Temporal and Potential Predictive Relationships between Sleep Spindle Density and Spike-and-Wave Discharges.

Spike-and-wave discharges (SWDs) and sleep spindles are characteristic electroencephalographic (EEG) hallmarks of absence seizures and nonrapid eye movement sleep, respectively. They are commonly generated by the cortico-thalamo-cortical network including the thalamic reticular nucleus (TRN). It has been reported that SWD development is accompanied by a decrease in sleep spindle density in absence seizure patients and animal models. However, whether the decrease in sleep spindle density precedes, coincides with, or follows, the SWD development remains unknown. To clarify this, we exploited Pvalb-tetracycline transactivator (tTA)::tetO-ArchT (PV-ArchT) double-transgenic mouse, which can induce an absence seizure phenotype in a time-controllable manner by expressing ArchT in PV neurons of the TRN. In these mice, EEG recordings demonstrated that a decrease in sleep spindle density occurred 1 week before the onset of typical SWDs, with the expression of ArchT. To confirm such temporal relationship observed in these genetic model mice, we used a gamma-butyrolactone (GBL) pharmacological model of SWDs. Prior to GBL administration, we administered caffeine to wild-type mice for 3 consecutive days to induce a decrease in sleep spindle density. We then administered low-dose GBL, which cannot induce SWDs in normally conditioned mice but led to the occurrence of SWDs in caffeine-conditioned mice. These findings indicate a temporal relationship in which the decrease in sleep spindle density consistently precedes SWD development. Furthermore, the decrease in sleep spindle activity may have a role in facilitating the development of SWDs. Our findings suggest that sleep spindle reductions could serve as early indicators of seizure susceptibility.

Serotonergic regulation of cortical neurovascular coupling and hemodynamics upon awakening from sleep in mice.

Neurovascular coupling (NVC) is the functional hyperemia of the brain responding to local neuronal activity. It is mediated by astrocytes and affected by subcortical ascending pathways in the cortex that convey information, such as sensory stimuli and the animal condition. Here, we investigate the influence of the raphe serotonergic system, a subcortical ascending arousal system in animals, on the modulation of cortical NVC and cerebral blood flow (CBF). Raphe serotonergic neurons were optogenically activated for 30 s, which immediately awakened the mice from non-rapid eye movement sleep. This caused a biphasic cortical hemodynamic change: a transient increase for a few seconds immediately after photostimulation onset, followed by a large progressive decrease during the stimulation period. Serotonergic neuron activation increased intracellular Ca2+ levels in cortical pyramidal neurons and astrocytes, demonstrating its effect on the NVC components. Pharmacological inhibition of cortical neuronal firing activity and astrocyte metabolic activity had small hypovolemic effects on serotonin-induced biphasic CBF changes, while blocking 5-HT1B receptors expressed primarily in cerebral vasculature attenuated the decreasing CBF phase. This suggests that serotonergic neuron activation leading to animal awakening could allow the NVC to exert a hyperemic function during a biphasic CBF response, with a predominant decrease in the cortex.

Dopamine Receptor Type 2-Expressing Medium Spiny Neurons in the Ventral Lateral Striatum Have a Non-REM Sleep-Induce Function.

Dopamine receptor type 2-expressing medium spiny neurons (D2-MSNs) in the medial part of the ventral striatum (VS) induce non-REM (NREM) sleep from the wake state in animals. However, it is unclear whether D2-MSNs in the lateral part of the VS (VLS), which is anatomically and functionally different from the medial part of the VS, contribute to sleep-wake regulation. This study aims to clarify whether and how D2-MSNs in the VLS are involved in sleep-wake regulation. Our study found that specifically removing D2-MSNs in the VLS led to an increase in wakefulness time in mice during the dark phase using a diphtheria toxin-mediated cell ablation/dysfunction technique. D2-MSN ablation throughout the VS further increased dark phase wakefulness time. These findings suggest that VLS D2-MSNs may induce sleep during the dark phase with the medial part of the VS. Next, our fiber photometric recordings revealed that the population intracellular calcium (Ca2+) signal in the VLS D2-MSNs increased during the transition from wake to NREM sleep. The mean Ca2+ signal level of VLS D2-MSNs was higher during NREM and REM sleep than during the wake state, supporting their sleep-inducing role. Finally, optogenetic activation of the VLS D2-MSNs during the wake state always induced NREM sleep, demonstrating the causality of VLS D2-MSNs activity with sleep induction. Additionally, activation of the VLS D1-MSNs, counterparts of D2-MSNs, always induced wake from NREM sleep, indicating a wake-promoting role. In conclusion, VLS D2-MSNs could have an NREM sleep-inducing function in coordination with those in the medial VS.

Serotonergic neurons control cortical neuronal intracellular energy dynamics by modulating astrocyte-neuron lactate shuttle.

The central serotonergic system has multiple roles in animal physiology and behavior, including sleep-wake control. However, its function in controlling brain energy metabolism according to the state of animals remains undetermined. Through in vivo monitoring of energy metabolites and signaling, we demonstrated that optogenetic activation of raphe serotonergic neurons increased cortical neuronal intracellular concentration of ATP, an indispensable cellular energy molecule, which was suppressed by inhibiting neuronal uptake of lactate derived from astrocytes. Raphe serotonergic neuronal activation induced cortical astrocytic Ca2+ and cAMP surges and increased extracellular lactate concentrations, suggesting the facilitation of lactate release from astrocytes. Furthermore, chemogenetic inhibition of raphe serotonergic neurons partly attenuated the increase in cortical neuronal intracellular ATP levels as arousal increased in mice. Serotonergic neuronal activation promoted an increase in cortical neuronal intracellular ATP levels, partly mediated by the facilitation of the astrocyte-neuron lactate shuttle, contributing to state-dependent optimization of neuronal intracellular energy levels.

Region-specific involvement of ventral striatal dopamine D2 receptor-expressing medium spiny neurons in nociception.

The ventrolateral striatum (VLS), a subregion of the ventral striatum (VS), possesses distinct neuronal Ca2+ activities and functions in reward-oriented behavior, compared with the ventromedial striatum (VMS) based on the anatomical feature. We hypothesized that the VLS exhibits unique neuronal activity and function in nociceptive processing, a part of aversive processing. Using fiber photometry to monitor the neuronal Ca2+ activities, we demonstrated that acute noxious mechanical stimuli like tail-pinch increased the Ca2+ activity of dopamine D2 receptor-expressing medium spiny neurons (D2-MSNs) in the VLS in correlation with the stimulus intensities in mice, whereas mechanical stimuli increased the VMS D2-MSN activity independent of the stimulus intensities. Likewise, thermal stimuli decreased the VLS and VMS D2-MSN Ca2+ activities during nociceptive behaviors in the hot plate test. Furthermore, the VLS D2-MSNs increased their Ca2+ activity accompanied by formalin-induced nociceptive behaviors in mice, whereas the VMS D2-MSNs decreased it. The optogenetic inhibition of VLS D2-MSN activity increased the formalin-induced pain-related behavior in mice, thus suggesting the inhibitory effect of VLS D2-MSN activity on chemical nociceptive behavior, in contrast to previous reports that the VMS D2-MSNs could not involve the behavior. Therefore, the VLS D2-MSNs exhibited region-specific roles in nociception.

The Mlc1 Promoter Directs Müller Cell-specific Gene Expression in the Retina.

Purpose: Because the importance of glia in regulating brain functions has been demonstrated, genetic technologies that manipulate glial cell-specific gene expression in the brain have become essential and have made great progress. However, it is unknown whether the same strategy that is used in the brain can be applied to the retina because retinal glia differs from glia in the brain. Here, we aimed to find a method for selective gene expression in Müller cells (characteristic glial cells in the retina) and identified Mlc1 as a specific promoter of Müller cells. Methods: Mlc1-tTA::Yellow-Cameleon-NanotetO/tetO (YC-Nano) mice were used as a reporter line. YC-Nano, a fluorescent protein, was ectopically expressed in the cell type controlled by the Mlc1 promotor. Immunofluorescence staining was used to identify the cell type expressing YC-Nano protein. Results: YC-Nano-positive (+) signals were observed as vertical stalks in the sliced retina and spanned from the nerve fiber layer through the outer nuclear layer. The density of YC-Nano+ cells was higher around the optic nerve head and lower in the peripheral retina. The YC-Nano+ signals colocalized with vimentin, a marker of Müller cells, but not with the cell markers for blood vessels, microglia, neurons, or astrocytes. Conclusions: The Mlc1 promoter allows us to manipulate gene expression in Müller cells without affecting astrocytes in the retina. Translational Relevance: Gene manipulation under control of Mlc1 promoter offers novel technique to investigate the role of Müller cells.

FoxG1 regulates the formation of cortical GABAergic circuit during an early postnatal critical period resulting in autism spectrum disorder-like phenotypes.

Abnormalities in GABAergic inhibitory circuits have been implicated in the aetiology of autism spectrum disorder (ASD). ASD is caused by genetic and environmental factors. Several genes have been associated with syndromic forms of ASD, including FOXG1. However, when and how dysregulation of FOXG1 can result in defects in inhibitory circuit development and ASD-like social impairments is unclear. Here, we show that increased or decreased FoxG1 expression in both excitatory and inhibitory neurons results in ASD-related circuit and social behavior deficits in our mouse models. We observe that the second postnatal week is the critical period when regulation of FoxG1 expression is required to prevent subsequent ASD-like social impairments. Transplantation of GABAergic precursor cells prior to this critical period and reduction in GABAergic tone via Gad2 mutation ameliorates and exacerbates circuit functionality and social behavioral defects, respectively. Our results provide mechanistic insight into the developmental timing of inhibitory circuit formation underlying ASD-like phenotypes in mouse models.

Intracellular ATP levels in mouse cortical excitatory neurons varies with sleep-wake states.

Whilst the brain is assumed to exert homeostatic functions to keep the cellular energy status constant under physiological conditions, this has not been experimentally proven. Here, we conducted in vivo optical recordings of intracellular concentration of adenosine 5'-triphosphate (ATP), the major cellular energy metabolite, using a genetically encoded sensor in the mouse brain. We demonstrate that intracellular ATP levels in cortical excitatory neurons fluctuate in a cortex-wide manner depending on the sleep-wake states, correlating with arousal. Interestingly, ATP levels profoundly decreased during rapid eye movement sleep, suggesting a negative energy balance in neurons despite a simultaneous increase in cerebral hemodynamics for energy supply. The reduction in intracellular ATP was also observed in response to local electrical stimulation for neuronal activation, whereas the hemodynamics were simultaneously enhanced. These observations indicate that cerebral energy metabolism may not always meet neuronal energy demands, consequently resulting in physiological fluctuations of intracellular ATP levels in neurons.

Neuroimaging correlates of narcolepsy with cataplexy: A systematic review.

Recent developments in neuroimaging techniques have advanced our understanding of biological mechanisms underpinning narcolepsy. We used MEDLINE to retrieve neuroimaging studies to compare patients with narcolepsy and healthy controls. Thirty-seven studies were identified and demonstrated several replicated abnormalities: (1) gray matter reductions in superior frontal, superior and inferior temporal, and middle occipital gyri, hypothalamus, amygdala, insula, hippocampus, cingulate cortex, thalamus, and nucleus accumbens, (2) decreased fractional anisotropy in white matter of fronto-orbital and cingulate area, (3) reduced brain metabolism or cerebral blood flow in middle and superior frontal, and cingulate cortex (4) increased activity in inferior frontal gyri, insula, amygdala, and nucleus accumbens, and (5) N-acetylaspartate/creatine-phosphocreatine level reduction in hypothalamus. In conclusion, all the replicated findings are still controversial due to the limitations such as heterogeneity or size of the samples and lack of multimodal imaging or follow-up. Thus, future neuroimaging studies should employ multimodal imaging methods in a large sample size of patients with narcolepsy and consider age, duration of disease, age at onset, severity, human leukocyte antigen type, cerebrospinal fluid hypocretin levels, and medication intake in order to elucidate possible neuroimaging characteristic of narcolepsy and identify therapeutic targets.

Distinct Roles of Ventromedial versus Ventrolateral Striatal Medium Spiny Neurons in Reward-Oriented Behavior.

The ventral striatum (VS) is a key brain center regulating reward-oriented behavior [1-4]. The VS can be anatomically divided into medial (VMS) and lateral (VLS) portions based on cortical input patterns. The VMS receives inputs from medial pallium-originated limbic structures (e.g., the medial prefrontal cortex [mPFC]), and the VLS receives inputs from the lateral pallium-originated areas (e.g., the insula) [5, 6]. This anatomical feature led us to hypothesize a functional segregation within the VS in terms of the regulation of reward-oriented behavior. Here, we engineered a fiber photometry system [4] and monitored population-level Ca2+ activities of dopamine D2-receptor-expressing medium spiny neurons (D2-MSNs), one of the major cell types in the striatum, during a food-seeking discrimination task. We found that VLS D2-MSNs were activated at the time of cue presentation. In stark contrast, VMS D2-MSNs were inhibited at this time point. Optogenetic counteraction of those changes in the VLS and VMS impaired action initiation and increased responding toward non-rewarded cues, respectively. During lever-press reversal training, VMS inhibition at the time of cue presentation temporarily ceased and optogenetic activation of VMS D2-MSNs facilitated acquisition of the new contingency. These data indicate that the opposing inhibition and excitation in VMS and VLS are important for selecting and initiating a proper action in a reward-oriented behavior. We propose distinct subregional roles within the VS in the execution of successful reward-oriented behavior.

Ventrolateral Striatal Medium Spiny Neurons Positively Regulate Food-Incentive, Goal-Directed Behavior Independently of D1 and D2 Selectivity.

The ventral striatum is involved in motivated behavior. Akin to the dorsal striatum, the ventral striatum contains two parallel pathways: the striatomesencephalic pathway consisting of dopamine receptor Type 1-expressing medium spiny neurons (D1-MSNs) and the striatopallidal pathway consisting of D2-MSNs. These two genetically identified pathways are thought to encode opposing functions in motivated behavior. It has also been reported that D1/D2 genetic selectivity is not attributed to the anatomical discrimination of two pathways. We wanted to determine whether D1- and D2-MSNs in the ventral striatum functioned in an opposing manner as previous observations claimed, and whether D1/D2 selectivity corresponded to a functional segregation in motivated behavior of mice. To address this question, we focused on the lateral portion of ventral striatum as a region implicated in food-incentive, goal-directed behavior, and recorded D1 or D2-MSN activity by using a gene-encoded ratiometric Ca2+ indicator and by constructing a fiberphotometry system, and manipulated their activities via optogenetic inhibition during ongoing behaviors. We observed concurrent event-related compound Ca2+ elevations in ventrolateral D1- and D2-MSNs, especially at trial start cue-related and first lever press-related times. D1 or D2 selective optogenetic inhibition just after the trial start cue resulted in a reduction of goal-directed behavior, indicating a shared coding of motivated behavior by both populations at this time. Only D1-selective inhibition just after the first lever press resulted in the reduction of behavior, indicating D1-MSN-specific coding at that specific time. Our data did not support opposing encoding by both populations in food-incentive, goal-directed behavior.SIGNIFICANCE STATEMENT An opposing role of dopamine receptor Type 1 or Type 2-expressing medium spiny neurons (D1-MSNs or D2-MSNs) on striatum-mediated behaviors has been widely accepted. However, this idea has been questioned by recent reports. In the present study, we measured concurrent Ca2+ activity patterns of D1- and D2-MSNs in the ventrolateral striatum during food-incentive, goal-directed behavior in mice. According to Ca2+ activity patterns, we conducted timing-specific optogenetic inhibition of each type of MSN. We demonstrated that both D1- and D2-MSNs in the ventrolateral striatum commonly and positively encoded action initiation, whereas only D1-MSNs positively encoded sustained motivated behavior. These findings led us to reconsider the prevailing notion of a functional segregation of MSN activity in the ventral striatum.

Observation and manipulation of glial cell function by virtue of sufficient probe expression.

The development of gene-encoded indicators and actuators to observe and manipulate cellular functions is being advanced and investigated. Expressing these probe molecules in glial cells is expected to enable observation and manipulation of glial cell activity, leading to elucidate the behaviors and causal roles of glial cells. The first step toward understanding glial cell functions is to express the probes in sufficient amounts, and the Knockin-mediated ENhanced Gene Expression (KENGE)-tet system provides a strategy for achieving this. In the present article, three examples of KENGE-tet system application are reviewed: depolarization of oligodendrocytes, intracellular acidification of astrocytes, and observation of intracellular calcium levels in the fine processes of astrocytes.

Identification of the extent of cortical spreading depression propagation by Npas4 mRNA expression.

Cortical spreading depression (CSD) is a phenomenon associated with a propagating large shift in direct current (DC) potential followed by suppression of electrophysiological activity. For temporal analysis of CSD propagation, electrophysiological recording is the most reliable tool. However, it is difficult to completely identify the spatial area of the brain influenced by CSD, because recording sites are technically limited. Histological post hoc identification of activated neurons by labeling the induction of an immediate early gene (IEG) could determine areas of CSD propagation. We found that cortical application of potassium chloride induced expression of Npas4 IEG mRNA in the ipsilateral dorsal cortex. Interestingly, induction of Npas4 was never observed in the ipsilateral hippocampus and there was a clear boundary to the area of Npas4 expression. To determine whether the boundary of the area of Npas4 mRNA expression was the limit of CSD propagation, we recorded local field potentials from multiple sites that crossed the boundary of Npas4 expression. We found that the area of Npas4 mRNA expression coincided with the area of DC-potential shift propagation. We propose that induction of Npas4 identifies the area influenced by CSD propagation.

Differential regulation of circadian melatonin rhythm and sleep-wake cycle by bright lights and nonphotic time cues in humans.

Our previous study demonstrated that physical exercise under dim lights (<10 lux) accelerated reentrainment of the sleep-wake cycle but not the circadian melatonin rhythm to an 8-h phase-advanced sleep schedule, indicating differential effects of physical exercise on the human circadian system. The present study examined the effects of bright light (>5,000 lux) on exercise-induced acceleration of reentrainment because timed bright lights are known to reset the circadian pacemaker. Fifteen male subjects spent 12 days in temporal isolation. The sleep schedule was advanced from habitual sleep times by 8 h for 4 days, which was followed by a free-run session. In the shift session, bright lights were given during the waking time. Subjects in the exercise group performed 2-h bicycle running twice a day. Subjects in the control kept quiet. As a result, the sleep-wake cycle was fully entrained by the shift schedule in both groups. Bright light may strengthen the resetting potency of the shift schedule. By contrast, the circadian melatonin rhythm was phase-advanced by 6.9 h on average in the exercise group but only by 2.0 h in the control. Thus physical exercise prevented otherwise unavoidable internal desynchronization. Polysomnographical analyses revealed that deterioration of sleep quality by shift schedule was protected by physical exercise under bright lights. These findings indicate differential regulation of sleep-wake cycle and circadian melatonin rhythm by physical exercise in humans. The melatonin rhythm is regulated primarily by bright lights, whereas the sleep-wake cycle is by nonphotic time cues, such as physical exercise and shift schedule.

Dual regulation of clock gene Per2 expression in discrete brain areas by the circadian pacemaker and methamphetamine-induced oscillator in rats.

Behavioral rhythms induced by methamphetamine (MAP) treatment in rats are independent of the circadian pacemaker in the suprachiasmatic nucleus (SCN). To know the site and mechanism of an underlying oscillation (MAP-induced oscillator; MAO), extra-SCN circadian rhythms in the discrete brain areas were examined in rats with and without the SCN. To fix the phase of MAO, MAP was supplied in drinking water at a restricted time of day for 14 days (R-MAP) and subsequently given ad libitum (ad-MAP). Plain water was given to the controls at the same restricted time (R-Water). Clock gene Per2 expression was measured by a bioluminescence reporter in cultured brain tissues. In SCN-intact rats, MAO was induced by R-MAP and behavioral rhythms were phase-delayed from the restricted time under ad-MAP with relative coordination. Circadian Per2 rhythms in R-MAP rats were not affected in the SCN but were slightly phase-advanced in the olfactory bulb (OB), caudate-putamen (CPU) and substantia nigra (SN) as compared with R-Water rats. Following SCN lesion, R-MAP-induced MAO phase-shifted more slowly and did not show a sign of relative coordination. In these rats, circadian Per2 rhythms were significantly phase-shifted in the OB and SN as compared with SCN-intact rats. These findings indicate that MAO was induced by MAP given at a restricted time of day in association with phase-shifts of the extra-SCN circadian oscillators in the brain dopaminergic areas. The findings also suggest that these extra-SCN oscillators are the components of MAO and receive dual regulation by MAO and the SCN circadian pacemaker.

Differential responses of circadian Per2 rhythms in cultured slices of discrete brain areas from rats showing internal desynchronisation by methamphetamine.

Chronic methamphetamine (MAP) treatment desynchronises the behavior rhythms of rats from light-dark cycles. Our previous study (Masubuchi et al., 2000) demonstrated the phase reversal of circadian rhythms in clock gene expression in several brain areas of rats treated with MAP. However, for technical reasons, it was not clear whether the phase shifts were the consequence of phase-shifted behavior rhythms or reflected phase shifts of extra-suprachiasmatic nucleus (SCN) oscillators in these areas. In the present study, circadian gene expression rhythms in discrete brain areas were continuously monitored in slice cultures of MAP-treated rats. Methamphetamine was given to rats carrying a Period2-dLuciferase reporter system via the drinking water for more than 2 weeks. When behavior rhythms were completely phase reversed, the brain was sampled for slice cultures and circadian bioluminescence rhythms were measured for 5 days in the SCN and four areas of the dopaminergic system, the olfactory bulb, caudate putamen, parietal cortex and substantia nigra. The circadian rhythms in the SCN and caudate putamen were not significantly phase shifted, whereas those in the parietal cortex and substantia nigra showed significant phase-delay shifts of 6-8 h and that in the olfactory bulb showed phase-advance shifts of ca. 8 h. Neither the period nor the amplitude of the circadian rhythm was changed by MAP treatment. These findings indicate that the extra-SCN oscillators in several brain areas are desynchronised from the SCN circadian pacemaker by MAP treatment in parallel with the desynchronisation of behavior rhythms in rats. As the direction and extent of phase shifts of circadian rhythms were different among the areas examined, the brain extra-SCN oscillators responded differentially to MAP.

Differential responses of circadian Per2 expression rhythms in discrete brain areas to daily injection of methamphetamine and restricted feeding in rats.

Behavioral rhythms induced by methamphetamine (MAP) and daily restricted feeding (RF) in rats are independent of the circadian pacemaker in the suprachiasmatic nucleus (SCN), and have been regarded to share a common oscillatory mechanism. In the present study, in order to examine the responses of brain oscillatory systems to MAP and RF, circadian rhythms in clock gene, Period2, expression were measured in several brain areas in rats. Transgenic rats carrying a bioluminescence reporter of Period2-dLuciferase were subjected to either daily injection of MAP or RF of 2 h at a fixed time of day for 14 days. As a result, spontaneous movement and wheel-running activity were greatly enhanced following MAP injection and prior to daily meal under RF. Circadian Per2 rhythms were measured in the cultured brain tissues containing one of the following structures: the olfactory bulb; caudate-putamen; parietal cortex; substantia nigra; and SCN. Except for the SCN, the circadian Per2 rhythms in the brain tissues were significantly phase-delayed by 1.9 h on average in MAP-injected rats as compared with the saline-controls. On the other hand, the circadian rhythms outside the SCN were significantly phase-advanced by 6.3 h on average in rats under RF as compared with those under ad libitum feeding. These findings indicate that the circadian rhythms in specific brain areas of the central dopaminergic system respond differentially to MAP injection and RF, suggesting that different oscillatory mechanisms in the brain underlie the MAP-induced behavior and pre-feeding activity under RF.