Sleep deprivation effects on circadian clock gene expression in the cerebral cortex parallel electroencephalographic differences among mouse strains.

Sleep deprivation (SD) results in increased electroencephalographic (EEG) delta power during subsequent non-rapid eye movement sleep (NREMS) and is associated with changes in the expression of circadian clock-related genes in the cerebral cortex. The increase of NREMS delta power as a function of previous wake duration varies among inbred mouse strains. We sought to determine whether SD-dependent changes in circadian clock gene expression parallel this strain difference described previously at the EEG level. The effects of enforced wakefulness of incremental durations of up to 6 h on the expression of circadian clock genes (bmal1, clock, cry1, cry2, csnk1epsilon, npas2, per1, and per2) were assessed in AKR/J, C57BL/6J, and DBA/2J mice, three strains that exhibit distinct EEG responses to SD. Cortical expression of clock genes subsequent to SD was proportional to the increase in delta power that occurs in inbred strains: the strain that exhibits the most robust EEG response to SD (AKR/J) exhibited dramatic increases in expression of bmal1, clock, cry2, csnkIepsilon, and npas2, whereas the strain with the least robust response to SD (DBA/2) exhibited either no change or a decrease in expression of these genes and cry1. The effect of SD on circadian clock gene expression was maintained in mice in which both of the cryptochrome genes were genetically inactivated. cry1 and cry2 appear to be redundant in sleep regulation as elimination of either of these genes did not result in a significant deficit in sleep homeostasis. These data demonstrate transcriptional regulatory correlates to previously described strain differences at the EEG level and raise the possibility that genetic differences underlying circadian clock gene expression may drive the EEG differences among these strains.

Genes involved in the astrocyte-neuron lactate shuttle (ANLS) are specifically regulated in cortical astrocytes following sleep deprivation in mice.

STUDY OBJECTIVES: There is growing evidence indicating that in order to meet the neuronal energy demands, astrocytes provide lactate as an energy substrate for neurons through a mechanism called "astrocyte-neuron lactate shuttle" (ANLS). Since neuronal activity changes dramatically during vigilance states, we hypothesized that the ANLS may be regulated during the sleep-wake cycle. To test this hypothesis we investigated the expression of genes associated with the ANLS specifically in astrocytes following sleep deprivation. Astrocytes were purified by fluorescence-activated cell sorting from transgenic mice expressing the green fluorescent protein (GFP) under the control of the human astrocytic GFAP-promoter. DESIGN: 6-hour instrumental sleep deprivation (TSD). SETTING: Animal sleep research laboratory. PARTICIPANTS: Young (P23-P27) FVB/N-Tg (GFAP-GFP) 14Mes/J (Tg) mice of both sexes and 7-8 week male Tg and FVB/Nj mice. INTERVENTIONS: Basal sleep recordings and sleep deprivation achieved using a modified cage where animals were gently forced to move. MEASUREMENTS AND RESULTS: Since Tg and FVB/Nj mice displayed a similar sleep-wake pattern, we performed a TSD in young Tg mice. Total RNA was extracted from the GFP-positive and GFP-negative cells sorted from cerebral cortex. Quantitative RT-PCR analysis showed that levels of Glut1, α-2-Na/K pump, Glt1, and Ldha mRNAs were significantly increased following TSD in GFP-positive cells. In GFP-negative cells, a tendency to increase, although not significant, was observed for Ldha, Mct2, and α-3-Na/K pump mRNAs. CONCLUSIONS: This study shows that TSD induces the expression of genes associated with ANLS specifically in astrocytes, underlying the important role of astrocytes in the maintenance of the neuro-metabolic coupling across the sleep-wake cycle.

Regulation of glucagon secretion by glucose transporter type 2 (glut2) and astrocyte-dependent glucose sensors.

Ripglut1;glut2-/- mice have no endogenous glucose transporter type 2 (glut2) gene expression but rescue glucose-regulated insulin secretion. Control of glucagon plasma levels is, however, abnormal, with fed hyperglucagonemia and insensitivity to physiological hypo- or hyperglycemia, indicating that GLUT2-dependent sensors control glucagon secretion. Here, we evaluated whether these sensors were located centrally and whether GLUT2 was expressed in glial cells or in neurons. We showed that ripglut1;glut2-/- mice failed to increase plasma glucagon levels following glucoprivation induced either by i.p. or intracerebroventricular 2-deoxy-D-glucose injections. This was accompanied by failure of 2-deoxy-D-glucose injections to activate c-Fos-like immunoreactivity in the nucleus of the tractus solitarius and the dorsal motor nucleus of the vagus. When glut2 was expressed by transgenesis in glial cells but not in neurons of ripglut1;glut2-/- mice, stimulated glucagon secretion was restored as was c-Fos-like immunoreactive labeling in the brainstem. When ripglut1;glut2-/- mice were backcrossed into the C57BL/6 genetic background, fed plasma glucagon levels were also elevated due to abnormal autonomic input to the alpha cells; glucagon secretion was, however, stimulated by hypoglycemic stimuli to levels similar to those in control mice. These studies identify the existence of central glucose sensors requiring glut2 expression in glial cells and therefore functional coupling between glial cells and neurons. These sensors may be activated at different glycemic levels depending on the genetic background.

Macrophage migration inhibitory factor deficiency leads to age-dependent impairment of glucose homeostasis in mice.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by many cells and tissues including pancreatic beta-cells, liver, skeletal muscle, and adipocytes. This study investigates the potential role of MIF in carbohydrate homeostasis in a physiological setting outside of severe inflammation, utilizing Mif knockout (MIF-/-) mice. Compared with wild-type (WT) mice, MIF-/- mice had a lower body weight, from birth until 4 months of age, but subsequently gained weight faster, resulting in a higher body weight at 12 months of age. The lower weight in young mice was related to a higher energy expenditure, and the higher weight in older mice was related to an increased food intake and a higher fat mass. Fasting blood insulin level was higher in MIF-/- mice compared with WT mice at any age. After i.p. glucose injection, the elevation of blood insulin level was higher in MIF-/- mice compared with WT mice, at 2 months of age, but was lower in 12-month-old MIF-/- mice. As a result, the glucose clearance during intraperitoneal glucose tolerance tests was higher in MIF-/- mice compared with WT mice until 4 months of age, and was lower in 12-month-old MIF-/- mice. Insulin resistance was estimated (euglycemic-hyperinsulinemic clamp tests), and the phosphorylation activity of AKT was similar in MIF-/- mice and WT mice. In conclusion, this mouse model provides evidence for the role of MIF in the control of glucose homeostasis.

Deficiency of glucose-dependent insulinotropic polypeptide receptor prevents ovariectomy-induced obesity in mice.

Menopause and premature gonadal steroid deficiency are associated with increases in fat mass and body weight. Ovariectomized (OVX) mice also show reduced locomotor activity. Glucose-dependent-insulinotropic-polypeptide (GIP) is known to play an important role both in fat metabolism and locomotor activity. Therefore, we hypothesized that the effects of estrogen on the regulation of body weight, fat mass, and spontaneous physical activity could be mediated in part by GIP signaling. To test this hypothesis, C57BL/6 mice and GIP-receptor knockout mice (Gipr(-/-)) were exposed to OVX or sham operation (n = 10 per group). The effects on body composition, markers of insulin resistance, energy expenditure, locomotor activity, and expression of hypothalamic anorexigenic and orexigenic factors were investigated over 26 wk in all four groups of mice. OVX wild-type mice developed obesity, increased fat mass, and elevated markers of insulin resistance as expected. This was completely prevented in OVX Gipr(-/-) animals, even though their energy expenditure and spontaneous locomotor activity levels did not significantly differ from those of OVX wild-type mice. Cumulative food intake in OVX Gipr(-/-) animals was significantly reduced and associated with significantly lower hypothalamic mRNA expression of the orexigenic neuropeptide Y (NPY) but not of cocaine-amphetamine-related transcript (CART), melanocortin receptors (MCR-3 and MCR-4), or thyrotropin-releasing hormone (TRH). GIP receptors thus interact with estrogens in the hypothalamic regulation of food intake in mice, and their blockade may carry promising potential for the prevention of obesity in gonadal steroid deficiency.

CD40 activation induces NREM sleep and modulates genes associated with sleep homeostasis.

The T-cell derived cytokine CD40 ligand is overexpressed in patients with autoimmune diseases. Through activation of its receptor, CD40 ligand leads to a tumor necrosis factor (TNF) receptor 1 (TNFR1) dependent impairment of locomotor activity in mice. Here we report that this effect is explained through a promotion of sleep, which was specific to non-rapid eye movement (NREM) sleep while REM sleep was suppressed. The increase in NREM sleep was accompanied by a decrease in EEG delta power during NREM sleep and by a decrease in the expression of transcripts in the cerebral cortex known to be associated with homeostatic sleep drive, such as Homer1a, Early growth response 2, Neuronal pentraxin 2, and Fos-like antigen 2. The effect of CD40 activation was mimicked by peripheral TNF injection and prevented by the TNF blocker etanercept. Our study indicates that sleep-wake dysregulation in autoimmune diseases may result from CD40 induced TNF:TNFR1 mediated alterations of molecular pathways, which regulate sleep-wake behavior.

NPAS2 as a transcriptional regulator of non-rapid eye movement sleep: genotype and sex interactions.

Because the transcription factor neuronal Per-Arnt-Sim-type signal-sensor protein-domain protein 2 (NPAS2) acts both as a sensor and an effector of intracellular energy balance, and because sleep is thought to correct an energy imbalance incurred during waking, we examined NPAS2's role in sleep homeostasis using npas2 knockout (npas2-/-) mice. We found that, under conditions of increased sleep need, i.e., at the end of the active period or after sleep deprivation (SD), NPAS2 allows for sleep to occur at times when mice are normally awake. Lack of npas2 affected electroencephalogram activity of thalamocortical origin; during non-rapid eye movement sleep (NREMS), activity in the spindle range (10-15 Hz) was reduced, and within the delta range (1-4 Hz), activity shifted toward faster frequencies. In addition, the increase in the cortical expression of the NPAS2 target gene period2 (per2) after SD was attenuated in npas2-/- mice. This implies that NPAS2 importantly contributes to the previously documented wake-dependent increase in cortical per2 expression. The data also revealed numerous sex differences in sleep; in females, sleep need accumulated at a slower rate, and REMS loss was not recovered after SD. In contrast, the rebound in NREMS time after SD was compromised only in npas2-/- males. We conclude that NPAS2 plays a role in sleep homeostasis, most likely at the level of the thalamus and cortex, where NPAS2 is abundantly expressed.

Reversal of activity-mediated spine dynamics and learning impairment in a mouse model of Fragile X syndrome.

Fragile X syndrome (FXS) is characterized by intellectual disability and autistic traits, and results from the silencing of the FMR1 gene coding for a protein implicated in the regulation of protein synthesis at synapses. The lack of functional Fragile X mental retardation protein has been proposed to result in an excessive signaling of synaptic metabotropic glutamate receptors, leading to alterations of synapse maturation and plasticity. It remains, however, unclear how mechanisms of activity-dependent spine dynamics are affected in Fmr knockout (Fmr1-KO) mice and whether they can be reversed. Here we used a repetitive imaging approach in hippocampal slice cultures to investigate properties of structural plasticity and their modulation by signaling pathways. We found that basal spine turnover was significantly reduced in Fmr1-KO mice, but markedly enhanced by activity. Additionally, activity-mediated spine stabilization was lost in Fmr1-KO mice. Application of the metabotropic glutamate receptor antagonist α-Methyl-4-carboxyphenylglycine (MCPG) enhanced basal turnover, improved spine stability, but failed to reinstate activity-mediated spine stabilization. In contrast, enhancing phosphoinositide-3 kinase (PI3K) signaling, a pathway implicated in various aspects of synaptic plasticity, reversed both basal turnover and activity-mediated spine stabilization. It also restored defective long-term potentiation mechanisms in slices and improved reversal learning in Fmr1-KO mice. These results suggest that modulation of PI3K signaling could contribute to improve the cognitive deficits associated with FXS.

Key electrophysiological, molecular, and metabolic signatures of sleep and wakefulness revealed in primary cortical cultures.

Although sleep is defined as a behavioral state, at the cortical level sleep has local and use-dependent features suggesting that it is a property of neuronal assemblies requiring sleep in function of the activation experienced during prior wakefulness. Here we show that mature cortical cultured neurons display a default state characterized by synchronized burst-pause firing activity reminiscent of sleep. This default sleep-like state can be changed to transient tonic firing reminiscent of wakefulness when cultures are stimulated with a mixture of waking neurotransmitters and spontaneously returns to sleep-like state. In addition to electrophysiological similarities, the transcriptome of stimulated cultures strikingly resembles the cortical transcriptome of sleep-deprived mice, and plastic changes as reflected by AMPA receptors phosphorylation are also similar. We used our in vitro model and sleep-deprived animals to map the metabolic pathways activated by waking. Only a few metabolic pathways were identified, including glycolysis, aminoacid, and lipids. Unexpectedly large increases in lysolipids were found both in vivo after sleep deprivation and in vitro after stimulation, strongly suggesting that sleep might play a major role in reestablishing the neuronal membrane homeostasis. With our in vitro model, the cellular and molecular consequences of sleep and wakefulness can now be investigated in a dish.

Circadian and wake-dependent effects on the pupil light reflex in response to narrow-bandwidth light pulses.

PURPOSE: Nonvisual light-dependent functions in humans are conveyed mainly by intrinsically photosensitive retinal ganglion cells, which express melanopsin as photopigment. We aimed to identify the effects of circadian phase and sleepiness across 24 hours on various aspects of the pupil response to light stimulation. METHODS: We tested 10 healthy adults hourly in two 12-hour sessions covering a 24-hour period. Pupil responses to narrow bandwidth red (635 ± 18 nm) and blue (463 ± 24 nm) light (duration of 1 and 30 seconds) at equal photon fluxes were recorded, and correlated with salivary melatonin concentrations at the same circadian phases and to subjective sleepiness ratings. The magnitude of pupil constriction was determined from minimal pupil size. The post-stimulus pupil response was assessed from the pupil size at 6 seconds following light offset, the area within the redilation curve, and the exponential rate of redilation. RESULTS: Among the measured parameters, the pupil size 6 seconds after light offset correlated with melatonin concentrations (P < 0.05) and showed a significant modulation over 24 hours with maximal values after the nocturnal peak of melatonin secretion. In contrast, the post-stimulus pupil response following red light stimulation correlated with subjective sleepiness (P < 0.05) without significant changes over 24 hours. CONCLUSIONS: The post-stimulus pupil response to blue light as a marker of intrinsic melanopsin activity demonstrated a circadian modulation. In contrast, the effect of sleepiness was more apparent in the cone contribution to the pupil response. Thus, pupillary responsiveness to light is under influence of the endogenous circadian clock and subjective sleepiness.

Telling the truth: medical students' progress with an ethical skill.

BACKGROUND: Truth-telling is a complex task requiring multiple skills in communication, understanding, and empathy. Its application in the context of breaking bad news (BBN) is distressing and problematic if conducted with insufficient skills. PURPOSE: We investigated the long-term influence of a simulated patient-based teaching intervention integrating the learning of communication skills within an ethical reflection on students' ethical attitudes towards truth-telling, perceived competence and comfort in BBN. METHODS: We followed two cohorts of medical students from the preclinical third year to their clinical rotations (fifth year). We analysed their ethical attitudes and level of comfort and competence in BBN before, after the intervention, and during clinical rotations. RESULTS: Students' ethical attitudes towards truth-telling remained stable. Students feeling uncomfortable or incompetent improved their level of perceived comfort or competence after the intervention, but those feeling comfortable or competent became more aware of the difficulty of the situation, and consequently decreased their level of comfort and competence. CONCLUSIONS: Confronting students with a realistic situation and integrating the practice of communication skills within an ethical reflection may be effective in maintaining ethical attitudes towards truth-telling, in developing new skills and increasing awareness about the difficulty and challenges of a BBN situation.

In vivo assessment of use-dependent brain plasticity--beyond the "one trick pony" imaging strategy.

This article has been written as a comment to Dr Thomas and Dr Baker's article "Teaching an adult brain new tricks: A critical review of evidence for training-dependent structural plasticity in humans". We deliberately expand on the key question about the biological substrates underlying use-dependent brain plasticity rather than reiterating the authors' main points of criticism already addressed in more general way by previous publications in the field. The focus here is on the following main issues: i) controversial brain plasticity findings in voxel-based morphometry studies are partially due to the strong dependency of the widely used T1-weighted imaging protocol on varying magnetic resonance contrast contributions; ii) novel concepts in statistical analysis allow one to directly infer topological specificity of structural brain changes associated with plasticity. We conclude that iii) voxel-based quantification of relaxometry derived parameter maps could provide a new perspective on use-dependent plasticity by characterisation of brain tissue property changes beyond the estimation of volume and cortical thickness changes. In the relevant sections we respond to the concerns raised by Dr Thomas and Dr Baker from the perspective of the proposed data acquisition and analysis strategy.

Stability-dependent behavioural and electro-cortical reorganizations during intentional switching between bimanual tapping modes

This study investigated behavioural and electro-cortical reorganizations accompanying intentional switching between two distinct bimanual coordination tapping modes (In-phase and Anti-phase) that differ in stability when produced at the same movement rate. We expected that switching to a less stable tapping mode (In-to-Anti switching) would lead to larger behavioural perturbations and require supplementary neural resources than switching to a more stable tapping mode (Anti-to-In switching). Behavioural results confirmed that the In-to-Anti switching lasted longer than the Anti-to-In switching. A general increase in attention-related neural activity was found at the moment of switching for both conditions. Additionally, two condition-dependent EEG reorganizations were observed. First, a specific increase in cortico-cortical coherence appeared exclusively during the In-to-Anti switching. This result may reflect a strengthening in inter-regional communication in order to engage in the subsequent, less stable, tapping mode. Second, a decrease in motor-related neural activity (increased beta spectral power) was found for the Anti-to-In switching only. The latter effect may reflect the interruption of the previous, less stable, tapping mode. Given that previous results on spontaneous Anti-to-In switching revealing an inverse pattern of EEG reorganization (decreased beta spectral power), present findings give new insight on the stability-dependent neural correlates of intentional motor switching. © 2010 Elsevier Ireland Ltd. All rights reserved

Anesthesia prevents auditory perceptual learning.

BACKGROUND: An auditory perceptual learning paradigm was used to investigate whether implicit memories are formed during general anesthesia. METHODS: Eighty-seven patients who had an American Society of Anesthesiologists physical status of I-III and were scheduled to undergo an elective surgery with general anesthesia were randomly assigned to one of two groups. One group received auditory stimulation during surgery, whereas the other did not. The auditory stimulation consisted of pure tones presented via headphones. The Bispectral Index level was maintained between 40 and 50 during surgery. To assess learning, patients performed an auditory frequency discrimination task after surgery, and comparisons were made between the groups. General anesthesia was induced with thiopental and maintained with a mixture of fentanyl and sevoflurane. RESULTS: There was no difference in the amount of learning between the two groups (mean +/- SD improvement: stimulated patients 9.2 +/- 11.3 Hz, controls 9.4 +/- 14.1 Hz). There was also no difference in initial thresholds (mean +/- SD initial thresholds: stimulated patients 31.1 +/- 33.4 Hz, controls 28.4 +/- 34.2 Hz). These results suggest that perceptual learning was not induced during anesthesia. No correlation between the bispectral index and the initial level of performance was found (Pearson r = -0.09, P = 0.59). CONCLUSION: Perceptual learning was not induced by repetitive auditory stimulation during anesthesia. This result may indicate that perceptual learning requires top-down processing, which is suppressed by the anesthetic.

Activity-dependent phosphorylation of SNAP-25 in hippocampal organotypic cultures.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is thought to play a key role in vesicle exocytosis and in the control of transmitter release. However, the precise mechanisms of action as well as the regulation of SNAP-25 remain unclear. Here we show by immunoprecipitation that activation of protein kinase C (PKC) by phorbol esters results in an increase in SNAP-25 phosphorylation. In addition, immunochemical analysis of two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels shows that SNAP-25 focuses as three or four distinct spots in the expected range of molecular weight and isoelectric point. Changing the phosphorylation level of the protein by incubating the slices in the presence of either a PKC agonist (phorbol 12,13-dibutyrate) or antagonist (chelerythrine) modified the distribution of SNAP-25 among these spots. Phorbol 12,13-dibutyrate increased the intensity of the spots with higher molecular weight and lower isoelectric point, whereas chelerythrine produced the opposite effect. This effect was specific for regulators of PKC, as agonists of other kinases did not produce similar changes. Induction of long-term potentiation, a property involved in learning mechanisms, and production of seizures with a GABA(A) receptor antagonist also increased the intensity of the spots with higher molecular weight and lower isoelectric point. This effect was prevented by the PKC inhibitor chelerythrine. We conclude that SNAP-25 can be phosphorylated in situ by PKC in an activity-dependent manner.