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*V. Naumenko, Laboratory of Behavioral Neurogenomics, Institute of Cytology and Genetics, Siberian Division of Russian Academy of Sciences, Lavrentyev Avenue 10, 630090 Novosibirsk, Russia. E-mail: firstname.lastname@example.org
Hibernation is a unique physiological state characterized by profound reversible sleep-like state, depression in body temperature and metabolism. The serotonin 5-hydroxytryptamine1A (5-HT1A) receptor gene sequence in typical seasonal hibernator, ground squirrel (Spermophilus undulatus), was specified. It was found that the fragment encoding the fifth transmembrane domain showed 93.6% of homology with the analogous fragment of the mouse and rat genes and displayed 88.5% homology with the human 5-HT1A receptor gene. Using primers designed on the basis of obtained sequence, the expression of 5-HT1A receptor gene in the brain regions in active, entering into hibernation, hibernating and coming out of hibernation ground squirrels was investigated. Significant structure-specific changes were revealed in the 5-HT1A messenger RNA (mRNA) level in entry into hibernation and in arousal. An increase in the 5-HT1A gene expression was found in the hippocampus during the prehibernation period and in ground squirrels coming out of hibernation, thus confirming the idea of the hippocampus trigger role in the hibernation. Significant decrease in 5-HT1A receptor mRNA level in the midbrain was found in animals coming out of hibernation. There was no considerable changes in 5-HT1A receptor mRNA level in different stages of sleep–wake cycle in the frontal cortex. Despite drastically decreased body temperature in hibernating animals (about 37°C in active and 4–5°C in hibernation), 5-HT1A receptor mRNA level in all examined brain regions remained relatively high, suggesting the essential role of this 5-HT receptor subtype in the regulation of hibernation and associated hypothermia.
Mammalian hibernation represents a unique model for studies of the mechanisms regulating physiological systems, behavior and natural tolerance to ischemia and hypothermia. The brain mechanisms that are used by hibernators to make the transition from normal, euthermic state to hibernation characterized by profound alteration of body temperature are of particular interest. In the hibernating ground squirrel, body temperature decreases to near ambient, sometimes as low as 1.5–3°C, and there is a concomitant reduction (up to 1–5% of euthermic rate) in heart, respiratory and metabolic rates (Barnes 1989; Popova 1986). The brain of a hibernating mammal withstands physiological extremes that are mortal for nonhibernators. The identification of the genes and the neurotransmitter mechanisms underlying this natural adaptive behavior is the key first step toward understanding the brain plasticity and central mechanism for survival.
The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) has been implicated in various physiological functions, including sleep (Jouvet 1969) and thermoregulation (Popova & Konusova 1985). It has been shown previously that entry into and coming out of hibernation were associated with changes in the brain 5-HT metabolism and level. The increase in brain 5-HT level and in the activity of key enzyme in 5-HT biosynthesis, tryptophan hydroxylase, in the midbrain, hippocampus and striatum (Popova et al. 1993), and the decrease in monoamine oxidase A (MAO A) activity and 5-HT catabolism in the brain in ground squirrels entering into hibernation (Popova & Voitenko 1981; Semenova et al. 2004) were shown. The most pronounced changes were revealed in hippocampus, where the increase of tryptophan hydroxylase activity and 5-HT level preceded entry into hibernation (Popova et al. 1993). Opposite changes were found in brain of animals coming out of hibernation. The arousal was associated with an increase in MAO A activity, a decrease in 5-HT level and a rise of its metabolite, 5-hydroxyindoleacetic acid. The data suggested that brain 5-HT was involved as an inhibitory factor in the control of hibernation. This hypothesis was confirmed by the experiments showing that the decrease in brain 5-HT level as a result of midbrain raphe nuclei lesion or pretreatment with inhibitor of 5-HT biosynthesis p-CPA evoked the arousal from hibernation (Popova et al. 1978; Spafford & Pengelley 1971), whereas 5-HT precursor 5-hydroxytryptophan administered into brain ventricle III prolonged the time taken for arousal (Popova & Kudryavtseva 1985).
In the past decades, 14 different subtypes of 5-HT receptors were described, which were subdivided into seven families based on operational (drug related), transductional (receptor coupling) and structural (primary amino acid sequence) characteristics. All 5-HT receptors except one (5-HT3 type) are metabotropic G protein-coupled receptors; structurally and functionally distinct from all the other 5-HT receptor types, the 5-HT3 receptor is ionotropic ligand-gated ion-channel receptor (Barnes & Sharp 1999).
Among the amazing variety of cloned and identified 5-HT receptors, particular attention has focused on the 5-HT1A receptor because of the data on its involvement in the control of anxiety (Heisler et al. 1998; Nutt & Glue 1991), sleep (Derry et al. 2006; Wilson et al. 2005) and hypothermia (Hjorth 1985; Overstreet et al. 1996), suggesting that 5-HT1A receptor can take part in the maintenance of deep hypothermia and torpor typical for hibernation. At the same time, there was no data on the brain 5-HT1A receptors in sleep–waking cycle as well as there was no information on the 5-HT1A receptor gene in hibernators.
The aim of the present work was to investigate the expression of 5-HT1A receptor gene in the brain of hibernating ground squirrels in diverse stages of the sleep–waking cycle. There was no data on the 5-HT1A receptor gene sequences in ground squirrel in the databases, so to determine the expression of the receptor, the first stage of our research was the determination of the sequence of 5-HT1A receptor gene in the ground squirrel.
Materials and methods
The experiments were carried out on male ground squirrels (Spermophilus undulatus) weighing 600–800 g. Animals were trapped in Yakutia at the end of August. In the Institute of Biophysics, Russian Academy of Sciences, Pushchino, ground squirrels were under veterinary observation in the special vivarium. Animals were individually kept in the 35 × 40 × 20 cm cages with free access to food and water. The light was switched on from 0700 h to 1900 h. In the end of October, animals were placed in a special chamber with constant temperature approximating natural conditions (+4°C). Experiments were performed on four groups of ground squirrels (10 animals in each group), decapitated in the different stages of sleep–waking cycle: (1) in July – active animals (body temperature 37°C); (2) in October – prepared to hibernation but yet euthermic animals (body temperature 37°C); (3) in January – hibernating animals (body temperature 4°C) and (4) in April – animals coming out of hibernation (body temperature 28°C). Arousal was provoked by transferring animals to laboratory room with an ambient temperature of 21–22°C. Body temperature was measured in the colon with an electric thermometer.
After a rapid decapitation, brains were removed on ice and then the frontal cortex, hippocampus and midbrain were isolated. The cortical samples from symmetrical areas of the frontal regions bilaterally (Schober 1986), all of right hippocampus, and caudal part of the brain stem, including n. raphe dorsalis and n. raphe medianus (Konig & Klippel 1963), were taken. The brain structures were immediately frozen in liquid nitrogen and stored at −65°C until RNA extraction.
All experimental procedures were made in compliance with the Guidelines for Ethical Conduct in the Care and Use of Animals (developed by Committee on Animal Research and Ethics, 1991; http://www.apa.org/science/anguide.html).
Reverse transcriptase–polymerase chain reaction and sequencing
The total RNA was isolated using extraction with guanidine thiocyanate, phenol and chloroform (Chomczynski & Sacchi 1987) with modifications (Naumenko & Kulikov 2006). The obtained total RNA was stored in 70% ethanol at −20°C for transferring to the Institute of Cytology and Genetics, Novosibirsk, and then total RNA was precipitated, dried and dissolved in diethyl pyrocarbonate-treated water to concentration of 0.125 μg/μl and stored at −65°C.
The reverse transcription was performed as described elsewhere (Naumenko & Kulikov 2006). The synthesized complementary DNA (cDNA) was stored at −20°C.
The polymerase chain reaction (PCR) was carried out using primers directed to the murine 5-HT1A receptor gene according to the following conditions: (1) 5 min at 94°C, 1 cycle, (2) 40 seconds at 94°C, 40 seconds at 59°C and 40 seconds at 72°C, 36 cycles and (3) 4 min at 72°C, 1 cycle (Table 1).
Table 1. Nucleotide sequences and characteristics of the primers used to amplify fragments of the 5-HT1A receptor and β-actin
Annealing temperature (°C)
PCR product size (bp)
F, forward; R, reverse.The primers directed to the mouse 5-HT1A receptor genes were designed on the basis of published sequences (Charest et al. 1993), using the European Molecular Biology Laboratory Nucleotide database, especially for estimation of this receptor gene sequence in the ground squirrels. The primers directed to the β-actin gene were as reported previously (Zamorano et al. 1996). All primers used in this work were synthesized by Biosan (Novosibirsk, Russia).
Murine 5-HT1A receptor gene
Ground squirrels 5-HT1A receptor gene
F 5′- tactccactttcggcgctttctatat-3′
R 5′- cagtggcaggtgctctttggagtt-3′
The PCR products were separated by electrophoresis in 6% polyacrylamide gel (acrylamide/bisacrylamide ratio = 30:1) in Tris–acetate buffer. The gel was stained with ethidium bromide. Then the bands corresponded to the expected size of the PCR product of 5-HT1A receptor [602 base pair (bp)] were cut out. The target PCR product was extracted by passive elution and reamplified with 65 pmol of both direct and reverse primers under the conditions given above. Purification of the obtained PCR products was carried out by gel filtration with Sefadex G-100 superfine resin.
For sequencing, the same primer pair that used earlier for the PCR was taken. We mixed 500 fmol of purified PCR product with 2 pmol of corresponding direct or reverse primer and 3 μl of ABI Prism® dGTP BigDye™ version 3.0 mix (Applied Biosystems, Foster City, CA, USA) was added and then water added to the final volume of 8 μl. The sequencing reaction was carried out in Eppendorf MasterCycler (Eppendorf, Hamburg, Germany) with the following conditions: 96°C for 10 seconds, 96°C for 8 seconds and 64°C for 4 min × 3 cycles, 96°C for 8 seconds and 60°C for 4 min × 5 cycles, 96°C for 10 seconds, 50°C for 5 min and 60°C for 4 min × 19 cycles, 96°C for 3 min and storage at 4°C. The products of the reaction were purified using gel filtration in columns with Sefadex G-50 superfine resin.
After purification, the sequencing reaction products were analyzed with ABI PRISM® 310 Genetic Analyzer (Applied Biosystems) in the Inter-institute Sequencing Center of Siberian Division of the Russian Academy of Sciences (Novosibirsk, Russia). We carried out the molecular genetic analysis of the obtained sequences using programs Fasta v. 3 Nucleotide Database Query (European Bioinformatics Institute) (for homology search) and AlignX included in Vector NTI Suite version 8.0 (InforMax, Inc., Bethesda, MA, USA) (for sequences alignment). The data have been submitted to the GenBank database at 21 June 2006. The accession number is DQ832326.
Analysis of 5-HT1A gene expression
Based on this sequence, the primer pair specifically annealing on the ground squirrels 5-HT1A receptor gene was designed (Table 1). These primers were used for 5-HT1A receptor gene expression estimation in the brains of hibernating ground squirrels in diverse stages of the sleep–waking cycle.
The quantitative PCR was performed as described earlier (Naumenko & Kulikov 2006). The messenger RNA (mRNA) of β-actin served as an internal endogenous standard, but instead of the genomic DNA, the amplified cDNA fragments served as external standards. These amplicons were obtained using the same primers that were used for β-actin and 5-HT1A receptor mRNA quantification correspondingly (Table 1). The primers for β-actin were used because of the homology of this gene between mammals, and the use of the external standard allows us to avoid mistakes connected with mouse and ground squirrels β-actin gene diversity.
The PCR products were electrophoretically resolved in 2% agarose gel. Gels were stained with ethidium bromide and scanned using a Biometra TI3 system (Götingen, Germany). Fluorescent intensities of the PCR products amplified from cDNA or external standards were measured using the scion image v. beta 4.0.2 program. The fluorescent intensities of the PCR products amplified from external standards were used for calibration; this made it possible to estimate the copy numbers per microliter of cDNA preparation for the cDNAs of the 5-HT1A receptor and β-actin. The level of 5-HT1A receptor gene expression was normalized with respect to 100 copies of the β-actin cDNA.
Statistical treatment of the data was performed using one-way anova followed by post hoc comparison according to Fisher’s exact test. The effects of hibernation (active vs. hibernating animals) and the brain region on the 5-HT1A receptor mRNA level were analyzed by repeated measures anova. The data were presented as means ± SEM.
The sequence of the 5-HT1A receptor gene fragment including the fifth transmembrane domain (79 bp) and the third intracellular loop (385 bp) was specified (Fig. 1). The comparison of the obtained 5-HT1A receptor gene fragment sequence with the sequences of other species presented in the GenBank database (http://www.ncbi.nih.gov/Genbank/index.html) revealed significant homology with corresponding receptors (Fig. 2).
It has been found that the sequence of 5-HT1A receptor gene fragment of the hibernating ground squirrels that encodes the fifth transmembrane domain showed 93.6% homology with the analogous fragment of the mouse and rat genes and it showed 88.5% of homology with the human 5-HT1A receptor gene. Furthermore, about 70% of homology with the sequences of the genes encoding other 5-HT receptors (5-HT1B, 5-HT7, 5-HT1D) was revealed. At the same time, the insertion of three nucleotides (underlined and marked in bold in the Fig. 2) absent in rat, mouse and human 5-HT1A receptor genes was found in the area encoding third intracellular loop of the ground squirrel 5-HT1A receptor gene.
Despite extraordinary decrease in animal activity and the body temperature, there was no significant depression in the 5-HT1A receptor mRNA level in the brain of hibernating ground squirrels compared with active summer animals (F1,9 = 4.008, P > 0.05). At the same time, significant changes in 5-HT1A receptor gene expression were shown in critical time-points, such as entry into hibernation and coming out of hibernation. Substantial impact of brain region on 5-HT1A receptor gene responses was revealed (F2,18 = 16.825, P < 0.001). Distinct changes in 5-HT1A receptor mRNA level were found in the hippocampus (Fig. 3). A pronounced increase was shown in the prehibernation period (Fig. 4). In inactive but yet normothermic ground squirrels prepared to enter into hibernation, the 5-HT1A receptor mRNA level in the hippocampus was almost threefold higher than in active animals (P < 0.05). There was no significant changes in prehibernation in the 5-HT1A receptor mRNA expression in the midbrain and frontal cortex.
In arousal from hibernation, 5-HT1A receptor mRNA level in the midbrain decreased (P < 0.05), whereas it increased in the hippocampus (P < 0.05).
So, during all phases of the hibernation cycle 5-HT1A, mRNA level in the frontal cortex was not changed noticeably, whereas in the hippocampus and midbrain, the significant alterations in the 5-HT1A mRNA level took place in animals as they entered hibernation or as they aroused from it (Fig. 4).
The alignment of obtained sequence and corresponding fragment of 5-HT1A receptor gene in another species showed the significant similarity of the conservative transmembrane domain reflecting the phylogenetic links between them. Indeed, the fragment encoding fifth transmembrane domain of the 5-HT1A receptor gene showed 93.6% of homology with the analogous fragments of the mouse and rat genes and displayed 88.5% of homology with the human 5-HT1A receptor gene. The differences between hibernator and nonhibernators 5-HT1A receptor genes were found in the third intracellular loop that controls the 5-HT1A receptor coupling with G protein to initiate signal transduction (Baez et al. 1995; Sanders-Bush & Canton 1995). The 5-HT1A receptor gene fragment encoding the third intracellular loop in ground squirrels was different from that in rat, mouse and human 5-HT1A receptor genes by the insertion of three nucleotides, suggesting specific adaptive role of this receptor in mechanism of hibernation. Evidence accumulated within past years indicates that physiological and molecular mechanisms for highly regulated long-lasting physiological state characterized by profound decrease in body temperature, metabolism and torpor are not based on some extraordinary principle but on the use the same mechanisms as those in nonhibernators, although undergoing structural remodeling. 5-HT1A receptor is now known to contribute to the sleep architecture (Wilson et al. 2005) and regulation of body temperature, particularly hypothermic response (Hjorth 1985). The differences between 5-HT1A receptor gene in hibernator and nonhibernators may be attributable to much longer (up to 7 months) and more marked hypothermia and sleep-like state in hibernating ground squirrel. However, an understanding of the significance of this difference will depend on more information about functional peculiarities and the role of 5-HT1A receptor in the mechanism of hibernation.
Our study of the 5-HT1A receptor mRNA demonstrates rather high expression of 5-HT1A receptor gene in the brain regions in hibernation. Despite drastically decreased body temperature and general metabolism, 5-HT1A receptor mRNA level in the hippocampus, midbrain and the frontal cortex did not differ significantly from the mRNA level in active ground squirrels. At the same time, significant changes in the 5-HT1A receptor gene expression were shown in prehibernation period and in ground squirrels coming out of hibernation. Importantly, the 5-HT1A receptor gene was activated before the onset of the hypothermic state characteristic of hibernation. Increased 5-HT1A receptor mRNA level was found in the hippocampus in a period in which animals were prepared to enter hibernation but still maintained euthermic body temperature. These results are in good agreement with our earlier data showing that the most marked increase in 5-HT1 receptor density, 5-HT level and in the activity of key enzyme in 5-HT biosynthesis, tryptophan hydroxylase, was found in the hippocampus, and these changes preceded the entry into hibernation (Pak et al. 1987; Popova et al. 1993). These results indicate three important points: first, the alterations in the brain 5-HT metabolism and 5-HT1A receptor were not a consequence of body temperature change nor were they induced by low temperature; second, alterations in 5-HT metabolism, 5-HT1A receptor mRNA level and the 5-HT1 receptor density preceded the entry into hibernation, strongly supporting the paradigm of participation of the brain 5-HT system in the mechanism controlling the preparation for and the induction of the forthcoming hibernation state and third, the results indicate the involvement of 5-HT1A receptor gene into these mechanisms.
Attention is drawn to the increase in 5-HT1A receptor mRNA level during prehibernation period and arousal from hibernation in hippocampus. These results are in good agreement with several lines of electrophysiological evidence suggesting that the hippocampus could be a pacemaker for the induction and maintenance of the hibernation state (South et al. 1972). The significant increase in 5-HT1A receptor gene expression in the prehibernation period and in arousal from hibernation strongly suggests a functional significance of 5-HT1A receptor in the hippocampus during the transition to both the hibernation and the active state. The significance and mode of action of increased 5-HT1A receptor gene expression in hippocampus in entering hibernation and arousing animals as well as maintained rather high gene expression in hibernation has yet to be determined. At the same time, it has to be noted that a highly selective 5-HT1A receptor agonist repinotan was found to decrease ischemic brain injury, suggesting neuroprotective effect of 5-HT1A receptors (Berends et al. 2005).
In contrast to hippocampus, significant decrease in 5-HT1A receptor mRNA level was shown in the midbrain, the primary site for brain 5-HT synthesis in the raphe nuclei. It is well known that 5-HT1A receptors are localized both presynaptically in midbrain raphe nuclei, and postsynaptically and according to their localization, they exert different effect on the functional state of the 5-HT system. Stimulation of presynaptic receptors inhibits this system, whereas stimulation of postsynaptic receptors produces effects typical for the functional activation of the 5-HT system. There appears to be a species differences in prevailing pre- or postsynaptic mechanism of 5-HT1A-induced hypothermia in mouse and rats (Barnes & Sharp 1999). There is no data on the presynaptic/postsynaptic 5-HT1A receptor ratio in ground squirrels. So it is rather difficult to evaluate functional significance of 5-HT1A receptor mRNA level reduction in animals coming out of hibernation. Earlier, a decrease in tryptophan hydroxylase activity during coming out of hibernation in the midbrain of ground squirrel was shown (Popova et al. 1993), suggesting that arousal from hibernation was characterized by inactive 5-HTergic system in some brain regions.
In conclusion, the present studies demonstrate, for the first time, the significant similarity of the conservative transmembrane domain and the differences in the third intracellular loop in 5-HT1A receptor gene of hibernating ground squirrel and nonhibernators, mouse, rats and humans. In the future, it will be important to understand the functional significance of hibernator distinction for 5-HT1A receptor signal transduction and its role in unique hibernator’s survival strategy at low environmental temperature. The findings that the overexpression of 5-HT1A receptor gene in hippocampus precedes the entry into hibernation are in accordance with our previous results, indicating that 5-HT is involved in the transition to the hibernation as well as the idea that hippocampus could be a pacemaker for the induction and the maintenance of the hibernation state.
The study was supported by Russian Foundation for Basic Research (grant 04-49098), grants for Young Scientists of International Society for Neurochemistry and the Lavrentiev Foundation (Grant 109).