BACKGROUND
at the cellular level, circadian rhythms are thought to be generated by transcriptional-translational feedback loops made up of positively and negatively acting transcriptional regulators  <cit> .
in mammals, the core positive elements are clock and npas <dig> and their obligate dimerization partner bmal <dig>  these transcription factors drive per and cry transcription.
per and cry proteins, in turn, interact with clock/npas2:bmal <dig> heterodimers to inhibit their own transcription, thus constituting the negative feedback elements.
the circuitry is clearly more complex as additional feedback loops and post-translational modifications are involved  <cit> .
clock genes underlie circadian rhythm generation in many tissues, but the circadian rhythm in the suprachiasmatic nucleus  is required for the manifestation of overt physiological and behavioral rhythms, and is therefore considered the master circadian pacemaker  <cit> .
although the role of clock genes in generating circadian rhythms is firmly established, we and others have found that they also play a role in the homeostatic regulation of non-rem sleep   <cit> .
this notion is based on the observation that mice deficient for one or more clock genes show an altered response to sleep deprivation  and on the observation that clock-gene expression in brain areas outside the scn, notable the cerebral cortex, is influenced by the prior occurrence of sleep and wake.
for instance, the typical responses in nrems to sd were reduced or absent in mice lacking the cry <dig> and - <dig>   <cit>  or bmal <dig>   <cit>  genes compared to wild-type controls.
the diminished nrems variables included duration, consolidation, and eeg delta power.
eeg delta power quantifies the prevalence and amplitude of delta oscillations and is widely used to index the homeostatic need for nrems  <cit> .
sleep under undisturbed, baseline conditions was also altered in mutant mice.
clock-mutant   <cit>  and npas2-/- <cit>  mice spent less time in nrems whereas cry <dig> -/- <cit>  and bmal1-/-  <cit>  mice slept more.
nrems consolidation was increased in cry <dig> -/- mice while the opposite was observed in clockΔ/Δ  <cit>  and bmal1-/-  <cit>  mice and in mice lacking albumin d-binding protein  <cit> ; a gene under the transcriptional control of clock  <cit>  and that also has been implicated in circadian rhythm generation  <cit> .
average levels of eeg delta power were reduced in clockΔ/Δ  <cit> , dbp-/- <cit> , and bmal1-/-  <cit>  mice and increased in cry <dig> -/-  <cit>  mice.
clock-gene expression in the brain, notably the cerebral cortex, depends on the sleep-wake  distribution.
cortical levels of per are high at times when sleep drive is high, irrespective of the phase at which the circadian expression of per peaks in the scn.
thus, in both nocturnal and diurnal species, per expression in the cortex is maximal in conjunction with the major waking episode  <cit> .
under conditions where the phase  or distribution  of locomotor activity is altered, per expression in the cortex parallels the overt rhythm of wakefulness, while per expression in the scn remains unaffected  <cit> .
consistent with these observations, our data demonstrate that per expression increases when mice are kept awake  <cit> .
the lack or alteration of particular clock genes in the various mouse models mentioned above, changes the expression levels of other clock genes in the circuitry.
for instance, basal per expression levels are decreased in clockΔ/Δ mice  <cit>  and increased in cry <dig> -/- mice  <cit>  which corresponds to their opposing sleep phenotypes .
also, the sd-induced increase in per expression is reduced in npas2-/- mice  <cit>  which suggest that npas <dig> is implicated in linking per <dig> expression to the sleep-wake distribution.
likewise, npas2-/- mice lack circadian rhythms in cortical per <dig> expression despite an intact circadian organization of sleep and waking  <cit> .
the finding that npas <dig> couples wakefulness to clock gene expression is of particular interest given the evidence that npas <dig>  -mediated transcription is redox sensitive  <cit> .
redox sensitivity suggests that this clock gene network may be linked to cellular energy metabolism  <cit> .
since restoration of an optimal neural energy state has long been considered a possible function of sleep  <cit> , this gene network might underlie a fundamental aspect of sleep homeostasis.
if so, another critical gene in this network is likely to be ldha .
ldha can be induced in cell culture by npas <dig>  has multiple e-box sites, is a critical enzyme for neuronal energy metabolism, and utilizes nadh as a co-factor  <cit> .
therefore, we decided to investigate gene expression in the forebrain in greater detail over the course of a sd and after recovery sleep for the clock and npas <dig> target genes per <dig> per <dig>  dbp, and ldha.
futhermore, we undertook this examination of the sleep-wake dependent changes in gene expression in three inbred strains of mice known to differ in the rate at which sleep need, quantified as eeg delta power, increases during wakefulness  <cit> .
we reasoned that if clock genes are implicated in sleep homeostasis, then the time course of changes in gene expression during a sd in these strains should vary according to the published differences in eeg delta power dynamics.
RESULTS
control conditions
under undisturbed control conditions, forebrain levels of per <dig>  per <dig>  and dbp expression varied both as a function of time-of-day and of genotype .
generally, all three genes reached highest expression levels at zt <dig> , but there were time-course differences.
the values for dbp expression were lowest at zt <dig> and zt <dig> and then increased throughout the light period.
in contrast, the expression of per <dig> and per <dig> first decreased from zt <dig> reaching lowest levels at zt <dig> or - <dig> before increasing.
the time courses observed here match well those previously published for most brain areas outside the scn and for certain other peripheral tissues  <cit>  with lowest mrna values observed early  or mid  day  and peak levels being reached around zt <dig> for dbp or around zt12- <dig> for per <dig> and per <dig> .
no systematic variation in ldha expression levels was observed across the  <dig> time points in any strain.
although the time-dependent changes in gene expression did not differ among the  <dig> inbred strains, genetic background did affect overall expression levels within the control group.
with few exceptions, expression of per <dig>  per <dig>  and dbp was higher in d <dig> compared to ak and b <dig> mice at all four time-points .
elevated expression levels for d <dig> were maintained even during sd-conditions .
genotype did not affect the level of ldha expression.
sleep deprivation
against this varying background the effects of enforced wakefulness on the expression of clock and clock-controlled genes was evaluated.
confirming our previous results in b <dig> mice  <cit> ,  <dig> h sd increased per <dig> and per <dig> expression and decreased dbp expression in comparison to time controls .
the present experiment allowed us to follow the time course of gene expression during the  <dig> h sd and also enabled us to assess and compare the dynamics among mice of different genetic backgrounds.
sd changed the expression of these genes in the same direction in all three strains, however, significant genotype differences were observed in the dynamics and the magnitude of these changes .
in ak mice per <dig> and per <dig> expression was significantly increased after only  <dig> h of sd compared to control levels at zt <dig>  in d <dig> mice only per <dig> was increased after  <dig> h of sd, and in b <dig> mice neither per <dig> nor per <dig> were increased by  <dig> h of sd.
however, per <dig> and per <dig> mrna in ak mice did not increase further as sd progressed whereas in d <dig> mice  a progressive increase was observed from 1- to 3- to  <dig> h sd .
forebrain mrna levels of dbp increased over the  <dig> h of the experiment in both control and sd groups of d <dig> and b <dig> mice, but the sd values tended to be lower than the control values after  <dig> and  <dig> h sd.
the increase of per <dig>  per <dig>  and dbp over the course of the sd was quantified by linear regression analysis which demonstrated that the rate of increase was significantly greater in d <dig> mice compared to ak mice .
the largest changes  in mrna levels were observed after  <dig> h sd.
for per <dig> and per <dig> expression the increase at this time point differed significantly among inbred strains .
in d <dig> mice the per <dig> increase  was larger than in b <dig> mice  and the per <dig> increase  larger than in ak mice .
sleep deprivation did not affect ldha expression.
the genotype specific changes in clock-gene expression during sd , the genotype differences in the average expression levels during both control and sd , and the larger relative increase in per <dig> and per <dig> after  <dig> h sd are all opposite to the strain differences we previously reported in the dynamics of the homeostatic regulation of sleep  <cit> .
for this phenotype the rate of increase in sleep need during wakefulness  was fastest in ak mice and slowest in d <dig> mice .
this suggests a negative correlation between the magnitude of the increase in these clock genes and the homeostatic regulation of sleep.
to facilitate a direct comparison between our present and our previous study we made every effort to follow the same experimental protocol and conditions including the ld cycle, light intensity, ambient temperature, animal provider, sex and age of the mice, acclimatization time, start time and method of sleep deprivation.
recovery sleep
two hours of recovery sleep affected per <dig> and per <dig> forebrain mrna levels in a strain specific manner .
per <dig> expression sharply decreased from the levels reached at the end of the  <dig> h sd and in ak mice values reached at zt <dig> even fell below control levels.
a similar recovery pattern was observed for per <dig> expression with the exception of d2; in this strain, recovery sleep could not lower per <dig> mrna levels and values remained significantly elevated compared to control.
conversely, only in d <dig> mice did the decrease in dbp expression revert to control levels.
correlations
the expression of all four genes studied here are regulated by clock and npas <dig> through e-boxes  <cit>  although other cis-acting elements are known to be involved; e.g., camp-response and dbp-binding elements  <cit> .
given the likelihood of shared transcriptional regulation through e-boxes, we tested expression patterns for similarities among these four genes and how these relationships were affected by the sd .
under both control and sd conditions the two period genes were strongly correlated sharing  <dig> and 42% of the variance, respectively .
other significant correlations observed during control conditions were between per <dig> and dbp  and between dbp and ldha  expression.
these relationships changed dramatically with sd; per <dig> expression, which was affected the most by sd, now became correlated with that of the other  <dig> genes while the per1-dbp and dbp-ldha correlations no longer reached significance levels and the per2-dbp correlation, that was not significant during control, became equally important as the per1-per <dig> correlation .
the sd-induced increase in the correlation coefficient between per <dig> and dbp expression  and the decrease observed for the per1-dbp correlation  were significant .
within strains, the same relationships were observed, although they did not always reach significance levels .
in situ hybridization
to gain insight into which forebrain regions contributed to the sd-induced changes in per <dig> mrna levels reported above, we performed in situ hybridizations.
during control conditions, forebrain expression levels were low, especially in the cerebral cortex, while expression in the cerebellum was high , matching previously published studies  <cit> .
following  <dig> h sd, per <dig> expression increased throughout the brain .
this increase was especially pronounced in the cerebral cortex, consistent with our qpcr data .
within the cortex, increases were especially notable in the frontal cortex and in and around the cingulate cortex.
expression in the cerebellum was also importantly increased.
expression patterns and effects of sd described here for per <dig> match those of per <dig> and per <dig> publicly available from the allen institute for brain science website  <cit>  .
discussion
we previously found that cortical mrna levels of the clock genes per <dig> and per <dig> were increased after sd in rats and b <dig> mice  <cit> .
here we have extended that observation in two additional inbred strains; ak and d <dig>  furthermore, by following the time course of these changes during sd and recovery sleep, we established that per expression changes as a function of the time-spent-awake and asleep.
such a time course is consistent with a role for these genes in the homeostatic regulation of sleep since the need for sleep increases with duration of wake and decreases with duration of sleep.
a novel aspect of this study is the comparison of the sd-induced changes in gene expression among three inbred strains for which the homeostatic regulation of sleep differs  <cit> .
during both spontaneous and enforced periods of wakefulness the need for sleep, indexed by the level of eeg delta power, increased fastest in ak, intermediate in b <dig>  and slowest in d <dig> mice.
if indeed per expression would simply parallel eeg delta power we would expect to find the largest increase in per expression after sd in ak mice and the smallest in d <dig>  here we found evidence for the opposite; although per expression was consistently increased with sd, this increase was modulated by genotype and was found to be highest in d <dig> and lowest in ak.
in addition, per <dig> expression remained elevated during recovery sleep in d <dig> mice only.
thus high, sustained levels of per <dig> expression might negatively impact the sleep recovery process and/or expression of eeg delta oscillations during nrems.
an implicit assumption in our interpretation of our results is that the sleep-wake dependent changes in the expression of per <dig>  per <dig>  and dbp are mediated by clock and/or npas <dig> induced transcription.
the observation in npas2-/- mice that cortical per <dig> expression no longer follows the circadian sleep-wake distribution  <cit> , together with the observations that the increase in per <dig> expression after sd is reduced in these mice  <cit> , and that the homeostatic response to sd is altered in npas2-/-, cry <dig> -/-, and bmal1-/- mice  strongly support this assumption.
nevertheless, expression of the genes we examined can be induced by other transcriptional regulators as well.
e.g., per <dig> can be readily induced by camp-responsive element-binding protein   <cit> .
creb signaling also increases during prolonged wakefulness  <cit>  and thus might have contributed to the sd-dependent changes in per <dig> reported here.
another example is the close link between glucocorticoid signaling and per <dig> and per <dig> expression  <cit>  through which sd, which is accompanied by an increase in plasma corticosterone levels, could have affected per expression.
in fact, we found that the sd-induced increase in corticosterone levels was 3-fold higher in d <dig> mice compared to the other two strains  consistent with the larger relative increase in per expression in this strain observed here.
evidence for an altered transcriptional regulation of the four genes during sd  is provided by the altered relationship between the expression patterns we report here.
the cortical expression of dbp was decreased after sd confirming previously published data  <cit> .
given the fact that per <dig>  per <dig>  and dbp transcription are, in part, under the same transcriptional control, these opposite effects of the sd are not easily explained.
per proteins are co-repressors of clock and npas <dig> induced transcription.
thus, assuming that increased per expression is followed by increased per levels within the time frame of the sd, per-mediated transcriptional repression is an obvious possible pathway by which dbp expression could be decreased.
in line with this assertion, we found that the decrease in dbp expression to be largest in the strain with the largest increase in per expression; i.e.
d <dig>  the observation that per and dbp expression were nevertheless positively correlated during the sd is, however, inconsistent with this scenario.
in addition, per expression itself could be expected to decrease as well, at least the contribution of clock and npas <dig> mediated transcription to the total per mrna.
finally, the time course of the peripheral changes in dbp and per expression under control conditions also doesn't support a dbp suppressing role for per.
dbp mrna levels peak at zt <dig> and are declining when per mrna starts to increase  <cit> .
the in situ hybridization studies indicated that sd induced pronounced changes in per expression in cerebellum and cerebral cortex.
this is in line with our previous findings using qpcr showing that per expression was increased in the cerebral cortex but not in brainstem and hypothalamus  <cit>  and with the per <dig> increase observed after sd both in cerebellum and cerebral cortex  <cit> .
while the in situ data are limited, we observed some of the highest expression and largest increases in per <dig> mrna in the most frontal area of the cortex.
eeg delta power is generally higher and its increase after sd more pronounced in frontal cortex relative to other cortical areas such as occipital regions  <cit>  in accordance with the concept that frontal areas in particular are affected by sd  <cit> .
in mice lacking a functional per <dig> protein  eeg delta power was decreased in the frontal derivation specifically and the sd-induced increase in eeg delta power was smaller in per1Δ/Δ and per2Δ/Δ mice also only in the frontal derivation  <cit> .
already clear from the present data is that not all changes in forebrain per expression can be attributed to changes in the sleep-wake distribution; expression increases during the latter part of the light period while animals remain mostly asleep.
this increase precedes dark onset after which maximal per expression levels are reached when mice are the most active  <cit> .
a major adaptive advantage provided by circadian rhythms is that it allows organisms to anticipate and prepare for predictable periodic changes in the environment  <cit> .
the anticipatory increase in per expression might be part of a molecular repertoire preparing the animal for the active period.
some types of anticipatory behavior, however, don't require an intact circadian system, as food-restriction studies have shown  <cit> .
moreover, clock-genes in the forebrain seem to play a permissive role in such anticipatory behaviors.
for example, npas2-/- mice, which have intact circadian rhythms in overt behavior, have difficulty adapting to and anticipating altered feeding schedules  <cit> .
besides food intake, other homeostatically regulated behaviors such as sleep seem to be similarly affected  <cit> .
thus, the same molecular network of transcriptional regulators that sets internal time-of-day in the scn might, in the forebrain, track and signal time without sleep .
conceptually the distinction between homeostatic and circadian processes at the molecular level thus becomes unclear.
mechanistically we proposed that changes in clock-gene expression could be tied directly to cellular energy state based mainly on the observation that clock and npas <dig> mediated transcription is sensitive to redox state  <cit> .
in this respect the lack of an effect of sd on ldha expression is disappointing.
although ldha has a canonical e-box and has been shown to be activated by both npas <dig> and clock in a neuroblastoma cell line  <cit> , mrna levels of this gene were surprisingly stable across time-of-day, experimental condition, and genetic background.
CONCLUSIONS
we found that the expression of three circadian clock genes changes as a function of time-spent-awake and that these changes were influenced by strain.
these results further indicate that circadian clock genes play a role in the homeostatic regulation of sleep.
additional evidence in support of our hypothesis has recently been obtained in humans homozygous for one of two known polymorphisms in hper <dig>  <cit> .
based on scn lesion experiments in rats  <cit>  and forced desynchrony studies in humans  <cit>  it is generally thought that homeostatic and circadian processes operate separately and have different neurophysiological substrates.
our current results underscore that, at least at a molecular level, these two processes are difficult to separate.
recent data also show that at the neuro-physiological level, sd can affect the activity of scn neurons previously thought to be dedicated exclusively to setting internal time-of-day <cit> .
finally, the present results might be of relevance for research on the biology of mood disorders.
in this field there is increasing interest in the relation between clock-genes and e.g.
seasonal affective disorders  <cit>  or bipolar disorders  <cit> .
the effects of sleep deprivation on clock-gene expression demonstrated here combined with the fact that sleep deprivation is a well known antidepressant add a molecular component to the intricate relationship between mood, circadian rhythms, and sleep .

 at the cellular level, circadian rhythms are thought to be generated by transcriptional-translational feedback loops made up of positively and negatively acting transcriptional regulators. in mammals, the core positive elements are clock and npas and their obligate dimerization partner bmal these transcription factors drive per and cry transcription. per and cry proteins, in turn, interact with clock/npas2:bmal heterodimers to inhibit their own transcription, thus constituting the negative feedback elements. the circuitry is clearly more complex as additional feedback loops and post-translational modifications are involved. clock genes underlie circadian rhythm generation in many tissues, but the circadian rhythm in the suprachiasmatic nucleus is required for the manifestation of overt physiological and behavioral rhythms, and is therefore considered the master circadian pacemaker. although the role of clock genes in generating circadian rhythms is firmly established, we and others have found that they also play a role in the homeostatic regulation of non-rem sleep. this notion is based on the observation that mice deficient for one or more clock genes show an altered response to sleep deprivation and on the observation that clock-gene expression in brain areas outside the scn, notable the cerebral cortex, is influenced by the prior occurrence of sleep and wake. for instance, the typical responses in nrems to sd were reduced or absent in mice lacking the cry and - or bmal genes compared to wild-type controls. the diminished nrems variables included duration, consolidation, and eeg delta power. eeg delta power quantifies the prevalence and amplitude of delta oscillations and is widely used to index the homeostatic need for nrems. sleep under undisturbed, baseline conditions was also altered in mutant mice. clock-mutant and npas2-/- mice spent less time in nrems whereas cry -/- and bmal1-/- mice slept more. nrems consolidation was increased in cry -/- mice while the opposite was observed in clockΔ/Δ and bmal1-/- mice and in mice lacking albumin d-binding protein; a gene under the transcriptional control of clock and that also has been implicated in circadian rhythm generation. average levels of eeg delta power were reduced in clockΔ/Δ, dbp-/-, and bmal1-/- mice and increased in cry -/- mice. clock-gene expression in the brain, notably the cerebral cortex, depends on the sleep-wake distribution. cortical levels of per are high at times when sleep drive is high, irrespective of the phase at which the circadian expression of per peaks in the scn. thus, in both nocturnal and diurnal species, per expression in the cortex is maximal in conjunction with the major waking episode. under conditions where the phase or distribution of locomotor activity is altered, per expression in the cortex parallels the overt rhythm of wakefulness, while per expression in the scn remains unaffected. consistent with these observations, our data demonstrate that per expression increases when mice are kept awake. the lack or alteration of particular clock genes in the various mouse models mentioned above, changes the expression levels of other clock genes in the circuitry. for instance, basal per expression levels are decreased in clockΔ/Δ mice and increased in cry -/- mice which corresponds to their opposing sleep phenotypes. also, the sd-induced increase in per expression is reduced in npas2-/- mice which suggest that npas is implicated in linking per expression to the sleep-wake distribution. likewise, npas2-/- mice lack circadian rhythms in cortical per expression despite an intact circadian organization of sleep and waking. the finding that npas couples wakefulness to clock gene expression is of particular interest given the evidence that npas -mediated transcription is redox sensitive. redox sensitivity suggests that this clock gene network may be linked to cellular energy metabolism. since restoration of an optimal neural energy state has long been considered a possible function of sleep, this gene network might underlie a fundamental aspect of sleep homeostasis. if so, another critical gene in this network is likely to be ldha. ldha can be induced in cell culture by npas has multiple e-box sites, is a critical enzyme for neuronal energy metabolism, and utilizes nadh as a co-factor. therefore, we decided to investigate gene expression in the forebrain in greater detail over the course of a sd and after recovery sleep for the clock and npas target genes per per dbp, and ldha. futhermore, we undertook this examination of the sleep-wake dependent changes in gene expression in three inbred strains of mice known to differ in the rate at which sleep need, quantified as eeg delta power, increases during wakefulness. we reasoned that if clock genes are implicated in sleep homeostasis, then the time course of changes in gene expression during a sd in these strains should vary according to the published differences in eeg delta power dynamics.  control conditions under undisturbed control conditions, forebrain levels of per per and dbp expression varied both as a function of time-of-day and of genotype. generally, all three genes reached highest expression levels at zt, but there were time-course differences. the values for dbp expression were lowest at zt and zt and then increased throughout the light period. in contrast, the expression of per and per first decreased from zt reaching lowest levels at zt or - before increasing. the time courses observed here match well those previously published for most brain areas outside the scn and for certain other peripheral tissues with lowest mrna values observed early or mid day and peak levels being reached around zt for dbp or around zt12- for per and per. no systematic variation in ldha expression levels was observed across the time points in any strain. although the time-dependent changes in gene expression did not differ among the inbred strains, genetic background did affect overall expression levels within the control group. with few exceptions, expression of per per and dbp was higher in d compared to ak and b mice at all four time-points. elevated expression levels for d were maintained even during sd-conditions. genotype did not affect the level of ldha expression. sleep deprivation against this varying background the effects of enforced wakefulness on the expression of clock and clock-controlled genes was evaluated. confirming our previous results in b mice, h sd increased per and per expression and decreased dbp expression in comparison to time controls. the present experiment allowed us to follow the time course of gene expression during the h sd and also enabled us to assess and compare the dynamics among mice of different genetic backgrounds. sd changed the expression of these genes in the same direction in all three strains, however, significant genotype differences were observed in the dynamics and the magnitude of these changes. in ak mice per and per expression was significantly increased after only h of sd compared to control levels at zt in d mice only per was increased after h of sd, and in b mice neither per nor per were increased by h of sd. however, per and per mrna in ak mice did not increase further as sd progressed whereas in d mice a progressive increase was observed from 1- to 3- to h sd. forebrain mrna levels of dbp increased over the h of the experiment in both control and sd groups of d and b mice, but the sd values tended to be lower than the control values after and h sd. the increase of per per and dbp over the course of the sd was quantified by linear regression analysis which demonstrated that the rate of increase was significantly greater in d mice compared to ak mice. the largest changes in mrna levels were observed after h sd. for per and per expression the increase at this time point differed significantly among inbred strains. in d mice the per increase was larger than in b mice and the per increase larger than in ak mice. sleep deprivation did not affect ldha expression. the genotype specific changes in clock-gene expression during sd, the genotype differences in the average expression levels during both control and sd, and the larger relative increase in per and per after h sd are all opposite to the strain differences we previously reported in the dynamics of the homeostatic regulation of sleep. for this phenotype the rate of increase in sleep need during wakefulness was fastest in ak mice and slowest in d mice. this suggests a negative correlation between the magnitude of the increase in these clock genes and the homeostatic regulation of sleep. to facilitate a direct comparison between our present and our previous study we made every effort to follow the same experimental protocol and conditions including the ld cycle, light intensity, ambient temperature, animal provider, sex and age of the mice, acclimatization time, start time and method of sleep deprivation. recovery sleep two hours of recovery sleep affected per and per forebrain mrna levels in a strain specific manner. per expression sharply decreased from the levels reached at the end of the h sd and in ak mice values reached at zt even fell below control levels. a similar recovery pattern was observed for per expression with the exception of d2; in this strain, recovery sleep could not lower per mrna levels and values remained significantly elevated compared to control. conversely, only in d mice did the decrease in dbp expression revert to control levels. correlations the expression of all four genes studied here are regulated by clock and npas through e-boxes although other cis-acting elements are known to be involved; e.g., camp-response and dbp-binding elements. given the likelihood of shared transcriptional regulation through e-boxes, we tested expression patterns for similarities among these four genes and how these relationships were affected by the sd. under both control and sd conditions the two period genes were strongly correlated sharing and 42% of the variance, respectively. other significant correlations observed during control conditions were between per and dbp and between dbp and ldha expression. these relationships changed dramatically with sd; per expression, which was affected the most by sd, now became correlated with that of the other genes while the per1-dbp and dbp-ldha correlations no longer reached significance levels and the per2-dbp correlation, that was not significant during control, became equally important as the per1-per correlation. the sd-induced increase in the correlation coefficient between per and dbp expression and the decrease observed for the per1-dbp correlation were significant. within strains, the same relationships were observed, although they did not always reach significance levels. in situ hybridization to gain insight into which forebrain regions contributed to the sd-induced changes in per mrna levels reported above, we performed in situ hybridizations. during control conditions, forebrain expression levels were low, especially in the cerebral cortex, while expression in the cerebellum was high, matching previously published studies. following h sd, per expression increased throughout the brain. this increase was especially pronounced in the cerebral cortex, consistent with our qpcr data. within the cortex, increases were especially notable in the frontal cortex and in and around the cingulate cortex. expression in the cerebellum was also importantly increased. expression patterns and effects of sd described here for per match those of per and per publicly available from the allen institute for brain science website. discussion we previously found that cortical mrna levels of the clock genes per and per were increased after sd in rats and b mice. here we have extended that observation in two additional inbred strains; ak and d furthermore, by following the time course of these changes during sd and recovery sleep, we established that per expression changes as a function of the time-spent-awake and asleep. such a time course is consistent with a role for these genes in the homeostatic regulation of sleep since the need for sleep increases with duration of wake and decreases with duration of sleep. a novel aspect of this study is the comparison of the sd-induced changes in gene expression among three inbred strains for which the homeostatic regulation of sleep differs. during both spontaneous and enforced periods of wakefulness the need for sleep, indexed by the level of eeg delta power, increased fastest in ak, intermediate in b and slowest in d mice. if indeed per expression would simply parallel eeg delta power we would expect to find the largest increase in per expression after sd in ak mice and the smallest in d here we found evidence for the opposite; although per expression was consistently increased with sd, this increase was modulated by genotype and was found to be highest in d and lowest in ak. in addition, per expression remained elevated during recovery sleep in d mice only. thus high, sustained levels of per expression might negatively impact the sleep recovery process and/or expression of eeg delta oscillations during nrems. an implicit assumption in our interpretation of our results is that the sleep-wake dependent changes in the expression of per per and dbp are mediated by clock and/or npas induced transcription. the observation in npas2-/- mice that cortical per expression no longer follows the circadian sleep-wake distribution, together with the observations that the increase in per expression after sd is reduced in these mice, and that the homeostatic response to sd is altered in npas2-/-, cry -/-, and bmal1-/- mice strongly support this assumption. nevertheless, expression of the genes we examined can be induced by other transcriptional regulators as well. e.g., per can be readily induced by camp-responsive element-binding protein. creb signaling also increases during prolonged wakefulness and thus might have contributed to the sd-dependent changes in per reported here. another example is the close link between glucocorticoid signaling and per and per expression through which sd, which is accompanied by an increase in plasma corticosterone levels, could have affected per expression. in fact, we found that the sd-induced increase in corticosterone levels was 3-fold higher in d mice compared to the other two strains consistent with the larger relative increase in per expression in this strain observed here. evidence for an altered transcriptional regulation of the four genes during sd is provided by the altered relationship between the expression patterns we report here. the cortical expression of dbp was decreased after sd confirming previously published data. given the fact that per per and dbp transcription are, in part, under the same transcriptional control, these opposite effects of the sd are not easily explained. per proteins are co-repressors of clock and npas induced transcription. thus, assuming that increased per expression is followed by increased per levels within the time frame of the sd, per-mediated transcriptional repression is an obvious possible pathway by which dbp expression could be decreased. in line with this assertion, we found that the decrease in dbp expression to be largest in the strain with the largest increase in per expression; i.e. d the observation that per and dbp expression were nevertheless positively correlated during the sd is, however, inconsistent with this scenario. in addition, per expression itself could be expected to decrease as well, at least the contribution of clock and npas mediated transcription to the total per mrna. finally, the time course of the peripheral changes in dbp and per expression under control conditions also doesn't support a dbp suppressing role for per. dbp mrna levels peak at zt and are declining when per mrna starts to increase. the in situ hybridization studies indicated that sd induced pronounced changes in per expression in cerebellum and cerebral cortex. this is in line with our previous findings using qpcr showing that per expression was increased in the cerebral cortex but not in brainstem and hypothalamus and with the per increase observed after sd both in cerebellum and cerebral cortex. while the in situ data are limited, we observed some of the highest expression and largest increases in per mrna in the most frontal area of the cortex. eeg delta power is generally higher and its increase after sd more pronounced in frontal cortex relative to other cortical areas such as occipital regions in accordance with the concept that frontal areas in particular are affected by sd. in mice lacking a functional per protein eeg delta power was decreased in the frontal derivation specifically and the sd-induced increase in eeg delta power was smaller in per1Δ/Δ and per2Δ/Δ mice also only in the frontal derivation. already clear from the present data is that not all changes in forebrain per expression can be attributed to changes in the sleep-wake distribution; expression increases during the latter part of the light period while animals remain mostly asleep. this increase precedes dark onset after which maximal per expression levels are reached when mice are the most active. a major adaptive advantage provided by circadian rhythms is that it allows organisms to anticipate and prepare for predictable periodic changes in the environment. the anticipatory increase in per expression might be part of a molecular repertoire preparing the animal for the active period. some types of anticipatory behavior, however, don't require an intact circadian system, as food-restriction studies have shown. moreover, clock-genes in the forebrain seem to play a permissive role in such anticipatory behaviors. for example, npas2-/- mice, which have intact circadian rhythms in overt behavior, have difficulty adapting to and anticipating altered feeding schedules. besides food intake, other homeostatically regulated behaviors such as sleep seem to be similarly affected. thus, the same molecular network of transcriptional regulators that sets internal time-of-day in the scn might, in the forebrain, track and signal time without sleep. conceptually the distinction between homeostatic and circadian processes at the molecular level thus becomes unclear. mechanistically we proposed that changes in clock-gene expression could be tied directly to cellular energy state based mainly on the observation that clock and npas mediated transcription is sensitive to redox state. in this respect the lack of an effect of sd on ldha expression is disappointing. although ldha has a canonical e-box and has been shown to be activated by both npas and clock in a neuroblastoma cell line, mrna levels of this gene were surprisingly stable across time-of-day, experimental condition, and genetic background.  we found that the expression of three circadian clock genes changes as a function of time-spent-awake and that these changes were influenced by strain. these results further indicate that circadian clock genes play a role in the homeostatic regulation of sleep. additional evidence in support of our hypothesis has recently been obtained in humans homozygous for one of two known polymorphisms in hper. based on scn lesion experiments in rats and forced desynchrony studies in humans it is generally thought that homeostatic and circadian processes operate separately and have different neurophysiological substrates. our current results underscore that, at least at a molecular level, these two processes are difficult to separate. recent data also show that at the neuro-physiological level, sd can affect the activity of scn neurons previously thought to be dedicated exclusively to setting internal time-of-day. finally, the present results might be of relevance for research on the biology of mood disorders. in this field there is increasing interest in the relation between clock-genes and e.g. seasonal affective disorders or bipolar disorders. the effects of sleep deprivation on clock-gene expression demonstrated here combined with the fact that sleep deprivation is a well known antidepressant add a molecular component to the intricate relationship between mood, circadian rhythms, and sleep. 
we reasoned that if clock genes are functionally implicated in sleep homeostasis then the sd-induced changes in gene expression should vary according to the genotypic differences in the sleep rebound. we have previously reported that the expression of circadian clock-genes increases in the cerebral cortex after sleep deprivation and that the sleep rebound following sd is attenuated in mice deficient for one or more clock-genes. here we follow the time course of the forebrain changes in the expression of the clock-genes period - per and of the clock-controlled gene albumin d-binding protein during a h sd and subsequent recovery sleep in three inbred strains of mice for which the homeostatic sleep rebound following sd differs. moreover, whereas in the other two strains per and per reverted to control levels with recovery sleep, per expression specifically, remained elevated in d mice. in contrast to per dbp expression reverted to control levels with recovery sleep in d only, whereas in the two other strains expression remained decreased. these findings support and extend our previous findings that clock genes in the forebrain are implicated in the homeostatic regulation of sleep and suggest that sustained, high levels of per expression may negatively impact recovery sleep. dbp expression increased during the light period both during baseline and during sd although levels were reduced during the latter condition compared to baseline. we hypothesized that besides generating circadian rhythms, clock-genes also play a role in the homeostatic regulation of sleep. in all three strains per expression was increased when animals were kept awake but the rate of increase during the sd as well as the relative increase in per after h sd were highest in the strain for which the sleep rebound was smallest; i.e., dba/2j.
 we have previously reported that the expression of circadian clock-genes increases in the cerebral cortex after sleep deprivation and that the sleep rebound following sd is attenuated in mice deficient for one or more clock-genes. we hypothesized that besides generating circadian rhythms, clock-genes also play a role in the homeostatic regulation of sleep. here we follow the time course of the forebrain changes in the expression of the clock-genes period - per and of the clock-controlled gene albumin d-binding protein during a h sd and subsequent recovery sleep in three inbred strains of mice for which the homeostatic sleep rebound following sd differs. we reasoned that if clock genes are functionally implicated in sleep homeostasis then the sd-induced changes in gene expression should vary according to the genotypic differences in the sleep rebound.  in all three strains per expression was increased when animals were kept awake but the rate of increase during the sd as well as the relative increase in per after h sd were highest in the strain for which the sleep rebound was smallest; i.e., dba/2j. moreover, whereas in the other two strains per and per reverted to control levels with recovery sleep, per expression specifically, remained elevated in d mice. dbp expression increased during the light period both during baseline and during sd although levels were reduced during the latter condition compared to baseline. in contrast to per dbp expression reverted to control levels with recovery sleep in d only, whereas in the two other strains expression remained decreased.  these findings support and extend our previous findings that clock genes in the forebrain are implicated in the homeostatic regulation of sleep and suggest that sustained, high levels of per expression may negatively impact recovery sleep. 