上田 壮志

Rapid eye movement (REM) sleep is primarily regulated by the brainstem pons. In particular, the sublaterodorsal tegmentum (SubLDT) in the dorsal pons contains neurons whose activity is selective to REM sleep. Elucidation of the precise identities of these neurons and their roles in REM sleep regulation is challenging, however, due to the functional and molecular heterogeneity of the SubLDT. A recent study revealed that corticotropin-releasing hormone-binding protein (Crhbp)-positive neurons in the SubLDT projecting to the medulla play a crucial role in REM sleep regulation and that loss of these Crhbp-positive neurons underlies sleep deficits observed in Parkinson's disease. The firing patterns of these neurons during sleep/wake, however, remained unknown. Here, we used an opto-tagging method and conducted cell-type-specific recordings from Crhbp-positive neurons using a glass pipette microelectrode in unanesthetized male mice. We recorded 58 Crhbp-positive neurons and found that many of these neurons are REM sleep-active neurons (41.4%) and that the remaining neurons are mostly either wake-active, wake/REM sleep-active, or NREM sleep-active. In addition, projection-specific recordings revealed that the medulla-projecting Crhbp-positive neurons are mostly REM sleep-active neurons (75.0%). Based on clustering analysis and spike waveform analysis, REM sleep-active Crhbp-positive neurons can be further divided into different subtypes according to their electrophysiological properties, suggesting that Crhbp-positive neurons play diverse roles in REM sleep regulation.Significance statement Reduced REM sleep is a risk for dementia and mortality, suggesting it has critical roles in health. The mechanisms and functions of REM sleep, however, remain largely elusive. Classical electrophysiological studies identified neurons in the pons that are active during REM sleep, and a recent study revealed that Crhbp-positive neurons within the same area contribute to REM sleep regulation. The relationship between the neurons identified in each study, however, remained unknown. Loss of Crhbp-positive neurons underlies sleep deficits in Parkinson's disease, underscoring the importance of characterizing these neurons. Our study revealed that many of the Crhbp-positive neurons are REM sleep-active and comprise distinct subtypes in regard to firing patterns, suggesting their diverse roles in REM sleep regulation.

Summary The neural mechanisms regulating sequential transitions of male sexual behaviors, such as mounting, intromission, and ejaculation, in the brain remain unclear. Here, we report that dopamine (DA) and acetylcholine (ACh) dynamics in the ventral shell of the nucleus accumbens (vsNAc) closely aligns with serial transitions of sexual behaviors in male mice. During intromission, the vsNAc exhibits dual ACh-DA rhythms generated by reciprocal regulation between ACh and DA signaling via nicotinic acetylcholine (nAChR) and dopamine D2 (D2R) receptors. Knockdown of choline acetyl transferase (ChAT) or D2R in the vsNAc diminished the likelihood of intromission and ejaculation. Optogenetic manipulations reveal that DA signaling sustains male sexual behaviors by suppressing activities of D2R^vsNAcneurons. Moreover, ACh signaling promotes the initiation of mounting and intromission, but also induces the intromission-to-ejaculation transition by triggering a slowdown of DA rhythm. Therefore, dual ACh-DA dynamics harmonize in the vsNAc to drive sequential transitions of male mating behaviors.

Abstract Despite the importance of sleep to the cerebral cortex, how much sleep changes cortical neuronal firing remains unclear due to complicated firing behaviors. Here we quantified firing of cortical neurons using Hawkes process modeling that can model sequential random events exhibiting temporal clusters. “Intensity” is a parameter of Hawkes process that defines the probability of an event occurring. We defined the appearance of repetitive firing as the firing intensity corresponding to “intensity” in Hawkes process. Firing patterns were quantified by the magnitude of firing intensity, the time constant of firing intensity, and the background firing intensity. The higher the magnitude of firing intensity, the higher the likelihood that the spike will continue. The larger the time constant of firing intensity, the longer the repetitive firing lasts. The higher the background firing intensity, the more likely neurons fire randomly. The magnitude of firing intensity was inversely proportional to the time constant of firing intensity, and non-REM sleep increased the magnitude of firing intensity and decreased the time constant of firing intensity. The background firing intensity was not affected by the sleep/wake state. Our findings suggest that the cortex is organized such that neurons with a higher probability of repetitive firing have shorter repetitive firing periods. In addition, our results suggest that repetitive firing is ordered to become high frequency and short term during non-REM sleep, while unregulated components of firing are independent of the sleep/wake state in the cortex. Hawkes process modeling of firing will reveal novel properties of the brain.

We recently determined that the excitatory manipulation of Qrfp-expressing neurons in the preoptic area of the hypothalamus (quiescence-inducing neurons [Q neurons]) induced a hibernation-like hypothermic/hypometabolic state (QIH) in mice. To control the QIH with a higher time resolution, we develop an optogenetic method using modified human opsin4 (OPN4; also known as melanopsin), a G protein-coupled-receptor-type blue-light photoreceptor. C-terminally truncated OPN4 (OPN4dC) stably and reproducibly induces QIH for at least 24 h by illumination with low-power light (3 μW, 473 nm laser) with high temporal resolution. The high sensitivity of OPN4dC allows us to transcranially stimulate Q neurons with blue-light-emitting diodes and non-invasively induce the QIH. OPN4dC-mediated QIH recapitulates the kinetics of the physiological changes observed in natural hibernation, revealing that Q neurons concurrently contribute to thermoregulation and cardiovascular function. This optogenetic method may facilitate identification of the neural mechanisms underlying long-term dormancy states such as sleep, daily torpor, and hibernation.

Sleep is generally viewed as a period of recovery, but how the supply of cerebral blood flow (CBF) changes across sleep/wake states has remained unclear. Here, we directly observe red blood cells (RBCs) within capillaries, where the actual substance exchange between the blood and neurons/glia occurs, by two-photon microscopy. Across multiple cortical areas, average capillary CBF is largely increased during rapid eye movement (REM) sleep, whereas it does not differ between periods of active wakefulness and non-REM sleep. Capillary RBC flow during REM sleep is further elevated following REM sleep deprivation, suggesting that capillary CBF reflects REM sleep pressure. At the molecular level, signaling via adenosine A2a receptors is crucial; in A2a-KO mice, capillary CBF upsurge during REM sleep is dampened, and effects of REM sleep pressure are abolished. These results provide evidence regarding the dynamics of capillary CBF across sleep/wake states and insights to the underlying mechanisms.

Cortical neurons fire intermittently and synchronously during non-rapid eye movement sleep (NREMS), in which active and silent periods are referred to as ON and OFF periods, respectively. Neuronal firing rates during ON periods (NREMS-ON-activity) are similar to those of wakefulness (W-activity), raising the possibility that NREMS-ON neuronal-activity is fragmented W-activity. To test this, we investigated the patterning and organization of cortical spike trains and of spike ensembles in neuronal networks using extracellular recordings in mice. Firing rates of neurons during NREMS-ON and W were similar, but showed enhanced bursting in NREMS with no apparent preference in occurrence, relative to the beginning or end of the on-state. Additionally, there was an overall increase in the randomness of occurrence of sequences comprised of multi-neuron ensembles in NREMS recorded from tetrodes. In association with increased burst firing, somatic calcium transients were increased in NREMS. The increased calcium transients associated with bursting during NREM may activate calcium-dependent, cell-signaling pathways for sleep related cellular processes.

The amount, quality and diurnal pattern of sleep change greatly during development. Developmental changes of sleep/wake architecture are in a close relationship to brain development. The fragmentation of wake episodes is one of the salient features in the neonatal period, which is also observed in mature animals and human individuals lacking neuropeptide orexin/hypocretin signaling. This raises the possibility that developmental changes of lateral hypothalamic orexin neurons are relevant to the development of sleep/wake architecture. However, little information is available on morphological and physiological features of developing orexin neurons. To address the cellular basis for maturation of the sleep/wake regulatory system, we investigated the functional development of orexin neurons in the lateral hypothalamus. The anatomical development as well as the changes in the electrophysiological characteristics of orexin neurons was examined from embryonic to postnatal stages in Orexin-EGFP mice. Prepro-orexin promoter activity was detectable at embryonic day (E) 12.0, followed by expression of orexin A after E14.0. The number of orexin neurons and their membrane capacitance reached similar levels to adults by postnatal day (P) 7, while their membrane potentials, firing rates, and action potential waveforms were developed by P21. The hyperpolarizing effect of serotonin, which is a major inhibitory signal for adult orexin neurons, was detected after E18.0 and matured at P1. These results suggest that the expression of orexin peptides precedes the maturation of electrophysiological activity of orexin neurons. The function of orexin neurons gradually matures by three weeks after birth, coinciding with maturation of sleep/wake architecture. This article is protected by copyright. All rights reserved.