Search for rhythmicity during hibernation in the European hamster

Temporal patterns of hibernation were studied by continuous monitoring of body temperature by radiotelemetry over 6 months in European hamsters, Cricetus cricetus, at constant temperature and photoperiod. Entrances into hibernation occurred mostly at the end of the night (0000–0800 hours), while arousals were randomly distributed between day and night. This is at variance with a control of bout duration by a clock with a period of 24 h. Consequently, the timing of entrances implies a phase-resetting of the circadian clock on each arousal. Persistence of circadian rhythmicity with a period different from 24 h during deep hibernation was investigated examining whether the durations of torpor bouts were integer multiples of a constant period. A non-parametric version of the classical contingency test of periodicity was developed for this purpose. Periods ranging from 21 to 29 h were tested. Nine animals out of ten showed at least one significant period in this range (P<0.01), either below 24 h (21.8±0.5 h, n=4) or above (27.3±0.5 h, n=7). However, we have found a theoretical model of bout durations for which the contingency test of periodicity sometimes gives false significant results. This indicates that the power of the test is weak. With this reservation our results suggest that a circadian oscillator controls the duration of a bout of hibernation, which would occur after an integer, but variable and possibly temperature-dependent number of cycles.Abbreviations _b a contingency test (see Appendix) - SCN suprachiasmatic nuclei - period - T _b body temperature     

Enhanced antioxidant defense due to extracellular catalase activity in Syrian hamster during arousal from hibernation

Mammalian hibernators are considered a natural model for resistance to ischemia-reperfusion injuries, and protective mechanisms against oxidative stress evoked by repeated hibernation-arousal cycles in these animals are increasingly the focus of experimental investigation. Here we show that extracellular catalase activity provides protection against oxidative stress during arousal from hibernation in Syrian hamster. To examine the serum antioxidant defense system, we first assessed the hibernation-arousal state-dependent change in serum attenuation of cytotoxicity induced by hydrogen peroxide. Serum obtained from hamsters during arousal from hibernation at a rectal temperature of 32 degrees C, concomitant with the period of increased oxidative stress, attenuated the cytotoxicity four-fold more effectively than serum from cenothermic control hamsters. Serum catalase activity significantly increased during arousal, whereas glutathione peroxidase activity decreased by 50%, compared with cenothermic controls. The cytoprotective effect of purified catalase at the concentration found in serum was also confirmed in a hydrogen peroxide-induced cytotoxicity model. Moreover, inhibition of catalase by aminotriazole led to an 80% loss of serum hydrogen peroxide scavenging activity. These results suggest that extracellular catalase is effective for protecting hibernators from oxidative stress evoked by arousal from hibernation.     

Remodeling mitochondrial membranes during arousal from hibernation

During arousal from hibernation, body temperature (T(b)) increases by ～30°C and liver mitochondrial respiration increases threefold in as little as 2 h. We analyzed liver mitochondria purified from ground squirrels (Ictidomys tridecemlineatus) to see whether membrane phospholipids were remodeled during spontaneous arousal. Cardiolipin content did not change among animals in torpor (T ～ 5°C), the early phase of arousal (T ～ 15°C), late arousal (T ～ 30°C), interbout euthermia (T ～ 37°C), and summer-active animals (T ～ 37°C) that do not hibernate. Phosphatidylcholine content increased in late arousal relative to interbout euthermia, while phosphatidylethanolamine decreased. Phospholipid monounsaturated fatty acids (MUFAs) did not change throughout arousal, but polyunsaturated fatty acids (PUFAs) and MUFA/PUFA decreased and increased, respectively. In the fatty acid conjugates of phospholipids, neither unsaturation index nor n-3/n-6 differed. Few changes in individual fatty acids were noted, but palmitoleic acid (16:1, n-7) was higher in interbout euthermia and summer. Although 16:1 accounted for less than 1.5% of phospholipid fatty acids, it correlated strongly and positively with succinate-fueled state 3 mitochondrial respiration. No other phospholipid characteristic measured here correlated with mitochondrial respiration. These data show that mitochondrial membranes are remodeled rapidly during arousal, but the contribution to reversible suppression of mitochondrial respiration remains unclear.     

Activation of stress signaling molecules in bat brain during arousal from hibernation

Induction of glucose-regulated proteins (GRPs) is a ubiquitous intracellular response to stresses such as hypoxia, glucose starvation and acidosis. The induction of GRPs offers some protection against these stresses in vitro, but the specific role of GRPs in vivo remains unclear. Hibernating bats present a good in vivo model to address this question. The bats must overcome local high oxygen demand in tissue by severe metabolic stress during arousal thermogenesis. We used brain tissue of a temperate bat Rhinolopus ferrumequinum to investigate GRP induction by high metabolic oxygen demand and to identify associated signaling molecules. We found that during 30 min of arousal, oxygen consumption increased from nearly zero to 11.9/kg/h, which was about 8.7-fold higher than its active resting metabolic rate. During this time, body temperature rose from 7 degrees C to 35 degrees C, and levels of TNF-alpha and lactate in brain tissue increased 2-2.5-fold, indicating a high risk of oxygen shortage. Concomitantly, levels of GRP75, GRP78 and GRP94 increased 1.5-1.7-fold. At the same time, c-Jun N-terminal protein kinase (JNK) activity increased 6.4-fold, and extracellular signal-regulated protein kinase (ERK) activity decreased to a similar degree (6.1-fold). p38 MAPK activity was very low and remained unchanged during arousal. In addition, survival signaling molecules protein kinase B (Akt) and protein kinase C (PKC) were activated 3- and 5-fold, respectively, during arousal. Taken together, our results showed that bat brain undergoes high oxygen demand during arousal from hibernation. Up-regulation of GRP proteins and activation of JNK, PKCgamma and Akt may be critical for neuroprotection and the survival of bats during the repeated process.     

Heat production in the ground squirrel Citellus pygmaeus during arousal from hibernation]

Heat production has been both measured experimentally (Qcal) and calculated from oxygen consumption (QO2) in arousing ground squirrels during the rise of their body temperature. Studies were made on total heat production during all the period of their warming as well as on the heat production at various stages of arousal. Qcal was evaluated by changes in body temperature and those in heat losses via convection and irradiation (calorimetrically). During arousal of animals, their body temperature, heat losses, Qcal and QO2 Gradually increase. However, the increase in heat losses is 3-4 times lower as compared to the intrinsic heat production measured both calorimetrically and by oxygen consumption. Limitation of heat losses (due to the constriction of subcutaneous blood vessels) together with activation of the metabolism in muscles and other tissues provide for significant heat accumulation and the increase in body temperature of arousing ground squirrels.     

Intracellular pH in hibernation and respiratory acidosis in the European hamster

Intracellular pH was determined (DMO method) in European hamsters, in the spontaneously-occurring respiratory acidosis of hibernation, in hypercapnia due to breathing 12% CO2 in air in euthermy in spring, and in euthermicnormocapnic controls. From euthermy to hibernation, the temperature coefficient of pH was lowest in blood plasma and brain, intermediate in striated muscles (thigh muscles and diaphragm), and highest in heart and liver (Fig. 1). Correspondingly, the estimated dissociation ratio of the protein imidazole buffer groups, alpha Im, decreased markedly in plasma and brain, denoting an acid titration, but varied little in liver and heart. Striated muscles were intermediate (Fig. 2). Like in other mammals, intracellular responses to short-term euthermic respiratory acidosis were characterized by a partial metabolic compensation in the brain and a small metabolic acidification in striated muscles. In hibernation, a powerful metabolic compensation took place in liver and heart, nearly restoring alpha Im, but none occurred in brain (Figs. 3 to 5). The existence of an intracellular acidosis in brain and striated muscles during hibernation is in keeping with an inhibitory role of acidosis, whereas the homeostasis of intracellular alpha Im in liver and heart would subserve the eurythermal functioning of metabolic regulations in these organs, like in most organs of ectotherms.     

Carboxyatractylate-sensitive uncoupling in liver mitochondria from ground squirrels during hibernation and arousal

Energy coupling parameters of liver mitochondria from hibernating and arousing ground squirrels have been studied. In the oligomycin-treated mitochondria, carboxyatractylate, an inhibitor of the ATP/ADP-antiporter, is shown to decrease the respiration rate, to increase the membrane potential and to lower the rate of the membrane-potential discharge after the addition of cyanide to liver mitochondria from hibernating and arousing animals. BSA effectively substitutes for carboxyactactylate so that carboxyactactylate, added after BSA, has no effect. In mitochondria from hibernating animals, the maximal respiration rate in the presence of DNP and the rate of the membrane potential discharge in its absence are much lower than in those from arousing animals. It has been concluded that upon arousal of the animals from hibernation, the uncoupling of oxidative phosphorylation, mediated by free fatty acids and ATP/ADP-antiporter, parallels the respiratory chain activation.     

Influence of photoperiod and gonadal steroids on hibernation in the European hamster

Torpor was monitored daily in adult male and female European hamsters (Cricetus cricetus) induced to hibernate by exposure to a cold environment (6 degrees C). The effect of photoperiodic manipulations or administration of exogenous gonadal steroids was examined in gonadectomized or intact hamsters. 1. Gonadal regression occurred in all short day, but only in some long day, cold-exposed hamsters. Entry into hibernation was not observed until reproductive regression had occurred. Thus, gonadal atrophy appears to be a necessary precondition for hibernation. 2. Castrated hamsters in the short day cold condition showed a significantly greater incidence of torpor than those in the long day cold condition. Hence, photoperiod affected torpor independently of its effect on the gonadal cycle. 3. Testosterone, when administered via silastic capsules at near physiological levels, completely inhibited torpor in gonadectomized male and female hamsters hibernating in the short day cold condition. 4. In ovariectomized females, torpor was unaffected by progesterone treatment, but partially inhibited by estradiol. A greater inhibition of torpor was observed when estradiol-primed females were administered both estradiol and progesterone simultaneously. Thus, the effect of both hormones may be functionally comparable to that of the single testicular hormone. 5. Estradiol inhibited torpor to a greater extent in intact and ovariectomized female hamsters hibernating in long days than those in short days, suggesting an effect of photoperiod on responsiveness to estradiol. These results indicate an inverse relationship between the gonadal and hibernation cycles, and a probable role for gonadal steroids to influence the timing of the hibernation season. However, non-gonadal factors must also be involved in controlling hibernation, since photoperiod affected the incidence of torpor in gonadectomized animals and because hamsters were able to terminate hibernation in the absence of gonadal hormones.