Plasma lipoprotein cholesterol concentrations in the golden-mantled ground squirrel (Spermophilus lateralis): a comparison between pre-hibernators and hibernators.

1. The concentrations of total cholesterol (free cholesterol plus cholesteryl ester) in the plasma and in two lipoprotein fractions of golden-mantled ground squirrels (Spermophilus lateralis) were measured during pre-hibernation and compared to those values measured during hibernation. 2. Hibernating ground squirrels had significantly higher (P less than 0.005) very low density lipoprotein plus low density lipoprotein cholesterol (VLDL + LDL-C) concentrations than did pre-hibernating ground squirrels. 3. Hibernating squirrels additionally exhibited significantly higher (P less than 0.005) total plasma cholesterol concentration per high density lipoprotein cholesterol concentration (TPC/HDL-C) ratios than did pre-hibernating squirrels. 4. The significant differences in the lipoprotein cholesterol concentrations observed in this study suggest that lipoprotein metabolism in pre-hibernators was significantly different from that in hibernators and was a reflection of the marked biochemical and physiological adjustments these animals must undergo during their transition from pre-hibernation to hibernation.     

Membrane lipids and morphology of brain cortex synaptosomes isolated from hibernating Yakutian ground squirrel

Synaptosomes were isolated from Yakutian ground squirrel brain cortex of summer and winter hibernating animals in active and torpor states. Synaptosomal membrane cholesterol and phospholipids were determined. The seasonal changes of synaptosomal lipid composition were found. Synaptosomes isolated from hibernating Yakutian ground squirrel brain cortex maintained the cholesterol sphingomyelin, phosphatidylethanolamine, lysophosphatidylcholine, cardiolipin, phosphatidylinositol and phosphatidylserine contents 2.5, 1.8, 2.6, 1.8, 1.6, and 1.3 times less, respectively, and the content of phosphatidylcholine twice as much as the one in summer season. The synaptosomal membrane lipid composition of summer animals was shown to be markedly different from that as hibernating ground squirrels and non-hibernating rodents. It is believed that phenotypic changes of synaptosomal membrane lipid composition in summer Yakutian ground squirrel are the important preparation step for hibernation. The phosphatidylethanolamine content was increased in torpor state compared with winter-active state and the molar ratio of cholesterol/phospholipids in synaptosomal membrane of winter torpor ground squirrels was lower than that in active winter and summer animals. These events were supposed to lead to increase of the synaptosomal membrane fluidity during torpor. Synaptosomes isolated from torpor animals have larger sizes and contain a greater number of synaptic vesicles on the synaptosomal profile area. The synaptosomal membrane lipid composition and synaptosome morphology were involved in phenotypic adaptation of Yakutian ground squirrel to hibernation.     

The excitability of neurons of the medial septal area in brain slices from hibernating and active Citellus undulatus susliks]

Spontaneous and evoked neuronal activity of the medical septum-diagonal band complex (MS-DB) has been investigated in slices from the brain of hibernating and active ground squirrels, as well as guinea pigs. In all experimental groups, the majority of the MS-DB neurones exhibited high regular of rhythmic burst spontaneous activity which persisted even after synaptic blockade in half of the neuronal population. Under the same conditions, the activity of the surrounding structures was completely suppressed. The density of the spontaneously active neurones in the slices, as well as the mean frequency of discharges in the MS-DB of hibernating ground squirrels, were significantly higher than in active ground squirrels and guinea pigs. Stimulation of the medial forebrain bundle evoked initial suppression of the activity in the majority of MS-DB units; in many of them, the suppression was followed by a burst discharge. Neurones with background rhythmic burst activity always reacted by resetting the spontaneous bursts. In total, 50-60% of the MS-DB neurones in active ground squirrels and guinea pigs reacted by post-inhibitory bursts, whereas in hibernating animals these responses were observed nearly in all neurones. Threshold values of the stimulating current were lower in hibernating animals; the intraburst density of spikes was increased.     

The effect of medium tonicity on the rate of respiration and oxidative phosphorylation in liver mitochondria of active and hibernating ground squirrels

The rate of respiration and ATP synthesis in liver mitochondria (M) isolated from hibernating ground squirrels and incubated in the medium with normal tonicity (250 mosm) was shown to be considerably lower than the rate of respiration and ATP synthesis in liver M from active animals. The increase of the medium tonicity to 600 mosm simulated the state of M from hibernating animals, resulting in a decrease of the respiration rate of M from active ground squirrels. On the contrary, the decrease of the tonicity to 60 mosm caused the activation of the respiration and increase of the ATP synthesis in M from hibernating ground squirrels. Bromophenacylbromide (BPhB), an inhibitor of phospholipase A2, prevented the activation of the respiration of M from hibernating animals incubated in the medium with low tonicity. BPhB had practically no effect on the respiration of M from both hibernating and active ground squirrels as well as on the swelling of M in hypotonic medium. It was concluded that the activation of the respiration and increase of the ATP synthesis rate in M from hibernating ground squirrels incubated in the medium with low tonicity is related to the activation of phospholipase A2. It was assumed that decrease of phospholipase A2 activity and change in the lipid composition of mitochondrial membrane may be one of the reasons for inhibition of the respiration rate in M from hibernating ground squirrels.     

Ca2+ cycling in heart cells from ground squirrels: adaptive strategies for intracellular Ca2+ homeostasis

Heart tissues from hibernating mammals, such as ground squirrels, are able to endure hypothermia, hypoxia and other extreme insulting factors that are fatal for human and nonhibernating mammals. This study was designed to understand adaptive mechanisms involved in intracellular Ca(2+) homeostasis in cardiomyocytes from the mammalian hibernator, ground squirrel, compared to rat. Electrophysiological and confocal imaging experiments showed that the voltage-dependence of L-type Ca(2+) current (I(Ca)) was shifted to higher potentials in ventricular myocytes from ground squirrels vs. rats. The elevated threshold of I(Ca) did not compromise the Ca(2+)-induced Ca(2+) release, because a higher depolarization rate and a longer duration of action potential compensated the voltage shift of I(Ca). Both the caffeine-sensitive and caffeine-resistant components of cytosolic Ca(2+) removal were more rapid in ground squirrels. Ca(2+) sparks in ground squirrels exhibited larger amplitude/size and much lower frequency than in rats. Due to the high I(Ca) threshold, low SR Ca(2+) leak and rapid cytosolic Ca(2+) clearance, heart cells from ground squirrels exhibited better capability in maintaining intracellular Ca(2+) homeostasis than those from rats and other nonhibernating mammals. These findings not only reveal adaptive mechanisms of hibernation, but also provide novel strategies against Ca(2+) overload-related heart diseases.     

Content of DSIP, enkephalins and ACTH in some tissues of active and hibernating ground squirrels (Citellus suslicus)

Seasonal changes in the amount of DSIP-like material have been measured in extracts from the brain of Citellus suslicus. 2. Analysis of extracts from the brain of deeply sleeping ground squirrels shows that the level of enkephalin-like material reliably increases while that of ACTH-like material decreases. 3. It is concluded that the opiate brain activation system is among the factors participating in the induction of hibernation in ground squirrels.     

The hypometabolic effect of the blood plasma from a hibernating suslik Citellus undulatus. Neokyotorphin activates cardiac and respiratory activities in the suslik during arousal from hibernation]

The effect of intraabdominal injection of the blood serum from ground squirrels Citellus undulatus obtained at various stages of the baut has been studied on the level of oxygen consumption in albino mice. The strongest hypometabolic effect exhibited the blood plasma from animals at the beginning of the baut. The onset of baut was used in further experiments on C. undulatus for investigation of activating effects of neokiotorphin (Thr-Ser-Lys-Tyr-Arg) which was earlier found in the brain of the ground squirrel. It was shown that intraabdominal injection of this peptide to hibernating animals facilitates their arousal stimulating both the cardiac activity and respiration. The strongest effect was noted with neokiotorphin of [D-Ser] 2-modification which is more stable to proteolysis.     

Lipids of the liver microsomal fraction in the ground squirrel <Emphasis Type="Italic">Spermophilus undulatus</Emphasis> during hibernation

Winter sleep of the ground squirrel Spermophilus undulatus was accompanied by a 20% decrease in phospholipid content (µg phospholipid per 1 mg protein) in microsomal fractions of the liver as compared with summer-active squirrels. The phosphatidylcholine level (mol %) in hibernating squirrels was lower than in summer-active squirrels, and the content of sphingomyelin (mol %) during the torpor bout was higher than in winter- and summer-active squirrels. The cholesterol, fatty acid, monoglyceride, and diglyceride levels in the microsomal fraction of the liver were elevated during hibernation. Pronounced seasonal changes in the lipid/protein ratio implicate the lipids of the liver microsomal fraction in adaptation of the ground squirrel to hibernation.     

Temperature-independence of circannual variations in circadian rhythms of golden-mantled ground squirrels

In golden-mantled ground squirrels, phase angles of entrainment of circadian locomotor activity to a fixed light-dark cycle differ markedly between subjective summer and winter. A change in ambient temperature affects entrainment only during subjective winter when it also produces pronounced effects on body temperature (Tb). It was previously proposed that variations in Tb are causally related to the circannual rhythm in circadian entrainment. To test this hypothesis, wheel-running activity and Tb were monitored for 12 to 14 months in castrated male ground squirrels housed in a 14:10 LD photocycle at 21 degrees C. Animals were treated with testosterone implants that eliminated hibernation and prevented the marked winter decline in Tb; these squirrels manifested circannual changes in circadian entrainment indistinguishable from those of untreated animals. Both groups exhibited pronounced changes in phase angle and alpha of circadian wheel-running and Tb rhythms. Seasonal variation in Tb is not necessary for circannual changes in circadian organization of golden-mantled ground squirrels.     

Uridine uptake and RNA synthesis in the brain of torpid and awakened ground squirrels.

1. Uptake of [3H]uridine into the nucleotide precursor pool after intraventricular injection occurs with the same intensity in the brain of torpid and normothermic awakened ground squirrels. This indicates that the membrane uridine transporters and uridine kinases operate in the hibernator's brain in a hypothermia-tolerant way. 2. Utilization of the [3H]uridine pool for synthesis of the rapidly labelled RNA in the brain of torpid ground squirrels falls more than eight times against RNA labelling in the brain of the active animals between bouts of hibernation. 3. Two hours from the beginning of the artificially provoked awakening, RNA uridine incorporation in the brain of ground squirrels has risen 6.5 times. 4. Drastic changes in [3H]uridine RNA labelling under the stable uridine uptake exclude the precursors and energy supply as the main factors determining changes in intensity of the brain RNA synthesis in the different stages of hibernation.     

Role of lipids in the assembly of endoplasmic reticulum and dictyosomes in neuronal cells from the cerebral cortex of Yakutian ground squirrel (Citellus undulatus) during hibernation

Phospholipids and cholesterol were assayed in homogenates and microsomal fractions from the cerebral cortex of summer-active, winter-torpid, and winter-active Yakutian ground squirrels (Citellus undulatus). Ultrastructural analysis of both microsomal fraction and intact neurons was performed by serial ultramicrotomy. The levels of sphingomyelin (SM), phosphatidylserine (PS), and phosphatidylethanolamine (PEA) were decreased in homogenates from the cerebral cortex of winter ground squirrels compared with the summer-active animals, while the levels of phosphatidylcholine (PC) and cardiolipin (CL) were increased. The level of cholesterol was decreased in the cerebral cortex of winter-torpid animals compared with both winter-active and summer-active animals, and the level of total phospholipids was decreased in comparison to the summer-active animals. Three-dimensional reconstruction of serial membrane profiles displayed the microsomal fraction to be an interconnected system of cisterns and vesicles, which corresponds to endoplasmic reticulum and dictyosomes (Golgi stacks) of intact neurons. In winter the content of PC was increased in the microsomal fraction, while the contents of lysophosphatidylcholine (LPC), PS, phosphatidylinositol (PI), and SM were decreased. In winter-torpid animals compared with the winter-active ones the contents of total phospholipids, PEA, LPC, and cholesterol were decreased. As for the winter-active ground squirrels, their lipid contents did not differ from those in the summer-active animals, but LPC content was decreased. The changes in microsomal lipid contents in intact pyramidal neurons throughout the hibernation were accompanied by disassembly of dictyosomes and endoplasmic reticulum (ER), including the decomposition of polyribosomes to monosomes. The ultrastructural analysis of nucleoli, ER, and dictyosomes of both winter-active and torpid ground squirrels showed a direct correlation between the increasing contents of both cholesterol and total phospholipids (mainly PEA and LPC) in microsomes and the structural recovery of endoplasmic reticulum, Golgi stacks, and nucleoli in intact pyramidal neurons. A role of seasonal variations in lipid contents of brain cells in their adaptation to low temperature is discussed. We also propose an involvement of cholesterol in the activation of protein-synthesizing function of endoplasmic reticulum and Golgi stacks in intact neurons.     

Hibernation confers resistance to intestinal ischemia-reperfusion injury

The damaging effects of intestinal ischemia-reperfusion (I/R) on the gut and remote organs can be attenuated by subjecting the intestine to a prior, less severe I/R insult, a process known as preconditioning. Because intestines of hibernating ground squirrels experience repeated cycles of hypoperfusion and reperfusion, we examined whether hibernation serves as a model for natural preconditioning against I/R-induced injury. We induced intestinal I/R in either the entire gut or in isolated intestinal loops using rats, summer ground squirrels, and hibernating squirrels during natural interbout arousals (IBA; body temperature 37-39 degrees C). In both models, I/R induced less mucosal damage in IBA squirrels than in summer squirrels or rats. Superior mesenteric artery I/R increased MPO activity in the gut mucosa and lung of rats and summer squirrels and the liver of rats but had no effect in IBA squirrels. I/R in isolated loops increased luminal albumin levels, suggesting increased gut permeability in rats and summer squirrels but not IBA squirrels. The results suggest that the hibernation phenotype is associated with natural protection against intestinal I/R injury.     

EVOLUTIONARY RELATIONSHIPS AMONG COLONIES OF COLUMBIAN GROUND SQUIRRELS AS SHOWN BY MITOCHONDRIAL DNA

Mitochondrial DNA (mtDNA) from 71 Columbian ground squirrels (Spermophilus columbianus) collected in 12 locations in western Canada were assayed for restriction-site variation with 10 endonucleases. Five of these endonucleases revealed variant patterns, and the composite genotypes were used to develop a linear transformation series among the mtDNA genotypes. Two of the four clones had a wide distribution, while the remaining two clones were geographically restricted. The mtDNA of Columbian ground squirrels was also compared to two other species of Sciuridae: Richardson's ground squirrels (S. richardsonii) and Arctic ground squirrels (S. parryii). Calculation of divergences from fragment length and restriction-site data indicated that Arctic ground squirrels and Richardson's ground squirrels were more closely related to each other than either was to Columbian ground squirrels. The transformation series among clones within the Columbian ground squirrels was rooted using Richardson's and Arctic ground squirrels as out-groups. From these data, we conclude that the colonization by female founders of Columbian ground squirrel populations occurred after deglaciation along the eastern ranges of the Rocky Mountains, while colonies on the western ranges may have been present before extensive deglaciation occurred, having existed in refugia in northwestern Alberta.     

Opioids and hibernation. II. Effects of kappa opioid U69593 on induction of hibernation in summer-active ground squirrels by "hibernation induction trigger" (HIT)

A "Hibernation Induction Trigger" (HIT) isolated from plasma of winter-hibernating woodchucks induced hibernation in summer-active ground squirrels (Citellus tridecemlineatus). Effects of kappa opioid U69593 on the HIT-induced hibernation were examined. U69593 alone did not elicit marked behavioral alteration or hibernation in summer-active ground squirrels. U69593, however, antagonized hibernation induced by HIT in summer active ground squirrels. In the guinea pig ileum myenteric plexus-longitudinal muscle preparation, woodchuck HIT depressed the electrically-induced contraction. The depression was, however, neither reversed nor blocked by naloxone even when naloxone was used at high doses. This study demonstrates that kappa opioid, at least in the case of U69593, was unable to induce hibernation in the summer-active ground squirrels. The results also demonstrate that woodchuck HIT, like the bear HIT, did not act directly at opioid receptors. Together with our previous observation that naloxone blocked summer hibernation induced by HIT (Bruce et al., Life Sci.., this issue), it is tempting to suggest that HIT may not mediate its effects through kappa opioid receptors but may do so through other types of opioid receptors such as mu or delta. U69593 may antagonize HIT-induced hibernation as a mu or delta receptor antagonist.     

The state-dependent action of neuropeptides and monoamines on the background neuronal activity of the medial septal area in hibernating animals

Neuronal activity of the medial septal area (MS-DB) was recorded extracellularly in brain slices from two groups of Yakutian ground squirrels Citellus undulatus--hibernating (winter period) and actively waking (summer period). Effects of three neuropeptides identified in the brain of hibernators (TSKYR, TSKY, and DY) and of two monoamines (serotonin and noradrenaline) on spontaneous activity were analyzed. All neuropeptides reversibly changed the levels of the background activity, but in the hibernating ground squirrels (HGS) the level of reactivity (47-56%) was significantly higher than in the waking ground squirrels (WGS, 25-30%). Serotonin also showed some tendency to higher efficacy in the HGS. Only noradrenaline was equally effective and had absolutely dominating excitatory effect in both states, although the level of excitation in the HGS was higher. All other substances evoked excitatory and inhibitory effects in various proportions. Their distribution was state-dependent, the rate of development, intensity and duration of the effects were greater in the HGS. The experiments confirmed the data on higher excitability and reactivity of the septal neurons in the state of hibernation. It is suggested that the tested neuropeptides may participate in the control of hibernation.     

Effect of experimental ectoparasite control on bartonella infections in wild Richardson's ground squirrels

The purpose of this study was to investigate the role of ectoparasites in transmitting Bartonella infections in wild Richardson's ground squirrels (Spermophilus richardsonii). Richardson's ground squirrels were trapped, examined for fleas, and tested for Bartonella bacteremia once monthly, at six sites, from April to September 2004. After the initial trapping session in April, burrows at three sites were treated with deltamethrin insecticide. Richardson's ground squirrels trapped on treated sites were less likely to have fleas and had fewer fleas than squirrels on control sites in all months following treatment. We found no difference in the prevalence of Bartonella infections on control and treated sites in May, immediately following treatment; however, significantly fewer squirrels were infected with Bartonella on treated sites in June and July. We conclude that ectoparasites are a main route of transmission for Bartonella infections in Richardson's ground squirrels.     

Seasonal changes in the isoform composition of the myosin heavy chains in skeletal muscles of hibernating ground squirrels Spermophilus undulatus

Seasonal changes of the isoform composition of myosin heavy chains in skeletal muscles (m. triceps, m. longissimus dorsi, m. soleus, m. gastrocnemius, m. vastus lateralis) of hibernating ground squirrels Spermophilus undulatus were studied. Functional properties of myosin (the actin-activated ATPase activity and its Ca²⁺-sensitivity in vitro) were also examined. It was observed that the content of slow myosin heavy chain I isoform increased and the content of fast IIx/d isoform decreased in muscles of torpid ground squirrels and animals which are active in autumn and winter. In muscles of these animals the content of N2A-titin isoform decreased although the relative content of NT-titin isoform, observed in striated muscles of mammals in our previous experimental works, increased. Actin-activated ATPase activity and Ca²⁺-sensitivity of myosin isolated from skeletal muscles of torpid and interbout ground squirrels were found to reduce. The changes observed are discussed in the context of adaptation of skeletal muscles of ground squirrels to hibernation conditions.     

Evaluation of the role of AMP-activated protein kinase and its downstream targets in mammalian hibernation

Mammalian hibernation requires an extensive reorganization of metabolism that typically includes a greater than 95% reduction in metabolic rate, selective inhibition of many ATP-consuming metabolic activities and a change in fuel use to a primary dependence on the oxidation of lipid reserves. We investigated whether the AMP-activated protein kinase (AMPK) could play a regulatory role in this reorganization. AMPK activity and the phosphorylation state of multiple downstream targets were assessed in five organs of thirteen-lined ground squirrels (Spermophilus tridecemlineatus) comparing euthermic animals with squirrels in deep torpor. AMPK activity was increased 3-fold in white adipose tissue from hibernating ground squirrels compared with euthermic controls, but activation was not seen in liver, skeletal muscle, brown adipose tissue or brain. Immunoblotting with phospho-specific antibodies revealed an increase in phosphorylation of eukaryotic elongation factor-2 at the inactivating Thr56 site in white adipose tissue, liver and brain of hibernators, but not in other tissues. Acetyl-CoA carboxylase phosphorylation at the inactivating Ser79 site was markedly increased in brown adipose tissue from hibernators, but no change was seen in white adipose tissue. No change was seen in the level of phosphorylation of the Ser565 AMPK site of hormone-sensitive lipase in adipose tissues of hibernating animals. In conclusion, AMPK does not appear to participate in the metabolic re-organization and/or the metabolic rate depression that occurs during ground squirrel hibernation.     

Diabetes mellitus in the 13-lined ground squirrel (Citellus tridecemlineatus).

Spontaneous diabetes mellitus was diagnosed in six of 126, 13-lined ground squirrels, Citellus tridecemlineatus. Serum glucose values were significantly higher in the diabetic ground squirrels than in the non-diabetic ground squirrels, while serum insulin values of fasted diabetic squirrels were significantly lower than fasted nondiabetic ground squirrels. In addition, the classic diabetic signs of poly-dipsia, polyuria, glycosuria, ketonuria, polyphasia, and weight loss were present. The proportion of islet tissue to total pancreatic area in diabetic ground squirrels was less than 25% of that in the nondiabetic ground squirrels. Both the number and size of the islets of Langerhans in diabetic ground squirrels were less than those in nondiabetic ground squirrels.     

Biosynthesis of dolichyl pyrophosphate N-acetylglucosaminyl intermediates in liver microsomes from hibernating ground squirrels (Citellus citellus L.)

Dolichyl pyrophosphate N-acetyl[14C]glucosamine was synthesized after incubation of liver microsomes from hibernating ground squirrels with UDP-N-acetyl[14C )glucosamine. The radioactivity of glycolipid formed by liver microsomes from hibernating ground squirrels was about 2-fold greater than by liver microsomes from active animals. Addition of exogenous dolichyl phosphate to the incubation mixture increased the formation of dolichyl pyrophosphate N-acetyl[14C]glucosamine by microsomes from both active and hibernating ground squirrels about 6 times. Liver microsomes from hibernating ground squirrels converted dolichyl pyrophosphate N-acetyl[14C]glucosamine into dolichyl pyrophosphate N,N'-diacetyl[14C]chitobiose in the presence of unlabelled UDP-N-acetylglucosamine. This conversion was maximal at 1.0 M concentration of unlabelled UDP-N-acetylglucosamine. The level of dolichyl phosphate assessed by the level of dolichyl pyrophosphate N-acetylglucosamine formation was nearly 2 times greater in liver microsomes from hibernating ground squirrels than from active animals.