hibernation is a behavioural and physiological adaptation of endothermic animals which enhances survival during extended seasonal periods of reduced food supply and low ambient temperature when core body temperatures can be close to 0c .
the ability to hibernate is found throughout the class mammalia and involves differential expression of genes common to all mammals , rather than induction of novel gene products unique to hibernation .
most hibernators periodically interrupt the state of hibernation ( torpor ) by euthermic episodes or arousal , a process responsible for up to 90% of the energy consumed during hibernation . previously , using golgi and electron microscope studies in hippocampal tissue of hibernating ground squirrels
we have shown marked structural alterations in the components of neural circuitry , with the reversible retraction of dendritic spines and synapses in ca1 and ca3 [ 1 , 2 ] . with the exception of hibernators , mammals as a whole are much more sensitive to hypothermia , than are their individual constituent cells .
even though the optimum growth of mammalian cells occurs at 3537c , in culture many continue to grow and divide at 2533c , although substantially more slowly than at optimal body temperatures .
prolongation of the cell cycle at  suboptimal  ( 25c33c ) temperatures is attributed to a temporal expansion of the g1 and s phases , with g2 being the least sensitive . however , research into the process by which hypothermia affects the cell cycle has been largely neglected .
this is despite the fact that work on the response of mammalian cells to lower than optimal temperatures , first published in the mid 20th century , suggested several intriguing effects on cell cycle progression .
although the mechanisms involved remain largely unexplored , a sudden but severe cold shock can be used to generate a high degree of cell cycle synchrony in some mammalian cultures .
other hypothermic regimes can also be used to enrich cultures for g2 cells and to significantly slow otherwise rapid cellular processes , including mitosis . in this respect
hibernating mammals provide a rich resource to study the process of cell division and neurogenesis .
neurogenesis in the mammalians in hippocampus persists through adulthood mainly within the two neurogenic structures , the dentate gyrus of the hippocampus and the subventricular zone of the forebrain [ 58 ] . in these areas
neural progenitor cells continuously divide and give birth to new neurons [ 5 , 6 ] .
previous studies have demonstrated that behavioural and physiological stimuli , such as learning , voluntary wheel running exercise , kindling , and environmental enrichment , enhance hippocampal neurogenesis .
however , although the veracity of hippocampal neurogenesis is now generally accepted , there is uncertainty surrounding the extent of adult neurogenesis .
moreover , whether neurogenesis involves only progenitor cells or also more mature hippocampal cells is unclear and the absence of definitive phenotypic markers at ultrastructural level has limited the development of the field .  
investigations initiated in the 1970s by kaplan using h - thymidine light microscopic autoradiography and ultrathin sections at electron microscope level reexamined the initial observations and provided evidence not only that could neurogenesis occur in the adult brain , but also that the cells appear ultrastructurally , similar to sister cells in the dentate gyrus of the hippocampus , one of the structures shown to be neurogenic [ 1214 ] .
also , studies performed by gould and mcewen demonstrated that mitosis and apoptosis in hippocampus are regulated by adrenal steroids , possibly through excitatory amino acids which may also participate in the development and maintenance of the dentate gyrus . the siberian ground squirrel ( spermophilus undulatus ) which has adapted to survive under very low environmental temperatures and a short  summer season  is a unique animal for investigation of phenotypic metabolism changes [ 1618 ]
this is because there are 2 euthermic states : ( i ) a winter interbout state and ( ii ) summer animals , with typical features for nonhibernating rodents . in the present study we have utilised this natural hibernation model in the adult arctic ground squirrel to study mitosis at different stages of cell cycle .
our data suggest that immature cells exist in a mitotic state and may represent a renewable pool for generation of new neurons within the dentate gyrus .
adult ground squirrels , spermophilus undulatus , of both sexes and 600700  g in weight , estimated as between 2 and 3 years of age were caught in yakutiya ( siberia ) and kept in individual cages in a cold vivarium under natural photoperiodicity .
food was supplemented with sunflower seeds and carrots , and nesting material was provided ad libitum . in november , the animals were individually placed in wooden hibernation boxes ( 20  20  25  cm ) and transferred to a dark room having a temperature of   13c .
functional states were initially characterized by telemetry of heart rate ( hr ) and body temperature ( tb ) , by using a special transducer .
later , continuous monitoring of functional states was performed simply by recording the nest temperature . at the bottom of the wooden animal box , a thermistor ( sensitivity 0.2c )
the temperature of the bedding was in the range 14c , whereas in interbout events it increased to 1420c .
the course of torpor - activity cycles could thus be monitored in individual animals and allowed us to predict the length of a subsequent bout . 
samples were taken from animals killed in the predicted middle of a hibernation bout . in december ,
hibernation bouts had a 1-week duration , but this became 1.52 weeks in january - february .
hr was determined by using electrodes fixed onto the skin of the shoulder blade and the left foreleg .
directly after the start of perfusion procedure , the heart temperature ( th ) was determined with an electrical point thermometer ( sensitivity 0.2c ) .
the functional states were defined as follows : ( i ) deep torpor , in the middle of an extended bout of deep hibernation ( usually 3 to 4 days of hibernation bout ; brain temperature 24c ; hr 68 beats per min ) ; ( ii ) interbout ( usually at the second day after natural arousal ) , quasiactive animals , which had increased their brain temperature spontaneously to about 36c between two hibernation bouts ; s. undulatus relies on its fat depots and does not need food in these brief intervals of normothermic arousal ( hr 150300 beats per min ) ; ( iii ) 2.5 h provoked arousal : 22.5  h awakening from the middle of hibernation bout , provoked by transfer to a warm room . in the last case the provoked awakened ground squirrels had a tb of 34c ( hr 300400 beats per min ) .
three animals from each group received an intraperitoneal injection of brdu ( 5-bromo-2-deoxyuridine ; sigma ) in dose 150  ug / g body weight in sterile 0.9% nacl solution .
the animals were injected according to their group ( 3 - 4 animals per group ) as follows . 

using nest temperature monitoring ( as above ) 4 animals were chosen with interbout normothermic interval 2 days . 1 day before entrance in hibernation brdu injection was done in interbout / normothermic animals ( tb 35 - 36c ) .
after 3 days of hibernation the animals were perfused with solution temperature similar to tb 46c .
animals were sacrificed with an overdose of ketamine and perfused transcardially with 100  ml physiological saline in 0.01  m phosphate buffer ( ph 7.4 ) ( pbs ) , followed by 300  ml of 0.5% glutaraldehyde and 3% paraformaldehyde in 0.05  m cacodylate buffer ( ph 7.4 ) . the brains were stored in the fixative overnight .
all antibodies were diluted in 0.1  m phosphate buffer ( ph 7.4 ) containing 0.05% triton x-100 and 2% fish gelatin ( sigma ) ( incubation buffer ) .
the primary antibodies used in this study were monoclonal rat anti - brdu ( cat .
ab6326 , abcam , uk ) , 1  :  300 , and polyclonal rabbit anti - dcx ( cat .
ab18723 , abcam , uk ) , 1  :  700 . for immunohistochemistry with the peroxidase technique , biotinylated donkey anti - rat igg   ( cat .
712 - 065 - 150 , jackson , usa ) , 1  :  200 and biotinylated donkey antirabbit igg ( cat .
711 - 065 - 152 , jackson , usa ) , 1  :  100 , were used as secondary antibodies and detected with avidinbiotin - peroxidase complex ( abc , vectastain elite , vector laboratories , uk ) ( 9  l / ml ) .
after quenching excess of aldehyde groups with 1% sodium borohydride in pb for 30 minutes the 50  um sections were incubated in 2  m hcl for 30  min at 37c and washed in borate for 5 minutes .
the sections were blocked in incubation buffer ( ib ) for 2 hours , followed by incubation in primary antibody in ib overnight at 4c .
after rinses in pb , the sections were incubated in the secondary antibody in ib for 4 hours at room temperature .
after another set of rinses , abc elite reagent ( vector laboratories , uk ) was applied for 1 hour . as substrate for the peroxidase reaction ,
diaminobenzidine ( dab , sigma , usa ) was applied for 5 minutes at a concentration of 0.22  mg / ml in tris buffer ( ph 7.4 ) with 0.01% hydrogen peroxide .
sections were thoroughly washed , mounted , air - dried , dehydrated , and cover - slipped . to control for nonspecific labelling
, adjacent sections were incubated without primary or secondary antibody ; no labelling was detected following this procedure .
statistica ( statsoft , tulsa , okla , usa ) was used to obtain means sds to perform statistical analyses , and the kolmogorov - smirnov and shapiro - wilk normality tests to compare distributions ( criterion p < .05 ) . one - way anova test followed by bonferroni 's or tukey 's unequal n honest significant differences tests was performed with the originpro 7.5 .
figures 1(a ) , 1(b ) , 1(c ) and 1(d ) show the distribution of immature neurons with dcx - immunoperoxidase staining in the dorsal portion of dentate gyrus in the 4 different functional states as previously described in , which corresponds to the distribution of newborn neurons in this brain region observed using other markers for newly generated neurons [ 1923 ] .
the sgz appears discontinuous because dcx - labelled granule cells formed clusters [ 3 , 19 ] and the appearance of the labelled cells was similar to mature granule cells .
dcx - positive cells were mainly found in the sgz at the hilar border . labelled cells with branched apical dendrites
were found only in summer animals , and many had bifurcating and trifurcating processes ( figure 1(a ) ) .
comparative analysis of dcx - labelled dentate gyrus ( dg ) cells in summer ( figure 1(a ) ) , and hibernating animals ( figure 1(d ) ) , shows retraction of dendritic branches during hibernation supporting previous data on reversible retraction of dendritic branches .
the appearance of these dcx - labelled neurons was similar to that of dg cells .
it is notable that in summer , normothermic animals showed intensive labelling of neurons in layer ii of entorhinal cortex ( figure 1(e ) ) .
axons of neurons from layer ii of the perforant path input directly to the dg and both entorhinal cortex and dg participate in spatial memory .
these data suggest that immature neurons may persist into adulthood rodents and that these cells appear to undergo development and differentiation .
it is possible that these dcx immunoreactive neurons enable the high degree of plasticity of the neuronal network .
our study was designed only to show that mitotically active granule cells were localized in the dg .
theoretically brdu insertion occurs during dna repair and may not detect a distinct proliferative state .
however ; we did not observe brdu labelling of either pyramidal neurons or interneurons in different brain regions where there could be intensive dna repair .
hypothermia can stop the cell in any phase of the cell cycle , though mainly the g2 phase . for mitotically active intestinal epithelial cells during hibernation the mitotic index in the intestinal crypts of ground squirrels
entrance into hibernation stimulated suspension of the cells in different phases of the cell cycle .
figure 2 shows brdu immunoreactive cells in 3 different phases of mitosis : metaphase , anaphase , and telophase including ultrastructural patterns of metaphase immature granule neuron .
figure 3 shows that after telophase many brdu - labelled cells in the dg formed cellular doublets .
brdu - labelled cells such as those shown in figures 2 and 3 originate apparently from the dg layer supporting the idea   that mitosis occurs within dcx clusters . previously using serial ultrathin sections we found   that , within dcx - labelled clusters of immature granule cells , neurons are present during mitosis .
show interphase and metaphase granule cells where definitive markers are somatic synapses that are absent in mature granule cells .
uncovering such an event is exceptional because the cell cycle time is approximately 24.7 hours [ 25 , 27 ] whilst the duration of mitosis has been assessed as only 10% of total time of the cell cycle . by determining the length of the cell cycle for dividing cells and the total number of dividing cells
, it may be estimated that approximately 9,000 new cells are generated in the adult rat dentate gyrus each day .
the daily regulation of the hippocampal neurons , an apparent daily change in the number of s - phase cells , has been reported in the hilus .
thus , proliferation in the hilus seems to depend on the time of the day .
cell divisions in other mammalian tissues ( e.g. , tongue epithelium , intestinal epithelium , and skin ) are associated with specific periods in the day [ 2931 ] . in the hippocampus , it has been observed that m - phase cells show a clear day / night variation , with a significant increase during the night but the number of s - phase progenitors remains unchanged across the day .
it is therefore very difficult to demonstrate mitotic cells in dentate gyrus of adult rodents amongst more than 2 millions   granule cells .
however , in this connection hibernation of arctic ground squirrel presents unique model for accumulation and the study of different stages of cell cycle including mitosis .
our immunohistochemical studies of doublecortin- ( dcx ) and brdu - labelled cells in the dentate gyrus of the adult arctic ground squirrel hippocampus have demonstrated a population of immature cells which appear to exist in a mitotic state and may potentially represent a renewable pool for generation of new neurons within the dentate gyrus .
 neurogenesis occurs in the adult mammalian hippocampus , a region of the brain important for learning and memory . 
 hibernation in siberian ground squirrels provides a natural model to study mitosis as the rapid fall in body temperature in 24  h ( from 35 - 36c to + 46c ) permits accumulation of mitotic cells at different stages of the cell cycle . 
 histological methods used to study adult neurogenesis are limited largely to fixed tissue , and the mitotic state elucidated depends on the specific phase of mitosis at the time of day . however , using an immunohistochemical study of doublecortin ( dcx ) and brdu - labelled neurons , we demonstrate that the dentate gyrus of the ground squirrel hippocampus contains a population of immature cells which appear to possess mitotic activity . 
 our data suggest that doublecortin - labelled immature cells exist in a mitotic state and may represent a renewable pool for generation of new neurons within the dentate gyrus . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusion