Neuronal subtype-specific transcriptomic changes in the cerebral neocortex associated with sleep pressure.

Sleep is homeostatically regulated by sleep pressure, which increases during wakefulness and dissipates during sleep. Recent studies have suggested that the cerebral neocortex, a six-layered structure composed of various layer- and projection-specific neuronal subtypes, is involved in the representation of sleep pressure governed by transcriptional regulation. Here, we examined the transcriptomic changes in neuronal subtypes in the neocortex upon increased sleep pressure using single-nucleus RNA sequencing datasets and predicted the putative intracellular and intercellular molecules involved in transcriptome alterations. We revealed that sleep deprivation (SD) had the greatest effect on the transcriptome of layer 2 and 3 intratelencephalic (L2/3 IT) neurons among the neocortical glutamatergic neuronal subtypes. The expression of mutant SIK3 (SLP), which is known to increase sleep pressure, also induced profound changes in the transcriptome of L2/3 IT neurons. We identified Junb as a candidate transcription factor involved in the alteration of the L2/3 IT neuronal transcriptome by SD and SIK3 (SLP) expression. Finally, we inferred putative intercellular ligands, including BDNF, LSAMP, and PRNP, which may be involved in SD-induced alteration of the transcriptome of L2/3 IT neurons. We suggest that the transcriptome of L2/3 IT neurons is most impacted by increased sleep pressure among neocortical glutamatergic neuronal subtypes and identify putative molecules involved in such transcriptional alterations.

Kinase signalling in excitatory neurons regulates sleep quantity and depth.

Progress has been made in the elucidation of sleep and wakefulness regulation at the neurocircuit level1,2. However, the intracellular signalling pathways that regulate sleep and the neuron groups in which these intracellular mechanisms work remain largely unknown. Here, using a forward genetics approach in mice, we identify histone deacetylase 4 (HDAC4) as a sleep-regulating molecule. Haploinsufficiency of Hdac4, a substrate of salt-inducible kinase 3 (SIK3)3, increased sleep. By contrast, mice that lacked SIK3 or its upstream kinase LKB1 in neurons or with a Hdac4S245A mutation that confers resistance to phosphorylation by SIK3 showed decreased sleep. These findings indicate that LKB1-SIK3-HDAC4 constitute a signalling cascade that regulates sleep and wakefulness. We also performed targeted manipulation of SIK3 and HDAC4 in specific neurons and brain regions. This showed that SIK3 signalling in excitatory neurons located in the cerebral cortex and the hypothalamus positively regulates EEG delta power during non-rapid eye movement sleep (NREMS) and NREMS amount, respectively. A subset of transcripts biased towards synaptic functions was commonly regulated in cortical glutamatergic neurons through the expression of a gain-of-function allele of Sik3 and through sleep deprivation. These findings suggest that NREMS quantity and depth are regulated by distinct groups of excitatory neurons through common intracellular signals. This study provides a basis for linking intracellular events and circuit-level mechanisms that control NREMS.

Surface-potential-modulated piezoresistive effect of core-shell 3C-SiC nanowires.

The effect of surface potential on the carrier mobility and piezoresistance of core-shell silicon carbide nanowires (SiC NWs) was investigated to realize small and sensitive SiC-microelectromechanical systems sensors. The p-type cubic crystalline SiC (3C-SiC) NWs were synthesized via the vapor-liquid-solid method and coated with silicon dioxide (SiO2) or aluminum oxide (Al2O3) dielectric shells to form core-shell structured NWs with different surface potentials. Four-point bending devices (FBDs) with a field-effect transistor (FET) configuration integrating a single core-shell 3C-SiC NW as the FET channel were fabricated to apply an additional electric field and strain to the core-shell 3C-SiC NWs. The fixed oxide charge densities of the SiO2and Al2O3shells showed positive and negative values, respectively, which were equivalent to electric fields of the order of several hundred thousand volt per centimeter in absolute values. In the core-shell 3C-SiC NWs with originally low impurity concentrations, the electric field induced by the fixed oxide charge of the shells can determine not only the electrical conduction but also the charge carriers in the NWs. Bending tests using the FBDs showed that the piezoresistive effect of the SiO2-coated NW was almost the same as that of the as-grown 3C-SiC NW reported previously, regardless of the gate voltage, whereas that of the Al2O3-coated NW was considerably enhanced at negative gate voltages. The enhancement of the piezoresistive effect was attributed to the piezo-pinch effect, which was more pronounced in the NW, where the carrier density at the core-shell interface is enhanced by the electric field of the dielectric.

Metabolomic and pharmacologic analyses of brain substances associated with sleep pressure in mice.

Sleep pressure, the driving force of the homeostatic sleep regulation, is accumulated during wakefulness and dissipated during sleep. Sleep deprivation (SD) has been used as a method to acutely increase animal's sleep pressure for investigating the molecular changes under high sleep pressure. However, SD induces changes not only reflecting increased sleep pressure but also inevitable stresses and prolonged wake state itself. The Sik3Sleepy mutant mice (Sleepy) exhibit constitutively high sleep pressure despite sleeping longer, and have been useful as a model of increased sleep pressure. Here we conducted a cross-comparison of brain metabolomic profiles between SD versus ad lib slept mice, as well as Sleepy mutant versus littermate wild-type mice. Targeted metabolome analyses of whole brains quantified 203 metabolites in total, of which 43 metabolites showed significant changes in SD, whereas three did in Sleepy mutant mice. The large difference in the number of differential metabolites highlighted limitations of SD as methodology. The cross-comparison revealed that a decrease in betaine and an increase in imidazole dipeptides are associated with high sleep pressure in both models. These metabolites may be novel markers of sleep pressure at the whole-brain level. Furthermore, we found that intracerebroventricular injection of imidazole dipeptides increased subsequent NREM sleep time, suggesting the possibility that imidazole dipeptides may participate in the regulation of sleep in mice.

Induction of Mutant Sik3Sleepy Allele in Neurons in Late Infancy Increases Sleep Need.

Sleep is regulated in a homeostatic manner. Sleep deprivation increases sleep need, which is compensated mainly by increased EEG δ power during non-rapid eye movement sleep (NREMS) and, to a lesser extent, by increased sleep amount. Although genetic factors determine the constitutive level of sleep need and sleep amount in mice and humans, the molecular entity behind sleep need remains unknown. Recently, we found that a gain-of-function Sleepy (Slp) mutation in the salt-inducible kinase 3 (Sik3) gene, which produces the mutant SIK3(SLP) protein, leads to an increase in NREMS EEG δ power and sleep amount. Since Sik3Slp mice express SIK3(SLP) in various types of cells in the brain as well as multiple peripheral tissues from the embryonic stage, the cell type and developmental stage responsible for the sleep phenotype in Sik3Slp mice remain to be elucidated. Here, we generated two mouse lines, synapsin1CreERT2 and Sik3ex13flox mice, which enable inducible Cre-mediated, conditional expression of SIK3(SLP) in neurons on tamoxifen administration. Administration of tamoxifen to synapsin1CreERT2 mice during late infancy resulted in higher recombination efficiency than administration during adolescence. SIK3(SLP) expression after late infancy increased NREMS and NREMS δ power in male synapsin1CreERT2; Sik3ex13flox/+ mice. The expression of SIK3(SLP) after adolescence led to a higher NREMS δ power without a significant change in NREMS amounts. Thus, neuron-specific expression of SIK3(SLP) after late infancy is sufficient to increase sleep.SIGNIFICANCE STATEMENT The propensity to accumulate sleep need during wakefulness and to dissipate it during sleep underlies the homeostatic regulation of sleep. However, little is known about the developmental stage and cell types involved in determining the homeostatic regulation of sleep. Here, we show that Sik3Slp allele induction in mature neurons in late infancy is sufficient to increase non-rapid eye movement sleep amount and non-rapid eye movement sleep δ power. SIK3 signaling in neurons constitutes an intracellular mechanism to increase sleep.

Strain engineering of core-shell silicon carbide nanowires for mechanical and piezoresistive characterizations.

This study evaluated the mechanical properties and piezoresistivity of core-shell silicon carbide nanowires (C/S-SiCNWs) synthesized by a vapor-liquid-solid technique, which are a promising material for harsh environmental micro electromechanical systems (MEMS) applications. The C/S-SiCNWs were composed of a crystalline cubic (3C) SiC core wrapped by an amorphous silicon dioxide (SiO x ) shell; however, TEM observations of the NWs showed that hexagonal polytypes (2H, 4H , and 6H) were partially induced in the core by a stacking fault owing to a Shockley partial dislocation. The stress-strain relationship of the C/S-SiCNWs and SiC cores without an SiO x shell was examined using MEMS-based nanotensile tests. The tensile strengths of the C/S-SiCNWs and SiC cores were 7.0 GPa and 22.4 GPa on average, respectively. The lower strength of the C/S-SiCNWs could be attributed to the SiO x shell with the surface roughness as the breaking point. The Young's modulus of the C/S-SiCNWs was 247.2 GPa on average, whereas that of the SiC cores had a large value with scatter data ranging from 450 to 580 GPa. The geometrical model of the SiC core based on the transmission electron microscopy observations rationalized this scatter data by the volume content of the stacking fault in the core. The piezoresistive effects of the C/S-SiCNW and SiC core were also evaluated from the I-V characteristics under uniaxial tensile strain. The gauge factor of -30.7 at 0.008 ε for the C/S-SiCNW was approximately two-times larger than that of -15.8 at 0.01 ε for the SiC core. This could be caused by an increase of the surface state density at the SiO x /SiC interface owing to the positive fixed oxide charge of the SiO x shell.