Mammalian sleep comprises rapid eye movement (REM) sleep and non-REM (NREM) sleep. To functionally isolate from the complex mixture of neurons populating the brainstem pons those involved in switching between REM and NREM sleep, we chemogenetically manipulated neurons of a specific embryonic cell lineage in mice. We identified excitatory glutamatergic neurons that inhibit REM sleep and promote NREM sleep. These neurons shared a common developmental origin with neurons promoting wakefulness; both derived from a pool of proneural hindbrain cells expressing Atoh1 at embryonic day 10.5. We also identified inhibitory γ-aminobutyric acid-releasing neurons that act downstream to inhibit REM sleep. Artificial reduction or prolongation of REM sleep in turn affected slow-wave activity during subsequent NREM sleep, implicating REM sleep in the regulation of NREM sleep.

Here we describe “Supernova” series of vector systems that enable single-cell labeling and labeled cell-specific gene manipulation, when introduced by in utero electroporation (IUE) or adeno-associated virus (AAV)-mediated gene delivery. In Supernova, sparse labeling relies on low TRE leakage. In a small population of cells with over-threshold leakage, initial tTA-independent weak expression is enhanced by tTA/TRE-positive feedback along with a site-specific recombination system (e.g., Cre/loxP, Flpe/FRT). Sparse and bright labeling by Supernova with little background enables the visualization of the morphological details of individual neurons in densely packed brain areas such as the cortex and hippocampus, both during development and in adulthood. Sparseness levels are adjustable. Labeled cell-specific gene knockout was accomplished by introducing Cre/loxP-based Supernova vectors into floxed mice. Furthermore, by combining with RNAi, TALEN, and CRISPR/Cas9 technologies, IUE-based Supernova achieved labeled cell-specific gene knockdown and editing/knockout without requiring genetically altered mice. Thus, Supernova system is highly extensible and widely applicable for single-cell analyses in complex organs, such as the mammalian brain.

In understanding the mechanism of schizophrenia pathogenesis, a significant finding is that drug abuse of phencyclidine or its analog ketamine causes symptoms similar to schizophrenia. Such drug effects are triggered even by administration at post-adolescent stages. Both drugs are N-methyl-d-aspartate receptor (NMDAR) antagonists, leading to a major hypothesis that glutamate hypofunction underlies schizophrenia pathogenesis. The precise region that depends on NMDAR function, however, is unclear. Here, we developed a mouse strain in which NMDARs in the intralaminar thalamic nuclei (ILN) were selectively disrupted. The mutant mice exhibited various schizophrenia-like phenotypes, including deficits in working memory, long-term spatial memory, and attention, as well as impulsivity, impaired prepulse inhibition, hyperlocomotion and hyperarousal. The electroencephalography analysis revealed that the mutant mice had a significantly reduced power in a wide range of frequencies including the alpha, beta and gamma bands, both during wake and rapid eye movement (REM) sleep, and a modest decrease of gamma power during non-REM sleep. Notably, restoring NMDARs in the adult ILN rescued some of the behavioral abnormalities. These findings suggest that NMDAR dysfunction in the ILN contributes to the pathophysiology of schizophrenia-related disorders. Furthermore, the reversal of inherent schizophrenia-like phenotypes in the adult mutant mice supports that ILN is a potential target site for a therapeutic strategy.

Morphological characteristics of dendritic spines form the basis of cognitive ability. However, molecular mechanisms involved in finetuning of spine morphology during development are not fully understood. Moreover, it is unclear whether, and to what extent, these developmental mechanisms determine the normal adult spine morphological features. Here, we provide evidence that ␣2-isoform of Rac-specific GTPase-activating protein ␣-chimaerin (␣2-chimaerin) is involved in spine morphological refinement during late postnatal period, and furthermore show that this developmental ␣2-chimaerin function affects adult spine morphologies. We used a series of mice with global and conditional knock-out of ␣-chimaerin isoforms (␣1-chimaerin and ␣2-chimaerin). ␣2-Chimaerin disruption, but not ␣1-chimaerin disruption, in the mouse results in an increased size (and density) of spines in the hippocampus. In contrast, overexpression of ␣2-chimaerin in developing hippocampal neurons induces a decrease of spine size. Disruption of ␣2-chimaerin suppressed EphA-mediated spine morphogenesis in cultured developing hippocampal neurons. ␣2-Chimaerin disruption that begins during the juvenile stage results in an increased size of spines in the hippocampus. Meanwhile, spine morphologies are unaltered when ␣2-chimaerin is deleted only in adulthood. Consistent with these spine morphological results, disruption of ␣2-chimaerin beginning in the juvenile stage led to an increase in contextual fear learning in adulthood; whereas contextual learning was recently shown to be unaffected when ␣2-chimaerin was deleted only in adulthood. Together, these results suggest that ␣2-chimaerin signaling in developmental stages contributes to determination of the morphological features of adult spines and establishment of normal cognitive ability.