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.

Identification of region-specific gene isoforms in the human brain using long-read transcriptome sequencing.

In neurological and neuropsychiatric diseases, different brain regions are affected, and differences in gene expression patterns could potentially explain this mechanism. However, limited studies have precisely explored gene expression in different regions of the human brain. In this study, we performed long-read RNA sequencing on three different brain regions of the same individuals: the cerebellum, hypothalamus, and temporal cortex. Despite stringent filtering criteria excluding isoforms predicted to be artifacts, over half of the isoforms expressed in multiple samples across multiple regions were found to be unregistered in the GENCODE reference. We then especially focused on genes with different major isoforms in each brain region, even with similar overall expression levels, and identified that many of such genes including GAS7 might have distinct roles in dendritic spine and neuronal formation in each region. We also found that DNA methylation might, in part, drive different isoform expressions in different regions. These findings highlight the significance of analyzing isoforms expressed in disease-relevant sites.

Repeated Acute Exacerbations of Chronic Inflammatory Demyelinating Polyradiculoneuropathy Accompanied by Pain and Swelling in Distal Extremities.

An 81-year-old man experienced acute progression of weakness in the extremities accompanied by a fever, tenderness, and swelling in distal parts of the extremities. He had flaccid tetraparesis with fasciculations and general hyporeflexia. Nerve conduction studies indicated demyelinating sensorimotor neuropathy. A cerebrospinal fluid examination revealed elevated proteins without pleocytosis. Immunological treatments were effective, but his symptoms exhibited repeated relapse and remission phases. He was diagnosed with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) with an acute onset. The highlight of this case is pain with inflammatory reaction recognized as red flags of CIDP, with the clinical course and electrophysiological findings compatible with CIDP.

Impaired long-term potentiation-like motor cortical plasticity in progressive supranuclear palsy.

OBJECTIVE: To elucidate long-term potentiation (LTP)-like effects on the primary motor cortical (M1) in progressive supranuclear palsy (PSP) and its relationships with clinical features. METHODS: Participants were 18 probable/possible PSP Richardson syndrome (PSP-RS) patients and 17 healthy controls (HC). We used quadripulse stimulation (QPS) over the M1 with an interstimulus interval of 5 ms (QPS-5) to induce LTP-like effect and analyzed the correlations between the degree of LTP-like effect and clinical features. We also evaluated cortical excitability using short interval intracortical inhibition (SICI), intracortical facilitation (ICF) and short interval intracortical facilitation (SICF) in 15 PSP patients and 17 HC. RESULTS: LTP-like effect after QPS in PSP was smaller than HC and negatively correlated with Unified Parkinson's Disease Rating Scale Part III (UPDRS-III) score, especially bradykinesia, but not with either age or any scores of cognitive functions. The SICI was abnormally reduced in PSP, but neither ICF nor SICF differed from those of normal subjects. None of these cortical excitability parameters correlated with any clinical features. CONCLUSIONS: LTP induction was impaired in PSP. The degree of LTP could reflect the severity of bradykinesia. The bradykinesia may partly relate with the motor cortical dysfunction. SIGNIFICANCE: The degree of motor cortical LTP could relate with the severity of motor symptoms in PSP.

Narcolepsy type I-associated DNA methylation and gene expression changes in the human leukocyte antigen region.

Narcolepsy type 1 (NT1) is caused by a loss of hypothalamic orexin-producing cells, and autoreactive CD4+ and CD8+ T cells have been suggested to play a role in the autoimmune mechanism. Although NT1 showed a strong association with human leukocyte antigen (HLA)-DQB1*06:02, the responsible antigens remain unidentified. We analyzed array-based DNA methylation and gene expression data for the HLA region in CD4+ and CD8+ T cells that were separated from the peripheral blood mononuclear cells of Japanese subjects (NT1, N = 42; control, N = 42). As the large number of SNPs in the HLA region might interfere with the affinity of the array probes, we conducted a comprehensive assessment of the reliability of each probe. The criteria were based on a previous study reporting that the presence of frequent SNPs, especially on the 3' side of the probe, makes the probe unreliable. We confirmed that 90.3% of the probes after general filtering in the HLA region do not include frequent SNPs, and are thus suitable for analysis, particularly in Japanese subjects. We then performed an association analysis, and found that several CpG sites in the HLA class II region of the patients were significantly hypomethylated in CD4+ and CD8+ T cells. This association was not detected when the effect of HLA-DQB1*06:02 was considered, suggesting that the hypomethylation was possibly derived from HLA-DQB1*06:02. Further RNA sequencing revealed reduced expression levels of HLA-DQB1 alleles other than HLA-DQB1*06:02 in the patients with NT1. Our results suggest the involvement of epigenetic and expressional changes in HLA-DQB1 in the pathogenesis of NT1.

Epigenetic silencing of selected hypothalamic neuropeptides in narcolepsy with cataplexy.

Narcolepsy with cataplexy is a sleep disorder caused by deficiency in the hypothalamic neuropeptide hypocretin/orexin (HCRT), unanimously believed to result from autoimmune destruction of hypocretin-producing neurons. HCRT deficiency can also occur in secondary forms of narcolepsy and be only temporary, suggesting it can occur without irreversible neuronal loss. The recent discovery that narcolepsy patients also show loss of hypothalamic (corticotropin-releasing hormone) CRH-producing neurons suggests that other mechanisms than cell-specific autoimmune attack, are involved. Here, we identify the HCRT cell-colocalized neuropeptide QRFP as the best marker of HCRT neurons. We show that if HCRT neurons are ablated in mice, in addition to Hcrt, Qrfp transcript is also lost in the lateral hypothalamus, while in mice where only the Hcrt gene is inactivated Qrfp is unchanged. Similarly, postmortem hypothalamic tissues of narcolepsy patients show preserved QRFP expression, suggesting the neurons are present but fail to actively produce HCRT. We show that the promoter of the HCRT gene of patients exhibits hypermethylation at a methylation-sensitive and evolutionary-conserved PAX5:ETS1 transcription factor-binding site, suggesting the gene is subject to transcriptional silencing. We show also that in addition to HCRT, CRH and Dynorphin (PDYN) gene promoters, exhibit hypermethylation in the hypothalamus of patients. Altogether, we propose that HCRT, PDYN, and CRH are epigenetically silenced by a hypothalamic assault (inflammation) in narcolepsy patients, without concurrent cell death. Since methylation is reversible, our findings open the prospect of reversing or curing narcolepsy.

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.

Motor cortical plasticity and its correlation with motor symptoms in Parkinson's disease.

Background: The relationship between abnormal cortical plasticity and parkinsonian symptoms remains unclear in Parkinson's disease (PD). Objective: We studied the relationship between their symptoms and degree of Long-term potentiation (LTP)-like effects induced by quadripulse magnetic stimulation (QPS) over the primary motor cortex, which has a small inter-individual variability in humans. Methods: Participants were 16 PD patients (drug-naïve or treated with L-DOPA monotherapy) and 13 healthy controls (HC). LTP-like effects by QPS were compared between three conditions (HC､PD with or without L-DOPA). In PD, correlation analyses were performed between clinical scores (MDS-UPDRS, MMSE and MoCA-J) and the degree of LTP-like effects induced by QPS. Results: In PD, QPS-induced LTP-like effect was reduced and restored by L-DOPA. The degree of the LTP was negatively correlated with MDS-UPDRS Part I and III scores, but not with MMSE and MoCA-J. In the sub-scores, upper limb bradykinesia and rigidity showed a negative correlation with the LTP-like effect whereas the tremor had no correlation. Conclusions: Our results suggest that motor cortical plasticity relate with mechanisms underlying bradykinesia and rigidity in the upper limb muscles. LTP induced by QPS may be used as an objective marker of parkinsonian symptoms.

Low carnitine palmitoyltransferase 1 activity is a risk factor for narcolepsy type 1 and other hypersomnia.

STUDY OBJECTIVES: Narcolepsy type 1 (NT1) is associated with metabolic abnormalities but their etiology remains largely unknown. The gene for carnitine palmitoyltransferase 1B (CPT1B) and abnormally low serum acylcarnitine levels have been linked to NT1. To elucidate the details of altered fatty acid metabolism, we determined levels of individual acylcarnitines and evaluated CPT1 activity in patients with NT1 and other hypersomnia. METHODS: Blood samples from 57 NT1, 51 other hypersomnia patients, and 61 healthy controls were analyzed. The levels of 25 major individual acylcarnitines were determined and the C0/(t[C16] + t[C18]) ratio was used as a CPT1 activity marker. We further performed transcriptome analysis using independent blood samples from 42 NT1 and 42 healthy controls to study the relevance of fatty acid metabolism. NT1-specific changes in CPT1 activity and in expression of related genes were investigated. RESULTS: CPT1 activity was lower in patients with NT1 (p = 0.00064) and other hypersomnia (p = 0.0014) than in controls. Regression analysis revealed that CPT1 activity was an independent risk factor for NT1 (OR: 1.68; p = 0.0031) and for other hypersomnia (OR: 1.64; p = 0.0042). There was a significant interaction between obesity (BMI <25, ≥25) and the SNP rs5770917 status such that nonobese NT1 patients without risk allele had better CPT1 activity (p = 0.0089). The expression levels of carnitine-acylcarnitine translocase (CACT) and CPT2 in carnitine shuttle were lower in NT1 (p = 0.000051 and p = 0.00014, respectively). CONCLUSIONS: These results provide evidences that abnormal fatty acid metabolism is involved in the pathophysiology of NT1 and other hypersomnia.

A rare genetic variant in the cleavage site of prepro-orexin is associated with idiopathic hypersomnia.

Idiopathic hypersomnia (IH) is a rare, heterogeneous sleep disorder characterized by excessive daytime sleepiness. In contrast to narcolepsy type 1, which is a well-defined type of central disorders of hypersomnolence, the etiology of IH is poorly understood. No susceptibility loci associated with IH have been clearly identified, despite the tendency for familial aggregation of IH. We performed a variation screening of the prepro-orexin/hypocretin and orexin receptors genes and an association study for IH in a Japanese population, with replication (598 patients and 9826 controls). We identified a rare missense variant (g.42184347T>C; p.Lys68Arg; rs537376938) in the cleavage site of prepro-orexin that was associated with IH (minor allele frequency of 1.67% in cases versus 0.32% in controls, P = 2.7 × 10-8, odds ratio = 5.36). Two forms of orexin (orexin-A and -B) are generated from cleavage of one precursor peptide, prepro-orexin. The difference in cleavage efficiency between wild-type (Gly-Lys-Arg; GKR) and mutant (Gly-Arg-Arg; GRR) peptides was examined by assays using proprotein convertase subtilisin/kexin (PCSK) type 1 and PCSK type 2. In both PCSK1 and PCSK2 assays, the cleavage efficiency of the mutant peptide was lower than that of the wild-type peptide. We also confirmed that the prepro-orexin peptides themselves transmitted less signaling through orexin receptors than mature orexin-A and orexin-B peptides. These results indicate that a subgroup of IH is associated with decreased orexin signaling, which is believed to be a hallmark of narcolepsy type 1.

Associations Between the 2011 Great East Japan Earthquake and Tsunami and the Sleep and Mental Health of Japanese People: A 3-Wave Repeated Survey.

PURPOSE: Large-scale natural disasters have an enormous physical and mental impact, immediately after they occur, on people living near the central disaster areas. It is known that, in the early stages, a seismic disaster triggers high rates of symptoms for insomnia, depression, and anxiety. However, little information is available about their medium- to long-term clinical outcomes. In this study, we conducted a repeated cross-sectional nationwide questionnaire survey to clarify changes in the prevalence of insomnia and its background factors after the Great East Japan Earthquake, a huge earthquake with a moment magnitude of 9.0 that occurred on March 11, 2011. METHODS: We conducted a repeated cross-sectional survey in November 2009 (pre-earthquake, 1224 participants), July 2011 (4 months post-earthquake, 1259 participants), and August 2012 (18 months post-earthquake, 1289 participants) using stratified random sampling from 157 Japanese sites. RESULTS: Compared to 2009, the prevalence of insomnia statistically increased nationwide immediately post-disaster (11.7% vs 21.2%; p < 0.001) but significantly decreased in 2012 compared to immediately after the earthquake (10.6% vs 21.2%; p < 0.001). In 2011, insomnia was most frequent in the central disaster area. Multivariable logistic regression models demonstrated the association between the following factors and increased risk of insomnia: being a woman (odds ratio [OR] 1.48, 95% confidence interval [CI]: 1.00-2.19), being employed in 2009 (OR 1.74, 95% CI: 1.15-2.62), and being of younger age group (20-64 years) in 2011 (OR 1.64, 95% CI: 1.12-2.42) and 2012 (OR 2.50 95% CI: 1.47-4.23). Post-earthquake, the prevalence of insomnia symptoms in men increased, while the gender difference decreased and was no longer statistically significant. Additionally, insomnia was associated with psychological distress (scores ≥5 on the Kessler Psychological Distress Scale) in 2011 and 2012. CONCLUSION: This study demonstrated that the prevalence of insomnia was significantly higher after the earthquake. Moreover, individuals with insomnia were more likely to experience psychological distress after the earthquake that continued until 2012.

A variant in orexin receptor-2 is associated with self-reported daytime sleepiness in the Japanese population.

Excessive daytime sleepiness is characterized by a persistent feeling of having trouble staying awake, typically with inappropriate sleep episodes. Orexin (hypocretin) is a neuropeptide that regulates sleep-wake cycles and rapid eye movement sleep. Several large-scale genome-wide association studies (GWASs) in European populations have found genetic variants in orexin receptor-1 (OX1R) and -2 (OX2R) that are associated with sleep traits including daytime sleepiness. To identify genetic variants associated with daytime sleepiness, we performed an association study of genetic variants in prepro-orexin, OX1R, and OX2R in 14,329 Japanese individuals from the Tohoku Medical Megabank Project cohort. A genetic variant in OX2R was significantly associated with self-reported daytime sleepiness after Bonferroni correction (rs188018846: P = 8.4E-05). In addition, a missense variant in OX2R identified by the European GWASs showed a nominally significant association with daytime sleepiness in a Japanese population (p.Ile308Val, rs2653349: P = 0.044). Multiple genetic variants in OX2R can affect daytime sleepiness in general populations.

Absence of multiple sleep-onset rapid eye movement periods (SOREMPs) is not a specific feature of patients with pathological sleep prolongation.

Purpose: Multiple sleep-onset rapid eye movement periods (SOREMPs) are involved in the pathophysiology of narcolepsy, but it is not clear whether the lack of multiple SOREMPs is associated with the pathophysiology of idiopathic hypersomnia or not. We examined the significance of multiple SOREMPs in patients with pathological sleep prolongation. Methods: Participants were consecutive patients complaining of unexplained sleepiness and agreed to a 3-day-sleep studies; 24 h polysomnography (PSG) followed by standard PSG and multiple sleep latency test (MSLT). Forty-one (26 females, 21.9 ± 8.1 years old, BMI 20.4 ± 2.3 kg/m2) of 54 eligible patients without other sleep pathologies showed pathological sleep prolongation. We subdivided them into those with and without multiple SOREMPs on MSLT and compared clinical and PSG variables between groups. Results: Six of 41 (14.6%) patients showed multiple SOREMPs on MSLT. There were almost no differences in sleep variables between those with and without multiple SOREMPs. We only found shorter mean sleep latency on MSLT and more REM cycles on 24 h PSG in those with multiple SOREMPs (adjusted p = 0.016 and 0.031). The frequencies of REM-related phenomena and clinical symptoms related to idiopathic hypersomnia were not different between groups. Conclusion: Our results indicated that patients with pathological sleep prolongation had the same clinical profiles regardless of the status of SOREMPs, suggesting the absence of multiple SOREMPs, prerequisite for the diagnosis of idiopathic hypersomnia, is not a specific feature of pathological sleep prolongation. Confirmation of sleep prolongation alone could be a diagnostic tool for idiopathic hypersomnia.

Genome-wide association study of idiopathic hypersomnia in a Japanese population.

Idiopathic hypersomnia (IH) is a rare sleep disorder characterized by excessive daytime sleepiness, great difficulty upon awakening, and prolonged sleep time. In contrast to narcolepsy type 1, which is a well-recognized hypersomnia, the etiology of IH remains poorly understood. No susceptibility loci for IH have been identified, although familial aggregations have been observed among patients with IH. Narcolepsy type 1 is strongly associated with human leukocyte antigen (HLA)-DQB1*06:02; however, no significant associations between IH and HLA alleles have been reported. To identify genetic variants that affect susceptibility to IH, we performed a genome-wide association study (GWAS) and two replication studies involving a total of 414 Japanese patients with IH and 6587 healthy Japanese individuals. A meta-analysis of the three studies found no single-nucleotide polymorphisms (SNPs) that reached the genome-wide significance level. However, we identified several candidate SNPs for IH. For instance, a common genetic variant (rs2250870) within an intron of PDE9A was suggestively associated with IH. rs2250870 was significantly associated with expression levels of PDE9A in not only whole blood but also brain tissues. The leading SNP in the PDE9A region was the same in associations with both IH and PDE9A expression. PDE9A is a potential target in the treatment of several brain diseases, such as depression, schizophrenia, and Alzheimer's disease. It will be necessary to examine whether PDE9A inhibitors that have demonstrated effects on neurophysiologic and cognitive function can contribute to the development of new treatments for IH, as higher expression levels of PDE9A were observed with regard to the risk allele of rs2250870. The present study constitutes the first GWAS of genetic variants associated with IH. A larger replication study will be required to confirm these associations. Supplementary Information: The online version contains supplementary material available at 10.1007/s41105-021-00349-2.

Kleine-Levin syndrome is associated with birth difficulties and genetic variants in the TRANK1 gene loci.

Kleine-Levin syndrome (KLS) is a rare disorder characterized by severe episodic hypersomnia, with cognitive impairment accompanied by apathy or disinhibition. Pathophysiology is unknown, although imaging studies indicate decreased activity in hypothalamic/thalamic areas during episodes. Familial occurrence is increased, and risk is associated with reports of a difficult birth. We conducted a worldwide case-control genome-wide association study in 673 KLS cases collected over 14 y, and ethnically matched 15,341 control individuals. We found a strong genome-wide significant association (rs71947865, Odds Ratio [OR] = 1.48, P = 8.6 × 10-9) within the 3'region of TRANK1 gene locus, previously associated with bipolar disorder and schizophrenia. Strikingly, KLS cases with rs71947865 variant had significantly increased reports of a difficult birth. As perinatal outcomes have dramatically improved over the last 40 y, we further stratified our sample by birth years and found that recent cases had a significantly reduced rs71947865 association. While the rs71947865 association did not replicate in the entire follow-up sample of 171 KLS cases, rs71947865 was significantly associated with KLS in the subset follow-up sample of 59 KLS cases who reported birth difficulties (OR = 1.54, P = 0.01). Genetic liability of KLS as explained by polygenic risk scores was increased (pseudo R2 = 0.15; P < 2.0 × 10-22 at P = 0.5 threshold) in the follow-up sample. Pathway analysis of genetic associations identified enrichment of circadian regulation pathway genes in KLS cases. Our results suggest links between KLS, circadian regulation, and bipolar disorder, and indicate that the TRANK1 polymorphisms in conjunction with reported birth difficulties may predispose to KLS.

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.