Spermophilus Tridecemlineatus Research Articles

Deep sequencing the transcriptome reveals seasonal adaptive mechanisms in a hibernating mammal.

Mammalian hibernation is a complex phenotype involving metabolic rate reduction, bradycardia, profound hypothermia, and a reliance on stored fat that allows the animal to survive for months without food in a state of suspended animation. To determine the genes responsible for this phenotype in the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) we used the Roche 454 platform to sequence mRNA isolated at six points throughout the year from three key tissues: heart, skeletal muscle, and white adipose tissue (WAT). Deep sequencing generated approximately 3.7 million cDNA reads from 18 samples (6 time points ×3 tissues) with a mean read length of 335 bases. Of these, 3,125,337 reads were assembled into 140,703 contigs. Approximately 90% of all sequences were matched to proteins in the human UniProt database. The total number of distinct human proteins matched by ground squirrel transcripts was 13,637 for heart, 12,496 for skeletal muscle, and 14,351 for WAT. Extensive mitochondrial RNA sequences enabled a novel approach of using the transcriptome to construct the complete mitochondrial genome for I. tridecemlineatus. Seasonal and activity-specific changes in mRNA levels that met our stringent false discovery rate cutoff (1.0×10−11) were used to identify patterns of gene expression involving various aspects of the hibernation phenotype. Among these patterns are differentially expressed genes encoding heart proteins AT1A1, NAC1 and RYR2 controlling ion transport required for contraction and relaxation at low body temperatures. Abundant RNAs in skeletal muscle coding ubiquitin pathway proteins ASB2, UBC and DDB1 peak in October, suggesting an increase in muscle proteolysis. Finally, genes in WAT that encode proteins involved in lipogenesis (ACOD, FABP4) are highly expressed in August, but gradually decline in expression during the seasonal transition to lipolysis.

Mitochondrial metabolic suppression and reactive oxygen species production in liver and skeletal muscle of hibernating thirteen-lined ground squirrels

During hibernation, animals cycle between periods of torpor, during which body temperature (T(b)) and metabolic rate (MR) are suppressed for days, and interbout euthermia (IBE), during which T(b) and MR return to resting levels for several hours. In this study, we measured respiration rates, membrane potentials, and reactive oxygen species (ROS) production of liver and skeletal muscle mitochondria isolated from ground squirrels (Ictidomys tridecemlineatus) during torpor and IBE to determine how mitochondrial metabolism is suppressed during torpor and how this suppression affects oxidative stress. In liver and skeletal muscle, state 3 respiration measured at 37°C with succinate was 70% and 30% lower, respectively, during torpor. In liver, this suppression was achieved largely via inhibition of substrate oxidation, likely at succinate dehydrogenase. In both tissues, respiration by torpid mitochondria further declined up to 88% when mitochondria were cooled to 10°C, close to torpid T(b). In liver, this passive thermal effect on respiration rate reflected reduced activity of all components of oxidative phosphorylation (substrate oxidation, phosphorylation, and proton leak). With glutamate + malate and succinate, mitochondrial free radical leak (FRL; proportion of electrons leading to ROS production) was higher in torpor than IBE, but only in liver. With succinate, higher FRL likely resulted from increased reduction state of complex III during torpor. With glutamate + malate, higher FRL resulted from active suppression of complex I ROS production during IBE, which may limit ROS production during arousal. In both tissues, ROS production and FRL declined with temperature, suggesting ROS production is also reduced during torpor by passive thermal effects.

Multistate proteomics analysis reveals novel strategies used by a hibernator to precondition the heart and conserve ATP for winter heterothermy.

The hibernator's heart functions continuously and avoids damage across the wide temperature range of winter heterothermy. To define the molecular basis of this phenotype, we quantified proteomic changes in the 13-lined ground squirrel heart among eight distinct physiological states encompassing the hibernator's year. Unsupervised clustering revealed a prominent seasonal separation between the summer homeotherms and winter heterotherms, whereas within-season state separation was limited. Further, animals torpid in the fall were intermediate to summer and winter, consistent with the transitional nature of this phase. A seasonal analysis revealed that the relative abundances of protein spots were mainly winter-increased. The winter-elevated proteins were involved in fatty acid catabolism and protein folding, whereas the winter-depleted proteins included those that degrade branched-chain amino acids. To identify further state-dependent changes, protein spots were re-evaluated with respect to specific physiological state, confirming the predominance of seasonal differences. Additionally, chaperone and heat shock proteins increased in winter, including HSPA4, HSPB6, and HSP90AB1, which have known roles in protecting against ischemia-reperfusion injury and apoptosis. The most significant and greatest fold change observed was a disappearance of phospho-cofilin 2 at low body temperature, likely a strategy to preserve ATP. The robust summer-to-winter seasonal proteomic shift implies that a winter-protected state is orchestrated before prolonged torpor ensues. Additionally, the general preservation of the proteome during winter hibernation and an increase of stress response proteins, together with dephosphorylation of cofilin 2, highlight the importance of ATP-conserving mechanisms for winter cardioprotection.

Myostatin levels in skeletal muscle of hibernating ground squirrels

Myostatin, a negative regulator of muscle mass, is elevated during disuse and starvation. Mammalian hibernation presents a unique scenario, where animals are hypocaloric and in torpor, but the extent of muscle protein loss is minimized. We hypothesized that myostatin expression, which is usually increased early in disuse and under hypocaloric conditions, could be suppressed in this unique model. Skeletal muscle was collected from thirteen-lined ground squirrels, Spermophilus tridecemlineatus, at six time points during hibernation: control euthermic (CON); entrance into hibernation (ENT), body temperature (T(b)) falling; early hibernation (EHib), stable T(b) in torpor for 24 h; late hibernation (LHib), stable T(b) in torpor for 3 days; early arousal (EAr), T(b) rising; and arousal (AR), T(b) restored to 34-37°C for about 18 h. There was no significant increase of myostatin during ENT, EHib or LHib. Unexpectedly, there were approximately sixfold increases in myostatin protein levels as squirrels arose from torpor. The elevation during EAr remained high in AR, which represented an interbout time period. Mechanisms that could release the suppression or promote increased levels of myostatin were assessed. SMAD2 and phosphorylated SMAD2 were increased during EHib, but only the phosphorylated SMAD2 during AR mirrored increases in myostatin. Follistatin, a negative regulator of myostatin, did not follow the same time course as myostatin or its signaling pathway, indicating more control of myostatin at the signaling level. However, SMAD7, an inhibitory SMAD, did not appear to play a significant role during deep hibernation. Hibernation is an excellent natural model to study factors involved in the endogenous intracellular mechanisms controlling myostatin.

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.

Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) show microstructural bone loss during hibernation but preserve bone macrostructural geometry and strength

Lack of activity causes bone loss In most animals. Hibernating bears have physiological processes to prevent cortical and trabecular bone loss associated with reduced physical activity, but different mechanisms of torpor among hibernating species may lead to differences in skeletal responses to hibernation. There are conflicting reports regarding whether small mammals experience bone loss during hibernation. To investigate this phenomenon, we measured cortical and trabecular bone properties in physically active and hibernating juvenile and adult 13-lined ground squirrels (Ictidomys tridecemlineatus, previous genus name Spermophilus). Cortical bone geometry, strength and mineral content were similar in hibernating compared with active squirrels, suggesting that hibernation did not cause macrostructural cortical bone loss. Osteocyte lacunar size increased (linear regression, P=0.001) over the course of hibernation in juvenile squirrels, which may indicate an osteocytic role in mineral homeostasis during hibernation. Osteocyte lacunar density and porosity were greater (+44 and +59%, respectively; P<0.0001) in hibernating compared with active squirrels, which may reflect a decrease in osteoblastic activity (per cell) during hibernation. Trabecular bone volume fraction in the proximal tibia was decreased (-20%; P=0.028) in hibernating compared with physically active adult squirrels, but was not different between hibernating and active juvenile squirrels. Taken together, these data suggest that 13-lined ground squirrels may be unable to prevent microstructural losses of cortical and trabecular bone during hibernation, but importantly may possess a biological mechanism to preserve cortical bone macrostructure and strength during hibernation, thus preventing an increased risk of bone fracture during remobilization in the spring.

Seasonal liver protein differences in a hibernator revealed by quantitative proteomics using whole animal isotopic labeling

Hibernation is an energy-saving strategy used by diverse species of mammals to survive winter. It is characterized by cycles between multi-day periods of torpor with low body temperature (T b), and short periods of rapid, spontaneous rewarming. The ability to retain cellular integrity and function throughout torpor and rewarming is a key attribute of hibernation. Livers from winter hibernators are resistant to cellular damage induced by cold storage followed by warm reperfusion. Identifying proteins that differ between the summer-sensitive and winter-protected phenotypic states is one useful approach that may elucidate the molecular mechanisms that underlie this protection. Here we employ a novel quantitative proteomics screening strategy whereby a newly-weaned 13-lined ground squirrel was metabolically labeled by ingesting heavy-isotope substituted ( 15N) Spirulina. The liver protein extract from this animal provided a common reference for quantitative evaluation of protein differences by its addition to extracts from pooled samples of summer active (SA) or winter entrance (Ent) phase hibernating ground squirrels. We identified 61 significantly different proteins between the two groups and compared them to proteins identified previously in the same samples using 2D gels. Of the 20 proteins common to the two datasets, the direction and magnitude of their differences were perfectly concordant for 18, providing confidence that both sets of altered proteins reflect bona fide differences between the two physiological states. Furthermore, the 41 novel proteins recovered in this study included many new enzymes in pathways identified previously: specifically, additional enzymes belonging to the urea cycle, amino acid and carbohydrate degradation, and lipid biosynthetic pathways were decreased, whereas enzymes involved in ketone body synthesis, fatty acid utilization, protein synthesis and gluconeogenesis were increased in the samples from entrance hibernators compared to summer active animals, providing additional specific evidence for the importance of these pathways in the hibernating phenotype.

Cholecystokinin activation of central satiety centers changes seasonally in a mammalian hibernator

Hibernators that rely on lipids during winter exhibit profound changes in food intake over the annual cycle. The mechanisms that regulate appetite changes in seasonal hibernators remain unclear, but likely consist of complex interactions between gut hormones, adipokines, and central processing centers. We hypothesized that seasonal changes in the sensitivity of neurons in the nucleus tractus solitarius (NTS) to the gut hormone cholecystokinin (CCK) may contribute to appetite regulation in ground squirrels. Spring (SPR), late summer (SUM), and winter euthermic hibernating (HIB) 13-lined ground squirrels ( Ictidomys tridecemlineatus) were treated with intraperitoneal CCK (100 μg/kg) or vehicle (CON) for 3 h and Fos expression in the NTS was quantified. In CON squirrels, numbers of Fos-positive neurons in HIB were low compared to SPR and SUM. CCK treatment increased Fos-positive neurons in the NTS at the levels of the area postrema (AP) and pre AP during all seasons and at the level of the rostral AP in HIB squirrels. The highest absolute levels of Fos-positive neurons were found in SPR CCK squirrels, but the highest relative increase from CON was found in HIB CCK squirrels. Fold-changes in Fos-positive neurons in SUM were intermediate between SPR and HIB. Thus, CCK sensitivity falls from SPR to SUM suggesting that seasonal changes in sensitivity of NTS neurons to vagally-derived CCK may influence appetite in the active phase of the annual cycle in hibernating squirrels. Enhanced sensitivity to CCK signaling in NTS neurons of hibernators indicates that changes in gut–brain signaling may contribute to seasonal changes in food intake during the annual cycle.

Pulmonary Surfactant Membranes of Hibernating Ground Squirrels Possess Increased Fluidity but are Capable of Maintaining an Ordered Membrane Structure at Low Temperatures

Pulmonary surfactant, a mixture of lipids and proteins, regulates the surface tension at the air-liquid interface of the lung. Reduced body temperature during hibernation is accompanied in 13-lined ground squirrels (Ictidomys tridecemlineatus) by an increase in fluid monounsaturated phosphatidylcholine (PC) species (e.g. PC 16:0/16:1, PC 16:0/18:1) and phosphatidylglycerol (PG) species (e.g. PG 16:0/18:1, PG 18:0/18:2), but fewer disaturated PC and PG species. Previously we speculated that altered surfactant lipid composition during metabolic depression states such as torpor or hibernation will reduce the phase transition temperature (Tm) of the mixture, enabling pulmonary surfactant to remain fluid over a broader range of temperatures and thereby maintaining respiratory function. Here we analyze thermodynamic properties and behavior of surfactant from hibernating and summer-active 13-lined ground squirrels in relation to natural porcine surfactant, using differential scanning calorimetry and LAURDAN fluorescence spectroscopy. In addition we conducted epifluorescence studies to visualize changes in phase coexistence of surfactant films of hibernating and summer-active animals. Surfactant membranes of hibernators showed gel-to-fluid transitions at lower Tm with reduced enthalpy relative to membranes from summer-active squirrels. Both exhibited lower enthalpy than porcine surfactant. LAURDAN fluorescence and epifluoresence suggested possible structural re-arrangements of surfactant membrane lipids and films, respectively, in hibernators. These exhibited a similarly dehydrated and condensed highly packed ordered phase as for summer active squirrels, despite differences in composition and Tm. In conclusion, pulmonary surfactant composition changes in hibernating squirrels to increase overall fluidity, but to maintain an ordered membrane structure at low temperature.

Mitochondrial respiration and succinate dehydrogenase are suppressed early during entrance into a hibernation bout, but membrane remodeling is only transient

We examined respiration and lipid composition of liver mitochondria purified from a hibernator (Ictidomys tridecemlineatus) in different stages of a torpor bout. Between interbout euthermia (body temperature, T (b), 37°C) and early entrance (T (b) 30°C), state 3 and state 4 respirations, fueled by 6mM succinate, fell by over 50%. Mitochondrial respiration did not decline any further in the late entrance and torpor stages (T (b) 15 and 5°C, respectively). Succinate dehydrogenase (SDH) activity declined in a similar pattern as mitochondrial respiration, and there was a significant positive correlation between state 3 respiration and SDH activity. However, unlike during arousal from torpor, oxaloacetate was not a major factor in inhibition of SDH. Analysis of mitochondrial lipids showed little change in neutral lipids or phospholipid classes, except for a transient decrease in phosphatidylethanolamine content in early entrance. In the transition from interbout euthermia to early entrance, we found transient increases in some saturated phospholipid fatty acids (16:0, 18:0) and decreases in some unsaturates (18:2, 20:4). These changes resulted in transient increases in total saturates and the ratio of saturates to unsaturates, and transient decreases in total unsaturates, total polyunsaturates, total n-6, the ratio of monounsaturates to polyunsaturates, and unsaturation index. None of these changes persisted into late entrance or torpor, nor did they correlate with mitochondrial respiration. We conclude that mitochondrial metabolic suppression during entrance into a torpor bout occurs very early and is likely related to acute regulation of electron transport chain enzymes rather than changes in membrane phospholipid composition.

Changes in ventral respiratory column GABAaR ε- and δ-subunits during hibernation mediate resistance to depression by EtOH and pentobarbital

During hibernation in the 13-lined ground squirrel, Ictidomys tridecemlineatus, the cerebral cortex is electrically silent, yet the brainstem continues to regulate cardiorespiratory function. Previous work showed that neurons in slices through the medullary ventral respiratory column (VRC) but not the cortex are insensitive to high doses of pentobarbital during hibernation, leading to the hypothesis that GABA(A) receptors (GABA(A)R) in the VRC undergo a seasonal modification in subunit composition. To test whether alteration of GABA(A)R subunits are responsible for hibernation-associated pentobarbital insensitivity, we examined an array of subunits using RT-PCR and Western blots and identified changes in ε- and δ-subunits in the medulla but not the cortex. Using immunohistochemistry, we confirmed that during hibernation, the expression of ε-subunit-containing GABA(A)Rs nearly doubles in the VRC. We also identified a population of δ-subunit-containing GABA(A)Rs adjacent to the VRC that were differentially expressed during hibernation. As δ-subunit-containing GABA(A)Rs are particularly sensitive to ethanol (EtOH), multichannel electrodes were inserted in slices of medulla and cortex from hibernating squirrels and EtOH was applied. EtOH, which normally inhibits neuronal activity, excited VRC but not cortical neurons during hibernation. This excitation was prevented by bicuculline pretreatment, indicating the involvement of GABA(A)Rs. We propose that neuronal activity in the VRC during hibernation is unaffected by pentobarbital due to upregulation of ε-subunit-containing GABA(A)Rs on VRC neurons. Synaptic input from adjacent inhibitory interneurons that express δ-subunit-containing GABA(A)Rs is responsible for the excitatory effects of EtOH on VRC neurons during hibernation.

Land Use and Small Mammal Predation Effects on Shortgrass Prairie Birds

ABSTRACT Grassland birds endemic to the central shortgrass prairie ecoregion of the United States have experienced steep and widespread declines over the last 3 decades, and factors influencing reproductive success have been implicated. Nest predation is the major cause of nest failure in passerines, and nesting success for some shortgrass prairie birds is exceptionally low. The 3 primary land uses in the central shortgrass prairie ecoregion are native shortgrass prairie rangeland (62%), irrigated and nonirrigated cropland (29%), and Conservation Reserve Program (CRP, 8%). Because shortgrass–cropland edges and CRP may alter the community of small mammal predators of grassland bird nests, I sampled multiple sites on and near the Pawnee National Grasslands in northeast Colorado, USA, to evaluate 1) whether small mammal species richness and densities were greater in CRP fields and shortgrass prairie–cropland edges compared to shortgrass prairie habitats, and 2) whether daily survival probabilities of ground‐nesting grassland bird nests were negatively correlated with densities of small mammals. Small mammal species richness and densities, estimated using trapping webs, were generally greater along edges and on CRP sites compared to shortgrass sites. Vegetation did not differ among edges and shortgrass sites but did differ among CRP and shortgrass sites. Daily survival probabilities of artificial nests at edge and CRP sites and natural nests at edge sites did not differ from shortgrass sites, and for natural nests small mammal densities did not affect nest survival. However, estimated daily survival probability of artificial nests was inversely proportional to thirteen‐lined ground squirrel (Spermophilus tridecemlineatus) densities. In conclusion, these data suggest that although land‐use patterns on the shortgrass prairie area in my study have substantial effects on the small mammal community, insufficient data existed to determine whether land‐use patterns or small mammal density were affecting grassland bird nest survival. These findings will be useful to managers for predicting the effects of land‐use changes in the shortgrass prairie on small mammal communities and avian nest success.

Thomas George Lee – Implantation and early development of North American rodents

A century ago Thomas G. Lee amassed an unparalleled collection of developmental series of North American rodents such as the thirteen-lined ground squirrel, the Plains pocket gopher and Merriam’s kangaroo rat. He was the first to describe the initial attachment of the squirrel blastocyst to the antimesometrial side of the uterus. The full potential of Lee’s material was not realized until after his death, when it came into the possession of Mossman. The latter relied heavily on Lee’s collection when writing his seminal monograph on the comparative morphogenesis of fetal membranes and much of Lee’s material was subsequently described in detail by Mossman and others. It now forms part of the Harland W. Mossman Collection at the University of Wisconsin.

Circulation and metabolic rates in a natural hibernator: an integrative physiological model.

Small hibernating mammals show regular oscillations in their heart rate and body temperature throughout the winter. Long periods of torpor are abruptly interrupted by arousals with heart rates that rapidly increase from 5 beats/min to over 400 beats/min and body temperatures that increase by ∼30°C only to drop back into the hypothermic torpid state within hours. Surgically implanted transmitters were used to obtain high-resolution electrocardiogram and body temperature data from hibernating thirteen-lined ground squirrels (Spermophilus tridecemlineatus). These data were used to construct a model of the circulatory system to gain greater understanding of these rapid and extreme changes in physiology. Our model provides estimates of metabolic rates during the torpor-arousal cycles in different model compartments that would be difficult to measure directly. In the compartment that models the more metabolically active tissues and organs (heart, brain, liver, and brown adipose tissue) the peak metabolic rate occurs at a core body temperature of 19°C approximately midway through an arousal. The peak metabolic rate of the active tissues is nine times the normothermic rate after the arousal is complete. For the overall metabolic rate in all tissues, the peak-to-resting ratio is five. This value is high for a rodent, which provides evidence for the hypothesis that the arousal from torpor is limited by the capabilities of the cardiovascular system.

Expression of myocyte enhancer factor-2 and downstream genes in ground squirrel skeletal muscle during hibernation

Myocyte enhancer factor-2 (MEF2) transcription factors regulate the expression of a variety of genes encoding contractile proteins and other proteins associated with muscle performance. We proposed that changes in MEF2 levels and expression of selected downstream targets would aid the skeletal muscle of thirteen-lined ground squirrels (Spermophilus tridecemlineatus) in meeting metabolic challenges associated with winter hibernation; e.g., cycles of torpor-arousal, body temperature that can fall to near 0°C, long periods of inactivity that could lead to atrophy. MEF2A protein levels were significantly elevated when animals were in torpor (maximally 2.8-fold higher than in active squirrels) and the amount of phosphorylated active MEF2A Thr312 increased during entrance into torpor. MEF2C levels also rose significantly during entrance and torpor as did the amount of phosphorylated MEF2C Ser387. Furthermore, both MEF2 members showed elevated amounts in the nuclear fraction during torpor as well as enhanced binding to DNA indicating that MEF2-mediated gene expression was up-regulated in torpid animals. Indeed, the protein products of two MEF2 downstream gene targets increased in muscle during torpor (glucose transporter isoforms 4; GLUT4) or early arousal (myogenic differentiation; MyoD). Significant increases in Glut4 and MyoD mRNA transcript levels correlated with the rise in protein product levels and provided further support for the activation of MEF2-mediated gene expression in the hibernator. Transcript levels of Mef2a and Mef2c also showed time-dependent patterns with levels of both being highest during arousal from torpor. The data suggest a significant role for MEF2-mediated gene transcription in the selective adjustment of muscle protein complement over the course of torpor-arousal cycles.

Extensive use of torpor in 13-lined ground squirrels in the fall prior to cold exposure

Mammalian hibernation is characterized by profound reductions in body temperature (T(b)) and metabolic, heart and respiratory rates. These reductions are characteristic of torpor, which is temporally confined to winter. Hibernators including ground squirrels are heterothermic in winter, cycling between multiday periods of torpor with low T(b) and brief periods of rewarming. In contrast, ground squirrels remain homeothermic during summer, like non-hibernating mammals. The transition between the homeothermic and heterothermic phases of the circannual rhythm of hibernation is often overlooked in hibernation studies. Here, we examined the use of torpor throughout the fall transition in laboratory-housed 13-lined ground squirrels by recording core body temperature with an implanted data logger. As is typical of laboratory-based hibernation studies, animals were kept in standard housing prior to being moved into a cold, dark room to simulate natural hibernation conditions. Significantly, the vast majority of both male and female ground squirrels expressed torpor in the fall while still housed conventionally and prior to cold exposure. The expression of torpor was not predicted by body weight or age, rather it appears to be preprogrammed in a time-dependent manner that is independent of, yet enhanced by, environmental cues. The timing and duration of these torpor bouts occurring prior to cold exposure were also remarkably sporadic. Thus, it is not possible to know with certainty which animals are torpor-naive before cold exposure in the absence of continuous measurement of body temperature. We conclude that fall animals encompass variable points in the transition between summer and winter phases of the circannual cycle of hibernation, thereby confounding studies in which they are used as non-hibernating controls. Conversely, these fall transition animals offer unique opportunities to define the molecular changes that accompany and enable hibernation.

Conspecific Reaction to Playback of Thirteen-lined Ground Squirrel Vocalizations

Abstract Recent studies have revealed that in some ground squirrel species alarm calling behavior is most prevalent among individuals with nearby genetic relatives. A playback experiment was conducted to determine if the predator-elicited vocalization of the thirteen-lined ground squirrel, Spermophilus tridecemlineatus, is capable of evoking escape or watchful behavior in conspecifics. The responses of juveniles and adults to playback of three of their vocalizations, the trill, the peep, and the squeal, and to silent control trials, were observed. The results are discussed in terms of the proposed function of the alarm calling behavior of the species. Zusammenfassung Junge und erwachsene Streifenziesel (Spermophilus tridecemlineatus) reagierten auf arteigene Alarmrufe signifikant häufiger mit Männchen machen und Laufen zum Baueingang als auf in sozialen Beziehungen benutzte Laute, und sie unterbrachen laufende Aktivitäten häufiger. Diese Ergebnisse stimmen mit der Hypothese überein, daß der durch Räuber ausgelöste Ruf als Warnung wirkt.

Ground squirrels as an animal research model for eating and metabolic disorders

Thirteen‐lined ground squirrels (Spermophilus tridecemlineatus) “binge” and stash food leading up to hibernation and “restrict” and ration food during hibernation. This requires a dramatic adaptation to changing environmental conditions. We have studied the transcription of appetite regulating hormone receptors in brains by quantitative PCR, and plasma hormone levels by ELISA, in hibernating and non‐hibernating squirrels. Transcription of ghrelin and leptin receptor genes were suppressed 10‐fold, and plasma leptin and ghrelin levels (n=5/condition) were significantly reduced by 20–25% during hibernation. In contrast, melanocortin and cholecystokinin receptor transcription was elevated during hibernation. Our preliminary studies suggest differential transcriptional regulation and hormone levels of key central (brain) and peripheral (gut and adipose tissue) appetite and metabolism regulatory systems. These differences warrant further investigation and correlation with nutrient intake during different seasonal phases. Moreover, comparison of these regulatory systems in different squirrel states and comparable human dietary conditions (such as binging and restricting) may provide insight for effective points of behavioral, nutritional or pharmaceutical intervention. Funded by University of Wisconsin‐System, WiSys, and NSF‐REU grants.

Adipose tissue responses to binge and restrictive eating in ground squirrels

Fat cells provide peripheral feedback to the brain for appetite regulation. Adiponectin, leptin, and resistin are adipocyte hormones and PPARs are diet (fatty acid) sensitive transcription factors that modulate glucose and fatty acid metabolism. The 13‐lined ground squirrel (Spermophilus tridecemlineatus) is a natural model of binge and restrictive eating associated with hibernation and coincident alterations in metabolism. We have compared key regulators of metabolism and appetite, in adipose tissue from non‐hibernating versus hibernating ground squirrels, with quantitative PCR. Amplification crossing points were determined for each gene and normalized to that of actin. Preliminary studies revealed adiponectin and PPAR alpha transcription were up‐regulated 6 and 16‐fold, respectively, while PPAR gamma transcription was down‐regulated by 3‐fold during hibernation. Adiponectin results are consistent with observations by others that levels of this hormone are inversely related to fat mass, which would be predicted to decline during hibernation. Future studies will address dietary versus endogenous nutrient contributions to these observations. Funded by University of Wisconsin‐System and WiSys grants.

Lipid synthesis and distribution during the annual cycle of a hibernator

Although the seasonal shift from carbohydrate to lipid metabolism in hibernators is well established, a comprehensive study of lipid regulation in one species is lacking. To address this we studied triglyceride (TG) and lipoprotein metabolism in spring (SPR), summer (SUM) and hibernating (HIB) 13‐lined ground squirrels (5–6 per group). Plasma TGs are carried in VLDL particles in all seasons. There are no differences among the groups in ApoB mRNA in liver and gut or ApoB protein in liver and plasma. Fasting decreases plasma TGs 2‐fold in SPR (p&lt;0.05), but not in SUM. Plasma TGs are lower in torpid HIB (76.7±43.9 mg/dl) than interbout arousal (IBA) HIB (178.0±17.4 mg/dl), and both are less than SUM (278.2±44.4 mg/dl) (P&lt;0.05). Plasma free fatty acids (FFAs) are highest in torpid HIB, equal in SUM and IBA HIB (P&lt;0.001), and are not affected by fasting in SPR or SUM. Liver TGs are higher in torpid HIB than in SPR or IBA HIB (P&lt;0.05), but liver FFAs are highest in torpid HIB (P&lt;0.05). White adipose tissue (WAT) TGs do not vary among states, but WAT FFAs are lower in torpid HIB than SUM (P&lt;0.05). mRNA levels of the TG synthesis enzymes MGAT2 and DGAT2 are greater in HIB than SUM (P&lt;0.05). The results provide insight into how hibernating species regulate synthesis and distribution of lipids throughout the body during their annual cycle of feeding and fasting.