eminence, while growth hormone-releasing factor-immunoreactive perikarya occurred only in the arcuate nucleus and projected into the posterior part of the median eminence.     

The origin of somatostatin fibers in the median eminence and neural lobe of Rana temporaria

Summary A light microscopic immunocytochemical study of the brain of frogs with hypothalamic lesions was performed in order to obtain evidence concerning the origin of somatostatin fibers in the median eminence and neural lobe of the hypophysis. The results indicate that the somatostatin fibers of the neural lobe originate from somatostatin perikarya located in the prechiasmatic part of the hypothalamus and possibly also in the telencephalon. The somatostatin neurons of the pars ventralis tuberis cinerei do not send axons to the neural lobe. The frog median eminence contains axon terminals of somatostatin neurons located in the pars ventralis of the tuber cinereum. Many other somatostatin fibers of the frog median eminence originate from somatostatin neurons located outside the tuber cinereum. Most of these neurons probably lie in the preoptic hypothalamic region.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Galanin-like immunoreactivity in the chicken brain

The anatomical distribution of neurons containing galanin has been studied in the central nervous system of the chicken by means of immunocytochemistry using antisera against rat galanin. Major populations of immunostained perikarya were detected in several brain areas. The majority of galanin-immunoreactive cell bodies was present in the hypothalamus and in the caudal brainstem. Extensive groups of labeled perikarya were found in the paraventricular, periventricular, dorsomedial and tuberal hypothalamic nuclei, and in the nucleus of the solitary tract in the medulla oblongata. In the telencephalon, immunoreactive perikarya were observed in the preoptic area, in the lateral septal nucleus and in the hippocampus. The mesencephalon contained only a few galanin-positive perikarya located in the interpeduncular nucleus. Immunoreactive nerve fibers of varying density were detected in all subdivisions of the brain. Dense accumulations of galanin-positive fibers were seen in the preoptic area, periventricular region of the diencephalon, the ventral hypothalamus, the median eminence, the central gray of the brainstem, and the dorsomedial caudal medulla. The distributional pattern of galanin-immunoreactive neurons suggests a possible involvement of a galanin-like peptide in several neuroregulatory mechanisms.     

Somatostatin-immunoreactive nerve fibers in close association with capillaries in the small intestine

Electron microscopic immunocytochemistry was used to identify somatostatin-immunoreactive nerve profiles around the capillary system of the small intestine in the rat and cat. The highest density of somatostatin-immunoreactive nerve profiles was seen in the mucosa, and 25-30% of all immunoreactive profiles were found immediately adjacent to the endothelial cells of capillary blood vessels. About half of the capillary endothelial cells were fenestrated. The majority of the immunoreactive nerve profiles were varicosities, containing synaptic vesicles. These observations suggest that somatostatin might be released from varicose nerve fibers and may contribute to the elevated levels of the peptide measured in portal blood as compared to peripheral blood.     

Hormonal control of lipolysis from the white adipose tissue of hibernating jerboa (Jaculus orientalis)

1. Plasma glucose, glycerol, free fatty acids and total lipid content of the white adipose tissue were measured in euthermic and hibernating jerboa. 2. During hibernation, plasma glucose and glycerol were low compared to the euthermic animals, whereas there was no obvious difference in plasma free fatty acids. The white adipose tissue lipid content was strongly reduced in the hibernating state. 3. The effect of lipolytic hormones (norepinephrine and glucagon) and antilipolytic hormone (insulin) on in vitro glycerol release by adipose tissue isolated from hibernating or euthermic jerboa has been studied. 4. The white adipose tissue from hibernating jerboa presented a higher sensitivity to norepinephrine and glucagon than that of euthermic jerboa; insulin did not modify either basal glycerol release or lipolysis induced by the two lipolytic hormones at low temperatures (7 degrees C) and during the rewarming (from 7 degrees C to 37 degrees C) of the tissue slices. 5. These results suggested that white adipose tissue constitutes an important source of substrates derived from lipolysis during hibernation.     

The serotonin-immunoreactive system of the suprachiasmatic nucleus in the hibernating ground squirrel,Spermophilus richardsonii

Summary In the suprachiasmatic nucleus (NSC) of hibernating and non-hibernating ground squirrels, the distribution of serotonin-immunoreactive (5HT-IR) fibers was studied by the use of the peroxidase-antiperoxidase technique. The cytology of perikarya giving rise to these suprachiasmatic 5HT-IR fibers was investigated in the anterior raphe nuclei. Differences in the immunoreactivity of suprachiasmatic fibers between hibernating and non-hibernating ground squirrels were determined by digital image analysis. The cellular activity was determined densitometrically after RNA-staining in anterior raphe neurons and suprachiasmatic perikarya. Abundant 5HT-IR fibers were observed in the medial and ventromedial portions of the NSC. Frequently, the fibers were found in close contact with perikarya of suprachiasmatic neurons. The central portion of the nucleus and the surrounding hypothalamic areas contained only a few scattered 5HT-IR fibers. Inside the raphe nuclei, 5HT-IR fibers and perikarya formed a dense network. In hibernating ground squirrels, the immunoreactivity to serotonin was approximately 45% higher than in non-hibernating controls. This difference is in accordance with signs of higher neuronal activity (40% higher RNA-content, 20% larger cell nuclei) in 5HT-IR perikarya of the raphe nucleus and the persisting activity of the NSC during hibernation; the activity of other brain regions dropped conspicuously in torpid animals.Supported by the Deutsche Forschungsgemeinschaft (Nu 36/2-1)     

Immunocytochemical evidence for anti-LHRH and anti-ACTH activity in the "F" antiserum.

An antiserum which has previously been thought to be specific for LHRH-like immunoreactive material was shown to contain two populations of antibodies, one demonstrating anti-LHRH activity and the other anti-ACTH(1-24) activity. In rat and mouse, ACTH(1-24)-like immunoreactive substance is present in perikarya within the basal hypothalamus and in fibers in arcuate, periventricular and dorsomedial nuclei. LHRH-like immunoreactivity is present in fibers within the median eminence and arcuate nucleus, in a few fibers running along the ventral border of the hypothalamus, and in a small number of cell bodies within the medial basal hypothalamus.