Peptidomic Analysis Reveals Seasonal Neuropeptide and Peptide Hormone Changes in the Hypothalamus and Pituitary of a Hibernating Mammal.

During the winter, hibernating mammals undergo extreme changes in physiology, which allow them to survive several months without access to food. These animals enter a state of torpor, which is characterized by decreased metabolism, near-freezing body temperatures, and a dramatically reduced heart rate. The neurochemical basis of this regulation is largely unknown. Based on prior evidence suggesting that the peptide-rich hypothalamus plays critical roles in hibernation, we hypothesized that changes in specific cell-cell signaling peptides (neuropeptides and peptide hormones) underlie physiological changes during torpor/arousal cycles. To test this hypothesis, we used a mass spectrometry-based peptidomics approach to examine seasonal changes of endogenous peptides that occur in the hypothalamus and pituitary of a model hibernating mammal, the thirteen-lined ground squirrel (Ictidomys tridecemlineatus). In the pituitary, we observed changes in several distinct peptide hormones as animals prepared for torpor in October, exited torpor in March, and progressed from spring (March) to fall (August). In the hypothalamus, we observed an overall increase in neuropeptides in October (pre-torpor), a decrease as the animal entered torpor, and an increase in a subset of neuropeptides during normothermic interbout arousals. Notable changes were observed for feeding regulatory peptides, opioid peptides, and several peptides without well-established functions. Overall, our study provides critical insight into changes in endogenous peptides in the hypothalamus and pituitary during mammalian hibernation that were not available from transcriptomic measurements. Understanding the molecular basis of the hibernation phenotype may pave the way for future efforts to employ hibernation-like strategies for organ preservation, combating obesity, and treatment of stroke.

Optogenetic induction of hibernation-like state with modified human Opsin4 in mice.

We recently determined that the excitatory manipulation of Qrfp-expressing neurons in the preoptic area of the hypothalamus (quiescence-inducing neurons [Q neurons]) induced a hibernation-like hypothermic/hypometabolic state (QIH) in mice. To control the QIH with a higher time resolution, we develop an optogenetic method using modified human opsin4 (OPN4; also known as melanopsin), a G protein-coupled-receptor-type blue-light photoreceptor. C-terminally truncated OPN4 (OPN4dC) stably and reproducibly induces QIH for at least 24 h by illumination with low-power light (3 µW, 473 nm laser) with high temporal resolution. The high sensitivity of OPN4dC allows us to transcranially stimulate Q neurons with blue-light-emitting diodes and non-invasively induce the QIH. OPN4dC-mediated QIH recapitulates the kinetics of the physiological changes observed in natural hibernation, revealing that Q neurons concurrently contribute to thermoregulation and cardiovascular function. This optogenetic method may facilitate identification of the neural mechanisms underlying long-term dormancy states such as sleep, daily torpor, and hibernation.

Seasonal changes in adenosine kinase in tanycytes of the Arctic ground squirrel (Urocitellus parryii).

Hibernation is a seasonal strategy to conserve energy, characterized by modified thermoregulation, an increase in sleep pressure and drastic metabolic changes. Glial cells such as astrocytes and tanycytes are the brain metabolic sensors, but it remains unknown whether they contribute to seasonal expression of hibernation. The onset of hibernation is controlled by an undefined endogenous circannual rhythm in which adenosine plays a role through the activation of the A1 adenosine receptor (A1AR). Seasonal changes in brain levels of adenosine may contribute to an increase in A1AR sensitivity leading to the onset of hibernation. The primary regulator of extracellular adenosine concentration is adenosine kinase, which is located in astrocytes. Using immunohistochemistry to localize and quantify adenosine kinase in Arctic ground squirrels' brain collected during different seasons, we report lower expression of adenosine kinase in the third ventricle tanycytes in winter compared to summer; a similar change was not seen in astrocytes. Moreover, for the first time, we describe adenosine kinase expression in tanycyte cell bodies in the hypothalamus and in the area postrema, both brain regions involved in energy homeostasis. Next we describe seasonal changes in tanycyte morphology in the hypothalamus. Although still speculative, our findings contribute to a model whereby adenosine kinase in tanycytes regulates seasonal changes in extracellular concentration of adenosine underling the seasonal expression of hibernation.

Characterization of Seasonal Pharmacokinetic Variability in Woodchucks.

The eastern woodchuck (Marmota monax) is a hibernating species extensively used as an in vivo efficacy model for chronic human hepatitis B virus infection. Under laboratory conditions, woodchucks develop a pseudohibernation condition; thus, the pharmacokinetics (PK) of small-molecule therapeutics may be affected by the seasonal change. The seasonal PK of four probe compounds were characterized over 12 months in seven male and nine female laboratory-maintained woodchucks. These compounds were selected to study changes in oxidative metabolism [antipyrine (AP)], glucuronidation [raltegravir (RTG)], renal clearance [lamivudine (3TC)], and hepatic function [indocyanine green (ICG)]. Seasonal changes in physiologic parameters and PK were determined. Seasonal body weight increases were ≥30%. Seasonal changes in body temperature and heart rate were <10%. The mean AP exposure remained unchanged from April to August 2017, followed by a significant increase (≥1.0-fold) from August to December and subsequent decrease to baseline at the end of study. A similar trend was observed in RTG and 3TC exposures. The ICG exposure remained unchanged. No significant sex difference in PK was observed, although female woodchucks appeared to be less susceptible to seasonal PK and body weight changes. Significant seasonal PK changes for AP, RTG, and 3TC indicate decreases in oxidative metabolism, phase II glucuronidation, and renal clearance during pseudohibernation. The lack of seasonal change in ICG exposure suggests there are no significant changes in hepatic function. This information can be used to optimize the scheduling of woodchuck studies to avoid seasonally driven variation in drug PK. SIGNIFICANCE STATEMENT: Woodchuck is a hibernating species and is commonly used as a nonclinical model of hepatitis B infection. Investigation of seasonal PK changes is perhaps of greater interest to pharmaceutical industry scientists, who use the woodchuck model to optimize the scheduling of woodchuck studies to avoid seasonally driven variation in drug PK and/or toxicity. This information is also valuable to drug metabolism and veterinary scientists in understanding woodchuck's seasonal metabolism and behavior under the pseudohibernation condition.

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.

CNGA3 acts as a cold sensor in hypothalamic neurons.

Most mammals maintain their body temperature around 37°C, whereas in hibernators it can approach 0°C without triggering a thermogenic response. The remarkable plasticity of the thermoregulatory system allowed mammals to thrive in variable environmental conditions and occupy a wide range of geographical habitats, but the molecular basis of thermoregulation remains poorly understood. Here we leverage the thermoregulatory differences between mice and hibernating thirteen-lined ground squirrels (Ictidomys tridecemlineatus) to investigate the mechanism of cold sensitivity in the preoptic area (POA) of the hypothalamus, a critical thermoregulatory region. We report that, in comparison to squirrels, mice have a larger proportion of cold-sensitive neurons in the POA. We further show that mouse cold-sensitive neurons express the cyclic nucleotide-gated ion channel CNGA3, and that mouse, but not squirrel, CNGA3 is potentiated by cold. Our data reveal CNGA3 as a hypothalamic cold sensor and a molecular marker to interrogate the neuronal circuitry underlying thermoregulation.

Perineuronal net expression in the brain of a hibernating mammal.

During hibernation, mammals like the 13-lined ground squirrel cycle between physiological extremes. Most of the hibernation season is spent in bouts of torpor, where body temperature, heart rate, and cerebral blood flow are all very low. However, the ground squirrels periodically enter into interbout arousals (IBAs), where physiological parameters return to non-hibernating levels. During torpor, neurons in many brain regions shrink and become electrically quiescent, but reconnect and regain activity during IBA. Previous work showed evidence of extracellular matrix (ECM) changes occurring in the hypothalamus during hibernation that could be associated with this plasticity. Here, we examined expression of a specialized ECM structure, the perineuronal net (PNN), in the forebrain of ground squirrels in torpor, IBA, and summer (non-hibernating). PNNs are known to restrict plasticity, and could be important for retaining essential connections in the brain during hibernation. We found PNNs in three regions of the hypothalamus: ventrolateral hypothalamus, paraventricular nucleus (PVN), and anterior hypothalamic area. We also found PNNs throughout the cerebral cortex, amygdala, and lateral septum. The total area covered by PNNs within the PVN was significantly higher during IBA compared to non-hibernating and torpor (P < 0.01). Additionally, the amount of PNN coverage area per Nissl-stained neuron in the PVN was significantly higher in hibernation compared to non-hibernating (P < 0.05). No other significant differences were found across seasons. The PVN is involved in food intake and homeostasis, and PNNs found here could be essential for retaining vital life functions during hibernation.

Sex-specific effects of food supplementation on hibernation performance and reproductive timing in free-ranging common hamsters.

Hibernation is characterized by reduced metabolism and body temperature during torpor bouts. Energy reserves available during winter play an important role for hibernation and some species respond to high energy reserves with reduced torpor expression. Common hamsters are food-storing hibernators and females hibernate for shorter periods than males, probably related to larger food stores. In this study, we provided free-ranging common hamsters with sunflower seeds shortly before winter and recorded body temperature using subcutaneously implanted data loggers. We compared hibernation patterns and body mass changes between individuals with and without food supplements and analysed reproductive onset in females. Supplemented males delayed hibernation onset, hibernated for much shorter periods, and emerged in spring with higher body mass than unsupplemented ones. Additional food did not affect hibernation performance in females, but supplemented females emerged earlier and preceded those without food supplements in reproductive onset. Thus, males and females differently responded to food supplementation: access to energy-rich food stores enabled males to shorten the hibernation period and emerge in better body condition, probably enhancing mating opportunities and reproductive success. Females did not alter hibernation patterns, but started to reproduce earlier than unsupplemented individuals, enabling reproductive benefits by an extended breeding period.

Adaptation of the FK506 binding protein 1B to hibernation in bats.

Hibernation is an adaptive strategy used by some animals to cope with cold and food shortage. The heart rate, overall energy need, body temperature, and many other physiological functions are greatly reduced during torpor but promptly return to normal levels upon arousal. The heartbeat of torpid bats can be hundreds fold lower than that of active bats, indicating that hibernating bats have a remarkable ability to control excitation-contraction coupling in cardiac muscle. FKBP1B (calstabin 2), a peptidyl-prolyl cis-trans isomerase, is critical for the regulation of excitation-contraction coupling. Whether FKBP1B is adapted to hibernation in bats is not known. Evolutionary analyses showed that the ω values of the Fkbp1b genes of 25 mammalian species are all less than 1, and amino acid sequence alignments revealed that FKBP1B proteins are highly conserved in mammals. The expression of the Fkbp1b gene was found to be elevated at both mRNA and protein levels in two distantly related bats (Rhinolophus ferrumequinum in Yinpterochiroptera and Myotis ricketti in Yangochiroptera) during torpor. Transcription factors such as YY1 and SPs were bioinformatically determined to have a higher binding affinity to the potential regulatory regions of Fkbp1b genes in hibernating than in non-hibernating mammals. This study provides new insights into the molecular evolution of Fkbp1b in adaptation to bat hibernation.

Effects of food store quality on hibernation performance in common hamsters.

Hibernating animals can adjust torpor expression according to available energy reserves. Besides the quantity, the quality of energy reserves could play an important role for overwintering strategies. Common hamsters are food-storing hibernators and show high individual variation in hibernation performance, which might be related to the quality of food hoards in the hibernacula. In this study, we tested the effects of food stores high in fat content, particularly polyunsaturated fatty acids (PUFAs), on hibernation patterns under laboratory conditions. Control animals received standard rodent pellets only, while in the other group pellets were supplemented with sunflower seeds. We recorded body temperature during winter using subcutaneously implanted data loggers, documented total food consumption during winter, and analysed PUFA proportions in white adipose tissue (WAT) before and after the winter period. About half of the individuals in both groups hibernated and torpor expression did not differ between these animals. Among the high-fat group, however, individuals with high sunflower seeds intake strongly reduced the time spent in deep torpor. PUFA proportions in WAT decreased during winter in both groups and this decline was positively related to the time an individual spent in deep torpor. Sunflower seeds intake dampened the PUFA decline resulting in higher PUFA levels in animals of the high-fat group after winter. In conclusion, our results showed that common hamsters adjusted torpor expression and food intake in relation to the total energy of food reserves, underlining the importance of food hoard quality on hibernation performance.

Effects of wintertime fasting and seasonal adaptation on AMPK and ACC in hypothalamus, adipose tissue and liver of the raccoon dog (Nyctereutes procyonoides).

The raccoon dog (Nyctereutes procyonoides) is a canid with autumnal fattening and passive wintering strategy. We examined the effects of wintertime fasting and seasonality on AMP-activated protein kinase (AMPK), a regulator of metabolism, and its target, acetyl-CoA carboxylase (ACC) on the species. Twelve farmed raccoon dogs (eleven females/one male) were divided into two groups: half were fasted for ten weeks in December-March (winter fasted) and the others were fed ad libitum (winter fed). A third group (autumn fed, eight females) was fed ad libitum and sampled in December. Total AMPK, ACC and their phosphorylated forms (pAMPK, pACC) were measured from hypothalamus, liver, intra-abdominal (iWAT) and subcutaneous white adipose tissues (sWAT). The fasted animals lost 32% and the fed 20% of their body mass. Hypothalamic AMPK expression was lower and pACC levels higher in the winter groups compared to the autumn fed group. Liver pAMPK was lower in the winter fasted group, with consistently decreased ACC and pACC. AMPK and pAMPK were down-regulated in sWAT and iWAT of both winter groups, with a parallel decline in pACC in sWAT. The responses of AMPK and ACC to fasting were dissimilar to the effects observed previously in non-seasonal mammals and hibernators. Differences between the winter fed and autumn fed groups indicate that the functions of AMPK and ACC could be regulated in a season-dependent manner. Furthermore, the distinctive effects of prolonged fasting and seasonal adaptation on AMPK-ACC pathway could contribute to the wintering strategy of the raccoon dog.

The effects of poly-unsaturated fatty acids on the physiology of hibernation in a South American marsupial, Dromiciops gliroides.

Many mammals hibernate, which is a profound lethargic state of several weeks or months during winter, that represents a transitory episode of hetherothermy. As with other cases of dormancy, the main benefit of hibernation seems to be energy saving. However, the depth and duration of torpor can be experimentally modified by the composition of food, especially by fattyacid composition. In eutherians, diets rich in unsaturated fatty acids (i.e., fatty acids with at least one double bond) lengthen torpor, reduce metabolism and permit hibernation at lower temperatures. Here we studied whether diets varying in fatty acid composition have an effect on the physiology of hibernation in a South American marsupial, Dromiciops gliroides. We designed a factorial experiment where thermal acclimation (two levels: natural versus constant temperature) was combined with diet acclimation: saturated (i.e., diets with high concentration of saturated fatty acids) versus unsaturated (i.e., diets with high concentration of unsaturated fatty acids). We measured energy metabolism in active and torpid individuals, as well as torpor duration, and a suite of 12 blood biochemical parameters. After a cafeteria test, we found that D. gliroides did not show any preference for a given diet. Also, we did not find effects of diet on body temperature during torpor, or its duration. However, saturated diets, combined with high temperatures provoked a disproportionate increase in fat utilization, leading to body mass reduction. Those animals were more active, and metabolized more fats than those fed with a high proportion of unsaturated fatty acids (="unsaturated diets"). These results contrast with previous studies, which showed a significant effect of fatty acid composition of diets on food preferences and torpor patterns in mammals.

Catestatin and orexin-A neuronal signals alter feeding habits in relation to hibernating states.

Hibernation is a physiological state that by putting vital biological processes at rest enables mammals to protect all organs, especially the brain against ischemic insults and reperfusion injuries. Earlier studies have highlighted the role of hypothalamic (HTH) sites like the periventricular nucleus (Pe) toward sleep-wake and cardiovascular activities of hibernators. In the present work, infusions of Pe with the orexigenic neuropeptide orexin-A (ORX-A) or the novel anti-obesity sympathoinhibitory neuroactive peptide catestatin (CST) have been correlated to differing feeding and motor behaviors in the facultative hibernating hamster Mesocricetus auratus. Behavioral observations showed that treatment with CST provided an anti-obesity activity via the reduction of food intake and body weight for all hibernating states, while ORX-A promoted orexigenic events during mainly the entrance phase. Moreover, hamsters treated with this neuropeptide during the entrance and the arousal hypertensive phases also featured elevated ORX 2 receptor (ORX2R) levels in the third layer of the parietal cortex and lateral HTH (LH), areas involved with feeding, motor plus sleep-wake rhythms. Conversely, ORX-A down-regulated ORX2Rs in the ventromedial (VMH) and supraoptic (SO) HTH nuclei that are associated with anorexigenic effects. Even CST induced mixed ORX2R expression patterns in mostly HTH areas like the evident down-regulation in LH along with the up-regulation in VMH and SO. Overall treatments, especially ORX-A+CST led to reduced neurodegenerative phenomena in HTH supporting their importance together with ORX2Rs in preserving hemodynamic activities, feeding and sleep-wake rhythms of this diencephalic station, which may supply useful therapeutic indications for treating cardiovascular disturbances linked with brain dysfunctions.

Regulation of mitochondrial bioenergetic function by hydrogen sulfide. Part II. Pathophysiological and therapeutic aspects.

Emerging work demonstrates the dual regulation of mitochondrial function by hydrogen sulfide (H2 S), including, at lower concentrations, a stimulatory effect as an electron donor, and, at higher concentrations, an inhibitory effect on cytochrome C oxidase. In the current article, we overview the pathophysiological and therapeutic aspects of these processes. During cellular hypoxia/acidosis, the inhibitory effect of H2 S on complex IV is enhanced, which may shift the balance of H2 S from protective to deleterious. Several pathophysiological conditions are associated with an overproduction of H2 S (e.g. sepsis), while in other disease states H2 S levels and H2 S bioavailability are reduced and its therapeutic replacement is warranted (e.g. diabetic vascular complications). Moreover, recent studies demonstrate that colorectal cancer cells up-regulate the H2 S-producing enzyme cystathionine ß-synthase (CBS), and utilize its product, H2 S, as a metabolic fuel and tumour-cell survival factor; pharmacological CBS inhibition or genetic CBS silencing suppresses cancer cell bioenergetics and suppresses cell proliferation and cell chemotaxis. In the last chapter of the current article, we overview the field of H2 S-induced therapeutic 'suspended animation', a concept in which a temporary pharmacological reduction in cell metabolism is achieved, producing a decreased oxygen demand for the experimental therapy of critical illness and/or organ transplantation.

Effect of dietary fatty acids on metabolic rate and nonshivering thermogenesis in golden hamsters.

Hibernating rodents prior to winter tend to select food rich in polyunsaturated fatty acids (PUFA). Several studies found that such diet may positively affect their winter energy budget by enhancing torpor episodes. However, the effect of composition of dietary fatty acids (FA) on metabolism of normothermic heterotherms is poorly understood. Thus we tested whether diets different in FA composition affect metabolic rate (MR) and the capacity for nonshivering thermogenesis (NST) in normothermic golden hamsters (Mesocricetus auratus). Animals were housed in outdoor enclosures from May 2010 to April 2011 and fed a diet enriched with PUFA (i.e., standard food supplemented weekly with sunflower and flax seeds) or with saturated and monounsaturated fatty acids (SFA/MUFA, standard food supplemented with mealworms). Since diet rich in PUFA results in lower MR in hibernating animals, we predicted that PUFA-rich diet would have similar effect on MR of normothermic hamsters, that is, normothermic hamsters on the PUFA diet would have lower metabolic rate in cold and higher NST capacity than hamsters supplemented with SFA/MUFA. Indeed, in winter resting metabolic rate (RMR) below the lower critical temperature was higher and NST capacity was lower in SFA/MUFA-supplemented animals than in PUFA-supplemented ones. These results suggest that the increased capacity for NST in PUFA-supplemented hamsters enables them lower RMR below the lower critical temperature of the thermoneural zone.

Homocysteine homeostasis and betaine-homocysteine S-methyltransferase expression in the brain of hibernating bats.

Elevated homocysteine is an important risk factor that increases cerebrovascular and neurodegenerative disease morbidity. In mammals, B vitamin supplementation can reduce homocysteine levels. Whether, and how, hibernating mammals, that essentially stop ingesting B vitamins, maintain homocysteine metabolism and avoid cerebrovascular impacts and neurodegeneration remain unclear. Here, we compare homocysteine levels in the brains of torpid bats, active bats and rats to identify the molecules involved in homocysteine homeostasis. We found that homocysteine does not elevate in torpid brains, despite declining vitamin B levels. At low levels of vitamin B6 and B12, we found no change in total expression level of the two main enzymes involved in homocysteine metabolism (methionine synthase and cystathionine ß-synthase), but a 1.85-fold increase in the expression of the coenzyme-independent betaine-homocysteine S-methyltransferase (BHMT). BHMT expression was observed in the amygdala of basal ganglia and the cerebral cortex where BHMT levels were clearly elevated during torpor. This is the first report of BHMT protein expression in the brain and suggests that BHMT modulates homocysteine in the brains of hibernating bats. BHMT may have a neuroprotective role in the brains of hibernating mammals and further research on this system could expand our biomedical understanding of certain cerebrovascular and neurodegenerative disease processes.

Seasonal and regional differences in gene expression in the brain of a hibernating mammal.

Mammalian hibernation presents a unique opportunity to study naturally occurring neuroprotection. Hibernating ground squirrels undergo rapid and extreme physiological changes in body temperature, oxygen consumption, and heart rate without suffering neurological damage from ischemia and reperfusion injury. Different brain regions show markedly different activity during the torpor/arousal cycle: the cerebral cortex shows activity only during the periodic returns to normothermia, while the hypothalamus is active over the entire temperature range. Therefore, region-specific neuroprotective strategies must exist to permit this compartmentalized spectrum of activity. In this study, we use the Illumina HiSeq platform to compare the transcriptomes of these two brain regions at four collection points across the hibernation season: April Active, October Active, Torpor, and IBA. In the cerebral cortex, 1,085 genes were found to be differentially expressed across collection points, while 1,063 genes were differentially expressed in the hypothalamus. Comparison of these transcripts indicates that the cerebral cortex and hypothalamus implement very different strategies during hibernation, showing less than 20% of these differentially expressed genes in common. The cerebral cortex transcriptome shows evidence of remodeling and plasticity during hibernation, including transcripts for the presynaptic cytomatrix proteins bassoon and piccolo, and extracellular matrix components, including laminins and collagens. Conversely, the hypothalamic transcriptome displays upregulation of transcripts involved in damage response signaling and protein turnover during hibernation, including the DNA damage repair gene RAD50 and ubiquitin E3 ligases UBR1 and UBR5. Additionally, the hypothalamus transcriptome also provides evidence of potential mechanisms underlying the hibernation phenotype, including feeding and satiety signaling, seasonal timing mechanisms, and fuel utilization. This study provides insight into potential neuroprotective strategies and hibernation control mechanisms, and also specifically shows that the hibernator brain exhibits both seasonal and regional differences in mRNA expression.

Regulatory mechanism of body temperature in the central nervous system during the maintenance phase of hibernation in Syrian hamsters: involvement of ß-endorphin.

We have shown previously that intracerebroventricular (icv) injection of naloxone (a non-selective opioid receptor antagonist) or naloxonazine (a selective µ1-opioid receptor antagonist) at the maintenance phase of hibernation arouses Syrian hamsters from hibernation. This study was designed to clarify the role of ß-endorphin (an endogenous µ-opioid receptor ligand) on regulation of body temperature (T(b)) during the maintenance phase of hibernation. The number of c-Fos-positive cells and ß-endorphin-like immunoreactivity increased in the arcuate nucleus (ARC) after hibernation onset. In contrast, endomorphin-1 (an endogenous µ-opioid receptor ligand)-like immunoreactivity observed on the anterior hypothalamus decreased after hibernation onset. In addition, hibernation was interrupted by icv injection of anti-ß-endorphin antiserum at the maintenance phase of hibernation. The mRNA expression level of proopiomelanocortin (a precursor of ß-endorphin) on ARC did not change throughout the hibernation phase. However, the mRNA expression level of prohormone convertase-1 increased after hibernation onset. [D-Ala2,N-MePhe4,Gly-ol5] enkephalin (DAMGO, a selective µ-opioid receptor agonist) microinjection into the dorsomedial hypothalamus (DMH) elicited the most marked T(b) decrease than other sites such as the preoptic area (PO), anterior hypothalamus (AH), lateral hypothalamus (LH), ventromedial hypothalamus and posterior hypothalamus (PH). However, microinjected DAMGO into the medial septum indicated negligible changes in T(b). These results suggest that ß-endorphin which synthesizes in ARC neurons regulates T(b) during the maintenance phase of hibernation by activating µ-opioid receptors in PO, AH, VMH, DMH and PH.

Distinct α subunit variations of the hypothalamic GABAA receptor triplets (αßγ) are linked to hibernating state in hamsters.

BACKGROUND: The structural arrangement of the γ-aminobutyric acid type A receptor (GABAAR) is known to be crucial for the maintenance of cerebral-dependent homeostatic mechanisms during the promotion of highly adaptive neurophysiological events of the permissive hibernating rodent, i.e the Syrian golden hamster. In this study, in vitro quantitative autoradiography and in situ hybridization were assessed in major hypothalamic nuclei. Reverse Transcription Reaction-Polymerase chain reaction (RT-PCR) tests were performed for specific GABAAR receptor subunit gene primers synthases of non-hibernating (NHIB) and hibernating (HIB) hamsters. Attempts were made to identify the type of αßγ subunit combinations operating during the switching ON/OFF of neuronal activities in some hypothalamic nuclei of hibernators. RESULTS: Both autoradiography and molecular analysis supplied distinct expression patterns of all α subunits considered as shown by a strong (p < 0.01) prevalence of α1 ratio (over total α subunits considered in the present study) in the medial preoptic area (MPOA) and arcuate nucleus (Arc) of NHIBs with respect to HIBs. At the same time α2 subunit levels proved to be typical of periventricular nucleus (Pe) and Arc of HIB, while strong α4 expression levels were detected during awakening state in the key circadian hypothalamic station, i.e. the suprachiasmatic nucleus (Sch; 60%). Regarding the other two subunits (ß and γ), elevated ß3 and γ3 mRNAs levels mostly characterized MPOA of HIBs, while prevalently elevated expression concentrations of the same subunits were also typical of Sch, even though this time during the awakening state. In the case of Arc, notably elevated levels were obtained for ß3 and γ2 during hibernating conditions. CONCLUSION: We conclude that different αßγ subunits are operating as major elements either at the onset of torpor or during induction of the arousal state in the Syrian golden hamster. The identification of a brain regional distribution pattern of distinct GABAAR subunit combinations may prove to be very useful for highlighting GABAergic mechanisms functioning at least during the different physiological states of hibernators and this may have interesting therapeutic bearings on neurological sleeping disorders.

[Comparative study of cardiac alternans of action potential duration in hypothermia in rabbits and ground squirrels].

Cardiac alternans is a promising predictor of sudden death, yet its role in the mechanism of hypothermic arrhythmia induction is unclear. We aimed to investigate the effect of hypothermia on spatial-temporal characteristics of repolarization pattern in the Langendorff-perfused hearts of summer active (SA, n = 6) and winter hibernating (WH, n = 7) ground squirrels Spermophilus undulatus and rabbits (n = 5), who were immobilized with the excitation-contraction uncoupler BDM (10 mM) and optically mapped using the voltage sensitive dye di-4-ANNEPS and CCD camera (128 x 128 pixels; 500 frames/sec). Action potential duration (APD) restitution was quantified over the posterior epicardial heart surface and estimated using Nolasco-Dahlen criterion. In rabbit hearts, hypothermia resulted in arrhythmogenic overshoots of APD alternans as well as increase of APD restitution curve steepness. In contrast, significant APD alternans were observed in SA hearts at 27 degrees C, and at 17 degrees C in WH hearts. Moreover, slope of APD restitution curve in ground squirrels hearts did not reached arrhythmogenic threshold (43 +/- 9 degrees and 39 +/- 5 degrees for SA and WH respectively). Our results demonstrate different resistance of hibernating and non-hibernating mammals against induction of arrhythmogenic cardiac alternans which is closely associated with adaptive changes in intracellular Ca2+ cycling during hibernation.