UNASSIGNED: N-Ethylmaleimide (NEM), an agonist of the potassium chloride cotransporters 2 (KCC2) receptor, has been correlated with neurosuppressive outcomes, including decreased pain perception and the prevention of epileptic seizures. Nevertheless, its relationship with sleep-inducing effects remains unreported.
UNASSIGNED: The present study aimed to investigate the potential enhancement of NEM on the sleep-inducing properties of alprazolam (Alp).
UNASSIGNED: The test of the righting reflex was used to identify the appropriate concentrations of Alp and NEM for inducing sleep-promoting effects in mice. Total sleep duration and sleep quality were evaluated through EEG/EMG analysis. The neural mechanism underlying the sleep-promoting effect was examined through c-fos immunoreactivity in the brain using immunofluorescence. Furthermore, potential CNS-side effects of the combination Alp and NEM were assessed using LABORAS automated home-cage behavioral phenotyping.
UNASSIGNED: Combination administration of Alp (1.84 mg/kg) and NEM (1.0 mg/kg) significantly decreased sleep latency and increased sleep duration in comparison to administering 1.84 mg/kg Alp alone. This effect was characterized by a notable increase in REM duration. The findings from c-fos immunoreactivity indicated that NEM significantly suppressed neuron activation in brain regions associated with wakefulness. Additionally, combination administration of Alp and NEM showed no effects on mouse neural behaviors during automated home cage monitoring.
UNASSIGNED: This study is the first to propose and demonstrate a combination therapy involving Alp and NEM that not only enhances the hypnotic effect but also mitigates potential CNS side effects, suggesting its potential application in treating insomnia.

Epilepsy is a chronic neurological disorder characterized by episodic dysfunction of central nervous system. The most basic mechanism of epilepsy falls to the imbalance between excitation and inhibition. In adults, GABAA receptor (GABAAR) is the main inhibitory receptor to prevent neurons from developing hyperexcitability, while its inhibition relies on the low intracellular chloride anion concentration ([Cl-]i). Neuronal-specific electroneutral K+-Cl- cotransporter (KCC2) can mediate chloride efflux to lower [Cl-]i for GABAAR mediated inhibition. Our previous study has revealed that the coordinated downregulation of KCC2 and GABAAR participates in epilepsy. According to a high-throughout screen for compounds that reduce [Cl-]i, CLP290 turns out to be a specific KCC2 functional modulator. In current study, we first confirmed that CLP290 could dose-dependently suppress convulsant-induced seizures in mice in vivo as well as the epileptiform burst activities in cultured hippocampal neurons in vitro. Then, we discovered that CLP290 functioned through preventing the downregulation of the KCC2 phosphorylation at Ser940 and hence the KCC2 membrane expression during convulsant stimulation, and consequently restored the GABA inhibition. In addition, while CLP290 was given in early epileptogenesis period, it also effectively decreased the spontaneous recurrent seizures. Generally, our current results demonstrated that CLP290, as a specific KCC2 modulator by enhancing KCC2 function, not only inhibits the occurrence of the ictal seizures, but also suppresses the epileptogenic process. Therefore, we believe KCC2 may be a suitable target for future anti-epileptic drug development.

The suppression of consciousness by anesthetics and the emergence of the brain from anesthesia are complex and elusive processes. Anesthetics may exert their inhibitory effects by binding to specific protein targets or through membrane-mediated targets, disrupting neural activity and the integrity and function of neural circuits responsible for signal transmission and conscious perception/subjective experience. Emergence from anesthesia was generally thought to depend on the elimination of the anesthetic from the body. Recently, studies have suggested that emergence from anesthesia is a dynamic and active process that can be partially controlled and is independent of the specific molecular targets of anesthetics. This article summarizes the fundamentals of anesthetics\' actions in the brain and the mechanisms of emergence from anesthesia that have been recently revealed in animal studies.

BACKGROUND: Spinal cord injury (SCI) is a destructive neurological and pathological state that causes major motor, sensory and autonomic dysfunctions. Bone marrow mesenchymal stem cells (BMSCs)-derived exosomes show great therapeutic potential for SCI. Exosomes derived from miR-26a-modified MSCs promote axonal regeneration following SCI. Our study aims to uncover the mechanisms by which BMSC-derived exosomes carrying miR-26a-5p regulate SCI.
METHODS: BMSCs and BMSC-derived exosomes were isolated and characterized by Oil Red O and alizarin red staining, transmission electron microscopy, flow cytometry, nanoparticle tracking analysis and Western blotting. PC12 cells were treated with lipopolysaccharides (LPS), and SCI was established through laminectomy with contusion injury in rats. Annexin-V staining, CCK-8 and EdU incorporation were applied to determine cell apoptosis, viability, and proliferation. Hematoxylin and Eosin, Nissl and TUNEL staining was used to evaluate SCI injury and apoptosis in the spinal cord. Luciferase and chromatin immunoprecipitation assays were applied to evaluate gene interaction.
RESULTS: BMSC-derived exosomes facilitated LPS-treated PC12 cell proliferation and inhibited apoptosis by delivering miR-26a-5p. Moreover, BMSC-derived exosomal miR-26a-5p alleviated SCI. Furthermore, miR-26a-5p inhibited EZH2 expression by directly binding to EZH2, and EZH2 inhibited BDNF expression via promoting H3K27me3. Increased phosphorylated CREB enhanced KCC2 transcription and expression by binding to its promoter. Knockdown of miR-26a-5p abrogated BMSC-derived exosome-mediated protection in LPS-treated PC12 cells, but it was reversed by KCC2 overexpression.
CONCLUSIONS: BMSC-derived exosomes carrying miR-26a-5p repressed EZH2 expression to promote BDNF and TrkB expression and CREB phosphorylation and subsequently increase KCC2 expression, thus protecting PC12 cells and ameliorating SCI.

Previous studies have shown that the hyperpolarized cyclic nucleotide gated (HCN) ion channels in the spinal dorsal horn (SDH) might be involved in the development of diabetic neuropathic pain (DNP). Additionally, other studies have shown that the decreased potassium-chloride cotransporter 2 (KCC2) expression in the SDH promotes pain hypersensitivity. Both HCN channels and KCC2 were highly expressed in spinal substantia gelatinosa neurons. However, whether the K+ efflux induced by the activation of HCN channels in DNP modulate KCC2 function and subsequently affect the role of γ-aminobutyric acid (GABA)/GABA-A receptors of neurons in the SDH remains to be clarified. The purpose of this work was to investigate the underlying mechanisms of KCC2 participating in HCN channels to promote DNP. Here, we found that the analgesic role of HCN channels blocker ZD7288 was associated with the up-regulated KCC2 expression and could be prevented by DIOA, a KCC2 blocker. Furthermore, the level of GABA in DNP rats significantly increased, which was decreased by ZD72288. Moreover, DIOA pretreatment could partly block the inhibitory effect of ZD7288 on the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signaling activation of DNP rats. Finally, inhibition of cAMP-PKA signaling alleviated allodynia and elevated KCC2 expression in DNP rats. Altogether, this study reveals that the role of cAMP-PKA signaling-regulated HCN channels in DNP associated with decreased KCC2 expression in the spinal cord and altered GABA nature.

Mechanical allodynia can be evoked by punctate pressure contact with the skin (punctate mechanical allodynia) and dynamic contact stimulation induced by gentle touching of the skin (dynamic mechanical allodynia). Dynamic allodynia is insensitive to morphine treatment and is transmitted through the spinal dorsal horn by a specific neuronal pathway, which is different from that for punctate allodynia, leading to difficulties in clinical treatment. K+-Cl- cotransporter-2 (KCC2) is one of the major determinants of inhibitory efficiency, and the inhibitory system in the spinal cord is important in the regulation of neuropathic pain. The aim of the current study was to determine whether neuronal KCC2 is involved in the induction of dynamic allodynia and to identify underlying spinal mechanisms involved in this process. Dynamic and punctate allodynia were assessed using either von Frey filaments or a paint brush in a spared nerve injury (SNI) mouse model. Our study discovered that the downregulated neuronal membrane KCC2 (mKCC2) in the spinal dorsal horn of SNI mice is closely associated with SNI-induced dynamic allodynia, as the prevention of KCC2 downregulation significantly suppressed the induction of dynamic allodynia. The over activation of microglia in the spinal dorsal horn after SNI was at least one of the triggers in SNI-induced mKCC2 reduction and dynamic allodynia, as these effects were blocked by the inhibition of microglial activation. Finally, the BDNF-TrkB pathway mediated by activated microglial affected SNI-induced dynamic allodynia through neuronal KCC2 downregulation. Overall, our findings revealed that activation of microglia through the BDNF-TrkB pathway affected neuronal KCC2 downregulation, contributing to dynamic allodynia induction in an SNI mouse model.

BACKGROUND: Glioblastoma multiforme (GBM) is the most prevalent and malignant intracranial tumor with significant features of dismal prognosis and limited therapeutic solutions. Consequently, the present studies are committed to exploring potential biomarkers through bioinformatics analysis, which may serve as valuable prognostic predictors or novel therapeutic targets and provide new insights into the pathogenesis of GBM.
METHODS: We filtered overlapping differentially expressed genes (DEGs) based on expression profilings from three GBM microarray datasets (GSE116520, GSE4290 and GSE68848) and combined RNA sequencing data from The Cancer Genome Atlas and the Genotype-Tissue Expression databases. Hub genes were prioritized from DEGs after performing protein-protein interaction (PPI) network analysis and weighted gene co-expression network analysis (WGCNA). This was followed by survival analysis to identify potential biomarkers among hub genes. Ultimately, the distributions of gene expressions, genetic alterations, upstream regulatory mechanisms and enrichments of gene functions of the identified biomarkers were analysed on public databases. QRT-PCR, immunohistochemical staining and western blotting was also used to confirm the gene expression patterns in GBM and normal brain tissues. CCK-8 assay clarified the effects of the genes on GBM cells.
RESULTS: A total of 322 common DEGs were determined and nine genes were subsequently considered as hub genes by the combination of PPI network analysis and WGCNA. Only SLC12A5 had prognostic significance, which was deficient in GBM whereas especially enriched in normal neural tissues. SLC12A5 overexpression would inhibit cell proliferation of U251MG. Genetic alterations of SLC12A5 were rarely seen in GBM patients, and there was no apparent association existed between SLC12A5 expression and DNA methylation. SLC12A5 was prominently involved in ion transport, synapse and neurotransmitter.
CONCLUSIONS: SLC12A5 shows promise to function as a novel effective biomarker for GBM and deserves further systematic research.

The balance of ion concentrations inside and outside the cell is an essential homeostatic mechanism in neurons and serves as the basis for a variety of physiological activities. In the central nervous system, NKCC1 and KCC2, members of the SLC12 cation-chloride co-transporter (CCC) family, participate in physiological and pathophysiological processes by regulating intracellular and extracellular chloride ion concentrations, which can further regulate the GABAergic system. Over recent years, studies have shown that NKCC1 and KCC2 are essential for the maintenance of Cl- homeostasis in neural cells. NKCC1 transports Cl- into cells while KCC2 transports Cl- out of cells, thereby regulating chloride balance and neuronal excitability. An imbalance of NKCC1 and KCC2 after spinal cord injury will disrupt CI- homeostasis, resulting in the transformation of GABA neurons from an inhibitory state into an excitatory state, which subsequently alters the spinal cord neural network and leads to conditions such as spasticity and neuropathic pain, among others. Meanwhile, studies have shown that KCC2 is also an essential target for motor function reconstruction after spinal cord injury. This review mainly introduces the physiological structure and function of NKCC1 and KCC2 and discusses their pathophysiological roles after spinal cord injury.

Alcohol use disorder (AUD) is a common and complex disorder resulting from repetitive alcohol drinking. The mesocorticolimbic dopamine (DA) system, originating from the ventral tegmental area (VTA) in the midbrain, is involved in the rewarding effect of ethanol. The γ-aminobutyric acid (GABA) neurons in VTA appear to be key substrates of acute and chronic ethanol, which regulates DA neurotransmission indirectly in the mesocorticolimbic system. Despite significant research on the relationship between brain-derived neurotrophic factor (BDNF) and reduced alcohol consumption in male rats involving tropomyosin-related kinase B (TrkB), the mechanisms of BDNF-TrkB regulating alcohol behavior remain scarce. K+-Cl- cotransporter 2 (KCC2) plays a crucial role in synaptic function in GABAergic neurons by modulating intracellular chlorine homeostasis. Here, we found that 4-week intermittent alcohol exposure impaired the function of KCC2 in VTA, evidenced by a lower expression level of phosphorylated KCC2 and decreased ratio of phosphorylated KCC2 to total KCC2, especially 72 h after withdrawal from 4-week ethanol exposure in male rats. CLP290 (a KCC2 activator) reduced excessive alcohol consumption after alcohol withdrawal, whereas VU0240551 (a specific KCC2 inhibitor) further enhanced alcohol intake. Importantly, VU0240551 reversed the attenuating effects of BDNF and 7,8-dihydroxyflavone (7,8-DHF) on alcohol consumption after withdrawal. Moreover, intraperitoneal injection of 7,8-DHF upregulated KCC2 expression and phosphorylated KCC2 in VTA 72 h after withdrawal from ethanol exposure in male rats. Collectively, our data indicate that KCC2 may be critical in the regulating action of BDNF-TrkB on ethanol consumption in AUD.

Neonatal sepsis can induce long-term cognitive impairment in adolescence or adulthood, but the underlying molecular mechanism is not fully understood. The expression of K+-Cl- co-transporter 2 (KCC2) plays a pivotal role in the GABAergic shift from depolarizing to hyperpolarizing during early postnatal development. In this study, we aimed to determine whether neonatal severe inflammation-induced cognitive impairment was associated with the expression of KCC2 during early development.
Neonatal severe inflammation was established by intraperitoneal injection of high dose lipopolysaccharide (LPS, 1 mg kg-1) in postnatal day 3 (P3) rats. The Morris water maze task and fear conditioning test were used to investigate long-term cognitive functions. ELISA, RT-PCR and Western blotting were used to examine the expression levels of proinflammatory cytokines and KCC2. Perforated patch-clamping recordings were used to determine the GABAergic shift.
Neonatal severe inflammation led to long-term cognitive impairment in rats. Meanwhile, sustained elevation of interleukin-1 beta (IL-1β) levels was found in the hippocampus until P30 after LPS injection. Elevated expression of KCC2 and hyperpolarized GABA reversal potential (EGABA) were observed in CA1 hippocampal pyramidal neurons from the P7-P10 and P14-P16 rats after LPS injection. Specific knockdown of IL-1β mRNA expression rescued the elevated expression of KCC2 and the hyperpolarized EGABA at P7-P10 and P14-P16. Accordingly, specific knockdown of IL-1β or KCC2 expression improved the cognitive impairment induced by neonatal severe inflammation.
Sustained elevation of IL-1β in the hippocampus may induce cognitive impairment by upregulation of KCC2 during early development.