Rational Engineering of XCaMPs, a Multicolor GECI Suite for In Vivo Imaging of Complex Brain Circuit Dynamics.

To decipher dynamic brain information processing, current genetically encoded calcium indicators (GECIs) are limited in single action potential (AP) detection speed, combinatorial spectral compatibility, and two-photon imaging depth. To address this, here, we rationally engineered a next-generation quadricolor GECI suite, XCaMPs. Single AP detection was achieved within 3-10 ms of spike onset, enabling measurements of fast-spike trains in parvalbumin (PV)-positive interneurons in the barrel cortex in vivo and recording three distinct (two inhibitory and one excitatory) ensembles during pre-motion activity in freely moving mice. In vivo paired recording of pre- and postsynaptic firing revealed spatiotemporal constraints of dendritic inhibition in layer 1 in vivo, between axons of somatostatin (SST)-positive interneurons and apical tufts dendrites of excitatory pyramidal neurons. Finally, non-invasive, subcortical imaging using red XCaMP-R uncovered somatosensation-evoked persistent activity in hippocampal CA1 neurons. Thus, the XCaMPs offer a critical enhancement of solution space in studies of complex neuronal circuit dynamics. VIDEO ABSTRACT.

Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response.

NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox-dependent manner. p62 bodies formed by liquid-liquid phase separation contain Ser349-phosphorylated p62, which participates in the redox-independent activation of NRF2. However, the regulatory mechanism and physiological significance of p62 phosphorylation remain unclear. Here, we identify ULK1 as a kinase responsible for the phosphorylation of p62. ULK1 colocalizes with p62 bodies, directly interacting with p62. ULK1-dependent phosphorylation of p62 allows KEAP1 to be retained within p62 bodies, thus activating NRF2. p62S351E/+ mice are phosphomimetic knock-in mice in which Ser351, corresponding to human Ser349, is replaced by Glu. These mice, but not their phosphodefective p62S351A/S351A counterparts, exhibit NRF2 hyperactivation and growth retardation. This retardation is caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis, a phenotype also observed in systemic Keap1-knockout mice. Our results expand our understanding of the physiological importance of the redox-independent NRF2 activation pathway and provide new insights into the role of phase separation in this process.

Inverse synaptic tagging of inactive synapses via dynamic interaction of Arc/Arg3.1 with CaMKIIß.

The Arc/Arg3.1 gene product is rapidly upregulated by strong synaptic activity and critically contributes to weakening synapses by promoting AMPA-R endocytosis. However, how activity-induced Arc is redistributed and determines the synapses to be weakened remains unclear. Here, we show targeting of Arc to inactive synapses via a high-affinity interaction with CaMKIIß that is not bound to calmodulin. Synaptic Arc accumulates in inactive synapses that previously experienced strong activation and correlates with removal of surface GluA1 from individual synapses. A lack of CaMKIIß either in vitro or in vivo resulted in loss of Arc upregulation in the silenced synapses. The discovery of Arc's role in "inverse" synaptic tagging that is specific for weaker synapses and prevents undesired enhancement of weak synapses in potentiated neurons reconciles essential roles of Arc both for the late phase of long-term plasticity and for reduction of surface AMPA-Rs in stimulated neurons.

A discrete neuronal circuit induces a hibernation-like state in rodents.

Hibernating mammals actively lower their body temperature to reduce energy expenditure when facing food scarcity1. This ability to induce a hypometabolic state has evoked great interest owing to its potential medical benefits2,3. Here we show that a hypothalamic neuronal circuit in rodents induces a long-lasting hypothermic and hypometabolic state similar to hibernation. In this state, although body temperature and levels of oxygen consumption are kept very low, the ability to regulate metabolism still remains functional, as in hibernation4. There was no obvious damage to tissues and organs or abnormalities in behaviour after recovery from this state. Our findings could enable the development of a method to induce a hibernation-like state, which would have potential applications in non-hibernating mammalian species including humans.

An intact pituitary vasopressin system is critical for building a robust circadian clock in the suprachiasmatic nucleus.

The circadian clock is a biological timekeeping system that oscillates with a circa-24-h period, reset by environmental timing cues, especially light, to the 24-h day-night cycle. In mammals, a "central" clock in the hypothalamic suprachiasmatic nucleus (SCN) synchronizes "peripheral" clocks throughout the body to regulate behavior, metabolism, and physiology. A key feature of the clock's oscillation is resistance to abrupt perturbations, but the mechanisms underlying such robustness are not well understood. Here, we probe clock robustness to unexpected photic perturbation by measuring the speed of reentrainment of the murine locomotor rhythm after an abrupt advance of the light-dark cycle. Using an intersectional genetic approach, we implicate a critical role for arginine vasopressin pathways, both central within the SCN and peripheral from the anterior pituitary.

Trans-synaptic interaction of GluRdelta2 and Neurexin through Cbln1 mediates synapse formation in the cerebellum.

Elucidation of molecular mechanisms that regulate synapse formation is required for the understanding of neural wiring, higher brain functions, and mental disorders. Despite the wealth of in vitro information, fundamental questions about how glutamatergic synapses are formed in the mammalian brain remain unanswered. Glutamate receptor (GluR) delta2 is essential for cerebellar synapse formation in vivo. Here, we show that the N-terminal domain (NTD) of GluRdelta2 interacts with presynaptic neurexins (NRXNs) through cerebellin 1 precursor protein (Cbln1). The synaptogenic activity of GluRdelta2 is abolished in cerebellar primary cultures from Cbln1 knockout mice and is restored by recombinant Cbln1. Knockdown of NRXNs in cerebellar granule cells also hinders the synaptogenic activity of GluRdelta2. Both the NTD of GluRdelta2 and the extracellular domain of NRXN1beta suppressed the synaptogenic activity of Cbln1 in cerebellar primary cultures and in vivo. These results suggest that GluRdelta2 mediates cerebellar synapse formation by interacting with presynaptic NRXNs through Cbln1.

Endocannabinoid-dependent formation of columnar axonal projection in the mouse cerebral cortex.

Columnar structure is one of the most fundamental morphological features of the cerebral cortex and is thought to be the basis of information processing in higher animals. Yet, how such a topographically precise structure is formed is largely unknown. Formation of columnar projection of layer 4 (L4) axons is preceded by thalamocortical formation, in which type 1 cannabinoid receptors (CB1R) play an important role in shaping barrel-specific targeted projection by operating spike timing-dependent plasticity during development (Itami et al., J. Neurosci. 36, 7039-7054 [2016]; Kimura & Itami, J. Neurosci. 39, 3784-3791 [2019]). Right after the formation of thalamocortical projections, CB1Rs start to function at L4 axon terminals (Itami & Kimura, J. Neurosci. 32, 15000-15011 [2012]), which coincides with the timing of columnar shaping of L4 axons. Here, we show that the endocannabinoid 2-arachidonoylglycerol (2-AG) plays a crucial role in columnar shaping. We found that L4 axon projections were less organized until P12 and then became columnar after CB1Rs became functional. By contrast, the columnar organization of L4 axons was collapsed in mice genetically lacking diacylglycerol lipase α, the major enzyme for 2-AG synthesis. Intraperitoneally administered CB1R agonists shortened axon length, whereas knockout of CB1R in L4 neurons impaired columnar projection of their axons. Our results suggest that endocannabinoid signaling is crucial for shaping columnar axonal projection in the cerebral cortex.

An RNA-binding protein, Qki5, regulates embryonic neural stem cells through pre-mRNA processing in cell adhesion signaling.

Cell type-specific transcriptomes are enabled by the action of multiple regulators, which are frequently expressed within restricted tissue regions. In the present study, we identify one such regulator, Quaking 5 (Qki5), as an RNA-binding protein (RNABP) that is expressed in early embryonic neural stem cells and subsequently down-regulated during neurogenesis. mRNA sequencing analysis in neural stem cell culture indicates that Qki proteins play supporting roles in the neural stem cell transcriptome and various forms of mRNA processing that may result from regionally restricted expression and subcellular localization. Also, our in utero electroporation gain-of-function study suggests that the nuclear-type Qki isoform Qki5 supports the neural stem cell state. We next performed in vivo transcriptome-wide protein-RNA interaction mapping to search for direct targets of Qki5 and elucidate how Qki5 regulates neural stem cell function. Combined with our transcriptome analysis, this mapping analysis yielded a bona fide map of Qki5-RNA interaction at single-nucleotide resolution, the identification of 892 Qki5 direct target genes, and an accurate Qki5-dependent alternative splicing rule in the developing brain. Last, our target gene list provides the first compelling evidence that Qki5 is associated with specific biological events; namely, cell-cell adhesion. This prediction was confirmed by histological analysis of mice in which Qki proteins were genetically ablated, which revealed disruption of the apical surface of the lateral wall in the developing brain. These data collectively indicate that Qki5 regulates communication between neural stem cells by mediating numerous RNA processing events and suggest new links between splicing regulation and neural stem cell states.

Fis1 ablation in the male germline disrupts mitochondrial morphology and mitophagy, and arrests spermatid maturation.

Male germline development involves choreographed changes to mitochondrial number, morphology and organization. Mitochondrial reorganization during spermatogenesis was recently shown to require mitochondrial fusion and fission. Mitophagy, the autophagic degradation of mitochondria, is another mechanism for controlling mitochondrial number and physiology, but its role during spermatogenesis is largely unknown. During post-meiotic spermatid development, restructuring of the mitochondrial network results in packing of mitochondria into a tight array in the sperm midpiece to fuel motility. Here, we show that disruption of mouse Fis1 in the male germline results in early spermatid arrest that is associated with increased mitochondrial content. Mutant spermatids coalesce into multinucleated giant cells that accumulate mitochondria of aberrant ultrastructure and numerous mitophagic and autophagic intermediates, suggesting a defect in mitophagy. We conclude that Fis1 regulates mitochondrial morphology and turnover to promote spermatid maturation.

Attempts for deriving extended pluripotent stem cells from common marmoset embryonic stem cells.

Extended pluripotent stem cells (EPSCs) derived from mice and humans showed an enhanced potential for chimeric formation. By exploiting transcriptomic approaches, we assessed the differences in gene expression profile between extended EPSCs derived from mice and humans, and those newly derived from the common marmoset (marmoset; Callithrix jacchus). Although the marmoset EPSC-like cells displayed a unique colony morphology distinct from murine and human EPSCs, they displayed a pluripotent state akin to embryonic stem cells (ESCs), as confirmed by gene expression and immunocytochemical analyses of pluripotency markers and three-germ-layer differentiation assay. Importantly, the marmoset EPSC-like cells showed interspecies chimeric contribution to mouse embryos, such as E6.5 blastocysts in vitro and E6.5 epiblasts in vivo in mouse development. Also, we discovered that the perturbation of gene expression of the marmoset EPSC-like cells from the original ESCs resembled that of human EPSCs. Taken together, our multiple analyses evaluated the efficacy of the method for the derivation of marmoset EPSCs.

Activation of Extrasynaptic Kainate Receptors Drives Hilar Mossy Cell Activity.

Mossy cells (MCs) of the dentate gyrus are key components of an excitatory associative circuit established by reciprocal connections with dentate granule cells (GCs). MCs are implicated in place field encoding, pattern separation, and novelty detection, as well as in brain disorders such as temporal lobe epilepsy and depression. Despite their functional relevance, little is known about the determinants that control MC activity. Here, we examined whether MCs express functional kainate receptors (KARs), a subtype of glutamate receptors involved in neuronal development, synaptic transmission, and epilepsy. Using mouse hippocampal slices, we found that bath application of submicromolar and micromolar concentrations of the KAR agonist kainic acid induced inward currents and robust MC firing. These effects were abolished in GluK2 KO mice, indicating the presence of functional GluK2-containing KARs in MCs. In contrast to CA3 pyramidal cells, which are structurally and functionally similar to MCs and express synaptic KARs at mossy fiber (MF) inputs (i.e., GC axons), we found no evidence for KAR-mediated transmission at MF-MC synapses, indicating that most KARs at MCs are extrasynaptic. Immunofluorescence and immunoelectron microscopy analyses confirmed the extrasynaptic localization of GluK2-containing KARs in MCs. Finally, blocking glutamate transporters, a manipulation that increases extracellular levels of endogenous glutamate, was sufficient to induce KAR-mediated inward currents in MCs, suggesting that MC-KARs can be activated by increases in ambient glutamate. Our findings provide the first direct evidence of functional extrasynaptic KARs at a critical excitatory neuron of the hippocampus.SIGNIFICANCE STATEMENT Hilar mossy cells (MCs) are an understudied population of hippocampal neurons that form an excitatory loop with dentate granule cells. MCs have been implicated in pattern separation, spatial navigation, and epilepsy. Despite their importance in hippocampal function and disease, little is known about how MC activity is recruited. Here, we show for the first time that MCs express extrasynaptic kainate receptors (KARs), a subtype of glutamate receptors critically involved in neuronal function and epilepsy. While we found no evidence for synaptic KARs in MCs, KAR activation induced strong action potential firing of MCs, raising the possibility that extracellular KARs regulate MC excitability in vivo and may also promote dentate gyrus hyperexcitability and epileptogenesis.

A new beta cell-specific mitophagy reporter mouse shows that metabolic stress leads to accumulation of dysfunctional mitochondria despite increased mitophagy.

AIMS/HYPOTHESIS: Mitophagy, the selective autophagy of mitochondria, is essential for maintenance of mitochondrial function. Recent studies suggested that defective mitophagy in beta cells caused diabetes. However, because of technical difficulties, the development of a convenient and reliable method to evaluate mitophagy in beta cells in vivo is needed. The aim of this study was to establish beta cell-specific mitophagy reporter mice and elucidate the role of mitophagy in beta cell function under metabolically stressed conditions induced by a high-fat diet (HFD). METHODS: Mitophagy was assessed using newly generated conditional mitochondrial matrix targeting mitophagy reporter (CMMR) mice, in which mitophagy can be visualised specifically in beta cells in vivo using a fluorescent probe sensitive to lysosomal pH and degradation. Metabolic stress was induced in mice by exposure to the HFD for 20 weeks. The accumulation of dysfunctional mitochondria was examined by staining for functional/total mitochondria and reactive oxygen species (ROS) using specific fluorescent dyes and antibodies. To investigate the molecular mechanism underlying mitophagy in beta cells, overexpression and knockdown experiments were performed. HFD-fed mice were examined to determine whether chronic insulin treatment for 6 weeks could ameliorate mitophagy, mitochondrial function and impaired insulin secretion. RESULTS: Exposure to the HFD increased the number of enlarged (HFD-G) islets with markedly elevated mitophagy. Mechanistically, HFD feeding induced severe hypoxia in HFD-G islets, which upregulated mitophagy through the hypoxia-inducible factor 1-ɑ (Hif-1ɑ)/BCL2 interacting protein 3 (BNIP3) axis in beta cells. However, HFD-G islets unexpectedly showed the accumulation of dysfunctional mitochondria due to excessive ROS production, suggesting an insufficient capacity of mitophagy for the degradation of dysfunctional mitochondria. Chronic administration of insulin ameliorated hypoxia and reduced ROS production and dysfunctional mitochondria, leading to decreased mitophagy and restored insulin secretion. CONCLUSIONS/INTERPRETATION: We demonstrated that CMMR mice enabled the evaluation of mitophagy in beta cells. Our results suggested that metabolic stress induced by the HFD caused the aberrant accumulation of dysfunctional mitochondria, which overwhelmed the mitophagic capacity and was associated with defective maintenance of mitochondrial function and impaired insulin secretion.

Insight into the function of a unique voltage-sensor protein (TMEM266) and its short form in mouse cerebellum.

Voltage-sensing proteins generally consist of voltage-sensor domains and pore-gate domains, forming the voltage-gated ion channels. However, there are several unconventional voltage-sensor proteins that lack pore-gate domains, conferring them unique voltage-sensing machinery. TMEM266, which is expressed in cerebellum granule cells, is one of the interesting voltage-sensing proteins that has a putative intracellular coiled-coil and a functionally unidentified cytosolic region instead of a pore-gate domain. Here, we approached the molecular function of TMEM266 by performing co-immunoprecipitation experiments. We unexpectedly discovered that TMEM266 proteins natively interact with the novel short form splice variants that only have voltage-sensor domains and putative cytosolic coiled-coil region in cerebellum. The crystal structure of coiled-coil region of TMEM266 suggested that these coiled-coil regions play significant roles in forming homodimers. In vitro expression experiments supported the idea that short form TMEM266 (sTMEM266) or full length TMEM266 (fTMEM266) form homodimers. We also performed proximity labeling mass spectrometry analysis for fTMEM266 and sTMEM266 using Neuro-2A, neuroblastoma cells, and fTMEM266 showed more interacting molecules than sTMEM266, suggesting that the C-terminal cytosolic region in fTMEM266 binds to various targets. Finally, TMEM266-deficient animals showed the moderate abnormality in open-field test. The present study provides clues about the novel voltage-sensing mechanism mediated by TMEM266.

Hypnotic effect of thalidomide is independent of teratogenic ubiquitin/proteasome pathway.

Thalidomide exerts its teratogenic and immunomodulatory effects by binding to cereblon (CRBN) and thereby inhibiting/modifying the CRBN-mediated ubiquitination pathway consisting of the Cullin4-DDB1-ROC1 E3 ligase complex. The mechanism of thalidomide's classical hypnotic effect remains largely unexplored, however. Here we examined whether CRBN is involved in the hypnotic effect of thalidomide by generating mice harboring a thalidomide-resistant mutant allele of Crbn (Crbn YW/AA knock-in mice). Thalidomide increased non-REM sleep time in Crbn YW/AA knock-in homozygotes and heterozygotes to a similar degree as seen in wild-type littermates. Thalidomide similarly depressed excitatory synaptic transmission in the cortical slices obtained from wild-type and Crbn YW/AA homozygous knock-in mice without affecting GABAergic inhibition. Thalidomide induced Fos expression in vasopressin-containing neurons of the supraoptic nucleus and reduced Fos expression in the tuberomammillary nuclei. Thus, thalidomide's hypnotic effect seems to share some downstream mechanisms with general anesthetics and GABAA-activating sedatives but does not involve the teratogenic CRBN-mediated ubiquitin/proteasome pathway.

Critical Role of the Presynaptic Protein CAST in Maintaining the Photoreceptor Ribbon Synapse Triad.

The cytomatrix at the active zone-associated structural protein (CAST) and its homologue, named ELKS, being rich in glutamate (E), leucine (L), lysine (K), and serine (S), belong to a family of proteins that organize presynaptic active zones at nerve terminals. These proteins interact with other active zone proteins, including RIMs, Munc13s, Bassoon, and the ß subunit of Ca2+ channels, and have various roles in neurotransmitter release. A previous study showed that depletion of CAST/ELKS in the retina causes morphological changes and functional impairment of this structure. In this study, we investigated the roles of CAST and ELKS in ectopic synapse localization. We found that the involvement of these proteins in ribbon synapse distribution is complex. Unexpectedly, CAST and ELKS, in photoreceptors or in horizontal cells, did not play a major role in ribbon synapse ectopic localization. However, depletion of CAST and ELKS in the mature retina resulted in degeneration of the photoreceptors. These findings suggest that CAST and ELKS play critical roles in maintaining neural signal transduction in the retina, but the regulation of photoreceptor triad synapse distribution is not solely dependent on their actions within photoreceptors and horizontal cells.

Pathogenesis, Clinical Features, and Treatment of Patients with Myelin Oligodendrocyte Glycoprotein (MOG) Autoantibody-Associated Disorders Focusing on Optic Neuritis with Consideration of Autoantibody-Binding Sites: A Review.

Although there is a substantial amount of data on the clinical characteristics, diagnostic criteria, and pathogenesis of myelin oligodendrocyte glycoprotein (MOG) autoantibody-associated disease (MOGAD), there is still uncertainty regarding the MOG protein function and the pathogenicity of anti-MOG autoantibodies in this disease. It is important to note that the disease characteristics, immunopathology, and treatment response of MOGAD patients differ from those of anti-aquaporin 4 antibody-positive neuromyelitis optica spectrum disorders (NMOSDs) and multiple sclerosis (MS). The clinical phenotypes of MOGAD are varied and can include acute disseminated encephalomyelitis, transverse myelitis, cerebral cortical encephalitis, brainstem or cerebellar symptoms, and optic neuritis. The frequency of optic neuritis suggests that the optic nerve is the most vulnerable lesion in MOGAD. During the acute stage, the optic nerve shows significant swelling with severe visual symptoms, and an MRI of the optic nerve and brain lesion tends to show an edematous appearance. These features can be alleviated with early extensive immune therapy, which may suggest that the initial attack of anti-MOG autoantibodies could target the structures on the blood-brain barrier or vessel membrane before reaching MOG protein on myelin or oligodendrocytes. To understand the pathogenesis of MOGAD, proper animal models are crucial. However, anti-MOG autoantibodies isolated from patients with MOGAD do not recognize mouse MOG efficiently. Several studies have identified two MOG epitopes that exhibit strong affinity with human anti-MOG autoantibodies, particularly those isolated from patients with the optic neuritis phenotype. Nonetheless, the relations between epitopes on MOG protein remain unclear and need to be identified in the future.

A Discrete Glycinergic Neuronal Population in the Ventromedial Medulla That Induces Muscle Atonia during REM Sleep and Cataplexy in Mice.

During rapid eye movement (REM) sleep, anti-gravity muscle tone and bodily movements are mostly absent, because somatic motoneurons are inhibited by descending inhibitory pathways. Recent studies showed that glycine/GABA neurons in the ventromedial medulla (VMM; GlyVMM neurons) play an important role in generating muscle atonia during REM sleep (REM-atonia). However, how these REM-atonia-inducing neurons interconnect with other neuronal populations has been unknown. In the present study, we first identified a specific subpopulation of GlyVMM neurons that play an important role in induction of REM-atonia by virus vector-mediated tracing in male mice in which glycinergic neurons expressed Cre recombinase. We found these neurons receive direct synaptic input from neurons in several brain stem regions, including glutamatergic neurons in the sublaterodorsal tegmental nucleus (SLD; GluSLD neurons). Silencing this circuit by specifically expressing tetanus toxin light chain (TeTNLC) resulted in REM sleep without atonia. This manipulation also caused a marked decrease in time spent in cataplexy-like episodes (CLEs) when applied to narcoleptic orexin-ataxin-3 mice. We also showed that GlyVMM neurons play an important role in maintenance of sleep. This present study identified a population of glycinergic neurons in the VMM that are commonly involved in REM-atonia and cataplexy.SIGNIFICANCE STATEMENT We identified a population of glycinergic neurons in the ventral medulla that plays an important role in inducing muscle atonia during rapid eye movement (REM) sleep. It sends axonal projections almost exclusively to motoneurons in the spinal cord and brain stem except to those that innervate extraocular muscles, while other glycinergic neurons in the same region also send projections to other regions including monoaminergic nuclei. Furthermore, these neurons receive direct inputs from several brainstem regions including glutamatergic neurons in the sublaterodorsal tegmental nucleus (SLD). Genetic silencing of this pathway resulted in REM sleep without atonia and a decrease of cataplexy when applied to narcoleptic mice. This work identified a neural population involved in generating muscle atonia during REM sleep and cataplexy.

CAPS2 Deficiency Impairs the Release of the Social Peptide Oxytocin, as Well as Oxytocin-Associated Social Behavior.

Ca2+-dependent activator protein for secretion 2 (CAPS2) regulates dense-core vesicle (DCV) exocytosis to facilitate peptidergic and catecholaminergic transmitter release. CAPS2 deficiency in mice has mild neuronal effects but markedly impairs social behavior. Rare de novo Caps2 alterations also occur in autism spectrum disorder, although whether CAPS2-mediated release influences social behavior remains unclear. Here, we demonstrate that CAPS2 is associated with DCV exocytosis-mediated release of the social interaction modulatory peptide oxytocin (OXT). CAPS2 is expressed in hypothalamic OXT neurons and localizes to OXT nerve projection and OXT release sites, such as the pituitary. Caps2 KO mice exhibited reduced plasma albeit increased hypothalamic and pituitary OXT levels, indicating insufficient release. OXT neuron-specific Caps2 conditional KO supported CAPS2 function in pituitary OXT release, also affording impaired social interaction and recognition behavior that could be ameliorated by exogenous OXT administered intranasally. Thus, CAPS2 appears critical for OXT release, thereby being associated with social behavior.SIGNIFICANCE STATEMENT The role of the neuropeptide oxytocin in enhancing social interaction and social bonding behavior has attracted considerable public and neuroscientific attention. A central issue in oxytocin biology concerns how oxytocin release is regulated. Our study provides an important insight into the understanding of oxytocin-dependent social behavior from the perspective of the CAPS2-regulated release mechanism.

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.

Forward-genetics analysis of sleep in randomly mutagenized mice.

Sleep is conserved from invertebrates to vertebrates, and is tightly regulated in a homeostatic manner. The molecular and cellular mechanisms that determine the amount of rapid eye movement sleep (REMS) and non-REMS (NREMS) remain unknown. Here we identify two dominant mutations that affect sleep and wakefulness by using an electroencephalogram/electromyogram-based screen of randomly mutagenized mice. A splicing mutation in the Sik3 protein kinase gene causes a profound decrease in total wake time, owing to an increase in inherent sleep need. Sleep deprivation affects phosphorylation of regulatory sites on the kinase, suggesting a role for SIK3 in the homeostatic regulation of sleep amount. Sik3 orthologues also regulate sleep in fruitflies and roundworms. A missense, gain-of-function mutation in the sodium leak channel NALCN reduces the total amount and episode duration of REMS, apparently by increasing the excitability of REMS-inhibiting neurons. Our results substantiate the use of a forward-genetics approach for studying sleep behaviours in mice, and demonstrate the role of SIK3 and NALCN in regulating the amount of NREMS and REMS, respectively.