The somatostatin system of the brain and hibernation ... - Springer Link

Cell Tissue Res (1997) 288:441–447

© Springer-Verlag 1997

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The somatostatin system of the brain and hibernation in the European hamster (Cricetus cricetus) Frank Nürnberger1, Klaus Pleschka2, Mireille Masson-Pévet3, Paul Pévet3 1

Dr. Senckenbergische Anatomie, Klinikum der J. W. Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt a.M., Germany MPI für physiologische und klinische Forschung, W. G. Kerckhoff-Institut, Parkstraße 1, D-61231 Bad Nauheim, Germany 3 CNRS-URA 1332, Neurobiologie des Fonctions Rhythmiques et Saisonnières, Université Louis Pasteur, F-67085 Strasbourg, France 2

&misc:Received: 20 May 1996 / Accepted: 15 December 1996

&p.1:Abstract The depression of physiological processes characteristic of mammalian hibernation is precisely regulated by the central nervous system, especially by the neuropeptidergic apparatus of the hypothalamus. Because of inhibitory influences on neuronal circuits within the brain and suppressive effects on the metabolism via the endocrine axis, somatostatin has been implicated in the regulation of hibernation. The somatostatin system of the brain was investigated with immunocytochemistry, in situ hybridization, and radioimmunoassays in euthermic summer, euthermic winter, and hibernating European hamsters (Cricetus cricetus). Numerous somatostatin-immunoreactive perikarya were observed in the periventricular hypothalamic nucleus. The striatum, amygdala, and cortex contained only scattered immunoreactive perikarya. These entities also contained immunoreactive fiber profiles, although the highest density of immunoreactive fibers was found in the median eminence. Immunocytochemistry and radioimmunoassays showed that the number of somatostatin-immunoreactive perikarya and fibers and the content of somatostatin in the hypothalamus and the median eminence was conspicuously lower in euthermic winter animals than in euthermic summer animals. This decrease was more pronounced in hibernating specimens. In situ hybridization also demonstrated a decrease in the expression and synthesis rate of somatostatin in euthermic winter animals; again, this was even more dramatic in hibernating hamsters. These changes were less pronounced or non-significant in the extrahypothalamic somatostatin-immunoreactive perikarya and fiber systems, as shown by immunocytochemistry and radioimmunoassay, respectively.

Dedicated to Professor A. Oksche in admiration and friendship on the occasion of his 70th birthday This work was supported by grants from the Deutsche Forschungsgemeinschaft (Nu 36/2) and the PROCOPE-program Correspondence to: F. Nürnberger&/fn-block:

&kwd:Key words: Growth hormone cells – Hibernation – Hypothalamo-hypophysial system – Somatostatin – Cricetus cricetus (Rodentia)

Introduction Mammalian hibernation represents a specialized physiological condition that allows survival in harsh winter conditions (Eisentraut 1956; Kayser 1961). By decreasing their body temperature (Tb) to a value close to the ambient temperature (~4°C inside the hibernation den of the European hamster), hibernators are able to save up to 90% of the amount of energy that euthermic animals need for survival in winter conditions (Wang 1989). Despite deep hypothermia, hibernation is a process that is precisely regulated at a low metabolic level. Accordingly, hibernators maintain their Tb well above 0°C and, moreover, are able to arouse rapidly because of their capacity to produce internal heat (cf. Lyman et al. 1982). The energy driving the physiological processes during hibernation is mainly provided by metabolizing white adipose tissue (Wang 1989), whereas heat production for periodic arousals from hibernation is based on the uncoupled decomposition of brown adipose tissue (non-shivering heat production; cf. Florant et al. 1993). During hibernation, the weight and volume of muscle tissue and inner organs remain almost constant (Wickler et al. 1993). Metabolic processes during hibernation are controlled by the neuropeptidergic hypothalamo-hypophysial system. Lipotropin is involved in the control of the activity of adipose tissue (Kuhn et al. 1984), thyrotropin generally activates the cell metabolism via the secretion of thyroid hormones (Lissitzky 1990), and somatotropin (STH=growth hormone) is involved in the regulation of the turnover of proteins (Schoenle et al. 1982). Accordingly, the maintenance of the weight and volume of muscle and parenchymal tissues, which are rich in protein, is mainly regulated by the STH system of the pituitary. The

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release of STH, in turn, is stimulated by hypothalamic growth hormone-releasing hormone (GRH=somatoliberin) and inhibited by somatostatin (SOM=somatotropin release-inhibiting factor). Therefore, the latter two peptides may actively participate in the control of hibernation (Nürnberger 1995; Nürnberger et al. 1986). Apart from their influence on the pituitary, these neuropeptides may control hibernation physiology via their effects within the brain itself (Nürnberger 1995). SOM may inhibit most functional systems during hibernation, since it suppresses the firing rate in various regions of the brain involved in vegetative regulation in non-hibernators (Renaud et al. 1975, 1976). The aim of the present study has been to elucidate the influence of the SOM system within the brain and hypothalamo-hypophysial axis on the control of hibernation. The distribution and reactivity of individual components of the SOM system have been studied in non-hibernating euthermic summer and winter animals and in hibernating European hamsters, by the use of immunocytochemistry (ICC), radioimmunoassay (RIA), and in situ hybridization (ISH). Materials and methods

was omitted, and buffer was used instead. (2) Non-specific staining was tested after adsorption of the SOM antiserum to a solidphase affinity matrix of SOM conjugated to cyanogen-bromideactivated Sepharose. (3) The conditions for optimal staining were tested by means of increasing dilutions of the SOM antiserum and subsequent selection of the concentration optimally staining perikarya and fibers of SOM-containing neurons with little or no background reaction.

Radioimmunoassay For RIA, the hamsters were decapitated, their brains were dissected, and tissue samples of the septum, hypothalamus, cortex, pituitary, and plasma were collected and assayed by a SOM-RIA kit based on rabbit antiserum to synthetic cyclic SOM-14 (Bühlmann Laboratories; Biermann, Bad Nauheim, Germany). SOM was extracted from the tissue samples by the use of 2 M acetic acid as described by Nürnberger et al. (1986). Plasma peptides were extracted by applying the acetone/petrol ether method of Peeters (1981). The antigen-antibody complex was precipitated by the combined application of anti-rabbit gammaglobulin and polyethylene glycol. This resulted in a resolution of up to 2.5 pg SOM per assay tube. Human plasma was used as a control. The concentration of 70–100 pg SOM/ml plasma detected in these samples was well within the normal range, thus, indicating the validity of the RIA applied in this study. At variance with an earlier approach (Nürnberger et al. 1986), high-performance liquid chromatography was not performed for the purification of SOM-14 vs SOM28, since both forms show identical functional behavior.

Animals and experimental groups The present investigation was performed with 42 adult male European hamsters (Cricetus cricetus, L. 1758, Cricetidae, Rodentia); 15 animals were used for ICC, 15 animals for RIA, and 12 hamsters for ISH. All animals were trapped, according to the legal regulations of France, in their natural habitat (agricultural land) to the south of Strasbourg during early spring. The hamsters were transferred to large Macrolon cages and kept under natural climatic conditions. Food (seeds, pellets) and water were provided ad libitum. For each type of investigation, three groups of animals were established: the first group, euthermic summer animals, consisted of sexually active hamsters studied during late spring and summer; the second group, euthermic winter animals, consisted of non-hibernating hamsters studied during winter; and the third group, hibernating animals, was composed of animals investigated during deep hibernation in winter. Five hamsters from each group were used for ICC and RIAs, respectively, and 3 hamsters from each group for ISH.

In situ hybidizatiom For ISH, the brains were collected by immediate dissection from decapitated hamsters and frozen on dry ice. Sections (20 µm thick) were cut on a cryostat and kept frozen (–80°C) until use. After fixation (4% paraformaldehyde) of the frozen sections on slides, ISH was performed by means of (1) the work station of British Bio-technology (distributed by Biermann, Bad Nauheim, Germany), and (2) the application of a digoxigenin-labeled singlestranded DNA-anti-sense probe complementary to 42 bases encoding for the whole amino-acid sequence of rat SOM-14 (British Bio-technology Products). The hybridization signal was detected with an anti-digoxigenin antiserum labeled with alkaline phosphatase, 5-bromo-4-chloro-3-indolyl phosphate, and nitroblue tetrazolium. For specificity controls, the specific anti-sense probe was substituted by the sense probe or completely omitted. Furthermore, the anti-sense probe was applied to sections pretreated with RNAse.

Immunocytochemistry For ICC, the animals were perfused with isotonic saline containing 5000 IU heparin/l and Bouin’s solution. The brains were dissected, postfixed in Bouin’s solution for 24 h, embedded in paraffin, and cut into 7-µm-thick serial sections. The sections were deparaffinized and then incubated consecutively with normal swine serum (1:20, 15 min), a rabbit antiserum to synthetic (rat-like) SOM linked to bovine serum albumin (1:1000, 18 h; UCB, Brussels, Belgium), anti-rabbit IgG (1:50, 30 min; Dako, Hamburg, Germany), peroxidase-antiperoxidase (1:80, 30 min; Dako), and finally diaminobenzidine (DAB; 0.02%; Sigma, Deisenhofen, Germany) plus hydrogen peroxide (0.02%, 10 min), according to the original method of Sternberger (1979). All incubations were performed in phosphate-buffered saline (0.1 M, pH 7.4), except for the DAB reaction, when TRIS buffer (0.1 M, pH 7.4) was used. The specificity and concentration tests were performed according to techniques described previously (Nürnberger et al. 1986). (1) SOM antiserum, anti-rabbit IgG, or peroxidase-antiperoxidase

Analysis and statistics All sections treated for ICC and ISH were analyzed and photographed by use of a Zeiss Axioskop microscope equipped with an MC 100 microscope camera (Zeiss, Jena, Germany). Within all sections of one parallel series from each animal studied, the num-

Fig. 1. Somatostatin-immunoreactive elements (a–c, g–m) and in situ hybridization product (d–f) in the hypothalamus of the European hamster at various functional stages. a–f Periventricular nucleus, g–i median eminence, k–m organum vasculosum of the lamina terminalis. BV, Blood vessel; E, ependyma; H, hibernating animal; PT, pars tuberalis; RI, infundibular recess; S, euthermic summer animal (sexually active); W, euthermic winter animal (sexually inactive); ZE, external zone; ZI, internal zone of median eminence; III, third ventricle (including long arrows); arrowheads, fiber boutons. ×400&ig.c:/f

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444 ber of periventricular SOM-immunoreactive (SOM-IR) perikarya was counted by means of the same microscope equipped with a drawing tube. The digoxigenin signal from the ISH was measured by densitometry (VIDAS, Kontron, Munich), and Student’s t-test was used to estimate statistical significance.

Results Immunocytochemistry The largest number and highest density of SOM-IR perikarya was observed in the hypothalamic periventricular nucleus (Fig. 1a-c). These neuronal perikarya were found in close spatial relationship to the medial preoptic, suprachiasmatic, and paraventricular nuclei. Extrahypothalamic SOM-IR perikarya were found in the tel-

Fig. 2. Stage-related changes in somatostatin-immunoreactive fibers (arrowheads) and perikarya (arrows) within the caudate nucleus (a–c), bed nucleus of the stria terminalis (d–f), and lateral septal nucleus (g–i) of the European hamster. E, Ependyma; H,

encephalic cortex, basal ganglia (nucl. caudatus: Fig. 2a–c), amygdala, and bed nucleus of the stria terminalis (Fig. 2d–f). The number of labeled perikarya in these areas was, however, considerably lower than that in the periventricular nucleus (3 or fewer perikarya per frontal section of the respective entity). SOM-IR fibers were observed in all regions containing SOM-IR perikarya (see above) and also in the telencephalic septum (Fig. 2g–i), the hypothalamic paraventricular, ventromedial, infundibular, and premammillary nuclei, the mesencephalic interpeduncular nucleus, the organum vasculosum of the lamina terminalis (Fig. 1k–m), and in the median eminence, which showed the highest density (Fig. 1g–i). Among hibernating and euthermic hamsters, which were sacificed either in the summer or in the winter, sev-

hibernating animal; LV, lateral ventricle; S, euthermic summer animal (sexually active); W, euthermic winter animal (sexually inactive). ×400&ig.c:/f

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periventricular nuclei of euthermic summer animals, that it was slightly decreased in euthermic winter animals (≈30% decrease), and that it was lowest in hibernating winter animals (≈70% decrease; Fig. 1d–f). No quantification was performed in extrahypothalamic regions, because they contained only a few perikarya (see above). Discussion

Fig. 3. Histogram of the content of somatostatin (SOM) in samples of brain tissue and blood plasma in hibernating winter (HIBERNATING), euthermic winter (EUTHERM), and euthermic summer, sexually active (SUMMERACTIVE) European hamsters, as measured by radioimmunoassay. CORT., Cortex; HYPOTH., hypothalamus; PROT., protein; * P=0.05, ** P=0.01&ig.c:/f

eral conspicuous differences in SOM immunoreactivity were observed. The most prominent SOM immunoreactivity was found in euthermic summer hamsters (cf. Figs. 1, 2). In euthermic winter animals, the SOM immunoreactivity was strongly decreased, especially in the periventricular nuclei. The weakest immunoreactivity was detected in hibernating European hamsters, which showed the smallest number of SOM-IR perikarya and fibers. From counts in serial sections, the total number of SOM-IR perikarya within the left and right periventricular nuclei was estimated to be approximately 1600 in euthermic summer animals (density ≈2900/mm³), ≈1100 in euthermic winter animals (density ≈2000/mm³), and ≈350 in hibernating hamsters (density ≈650/mm³). No immunostaining could be found after preadsorption of the antiserum with SOM-14. Radioimmunoassay The content of SOM-like material in the hypothalamus, septum, and pituitary was more than three times higher in euthermic summer hamsters than in euthermic winter animals (Fig. 3). In hibernating animals, the content of assayable SOM-like material was markedly decreased in the septum and pituitary (P≤0.05) and tended to be lower in the hypothalamus (P~0.05). The concentration of SOM-like material in plasma, however, did not vary significantly among animals from the three experimental groups (Fig. 3). In situ hybridization The anti-sense probe for the nucleotide sequence of SOM-14 labeled cell bodies in the hypothalamic periventricular nuclei (Fig. 1d–f), and only a few labeled elements were observed in those telencephalic regions shown to contain SOM-IR perikarya. The sense probe for SOM-14 did not hybridize. Densitometry performed on computerized images showed that the hybridization signal was strongest in the

The major aim of the present investigation has been to elucidate the functional relationship between hibernation and the SOM system in the brain of European hamsters. With regard to the spatial organization, this study shows that the SOM system in the brain of the European hamster is similar to that of other representative mammalian species (Insectivora: Nürnberger et al. 1986; Rodentia: Dubois et al. 1974; Finley et al. 1978; Krisch 1978; Bennett-Clarke et al. 1980; Richoux and Dubois 1980; Nürnberger et al. 1986; Carnivora: Hoffman and Hayes 1979; Primates: Bugnon et al. 1977). In all these species, the periventricular nucleus contains the highest density of SOM-IR neuronal perikarya. In accordance with the above-mentioned studies, the present investigation has also revealed the presence of SOM-IR cell bodies in several telencephalic areas (cortex, putamen, nucl. caudatus, amygdala, bed nucleus of the stria terminalis) of the European hamster. In contrast to findings in the rat (Hökfelt et al. 1978; Graybiel and Elde 1983) and hedgehog (Nürnberger et al. 1986), immunostained perikarya have not been detected in the reticular thalamic and supraoptic hypothalamic nucleus of Cricetus cricetus The distributional pattern of the SOM-IR fiber systems in the European hamster closely resembles that of the rat (Krisch 1978). The hypothalamo-hypophysial tract, which in part originates from the cell bodies of the periventricular nucleus (Hoffman et al. 1986), also contains the most prominent SOM-IR fiber tract in Cricetus cricetus. As in the rat, the European hamster is endowed with SOM-IR fiber terminals in various limbic and extrapyramidal motor areas (e.g., septum, amygdala, premammillary nucleus, striatum, habenula, interpeduncular nucleus) and in the organum vasculosum of the lamina terminalis. Irrespective of this general distributional pattern, the SOM-IR system shows conspicuous differences among euthermic summer, euthermic winter, and hibernating hamsters. The changes are uniform in the various brain areas studied and are readily observed with all three techniques applied in this investigation, i.e., ICC, RIA, and ISH. The most pronounced changes occur in the hypothalamo-hypophysial system. The changes are positively correlated with the activity stages: the euthermic summer animals, which are sexually active, display the strongest immunoreactivity; the euthermic winter animals, which are sexually inactive, show medium levels; and the hibernating hamsters display the lowest levels of SOM immunoreactivity. The data obtained with ISH are in good agreement with the immunocytochemical findings. The strong ISH signal in euthermic summer animals indicates a high level of SOM synthesis during the sexually active phase;

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the ISH signal is reduced by approximately 70% in hibernating hamsters, whereas no significant differences have been found between euthermic winter and euthermic summer animals. The changes in the hypothalamo-hypophysial system of hamsters are in good agreement with the findings in Richardson’s ground squirrel (Spermophilus richardsonii, Nürnberger et al. 1986) and the golden-mantled ground squirrel (Spermophilus lateralis, Muchlinski et al. 1983), which also show a dramatic decrease in SOM immunoreactivity in euthermic winter animals compared with those that are hibernating. In contrast, hedgehogs (Erinaceus europaeus, Insectivora) display an increase in SOM immunoreactivity during hibernation (Nürnberger et al. 1986). Accordingly, the hibernation-related changes in the brain SOM-IR system vary among hibernators of different mammalian taxa. As found by RIA, the content of SOM in blood plasma is constant among euthermic summer, euthermic winter, and hibernating hamsters. As in mammalian nonhibernators, the maintenance of a high content of plasma SOM during hibernation is probably caused by release from peripheral somatostatinergic neurons/cells (pancreas, gut; cf. Ohnishi et al. 1994). This interpretation is further substantiated by the observations of Zahn (1996) who has found the content of SOM to be strongly elevated in the enteric nervous system and the entero-endocrine system of hibernating Richardson’s ground squirrels (Spermophilus richardsonii). With respect to the functional significance of the changes in the SOM system of the rodent brain, two mechanisms may be involved. First, as known from mammalian non-hibernators, hypothalamic SOM and somatoliberin (=GRH) show antagonistic effects on the release of somatotropin (=STH) from the adenohypophysis; SOM inhibits and GRH enhances the secretion of STH into the peripheral blood stream (Vale et al. 1977; Plotsky and Vale 1985; Epelbaum 1986). Released into the blood stream, STH functions as a strong anabolic factor stimulating protein biosynthesis, with resulting high protein concentrations (Schoenle et al. 1982). Earlier studies in hibernating rodents have indicated that the protein concentration in muscle tissue, which is exceptionally rich in protein, is kept constant even during the long periods of hibernation (Wickler et al. 1993). This may be mediated by the decrease in hypothalamic SOM immunoreactivity during hibernation, a decrease that is accompanied by virtually unchanged immunoreactivity to GRH (Nürnberger and Heinrichs 1989; Nürnberger 1995; cf. McCann 1982). The present data do not clarify the question as to whether the drop in SOM immunoreactivity and gene expression reflects an endogenous action or a passive reaction of the brain SOM system. Secondly, the differences in the activity of the brainSOM system between euthermic summer and winter hamsters indicate a regulatory role for the SOM system in reproductive physiology. As described by Vale et al. (1974), SOM is a potent inhibitor of the synthesis of prolactin. Prolactin levels are also diminished when hypothalamic gonadotropin-releasing factor (GnRF) induces the release of luteinizing hormone and follicle-stimulating hormone from the adenohypophysis. The strong im-

munoreactivity to both SOM (present study; Muchlinski et al. 1983) and GnRF (Nürnberger 1995) during the sexually active phase may explain the decreased concentration of prolactin during the reproductive phase in rodent hibernators (Goldman et al. 1986). The concentration of prolactin increases at the end of the reproductive phase (Goldman et al. 1986), whereas the SOM immunoreactivity has begun to decline (Muchlinski et al. 1983). At present, the mechanism of the somatostatinergic control of prolactin secretion is not completely understood. (1) SOM directly stimulates the secretion of dopamine from neurons of the hypothalamus (Epelbaum 1986); dopamine is known to be the inhibiting factor for the release of prolactin. (2) A second mechanism may involve the raphe nuclei as a relay station. These nuclei receive SOM projections from the preoptic hypothalamic area, projections that inhibit serotonin production (Beitz et al. 1983). Since serotonin, which is released from hypothalamic projections of the raphe nucleus, inhibits the release of dopamine in the infundibular nucleus (cf. Steinbusch and Nieuwenhuys 1981), increased hypothalamic SOM production could finally result in the inhibition of prolactin release (Beitz et al. 1983; Bendotti et al. 1993). The increased SOM expression at the beginning of the reproductive phase may be caused because the content of SOM in the ventral preoptic hypothalamus, including the periventricular and suprachiasmatic nucleus (cf. Nürnberger et al. 1989, is elevated during the light phase of the diurnal cycle (Fukuhara et al. 1993, 1994; Yang et al. 1994). The increasing length of the light phase in spring may explain the elevated SOM concentration in euthermic, sexually active summer animals. Possible central effects of SOM remain a matter of debate. The comparatively smaller decrease in immunoreactivity and in the content of SOM within control regions of the extrapyramidal motor areas (e.g., striatum, interpeduncular nucleus) is in accord with the demands of hibernation; in all these regions, neuronal activity is dramatically diminished during the torpor state (Lyman et al. 1982; Heller et al. 1989). Only the septum, which represents a communication pathway between the limbic system and the endocrine hypothalamus (Staiger and Nürnberger 1991) and which is also a major target of SOM input (Köhler and Eriksson 1984), maintains its bioelectric and synthetic activity throughout the hibernation period (cf. Nürnberger 1995). However, in the light of the differential expression of activating and inactivating SOM receptors (Bell and Reisine 1993), clarification of the operational mode of SOM in the control of hibernation should be obtained by further investigations. References Beitz AJ, Shepard RD, Wells WE (1983) The periaqueductal gray-raphe magnus projection contains somatostatin, neurotensin and serotonin but not cholecystokinin. Brain Res 261:132–137 Bell GI, Reisine T (1993) Molecular biology of somatostatin receptors. Trends Neurosci 16:34–38 Bendotti C, Tarizzo G, Fumagalli F, Baldessari S, Samanin R (1993) Increased expression of preproneuropeptide Y and preprosomatostatin mRNA in striatum after selective serotoninergic lesion in rats. Neurosci Lett 160:197–200

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