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The Journal of Neuroscience, May 15, 1999, 19(10):3781-3790
Gene Expression in the Brain across the Hibernation Cycle
Bruce F.
O'Hara,
Fiona L.
Watson,
Hilary K.
Srere,
Himanshu
Kumar,
Steven W.
Wiler,
Susan K.
Welch,
Louise
Bitting,
H. Craig
Heller, and
Thomas S.
Kilduff
Center for Sleep and Circadian Neurobiology, Departments of
Psychiatry and Behavioral Sciences and Biological Sciences, Stanford
University, Stanford, California 94305-5020
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ABSTRACT |
The purpose of this study was to characterize changes in gene
expression in the brain of a seasonal hibernator, the golden-mantled ground squirrel, Spermophilus lateralis, during the
hibernation season. Very little information is available on molecular
changes that correlate with hibernation state, and what has been done focused mainly on seasonal changes in peripheral tissues. We produced over 4000 reverse transcription-PCR products from euthermic and hibernating brain and compared them using differential display. Twenty-nine of the most promising were examined by Northern
analysis. Although some small differences were observed across
hibernation states, none of the 29 had significant changes. However, a
more direct approach, investigating expression of putative
hibernation-responsive genes by Northern analysis, revealed an increase
in expression of transcription factors c-fos, junB, and c-Jun, but not
junD, commencing during late torpor and peaking during the
arousal phase of individual hibernation bouts. In contrast,
prostaglandin D2 synthase declined during late torpor and arousal but
returned to a high level on return to euthermia. Other genes that have putative roles in mammalian sleep or specific brain functions, including somatostatin, enkephalin, growth-associated protein 43, glutamate acid decarboxylases 65/67, histidine
decarboxylase, and a sleep-related transcript SD464 did not change
significantly during individual hibernation bouts. We also observed no
decline in total RNA or total mRNA during torpor; such a decline had
been previously hypothesized. Therefore, it appears that the dramatic changes in body temperature and other physiological variables that
accompany hibernation involve only modest reprogramming of gene
expression or steady-state mRNA levels.
Key words:
prostaglandin D2; enkephalin; c-fos; immediate early
genes; ddPCR; mRNA
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INTRODUCTION |
Hibernation has fascinated people
for centuries, but little is known about the mechanisms controlling the
hibernation process in mammals. Contrary to popular belief, small
hibernating mammals do not remain torpid throughout the hibernation
season. These hibernators exhibit periodic bouts of torpor throughout
the winter, marked by dramatic changes in physiological variables, such
as body temperature (Tb). Both entrance into
and arousal from torpor are regulated processes controlled by
hypothalamic nuclei and other brain structures (Heller et al., 1978 ;
Kilduff et al., 1990). Neural activity, as well as all metabolic
activity, is greatly reduced at the low temperatures associated with
deep torpor (Walker et al., 1977; Heller, 1979; Krilowicz et al.,
1988). Nevertheless, physiological regulation continues, and arousals
appear to be precisely timed (Grahn et al., 1994). Given the duration
and dramatic physiological changes that accompany hibernation events,
changes in gene expression are probably involved.
In this study, two approaches were used to detect changes in gene
expression during hibernation. First, random mRNAs were compared in
brain samples from euthermic versus hibernating animals using a
differential display PCR method (ddPCR). One advantage of this
technique is that no prior assumptions are made as to which mRNAs are
most likely to change. In the second approach, changes in abundance of
specific mRNAs were tested by Northern analysis. The genes examined
were selected based on expression patterns in brain and previous
evidence for involvement in arousal state control.
Specifically, we chose six categories of genes. The first category is
immediate early genes (IEGs). IEGs, including c-fos, junB,
c-jun, junD, NGFI-A, and others, are transcription factors that respond
to environmental stimulation within minutes. The role of IEG expression
in the CNS has been well documented (Sheng and Greenberg, 1990; Morgan
and Curran, 1991) and has been implicated in sleep, circadian rhythms,
and hibernation (Sutin and Kilduff, 1992; Grassi-Zucconi et al.,
1993; O'Hara et al., 1993, 1997; Bitting et al., 1994; Pompeiano et
al., 1994). The second category includes enkephalin and somatostatin.
Numerous studies have implicated the opioid system in hibernation and,
to a lesser extent, somatostatin (Beckman et al., 1981, 1986; Beckman
and Llados-Eckman, 1985; Nurnberger et al., 1991, 1997; Wang,
1993). The third is prostaglandin D2 (PGD2) synthase. PGD2 has been
shown to increase seasonally in a hibernator (Takahata et al., 1996),
induce sleep when injected intracerebroventricularly (Matsumura
et al., 1994; Hayaishi, 1997; Ram et al., 1997), and reduce
somatosensory pain perception (Minami et al., 1996). The fourth, the
SD464 clone, was isolated by Rhyner et al. (1990) for its possible role
in sleep homeostasis. The fifth category is glutamic acid
decarboxylases GAD65 and GAD67 and histidine decarboxylase. GAD65 and
GAD67 synthesize GABA (Erlander et al., 1991), the dominant inhibitory
neurotransmitter in brain. Histidine decarboxylase produces histamine
(Joseph et al., 1990), which may play a major role in arousal state
control (Sherin et al., 1998). The last is growth-associated protein 43 (GAP43). Changes in dendritic spines have been reported during torpor
(Popov and Bocharova, 1992; Popov et al., 1992), and GAP43 is strongly associated with synaptic plasticity (Skene et al., 1986; Benowitz and
Routtenberg, 1987; Neve et al., 1987).
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MATERIALS AND METHODS |
Animals. Golden-mantled ground squirrels,
Spermophilus lateralis, were trapped during the summer
months in Alpine and El Dorado Counties of the Sierra Nevada Mountains
of California. Both male and female animals were used in this study.
Animals were implanted with abdominal temperature transmitters
(Mini-Mitter Co., Sunriver, OR), and Tb was monitored
continuously during the hibernation season. At the time the animals
were killed, all Tb measurements were confirmed with
a thermocouple probe placed in one or more of the following locations:
rectal, abdominal, thoracic, and occasionally into the brain.
Temperature measurements in different locations were all similar,
although the head is known to warm first during arousal. Animals were
maintained at an ambient temperature of 5°C in a constant 12 hr
light/dark cycle (lights on at 8:00 A.M.). Animals were killed
by decapitation during a 3 hr "window" between 1:00 and 4:00 P.M.
during lights on. Although circadian rhythms of temperature persist
throughout the hibernation season (Grahn et al., 1994), the rhythms
appear to be primarily desynchronized from the light cycle (Pohl, 1987;
our unpublished observations). Most squirrels were killed in one
of five phases of the hibernation cycle as indicated in Figure
1: (1) euthermia (winter) (Tb
of 37°C); (2) midentrance (Tb of 20°C); (3) day 1 of
deep hibernation (Tb of 7-8°C); (4) day 4, 5, or 6 of
deep hibernation (Tb of 7-8°C); and (5) midarousal from
hibernation (Tb of 20°C), which typically occurs ~1 hr
after body temperature begins to rise. A smaller number of squirrels
were killed during the summer active phase (Tb of 37°C)
at early entrance (Tb of 30°C) or at 2, 5, or 9 hr after
arousal (from Tb of 20°C). The 2, 5, and 9 postarousal
animals all had a Tb of 37°C and were the same as other
winter euthermic animals (sometimes called interbout animals), except
that postarousal time was precisely determined. Summer active squirrels
had not hibernated for at least 3 weeks. Approximately 50 squirrels were continuously monitored to obtain animal(s) at each
specific phase, at the designated time of day.
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Figure 1.
Schematic representation of a bout of torpor
during the hibernation season. Bouts of torpor lasting several days to
>1 week are interspersed with periodic returns to euthermia, referred
to as winter euthermia or interbout euthermia. Arrows
indicate times at which samples were taken. WE, Winter
euthermic (Tb of 37°C); E30, entrance
(Tb of 30°C); E20, entrance
(Tb of 20°C); H1, hibernation day 1 (Tb of 7°C); H5, hibernation day 5 (Tb of 7°C); AR, arousal (Tb
of 20°C); 2 hr, 5 hr, and 9
hr, refer to postarousal time (Tb of 37°C) and
are abbreviated in other figures as P2,
P5, and P9.
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RNA isolation and Northern analysis. After decapitation,
brains were rapidly removed and dissected into 12 subregions
(hypothalamus, thalamus, cerebral cortex, basal forebrain, septum,
hippocampus, striatum, midbrain, cerebellum, pons, medulla, and
pineal). Many peripheral tissues were taken, as well. All tissues were
frozen on dry ice and stored at  70°C. Total RNA was extracted using a guanidinium-CsCl method or Trizol (BRL, Grand Island, NY),
fractionated on 1.2% formaldehyde-agarose gels, and transferred to
Nytran membranes (Schleicher & Schuell, Keene, NH). RNA was visualized
by ethidium bromide staining and cross-linked by UV irradiation. After
prehybridization, membranes were hybridized at 42°C in 5× SSC, 50%
formamide, 50 mM sodium phosphate, pH 6.8, 1% SDS,
1 mM EDTA, 2.5× Denhardt's solution, 200 mg/ml herring
sperm DNA, and 4-10 × 106 cpm/ml
32P-radiolabeled random-primed cDNA probe. After
hybridization, membranes were washed two times for 30 min at 58°C in
0.4× SSC and 0.5% SDS. Filters were then exposed to Kodak XAR5 film
(Eastman Kodak, Rochester, NY) for 1-12 d, and the band intensity was
quantitated using a computer-assisted image analysis system using the
film exposure with the optimal densities of each band (i.e., within the
linear range) (MCID; Imaging Research, Inc., St. Catharines, Ontario).
To control for variability in loading and transfer, all membranes were
probed with a 700 bp human  -actin probe. -Actin mRNA levels
appear to remain constant throughout the hibernation cycle based on our
comparisons of total RNA loaded and -actin band density of hundreds
of samples from different stages of hibernation. Multiple probings
of the same Northern filters were carefully monitored to ensure that no
residual radioactivity from previous probes remained and that no other
degradation of the filters was apparent.
Most hybridization probes of the known genes used in this study were
derived from gel-isolated fragments of rat, mouse, or human cDNA
sequences that are well conserved across mammalian species. Three
cDNAs, F1-ATPase, RNA poly(A) polymerase, and NADH-ubiquinone oxireductase, were isolated from a closely related species of squirrel,
Spermophilus richardsonii (Srere, 1995). The 29 ddPCR fragments were unknown cDNA sequences from Spermophilus
lateralis. The precise regions of each cDNA used to probe for
c-fos, junB, c-jun, junD, NGFI-A, enkephalin, somatostatin, GAD65,
GAD67, -actin, and GAP43 were described previously (O'Hara et al.,
1989, 1994, 1995, 1997). For other genes, cDNAs containing all or most
of the coding sequence were used and can be found in PGD2
synthase (Urade et al., 1993), SD464 (Rhyner et al., 1990), cyclophilin (Hasel et al., 1991), and histidine decarboxylase (Joseph et al., 1990).
ddPCR. All ddPCR experiments were performed with total RNA
derived from the cerebral cortex of Spermophilus lateralis
to have sufficient RNA for both ddPCR and for subsequent Northern
analyses to test putative positives. RNA derived from squirrels killed on day 4 of torpor (hibernation day 4), during the interbout euthermic period (winter euthermia), or during the summer active phase (summer euthermia) were compared. RNA samples were DNase treated and
phenol-chloroform extracted before reverse transcription. The reverse
transcription reaction was performed using 0.2 µg of DNA-free RNA in
a 20 µl volume containing 20 mM dNTPs, 125 mM
Tris-HCl, pH 8.3, 188 mM KCl, 7.5 mM
MgCl2, 25 mM DTT, and 100 U of
MMLV reverse transcriptase in separate reactions containing 0.2 mM 3' primers anchored to subsets of the RNA pool by single
base changes [poly-dT11A, poly-dT11G, poly-dT11C, and poly-dT11T supplied in kit form
(GenHunter Corp., Nashville, TN)]. Reactions were
performed at 37°C for 1 hr after an initial 65°C
denaturation step to allow annealing of the primers. One-tenth of the
reverse transcription reaction product from each reaction was used in
each subsequent PCR amplification using the original 3' primer, in
conjunction with random 13-mers as 5' primers (GenHunter Corp.). PCR
reactions contained 2 mM each 3' and 5' primers, 25 mM dNTPs, 2 µl of reverse transcription reaction product, PCR buffer (GenHunter Corp.), [ -35S]dATP (1200 Ci/mmol), and Amplitaq DNA polymerase (Perkin-Elmer, Emeryville, CA).
PCR conditions were 94°C for 30 sec, 40°C for 2 min, and 72°C for
30 sec, each for 40 cycles. PCR products were analyzed on 5%
nondenaturing polyacrylamide gels, and banding patterns were compared
between conditions. Forty-five primer combinations were used, and
~100 bands could be visualized with each primer set. Therefore, in
theory, ~4500 mRNA transcripts were compared between deep hibernation
and euthermic conditions, although there is likely to be some redundant
cDNAs amplified by different primer sets. Most analyses compared summer
euthermia versus deep hibernation.
Data analysis. The resultant data were analyzed by one-way
ANOVA. Wherever post hoc tests were indicated, the
data were analyzed by both Fisher's protected least significance
difference test and Scheffe's F test; the results
presented are only those in which these tests agree as to significance.
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RESULTS |
Changes in mRNA expression assayed by ddPCR
Samples of total RNA isolated from ground squirrel cortex
during summer euthermia, the winter interbout euthermic period, and the
fourth day of torpor, were used as templates to generate cDNA
fragments, which were then amplified by PCR and separated on
polyacrylamide gels. Differences in expression between test conditions
were visualized by autoradiography of 35S-labeled bands
(Fig. 2). Most comparisons were between
summer euthermia and winter torpor (day 4 of a single hibernation bout) because these two states were hypothesized to have the most differences in gene expression. The initial screen of 4500 transcripts (in theory)
produced 29 bands, indicating differences between these two states.
These bands were cut out and reamplified for verification by Northern
analysis on blots prepared from the initial RNA samples. All 29 probes
produced distinctive bands, but none of the 29 putative differences in
expression were significant when tested on Northern blots containing
samples of summer euthermia and winter hibernation (data not shown).
Therefore, no enrichment of hibernation-responsive genes was achieved
by this method. However, the fact that 29 of 29 selected cDNAs failed
to show any significant changes in steady-state mRNA levels suggests
that the majority of mRNAs do not dramatically change in abundance
across these conditions.
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Figure 2.
ddPCR. Samples of total RNA were isolated from
ground squirrel cortex during summer euthermia and hibernation day 4. The isolated RNAs were used as templates in duplicate to generate cDNA
fragments, which were then amplified by PCR and separated on
polyacrylamide gels. SE, Summer euthermic;
H4, hibernation day 4; c , bands that
appear to remain constant across the two conditions;
e, bands that appear to be more abundant during
euthermia;  h, bands that appear to be more abundant
during hibernation.
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Changes in mRNAs assayed by Northern analysis
In contrast to the ddPCR method, Northern analysis of selected,
well characterized genes was used as a directed approach to test
variability within the hibernation cycle. All cDNA probes produced one
or two distinctive bands, with sizes, intensity (reflecting abundance),
and expression patterns consistent with known data in the mouse, rat,
and human. Some Northern analyses were run with RNA derived from
multiple species to further support that each cDNA probe was
recognizing its homolog in the squirrel.
Differential expression among IEG family members was observed within
the hypothalamus across the hibernation cycle. Whereas c-fos, junB, and
c-Jun mRNAs appear to increase during torpor and peak during arousal in
hypothalamus, junD remains constant (Fig.
3). The increase in expression of c-fos
and c-Jun during arousal is not confined to the hypothalamus but occurs
in other brain regions, such as the cortex (Fig.
4), and in almost every other brain
region examined, including thalamus, basal forebrain, septum,
hippocampus, striatum, midbrain, cerebellum, pons, and medulla (our
unpublished observations). In a peripheral tissue, such as brown
fat (Fig. 5), c-fos expression is
dramatically higher during arousal but is virtually undetectable at
other times and does not exhibit the trend toward higher expression
during the latter part of torpor characteristic of hypothalamus (Fig.
3). Liver and spleen also exhibit sharp rises in c-fos mRNA during arousal but have somewhat higher basal levels (our unpublished observations). To investigate the time course of c-fos
expression in the hypothalamus in finer detail, samples were taken at
additional time points. Figure 6 confirms
that the peak in c-fos expression occurs during arousal from torpor,
with a return to basal levels within 2 hr after arousal.
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Figure 3.
Expression of IEGs in hypothalamus across
hibernation. A, Autoradiographs of Northern blots of
total RNA (20 µg/lane) isolated from hypothalamus of ground squirrels
killed during different phases of the hibernation cycle and hybridized
to [32P]-labeled c-fos, c-jun, junB, and junD cDNA
probes. Sizes of mRNAs are shown to the right in kilobases. Note the
increasing levels of both c-fos and c-jun mRNA during late torpor and
then peaking during arousal relative to other phases of hibernation.
The c-fos probing on the left and right
were done at different times with different specific activity and total
counts per minutes and do not represent differences in c-fos expression
between these groups. Each blot was normalized internally for
statistical analyses shown in the histograms in B.
SE, Summer euthermic; WE, winter
euthermic; EN, entrance; H1, hibernation
day 1; H5, hibernation day 5; AR,
arousal. -actin mRNA serves as a control for RNA loading in each
lane. n = 4 for each condition during the
hibernation season, and n = 2 for summer euthermia
(SE). B, Quantitation of the relative
mRNA levels are shown based on band density of the autoradiograms in
A. Each graph displays means ± SEs. To
control for variability in loading and transfer, all data are presented
relative to -actin, whose expression remains constant throughout the
hibernation cycle relative to total RNA and total mRNA (data not
shown). mRNA levels of c-fos, c-jun, and junB during arousal
(AR) are significantly elevated relative to baseline
winter euthermic levels. *p < 0.01.
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Figure 4.
Quantitation of relative c-fos mRNA levels in
cortex. Graphic representation of c-fos mRNA levels in the cerebral
cortex across different hibernation states as shown with hypothalamus
in this and Figure 3. n = 4 for WE,
EN, H1, H5, and
AR. n = 2 for SE. The
cortical samples were derived from the same animals as hypothalamus.
Abbreviations as in Figure 3. Note that c-fos mRNA levels are again
significantly elevated in AR relative to
WE, EN, H1, and
H5. *p < 0.05. SE levels of c-fos mRNA also appear to be elevated but
could not be supported statistically because n = 2.
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Figure 5.
Expression of c-fos and junB in brown fat.
Autoradiographs of Northern blots of total RNA (20 µg/lane) isolated
from brown adipose tissue of squirrels at different phases of the
hibernation cycle. Note the extremely low basal level of c-fos mRNA and
the very strong induction during arousal. P2, 2 hr after
arousal; P5, 5 hr after arousal; P9, 9 hr
after arousal (from a Tb of 20°C); SE,
summer euthermic; EN, entrance; H1,
hibernation day 1; H5, hibernation day 5;
AR, arousal. -actin mRNA serves as a control to
assess RNA loading in each lane.
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Figure 6.
Rapid return of c-fos mRNA levels after arousal.
Autoradiographs of Northern blots of total RNA (20 µg/lane) isolated
from hypothalamus of ground squirrels killed during different phases of
the hibernation cycle as in Figure 3, with the addition of several well
defined postarousal time points and one additional entrance time point
(N30). N30 is early entrance to torpor
(Tb of 30°C), and N20 is midentrance
(Tb of 20°C), the same as EN in other
figures. Note the increasing amount of c-fos mRNA during torpor and the
high peak during arousal as in Figure 3, and then the rapid return to
basal levels after arousal. Levels of c-fos mRNA during arousal are
significantly elevated over all other phases; *p < 0.01. n = 3 for each condition. Other abbreviations
as in Figures 3-5.
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In contrast to the IEGs, two neuropeptide mRNAs known to be expressed
in hypothalamus, somatostatin and enkephalin, did not undergo
significant changes across the hibernation cycle (Fig. 7), despite previous evidence for changes
in peptide or mRNA levels (Nurnberger et al., 1991, 1997).
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Figure 7.
Somatostatin and enkephalin mRNA levels show no
change across hibernation. A, Autoradiographs of
Northern blots of total RNA (20 µg/lane) isolated from the
hypothalamus of ground squirrels using the same filters as in Figure 3
and hybridized to [32P]-labeled somatostatin and
enkephalin cDNA probes. Abbreviations as in Figure 3. B,
Quantitation of relative mRNA levels of somatostatin and enkephalin
across hibernation. Graphic representation of band intensity was
quantitated by optical density. To control for variability in loading
and transfer, all data are presented relative to -actin as in other
figures. No significant change for either mRNA occurs across these
conditions.
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Figure 8 illustrates that PGD2 synthase
mRNA varies significantly over the hibernation cycle, declining sharply
in the hypothalamus after many days of hibernation, and returning to
its former level during arousal, whereas the cortex and basal forebrain
both show a more modest decline that does not quite reach statistical
significance.
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Figure 8.
PGD2 synthase mRNA declines during torpor.
Autoradiographs of Northern blots of total RNA (20 µg/lane) isolated
from hypothalamus along with graphic representations of hypothalamus,
cerebral cortex, and basal forebrain of ground squirrels killed during
different phases of hibernation cycle and hybridized to a
[32P]-labeled PGD2 synthase cDNA probe. Note the
decreased level of PGD2 synthase mRNA during late torpor in the
hypothalamus relative to all other conditions; *p < 0.05. The lower levels of PGD2 synthase mRNA in the cortex and basal
forebrain during late torpor and arousal do not quite reach statistical
significance. Filters for the hypothalamus are the same as used
previously, and the cortical and basal forebrain samples are from the
same animals as the hypothalamus shown in Figure 6.
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Several other genes were examined, including GAD65, GAD67, histidine
decarboxylase, SD464, GAP43, cyclophilin, and three housekeeping genes
whose expression in the liver varies across the hibernation season
(Srere, 1995). No significant changes in steady-state mRNA levels were
found in the brain for any of these genes across phases of hibernation.
These genes, along with those described above, are summarized in Table
1. Only some IEGs and PGD2 synthase were found to exhibit significant differences in steady-state mRNA levels
across different phases of hibernation. For most genes, the
within-group variation was modest, and differences in mRNA abundance
greater than twofold would usually be detected as significant.
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DISCUSSION |
We have investigated changes in mRNA levels in the brain from a
variety of genes across phases of hibernation in the golden-mantled ground squirrel. Considerable evidence suggests that the brain plays a
major role in regulating the extreme changes in body temperature, metabolism, and other physiological variables that accompany
hibernation (Mihailovic, 1972; Heller and Colliver, 1974; Heller, 1979;
Dark et al., 1990). The hypothalamus in particular appears to play a
critical role in both the entrance to and arousal from each hibernation
bout (Heller, 1979; Kilduff et al., 1990) and was studied most
extensively in this paper. The cerebral cortex was also of interest
because this brain region has provided measures of arousal state
variation across hibernation using conventional EEG monitoring
(Mihailovic, 1972; Walker et al., 1977), and suppression of metabolic
activity in this structure has been implicated in the entrance to
hibernation (Kilduff et al., 1990). In general, other brain regions,
such as hippocampus and basal forebrain, were similar to cortex for all
of the mRNAs studied (unpublished data). Brain regions aside from
hypothalamus, cortex, hippocampus, and basal forebrain were studied in
only a few samples because there did not appear to be any dramatic
regional differences. The hypothalamus, as the prime region of focus,
did display some potentially unique characteristics, such as increases
in IEG mRNAs as each torpor bout progressed, as opposed to increases
only at arousal. The hypothalamus also displayed the only significant change in PGD2 mRNA levels and the most rapid rise in PGD2 mRNA during arousal.
Across all mRNAs studied, we were somewhat surprised by the constancy
of mRNA levels given the extreme physiological changes that accompany
hibernation. Because the ddPCR compared summer euthermic versus deep
torpor, it could have identified genes that regulate the hibernation
process, genes that respond to the seasonal changes, or genes that
change with body temperature, metabolism, etc. The ddPCR method can
produce false positives (Debouck, 1995), and that appears to have
occurred in this study. Nonetheless, these experiments did provide a
sample of 29 different cDNAs. The fact that none of the ddPCR products
and only one known gene examined, PGD2 synthase, declined significantly
during torpor may be particularly worth noting. At the low body
temperatures achieved during hibernation (8°C or less under our
ambient conditions of 5°C), it seemed likely that RNA transcription
might virtually cease, and all RNAs would either be maintained or would
decrease in abundance. In fact, such a decline might explain the
mystery of periodic arousals throughout the hibernation season, which are metabolically costly and limit the energy conservation achieved (Lyman et al., 1982). However, we found no evidence for a general decline in total RNA yields, mRNA yields, or specific mRNAs tested, consistent with recent work in some peripheral tissues (Srere et al.,
1992; Carey and Martin, 1996). Instead, some mRNAs (e.g., c-fos, c-jun)
in our study appear to increase in the brain during torpor, although
these changes do not reach statistical significance until the arousal
phase. In peripheral tissues, a small number of mRNAs and proteins have
been found to vary between torpor and euthermia, with examples of both
increasing (Srere et al., 1992; Wilson et al., 1992; Andrews et al.,
1998; Gorham et al., 1998) and decreasing expression (Kondo and Kondo,
1992; Takamatsu et al., 1997), which are concordant at the mRNA and
protein levels in all cases examined. None of these other studies,
however, focus on the detailed time points across hibernation bouts,
especially the dramatic periods of arousal from and entrance to hibernation.
In this report, we found that basal levels of IEG mRNAs were clearly
higher in the brain than in brown fat and other peripheral tissues
(Figs. 3-5; our unpublished observations). These data
strengthen the argument that the brain is relatively more active during
hibernation than many peripheral tissues. Furthermore, because there
does not appear to be a general decline in most mRNAs (which might appear as relative increases in some genes), it is extremely likely that substantial new transcription of some genes does take place during
arousal from hibernation (as indicated by c-fos induction) and that, in
hypothalamus, this may occur even at the coldest body temperatures
during torpor. It does not seem likely that the large increases in IEG
mRNAs could occur only by changes in mRNA stability. In addition, our
data may be relevant to the fact that animals are increasingly
responsive to stimuli as each bout of hibernation progresses (Beckman
and Stanton, 1976), suggesting the possibility of increasing CNS
function and the need for new mRNA synthesis.
Our inability to detect significant changes in enkephalin or
somatostatin mRNA, despite changes at the protein level (Nurnberger et
al., 1991, 1997), suggests regulation at steps other than
transcription, although it is possible that our nonsignificant increase
in enkephalin mRNA does underlie the significant differences at the
protein level or that changes in enkephalin occur in a relatively small percentage of enkephalin neurons. Because of individual
variability, sample size, and technical limitations in the
quantification of mRNA levels, our study was designed to detect
relatively large changes in gene expression of greater than twofold.
There are probably many important smaller changes in gene expression
that, in concert, play an important or even dominant role. In fact, the
small number of genes we have found with large changes makes this
possibility likely.
In contrast to the majority of genes, IEG expression can vary widely
across experimental conditions (Bartel et al., 1989; Sheng and
Greenberg, 1990; Morgan and Curran, 1991; Sutin and Kilduff,
1992), as we found to occur across hibernation. This is especially
apparent in brown fat (Fig. 5) in which levels are barely detectable
but rise dramatically during arousal. Although c-fos levels in the
brain also exhibit this induction, basal levels are much higher and, as
noted above, appear to increase late in torpor in some brain regions,
such as hypothalamus. Both the higher basal level and the possible
earlier induction in the hypothalamus suggest a central role in
regulatory control. We have found previously that the suprachiasmatic
nucleus of the hypothalamus undergoes a dramatic induction of c-fos
mRNA during the arousal phase (Bitting et al., 1994); however, the
in situ methodology used was not sensitive enough to measure
c-fos in other brain regions. Our current study shows that c-fos
induction during arousal occurs throughout the brain and in most
peripheral tissues as well, although the hypothalamus continues to
stand out as the most dynamic brain region (both in terms of IEG and
PGD2 synthase mRNAs). The cerebral cortex has a relatively high basal
level of c-fos but shows no increase in late torpor. Aside from
hibernation, c-fos levels in the cortex correlate well with activity
and sleep (Grassi-Zucconi et al., 1993; Pompeiano et al., 1994; O'Hara
et al., 1997). Therefore, the higher levels of c-fos mRNA in the cortex
of summer active animals (Fig. 4) may reflect higher activity of the
squirrels at this time, although because of the availability of only
two summer active animals, this difference could not be established statistically. The rapid decline of c-fos mRNA in the hypothalamus after arousal may also be in part caused by sleep, because the initial
period of interbout euthermia almost invariably contains very deep
sleep (Daan et al., 1991; Trachsel et al., 1991; Kilduff et al., 1993).
Because IEGs are transcription factors, we are currently investigating
this postarousal period in greater detail to determine which genes may
be subsequently activated or inactivated. An important determining
factor in target gene activation by FOS/JUN heterodimers is which JUN
partner is paired with FOS (Ryseck and Bravo, 1991). Therefore,
it is relevant to note that c-jun and junB both increase along with
c-fos mRNA, but junD remains constant. In studies of IEG expression in
the rat across circadian time and arousal states, c-fos and junB both
increase with periods of high locomotor activity, whereas junD and
c-jun remain invariant (O'Hara et al., 1993, 1997). In contrast,
squirrels appear to use c-jun induction together with c-fos and junB
(O'Hara et al., 1997) (Fig. 3).
As with the rapid decline in c-fos mRNA after arousal, the change in
PGD2 synthase mRNA levels may also reflect relationships between
hibernation and sleep (Heller, 1979; Kilduff et al., 1993). Prostaglandins are locally acting hormones produced from metabolism of
arachidonic acid by the cyclooxygenase pathway. PGD2 both induces sleep
(Hayaishi, 1997; Ram et al., 1997) and reduces somatosensory pain perception (Minami et al., 1996). PGD2 circulates within the CNS
in the ventricular system and subarachnoid space and is produced
primarily by choroid plexus and leptomeninges, and to a lesser extent
by glia and neurons (Urade et al., 1993). We do not believe any of our
samples contain choroid plexus or leptomeninges; therefore, we are most
likely measuring the lower levels of expression in the neurons and glia
of each brain region. PGD2 has also been shown to increase
seasonally in the Asian chipmunk, another mammalian hibernator
(Takahata et al., 1996). Interestingly, not only is there a decline in
PGD2 synthase mRNA during late torpor but a subsequent rapid return to
high expression during arousal in the hypothalamus and during interbout
euthermia in the basal forebrain and cortex. The basal forebrain is the
most responsive brain region with respect to the sleep promoting
ability of PGD2 (Matsumura et al., 1994), and this may therefore
relate to the hypersomnia of the postarousal period (Trachsel et al.,
1991).
In summary, our findings suggest that only a small percentage of mRNAs
undergo large changes in steady-state levels in the brain across the
hibernation cycle. The mRNAs identified that do undergo large changes,
IEGs and PGD2 synthase, seem likely to play important roles in the
extreme physiological and arousal state changes that accompany
individual hibernation bouts.
| |
FOOTNOTES |
Received Oct. 5, 1998; revised Feb. 3, 1999; accepted March 9, 1999.
This work was supported by Army Research Office Grant DAAH04-95-1-0616,
National Institutes of Health Grants DA00187 and HL58985, and a grant
from the Upjohn Company. This article is dedicated to the memory of
Louise Bitting (1/20/49 to 1/9/99), whose creativity and love of
science and life were an inspiration to all who knew her.
Correspondence should be addressed to Dr. Bruce F. O'Hara, Department
of Biological Sciences, Stanford University, Stanford, CA 94305-5020.
Dr. Kilduff's present address: Molecular Neurobiology Laboratory, SRI
International, Menlo Park, CA 94025.
| |
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TOP
ABSTRACT
INTRODUCTION
