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With reference to observations in the hedgehog, Erinaceus europaeus*. Christoph U. Schindler and Frank Niirnberger. Department of Anatomy and Cytobiology, ...
Cell Tissue Res (1990) 262:293-300

andTissue Research 9 Springer-Verlag 1990

Hibernation-related changes in the immunoreactivity of neuropeptide systems in the suprachiasmatic nucleus of the ground squirrel, Spermophilus richardsonii With reference to observations in the hedgehog, Erinaceus europaeus* Christoph U. Schindler and Frank Niirnberger Department of Anatomyand Cytobiology,Justus LiebigUniversity,Giessen, Federal Republic of Germany Accepted June 15, 1990

Summary. The pattern of distribution and reactivity of the neuropeptides vasopressin (AVP), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), substance P (SP), and thyrotropin-releasing hormone (TRH) were studied in the suprachiasmatic nucleus (NSC) of 20 Richardson's ground squirrels (and 7 European hedgehogs) of both sexes during hibernation and euthermia. The total area of immunostained structures revealed by application of the individual immunocytochemical techniques was measured by means of computer-aided image analysis. In both species, elements of all peptide systems examined were related to particular subdivisions of the NSC. The pattern of immunoreactivity was strongly correlated with the physiological stage of hibernation or euthermia both in ground squirrels and hedgehogs. The immunoreactivities to AVP and SP increased in area during hibernation (AVP: 25%; SP: 25%), whereas the respective area immunoreactive to NPY and VIP decreased (NPY: 45%; VIP: 100%) in comparison to nonhibernating controls. The TRH-immunoreactive nerve fibers were rare and rather scattered; thus, the quantitative procedure was not applicable for this immunoreaction. Key words" Suprachiasmatic nucleus - Neuropeptide immunocytochemistry - Image analysis - Neuropeptide Y - Substance P - Thyrotropin-releasing hormone - Vasoactive intestinal peptide - Vasopressin Spermophilus richardsonii (Rodentia) - Erinaceus europaeus (Insectivora) * The results have been partly presented at the i0th International Symposium on Neurosecretion held in Bristol, UK, September 1987 Send offprint requests to: Dr. F. Nfirnberger, Department of Anatomy and Cytobiology,Aulweg123, D-6300 Giessen, FRG Abbreviations." AVP arginine vasopressin; N P Y neuropeptide Y; NSC suprachiasmaticnucleus; SP substance P; TRH thyrotropinreleasing hormone; VIP vasoactiveintestinalpeptide

The suprachiasmatic nucleus (NSC) is regarded to play a crucial role in the regulation of biorhythmic phenomena. As shown in a variety of investigations, the NSC particularly hosts the central nervous pacemaker of circadian cycles (Moore 1982). Its involvement in the entrainment of circannual rhythms could, however, not be clearly demonstrated (Moore 1979; Zucker et al. in press). By the use of immunocytochemical methods, the NSC of several species can be divided into different subdivisions, according to the respective subpopulations of neurons or patterned afferents to these neurons (see van den Pol und Tsujimoto 1985). To date, the functional role of this organizational pattern is still a matter of discussion. To elucidate the state of the neuropeptide-containing perikarya in and the afferents to the different subunits of the mammalian NSC, these aspects were studied during natural hibernation and euthermia; this functional model provides drastic variation in neuronal activity without producing experimental artifacts. On the other hand, hibernation is accompanied by characteristic physiological events, which clearly reveal activity stages of a number of regulatory processes (cf. Wang 1989). Since, to date, most of these regulatory mechanisms are only partly understood, it appears expedient to study the involvement of clearly defined functional components in the regulation of hibernation also with respect to the control of biorhythmicity. Especially for the NSC, which obviously represents one of the major integrative centers for peptidergic and aminergic control of biorhythmical processes, an account of their involvement during hibernation is still lacking (Niirnberger et al. 1989). In this respect, the maintenance of a relatively increased activity of suprachiasmatic neurons (Kilduff et al. 1982, 1986) may be explained by the interplay of specific sets of transmitters and peptides in the NSC. In the present study, the immunocytochemically detected distributional pattern of five neuropeptides in the

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NSC of two species of obligatory hibernators was studied with particular emphasis on differences in immunoreactivity among the individual stages of hibernation and euthermia. This functional approach is crucial for a better understanding of the excitatory or inhibitory effects of particular transmitters or modulators on the pacemaker system of the NSC (see also van den Pol, in press).

Materials and methods The present investigation is based on findings obtained from 14non-hibernating, euthermic, and 9 hibernating Richardson's ground squirrels (Spermophilus richardsonii; 1- to 2-year old males), which were compared with results obtained in 2 euthermic and 5 hibernating European hedgehogs (Erinaeeus europaeus; of both sexes, age > 2 years). The ground squirrels were caught in their natural habitat in the foothill region (north of Calgary, Alberta, Canada) during the summer months. They were kept under ambient conditions of temperature and photoperiod in Giessen (FRG, 50~ 8~ food (rats pellets, fruits, grain) and water were provided ad libitum. Unstained sections from the brains of hedgehogs were taken from the collection of the Department of Anatomy and Cytobiology, University of Giessen. These animals were trapped several decades ago either in the State of Hessen (FRG) or in Finland during the late summer months. They had been kept in large cages at ambient temperature and photoperiod in Giessen or Helsinki. The hedgehogs were reported to have free access to food (dog food, fruits) and water. Under natural ambient temperature and illumination, individuals of both species were reported to enter the state of hibernation. These animals were sacrificed for the investigations after a minimum of 3 days in deep hibernation (lethargy, body temperature Tb < 8~ C). The animals representing the group of non-hibernating, euthermic controls were prevented from hibernation by high ambient temperatures ( ~ 20~ C) in a vivarium and a daily photoperiod of 10L14D. To obtain brain material for immunocytochemical investigations, the ground squirrels were anesthetized with sodium pentobarbital (50 mg/kg body wt., i.p.) and perfused intracardially with a 0.9% sodium chloride solution (containing 15 000 IU heparine/1, perfusion pressure 85 Tort for ~ 3 min), followed by Bouin's solution (for ~ 15 min). In principle, the hedgehog brains were preserved accordingly, although with some variation in the protocol. To prevent problems caused by circadian variations of the peptide content within the NSC, all ground squirrels were sacrificed at the same time of day (10:00-11:00 a.m.). Subsequently, the brains were dissected out of the skull, postfixed in Bouin's solution (for ~48 h), embedded in paraffin, cut in serial sections (7 gm thick), and mounted in the form of 6 parallel serial sets. The sections were processed for the individual immunoreactions with primary antisera according to the peroxidase-antiperoxidase (PAP) method (Sternberger et al. 1970). The VIP antiserum was obtained from Cambridge Immunochemicals (Cambridge, UK). The antiserum against NPY was donated by Dr. J.K. McDonald (Atlanta, GA, USA); the antiserum against TRH was obtained from Dr. R. J6sza (P6cs, Hungary); the SP antiserum was contributed by Dr. G. Harti (Bad Nauheim, FRG); and the AVP antiserum was produced in our own laboratory. (As tested by RIA and immunocytochemistry, this AVP antiserum expresses only very weak cross-reactivities for oxytocin and vasotocin; it was generally used after preabsorption to oxyt0cin.) Goat-anti rabbit immunoglobulin (IgG) was purchased from Behringwerke (Marburg, FRG), and the peroxidase-anti-peroxidase complex from Dakopatts (Hamburg, FRG). For specificity tests, each single step of the staining procedure was omitted and replaced by buffer incubation. The specificity of the reactions was also verified by investigating reference slices

of different brain nuclei expressing a strong immunoreactivity to the different antigens and by preabsorbing the different antisera with their specific antigens (1 gM). For comparison of the patterns of immunoreactivity of the different peptide systems, the total area of immunostained elements (nerve fibers and perikarya) within a specific sample area of the NSC was measured by means of image analysis. These measurements of the area density were exclusively performed on comparable levels of frontal sections through the ground-squirrel NSC, since intraspecific variations in the immunostaining of each of the two physiological states were not observed. The individual variability of the immunostaining was more pronounced in hedgehogs probably due to sex-dependent and/or ecological factors. The computer-aided analysis of the area density and its validity for the interpretation of immunocytochemical findings have been extensively discussed earlier (Nfirnberger et al. 1989), Selected serial sections were stained for basophilic material according to Nissl (toluidine blue) or for Nissl material in combination with myelin staining according to Kliiver and Barrera (1953; cresyl fast violet+luxol fast blue) to allow the proper neuroanatomical localization of the immunocytochemical results.

Results

The suprachiasmatic nucleus (NSC) of ground squirrels contained perikarya and/or nerve fibers immunoreactive with antisera to all peptides examined (Fig. 2). All these peptide systems (with exception of the TRH-immunoreactive structures) displayed characteristic differences in their immunoreactivity between hibernating and euthermic animals (Fig. 3). For some of the peptides the reactivity was enhanced, for others it decreased during hibernation.

1. Neuropeptide Y (NPY). In the ground squirrel, a dense plexus of NPY-immunoreactive nerve fibers was located in the ventral and ventrolateral divisions of the NSC. The other subdivisions of the nucleus contained only very few stained fibers. The diameter of these fibers

Fig. 1 a-h. a Location of the NSC in the hypothalamus of the ground squirrel, Spermophilus richardsonii. Combined staining of Nissl substance and myelinated fibers (Klfiver and Barrera 1953). CO optic chiasm; III third ventricle; dashed line border of the NSC. • 80. b VIP-immunoreactive perikarya and fibers in the ventral subdivision of the NSC of a non-hibernating,euthermic ground squirrel. No immunoreacfive structures could be observed in hibernating animals. • 240. e, d AVP-immunoreaetive perikarya and fibers in the dorsomedial subdivision of the NSC of hibernating (e) and euthermic ground squirrels (d). Note the dense fiber network surrounding unstained suprachiasmatic perikarya within the dorsomedial portion of the ground squirrel NSC. E ependyma of the third ventricle (III); arrows indicating less intensely stained AVP-immunoreactive perikarya; dashed line dorsomedial border of the NSC. In euthermic animals, stained perikarya occur rarely. x 240. e, f SP-immunoreactive fibers within the lateral subdivision of the NSC of hibernating (e) and euthermic (f) ground squirrels. The fiber terminals are generally in close contact with suprachiasmatic perikarya (asterisks). x 500. g, h NPY-immunoreactive fibers in the lateroventral subdivision of the NSC of hibernating (g) and non-hibernating(h) ground squirrels. Boutons are mostly observed in neuropil areas distant from suprachiasmatic perikarya (asterisks), x 240

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2. Arginine vasopressin (AVP). AVP-immunoreactive neuronal somata were preferentially found in the dorsomedial subunit of the NSC (Fig. 1 c, d). In both the hibernating and euthermic stage, a dense meshwork o f AVP-immunoreactive fibers encompassed suprachiasmatic perikarya of the dorsomedial subunit. The arrangement of the numerous beaded fibers was suggestive of axo-somatic contacts. The area o f the immunostained terminal structures in hibernating ground squirrels exceeded that in euthermic controls by approximately 25% (see Fig. 3). In addi-

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subdivisions in the central portion of the NSC of the ground squirrel as observed in hibernating (left) and nonhibernating (right) animals. 9 Perikarya immunoreactive for AVP (dorsal portion) or VIP (ventral portion); CO optic chiasm; III third ventricle

tion, in hibernating animals, a considerable number of AVP-immunoreactive perikarya (displaying the typical cytoarchitectonic features of suprachiasmatic neurons) could be detected, whereas such perikarya were rare in euthermic animals; however, a rather large number of AVP-immunoreactive cell bodies were observed after intraventricular application of colchicine (50 gg/kg b.w.) also in non-hibernating ground squirrels (material obtained from another investigation not focused on the NSC).

3. Vasoactive intestinal peptide (VIP). A dense plexus of VIP-immunoreactive nerve fibers was regularly found in the ventral and ventromedial subdivision of the NSC of euthermic Richardson's ground squirrels. Scattered fibers were also distributed in the other subdivisions, These fibers formed numerous terminal knobs close to the perikarya. Furthermore, some perikarya of the ventral subdivision of the NSC expressed a strong immunoreactivity for VIP (Fig. 1 b). In hibernating animals, the VIP-immunoreactive elements were very rare or even undetectable; perikarya were never stained. Accordingly, the area immunoreactive for VIP was reduced by 100% in hibernating individuals (cf. Fig. 3).

4. Substance P ( SP). SP-immunoreactive fibers were distributed diffusely around perikarya in a basket-like fashion (Fig. 1 e, f). During hibernation, SP-immunoreactive fibers were more abundant and more densely arranged than in the euthermic controls. In hibernating animals, the immunoreactive elements covered an area 25% larger than that in controls (see Fig. 3). Suprachiasmatic perikarya were not stained for SP at any functional stage examined. 5. Thyrotropin-releasing hormone (TRH). The TRH-immunoreactive elements were sparsely represented in the

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Light synchronization NSC of the ground squirrel. Only a few terminals were stained and diffusely scattered over the entire area occupied by the NSC. A morphometric evaluation of these few fibers was not possible. The small number of stained structures did not allow to measure differences between hibernating and euthermic individuals. Observations in the hedgehog. In principle, the immunocytochemical observations in hedgehogs did not differ from those in ground squirrels. Although the immunostaining for the individual neuropeptides was more variable among animals representing one certain physiological stage, the differences observed between the group of hibernating and euthermic hedgehogs corresponded to the differences found between the respective groups of ground squirrels. However, the peculiar arrangement of VIP-immunoreactive perikarya in the NSC of the hedgehog should be mentioned; these perikarya are located in a linear fashion marking the borderline between the NSC and the optic chiasm.

Discussion The present results obtained from the immunocytochemical investigation of the NSC in euthermic ground squirrels and hedgehogs are consistent with findings in other mammalian species and also comparable to data concerning the suprachiasmatic organization in birds. They support the concept of distinct neuroanatomical subdivisions of the NSC (Mammals: golden hamster, Card and Moore 1984; man, Stopa et al. 1984; rat, van den Pol and Tsujimoto 1985; cat, Ueda et al. 1986; ground squirrel, Reuss et al. 1989; Schindler et al. 1989. Birds: house sparrow, Cassone and Moore 1987; dove, Norgren and Silver 1989.) Perikarya and/or nerve fibers immunoreactive for VIP were observed in the ventral subunit, for AVP in

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Fig. 4. Hypothetical diagram of the functional involvement of peptides (AVP, NPY, SP, VIP), serotonin (5HT), and melatonin in the control of the suprachiasmatic oscillator (central circle). NPY, VIP, melatonin and the unknown transmitter of the retinohypothalamic fibers probably act as input control synchronizing the oscillations of the NSC with the light cycle. AVP and 5HT are most likely involved in the output control. LGC lateral geniculate body; PVN paraventricular nucleus; RN raphe nuclei (for further details, see Discussion)

the dorsomedial subunit, for NPY in the ventrolateral subunit, and for SP in the lateral subunit of the NSC; T R H was diffusely distributed. As shown by tracer experiments (horseradish peroxidase: Meijer et al. 1989; Phaseolus vulgaris leucoagglutinin: our unpublished experiments), the retino-suprachiasmatic projection terminated mainly in the ventrolateral portion of the NSC, although a major input of optic fibers was also visible in the dorsomedial subdivision. In addition, serotoninimmunoreactive fibers extended to the medial and ventromedial portions of the suprachiasmatic nucleus (Ntirnberger et al. 1989), whereas GABA-immunoreactire fibers were found in all portions of the NSC (cf. Card and Moore 1984; van den Pol and Tsujimoto 1985). The analysis of the possible functional role of this specific organizational pattern with respect to suprachiasmatic integrative processes is a principal aim of NSC research. In the present study, the alterations in immunoreactivity of hibernating versus euthermic animals were elucidated: In the NSC of hibernating and euthermic, non-hibernating ground squirrels and hedgehogs certain striking variations in the reactivity pattern of the suprachiasmatic neuropeptide systems were observed. During hibernation, the immunoreactivity for AVP and SP was increased, whereas that for VIP and NPY was decreased compared to euthermic controls (see Fig. 3). Accordingly, the dorsal and medial portions of the NSC contain peptide systems, which show enhanced numbers of immunoreactive perikarya and/or nerve fibers during hibernation, whereas at the same time the ventrally and laterally located peptide systems show decreased immunoreactivity. In addition to the peptide systems, the serotonin-immunoreactive neural network, which is preferentially located in the medial subdivision of the NSC, shows increased immunoreactivity during the state of hibernation (Nfirnberger et al. 1989). Soma-

298 tostatin-14-immunoreactive nerve fibers, which have been described in the NSC of rat and hamster (Card and Moore 1984; van den Pol and Tsujimoto 1985), were almost undetectable within the NSC proper of the ground squirrel. However, dense networks of fibers immunoreactive for the mainly inhibitory somatostatin-14 were also found in the periventricular neuronal layers immediately dorsomedial to the NSC (Niirnberger et al. 1986). Comparable spatial and functional specializations among various peptide and transmitter systems of the NSC have been described in the literature. It is well documented that the retinohypothalamic fibers are spatially related to the NPY fibers, which originate from the lateral geniculate nuclei (cf. Moore 1973; Pickard 1985; Ueda et al. 1986; Cassone and Moore 1987). This wide overlap of NPY-immunoreactive afferents and retinohypothalamic terminals could also be verified in the ground squirrel; both components are located in the ventrolateral subdivision of the NSC. In Richardson's ground squirrels (own results) and thirteen-lined ground squirrels (Meijer et al. 1989), however, a conspicuous innervation by retinohypothalamic fibers occurs also in the dorsomedial subdivision of the NSC, which mainly houses the AVP-immunoreactive perikarya and fibers. Possibly, the distributional pattern of melatonin-binding sites might be spatially associated with these optic input systems; however, Vanecek et al. (1987) did not emphasize such a parcellation aspect. Spatial relationships have also been shown for NPY and serotonin (Guy et al. 1987); a suppression of the VIP synthesis by serotonin has been reported (Hery et al. 1986). These relationships are indicative of specialized subdivisions of the NSC, which may be capable of integration of the primary light signals from the retina (i) with the light information already processed via the lateral geniculate body or (ii) with the particular intrinsic situation in peptidergic or aminergic nuclei. As generally accepted on the basis of lesion studies, the NSC is the site of the circadian pacemaker (cf. Moore 1973, 1982). In addition to results from lesion studies, circadian changes have been observed in the content and release of AVP (Schwartz and Reppert 1985; Earnest and Sladek 1986), the gene expression of AVP (Reppert and Uhl 1988) and VIP/PHI (PHI=peptide histidine isoleucine; Albers et al. 1990), the general suprachiasmatic turnover (Schwartz, in press), and the electrophysiology of the nucleus in situ and in slice preparations (Newman and Hospod 1986; Gillette and Prosser 1988; Newman, in press). Most interestingly, these phenomena are conspicuously independent from general biochemical processes in suprachiasmatic neurons as indicated by the lack of circadian variations in protein synthesis (Scammell et al. 1989). Although the physiological capacity of the NSC is sufficiently documented, a well-defined morphological correlate of these functions is still missing. Ultrastructural investigations have revealed direct serial cell contacts in the NSC (Giildner 1976), which might be involved in the generation of circadian oscillations. The aim of the present study, however, was focussed

on the long-term biorhythms of hibernation. To avoid circadian effects, all animals were sacrificed at the same time of the day (10:00-11:00 a.m.). Thus, the observed changes in immunoreactivity reflect long-range phases in functional activity. Hibernation can be regarded as a chronobiological process based on long-term rhythms triggered by a series of endogenous and exogenous 'Zeitgeber'. Long-term pacemaker functions (circannual oscillators) of the NSC have been repeatedly discussed in the literature. However, a clear-cut indication of the existence of such oscillators has not yet been established. In hibernators, Dark et al. (1985) were unable to abolish the circannual rhythmicity by lesioning the NSC. Similarly, Zucker et al. (in press) observed persisting circannual rhythms after NSC lesions; however, the ability to enter hibernation was disturbed (golden-mantled ground squirrels) or even missing (Syrian hamsters) after lesioning of the NSC. These authors also showed that circadian rhythms persist during hibernation. The maintenance of the NSC activity was also described by Kilduff et al. (1982), who found an almost identical level of neuronal activity in this nucleus during hibernation and euthermia after application of ~4C-2-deoxyglucose. The relatively high suprachiasmatic activity during hibernation could also be shown by densitometric measurements of the content of R N A in the neurons of the NSC (Niirnberger et al. 1989). This activity pattern did not differ among the individual subdivisions of the NSC. Although the physiologically revealed activity patterns of the NSC do not completely correlate with the concept reflected by the immunoreactivity for different neuropeptides and transmitters, the present results suggest a general decrease in the extent of peptidergic deposits in the lateroventral division of the NSC, which also harbors the main optical inputs, and, in contrast, an increase of immunoreactivity in the mediodorsal subdivisions encompassing the AVP-, SP-, and serotonin-immunoreactive afferents (cf. Niirnberger et al. 1989). The implications of serotonin in the regulation of hibernation have been discussed elsewhere (Canguilhem 1977; Popova and Voitenko 1981; Nfirnberger et al. 1989). In this respect, the synergism of SP and serotonin should be mentioned (H6kfelt et al. 1987). The influence of the vasopressinergic system on the control of hibernation has been investigated with reference to several sites in the brain indicating hibernation-stage-dependent differences in the vasopressin system of the neurohypophysial axis and in accessory extrahypothalamic locations (Nfirnberger 1983; Nfirnberger etal. 1982, 1985; Hermes et al. 1989). The AVP-mediated effects in the NSC, however, are still enigmatic and, accordingly, the discussion of focal effects of this system on hibernation remains open to discussion. The present results and the data available from the literature reveal a parcellation of the suprachiasmatic nucleus (cf. van den Pol and Tsujimoto 1985; our Fig. 4). Its center is composed of neurons endowed with an unknown transmitter. The neuropeptide and transmitter systems in the marginal subdivisions show a varying pattern of immunoreactivity correlated to biorhythmical

299 p h y s i o l o g i c a l processes. A c c o r d i n g l y , the c e n t r a l p o r tions o f the N S C m a y r e p r e s e n t the m a i n i n t e g r a t i v e center, w h e r e a s the a d j a c e n t , s u r r o u n d i n g s u b d i v i s i o n s m a y be c o n s i d e r e d as the i n p u t o r r e l a y s t a t i o n s o f p e p t i dergic, a m i n e r g i c o r o t h e r c h e m i c a l l y m e d i a t e d i n f o r m a tion (cf. v a n d e n Pol a n d G o r c s 1986). A l t h o u g h this h y p o t h e s i s r e m a i n s to be verified on the basis o f u l t r a s t r u c t u r a l i n v e s t i g a t i o n s d e s c r i b i n g the intrinsic c i r c u i t r y o f the N S C , s o m e a r g u m e n t s s p e a k in f a v o r o f this m o d e o f i n t e g r a t i o n . A s s h o w n b y use o f slice t e c h n i q u e s a n d in h i b e r n a t i n g a n i m a l s in situ, the c i r c a d i a n o s c i l l a t o r r e m a i n s active w i t h o u t e x t e r n a l i n p u t s ( N e w m a n a n d H o s p o d 1986; Z u c k e r et al., in press). F o r s y n c h r o n i z i n g the persisting e n d o g e n o u s r h y t h m with the a m b i e n t p h y s i c a l c o n d i t i o n s , the r e t i n o - s u p r a c h i a s m a t i c a n d the g e n i c u l o - s u p r a c h i a s m a t i c n e u r a l i n p u t s a n d also the mel a t o n i n i n p u t a r e p o s s i b l e c a n d i d a t e s . To c o n t r o l the o u t flow o f the s u p r a c h i a s m a t i c c i r c a d i a n i n f o r m a t i o n , w h i c h is p r o b a b l y p e r m a n e n t l y g e n e r a t e d in the N S C , i n h i b i t o r y o u t p u t r e l a y s t a t i o n s are suggestive. T h e d o r s o m e d i a l p o r t i o n o f the N S C i n c l u d i n g its SP-, A V P - , a n d / o r s e r o t o n i n - i m m u n o r e a c t i v e systems c o u l d serve as a relay s t a t i o n for the o u t p u t c o n t r o l . This h y p o t h e s i s m i g h t be e x p l a n a t o r y for the missing signs o f p e r i p h e r a l c i r c a d i a n activities d u r i n g h i b e r n a t i o n .

Acknowledgements. The authors are grateful to Dr. G. Harti, Dr, R. Jdsza, and Dr. J, McDonald for providing the antisera to SP, TRH and NPY, respectively, to C. Tag for technical assistence, and to the Deutsche Forschungsgemeinschaft for financial support (Nu 36/2-2).

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