The evaluation of the sizes of erythrocytes of the hibernating ground squirrel Citellus undulatus Pallas

A topographical image of individual erythrocytes of the ground squirrel Citellus undulatus Pallas, in unfixed unstained smears was first obtained by scanning probe microscopy for two states of the animal: hibernation and the active state. The scannig of single discocytes, i.e. erythrocytes having a typical discal form, was fulfilled. For the active male, the diameter of the discocyte was found to be approximately 6500 nm. For the hibernating female, the diameter is approximately 6000 nm. According to the data of light microscopy, the discocyte diameters are: 6610 +/- 100 nm for the active state of animal and 6430 +/- 160 nm for the hibernating state.     

Properties of C protein from skeletal and cardiac muscles of the ground squirrel Citellus undulatus at various stages of hibernation

Changes in the molecular weight and functional properties of the C and X proteins from skeletal muscles and the C protein from the cardiac muscle of hibernating ground squirrels Citellus undulatus at different stages of the hibernation were studied. A decrease in the molecular weight of the C protein from fast fibers of skeletal muscles of hibernating ground squirrels compared with awakening and active animals was revealed. The appearance of shorter molecules of the C protein upon hibernation was accompanied by a lowering of its capacity to enhance the actin-activated ATPase activity of control myosin and by the inhibition of its Ca(2+)-sensitivity. No similar changes were observed for the skeletal X protein and the cardiac C protein. The influence of the skeletal C protein on the main functional properties of myosin allows one to draw a conclusion about its contribution to the inhibition of contractile activity of skeletal muscles upon hibernation. The physiological significance of the changes in the C protein upon hibernation is discussed in connection with similar changes in some cardiomyopathies.     

Antioxidant defenses in the ground squirrel Citellus citellus. 2. The effect of hibernation

In spring and autumn, the ground squirrel Citellus citellus is awake and active but in winter it usually hibernates. Reawakening from hibernation involves intense metabolic activity in the interscapular brown adipose tissue (IBAT). The IBAT of hibernation animals showed significant increases in the activities of superoxide dismutase (both copper-zinc and manganese-containing enzymes), glutathione peroxidase, and in the amount of ascorbate present. Glutathione peroxidase also increased in the liver, as did ascorbate in the plasma. These changes were not merely a consequence of exposure to low environmental temperatures. It is proposed that antioxidant defenses are increased in the IBAT of ground squirrels at the onset of hibernation in order to protect the tissue from reactive oxygen species generated as a result of the intense metabolic activity sustained by this tissue during reawakening.     

The hypothalamo-hypophysial system in the ground squirrels, Citellus erythrogenys Brandt and Citellus undulatus Pallas. I. Microanatomy and cytomorphology of the gomori-positive neurosecretory system with special reference to its state during hibernation.

Microanatomy of the peptidergic hypothalamo-hypophysial neurosecretory system in the ground squirrels, C. erythrogenys and C. undulatus, is similar to that of other rodents, but differs in details. Bundles of neurosecretory fibres with single neurosecretory cells along their course connect the hypothalamic neurosecretory centers into the one system. The dorso-caudal portions of the NPV reach the level of the columna fornix. The NPO is a special indepedent formation and appears to have some specific functions in the HHNS. The infundibular recess penetrates deeply ihto the hypophysial stem, but does not reach the posterior pituitary. The glandular epithelium of the tuberal portions of the adenohypophysis surrounds the median eminence and a considerable part of the lateral surface of the tuber cinereum. Different functional types of neurosecretory cells reflect certain phases of their secretory cycle. Correlations between their size, structure and the content of NSM are found. A low activity of the HHNS is observed in both C. erythrogenys and C. undulatus during December-January. A higher activity of the HHNS in torpid C. undulatus than in C. erythrogenys is established. This correlates with a difference in deepness of torpor in C. erythrogenys and C. undulatus. An important role of the HHNS in hibernation, i.e. in realization of adaption of species in ontogenesis is suggested.     

Changes of activity of the protein-synthesizing system of brain neurons of the ground squirrel Citellus undulatus during hibernation and hypothermia

Using fluorescent and electron microscopy a comparative analysis was performed of components of the protein-synthesizing system of hippocampal neurons both in ground squirrels in various phases of the torpor-activity cycle and in rats cooled under the hypoxia-hypercapnia conditions. Results of the study have shown that in hippocampal neurons of the ground squirrels entering the natural torpor state and of rats under conditions of artificial hypothermia to 17°C, similar mechanisms might be possible to function, one of their obligatory components being a generalized decrease of activity of the protein-synthesizing system with its subsequent restoration at the exit from hypothermia. Cessation of hypoxia-hypercapnia even under conditions of a further temperature decrease restored the rat neuronal protein-synthesizing activity, which seems to indicate the presence of a potential possibility of adaptation of brain neurons in vivo to low temperatures, at which the integral organism of non-hibernating homoeothermic animals does not survive.     

Characteristics of force-frequency relations in the myocardium of the squirrel Citellus undulatus

The force-frequency relationship (FFR) in papillary muscles of the heart of active ground squirrel in different seasons was studied. For comparison, similar preparations from rat and rabbit were used. It was shown that the FFR of papillary muscles of active ground squirrel undergo significant seasonal changes. In summer and a part of autumn squirrels, a negative staircase (a decrease in the isometric force with increasing stimulation frequency) similar to that in adult rat was revealed. The FFR of the majority of autumn, winter and spring squirrels were polyphasic and contained both positive and negative components. Changes in the force in response to the introduction of pauses at a constant stimulation frequency were recorded. Two types of the post-rest recovery pattern were revealed in the myocardium of ground squirrels. For frequencies range with the negative direction of FFR, a typical pattern of rest-potentiation similar to that in rat papillary muscles was observed. The amplitude of the first post-rest contraction (F1) was usually higher than that of the preceding steady-state contraction. In papillary muscles of autumn animals the F1 value was greater that in summer, which suggests an enhanced release of Ca2+ from the sarcoplasmic reticulum. There was no post-rest potentiation in the range of frequencies with positive direction of FFR, and the post-rest recovery pattern in these cases was principally different from those of rat and rabbit preparations. It was proposed that seasonal differences of the FFR of active ground squirrel heart are associated with changes in the ratio of activities of the calcium-transporting system in the hibernation period.     

Analysis of changes in heart rate and temperature of the ground squirrel Citellus undulatus in various physiological states

Heart rate (HR) of ground squirrel C. undulatus was studied in dependence of season, level of activity, physiological state and air temperature (T). In summer HR varies from 110-130 beat/min in sleep up to 420 beat/min at flight from danger. During winter hibernation HP was minimal (3-5 beat/min) at T 1-4 degrees C, the increase in T induced the growth of HP in correspondence to the Arrenius van't Hoff law. The temperature of the body in hibernation exceeded T on 1.5-3 degrees C. The time of getting off the hibernation increased with the decrease in T (6-7 hours at -1 degree C and 2.5-3 hours at +18 degrees C). At phase of increased thermogenesis during arousal heart temperature exceeded rectal one on 10-12 degrees C and heart rate run up to 360-420 beat/min i.e. 2-3 time higher than in active state. The decrease in T stimulated the increase in HP up to 3.8 in winter and 5.3 beat/min/degree C in summer. The highest values of Q10 for HP were revealed at the beginning of hibernation (15-20) and at the beginning of arousal (6-7), in other periods Q10 was similar to the normal biological values (2-2.5). Thus, at the beginning of transitional periods changes of HP were determined mainly by endogenic mechanisms that inhibited myocardium at the beginning of hibernation and activated in arousal. Some mechanisms of coordination between activities of heart and other systems of organism are considered. The features of hit exchange providing the hibernation in wide range of T are discussed.     

The intestinal epithelial cells of ground squirrel (Citellus undulatus) accumulate at G2 phase of the cell cycle throughout a bout of hibernation

1. The mitotic index was found to be greatly reduced in the intestinal crypt cells of ground squirrel during bout of hibernation. The percentage of mitosis rose abruptly at least 2 hr after arousal. 2. An increase in the number of G2 cells was found in the intestinal tract of ground squirrel during bouts of hibernation. 3. The conclusion can be drawn that the cells are progressing steadily through the cell cycle. The cells accumulate at G2 in hibernation. 4. It was assumed that the block in G2 prevents the cells from possible damage in mitosis under hypothermia accompanying hibernation and, therefore, it represents an adaptive reaction.     

Induction of summer hibernation in the 13-lined ground squirrel, Citellus tridecemlineatus

The induction of summer hibernation in the 13-lined ground squirrel (Citellus tridecemlineatus) by intravenous injection of plasma obtained from winter hibernating ground squirrels was confirmed. Hibernation was also induced by injection of urine from arousing winter ground squirrels. Results support the “trigger” theory of hibernation proposed by Dawe and Spurrier (3) and also suggest that tissues are set free from “trigger” influence during winter arousal by the excretion of “trigger.”     

Dynamics of 3H-lysine incorporation into the retinal ganglion cells of the ground squirrel (Citellus pygmaeus Pallas) in active state and during hibernation

Circahoralian rhythm of the intensity of 3H-lysine incorporation into proteins of the retina ganglionic cells and of the protein synthesis rate in the isolated retina with an amplitude of fluctuations of 35-36% of the mean has been found in active adult susliks. During hibernation, no circahoralian fluctuations of protein metabolism were recorded. Circahoralian rhythms of protein metabolism can serve as a marker of the tissue functional activity.     

Phosphorylation of myosin light chains and C-protein in the myocardium of hibernating ground squirrel Citellus undulatus

A comparative study was made of the extent of phosphorylation of myosin regulatory light chains and C-protein from the left ventricle of the hibernant ground squirrel Citellus undulatus during the periods of hibernation and activity. During hibernation, the light chains were found to be completely dephosphorylated. In active animals, the share of phosphorylated light chains averaged 40–45%. The extent of cardiac C-protein phosphorylation in hibernation was about twice higher than in the active state. Seasonal differences in phosphorylation of the two proteins of ground squirrel myocardium are discussed in the context of adaptation to 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.     

Thyroid hormone levels in the peripheral blood of the ground squirrel Citellus parryi during winter hibernation

In the Arctic ground squirrel Citellus parryi, thyroxine and triiodothyronine concentrations in the peripheral blood have been determined by RIA in various seasons and during a short hibernation period. It was shown that during hibernation (December-February), hormonal concentration is higher than during homoiothermal period (August-October). The highest concentration of the thyroid hormones was found in hibernating animals in January. The data obtained indicate that thyroid hormones are involved in control of hibernation of the species investigated.     

The behavior of titin and the proteins of its family from skeletal muscles of ground squirrel (Citellus undulatus) during hibernation and rats under conditions of simulated microgravity

By the use of SDS PAGE, the behavior of titin and MyBP-C in fast (m. psoas) as well as titin and MyBP-X in slow (m. soleus) muscles of ground squirrels (Citellus undulatus) during hibernation was compared with the behavior of titin and MyBP-X in rat m. soleus under conditions of simulated microgravity. A decrease in the amount of titin 1 and MyBP-C relative to that of myosin heavy chains by approximately 30% and approximately 40%, correspondingly, in muscles of hibernating and arousing ground squirrels was revealed in comparison with active animals. No differences in the relative amount of MyBP-X in m. soleus of hibernating, arousing and active ground squirrels were found. Under conditions of simulated microgravity, a decrease in the amount of titin 1 by approximately 2 times and MyBP-X by approximately1.5 times relative to that of myosin heavy chains in rat m. soleus was observed. By the method of SDS PAGE modified by us, an almost twofold decrease in the amount of short isovariants of the titin N2A isoform relative to that of myosin heavy chains was shown in muscles of hibernating and arousing ground squirrels, whereas no changes were found in the amount of long titin isovariants. The conditions of simulated microgravity resulted in a twofold decrease in the relative amount of both short and long titin isovariants in rat m. soleus. The results indicate that hibernating ground squirrels have an evolutionarily determined adaptive mechanism of selective degradation of fast muscle fibers and preservation or increase of slow fibers, as the most economic and energetically advantageous, with proteins typical of them. The microgravitation of nonhibernating animals (rats) leads to a non-selective degradation of MyBP-X and titin isovariants, which contributes to considerable atrophy of soleus fibers.     

Seasonal variations of DNA synthesis in intestinal epithelial cells of hibernating animals--I. DNA synthesis in intestinal epithelial cells of ground squirrel (Citellus undulatus) during deep hibernation

The conditions for obtaining crypt cells from ground squirrel small intestine were chosen which allow flow-through cytofluorometric analysis of the DNA synthesis of this tissue. DNA synthesis was found to be greatly reduced in the intestinal crypt cells of ground squirrel during deep hibernation in torpid animals, in animals during spontaneous arousals and in animals prevented from hibernation. The conclusion is made about endogenous control of the DNA synthesis in the cells of true hibernators.