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Changes in hypothalamic catecholamines and serotonin during hibernation and arousal in the arctic ground squirrel
Camp. Biochem. Physiol.,1974, Vol. 48A, pp. 623 to 662. Pergamon Press. Printed in Great Britain
CHANGES IN HYPOTHALAMIC CATECHOLAMINES AND SEROTONIN DURING HIBERNATION AND AROUSAL IN THE ARCTIC GROUND SQUIRREL DALE
D. FEIST
and WILLIAM
A. GALSTER
Institute of Arctic Biology, University of Alaska, Fairbanks, Alaska 99701, U.S.A. (Received 24 July 1973) Abstract-l. Hypothalamic norepinephrine (NE), epinephrine (E) and serotonin (5HT) were assayed and compared in the Arctic ground squirrel (Cit.&s undulutus) in the prehibernation summer season and different phases of the hibernation cycles during the winter. 2. From a high level in the prehibernation season, NE fell (by 3.5 per cent) to a lower level in the hibernation period, active phase and re-entry phase of the hibernation cycle. In early and mid arousal, NE increased by 50 per cent over that in deep hibernation. By the end of arousal, NE returned to the level found in other phases of the hibernation cycle. 3. During the hibernation cycle, E increased in late arousal and the active phase. The concentration of S-HT remained similar in all groups except in early arousal when the level was 34 per cent lower than that in hibernation. 4. The ratio of NE/S-HT in the hypothalamus was highest during early arousal from hibernation. 5. The results support the hypothesis that changes in the activity of hypothalamic noradrenergic and serotonergic systems play an important role in maintenance of and arousal from hibernation. INTRODUCTION THE ANNUAL cycle of mammalian hibernation includes an active summer season and a hibernation season in fall and winter. During the hibernation season, hibernators undergo periodic entrance into and arousal from deep torpidity. A brief active phase separates each hibernation period (Lyman & Chatfield, 1955). Dramatic shifts in body temperature and thermoregulatory processes occur during periodic entrance and arousal (Lyman & O’Brien, 1963 ; Hayward & Lyman, 1967). It has been suggested that hypothalamic monoamines play an important role in triggering the periodic entrance into and arousal from hibernation (Draskoczy & Lyman, 1967; Feist, 1970; Myers & Yaksh, 1971; Spafford & Pengelley, 1971). Considerable evidence indicates that norepinephrine (NE) and serotonin (5hydroxytryptamine = 5-HT) act as hypothalamic neurotransmitters in the mediation of thermoregulation in non-hibernators (Feldberg & Myers, 1965; Bligh, 1966; Lomax, 1970). Some evidence from studies of monoamine concentrations and turnover in the brain and of the effects of intrahypothalamic injections on NE and 5-HT suggest that central noradrenergic and serotonergic systems may also
653
654
DALE D. FEIST AND WILLIAM A. GALSTER
be important for triggering and/or mediating events in the hibernation cycle. Usspaa (1963a, b) f ound that whole brain content of NE increased and S-HT decreased in the hibernating hedgehog. He concluded that NE and 5-HT belong to different functional systems in the brain. Spafford & Pengelley (1971) found that whole brain 5-HT decreased during entrance into hibernation in the golden mantled ground squirrel. They also found that an inhibitor of serotonin synthesis temporarily blocked hibernation while lesions in the median raphe nucleus of the midbrain (a major locus of 5-HT synthesis) disrupted hibernation. They concluded that serotonin was involved in a functional way in the process of mammalian hibernation. Levels of NE and epinephrine (E) in hypothalamus were found to be constant during the hibernation season in both active and hibernating golden mantled ground squirrels (Twente et al. 1970). Thus, Twente & Twente could not account for possible progressive irritability (Twente & Twente, 1968) during the hibernation period by a generalized accumulation of catecholamines in the hypothalamus which would lead to arousal. The discovery that catecholamine turnover in the whole brain of the thirteenlined ground squirrel stopped immediately prior to entrance into hibernation led to the suggestion that a lack of central adrenergic activity may initiate hibernation (Draskoczy SCLyman, 1967). Recently, injections of low doses of NE or 5-HT into the preoptic and anterior area of the hypothalamus in the hibernating golden mantled ground squirrel caused arousal from hibernation (Beckman & Satinoff, 1972). In contrast, injections of NE or 5-HT into the third ventricle or the preoptic area in the awake marmot caused a decline in body temperature (Jacobs et al., 1971). Based primarily on observed effects of NE and 5-HT on thermoregulation in non-hibernators, Myers & Yaksh (1971) have suggested that the sustaining mechanism for hibernation as well as the arousal stimulus for restoration of normothermia could both depend on the balance in the release of NE or 5-HT in the anterior hypothalamus. The research reported here was initiated as a first step toward assessing the involvement of hypothalamic adrenergic and serotonergic mechanisms in hibernation and arousal of the Arctic ground squirrel (Citellus undulatus). The results include the first reported simultaneous measurements of NE and 5-HT in the hypothalamus of a hibernator during various phases of the hibernation cycle and in the active season. MATERIALS
Experimental
AND
METHODS
groups
Arctic ground squirrels (Citehs undulatus) of both sexes were collected during the summer months from the Thompson Lake, Fielding Lake and Paxson Lake areas of Alaska. The animals were individually caged in the laboratory in a metal cage with a wooden nest box and a constant supply of rat pellets and water. In summer, the active ground squirrels were kept at 18°C with a seasonal light cycle. In the fall and winter, they were kept at 5°C with no light. Hibernation was monitored by temperature changes detected with a thermocouple positioned in the nest under the animal and recorded hourly.
HIBJXNATION
AND
AROUSAL
IN
THE
ARCTIC
GROUND
SQUIRREL
655
The mean body weight of all animals as recorded at the time of killing was 367 + 16 g (mean + SE.). The following groups of ground squirrels were studied (see Fig. 1 also). Prehibernation: one group of animals from the active season in late summer (a) (i.e. September) which had not yet started their hibernation season. Hibernation cycle: eight groups of animals from the mid-late hibernation season (b) (i.e. March-April) in different phases of the hibernation cycle and designated as follows : (1) Early hibernation period-one group of animals in deep hibernation having spent less than 45 per cent (mean = 24 per cent) of their expected hibernation period (the expected period was determined from the average of the three previous hibernation periods, Galster & Morrison, 1970). (2) Late hibernation period-one group of animals in deep hibernation having spent more than 60 per cent (mean = 82 per cent) of their expected hibernation period. groups of animals at different times during (31, (4), (S), (6) Arousing-four arousal after having been artificially stimulated by insertion of a rectal temperature probe while in deep hibernation; these squirrels completed at least 86 per cent (mean = 92 per cent) of their expected hibernation period; the four groups were defined on the basis of body temperature as shown in the following table. TBF (“C)
3
105 25.2 34.7 35.9
8 13.8 21.1 35.3
Time after start of arousal (min) 48 100 130 180
TBF, Axillary brown fat temperature. TWl, Deep colomc temperature. (7) (8)
Active phase-one group of squirrels having aroused completely and active prior to re-entry into hibernation. Re-entry phase-one group of squirrels in the process of re-entering deep hibernation with body temperatures (coionic = 7_4”C, brown fat = 13.7”C) falling toward those attained in deep hibernation.
Measurements of body temperature Deep colonic temperature was measured with a telethermometer probe inserted to a depth of 7-8 cm. The axillary brown fat temperature was determined with a fine wire probe inserted into the brown fat. Temperatures were viewed and recorded with a multichannel telethermometer (YSI). Dissection of the brain Animals were killed by rapid decapitation between 9 a.m. and 1 p.m. Immediately after decapitation, the brain was quickly removed and dissected into regions essentially according to the procedure of Glowinski & Iversen (1966). The hypothalamus and other regions were quickly frozen in liquid nitrogen for storage until extraction of amines. Tissues were weighed on a Mettler balance to the nearest tenth of a mg immediately prior to extraction of amines (see Table 1 for weights of brain regions),
DALE D. FEIST ANDWILLIAM A. GALSTER
656
TABLE I-WEIGHTS OF BRAINREGIONSFROMARCTICGROUND SQUIRRELS Weight (mg)
Region Hypothalamus Medulla + pons Cerebellum Cerebral cortex and remainder of brain
311 532 533 2663
f26 + 17 f 24 ?I 55
Sample No. 42 12 12 12
Values represent the mean f S.E. Extraction,
isolation and assays of amines
Tissues were homogenized in ice-cold 0.4 M perchloric acid (0.1 y0 EDTA) in a motordriven glass homogenizer. After centrifugation, the supernatant was divided into 2.0 ml for the organic solvent extraction and isolation and fluorometric assay of S-HT (Snyder et al., 1965) and 4-O ml for the aluminium oxide isolation and fluorometric assay of NE and E (von Euler & Lishajko, 1961). Recovery studies showed a mean recovery of 70 per cent for NE, 64 per cent for E and 80 per cent for S-HT. Values given for NE, E and 5-HT represent the free amines but are uncorrected for losses during extraction and isolation. Concentrations are expressed in terms of tissue wet weight. Analysis of water content of the brain tissues revealed a constant water content (percentage of total weight) through all phases of the hibernating cycle and active season (Galster, unpublished data). Statistical
analyses The Student t-test (Simpson et al., 1960) was applied to test the significance of the difference between mean values for different groups. Values for amines are expressed as the mean + the standard error (S.E.). RESULTS
Hypothalamic
norepinephrine
As shown in Fig. 1, during the active season the concentration of NE in the hypothalamus of the active prehibernating ground squirrel was 1.687 + 0.186 pg/g. During the hibernation period, of the hibernation cycle, the level of NE was lower by 3.5 per cent (early hibernation P < O-07; late hibernation PC 0.05). In early arousal (45 min after onset of arousal) and mid arousal (100 min after onset of arousal) the concentration of NE increased by 50 per cent (early arousal P-C 0.07; mid arousal P< O-01) over that in deep hibernation. By the end of arousal (3 hr after onset of arousal) when body temperatures approached the normal homeothermic values the NE declined to the level found in deep hibernation. Hypothalamic
Shydroxytryptamine
The concentration of 5-HT in the hypothalamus of the active prehibernating squirrel (Fig. 1) was 0.593 f 0.127 pg/g. Comparable levels were found during the hibernation cycle in all groups except in early arousal (45 min after the onset) in which the level was 34 per cent (not significant ; P = 0.1) lower (0.354 + 0.077 pg/g) than that in hibernation.
HIBERNATION
AND
othalamic
0.00 ReHib.
AROUSAL
Amines
Early Hib.
Late
IN
THE
ARCTIC
GROUND
in Active Season and Phasesof
Hib.
Hibernation
Active Phase
-Arousal-
657
SQUIRREL
Active
ReEntry
FIG. 1. Hypothalamic monoamines in the active season and during different phases of the hibernation cycle of the Arctic ground squirrel. The upper section describes the time course and temperature changes during the hibernation cycle. The lower section shows changes in monoamine values. Values expressed as mean + S.E. * NE hibernation value lower than in prehibernation (P
Ratio of NE/SHT Comparison of the ratio of NE/SHT in the hypothalamus of all groups (see Fig. 2) shows that this ratio was higher in the prehibernating active group (NE/SHT = 3) than the general ratio during most phases of the hibernating cycle Ratioof
(1
Norepinephrine/5-Hydroxytryptamine
.
Pre H!b.
Early Hlb.
.
in Hypothalamus
l
Late Hib.-ArouSOI-
**.
l
Actwe
l
Phase
k-entry
FIG. 2. The ratio of norepinephrine over 5-hydroxytryptamine in the hypothalamus of the Arctic ground squirrel in prehibemation and different phases of the hibernation cycle.
658
DALE D. FEIST AND WILLIAM
about 2). However, the ratio was highest increased and 5-HT decreased.
(=
A. GALSTER
( = 4) during
early arousal
when NE
Hypothalamic epinephrine The concentration of E in the hypothalamus in all groups was lower than the concentrations of NE and 5-HT (see Table 2). The level of E was quite low or not detectable in the prehibernating group, higher during hibernation, early to TABLE
%-HYPOTHALAMIC EPINEPHRINE &g/g tissue) IN PRBHIBERNATION THEHIBERNATION CYCLE IN THE ARCTICGROUND SQUIRREL
N
Group
3 7 9
Prehibernation active Early hibernation Late hibernation Arousal 48 min 100 min 130 min 180 min Active phase Re-entry
AND
PHASES OF
Epinephrine 0.015 * 0.015 0.183 + O-027* 0.129 + 0.030 0.181 0.128 0.248 0.251 0.227 0,162
f 0.104 f 0.050 f 0.004t _I 0.052 + 0.004 f 0.019
N, Number of animals. * Significant increase (P < 0.01) over prehibernation value. t Mean of combined values for 130 and 180 min arousal higher (P< 0.02) than mean of values from early and late hibernation. Values represent the mean f S.E. expressed in pg/g tissue wet weight.
mid arousal and re-entry, and was highest during the last hour (130 and 180 min) of arousal and in the active phase. A larger sample size is needed to confirm the very low values for E found in the prehibernating group since other groups with higher mean values (e.g. late hibernation) also contain animals with non-detectable E. The mean of combined values from 130 and 180 min of arousal was significantly higher (PC 0.02) than the mean of combined values from early and late hibernation. DISCUSSION These
results
support
the concept
noradrenergic and serotonergic role in entry into, maintenance The concentrations of NE squirrel another
are comparable hibernator, the
No values
for hypothalamic
that changes
in the activity
of hypothalamic
systems accompany and may play an important of and arousal from hibernation. and E in the hypothalamus of the Arctic ground
to those previously reported for the hypothalamus of golden mantled ground squirrel (Twente et al. 1970).
5-HT
appear
available
for other
hibernators
but the
HIBERNATION
5-HT values found (Quay, 1968).
here
AND
AROUSAL
are quite
IN
THF? ARCTIC
similar
to those
GROUND
SQUIRREL
reported
for laboratory
659
rats
Decline in NE during hibernation season The steady-state level of NE in the hypothalamus during the hibernation season was lower than that in the prehibernation summer animals. The NE concentration in the hypothalamus appears to remain constant during the active, re-entry and hibernation phases of the hibernation cycle. These results concur with previous findings of a constant level of NE in the hypothalamus of the active and hibernating golden mantled ground squirrel during the hibernation season (Twente et al. 1970). The biological significance of a lower steady-state level during the hibernation season is not clear but may be related to the resulting ratio of NE to 5-HT or other amines (or transmitters) in the hypothalamus. Increased NE in hypothalamus in arousal The dramatic increase in NE during arousal accompanies very active thermogenesis (see Fig. 1). Previous studies show strong activation of the sympathetic nervous system during arousal to support cardiovascular and thermogenic changes (Lyman & O’Brien, 1963). There is considerable evidence that increased sympathetic activity is associated with accelerated NE synthesis in viva (Gordon et al., 1966; Sedvall et al., 1968; Weiner et al., 1972) and in vitro (Roth et al., 1967; Kopin et al., 1968; Weiner & Rabadjija, 1968; Weiner et al., 1972). Inhibition of tyrosine hydroxylase (the rate-limiting enzyme in the synthesis of NE) by injection of ar-methyl-p-tyrosine in the hibernating hamster prevents arousal from hibernation (Feist, 1970). Thus synthesis of new NE seems essential in certain tissues for normal arousal. In the present study, an increase in noradrenergic neuron firing in the hypothalamus or connecting regions probably resulted in stimulation of NE synthesis along with increased turnover. Synthesis and/or uptake apparently exceeded release and catabolism to give a higher level of NE until midway through arousal. It would seem reasonable to assume that as arousal proceeds a decline in sympathetic activity and increased inhibition of tyrosine hydroxylase (end product feedback inhibition by NE; Spector et al., 1967) resulted in a lower steady-state NE level by the end of arousal. The synthesis of new NE in the hypothalamus for arousal must depend on enzyme systems which are cold insensitive and capable of functioning over a wide range of temperatures (e.g. 0-38°C). These properties of tyrosine hydroxylase should be important adaptations in tissues of hibernators and may in part distinguish them from non-hibernators. Currently, we are investigating the thermal and kinetic properties of tyrosine hydroxylase in tissues of the Arctic ground squirrel. Changes in S-FIT Spafford & Pengelley (1971) found a decline golden mantled ground squirrel during entrance
in 5-HT in the whole brain of the into hibernation. Ussplia (1963a)
660
DALE D. FEIST AND WILLIAM A. GALSTER
reported an increase in whole brain 5-HT during hibernation in the hedgehog. The levels of 5-HT in the hypothalamus of our animals did not show either of these shifts. The apparent decline of 5-HT in the hypothalamus during early arousal may reflect a reduction in serotonergic neuron activity and concomitant imbalance of synthesis and loss which resulted in a lower steady-state 5-HT. Hypothalamic epinephrine The increased level of E during the second half of arousal in these ground squirrels could represent a non-functional by-product of the high concentrations of NE in arousal. Conversely, the increased E could reflect a functional role of E necessary for completion of arousal. There is still considerable conjecture as to whether or not E acts as a neurotransmitter in the central nervous system (Pohorecky et al., 1969). T wente et al. (1970) reported on unequal regional distribution of E in the brain of the golden mantled ground squirrel. This distribution paralleled that of NE and was highest in the hypothalamus. In contrast to the present results, they found no differences in hypothalamic E during the hibernation cycle in hibernating and active animals. However, they did not study animals during arousal. Ratio of NEI5-HT
in the hypothalamus
The high ratio of NE/5-HT during early arousal when the body temperature was 10°C or less could be interpreted as support for the hypothesis that the sustaining mechanism for hibernation as well as the arousal stimulus for restoration of normothermia depend on the balance in release of NE or 5-HT in the anterior hypothalamus (Myers & Yaksh, 1971). For esample, during hibernation a certain ratio of these neurotransmitters acting on hypothalamic thermoregulatory neurons could result in maintenance of torpor. Whereas, at the onset of arousal or during early arousal, an increase in the production and release of NE (with absolutely or relatively less 5-HT production) could result in stimulation or mediation of arousal mechanisms. If the high ratio of NE/5-HT was important for triggering arousal, we might expect to find a gradual increase in the ratio in late hibernation. But this was not found. Preliminary data on NE and 5-HT concentrations in the pons-medulla, an area of autonomic centers which could be important in hibernation and arousal, indicate lower levels of these amines than in the hypothalamus and NE/S-HT ratios very similar to those in the hypothalamus during hibernation (Feist & Galster, unpublished data). Recent studies of intrahypothalamic injections of NE or 5-HT in the hibernating golden mantled ground squirrel have shown that both of these amines cause arousal from hibernation (Beckman & Satinoff, 1972). As in rats (Beckman, 1970; Crawshaw, 1970), both NE and 5-HT show a common thermoregulatory effect in the golden mantled ground squirrel (Beckman & Satinoff, 1972). However, in some species of mammals intrahypothalamic injections of NE and 5-HT produce opposite effects (Lomax, 1970). It remains to be shown, but seems likely, that the Arctic ground squirrel would respond in a fashion similar to the
HIB~ATIO~
ANDAROUSAL IN THEARCTICGROUNDSQUIRReL
661
golden mantled ground squirrel. If this is so, then the increase in the release of NE alone, as reflected by the increased level of NE in early and mid arousal, may be important for activating or mediating arousal thermoregulatory mechanisms, rather than the ratio of NE/S-HT. Further studies to clarify the role of NE and 5-HT as hypoth~amic neurotransmitters during the hibernation cycle in the Arctic ground squirrel should include at least the following: (1) regional changes of NE and 5-HT and ionic (2) turnover of NE and composition (Myers & Yaksh, 1971) in the hypothalamus; 5-HT in hypothalamic regions; and (3) localized intrahypothalamic injections of these and other possible hypothalamic neurotransmitters. Acknowledgements-~~ wish to thank Marilyn Ailes and Kerwin Tschetter for their technical assistance, Dr. Carol F. Feist and Dr. Darrell Williams for criticai reading and helpful suggestions and the National Institutes of Health for financial support via U.S.P.H.S. Grant No, GM-10402. REFERENCES BECKMANA. L. (1970) Effect of intr~~othalamic norepinephrine on thermoregulatory responses in the rat. Am. J. P~ys~o~. 218, 1596-l 604. BECKMANA. L. & SATINOFFE. (1972) Arousal from hibernation by intrahypothalamic injections of biogenic amines in ground squirrels. Am. J. Physiol., 222, 875-879. BLIGH J. (1966) The thermosensitivity of the hypothalamus and thermoregulation in mammals. Viol. Rev. 41, 317-367. CRAWSHAWL. (1970) The effects of intracerebral chemical injections on behavioral and physiological thermoregulation in the rat. Ph.D. thesis, University of California, Santa Barbara, CaIifornia. DRASKOCZYP. R. & LYMAN C. P. (1967) Turnover of catecholamines in active and hibernating ground squirrels. J. Pharmac. exp. Ther. 155, 101-111. VON EULERU. S. & LISHAJKOF. (1961) Improved technique for the fluorometric estimation of catecholamines. Actu physiol. stand. 51, 348-355. FEIST D. D. (1970) Blockade of arousal from hibernation by inhibition of norepinephrine synthesis in the golden hamster. Life Sci. 9, 1117-l 125. FELDBERG W, & MYERS R. D. (1965) Changes in temperature produced by microinjection of amines into the anterior hypothalamus of cats. J. Physiol., Land. 117, 239-245. GALSTERW. A. & MORRISONP. R. (1970) Cyclic changes in carbohydrate concentrations during hibernation in the Arctic ground squirrel. Am. J. Physiol. 218, 1228-1232. GLOWINSKIJ. & IVERSENL. (1966) Regional studies of catecholamines in rat brain-I. The disposition of (H3) norepinephrine and (H3) dopa in various regions of the brain. 3’. ~~roch~. 13, 655-669. GORDON R., SPECTORS., STOERDSMA H. & UDFZNFRIEND S. (1966) Increased synthesis of norepinephrine and epinephrine in the intact rat during exercise and exposure to cold. J. Pharmac. exp. They. 153, 440447. HAYWARD J. S. & LYMAN C. P. (1967) Nonshivering heat production during arousal from hibernation and evidence for the contribution of brown fat. In Mamma&an Hibernation (Edited by FISHERK. C., DARE A. R., LYMAN C. P., SCH~NBAUME. & SOUTHF. E., JR,), Vol. III. American Elsevier, New York. JACOBSH. K., SOUTHF. E., HARTNERW. C. & ZATSMANM. L. (1971) Thermoregulatory effects of administration of biogenic amines into the third ventricle and preoptic area of the marmot (M.fEawiventris) (Abstract). Symposium, Hibernation-Hypothermia-IV. Cryobiol. 8, 313-314.
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KOPIN I. J., BRFZESE G. R., KRAUSEK. R. & WEISEV. K. (1968) Selective release of newly synthesized norepinephrine-from cat spleen during sympathetic nerve stimulation. r. Pharmac. exp. They. 161, 271-278. LOMAX P. (1970) Drugs and body temperature. Intern. Rev. ~eurobjoZ. 12, l-43. LYMAN C. P. & CHATFIELDP. 0. (19.55) Physiology of hibernation in mammals. Physiol. Rev. 35, 403-425. LYMAN C. P. & O’BRIEN R. C. (1963) Autonomic control of circulation during the hibernating cycle in ground squirrels. J. Physiol., Lond. 168, 477-499. MYJZRSR. D. & YAKSH T. L. (1972) The role of hypothalamic monoamines in hibernation and hypothermia. In hibernation and ~ypotherm~u (Edited by SOUTH F. E., HAMMON J. P., WILLIS J. R., PENGELLEY E. T. & ALPERTN. R.). American Elsevier, New York. POHORECKYL. A., ZICMOND M. J., KARTENH. J. & WURTMAN R. J. (1969) Enzymatic conversion of norepinephrine to epinephrine by the brain. J. Pharmac. exp. Ther. 165, 190-19s. QUAY W. B. (1968) Differences in circadian rhythms in 5hydroxytryptamine according to brain regions. Am. J. Physiol. 215, 1448-1453. ROTH R. H., STJ;QRNE L. & VONEULERII. S. (1967) Factors influencing the rate of norepinephrine biosynthesis in nerve tissue. J. Pharmac. exp. Ther. 158, 373-377. SEDVALLG. C., WEISEV. K. & KOPIN I. J. (1968) The rate of norepinephrine synthesis measured in vivo during short intervals; influence of adrenergic nerve impulse activity. r, Pharmac. exp. Ther. 159, 274-282. SIMPSONG. G., ROE A. & LEWONTINR. C. (1960) Q uantitative Zoology. Harcourt, Brace, New York. SNYDERS. H., AXELRODJ. & ZWEIG M, (1965) A sensitive and specific fluorescence assay for tissue serotonin. Biochem. Pharmac. 14, 831-835. SPAFFORDD. C. & PENCELLEY E. T. (1971) The influence of the neurohumor serotonin on hibernation in the golden-mantled ground squirrel, Citellus lateralis. Comp. Biochem. Physiol. 38A, 239-250. SPECTORS., GORDON R., SJOERDSMAA. & WDENFRIEND S. (1967) End product inhibition of tyrosine hydroxylase as a possible mechanism for regulation of norepinephrine synthesis. Molec. Phar~&o~. 3, 549-555. TWENTEJ. W., CLINE W. H., JR. & TWENTEJ. A. (1970) Distribution of epinephrine and norepinephrine in the brain of Cite&s lateralis during the hibernating cycle. Corn@. gen. Pharmac. 1, 47-53. T~ENTE J. W. & TWENTEJ. A. (1968) Progressive irritability of hibernating Citellus lateralis. Camp. Biochem. Physiol. 25, 467-474. UsspiU V. J. (1963a) The S-hydroxytryptamine content of the brain and some other organs of the hedgehog (Erinaeeus europaeus) during activity and hibernation. ~xper~~t~a 19, 156-158. UsspiiX V. J. (1963b) The catecholamine content of the brain and heart of the hedgehog (Erinaceus europaeus) during hibernation and in an active state. Am. Med. exp. Fenn. 41, 340-348. WEINERN., CLOUTIERG., BJURR. & PFEFFER R. I. (1972) Modification of norepinephrine synthesis in intact tissue by drugs and during short-term adrenergic nerve stimulation. Pharmac. Rev. 24, 203-221. WEINER N. & RABADJIJAM. (1968) The effect of nerve stimulation on the synthesis and metabolism of norepinephrine in the isolated guinea-pig hypogastric nerve-vas deferens preparation. J. Pharmac. exp. Ther. 160, 61-71. Key Word Index---Hibernation cycle; arousal; hypothalamus; catecholamines; serotonin; Citellus undulatus.
CHANGES IN HYPOTHALAMIC CATECHOLAMINES AND SEROTONIN DURING HIBERNATION AND AROUSAL IN THE ARCTIC GROUND SQUIRREL DALE
D. FEIST
and WILLIAM
A. GALSTER
Institute of Arctic Biology, University of Alaska, Fairbanks, Alaska 99701, U.S.A. (Received 24 July 1973) Abstract-l. Hypothalamic norepinephrine (NE), epinephrine (E) and serotonin (5HT) were assayed and compared in the Arctic ground squirrel (Cit.&s undulutus) in the prehibernation summer season and different phases of the hibernation cycles during the winter. 2. From a high level in the prehibernation season, NE fell (by 3.5 per cent) to a lower level in the hibernation period, active phase and re-entry phase of the hibernation cycle. In early and mid arousal, NE increased by 50 per cent over that in deep hibernation. By the end of arousal, NE returned to the level found in other phases of the hibernation cycle. 3. During the hibernation cycle, E increased in late arousal and the active phase. The concentration of S-HT remained similar in all groups except in early arousal when the level was 34 per cent lower than that in hibernation. 4. The ratio of NE/S-HT in the hypothalamus was highest during early arousal from hibernation. 5. The results support the hypothesis that changes in the activity of hypothalamic noradrenergic and serotonergic systems play an important role in maintenance of and arousal from hibernation. INTRODUCTION THE ANNUAL cycle of mammalian hibernation includes an active summer season and a hibernation season in fall and winter. During the hibernation season, hibernators undergo periodic entrance into and arousal from deep torpidity. A brief active phase separates each hibernation period (Lyman & Chatfield, 1955). Dramatic shifts in body temperature and thermoregulatory processes occur during periodic entrance and arousal (Lyman & O’Brien, 1963 ; Hayward & Lyman, 1967). It has been suggested that hypothalamic monoamines play an important role in triggering the periodic entrance into and arousal from hibernation (Draskoczy & Lyman, 1967; Feist, 1970; Myers & Yaksh, 1971; Spafford & Pengelley, 1971). Considerable evidence indicates that norepinephrine (NE) and serotonin (5hydroxytryptamine = 5-HT) act as hypothalamic neurotransmitters in the mediation of thermoregulation in non-hibernators (Feldberg & Myers, 1965; Bligh, 1966; Lomax, 1970). Some evidence from studies of monoamine concentrations and turnover in the brain and of the effects of intrahypothalamic injections on NE and 5-HT suggest that central noradrenergic and serotonergic systems may also
653
654
DALE D. FEIST AND WILLIAM A. GALSTER
be important for triggering and/or mediating events in the hibernation cycle. Usspaa (1963a, b) f ound that whole brain content of NE increased and S-HT decreased in the hibernating hedgehog. He concluded that NE and 5-HT belong to different functional systems in the brain. Spafford & Pengelley (1971) found that whole brain 5-HT decreased during entrance into hibernation in the golden mantled ground squirrel. They also found that an inhibitor of serotonin synthesis temporarily blocked hibernation while lesions in the median raphe nucleus of the midbrain (a major locus of 5-HT synthesis) disrupted hibernation. They concluded that serotonin was involved in a functional way in the process of mammalian hibernation. Levels of NE and epinephrine (E) in hypothalamus were found to be constant during the hibernation season in both active and hibernating golden mantled ground squirrels (Twente et al. 1970). Thus, Twente & Twente could not account for possible progressive irritability (Twente & Twente, 1968) during the hibernation period by a generalized accumulation of catecholamines in the hypothalamus which would lead to arousal. The discovery that catecholamine turnover in the whole brain of the thirteenlined ground squirrel stopped immediately prior to entrance into hibernation led to the suggestion that a lack of central adrenergic activity may initiate hibernation (Draskoczy SCLyman, 1967). Recently, injections of low doses of NE or 5-HT into the preoptic and anterior area of the hypothalamus in the hibernating golden mantled ground squirrel caused arousal from hibernation (Beckman & Satinoff, 1972). In contrast, injections of NE or 5-HT into the third ventricle or the preoptic area in the awake marmot caused a decline in body temperature (Jacobs et al., 1971). Based primarily on observed effects of NE and 5-HT on thermoregulation in non-hibernators, Myers & Yaksh (1971) have suggested that the sustaining mechanism for hibernation as well as the arousal stimulus for restoration of normothermia could both depend on the balance in the release of NE or 5-HT in the anterior hypothalamus. The research reported here was initiated as a first step toward assessing the involvement of hypothalamic adrenergic and serotonergic mechanisms in hibernation and arousal of the Arctic ground squirrel (Citellus undulatus). The results include the first reported simultaneous measurements of NE and 5-HT in the hypothalamus of a hibernator during various phases of the hibernation cycle and in the active season. MATERIALS
Experimental
AND
METHODS
groups
Arctic ground squirrels (Citehs undulatus) of both sexes were collected during the summer months from the Thompson Lake, Fielding Lake and Paxson Lake areas of Alaska. The animals were individually caged in the laboratory in a metal cage with a wooden nest box and a constant supply of rat pellets and water. In summer, the active ground squirrels were kept at 18°C with a seasonal light cycle. In the fall and winter, they were kept at 5°C with no light. Hibernation was monitored by temperature changes detected with a thermocouple positioned in the nest under the animal and recorded hourly.
HIBJXNATION
AND
AROUSAL
IN
THE
ARCTIC
GROUND
SQUIRREL
655
The mean body weight of all animals as recorded at the time of killing was 367 + 16 g (mean + SE.). The following groups of ground squirrels were studied (see Fig. 1 also). Prehibernation: one group of animals from the active season in late summer (a) (i.e. September) which had not yet started their hibernation season. Hibernation cycle: eight groups of animals from the mid-late hibernation season (b) (i.e. March-April) in different phases of the hibernation cycle and designated as follows : (1) Early hibernation period-one group of animals in deep hibernation having spent less than 45 per cent (mean = 24 per cent) of their expected hibernation period (the expected period was determined from the average of the three previous hibernation periods, Galster & Morrison, 1970). (2) Late hibernation period-one group of animals in deep hibernation having spent more than 60 per cent (mean = 82 per cent) of their expected hibernation period. groups of animals at different times during (31, (4), (S), (6) Arousing-four arousal after having been artificially stimulated by insertion of a rectal temperature probe while in deep hibernation; these squirrels completed at least 86 per cent (mean = 92 per cent) of their expected hibernation period; the four groups were defined on the basis of body temperature as shown in the following table. TBF (“C)
3
105 25.2 34.7 35.9
8 13.8 21.1 35.3
Time after start of arousal (min) 48 100 130 180
TBF, Axillary brown fat temperature. TWl, Deep colomc temperature. (7) (8)
Active phase-one group of squirrels having aroused completely and active prior to re-entry into hibernation. Re-entry phase-one group of squirrels in the process of re-entering deep hibernation with body temperatures (coionic = 7_4”C, brown fat = 13.7”C) falling toward those attained in deep hibernation.
Measurements of body temperature Deep colonic temperature was measured with a telethermometer probe inserted to a depth of 7-8 cm. The axillary brown fat temperature was determined with a fine wire probe inserted into the brown fat. Temperatures were viewed and recorded with a multichannel telethermometer (YSI). Dissection of the brain Animals were killed by rapid decapitation between 9 a.m. and 1 p.m. Immediately after decapitation, the brain was quickly removed and dissected into regions essentially according to the procedure of Glowinski & Iversen (1966). The hypothalamus and other regions were quickly frozen in liquid nitrogen for storage until extraction of amines. Tissues were weighed on a Mettler balance to the nearest tenth of a mg immediately prior to extraction of amines (see Table 1 for weights of brain regions),
DALE D. FEIST ANDWILLIAM A. GALSTER
656
TABLE I-WEIGHTS OF BRAINREGIONSFROMARCTICGROUND SQUIRRELS Weight (mg)
Region Hypothalamus Medulla + pons Cerebellum Cerebral cortex and remainder of brain
311 532 533 2663
f26 + 17 f 24 ?I 55
Sample No. 42 12 12 12
Values represent the mean f S.E. Extraction,
isolation and assays of amines
Tissues were homogenized in ice-cold 0.4 M perchloric acid (0.1 y0 EDTA) in a motordriven glass homogenizer. After centrifugation, the supernatant was divided into 2.0 ml for the organic solvent extraction and isolation and fluorometric assay of S-HT (Snyder et al., 1965) and 4-O ml for the aluminium oxide isolation and fluorometric assay of NE and E (von Euler & Lishajko, 1961). Recovery studies showed a mean recovery of 70 per cent for NE, 64 per cent for E and 80 per cent for S-HT. Values given for NE, E and 5-HT represent the free amines but are uncorrected for losses during extraction and isolation. Concentrations are expressed in terms of tissue wet weight. Analysis of water content of the brain tissues revealed a constant water content (percentage of total weight) through all phases of the hibernating cycle and active season (Galster, unpublished data). Statistical
analyses The Student t-test (Simpson et al., 1960) was applied to test the significance of the difference between mean values for different groups. Values for amines are expressed as the mean + the standard error (S.E.). RESULTS
Hypothalamic
norepinephrine
As shown in Fig. 1, during the active season the concentration of NE in the hypothalamus of the active prehibernating ground squirrel was 1.687 + 0.186 pg/g. During the hibernation period, of the hibernation cycle, the level of NE was lower by 3.5 per cent (early hibernation P < O-07; late hibernation PC 0.05). In early arousal (45 min after onset of arousal) and mid arousal (100 min after onset of arousal) the concentration of NE increased by 50 per cent (early arousal P-C 0.07; mid arousal P< O-01) over that in deep hibernation. By the end of arousal (3 hr after onset of arousal) when body temperatures approached the normal homeothermic values the NE declined to the level found in deep hibernation. Hypothalamic
Shydroxytryptamine
The concentration of 5-HT in the hypothalamus of the active prehibernating squirrel (Fig. 1) was 0.593 f 0.127 pg/g. Comparable levels were found during the hibernation cycle in all groups except in early arousal (45 min after the onset) in which the level was 34 per cent (not significant ; P = 0.1) lower (0.354 + 0.077 pg/g) than that in hibernation.
HIBERNATION
AND
othalamic
0.00 ReHib.
AROUSAL
Amines
Early Hib.
Late
IN
THE
ARCTIC
GROUND
in Active Season and Phasesof
Hib.
Hibernation
Active Phase
-Arousal-
657
SQUIRREL
Active
ReEntry
FIG. 1. Hypothalamic monoamines in the active season and during different phases of the hibernation cycle of the Arctic ground squirrel. The upper section describes the time course and temperature changes during the hibernation cycle. The lower section shows changes in monoamine values. Values expressed as mean + S.E. * NE hibernation value lower than in prehibernation (P
Ratio of NE/SHT Comparison of the ratio of NE/SHT in the hypothalamus of all groups (see Fig. 2) shows that this ratio was higher in the prehibernating active group (NE/SHT = 3) than the general ratio during most phases of the hibernating cycle Ratioof
(1
Norepinephrine/5-Hydroxytryptamine
.
Pre H!b.
Early Hlb.
.
in Hypothalamus
l
Late Hib.-ArouSOI-
**.
l
Actwe
l
Phase
k-entry
FIG. 2. The ratio of norepinephrine over 5-hydroxytryptamine in the hypothalamus of the Arctic ground squirrel in prehibemation and different phases of the hibernation cycle.
658
DALE D. FEIST AND WILLIAM
about 2). However, the ratio was highest increased and 5-HT decreased.
(=
A. GALSTER
( = 4) during
early arousal
when NE
Hypothalamic epinephrine The concentration of E in the hypothalamus in all groups was lower than the concentrations of NE and 5-HT (see Table 2). The level of E was quite low or not detectable in the prehibernating group, higher during hibernation, early to TABLE
%-HYPOTHALAMIC EPINEPHRINE &g/g tissue) IN PRBHIBERNATION THEHIBERNATION CYCLE IN THE ARCTICGROUND SQUIRREL
N
Group
3 7 9
Prehibernation active Early hibernation Late hibernation Arousal 48 min 100 min 130 min 180 min Active phase Re-entry
AND
PHASES OF
Epinephrine 0.015 * 0.015 0.183 + O-027* 0.129 + 0.030 0.181 0.128 0.248 0.251 0.227 0,162
f 0.104 f 0.050 f 0.004t _I 0.052 + 0.004 f 0.019
N, Number of animals. * Significant increase (P < 0.01) over prehibernation value. t Mean of combined values for 130 and 180 min arousal higher (P< 0.02) than mean of values from early and late hibernation. Values represent the mean f S.E. expressed in pg/g tissue wet weight.
mid arousal and re-entry, and was highest during the last hour (130 and 180 min) of arousal and in the active phase. A larger sample size is needed to confirm the very low values for E found in the prehibernating group since other groups with higher mean values (e.g. late hibernation) also contain animals with non-detectable E. The mean of combined values from 130 and 180 min of arousal was significantly higher (PC 0.02) than the mean of combined values from early and late hibernation. DISCUSSION These
results
support
the concept
noradrenergic and serotonergic role in entry into, maintenance The concentrations of NE squirrel another
are comparable hibernator, the
No values
for hypothalamic
that changes
in the activity
of hypothalamic
systems accompany and may play an important of and arousal from hibernation. and E in the hypothalamus of the Arctic ground
to those previously reported for the hypothalamus of golden mantled ground squirrel (Twente et al. 1970).
5-HT
appear
available
for other
hibernators
but the
HIBERNATION
5-HT values found (Quay, 1968).
here
AND
AROUSAL
are quite
IN
THF? ARCTIC
similar
to those
GROUND
SQUIRREL
reported
for laboratory
659
rats
Decline in NE during hibernation season The steady-state level of NE in the hypothalamus during the hibernation season was lower than that in the prehibernation summer animals. The NE concentration in the hypothalamus appears to remain constant during the active, re-entry and hibernation phases of the hibernation cycle. These results concur with previous findings of a constant level of NE in the hypothalamus of the active and hibernating golden mantled ground squirrel during the hibernation season (Twente et al. 1970). The biological significance of a lower steady-state level during the hibernation season is not clear but may be related to the resulting ratio of NE to 5-HT or other amines (or transmitters) in the hypothalamus. Increased NE in hypothalamus in arousal The dramatic increase in NE during arousal accompanies very active thermogenesis (see Fig. 1). Previous studies show strong activation of the sympathetic nervous system during arousal to support cardiovascular and thermogenic changes (Lyman & O’Brien, 1963). There is considerable evidence that increased sympathetic activity is associated with accelerated NE synthesis in viva (Gordon et al., 1966; Sedvall et al., 1968; Weiner et al., 1972) and in vitro (Roth et al., 1967; Kopin et al., 1968; Weiner & Rabadjija, 1968; Weiner et al., 1972). Inhibition of tyrosine hydroxylase (the rate-limiting enzyme in the synthesis of NE) by injection of ar-methyl-p-tyrosine in the hibernating hamster prevents arousal from hibernation (Feist, 1970). Thus synthesis of new NE seems essential in certain tissues for normal arousal. In the present study, an increase in noradrenergic neuron firing in the hypothalamus or connecting regions probably resulted in stimulation of NE synthesis along with increased turnover. Synthesis and/or uptake apparently exceeded release and catabolism to give a higher level of NE until midway through arousal. It would seem reasonable to assume that as arousal proceeds a decline in sympathetic activity and increased inhibition of tyrosine hydroxylase (end product feedback inhibition by NE; Spector et al., 1967) resulted in a lower steady-state NE level by the end of arousal. The synthesis of new NE in the hypothalamus for arousal must depend on enzyme systems which are cold insensitive and capable of functioning over a wide range of temperatures (e.g. 0-38°C). These properties of tyrosine hydroxylase should be important adaptations in tissues of hibernators and may in part distinguish them from non-hibernators. Currently, we are investigating the thermal and kinetic properties of tyrosine hydroxylase in tissues of the Arctic ground squirrel. Changes in S-FIT Spafford & Pengelley (1971) found a decline golden mantled ground squirrel during entrance
in 5-HT in the whole brain of the into hibernation. Ussplia (1963a)
660
DALE D. FEIST AND WILLIAM A. GALSTER
reported an increase in whole brain 5-HT during hibernation in the hedgehog. The levels of 5-HT in the hypothalamus of our animals did not show either of these shifts. The apparent decline of 5-HT in the hypothalamus during early arousal may reflect a reduction in serotonergic neuron activity and concomitant imbalance of synthesis and loss which resulted in a lower steady-state 5-HT. Hypothalamic epinephrine The increased level of E during the second half of arousal in these ground squirrels could represent a non-functional by-product of the high concentrations of NE in arousal. Conversely, the increased E could reflect a functional role of E necessary for completion of arousal. There is still considerable conjecture as to whether or not E acts as a neurotransmitter in the central nervous system (Pohorecky et al., 1969). T wente et al. (1970) reported on unequal regional distribution of E in the brain of the golden mantled ground squirrel. This distribution paralleled that of NE and was highest in the hypothalamus. In contrast to the present results, they found no differences in hypothalamic E during the hibernation cycle in hibernating and active animals. However, they did not study animals during arousal. Ratio of NEI5-HT
in the hypothalamus
The high ratio of NE/5-HT during early arousal when the body temperature was 10°C or less could be interpreted as support for the hypothesis that the sustaining mechanism for hibernation as well as the arousal stimulus for restoration of normothermia depend on the balance in release of NE or 5-HT in the anterior hypothalamus (Myers & Yaksh, 1971). For esample, during hibernation a certain ratio of these neurotransmitters acting on hypothalamic thermoregulatory neurons could result in maintenance of torpor. Whereas, at the onset of arousal or during early arousal, an increase in the production and release of NE (with absolutely or relatively less 5-HT production) could result in stimulation or mediation of arousal mechanisms. If the high ratio of NE/5-HT was important for triggering arousal, we might expect to find a gradual increase in the ratio in late hibernation. But this was not found. Preliminary data on NE and 5-HT concentrations in the pons-medulla, an area of autonomic centers which could be important in hibernation and arousal, indicate lower levels of these amines than in the hypothalamus and NE/S-HT ratios very similar to those in the hypothalamus during hibernation (Feist & Galster, unpublished data). Recent studies of intrahypothalamic injections of NE or 5-HT in the hibernating golden mantled ground squirrel have shown that both of these amines cause arousal from hibernation (Beckman & Satinoff, 1972). As in rats (Beckman, 1970; Crawshaw, 1970), both NE and 5-HT show a common thermoregulatory effect in the golden mantled ground squirrel (Beckman & Satinoff, 1972). However, in some species of mammals intrahypothalamic injections of NE and 5-HT produce opposite effects (Lomax, 1970). It remains to be shown, but seems likely, that the Arctic ground squirrel would respond in a fashion similar to the
HIB~ATIO~
ANDAROUSAL IN THEARCTICGROUNDSQUIRReL
661
golden mantled ground squirrel. If this is so, then the increase in the release of NE alone, as reflected by the increased level of NE in early and mid arousal, may be important for activating or mediating arousal thermoregulatory mechanisms, rather than the ratio of NE/S-HT. Further studies to clarify the role of NE and 5-HT as hypoth~amic neurotransmitters during the hibernation cycle in the Arctic ground squirrel should include at least the following: (1) regional changes of NE and 5-HT and ionic (2) turnover of NE and composition (Myers & Yaksh, 1971) in the hypothalamus; 5-HT in hypothalamic regions; and (3) localized intrahypothalamic injections of these and other possible hypothalamic neurotransmitters. Acknowledgements-~~ wish to thank Marilyn Ailes and Kerwin Tschetter for their technical assistance, Dr. Carol F. Feist and Dr. Darrell Williams for criticai reading and helpful suggestions and the National Institutes of Health for financial support via U.S.P.H.S. Grant No, GM-10402. REFERENCES BECKMANA. L. (1970) Effect of intr~~othalamic norepinephrine on thermoregulatory responses in the rat. Am. J. P~ys~o~. 218, 1596-l 604. BECKMANA. L. & SATINOFFE. (1972) Arousal from hibernation by intrahypothalamic injections of biogenic amines in ground squirrels. Am. J. Physiol., 222, 875-879. BLIGH J. (1966) The thermosensitivity of the hypothalamus and thermoregulation in mammals. Viol. Rev. 41, 317-367. CRAWSHAWL. (1970) The effects of intracerebral chemical injections on behavioral and physiological thermoregulation in the rat. Ph.D. thesis, University of California, Santa Barbara, CaIifornia. DRASKOCZYP. R. & LYMAN C. P. (1967) Turnover of catecholamines in active and hibernating ground squirrels. J. Pharmac. exp. Ther. 155, 101-111. VON EULERU. S. & LISHAJKOF. (1961) Improved technique for the fluorometric estimation of catecholamines. Actu physiol. stand. 51, 348-355. FEIST D. D. (1970) Blockade of arousal from hibernation by inhibition of norepinephrine synthesis in the golden hamster. Life Sci. 9, 1117-l 125. FELDBERG W, & MYERS R. D. (1965) Changes in temperature produced by microinjection of amines into the anterior hypothalamus of cats. J. Physiol., Land. 117, 239-245. GALSTERW. A. & MORRISONP. R. (1970) Cyclic changes in carbohydrate concentrations during hibernation in the Arctic ground squirrel. Am. J. Physiol. 218, 1228-1232. GLOWINSKIJ. & IVERSENL. (1966) Regional studies of catecholamines in rat brain-I. The disposition of (H3) norepinephrine and (H3) dopa in various regions of the brain. 3’. ~~roch~. 13, 655-669. GORDON R., SPECTORS., STOERDSMA H. & UDFZNFRIEND S. (1966) Increased synthesis of norepinephrine and epinephrine in the intact rat during exercise and exposure to cold. J. Pharmac. exp. They. 153, 440447. HAYWARD J. S. & LYMAN C. P. (1967) Nonshivering heat production during arousal from hibernation and evidence for the contribution of brown fat. In Mamma&an Hibernation (Edited by FISHERK. C., DARE A. R., LYMAN C. P., SCH~NBAUME. & SOUTHF. E., JR,), Vol. III. American Elsevier, New York. JACOBSH. K., SOUTHF. E., HARTNERW. C. & ZATSMANM. L. (1971) Thermoregulatory effects of administration of biogenic amines into the third ventricle and preoptic area of the marmot (M.fEawiventris) (Abstract). Symposium, Hibernation-Hypothermia-IV. Cryobiol. 8, 313-314.
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DALE D. FEISTANDWILLIAM A. GALSTER
KOPIN I. J., BRFZESE G. R., KRAUSEK. R. & WEISEV. K. (1968) Selective release of newly synthesized norepinephrine-from cat spleen during sympathetic nerve stimulation. r. Pharmac. exp. They. 161, 271-278. LOMAX P. (1970) Drugs and body temperature. Intern. Rev. ~eurobjoZ. 12, l-43. LYMAN C. P. & CHATFIELDP. 0. (19.55) Physiology of hibernation in mammals. Physiol. Rev. 35, 403-425. LYMAN C. P. & O’BRIEN R. C. (1963) Autonomic control of circulation during the hibernating cycle in ground squirrels. J. Physiol., Lond. 168, 477-499. MYJZRSR. D. & YAKSH T. L. (1972) The role of hypothalamic monoamines in hibernation and hypothermia. In hibernation and ~ypotherm~u (Edited by SOUTH F. E., HAMMON J. P., WILLIS J. R., PENGELLEY E. T. & ALPERTN. R.). American Elsevier, New York. POHORECKYL. A., ZICMOND M. J., KARTENH. J. & WURTMAN R. J. (1969) Enzymatic conversion of norepinephrine to epinephrine by the brain. J. Pharmac. exp. Ther. 165, 190-19s. QUAY W. B. (1968) Differences in circadian rhythms in 5hydroxytryptamine according to brain regions. Am. J. Physiol. 215, 1448-1453. ROTH R. H., STJ;QRNE L. & VONEULERII. S. (1967) Factors influencing the rate of norepinephrine biosynthesis in nerve tissue. J. Pharmac. exp. Ther. 158, 373-377. SEDVALLG. C., WEISEV. K. & KOPIN I. J. (1968) The rate of norepinephrine synthesis measured in vivo during short intervals; influence of adrenergic nerve impulse activity. r, Pharmac. exp. Ther. 159, 274-282. SIMPSONG. G., ROE A. & LEWONTINR. C. (1960) Q uantitative Zoology. Harcourt, Brace, New York. SNYDERS. H., AXELRODJ. & ZWEIG M, (1965) A sensitive and specific fluorescence assay for tissue serotonin. Biochem. Pharmac. 14, 831-835. SPAFFORDD. C. & PENCELLEY E. T. (1971) The influence of the neurohumor serotonin on hibernation in the golden-mantled ground squirrel, Citellus lateralis. Comp. Biochem. Physiol. 38A, 239-250. SPECTORS., GORDON R., SJOERDSMAA. & WDENFRIEND S. (1967) End product inhibition of tyrosine hydroxylase as a possible mechanism for regulation of norepinephrine synthesis. Molec. Phar~&o~. 3, 549-555. TWENTEJ. W., CLINE W. H., JR. & TWENTEJ. A. (1970) Distribution of epinephrine and norepinephrine in the brain of Cite&s lateralis during the hibernating cycle. Corn@. gen. Pharmac. 1, 47-53. T~ENTE J. W. & TWENTEJ. A. (1968) Progressive irritability of hibernating Citellus lateralis. Camp. Biochem. Physiol. 25, 467-474. UsspiU V. J. (1963a) The S-hydroxytryptamine content of the brain and some other organs of the hedgehog (Erinaeeus europaeus) during activity and hibernation. ~xper~~t~a 19, 156-158. UsspiiX V. J. (1963b) The catecholamine content of the brain and heart of the hedgehog (Erinaceus europaeus) during hibernation and in an active state. Am. Med. exp. Fenn. 41, 340-348. WEINERN., CLOUTIERG., BJURR. & PFEFFER R. I. (1972) Modification of norepinephrine synthesis in intact tissue by drugs and during short-term adrenergic nerve stimulation. Pharmac. Rev. 24, 203-221. WEINER N. & RABADJIJAM. (1968) The effect of nerve stimulation on the synthesis and metabolism of norepinephrine in the isolated guinea-pig hypogastric nerve-vas deferens preparation. J. Pharmac. exp. Ther. 160, 61-71. Key Word Index---Hibernation cycle; arousal; hypothalamus; catecholamines; serotonin; Citellus undulatus.