Auditory brainstem responses in ground squirrels arousing from hibernation

Summary Auditory brainstem responses (ABRs) were recorded in ground squirrels (Citellus lateralis) arousing from hibernation. Squirrels implanted with recording screws to record ABRs, and a thermistor to record brain temperature, were placed in a cold room at 9 °C on a 2L:22D light-dark cycle. Hibernating animals were moved from the cold room and ABRs recorded during arousal. The responses showed a gradual development of all brainstem peaks.At low temperatures there were very long latencies to the peaks. The amplitudes of the peaks increased (with fluctuations) as brain temperature increased. The data indicate that neural generators on the brainstem auditory pathway were all activated early in arousal.These results do not support the hypothesis that successive peaks appear and grow in amplitude only after previous peaks are fully developed.     

Daily rhythmicity and hibernation in the Anatolian ground squirrel under natural and laboratory conditions

We studied daily rhythmicity of body temperature (T _b) before and during hibernation in Anatolian ground squirrels (Spermophilus xanthoprymnus) under natural and laboratory conditions using surgically implanted temperature loggers. Under both conditions, robust daily T _b rhythmicity with parameters comparable to those of other ground squirrel species was observed before but not during hibernation. Euthermic animals had robust daily T _b rhythms with a mean of 37.0°C and a range of excursion of approximately 4°C. No T _b rhythm was detected during torpor bouts, either because T _b rhythmicity was absent or because the daily range of excursion was smaller than 0.2°C. The general patterns of hibernation that we observed in Anatolian ground squirrels were similar to those previously observed by other investigators in other species of ground squirrels.     

The serotonin-immunoreactive system of the suprachiasmatic nucleus in the hibernating ground squirrel,Spermophilus richardsonii

Summary In the suprachiasmatic nucleus (NSC) of hibernating and non-hibernating ground squirrels, the distribution of serotonin-immunoreactive (5HT-IR) fibers was studied by the use of the peroxidase-antiperoxidase technique. The cytology of perikarya giving rise to these suprachiasmatic 5HT-IR fibers was investigated in the anterior raphe nuclei. Differences in the immunoreactivity of suprachiasmatic fibers between hibernating and non-hibernating ground squirrels were determined by digital image analysis. The cellular activity was determined densitometrically after RNA-staining in anterior raphe neurons and suprachiasmatic perikarya. Abundant 5HT-IR fibers were observed in the medial and ventromedial portions of the NSC. Frequently, the fibers were found in close contact with perikarya of suprachiasmatic neurons. The central portion of the nucleus and the surrounding hypothalamic areas contained only a few scattered 5HT-IR fibers. Inside the raphe nuclei, 5HT-IR fibers and perikarya formed a dense network. In hibernating ground squirrels, the immunoreactivity to serotonin was approximately 45% higher than in non-hibernating controls. This difference is in accordance with signs of higher neuronal activity (40% higher RNA-content, 20% larger cell nuclei) in 5HT-IR perikarya of the raphe nucleus and the persisting activity of the NSC during hibernation; the activity of other brain regions dropped conspicuously in torpid animals.Supported by the Deutsche Forschungsgemeinschaft (Nu 36/2-1)     

Effects of hibernation on somatostatin-like immunoreactivity in the brain of the ground squirrel (Spermophilus richardsonii) and European hedgehog (Erinaceus europaeus)

Summary The localization of the somatostatin system in the brains of Richardson's ground squirrels (Spermophilus richardsonii) and European hedgehogs (Erinaceus europaeus) was described by use of immunocytochemical methods. In addition, (i) chemically differing types of somatostatin and (ii) different activity phases of the somatostatin system during the hibernation cycle were investigated in the ground squirrel by means of high pressure liquid chromatography (HPLC) and radioimmunoassay (RIA).In both species, the hypothalamic component of the somatostatin system (periventricular nuclei, fiber projections to the median eminence) is more prominent than the widespread extrahypothalamic representation of the system displaying mainly scattered perikarya and nerve fibers. The reactivity pattern of the somatostatin system varied among hibernating, aroused, and non-hibernating animals; moreover, the interspecific differences were pronounced. The activity of the hypothalamic somatostatin system in the hibernating ground squirrel appeared to be suppressed when compared to non-hibernating controls, whereas in the hibernating hedgehog this system showed signs of increased activity in comparison to non-hibernating controls. In contrast, in the present material the extrahypothalamic components of the somatostatin system did not exhibit significant changes in their activity.     

Genetic diversity and population divergence in fragmented habitats: Conservation of Idaho ground squirrels

The Idaho ground squirrel, which consists of a northern (Spermophilus brunneus brunneus) and a southern subspecies (S. b. endemicus), has suffered from habitat loss and fragmentation, resulting in a reduction in both numbers and geographic range of the species. The northern Idaho ground squirrel (NIDGS) is listed as a threatened subspecies under the Endangered Species Act, and the southern Idaho ground squirrel (SIDGS) is a candidate. Because Idaho ground squirrel populations are small and often isolated, they are susceptible to inbreeding and loss of genetic diversity through drift. This research evaluates levels of genetic diversity and patterns of population divergence in both subspecies of Idaho ground squirrels. We hypothesized that NIDGS would exhibit lower genetic diversity and greater population divergence due to a longer period of population isolation relative to most SIDGS populations. Genetic diversity and divergence were quantified using 8 microsatellite loci. Contrary to expectations, SIDGS populations exhibited consistently lower levels of microsatellite diversity. Additionally, NIDGS exhibited only modest divergence among populations, while divergence levels among SIDGS populations were highly varied. Preliminary evaluations of mitochondrial DNA diversity and structure revealed lower diversity in NIDGS and some differences in gene flow that warrant further study. Based on our results, we suggest different management strategies for the two subspecies. Habitat restoration appears to be the most desirable conservation strategy for NIDGS populations. In contrast, low genetic diversity observed in SIDGS may warrant supplementation of isolated populations through translocations or captive breeding to mitigate further loss of genetic variability.     

Hibernation enhancesd-glucose uptake by intestinal brush border membrane vesicles in ground squirrels

The ability to actively transport nutrients is maintained in intestinal tissues of hibernating ground squirrels compared with their active counterparts, and shows apparent upregulation in hibernators when transport rates are normalized to tissue mass. To identify the mechanisms responsible for the preservation of transport function during the extended fast of hibernation, we studiedd-glucose uptake into jejunal brush border membrane vesicles prepared from active and hibernating 13-lined ground squirrels. Hibernators were without food and showing regular bouts of torpor for at least 6 weeks before sacrifice. Electron micrographs indicated similar microvillus heights of jejunal enterocytes in the two activity states, whereas microvillus density was slightly greater in the hibernators. Glucose uptake into brush border membrane vesicles was inversely related to medium osmolarity, indicating negligible binding of substrate to brush border membrane vesicles surfaces, and intravesicular spaces were similar in hibernating and active squirrels. Glucose uptake showed strong Na⁺ dependency in both groups, with equivalent overshoot values in the presence of Na⁺. Kinetic analysis revealed a significant increase in the maximal velocity of transport (J _max) in hibernators (55.9±5.6 nmol·min^-1·mg^-1) compared with active squirrels (36.7±5.1 nmol·min^-1·mg^-1,P<0.05), with no change inK _m. Thus, the structure and absorptive capacity of the intestinal brush border persists in fasted hibernators, and the increase inJ _max for glucose uptake during hibernation likely contributes to the enhanced Na⁺-dependent glucose absorption previously observed at the tissue level.Abbreviations BBM brush border membrane(s) - BBMV brush border membranes vesicles - SGLT1 Na⁺-glucose transporter - 3-OMG 3-orthomethylglucose - J _max maximal velocity of transport - K _m transporter affinity for substrate - T _b body temperature     

Regulation of Akt during hibernation in Richardson's ground squirrels

Akt (or protein kinase B) plays a central role in coordinating growth, survival and anti-apoptotic responses in cells and we hypothesized that changes in Akt activity and properties would aid the reprioritization of metabolic functions that occurs during mammalian hibernation. Akt was analyzed in skeletal muscle and liver of Richardson's ground squirrels, Spermophilus richardsonii, comparing the enzyme from euthermic and hibernating states. Akt activity, measured with a synthetic peptide substrate, decreased by 60–65% in both organs during hibernation. Western blotting showed that total Akt protein did not change in hibernation but active, phosphorylated Akt (Ser 473) was reduced by 40% in muscle compared with euthermic controls and was almost undetectable in liver. Kinetic analysis of muscle Akt showed that S_0.5 values for Akt peptide were 28% lower during hibernation, compared with the euthermic enzyme, whereas S_0.5 ATP increased by 330%. Assay at 10 °C also elevated S_0.5 ATP of euthermic Akt by 350%. Changes in ATP affinity would limit Akt function in the hibernator since the muscle adenylate pool size is also strongly suppressed during cold torpor. Other parameters of euthermic and hibernator Akt were the same including activation energy calculated from Arrhenius plots and sensitivity to urea denaturation. DEAE Sephadex chromatography of muscle extracts revealed three peaks of Akt activity in euthermia but only two during hibernation suggesting isozymes are differentially dephosphorylated during torpor. Altered enzyme properties and suppression of Akt activity would contribute to the coordinated suppression of energy-expensive anabolic and growth processes that is needed to maintain viability during over weeks of winter torpor.     

Body temperature and behaviour of golden hamsters (Mesocricetus auratus) and ground squirrels (Spermophilus tridecemlineatus) in a thermal gradient

Golden hamsters and thirteen-lined ground squirrels were maintained individually in a thermal gradient (14°C to 33°C) for several weeks under a 14L: 10D light-dark cycle. Animals of both species showed robust daily rhythms of body temperature and locomotor activity with acrophases consistent with the habits of the species (diurnal acrophases in the diurnal squirrels and nocturnal acrophases in the nocturnal hamsters). Hamsters showed a robust daily rhythm of temperature selection 180° out of phase with the rhythms of body temperature and locomotor activity. Squirrels did not show a daily rhythm of temperature selection. These results raise the hypothesis that a daily rhythm of temperature selection is exhibited by nocturnal but not by diurnal endotherms.     

Mechanisms of glycolytic control during hibernation in the ground squirrel Spermophilus lateralis

Summary The mechanisms of glycolytic rate control during hibernation in the ground squirrel Spermophilus lateralis were investigated in four tissues: heart, liver, kidney, and leg muscle. Overall glycogen phosphorylase activity decreased significantly in liver and kidney to give 50% or 75% of the activity found in the corresponding euthermic organs, respectively. The concentration of fructose-2,6-bisphosphate (F-2,6-P₂) decreased significantly in heart and leg muscle during hibernation to 50% and 80% of euthermic tissue concentrations, respectively, but remained constant in liver and kidney. The overall activity of pyruvate dehydrogenase (PDH) in heart and kidney from hibernators was only 4% of the corresponding euthermic values. Measurements of phosphofructokinase (PFK) and pyruvate kinase (PK) kinetic parameters in euthermic and hibernating animals showed that heart and skeletal muscle had typical rabbit skeletal M-type PFK and M₁-type PK. Liver and kidney PFK were similar to the L-type enzyme from rabbit liver, whereas liver and kidney PK were similar to the M₂ isozyme found primarily in rabbit kidney. The kinetic parameters of PFK and PK from euthermic vs hibernating animals were not statistically different. These data indicate that tissue-specific phosphorylation of glycogen phosphorylase and PDH, as well as changes in the concentration of F-2,6-P₂ may be part of a general mechanism to coordinate glycolytic rate reduction in hibernating S. lateralis.Abbreviations ADP adenosine diphosphate - AMP adenosine monophosphate - ATP adenonine triphoshate - EDTA ethylenediaminetetra-acetic acid - EGTA ethylene glycol tetra-acetic acid - F-6-P fructose 6-phosphate - F-1,6-P₂ fructose 1,6-bisphosphate - F-2,6-P₂ fructose-2,6-bisphosphate - K _a activation coefficient - I₅₀ concentration of inhibitor which reduces control activity by 50% - PDH pyruvate dehydrogenase - PEP phosphoenolpyruvate - PFK 6-phosphofructo-1-kinase - PK pyruvate kinase     

Regulation of Akt during hibernation in Richardson's ground squirrels

Akt (or protein kinase B) plays a central role in coordinating growth, survival and anti-apoptotic responses in cells and we hypothesized that changes in Akt activity and properties would aid the reprioritization of metabolic functions that occurs during mammalian hibernation. Akt was analyzed in skeletal muscle and liver of Richardson's ground squirrels, Spermophilus richardsonii, comparing the enzyme from euthermic and hibernating states. Akt activity, measured with a synthetic peptide substrate, decreased by 60-65% in both organs during hibernation. Western blotting showed that total Akt protein did not change in hibernation but active, phosphorylated Akt (Ser 473) was reduced by 40% in muscle compared with euthermic controls and was almost undetectable in liver. Kinetic analysis of muscle Akt showed that S(0.5) values for Akt peptide were 28% lower during hibernation, compared with the euthermic enzyme, whereas S(0.5) ATP increased by 330%. Assay at 10 degrees C also elevated S(0.5) ATP of euthermic Akt by 350%. Changes in ATP affinity would limit Akt function in the hibernator since the muscle adenylate pool size is also strongly suppressed during cold torpor. Other parameters of euthermic and hibernator Akt were the same including activation energy calculated from Arrhenius plots and sensitivity to urea denaturation. DEAE Sephadex chromatography of muscle extracts revealed three peaks of Akt activity in euthermia but only two during hibernation suggesting isozymes are differentially dephosphorylated during torpor. Altered enzyme properties and suppression of Akt activity would contribute to the coordinated suppression of energy-expensive anabolic and growth processes that is needed to maintain viability during over weeks of winter torpor.     

Characteristics of auditory brainstem responses in ground squirrels

Summary Auditory brainstem responses (ABRs) were characterized at 37 °C in ground squirrels (Citellus lateralis) which were implanted with recording screws to record ABRs, and a thermistor to record brain temperature. After two weeks ground squirrels were reanesthetized and tone pips and clicks were delivered through a TDH-49 headphone.Recorded ABRs were found to vary in a predictable manner as a function of stimulus frequency and intensity. At intensities above 50 dB SPL, ABRs could be recorded over the range tested (2–32 kHz). An 8 kHz tone pip was the best frequency for recording ABRs at the lowest stimulus intensities. Latencies decreased as stimulus frequencies increased from 4 kHz to 32 kHz.     

Changes in renal morphology and renin secretion in the golden-mantled ground squirrel (Spermophilus lateralis) during activity and hibernation

Summary Chronological changes in renal glomerular morphology and plasma renin activity were investigated during active and hibernating periods in the golden-mantled ground squirrel Spermophilus lateralis. The objective of this study was to determine whether the glomerular endothelium, visceral epithelium (podocytes), basement membrane, mesangial cells, proximal convoluted tubule cells and plasma renin activity exhibit measurable sequential differences between as well as within active and hibernating states at various time points. Limitations in the size of the experimental population prevented an evaluation of changes in these parameters during other important periods such as periodic arousal between hibernation bouts. In this study, glomerular endothelial pore number and epithelial filtration slit number significantly decreased by early hibernation when compared to those during summer activity, and then they increased back toward summer levels by late hibernation. In contrast, podocytic pedicel width along the glomerular basement membrane increased from summer activity to early hibernation, before significantly decreasing again by late hibernation. Mesangial cell and proximal convoluted tubule cell activity appeared increased during hibernation as compared to summer activity, whereas the width of the glomerular basement membrane showed no significant alterations throughout. Plasma renin activity significantly increased during early hibernation and mid-hibernation when compared to summer levels but had decreased by late hibernation toward summer values. The glomerular and plasma renin activity changes observed in this study clearly illustrate the drastic structural and functional adjustments which hibernating species make during torpor and also correlate well with the reported decrease in renal perfusion pressure and urine formation during hibernation. The observed morphological changes during hibernation do not appear to be temperature-dependent, because significant alterations in most of the parameters studied occurred during this period despite the fact that cold-room temperatures were kept constant throughout. The chronological approach to this study and its morphometric evaluation represent a pilot attempt at accurately documenting these changes during two critical states in the hibernator's cycle and may eventually lead to the characterization of these changes during the entire circannual cycle.     

Normalization of aortic function during arousal episodes in the hibernating ground squirrel

Hypothermia is commonly used to restrict organ damage during preservation of tissue, but does not offer complete protection. Organ damage after reperfusion/rewarming is amongst others caused by an impairment of vascular properties, particularly endothelium-dependent vasodilatation. We hypothesized that hibernating small animals, which frequently cycle through periods of deep cooling (torpor) and full rewarming (arousal), employ specific mechanisms to preserve vascular function after cooling and reperfusion. Therefore we measured contraction of aortic tissue of hibernating European ground squirrels after 24 h and 7 days of torpor, arousal (1.5 h) and in non-hibernating animals. To assess the role of nitric oxide (NO), experiments were performed in the absence and presence of the NO-synthesis inhibitor, L-NMMA (10(-4) M). Maximum contraction to phenylephrine and angiotensin II was doubled in 7-days torpid animals without a shift in EC50, compared to the other 3 groups. Maximum contraction to KCl was doubled in 7-days torpid animals compared to the arousal group and non-hibernating animals. Relaxation to acetylcholine (ACh) and sodium nitrite in phenylephrine precontracted rings did not differ between groups. In the presence of L-NMMA, the maximum of concentration-response curves for all three vasoconstrictors was increased by about 30% in the arousal group, but unaffected in other groups. L-NMMA completely inhibited ACh-induced relaxation in 24-h torpid animals and non-hibernating animals, but only partially in 7-days torpid animals and in the arousal group. From this we conclude that vascular adaptation proceeds during torpor. Further, increased contractility of aortic tissue during long torpor returns to normal within 1.5 hours of arousal, which is associated with an increased basal NO synthesis. In addition, involvement of NO in agonist-mediated relaxation differs between the various stages of hibernation.Thus, hibernating animals have effectively developed mechanisms to preserve vascular function after cooling and rewarming.     

Global analysis of circulating metabolites in hibernating ground squirrels

Hibernation in mammals involves major alterations in nutrition and metabolism that would be expected to affect levels of circulating molecules. To gain insight into these changes we conducted a non-targeted LC–MS based metabolomic analysis of plasma using hibernating ground squirrels in late torpor (LT, T_b ~ 5 °C) or during an interbout arousal period (IBA, T_b ~ 5 °C) and non-hibernating squirrels in spring (T_b ~ 37 °C). Several metabolites varied and allowed differentiation between hibernators and spring squirrels, and between torpid and euthermic squirrels. Methionine and the short-chain carnitine esters of propionate and butyryate/isobutyrate were reduced in LT compared with the euthermic groups. Pantothenic acid and several lysophosphatidylcholines were elevated in LT relative to the euthermic groups, whereas lysophosphatidylethanolamines were elevated during IBA compared to LT and spring animals. Two regulatory lipids varied among the groups: sphingosine 1-phosphate was lower in LT vs. euthermic groups, whereas cholesterol sulfate was elevated in IBA compared to spring squirrels. Levels of long-chain fatty acids (LCFA) and total NEFA tended to be elevated in hibernators relative to spring squirrels. Three long-chain acylcarnitines were reduced in LT relative to IBA; free carnitine was also lower in LT vs. IBA. Our results identified several biochemical changes not previously observed in the seasonal hibernation cycle, including some that may provide insight into the metabolic limitations of mammalian torpor.     

The effect of temperature on the pattern of torpor in a marsupial hibernator

Physiological variables of torpor are strongly temperature dependent in placental hibernators. This study investigated how changes in air temperature affect the duration of torpor bouts, metabolic rate, body temperature and weight loss of the marsupial hibernator Burramys parvus (50 g) in comparison to a control group held at a constant air temperature of 2°C. The duration of torpor bouts was longest (14.0±1.0 days) and metabolic rate was lowest (0.033±0.001 ml O₂·g^-1·h^-1) at2°C. At higher air temperatures torpor bouts were significantly shorter and the metabolic rate was higher. When air temperature was reduced to 0°C, torpor bouts also shortened to 6.4±2.9 days, metabolic rate increased to about eight-fold the values at 2°C, and body temperature was maintained at the regulated minimum of 2.1±0.2°C. Because air temperature had such a strong effect on hibernation, and in particular energy expenditure, a change in climate would most likely increase winter mortality of this endangered species.Abbreviationst STP standard temperature and pressure - T _a air temperature - T _b body temperature - VO₂ rate of oxygen consumption