Cellular, Molecular, and Physiological Adaptations of Hibernation: The Solution to Environmental Challenges.

Thriving in times of resource scarcity requires an incredible flexibility of behavioral, physiological, cellular, and molecular functions that must change within a relatively short time. Hibernation is a collection of physiological strategies that allows animals to inhabit inhospitable environments, where they experience extreme thermal challenges and scarcity of food and water. Many different kinds of animals employ hibernation, and there is a spectrum of hibernation phenotypes. Here, we focus on obligatory mammalian hibernators to identify the unique challenges they face and the adaptations that allow hibernators to overcome them. This includes the cellular and molecular strategies used to combat low environmental and body temperatures and lack of food and water. We discuss metabolic, neuronal, and hormonal cues that regulate hibernation and how they are thought to be coordinated by internal clocks. Last, we touch on questions that are left to be addressed in the field of hibernation research. Studies from the last century and more recent work reveal that hibernation is not simply a passive reduction in body temperature and vital parameters but rather an active process seasonally regulated at the molecular, cellular, and organismal levels.

A discrete neuronal circuit induces a hibernation-like state in rodents.

Hibernating mammals actively lower their body temperature to reduce energy expenditure when facing food scarcity1. This ability to induce a hypometabolic state has evoked great interest owing to its potential medical benefits2,3. Here we show that a hypothalamic neuronal circuit in rodents induces a long-lasting hypothermic and hypometabolic state similar to hibernation. In this state, although body temperature and levels of oxygen consumption are kept very low, the ability to regulate metabolism still remains functional, as in hibernation4. There was no obvious damage to tissues and organs or abnormalities in behaviour after recovery from this state. Our findings could enable the development of a method to induce a hibernation-like state, which would have potential applications in non-hibernating mammalian species including humans.

Molecular Physiology of Freeze Tolerance in Vertebrates.

Freeze tolerance is an amazing winter survival strategy used by various amphibians and reptiles living in seasonally cold environments. These animals may spend weeks or months with up to ∼65% of their total body water frozen as extracellular ice and no physiological vital signs, and yet after thawing they return to normal life within a few hours. Two main principles of animal freeze tolerance have received much attention: the production of high concentrations of organic osmolytes (glucose, glycerol, urea among amphibians) that protect the intracellular environment, and the control of ice within the body (the first putative ice-binding protein in a frog was recently identified), but many other strategies of biochemical adaptation also contribute to freezing survival. Discussed herein are recent advances in our understanding of amphibian and reptile freeze tolerance with a focus on cell preservation strategies (chaperones, antioxidants, damage defense mechanisms), membrane transporters for water and cryoprotectants, energy metabolism, gene/protein adaptations, and the regulatory control of freeze-responsive hypometabolism at multiple levels (epigenetic regulation of DNA, microRNA action, cell signaling and transcription factor regulation, cell cycle control, and anti-apoptosis). All are providing a much more complete picture of life in the frozen state.

Effects of hibernation on two important contractile tissues in tibetan frogs, Nanorana parkeri: a perspective from transcriptomics and metabolomics approaches.

BACKGROUND: In response to seasonal cold and food shortage, the Xizang plateau frogs, Nanorana parkeri (Anura: Dicroglossidae), enter a reversible hypometabolic state where heart rate and oxygen consumption in skeletal muscle are strongly suppressed. However, the effect of winter hibernation on gene expression and metabolic profiling in these two tissues remains unknown. In the present study, we conducted transcriptomic and metabolomic analyses of heart and skeletal muscle from summer- and winter-collected N. parkeri to explore mechanisms involved in seasonal hibernation. RESULTS: We identified 2407 differentially expressed genes (DEGs) in heart and 2938 DEGs in skeletal muscle. Enrichment analysis showed that shared DEGs in both tissues were enriched mainly in translation and metabolic processes. Of these, the expression of genes functionally categorized as "response to stress", "defense mechanisms", or "muscle contraction" were particularly associated with hibernation. Metabolomic analysis identified 24 and 22 differentially expressed metabolites (DEMs) in myocardium and skeletal muscle, respectively. In particular, pathway analysis showed that DEMs in myocardium were involved in the pentose phosphate pathway, glycerolipid metabolism, pyruvate metabolism, citrate cycle (TCA cycle), and glycolysis/gluconeogenesis. By contrast, DEMs in skeletal muscle were mainly involved in amino acid metabolism. CONCLUSIONS: In summary, natural adaptations of myocardium and skeletal muscle in hibernating N. parkeri involved transcriptional alterations in translation, stress response, protective mechanisms, and muscle contraction processes as well as metabolic remodeling. This study provides new insights into the transcriptional and metabolic adjustments that aid winter survival of high-altitude frogs N. parkeri.

Seasonal fluctuations in BDNF regulate hibernation and torpor in golden-mantled ground squirrels.

Aphagic hibernators such as the golden-mantled ground squirrel (GMGS; Callospermophilus lateralis) can fast for months and exhibit profound seasonal fluctuations in body weight, food intake, and behavior. Brain-derived neurotrophic factor (BDNF) regulates cellular and systemic metabolism via mechanisms that are conserved across mammalian species. In this study, we characterized regional changes in BDNF with hibernation, hypothermia, and seasonal cycle in GMGS. Analysis of BDNF protein concentrations by ELISA revealed overlapping seasonal patterns in the hippocampus and hypothalamus, where BDNF levels were highest in summer and lowest in winter. BDNF is the primary ligand for receptor tyrosine kinase B (TrkB), and BDNF/TrkB signaling in the brain potently regulates energy expenditure. To examine the functional relevance of seasonal variation in BDNF, hibernating animals were injected with the small molecule TrkB agonist 7,8-dihydroxyflavone (DHF) daily for 2 wk. When compared with vehicle, DHF-treated animals exhibited fewer torpor bouts and shorter bout durations. These results suggest that activating BDNF/TrkB disrupts hibernation and raise intriguing questions related to the role of BDNF as a potential regulatory mechanism or downstream response to seasonal changes in body temperature and environment.NEW & NOTEWORTHY Golden-mantled ground squirrels exhibit dramatic seasonal fluctuations in metabolism and can fast for months while hibernating. Brain-derived neurotrophic factor is an essential determinant of cellular and systemic metabolism, and in this study, we characterized seasonal fluctuations in BDNF expression and then administered the small molecule BDNF mimetic 7,8-dihydroxyflavone (DHF) in hibernating squirrels. The results indicate that activating BDNF/TrkB signaling disrupts hibernation, with implications for synaptic homeostasis in prolonged hypometabolic states.

Shifts in population density centers of a hibernating mammal driven by conflicting effects of climate change and disease.

Populations wax and wane over time in response to an organism's interactions with abiotic and biotic forces. Numerous studies demonstrate that fluctuations in local populations can lead to shifts in relative population densities across the geographic range of a species over time. Fewer studies attempt to disentangle the causes of such shifts. Over four decades (1983-2022), we monitored populations of hibernating Indiana bats (Myotis sodalis) in two areas separated by ~110 km. The number of bats hibernating in the northern area increased from 1983 to 2011, while populations in the southern area remained relatively constant. We used simulation models and long-term weather data to demonstrate the duration of time bats must rely on stored fat during hibernation has decreased in both areas over that period, but at a faster rate in the northern area. Likewise, increasing autumn and spring temperatures shortened the periods of sporadic prey (flying insect) availability at the beginning and end of hibernation. Climate change thus increased the viability of northern hibernacula for an increasing number of bats by decreasing energetic costs of hibernation. Then in 2011, white-nose syndrome (WNS), a disease of hibernating bats that increases energetic costs of hibernation, was detected in the area. From 2011 to 2022, the population rapidly decreased in the northern area and increased in the southern area, completely reversing the northerly shift in population densities associated with climate change. Energy balance during hibernation is the singular link explaining the northerly shift under a changing climate and the southerly shift in response to a novel disease. Continued population persistence suggests that bats may mitigate many impacts of WNS by hibernating farther south, where insects are available longer each year.

Sulfide catabolism in hibernation and neuroprotection.

The mammalian brain is exquisitely vulnerable to lack of oxygen. However, the mechanism underlying the brain's sensitivity to hypoxia is incompletely understood. In this narrative review, we present a case for sulfide catabolism as a key defense mechanism of the brain against acute oxygen shortage. We will examine literature on the role of sulfide in hypoxia/ischemia, deep hibernation, and leigh syndrome patients, and present our recent data that support the neuroprotective effects of sulfide catabolism and persulfide production. When oxygen levels become low, hydrogen sulfide (H2S) accumulates in brain cells and impairs the ability of these cells to use the remaining, available oxygen to produce energy. In recent studies, we found that hibernating ground squirrels, which can withstand very low levels of oxygen, have high levels of sulfide:quinone oxidoreductase (SQOR) and the capacity to catabolize hydrogen sulfide in the brain. Silencing SQOR increased the sensitivity of the brain of squirrels and mice to hypoxia, whereas neuron-specific SQOR expression prevented hypoxia-induced sulfide accumulation, bioenergetic failure, and ischemic brain injury in mice. Excluding SQOR from mitochondria increased sensitivity to hypoxia not only in the brain but also in heart and liver. Pharmacological agents that scavenge sulfide and/or increase persulfide maintained mitochondrial respiration in hypoxic neurons and made mice resistant to ischemic injury to the brain or spinal cord. Drugs that oxidize hydrogen sulfide and/or increase persulfide may prove to be an effective approach to the treatment of patients experiencing brain injury caused by oxygen deprivation or mitochondrial dysfunction.

Hibernating female big brown bats (Eptesicus fuscus) adjust huddling and drinking behaviour, but not arousal frequency, in response to low humidity.

Many mammals hibernate during winter, reducing energy expenditure via bouts of torpor. The majority of a hibernator's energy reserves are used to fuel brief, but costly, arousals from torpor. Although arousals likely serve multiple functions, an important one is to restore water stores depleted during torpor. Many hibernating bat species require high humidity, presumably to reduce torpid water loss, but big brown bats (Eptesicus fuscus) appear tolerant of a wide humidity range. We tested the hypothesis that hibernating female E. fuscus use behavioural flexibility during torpor and arousals to maintain water balance and reduce energy expenditure. We predicted: (1) E. fuscus hibernating in dry conditions would exhibit more compact huddles during torpor and drink more frequently than bats in high humidity conditions; and (2) the frequency and duration of torpor bouts and arousals, and thus total loss of body mass would not differ between bats in the two environments. We housed hibernating E. fuscus in temperature- and humidity-controlled incubators at 50% or 98% relative humidity (8°C, 110 days). Bats in the dry environment maintained a more compact huddle during torpor and drank more frequently during arousals. Bats in the two environments had a similar number of arousals, but arousal duration was shorter in the dry environment. However, total loss of body mass over hibernation did not differ between treatments, indicating that the two groups used similar amounts of energy. Our results suggest that behavioural flexibility allows hibernating E. fuscus to maintain water balance and reduce energy costs across a wide range of hibernation humidities.

c-fos induction in the choroid plexus, tanycytes and pars tuberalis is an early indicator of spontaneous arousal from torpor in a deep hibernator.

Hibernation is an extreme state of seasonal energy conservation, reducing metabolic rate to as little as 1% of the active state. During the hibernation season, many species of hibernating mammals cycle repeatedly between the active (aroused) and hibernating (torpid) states (T-A cycling), using brown adipose tissue (BAT) to drive cyclical rewarming. The regulatory mechanisms controlling this process remain undefined but are presumed to involve thermoregulatory centres in the hypothalamus. Here, we used the golden hamster (Mesocricetus auratus), and high-resolution monitoring of BAT, core body temperature and ventilation rate, to sample at precisely defined phases of the T-A cycle. Using c-fos as a marker of cellular activity, we show that although the dorsomedial hypothalamus is active during torpor entry, neither it nor the pre-optic area shows any significant changes during the earliest stages of spontaneous arousal. Contrastingly, in three non-neuronal sites previously linked to control of metabolic physiology over seasonal and daily time scales - the choroid plexus, pars tuberalis and third ventricle tanycytes - peak c-fos expression is seen at arousal initiation. We suggest that through their sensitivity to factors in the blood or cerebrospinal fluid, these sites may mediate metabolic feedback-based initiation of the spontaneous arousal process.

Integrated metabolomics and proteomics analysis to understand muscle atrophy resistance in hibernating Spermophilus dauricus.

Hibernating Spermophilus dauricus experiences minor muscle atrophy, which is an attractive anti-disuse muscle atrophy model. Integrated metabolomics and proteomics analysis was performed on the hibernating S. dauricus during the pre-hibernation (PRE) stage, torpor (TOR) stage, interbout arousal (IBA) stage, and post-hibernation (POST) stage. Time course stage transition-based (TOR vs. PRE, IBA vs. TOR, POST vs. IBA) differential expression analysis was performed based on the R limma package. A total of 14 co-differential metabolites were detected. Among these, l-cystathionine, l-proline, ketoleucine, serine, and 1-Hydroxy-3,6,7-Trimethoxy-2, 8-Diprenylxanthone demonstrated the highest levels in the TOR stage; Beta-Nicotinamide adenine dinucleotide, Dihydrozeatin, Pannaric acid, and Propionylcarnitine demonstrated the highest levels in the IBA stage; Adrenosterone, PS (18:0/14,15-EpETE), S-Carboxymethylcysteine, TxB2, and 3-Phenoxybenzylalcohol demonstrated the highest levels in the POST stage. Kyoto Encyclopedia of Genes and Genomes pathways annotation analysis indicated that biosynthesis of amino acids, ATP-binding cassette transporters, and cysteine and methionine metabolism were co-differential metabolism pathways during the different stages of hibernation. The stage-specific metabolism processes and integrated enzyme-centered metabolism networks in the different stages were also deciphered. Overall, our findings suggest that (1) the periodic change of proline, ketoleucine, and serine contributes to the hindlimb lean tissue preservation; and (2) key metabolites related to the biosynthesis of amino acids, ATP-binding cassette transporters, and cysteine and methionine metabolism may be associated with muscle atrophy resistance. In conclusion, our co-differential metabolites, co-differential metabolism pathways, stage-specific metabolism pathways, and integrated enzyme-centered metabolism networks are informative for biologists to generate hypotheses for functional analyses to perturb disuse-induced muscle atrophy.

Hibernation-Like Behavior Induced by 2-Methyl-2-Thiazoline and Its Organ-Protective Effects and Mechanisms.

Hibernation is a prolonged state of low metabolism that animals enter in response to extreme environmental conditions to enhance their survival in harsh environments. Recent studies have shown that non-hibernating species can also be induced to enter a hibernation-like state. 2-methyl-2-thiazoline (2MT), a potent analog of fox odor, can induce fear-related behavior in mice with low body temperature and low metabolism, and has specific organ-protective effects. A systematic understanding of 2MT-induced hibernation and its underlying mechanisms may aid in expanding its applications in medicine and other fields.

The effects of nickel chloride on papillary muscle contractility under normothermic and hypothermic conditions: Comparison of active and hibernating ground squirrels (Urocitellus undulatus) with Wistar rats.

Extracellular Ca2+ plays a pivotal role in the regulation of cardiac contractility under normal and extreme conditions. Here, by using nickel chloride (NiCl2), a non-specific blocker of extracellular Ca2+ influx, we studied the input of extracellular Ca2+ on the regulation of papillary muscle (PM) contractility under normal and hypothermic conditions in ground squirrels (GS), and rats. By measuring isometric force of contraction, we studied how NiCl2 affects force-frequency relationship and the rest effect in PM of these species at 30 °C and 10 °C. We found that at 30 °C 1.5 mM NiCl2 significantly reduced force of contraction across entire frequency range in active GS and rats, whereas in hibernating GS force of contraction was reduced at low and high frequency range. Additionally, NiCl2 evoked spontaneous contractility in rats but not GS PM. The rest effect was significantly reduced by NiCl2 for active GS and rats but not hibernating GS. At 10 °C, NiCl2 fully reduced contractility in active GS and, to a lesser extent, in rats, whereas in hibernating GS it was significant only at 0.3 Hz. The rest effect was significantly reduced by NiCl2 in both active and hibernating GS, whereas it was unmasked in rats that had high contractility under hypothermic conditions in control. Our results show a significant contribution of extracellular Ca2+ to myocardial contractility in GS not only in active but also in hibernating states, especially under hypothermic conditions, whereas limitation of extracellular Ca2+ influx in rats under hypothermia can play protective role for myocardial contractility.

Body temperature and locomotor activity of social subterranean mole voles (Ellobius talpinus) in winter.

To find out whether a social subterranean rodent-the northern mole vole (Ellobius talpinus)-hibernates in winter, nine individuals from Southern Siberia were captured in late autumn and implanted with loggers [that constantly recorded body temperature (Tb) and locomotor activity] and then released. Eight of them were recaptured the following spring. From October to April, the animals' Tb never dropped below 33 °C, although cosinor analysis revealed a decrease in mesor values and in the amplitude of daily fluctuations of Tb and activity in winter months. Spectral density of circadian rhythms of both indexes also diminished in winter. The magnitude of Tb and fluctuations of activity differed between the two studied familial groups, probably due to their unequal numbers of individuals, which could affect the total heat production. The levels and patterns of temperature and activity fluctuations observed in winter rule out the possibility of hibernation in this species.

Mitochondrial Metabolism in Hibernation: Regulation and Implications.

Hibernators rapidly and reversibly suppress mitochondrial respiration and whole animal metabolism. Posttranslational modifications likely regulate these mitochondrial changes, which may help conserve energy in winter. These modifications are affected by reactive oxygen species (ROS), so suppressing mitochondrial ROS production may also be important for hibernators, just as it is important for surviving ischemia-reperfusion injury.

High latitude northern bats (Eptesicus nilssonii) reveal adaptations to both high and low ambient temperatures.

Insectivorous bats at northern latitudes need to cope with long periods of no food for large parts of the year. Hence, bats which are resident at northern latitudes throughout the year will need to undergo a long hibernation season and a short reproductive season where foraging time is limited by extended daylight periods. Eptesicus nilssonii is the northernmost occurring bat species worldwide and hibernates locally when ambient temperatures (Ta) limit prey availability. Therefore, we investigated the energy spent maintaining normothermy at different Ta, as well as how much bats limit energy expenditure while in torpor. We found that, despite being exposed to Ta as low as 1.1°C, bats did not increase torpid metabolic rate, thus indicating that E. nilssonii can survive and hibernate at low ambient temperatures. Furthermore, we found a lower critical temperature (Tlc) of 27.8°C, which is lower than in most other vespertilionid bats, and we found no indication of any metabolic response to Ta up to 37.1°C. Interestingly, carbon dioxide production increased with increasing Ta above the Tlc, presumably caused by a release of retained CO2 in bats that remained in torpor for longer and aroused at Ta above the Tlc. Our results indicate that E. nilssonii can thermoconform at near-freezing Ta, and hence maintain longer torpor bouts with limited energy expenditure, yet also cope with high Ta when sun exposed in roosts during long summer days. These physiological traits are likely to enable the species to cope with ongoing and predicted climate change.

Seasonal plasticity in the adult somatosensory cortex.

Seasonal cycles govern life on earth, from setting the time for the mating season to influencing migrations and governing physiological conditions like hibernation. The effect of such changing conditions on behavior is well-appreciated, but their impact on the brain remains virtually unknown. We investigate long-term seasonal changes in the mammalian brain, known as Dehnel's effect, where animals exhibit plasticity in body and brain sizes to counter metabolic demands in winter. We find large seasonal variation in cellular architecture and neuronal activity in the smallest terrestrial mammal, the Etruscan shrew, Suncus etruscus Their brain, and specifically their neocortex, shrinks in winter. Shrews are tactile hunters, and information from whiskers first reaches the somatosensory cortex layer 4, which exhibits a reduced width (-28%) in winter. Layer 4 width (+29%) and neuron number (+42%) increase the following summer. Activity patterns in the somatosensory cortex show a prominent reduction of touch-suppressed neurons in layer 4 (-55%), the most metabolically active layer. Loss of inhibitory gating occurs with a reduction in parvalbumin-positive interneurons, one of the most active neuronal subtypes and the main regulators of inhibition in layer 4. Thus, a reduction in neurons in layer 4 and particularly parvalbumin-positive interneurons may incur direct metabolic benefits. However, changes in cortical balance can also affect the threshold for detecting sensory stimuli and impact prey choice, as observed in wild shrews. Thus, seasonal neural adaptation can offer synergistic metabolic and behavioral benefits to the organism and offer insights on how neural systems show adaptive plasticity in response to ecological demands.

Sodium current preserves electrical excitability in the heart of hibernating ground squirrel (Citellus undulatus).

Hibernating mammals are capable of maintaining normal cardiac function at low temperatures. Excitability of cardiac myocytes crucially depends on the fast sodium current (INa), which is decreased in hypothermia due to both depolarization of resting membrane potential and direct negative effect of low temperature. Therefore, INa in hibernating mammals should have specific features allowing to maintain excitability of myocardium at low temperatures. The current-voltage dependence of INa, its steady-state inactivation and activation and recovery from inactivation were studied in winter hibernating (WH) and summer active (SA) ground squirrels and in rats using whole-cell patch clamp at 10 °C and 20 °C. INa peak amplitude and the parameters of steady-state activation and inactivation curves did not differ between SA and WH ground squirrels at both temperatures. However, at both temperatures strong positive shift of activation and inactivation curves by 5-12 mV was observed in both WH and SA ground squirrels if compared to rats. This peculiarity of cardiac INa in ground squirrels helps to maintain excitability in conditions of depolarized resting membrane potential. The time course of INa recovery from inactivation at 10 °C was faster in WH than in SA ground squirrels, which could ensure normal activation of myocardium during hibernation.

Bone adaptation and osteoporosis prevention in hibernating mammals.

Hibernating bears and rodents have evolved mechanisms to prevent disuse osteoporosis during the prolonged physical inactivity that occurs during hibernation. Serum markers and histological indices of bone remodeling in bears indicate reduced bone turnover during hibernation, which is consistent with organismal energy conservation. Calcium homeostasis is maintained by balanced bone resorption and formation since hibernating bears do not eat, drink, urinate, or defecate. Reduced and balanced bone remodeling protect bear bone structure and strength during hibernation, unlike the disuse osteoporosis that occurs in humans and other animals during prolonged physical inactivity. Conversely, some hibernating rodents show varying degrees of bone loss such as osteocytic osteolysis, trabecular loss, and cortical thinning. However, no negative effects of hibernation on bone strength in rodents have been found. More than 5000 genes in bear bone tissue are differentially expressed during hibernation, highlighting the complexity of hibernation induced changes in bone. A complete picture of the mechanisms that regulate bone metabolism in hibernators still alludes us, but existing data suggest a role for endocrine and paracrine factors such as cocaine- and amphetamine-regulated transcript (CART) and endocannabinoid ligands like 2-arachidonoyl glycerol (2-AG) in decreasing bone remodeling during hibernation. Hibernating bears and rodents evolved the capacity to preserve bone strength during long periods of physical inactivity, which contributes to their survival and propagation by allowing physically activity (foraging, escaping predators, and mating) without risk of bone fracture following hibernation. Understanding the biological mechanisms regulating bone metabolism in hibernators may inform novel treatment strategies for osteoporosis in humans.

Transcriptomic and proteomic time-course analyses based on Metascape reveal mechanisms against muscle atrophy in hibernating Spermophilus dauricus.

Hibernating Spermophilus dauricus is resistant to muscle atrophy. Comprehensive transcriptome and proteome time-course analyses based on Metascape can further reveal the underlying processes (pre-hibernation stage, PRE; torpor stage, TOR; interbout arousal stage, IBA; and post-hibernation stage, POST). Transcriptome analysis showed that the cellular responses to growth factor stimulus and discrete oxygen levels continuously changed during hibernation. Proteomic analysis showed that neutrophil degranulation, sulfur compound metabolic process, and generation of precursor metabolites and energy continuously changed during hibernation. Molecular complex detection (MCODE) analysis in both transcriptome and proteome indicated that smooth muscle contraction was involved in the POST versus IBA stage, and peroxisome proliferator-activated receptor delta (Ppard), Myc proto-oncogene (Myc), Sp1 transcription factor (Sp1), and nuclear factor Kappa B subunit 1 (NFκB1) are the common TFs during the hibernation process. Integrated transcriptome and proteome analyses found 18 molecules in the TOR versus PRE stage, 1 molecule in the IBA versus TOR stage, and 16 molecules in the POST versus IBA stage. Among these molecules, carnitine palmitoyltransferase 1A (Cpt1a), SET and MYND domain containing 2 (Smyd2), four and a half LIM domains 1(Fhl1), reactive oxygen species modulator 1 (Romo1), and translocase of the inner mitochondrial membrane 50 (Timm50) were testified by Western blot. In conclusion, novel muscle atrophy resistance mechanisms can be deciphered by time-course transcriptome and proteome analyses based on Metascape.

Bat-derived cells use glucose as a cryoprotectant.

Evolution of heterothermy in environments with variable temperatures has allowed bats to survive food scarcity during seasonal climatic extremes by using torpor as a hibernation strategy. The controlled reduction of body temperature and metabolism through complex behavioural and physiological adaptations at organismal, organ, cellular and molecular levels includes the ability of tissues and cells to adapt to temperature alterations. Based on the prediction that cells of different tissues cultured in vitro would differ in their ability to withstand freezing and thawing of the medium, we determined the survival rate of bat-derived cells following exposure to -20 °C for 24 h in media with no cryoprotective agents or medium supplemented by glucose in concentration range 0-3333 mM. Cell survival rates were determined in relation to availability of glucose in the medium, organ origin, cell concentration and bat species. In general, increased glucose helped cells survive at sub-zero temperatures, though concentrations up to 80-fold higher than those found in chiropterans were needed. However, cells in glucose-free phosphate buffered saline also survived, suggesting that other mechanisms may be contributing to cell survival at low temperatures. Highest in vitro viability was observed in nervus olfactorius-derived cell cultures, with high survival rates and rapid re-growth under optimal conditions after exposure to -20 °C. Kidney cells from different bat species showed comparable overall survival rate patterns, though smaller chiropteran species appeared to utilise lower glucose levels as a cryoprotectant than larger species. Our in vitro data provide evidence that cells of heterothermic bats can survive sub-zero temperatures and that higher glucose levels in important tissues significantly improve hibernation survival at extremely low temperatures.