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Brain Research, 547 (1991) 115-121 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116529R BRES 16529
Seasonal changes in methionine-enkephalin immunoreactivity in the brain of a hibernator, Spermophilus columbianus E Nurnberger 1, T.E L e e 2, M.L. Jourdan 2 and L.C.H. Wang 2 l Department of Anatomy and Cytobiology, Justus Liebig University, Giessen (E R. G.) and 2Department of Zoology, University of Alberta, Edmonton, Alta. (Canada) (Accepted 13 November 1990) Key words: Hibernation; Methionine-enkephalin; Opioid; Immunocytochemistry; Septum; Hypothermia; Thermoregulation; Ground squirrel
To identify the actual location of central endogenous opioid systems which may be involved in regulating the hibernation cycle, differences in the pattern of central methionine-enkephalin (Met-EK) immunoreactivity were compared between hibernating (body temperature, Tb = 7 °C) and non-hibernating (T~ = 37 °C) Columbian ground squirrels using the peroxidase-antiperoxidase technique. In non-hibernating animals, Met-EK-immunoreactive perikarya were observed in telencephalic (putamen, caudate nucleus, medial septum-diagonal band complex, amygdala) and diencephalic (periventricular hypothalamic nucleus, lateral hypothalamic area) regions; whereas immunoreactive fibers were found in the lateral septum, stria terminalis nucleus, various hypothalamic areas, arcuate nucleus, median eminence, thalamic intralaminar, periventricular nucleus and lateral habenular nucleus. Compared to the non-hibernating animal, a marked increase in the number of Met-EK-immunoreactive fibers was found in the lateral septal nucleus, the periventricular nucleus, the intralaminar thalamus and the paraventricular hypothalamus of hibernating ground squirrels. Since these changes in immunoreactivity were not observed in the artificially induced hypothermic ground squirrels (Tb = 7 °C), it is unlikely that the dissimilarity in immunoreactivity between animals from different hibernating phases is due to differences in their Tb. In combination with our previous studies, these results tend to suggest that hibernation may be brought about by an increase in endogenous opioid activity, especially in the lateral septal region. INTRODUCTION Several mammalian species of various phylogenetic groups use hibernation as a strategy for survival under harsh conditions (such as extreme cold, shortage of food and/or water). Hibernation is characterized by a drastic decrease in metabolic rate, which is associated with a decrease in body temperature, heart rate and respiratory rate. Nevertheless, all physiological processes are precisely regulated at this low level. Unlike ectotherms in torpor, mammalian hibernators are capable of spontaneous recovery to euthermia using the endogenous heat generated by shivering and nonshivering thermogenesis. To date, the exact mechanisms regulating the hibernation processes are not well understood and remain to be elucidated 26. Because of the similarity between the depressed state of physiological systems during hibernation and after high doses of opioid, endogenous opioids have been postulated as one of the candidates involved in the regulation of hibernation processes. Several studies implicate that this may be the case. For instance, increased overall brain levels of Met- and Leu-enkephalins (EK) x° and increased M e t - E K immunoreactivity have been observed in specific
hypothalamic areas during hibernation 16. Administration of opioid receptor antagonist, such as naloxone or naltrexone, to hibernating animals either reduces the incidence 11 and duration of hibernation 2 or initiates premature termination of hibernation 13. Further, variations in thermoregulatory 28 and feeding responses 15 to exogenous opioids have been reported in ground squirrels during different hibernation cycles. From the evidence gathered above, it is apparent that there are seasonal changes in endogenous opioid activity commensurate with hibernation. However, the actual loci within the central nervous system (CNS) regulating these changes remain unknown. In order to identify some of the possible neuroanatomical locations within the CNS which may be involved in the hibernation process, the present study was carried out to compare the distribution and reactivity pattern of the central enkephalinergic system of the hibernator at different hibernation stages. To ascertain that any observed changes are not simply due to a difference in body temperature (Tb) alone 27, the changes of immunoreactivity was also measured in squirrels which were made hypothermic to the same Tb as that seen in hibernation.
Correspondence: L.C.H. Wang, Department of Zoology, University of Alberta, Edmonton, Alta. T6G 2E9, Canada.
116 MATERIALS AND METHODS
ing brain sections from animals belonging to different physiological groups. Differences in the immunostaining of such corresponding sections were measured using a computerized image analysis system (Kontron IBAS, Munich, F.R.G.). The total area of structures immunostained for Met-EK (fibers and perikarya) were determined within a reference square of 250 x 250/~m which was placed within the outline of brain nuclei selected for measurement 19. Although the measurements and the statistical analysis (Student's t-test) were based on the numbers of stained pixels vs unstained pixels within the reference square (total number of pixels is about 2.62 × 105), differences in immunostaining of corresponding brain sections of animals from different physiological stages were expressed as percentage change of the euthermic animal.
Animals used Sixteen mature Columbian ground squirrels (Spermophilus columbianus) of both sexes were used in this study. They were live-trapped in the foothills of Rocky mountains in Alberta during July and August, and kept individually at an ambient temperature of 22 °C under 12L:12D photoperiod with ad lib food (rat and dog chow mix supplemented with sunflower seeds) and water. The hibernation phase was characterized by a rapid weight gain followed by a weight plateau and anorexia. The completion of transition to the hibernation phase from the non-hibernation phase was further verified by the exhibition of hibernation when the animal was placed in the cold (5 °C) and dark without food for three days in a walk-in environmental chamber. Animals were used after having completed at least two hibernation bouts and were terminated while they were still hibernating (T b = 7 °C; n = 5). The non-hibernating phase was evident when animals showed no weekly weight increase for at least two months before use (T b = 37 °C; n = 5). In order to dispel the possibility that any observed changes in the immunostaining was simply due to the difference in Tb rather than the state of hibernation, euthermic ground squirrels (n = 4) were induced to become hypothermic, using the method of Jourdan and Wang 6, to about the same Tb as the hibernating animal (7 °C). The stable hypothermia was maintained for 72 h before sacrifice.
RESULTS By using the
specific a n t i s e r a
against
Met-EK
or
Leu-EK, Met-EK seemed to be the predominant opioid peptide
within
the
CNS
of
the
Columbian
ground
squirrel. Only very faintly stained neuronal fibers immunoreactive to Leu-EK
were
located within the same
r e g i o n s c o n t a i n i n g h e a v i l y s t a i n e d M e t - E K fibers. N u c l e i c o n t a i n i n g e l e m e n t s w h i c h w e r e less r e a c t i v e t o M e t - E K
lmmunocytochemical studies For histological investigations, the animals were anaesthetized with sodium pentobarbital (50 mg/kg, i.p.) and perfused via the heart with sodium chloride solution (0.9%, containing 15000 IU heparin/i) followed by Zamboni's solution. The brains were then removed from the skull and fixed with Bouin's fixatives for 48 h. After embedding in paraffin, serial sections (6 #m) were prepared and mounted in 6 parallel series. Colchicine was not used in the present study because of possible disturbance to the hibernating animal and the obscurance on immunostaining. For comparison with the results from other species, which were obtained after pretreating the animals with colchieine, two additional euthermic animals were pretreated with coichicine (100 #g/kg) intracerebroventrieularly 24 h before termination. The brain sections of the colchicine-treated animals were cut on a freezing microtome at a thickness of 40/~m and incubated in a free-floating process. The immunohistochemistry was performed according to the method described previously~. Briefly, the sections were treated with normal swine serum (1:10, 15 min), specific antiserum (1:1000, 48 h), goat anti-rabbit immunoglobulin (IgG, 1:20, 30 min), and peroxidase-antiperoxidase (PAP) complex (1:80, 30 min). The peroxidase reaction was visualized with diaminobenzidine (0.025% in 0.1 M Tris buffer, pH 7.4) containing 0.01% hydrogen peroxide (8 rain). Between each step, the sections were thoroughly rinsed in phosphate buffered saline (pH 7.4, 0.1 M). Met-EK antisera were purchased from UCB-Bioproducts (Brussels, Belgium), IgG from Behring-Werke (Marburg, F.R.G.), and PAP complex was obtained from Dako (Copenhagen, Denmark). Non-specific binding sites were covered with normal swine serum purchased from Dako (Copenhagen, Denmark). The specificity of the immunoreaction was tested by omitting Met-EK antiserum, anti-rabbit IgG, or PAP complex and using buffer instead. Furthermore, preadsorption of the antiserum to Met-EK (1/zM/ml diluted serum) abolished the immunoreaction in sections adjacent to sections stained with non-adsorbed antisera. Crossreactivity to Leu-EK was very low and to fl-endorphin could not be detected. The antibody concentration for optimal immunostaining was determined by the use of increasing dilutions of Met-EK antiserum until staining could no longer be detected. The concentration of the antibodies was selected to obtain optimal staining of perikarya and nerve fibers without reacting with the background. Goat anti-rabbit IgG as well as PAP complex were used in excess amount. Comparisons were made by simultaneously treating correspond-
did not show any Leu-EK immunoreactivity. In euthermic Met-EK
animals without coichicine treatment,
immunoreactive
p e r i k a r y a w e r e f o u n d in t h e
telencephalic striatum (caudate and accumbens nucleus, putamen), amygdala, pallidum, lateral septal nucleus and Broca's diagonal nucleus. Among
t h e d i e n c e p h a l i c re-
TABLE I
Distribution and reactivity of met-EK immunoreactive perikarya and fibers in the brain of hibernating and non-hibernating Spermophilus columbianus -
= absence; + = weak staining; + + = moderate staining; + + + = strong staining; + + + + = intensive staining.
Brain region or nucleus (N. )
Hibernating
Non-hibernating
Perikarya
Fibers
Perikarya
Fibers
Striatum N. septi lateralis N. diagonalis (Broca) N. Striae terminalis N. amygdaloideus N. periventricularis hypothalami N. paraventricularis N. ventromedialis N. infundibularis N. praemamillaris Area hypothalamica lateralis Zona interna eminentiae medianae Zona externa eminentiae medianae N. intralaminares thalami N. periventricularis thalami N. habenularis lateralis
++ + +
++ ++++ + ++ ++
++ ++ +
++ ++ ++ ++ +
++ -
++ +++ + + +
+ -
+ + + ++ ++
+
++
+
++
-
++++
-
++++
-
+ +++ ++ +
-
+++ + + +
117
Fig. 1. Changes in Met-EK-immunoreactiveelements in the septal area of (a) euthermic, (b) hypothermic and (c) hibernating Columbian ground squirrels; and in the amygdalaof (d) euthermic, (e) hypothermic and (f) hibernating Columbian ground squirrels (x 150). NSL, lateral septal nucleus; NSM, medial septal nucleus; dashed line, border between lateral and medial septal nuclei.
gions, the periventricular nucleus and the lateral hypothalamic area within the range of the lateral subdivision of the paraventricular nucleus contained Met-Ek-immunoreactive cell bodies. The Met-EK-immunoreactive cell bodies were also found in the central gray of the cerebral aqueduct and in the lateral tegmental nuclei. In addition to cell bodies, Met-EK-immunoreactive axonal processes were observed in the nucleus of the stria terminalis, the medial preoptic, paraventricular, ventromedial, arcuate and premammillary nuclei of the hypothalamus, the median eminence as well as in the thalamic, periventricular, intralaminar, and lateral habenular nuclei. The subthalamus (zona incerta and pallidum) also contained immunoreactive fibers. In the
brainstem, the fibers were mostly found in the central gray of the aqueduct, the raphe and interpeduncular nuclei. The most densely arranged Met-EK-immunoreactive fiber networks (by virtue of light microscopy), composed of typical terminal structures, were found in the intermediate lateral septum, amygdala, peri- and paraventricular hypothalamic and intralaminar thalamic nuclei. Within these latter nuclei, the fibers loosely surrounded the cell bodies in a basket like shape. The number of Met-EK immunostained fibers, which displayed the spine like elements characteristic of terminal structures, were rather small in other nuclei (e.g. the ventromedial hypothalamic nucleus). After intraventricular treatment with colchicine, the
118
8
Q
I
m Q
¢
Fig. 2. Changes in Met-EK-immunoreactive elements in the paraventricular nucleus of (a) euthermic and (b) hibernating Columbian ground squirrels; in the periventricular nucleus of (c) euthermic and (d) hibernating Columbian ground squirrels and in the median eminence of (e) euthermic and (f) hibernating (note the less abundant fibers in the external zone) Columbian ground squirrels (x 250). ZI, internal zone; ZE, external zone; III, third ventricle. distributional pattern of Met-EK-immunoreactive perikarya and fiber systems was not significantly different from those observed in the non-treated group. However, the number and the intensity of Met-EK-immunoreactive fibers and cell bodies were much more pronounced in the animal with colchicine pretreatment. Furthermore, some cell bodies were found in the ventromedial, arcuate, and
paraventricular nuclei of the hypothalamus, pallidum of the subthalamus, supramammillary nucleus and in the raphe region of the midbrain. Comparing the immunostaining between euthermic animals and hibernating animals, both the intensity and the density of immunostaining were different between samples collected from these two groups of animals
119 (Table I). One of the most prominent changes occurred in the intermediate part of the lateral septal nuclei (Fig. la,c). The immunoreactivity to Met-EK in the lateral septum of the hibernating ground squirrel was more intensive (area density increased by 45% (P < 0.01) in comparison to the euthermic control). In the hibernating ground squirrel, significant increases in the immunoreactivity to Met-EK were also observed in amygdala (about 15%, P < 0.05)(Fig. d,f), the paraventricular (about 25%, P < 0.05)(Fig. 2a,b) and periventricular nuclei (about 30%, P < 0.05)(Fig. 2c,d) of the hypothalamus, and the periventricular and intralaminar nuclei (about 25%, P < 0.05) of the thalamus. In contrast, both the distribution and intensity of the immunoreactivity to Met-EK of these regions (compare Fig. la and lb; Fig. ld,e) were about the same when comparing samples collected from euthermic and hypothermic animals. Further, the increase in Met-EK immunoreactivity appears to be site-specific as immunoreactivity was decreased in the median eminence (Fig. 2e,f), the arcuate and mammillary nuclei and in the lateral hypothalamic area of the hibernating animal as compared to euthermic controls (Table I). The immunoreactivity in other regions, for example in the striatum, ventromedial nucleus of the hypothalamus and lateral habenular nucleus (Table I), did not reveal any significant change between animals obtained at different hibernation and thermal states. DISCUSSION As demonstrated by application of antisera against Met-EK and Leu-EK, the predominant endogenous opioid pentapeptide of the Columbian ground squirrel appears to be Met-EK. This finding is in agreement with results obtained from other rodent species7'2°. Further, the distributional pattern of Met-EK immunoreactive perikarya and fibers in the Columbian ground squirrel were also similar to those described in other mammalian species 8'22'23. That is, Met-EK-immunoreactive perikarya were observed in telencephalic (putamen, caudate nucleus, medial septum-diagonal band complex, amygdala) and diencephalic (periventricular hypothalamic nucleus, lateral hypothalamic area) regions; whereas Met-EKimmunoreactive fibers were found in the lateral septum, stria terminalis nucleus, various hypothalamic areas including the arcuate nucleus and median eminence, thalamic intralaminar, periventricular nucleus and lateral habenular nucleus. The similarity in the regional distribution of Met-EK immunoreactivity between Columbian ground squirrels and other species was more noticable in animals pretreated with colchicine to maximize the immunostaining. Because of the possible obscurance of the immunocy-
tochemical staining with colchicine pretreatment, comparison of the Met-EK immunoreactivity was only made in animal from different physiological states without chochicine pretreatment. Although one may argue the validity of using immunoreactivity to assess the endogenous activity of an immunostained neuron, it may indicate the content of neuropeptide under particular circumstances. By using both sides of the same brain, it has been demonstrated that the actual content of the peptide within the tissue as measured by RIA is comparable with the immunocytochemical staining of the same region on the other side of the brain TM. Therefore, the analysis employed in the present study, which was based on the area covered by stained structures within an specific sample area (area density), should provide a reasonable estimate in quantitative comparison. One of the most interesting findings of the present study is the striking difference in Met-EK-immunoreactive patterns between active and hibernating animals. A marked increase in the number of Met-EK immunoreactive fibers was found in the lateral septum, the periventricular nucleus and the paraventricular hypothalamus, and the intralaminar thalamus of hibernating ground squirrels. The increase in Met-EK immunoreactivity appears to be site-specific as the intensity of immunostaining is either reduced in the median eminence, or remains constant in the striatum. Recently, it has been shown that the brain opioid receptors can be modified by low temperature even in non-hibernating species 29. This raises the possibility that changes in Met-EK immunoreactivity in the hibernating squirrel may be due to depression of Tb per se during hibernation. To test this, we compared Met-EK-immunoreactive patterns between non-hibernating squirrels in euthermia (Tb = 37 °C) and in hypothermia with Tb maintained at 7 °C for 72 h. Since no significant difference in the Met-EK-immunoreactive patterns was observed between these two groups of ground squirrels, it is unlikely that the regional increases in Met-EK immunoreactivity observed in hibernating squirrels is the result of low Tb. Among all Met-EK-immunoreactive changes during hibernation, an increase in the immunoreactivity in the lateral septum is of particular interest. This area has been shown to contain thermosensitive neurons 14 and remain relatively active during hibernation 9. Therefore, it is possible that the septal opioid system may have some functional roles in regulating the hibernation process. Previously, we showed that Columbian ground squirrels from the hibernating phase were less responsive to exogenous injections of Met-EK as compared with animals from the non-hibernating phase 12. The reduced thermoregulatory response to intraseptal injection of Met-EK observed during the hibernating phase could
120 have been caused by an increase in septal opioid activity, as indicated by the increase in the immunoreactivity in the present study, during this part of the annual cycle. This in turn might induce a down-regulation of opioid receptor efficacy. Further support for this suggestion is provided by recent findings that the overall opioid binding sites to dihydromorphine decrease in the septum and some other brain areas during hibernation 1. It has been proposed that a reduction in septal activity in hippocampus is a prerequisite for the maintenance of hibernation 5. Since the physiological exemplification of Met-enkephalinergic activity is primarily inhibitory, its enhanced activity in the septum during hibernation may play an important role in regulating hibernation. Apart from the lateral septum, changes in immunoreactivity were also observed in different hypothalamic areas, such as the paraventricular and periventricular nuclei. It has been shown that the hypothalamic vasopressin- and oxytocin-immunoreactive systems are generally reduced in hibernating ground squirrels 16'17 and the activities of these neurons, especially those containing oxytocin, are inhibited by opioid system 3'25. Recently, the reestablishment of endogenous vasopressinergic system has been suggested to be the factor causing arousal in the hibernating animal 21. It is, therefore, quite possible that Met-EK within the hypothalamic regions may suppress the hormonal activities in order to induce and/or
maintain hibernation. The differences in immunoreactivity observed in the intralaminar nuclei of the thalamus may be involved in inhibiting the ascending reticular activating system, which is responsible for the activation of the cortex during the circadian cycle4. However, further study has to be carried out to examine this possible functional role. In summary, the present results demonstrate a sitespecific increase in Met-EK immunoreactivity in certain brain areas, such as lateral septum, periventricular and paraventricular nucleus of the hypothalamus, and the amygdala, during hibernation. Whether such an increase in enkephalin activity is related to regulating the hibernation process remains unknown. It is, therefore, apparent that much needs to be learned about the functional role of changes in immunoreactive patterns during hibernation, these studies are presently underway in our laboratory by employing selective opioid agonists and/or antagonists. The combination of both immunocytochemical and physiological data may lead to a more precise understanding of the role(s) of enkephalinergic systems in regulating the diverse physiological processes which are manifest during the hibernation cycle.
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York, 1986, pp. 215-223. 10 Kramarova, L.I., Kolaeva, S.H., Yukhananov, R.Y. and Rozhanets, V.V., Content of DSIP, enkephalins, and ACTH in some tissues of active and hibernating ground squirrels (Citellus suslicus), Comp. Biochem. Physiol., 74C (1983) 31-33. 11 Kromer, W., Naitrexone influence on hibernation, Experientia, 36 (1980) 581-582. 12 Lee, T.E, Nurnberger, E, Jourdan, M.L. and Wang, L.C.H. Possible involvement of septum in seasonal changes in thermoregulatory responses to Met-enkephalinamide in ground squirrels. In P. Lomax and E. Schonbaum (Eds.), Thermoregulation: Research and Clinical Application, Karger, Basel, 1989, pp. 200-203. 13 Margules, D.L., Goldman, B. and Finck, A., Hibernation: An opioid-dependent state?, Brain Res. Bull., 4 (1979) 721-724. 14 Nakashima, T., Hod, T. and Kiyohara, T., lntraeellular recording analysis of septal thermosensitive neurons in the rat. In J.B. Mercer (Ed.), Thermal Physiology, Elsevier, New York, 1989, pp. 69-72. 15 Nizielski, S.E., Levine, A.S., Morley, J.E., Hall, K.A. and Gosnell, B.A., Seasonal variation in opioid modulation of feeding in the 13-lined ground squirrel, Physiol. Behav., 37 (1986) 5-9. 16 Nurnberger, E, Der Hypothalamus des lgeis (Erinaceus europaeus L.) unter besonderer Berucksichtigung des Winterschlafes, Cytoarchitekt. immuncytochem. Stud. Diss., Marburg, 1983, 162 pp. 17 Nurnberger, E, Rorstad, O.P. and Lederis, K., The hypothalamo-hypophysial system in the ground squirrel (Spermophilus richardsonii). In H. Kobayashi (Ed.) Neurosecretion and the Biology of Neuropeptides, Japanese Scientific Society Press, Tokyo, 1985, pp. 518-520. 18 Nurnberger, F., Lederis, K. and Rorstad, O.P., Effects of
1 Beckman, A.L., Functional aspects of brain opioid peptide systems in hibernation. In H.C. Heller, X.J. Musacchia and L.C.H. Wang (Eds.), Living in the Cold, Elsevier, New York, 1986, pp. 225-234. 2 Beckman, A.L. and Llados-Eckman, C., Antagonism of brain opioid peptide action reduced hibernation bout duration, Brain Research, 328 (1985) 201-205. 3 Bicknell, R.J., Zhao, B.G., Chapman, C., Heavens, R.P. and Sirinathsinghji, D.J.S., Opioid inhibition of secretion from oxytocin and vasopressin nerve terminals following selective depletion of neurohypophysial catecholamines, Endocrinology, 122 (1988) 1321-1327. 4 Bowsher, D., Some afferent and efferent connections of the parafascicular-center median complex. In D.P. Purpura and M.D. Yahr (Eds.), The Thalamus, Columbia University Press, New York, 1966, pp. 99-108. 5 Heller, H.C., Hibernation: neural aspects, Annu. Rev. Physiol., 41 (1979) 305-321. 6 Jourdan, M.L. and Wang, L.C.H., An improved technique for the study of hypothermia physiology, J. Therm. Biol., 12 (1987) 175-178. 7 Khachaturian, H., Lewis, M.E. and Watson, S.J., Enkephalin systems in diencephalon and brainstem of the rat, J. Comp. Neurol., 220 (1983) 310--320. 8 Khachaturian, H., Lewis, M.E., Haber, S.N., Akil, H. and Watson, S.J., Proopiomelanocortin peptide immunocytochemistry in rhesus monkey brain, Brain Res. Bull., 13 (1984) 785-800. 9 Kilduff, T.S., Radeke, C.D. and Heller, H.C., Neural activity during mammalian hibernation. In H.C. Heller, X.J. Musacchia and L.C.H. Wang (Eds.), Living in the Cold, Elsevier, New
Acknowledgements. The present study was supported by the Natural Sciences and Engineering Research Council of Canada to L.C.H.W. (Grant A6455) and the Deutsche Forschungsgemeinschaft to F.N. (Nu 36/2-2).
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hibernation on somatostatin-like immunoreactivity in the brain of the ground squirrel (Spermophilus richardsonii) and European hadgehog (Erinaceus europaeus), Cell Tissue Res., 243 (1986) 263-271. Nurnberger, E, Schindler, C.U. and Kriete, A., The serotoninimmunoreactive system of the suprachiasmaticnudeus in the hibernating ground squirrel, Spermophilus richardsonii, Cell Tissue Res., 256 (1989) 593-599. Petrusz, P., Merchenthaler, I. and Maderdrut, J.L. Distribution of enkephalin containing neurons in the central nervous system. In A. Bj6rklund and T. H6kfelt (Eds.), Handbook of Chemical Neuroanatomy, Vol. 4, Elsevier, Amsterdam, 1985, pp. 273334. Pevet, P., Masson-Pevet, M., Hermes, M.L.H.J., Buijs, R.M. and Canguilhem, B., Photoperiod, pineal gland, vasopressinerigc innervation and timing of hibernation. In A. Malan and B. Canguilhem (Eds.), Living in the Cold H, Libbey, Montrough, 1989, pp. 43-51. Sar, M., Stumpf, W.E., Miller, R.J., Chang, K.J. and Curatrecasas, P., Immunohistochemical localization of enkephalin in rat brain and spinal cord, J. Comp. Neurol., 182 (1978) 460-482. Simantov, R., Kuhar, M.J., Uhl, G.R. and Snyder, S.H. Opioid peptide enkephalin: Immnnohistochemical mapping in rat central nervous system, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 2167-2171.
24 Sternberger, L.A., Hardy, EH., Cuculis, J.J. and Meyer, H.G., The unlabeled antibody enzyme method of immunocytochemistry. Preparation and properties of soluble antigen antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes, J. Histochem. Cytochem., 18 (1970) 315-333. 25 Wakerley, J.B., Noble, R. and Clarke, G., Effects of morphine and D-AIa, D-Leu-enkephalin on the electrical activity of supraoptic neurosecretory cells in vitro, Neuroscience, 10 (1983) 73-81. 26 Wang, L.C.H., Mammalian hibernation: An escape from the cold. In R. Gilles (Ed.), Advances in Comparative and Environmental Physiology, Vol. 2, Springer, Berlin, 1988, pp. 1-46. 27 Wang, L.C.H. and Lee, T.F., Perspectives in hibernation research: concepts and executions. In A. Malan and B. Canguilhem (F.ds.), Living in The Cold H, Libbey, Montrough, 1989, pp. 509-519. 28 Wang, L.C.H., Lee, T.F. and Jourdan, M.L., Seasonal difference in thermoregulatory responses to opiates in a mammalian hibernator, Pharmacol. Biochem. Behav., 26 (1987) 565-571. 29 Wilkinson, M., Buchanan, G.D., Jacobson, W. and Younglai, E.V. Brain opioid receptors in the hibernating bat, Myotis lucifugus: modification by low temperature and comparison with rat, mouse and hamster, Pharmacol. Biochem. Behav., 25 (1986) 527-532.
Brain Research, 547 (1991) 115-121 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116529R BRES 16529
Seasonal changes in methionine-enkephalin immunoreactivity in the brain of a hibernator, Spermophilus columbianus E Nurnberger 1, T.E L e e 2, M.L. Jourdan 2 and L.C.H. Wang 2 l Department of Anatomy and Cytobiology, Justus Liebig University, Giessen (E R. G.) and 2Department of Zoology, University of Alberta, Edmonton, Alta. (Canada) (Accepted 13 November 1990) Key words: Hibernation; Methionine-enkephalin; Opioid; Immunocytochemistry; Septum; Hypothermia; Thermoregulation; Ground squirrel
To identify the actual location of central endogenous opioid systems which may be involved in regulating the hibernation cycle, differences in the pattern of central methionine-enkephalin (Met-EK) immunoreactivity were compared between hibernating (body temperature, Tb = 7 °C) and non-hibernating (T~ = 37 °C) Columbian ground squirrels using the peroxidase-antiperoxidase technique. In non-hibernating animals, Met-EK-immunoreactive perikarya were observed in telencephalic (putamen, caudate nucleus, medial septum-diagonal band complex, amygdala) and diencephalic (periventricular hypothalamic nucleus, lateral hypothalamic area) regions; whereas immunoreactive fibers were found in the lateral septum, stria terminalis nucleus, various hypothalamic areas, arcuate nucleus, median eminence, thalamic intralaminar, periventricular nucleus and lateral habenular nucleus. Compared to the non-hibernating animal, a marked increase in the number of Met-EK-immunoreactive fibers was found in the lateral septal nucleus, the periventricular nucleus, the intralaminar thalamus and the paraventricular hypothalamus of hibernating ground squirrels. Since these changes in immunoreactivity were not observed in the artificially induced hypothermic ground squirrels (Tb = 7 °C), it is unlikely that the dissimilarity in immunoreactivity between animals from different hibernating phases is due to differences in their Tb. In combination with our previous studies, these results tend to suggest that hibernation may be brought about by an increase in endogenous opioid activity, especially in the lateral septal region. INTRODUCTION Several mammalian species of various phylogenetic groups use hibernation as a strategy for survival under harsh conditions (such as extreme cold, shortage of food and/or water). Hibernation is characterized by a drastic decrease in metabolic rate, which is associated with a decrease in body temperature, heart rate and respiratory rate. Nevertheless, all physiological processes are precisely regulated at this low level. Unlike ectotherms in torpor, mammalian hibernators are capable of spontaneous recovery to euthermia using the endogenous heat generated by shivering and nonshivering thermogenesis. To date, the exact mechanisms regulating the hibernation processes are not well understood and remain to be elucidated 26. Because of the similarity between the depressed state of physiological systems during hibernation and after high doses of opioid, endogenous opioids have been postulated as one of the candidates involved in the regulation of hibernation processes. Several studies implicate that this may be the case. For instance, increased overall brain levels of Met- and Leu-enkephalins (EK) x° and increased M e t - E K immunoreactivity have been observed in specific
hypothalamic areas during hibernation 16. Administration of opioid receptor antagonist, such as naloxone or naltrexone, to hibernating animals either reduces the incidence 11 and duration of hibernation 2 or initiates premature termination of hibernation 13. Further, variations in thermoregulatory 28 and feeding responses 15 to exogenous opioids have been reported in ground squirrels during different hibernation cycles. From the evidence gathered above, it is apparent that there are seasonal changes in endogenous opioid activity commensurate with hibernation. However, the actual loci within the central nervous system (CNS) regulating these changes remain unknown. In order to identify some of the possible neuroanatomical locations within the CNS which may be involved in the hibernation process, the present study was carried out to compare the distribution and reactivity pattern of the central enkephalinergic system of the hibernator at different hibernation stages. To ascertain that any observed changes are not simply due to a difference in body temperature (Tb) alone 27, the changes of immunoreactivity was also measured in squirrels which were made hypothermic to the same Tb as that seen in hibernation.
Correspondence: L.C.H. Wang, Department of Zoology, University of Alberta, Edmonton, Alta. T6G 2E9, Canada.
116 MATERIALS AND METHODS
ing brain sections from animals belonging to different physiological groups. Differences in the immunostaining of such corresponding sections were measured using a computerized image analysis system (Kontron IBAS, Munich, F.R.G.). The total area of structures immunostained for Met-EK (fibers and perikarya) were determined within a reference square of 250 x 250/~m which was placed within the outline of brain nuclei selected for measurement 19. Although the measurements and the statistical analysis (Student's t-test) were based on the numbers of stained pixels vs unstained pixels within the reference square (total number of pixels is about 2.62 × 105), differences in immunostaining of corresponding brain sections of animals from different physiological stages were expressed as percentage change of the euthermic animal.
Animals used Sixteen mature Columbian ground squirrels (Spermophilus columbianus) of both sexes were used in this study. They were live-trapped in the foothills of Rocky mountains in Alberta during July and August, and kept individually at an ambient temperature of 22 °C under 12L:12D photoperiod with ad lib food (rat and dog chow mix supplemented with sunflower seeds) and water. The hibernation phase was characterized by a rapid weight gain followed by a weight plateau and anorexia. The completion of transition to the hibernation phase from the non-hibernation phase was further verified by the exhibition of hibernation when the animal was placed in the cold (5 °C) and dark without food for three days in a walk-in environmental chamber. Animals were used after having completed at least two hibernation bouts and were terminated while they were still hibernating (T b = 7 °C; n = 5). The non-hibernating phase was evident when animals showed no weekly weight increase for at least two months before use (T b = 37 °C; n = 5). In order to dispel the possibility that any observed changes in the immunostaining was simply due to the difference in Tb rather than the state of hibernation, euthermic ground squirrels (n = 4) were induced to become hypothermic, using the method of Jourdan and Wang 6, to about the same Tb as the hibernating animal (7 °C). The stable hypothermia was maintained for 72 h before sacrifice.
RESULTS By using the
specific a n t i s e r a
against
Met-EK
or
Leu-EK, Met-EK seemed to be the predominant opioid peptide
within
the
CNS
of
the
Columbian
ground
squirrel. Only very faintly stained neuronal fibers immunoreactive to Leu-EK
were
located within the same
r e g i o n s c o n t a i n i n g h e a v i l y s t a i n e d M e t - E K fibers. N u c l e i c o n t a i n i n g e l e m e n t s w h i c h w e r e less r e a c t i v e t o M e t - E K
lmmunocytochemical studies For histological investigations, the animals were anaesthetized with sodium pentobarbital (50 mg/kg, i.p.) and perfused via the heart with sodium chloride solution (0.9%, containing 15000 IU heparin/i) followed by Zamboni's solution. The brains were then removed from the skull and fixed with Bouin's fixatives for 48 h. After embedding in paraffin, serial sections (6 #m) were prepared and mounted in 6 parallel series. Colchicine was not used in the present study because of possible disturbance to the hibernating animal and the obscurance on immunostaining. For comparison with the results from other species, which were obtained after pretreating the animals with colchieine, two additional euthermic animals were pretreated with coichicine (100 #g/kg) intracerebroventrieularly 24 h before termination. The brain sections of the colchicine-treated animals were cut on a freezing microtome at a thickness of 40/~m and incubated in a free-floating process. The immunohistochemistry was performed according to the method described previously~. Briefly, the sections were treated with normal swine serum (1:10, 15 min), specific antiserum (1:1000, 48 h), goat anti-rabbit immunoglobulin (IgG, 1:20, 30 min), and peroxidase-antiperoxidase (PAP) complex (1:80, 30 min). The peroxidase reaction was visualized with diaminobenzidine (0.025% in 0.1 M Tris buffer, pH 7.4) containing 0.01% hydrogen peroxide (8 rain). Between each step, the sections were thoroughly rinsed in phosphate buffered saline (pH 7.4, 0.1 M). Met-EK antisera were purchased from UCB-Bioproducts (Brussels, Belgium), IgG from Behring-Werke (Marburg, F.R.G.), and PAP complex was obtained from Dako (Copenhagen, Denmark). Non-specific binding sites were covered with normal swine serum purchased from Dako (Copenhagen, Denmark). The specificity of the immunoreaction was tested by omitting Met-EK antiserum, anti-rabbit IgG, or PAP complex and using buffer instead. Furthermore, preadsorption of the antiserum to Met-EK (1/zM/ml diluted serum) abolished the immunoreaction in sections adjacent to sections stained with non-adsorbed antisera. Crossreactivity to Leu-EK was very low and to fl-endorphin could not be detected. The antibody concentration for optimal immunostaining was determined by the use of increasing dilutions of Met-EK antiserum until staining could no longer be detected. The concentration of the antibodies was selected to obtain optimal staining of perikarya and nerve fibers without reacting with the background. Goat anti-rabbit IgG as well as PAP complex were used in excess amount. Comparisons were made by simultaneously treating correspond-
did not show any Leu-EK immunoreactivity. In euthermic Met-EK
animals without coichicine treatment,
immunoreactive
p e r i k a r y a w e r e f o u n d in t h e
telencephalic striatum (caudate and accumbens nucleus, putamen), amygdala, pallidum, lateral septal nucleus and Broca's diagonal nucleus. Among
t h e d i e n c e p h a l i c re-
TABLE I
Distribution and reactivity of met-EK immunoreactive perikarya and fibers in the brain of hibernating and non-hibernating Spermophilus columbianus -
= absence; + = weak staining; + + = moderate staining; + + + = strong staining; + + + + = intensive staining.
Brain region or nucleus (N. )
Hibernating
Non-hibernating
Perikarya
Fibers
Perikarya
Fibers
Striatum N. septi lateralis N. diagonalis (Broca) N. Striae terminalis N. amygdaloideus N. periventricularis hypothalami N. paraventricularis N. ventromedialis N. infundibularis N. praemamillaris Area hypothalamica lateralis Zona interna eminentiae medianae Zona externa eminentiae medianae N. intralaminares thalami N. periventricularis thalami N. habenularis lateralis
++ + +
++ ++++ + ++ ++
++ ++ +
++ ++ ++ ++ +
++ -
++ +++ + + +
+ -
+ + + ++ ++
+
++
+
++
-
++++
-
++++
-
+ +++ ++ +
-
+++ + + +
117
Fig. 1. Changes in Met-EK-immunoreactiveelements in the septal area of (a) euthermic, (b) hypothermic and (c) hibernating Columbian ground squirrels; and in the amygdalaof (d) euthermic, (e) hypothermic and (f) hibernating Columbian ground squirrels (x 150). NSL, lateral septal nucleus; NSM, medial septal nucleus; dashed line, border between lateral and medial septal nuclei.
gions, the periventricular nucleus and the lateral hypothalamic area within the range of the lateral subdivision of the paraventricular nucleus contained Met-Ek-immunoreactive cell bodies. The Met-EK-immunoreactive cell bodies were also found in the central gray of the cerebral aqueduct and in the lateral tegmental nuclei. In addition to cell bodies, Met-EK-immunoreactive axonal processes were observed in the nucleus of the stria terminalis, the medial preoptic, paraventricular, ventromedial, arcuate and premammillary nuclei of the hypothalamus, the median eminence as well as in the thalamic, periventricular, intralaminar, and lateral habenular nuclei. The subthalamus (zona incerta and pallidum) also contained immunoreactive fibers. In the
brainstem, the fibers were mostly found in the central gray of the aqueduct, the raphe and interpeduncular nuclei. The most densely arranged Met-EK-immunoreactive fiber networks (by virtue of light microscopy), composed of typical terminal structures, were found in the intermediate lateral septum, amygdala, peri- and paraventricular hypothalamic and intralaminar thalamic nuclei. Within these latter nuclei, the fibers loosely surrounded the cell bodies in a basket like shape. The number of Met-EK immunostained fibers, which displayed the spine like elements characteristic of terminal structures, were rather small in other nuclei (e.g. the ventromedial hypothalamic nucleus). After intraventricular treatment with colchicine, the
118
8
Q
I
m Q
¢
Fig. 2. Changes in Met-EK-immunoreactive elements in the paraventricular nucleus of (a) euthermic and (b) hibernating Columbian ground squirrels; in the periventricular nucleus of (c) euthermic and (d) hibernating Columbian ground squirrels and in the median eminence of (e) euthermic and (f) hibernating (note the less abundant fibers in the external zone) Columbian ground squirrels (x 250). ZI, internal zone; ZE, external zone; III, third ventricle. distributional pattern of Met-EK-immunoreactive perikarya and fiber systems was not significantly different from those observed in the non-treated group. However, the number and the intensity of Met-EK-immunoreactive fibers and cell bodies were much more pronounced in the animal with colchicine pretreatment. Furthermore, some cell bodies were found in the ventromedial, arcuate, and
paraventricular nuclei of the hypothalamus, pallidum of the subthalamus, supramammillary nucleus and in the raphe region of the midbrain. Comparing the immunostaining between euthermic animals and hibernating animals, both the intensity and the density of immunostaining were different between samples collected from these two groups of animals
119 (Table I). One of the most prominent changes occurred in the intermediate part of the lateral septal nuclei (Fig. la,c). The immunoreactivity to Met-EK in the lateral septum of the hibernating ground squirrel was more intensive (area density increased by 45% (P < 0.01) in comparison to the euthermic control). In the hibernating ground squirrel, significant increases in the immunoreactivity to Met-EK were also observed in amygdala (about 15%, P < 0.05)(Fig. d,f), the paraventricular (about 25%, P < 0.05)(Fig. 2a,b) and periventricular nuclei (about 30%, P < 0.05)(Fig. 2c,d) of the hypothalamus, and the periventricular and intralaminar nuclei (about 25%, P < 0.05) of the thalamus. In contrast, both the distribution and intensity of the immunoreactivity to Met-EK of these regions (compare Fig. la and lb; Fig. ld,e) were about the same when comparing samples collected from euthermic and hypothermic animals. Further, the increase in Met-EK immunoreactivity appears to be site-specific as immunoreactivity was decreased in the median eminence (Fig. 2e,f), the arcuate and mammillary nuclei and in the lateral hypothalamic area of the hibernating animal as compared to euthermic controls (Table I). The immunoreactivity in other regions, for example in the striatum, ventromedial nucleus of the hypothalamus and lateral habenular nucleus (Table I), did not reveal any significant change between animals obtained at different hibernation and thermal states. DISCUSSION As demonstrated by application of antisera against Met-EK and Leu-EK, the predominant endogenous opioid pentapeptide of the Columbian ground squirrel appears to be Met-EK. This finding is in agreement with results obtained from other rodent species7'2°. Further, the distributional pattern of Met-EK immunoreactive perikarya and fibers in the Columbian ground squirrel were also similar to those described in other mammalian species 8'22'23. That is, Met-EK-immunoreactive perikarya were observed in telencephalic (putamen, caudate nucleus, medial septum-diagonal band complex, amygdala) and diencephalic (periventricular hypothalamic nucleus, lateral hypothalamic area) regions; whereas Met-EKimmunoreactive fibers were found in the lateral septum, stria terminalis nucleus, various hypothalamic areas including the arcuate nucleus and median eminence, thalamic intralaminar, periventricular nucleus and lateral habenular nucleus. The similarity in the regional distribution of Met-EK immunoreactivity between Columbian ground squirrels and other species was more noticable in animals pretreated with colchicine to maximize the immunostaining. Because of the possible obscurance of the immunocy-
tochemical staining with colchicine pretreatment, comparison of the Met-EK immunoreactivity was only made in animal from different physiological states without chochicine pretreatment. Although one may argue the validity of using immunoreactivity to assess the endogenous activity of an immunostained neuron, it may indicate the content of neuropeptide under particular circumstances. By using both sides of the same brain, it has been demonstrated that the actual content of the peptide within the tissue as measured by RIA is comparable with the immunocytochemical staining of the same region on the other side of the brain TM. Therefore, the analysis employed in the present study, which was based on the area covered by stained structures within an specific sample area (area density), should provide a reasonable estimate in quantitative comparison. One of the most interesting findings of the present study is the striking difference in Met-EK-immunoreactive patterns between active and hibernating animals. A marked increase in the number of Met-EK immunoreactive fibers was found in the lateral septum, the periventricular nucleus and the paraventricular hypothalamus, and the intralaminar thalamus of hibernating ground squirrels. The increase in Met-EK immunoreactivity appears to be site-specific as the intensity of immunostaining is either reduced in the median eminence, or remains constant in the striatum. Recently, it has been shown that the brain opioid receptors can be modified by low temperature even in non-hibernating species 29. This raises the possibility that changes in Met-EK immunoreactivity in the hibernating squirrel may be due to depression of Tb per se during hibernation. To test this, we compared Met-EK-immunoreactive patterns between non-hibernating squirrels in euthermia (Tb = 37 °C) and in hypothermia with Tb maintained at 7 °C for 72 h. Since no significant difference in the Met-EK-immunoreactive patterns was observed between these two groups of ground squirrels, it is unlikely that the regional increases in Met-EK immunoreactivity observed in hibernating squirrels is the result of low Tb. Among all Met-EK-immunoreactive changes during hibernation, an increase in the immunoreactivity in the lateral septum is of particular interest. This area has been shown to contain thermosensitive neurons 14 and remain relatively active during hibernation 9. Therefore, it is possible that the septal opioid system may have some functional roles in regulating the hibernation process. Previously, we showed that Columbian ground squirrels from the hibernating phase were less responsive to exogenous injections of Met-EK as compared with animals from the non-hibernating phase 12. The reduced thermoregulatory response to intraseptal injection of Met-EK observed during the hibernating phase could
120 have been caused by an increase in septal opioid activity, as indicated by the increase in the immunoreactivity in the present study, during this part of the annual cycle. This in turn might induce a down-regulation of opioid receptor efficacy. Further support for this suggestion is provided by recent findings that the overall opioid binding sites to dihydromorphine decrease in the septum and some other brain areas during hibernation 1. It has been proposed that a reduction in septal activity in hippocampus is a prerequisite for the maintenance of hibernation 5. Since the physiological exemplification of Met-enkephalinergic activity is primarily inhibitory, its enhanced activity in the septum during hibernation may play an important role in regulating hibernation. Apart from the lateral septum, changes in immunoreactivity were also observed in different hypothalamic areas, such as the paraventricular and periventricular nuclei. It has been shown that the hypothalamic vasopressin- and oxytocin-immunoreactive systems are generally reduced in hibernating ground squirrels 16'17 and the activities of these neurons, especially those containing oxytocin, are inhibited by opioid system 3'25. Recently, the reestablishment of endogenous vasopressinergic system has been suggested to be the factor causing arousal in the hibernating animal 21. It is, therefore, quite possible that Met-EK within the hypothalamic regions may suppress the hormonal activities in order to induce and/or
maintain hibernation. The differences in immunoreactivity observed in the intralaminar nuclei of the thalamus may be involved in inhibiting the ascending reticular activating system, which is responsible for the activation of the cortex during the circadian cycle4. However, further study has to be carried out to examine this possible functional role. In summary, the present results demonstrate a sitespecific increase in Met-EK immunoreactivity in certain brain areas, such as lateral septum, periventricular and paraventricular nucleus of the hypothalamus, and the amygdala, during hibernation. Whether such an increase in enkephalin activity is related to regulating the hibernation process remains unknown. It is, therefore, apparent that much needs to be learned about the functional role of changes in immunoreactive patterns during hibernation, these studies are presently underway in our laboratory by employing selective opioid agonists and/or antagonists. The combination of both immunocytochemical and physiological data may lead to a more precise understanding of the role(s) of enkephalinergic systems in regulating the diverse physiological processes which are manifest during the hibernation cycle.
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