Image_1_Sex-specific regulation of the cortical transcriptome in response to sleep deprivation.png

Multiple studies have documented sex differences in sleep behaviour, however, the molecular determinants of such differences remain unknown. Furthermore, most studies addressing molecular mechanisms have been performed only in males, leaving the current state of knowledge biased towards the male sex. To address this, we studied the differences in the transcriptome of the cerebral cortex of male and female C57Bl/6 J mice after 6 h of sleep deprivation. We found that several genes, including the neurotrophin growth factor Bdnf, immediate early genes Fosb and Fosl2, and the adenylate cyclase Adcy7 are differentially upregulated in males compared to females. We identified the androgen-receptor activating transcription factor EZH2 as the upstream regulatory element specifying sex differences in the sleep deprivation transcriptome. We propose that the pathways downstream of these transcripts, which impact on cellular re-organisation, synaptic signalling, and learning may underpin the differential response to sleep deprivation in the two sexes.</p

Table_1_Sex-specific regulation of the cortical transcriptome in response to sleep deprivation.XLSX

Multiple studies have documented sex differences in sleep behaviour, however, the molecular determinants of such differences remain unknown. Furthermore, most studies addressing molecular mechanisms have been performed only in males, leaving the current state of knowledge biased towards the male sex. To address this, we studied the differences in the transcriptome of the cerebral cortex of male and female C57Bl/6 J mice after 6 h of sleep deprivation. We found that several genes, including the neurotrophin growth factor Bdnf, immediate early genes Fosb and Fosl2, and the adenylate cyclase Adcy7 are differentially upregulated in males compared to females. We identified the androgen-receptor activating transcription factor EZH2 as the upstream regulatory element specifying sex differences in the sleep deprivation transcriptome. We propose that the pathways downstream of these transcripts, which impact on cellular re-organisation, synaptic signalling, and learning may underpin the differential response to sleep deprivation in the two sexes.</p

Image_2_Sex-specific regulation of the cortical transcriptome in response to sleep deprivation.png

Multiple studies have documented sex differences in sleep behaviour, however, the molecular determinants of such differences remain unknown. Furthermore, most studies addressing molecular mechanisms have been performed only in males, leaving the current state of knowledge biased towards the male sex. To address this, we studied the differences in the transcriptome of the cerebral cortex of male and female C57Bl/6 J mice after 6 h of sleep deprivation. We found that several genes, including the neurotrophin growth factor Bdnf, immediate early genes Fosb and Fosl2, and the adenylate cyclase Adcy7 are differentially upregulated in males compared to females. We identified the androgen-receptor activating transcription factor EZH2 as the upstream regulatory element specifying sex differences in the sleep deprivation transcriptome. We propose that the pathways downstream of these transcripts, which impact on cellular re-organisation, synaptic signalling, and learning may underpin the differential response to sleep deprivation in the two sexes.</p

Representative actograms of <i>Grm2/3</i>^+/+ and <i>Grm2/3</i>^-/- mice during a 12:12 h light/dark (12:12 LD) cycle.

<p>Each row depicts a single 24 h period. The light and dark grey shading corresponds to periods of (100 lux) light and dark, respectively. (A) Representative wheel-running actograms. The black bars denote periods of wheel-running activity, binned in 6 min epochs. The height of the bars corresponds to the number of wheel rotations within each epoch. (B) Representative passive-infrared (PIR) actograms. The black bars denote periods of home-cage activity, binned in 6 min epochs. The height of the bars corresponds to % time active within each epoch. ZT = zeitgeber time.</p

Light sensitivity is increased by the genetic ablation or pharmacological inhibition of mGlu2 & 3.

<p>All figures depict rest-activity or circadian parameters derived from wheel-running data, with the exception of Fig 2F, which is based on video-tracking data. (A) Free-running period length in constant dark (DD) does not differ between <i>Grm2/3</i>^<b>+/+</b> and <i>Grm2/3</i>^<b>-/-</b> mice. (B) Free-running period length in constant light (LL) is greater in <i>Grm2/3</i>^<b>-/-</b> than <i>Grm2/3</i>^<b>+/+</b> mice. (C & D) Phase delays induced by type I (Fig 2C) and type II (Fig 2D) phase-shifting light pulses are larger in <i>Grm2/3</i>^<b>-/-</b> than <i>Grm2/3</i>^<b>+/+</b> mice. (E & F) <i>Grm2/3</i>^<b>+/+</b> and <i>Grm2/3</i>^<b>-/-</b> mice show similar levels of negative masking during a type II light pulse, as assayed by wheel-running (Fig 2E) and video-tracking (Fig 2F). (G) In wildtype C57Bl/6J mice, phase delays induced by a type I light pulse are enhanced following the administration of the mGlu2/3 negative allosteric modulator (NAM) RO4432717.</p

Deletion of Metabotropic Glutamate Receptors 2 and 3 (mGlu2 & mGlu3) in Mice Disrupts Sleep and Wheel-Running Activity, and Increases the Sensitivity of the Circadian System to Light

<div><p>Sleep and/or circadian rhythm disruption (SCRD) is seen in up to 80% of schizophrenia patients. The co-morbidity of schizophrenia and SCRD may in part stem from dysfunction in common brain mechanisms, which include the glutamate system, and in particular, the group II metabotropic glutamate receptors mGlu2 and mGlu3 (encoded by the genes <i>Grm2</i> and <i>Grm3</i>). These receptors are relevant to the pathophysiology and potential treatment of schizophrenia, and have also been implicated in sleep and circadian function. In the present study, we characterised the sleep and circadian rhythms of <i>Grm2/3</i> double knockout (<i>Grm2/3</i>^-/-) mice, to provide further evidence for the involvement of group II metabotropic glutamate receptors in the regulation of sleep and circadian rhythms. We report several novel findings. Firstly, <i>Grm2/3</i>^-/- mice demonstrated a decrease in immobility-determined sleep time and an increase in immobility-determined sleep fragmentation. Secondly, <i>Grm2/3</i>^-/- mice showed heightened sensitivity to the circadian effects of light, manifested as increased period lengthening in constant light, and greater phase delays in response to nocturnal light pulses. Greater light-induced phase delays were also exhibited by wildtype C57Bl/6J mice following administration of the mGlu2/3 negative allosteric modulator RO4432717. These results confirm the involvement of group II metabotropic glutamate receptors in photic entrainment and sleep regulation pathways. Finally, the diurnal wheel-running rhythms of <i>Grm2/3</i>^-/- mice were perturbed under a standard light/dark cycle, but their diurnal rest-activity rhythms were unaltered in cages lacking running wheels, as determined with passive infrared motion detectors. Hence, when assessing the diurnal rest-activity rhythms of mice, the choice of assay can have a major bearing on the results obtained.</p></div

Dark phase wheel-running activity is reduced in Grm2/3^-/- mice, but general home-cage activity is not.

<p>Fig 3A-C depict activity parameters derived from 14 consecutive days of wheel-running data. Fig 3D-F depict activity parameters derived from 3 separate days of video-tracking data. Fig 3G-I depict activity parameters derived from 14 consecutive days of passive-infrared (PIR) data. (A, D & G) Average activity profiles, (B, E & H) light phase activity, and (C, F & I) dark phase activity for <i>Grm2/3</i>^<b>+/+</b> and <i>Grm2/3</i>^<b>-/-</b> mice during a 12:12 h light/dark (12:12 LD) cycle at 100 lux. Data in Fig 3A, D & G are presented in 2 h time bins. ZT = zeitgeber time. AU = arbitrary units.</p

The diurnal rest-activity rhythms of <i>Grm2/3</i>^-/- mice are dependent on the assay used to measure activity.

<p>Fig <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125523#pone.0125523.g004" target="_blank">4A</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125523#pone.0125523.g004" target="_blank">4D</a> depict rest-activity parameters derived from 14 consecutive days of wheel-running data. Fig <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125523#pone.0125523.g004" target="_blank">4E</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125523#pone.0125523.g004" target="_blank">4H</a> depict rest-activity parameters derived from 14 consecutive days of passive-infrared (PIR) data. (A & E) Daily activity bouts, (B & F) daily activity bout duration, (C & G) interdaily stability, and (D & H) chi-square periodogram amplitude in <i>Grm2/3</i>^<b>+/+</b> and <i>Grm2/3</i>^<b>-/-</b> mice during a 12:12 h light/dark (12:12 LD) cycle at 100 lux. Activity bouts were defined using pre-established criteria, as described in the materials and methods. AU = arbitrary units.</p

Sleep time is reduced and sleep fragmentation is increased in <i>Grm2/3</i>^-/- mice.

<p>Fig 1A-F depict immobility-determined sleep parameters derived from 3 separate days of video-tracking data. Fig 1G-I depict immobility-determined sleep parameters derived from 14 consecutive days of passive-infrared (PIR) data. (A & G) Average sleep profiles, (B & H) light phase sleep time, and (C & I) dark phase sleep time for <i>Grm2/3</i>^<b>+/+</b> and <i>Grm2/3</i>^<b>-/-</b> mice during a 12:12 h light/dark (12:12 LD) cycle at 100 lux. (D) Average temporal distribution of sleep bouts, (E) number of light phase sleep bouts, and (F) light phase sleep bout duration in <i>Grm2/3</i>^<b>+/+</b> and <i>Grm2/3</i>^<b>-/-</b> mice during 12:12 LD at 100 lux. Sleep was defined as a period of immobility of at least 40 s. Data in Fig 1A, D & G are presented in 2 h time bins. ZT = zeitgeber time. Note that methods do not yet exist to automatically compute parameters pertaining to the frequency and duration of sleep bouts from PIR data.</p

Descriptive statistics for selected rest-activity parameters derived from 14 consecutive days of wheel-running data.

<p>Mice were housed under a 12:12 h light/dark (12:12 LD) cycle at 100 lux. Units of measurement and sample sizes are indicated in brackets. AU = arbitrary units. SEM = standard error of the mean.</p><p>Descriptive statistics for selected rest-activity parameters derived from 14 consecutive days of wheel-running data.</p