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Lack in understanding of the mechanism on brachial plexus avulsion (BPA)-induced neuropathic pain (NP) is the key factor restricting its treatment. In the current investigation, we focused on the nociceptor-localized K+-Cl− cotransporter 2 (KCC2) to investigate its role in BPA-induced NP and related pain sensitization. A novel mice model of BPA on the middle trunk (C7) was established, and BPA mice showed a significant reduction in mechanical withdrawal threshold of the affected fore- and hind- paws without affecting the motor function through CatWalk Gait analysis. Decreased expression of KCC2 in dorsal root ganglion (DRG) was detected through Western blot and FISH technology after BPA. Overexpression of KCC2 in DRG could reverse the hyperexcitability of DRG neurons and alleviate the pain of BPA mice synchronously. Meanwhile, the calcium response signal of the affected SDH could be significantly reduced through above method using spinal cord fiber photometry. The synthesis and release of brain-derived neurotrophic factor (BDNF) was also proved reduction through overexpression of KCC2 in DRG, which indicates BDNF can also act as the downstream role in this pain state. As in human-derived tissues, we found decreased expression of KCC2 and increased expression of BDNF and TrκB in avulsed roots of BPA patients compared with normal human DRGs. Our results indicate that nociceptor-localized KCC2 can suppress BPA-induced NP, and peripheral sensitization can be regulated to reverse central sensitization by targeting KCC2 in DRG at the peripheral level through BDNF signaling. The consistent results in both humanity and rodents endow great potential to future transformation of clinical practice. [ABSTRACT FROM AUTHOR]

Introduction: The neuron-specific K-Cl cotransporter KCC2 maintains low intracellular chloride levels, which are crucial for fast GABAergic and glycinergic neurotransmission. KCC2 also plays a pivotal role in the development of excitatory glutamatergic neurotransmission by promoting dendritic spine maturation. The cytoplasmic C-terminal domain (KCC2-CTD) plays a critical regulatory role in the molecular mechanisms controlling the cotransporter activity through dimerization, phosphorylation, and protein interaction. Methods: To identify novel CTD regulatory motifs, we used the Mu transposon-based mutagenesis system to generate a library of KCC2 mutants with 5 amino acid insertions randomly distributed within the KCC2-CTD. We determined the insertion positions in 288 mutants by restriction analysis and selected clones with a single insertion site outside known KCC2 regulatory motifs. We analyzed the subcellular distribution of KCC2-CTD mutants in cultured cortical neurons using immunocytochemistry and selected ten mutants with ectopic expression patterns for detailed characterization. Results: A fluorescent Cl−-transport assay in HEK293 cells revealed mutants with both reduced and enhanced Cl−-extrusion activity, which overall correlated with their glycosylation patterns. Live-cell immunostaining analysis of plasma membrane expression of KCC2-CTD mutants in cultured cortical neurons corroborated the glycosylation data. Furthermore, the somatodendritic chloride gradient in neurons transfected with the KCC2-CTD mutants correlated with their Cl−-extrusion activity in HEK293 cells. Gain- and loss-of-function mutant positions were analyzed using available KCC2 cryo-EM structures. Discussion: Two groups of mutants were identified based on 3D structural analysis. The first group, located near the interface of transmembrane and cytoplasmic domains, may affect interactions with the N-terminal inhibitory peptide regulating KCC2 activity. The second group, situated on the external surface of the cytoplasmic domain, may disrupt interactions with regulatory proteins. Analyzing CTD mutations that modulate KCC2 activity enhances our understanding of its function and is essential for developing novel anti-seizure therapies. [ABSTRACT FROM AUTHOR]

Abstract Background Mood disorders, particularly depression and anxiety, are associated with zinc dyshomeostasis and aberrant GABAergic signaling. Activation of ZnR/GPR39 by synaptic zinc in the hippocampus triggers phosphorylation of extracellular regulated kinase (ERK1/2), which regulates the K+/Cl− cotransporter (KCC2) and thereby GABAergic inhibitory neurotransmission and seizure activity. Therefore, we studied whether impaired ZnR/GPR39 signaling is linked to anxiety-related behavior in male or female mice. Results Using the acoustic startle response, elevated plus maze, and open field test, we found increased anxiety-related behavior in ZnR/GPR39 knockout (KO) mice. Despite a well-established sex difference, where females are typically more prone to anxiety, both male and female ZnR/GPR39 KO mice exhibited increased anxiety-related behavior compared to wildtype (WT) mice. Additionally, ZnR/GPR39 KO mice displayed impaired motor coordination in the pole and rotarod tests but did not show reduced muscle strength, as indicated by a grip test. Finally, we found intrinsic alterations in the expression level of KCC2, a major Cl− transporter regulating GABAergic signaling, in the amygdala of naïve ZnR/GPR39 KO mice compared to controls. Conclusions Our findings indicate that loss of ZnR/GPR39 enhances anxiety-related behavior in both male and female mice. Moreover, ZnR/GPR39 KO mice exhibit impaired motor coordination, which may be associated with increased anxiety. Finally, we demonstrate that loss of ZnR/GPR39 modulates the expression of KCC2 in the amygdala. Thus, we propose that ZnR/GPR39 can serve as a target for regulating GABAergic signaling in anxiety treatment.

Background: Mood disorders, particularly depression and anxiety, are associated with zinc dyshomeostasis and aberrant GABAergic signaling. Activation of ZnR/GPR39 by synaptic zinc in the hippocampus triggers phosphorylation of extracellular regulated kinase (ERK1/2), which regulates the K+/Cl− cotransporter (KCC2) and thereby GABAergic inhibitory neurotransmission and seizure activity. Therefore, we studied whether impaired ZnR/GPR39 signaling is linked to anxiety-related behavior in male or female mice. Results: Using the acoustic startle response, elevated plus maze, and open field test, we found increased anxiety-related behavior in ZnR/GPR39 knockout (KO) mice. Despite a well-established sex difference, where females are typically more prone to anxiety, both male and female ZnR/GPR39 KO mice exhibited increased anxiety-related behavior compared to wildtype (WT) mice. Additionally, ZnR/GPR39 KO mice displayed impaired motor coordination in the pole and rotarod tests but did not show reduced muscle strength, as indicated by a grip test. Finally, we found intrinsic alterations in the expression level of KCC2, a major Cl− transporter regulating GABAergic signaling, in the amygdala of naïve ZnR/GPR39 KO mice compared to controls. Conclusions: Our findings indicate that loss of ZnR/GPR39 enhances anxiety-related behavior in both male and female mice. Moreover, ZnR/GPR39 KO mice exhibit impaired motor coordination, which may be associated with increased anxiety. Finally, we demonstrate that loss of ZnR/GPR39 modulates the expression of KCC2 in the amygdala. Thus, we propose that ZnR/GPR39 can serve as a target for regulating GABAergic signaling in anxiety treatment. [ABSTRACT FROM AUTHOR]

In most mammals, postural soleus muscles are involved in the maintenance of the stability of the body in the gravitational field of Earth. It is well established that immediately after a laboratory rat is exposed to conditions of weightlessness (parabolic flight) or simulated microgravity (hindlimb suspension/unloading), a sharp decrease in soleus muscle electrical activity occurs. However, starting from the 3rd day of mechanical unloading, soleus muscle electrical activity begins to increase and reaches baseline levels approximately by the 14th day of hindlimb suspension. This phenomenon, observed in the course of rat hindlimb suspension, was named the "spontaneous electrical activity of postural muscle". The present review discusses spinal mechanisms underlying the development of such spontaneous activity of rat soleus muscle and the effect of this activity on intracellular signaling in rat soleus muscle during mechanical unloading. [ABSTRACT FROM AUTHOR]

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. Conclusion: 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. [ABSTRACT FROM AUTHOR]

IntroductionThe neuron-specific K-Cl cotransporter KCC2 maintains low intracellular chloride levels, which are crucial for fast GABAergic and glycinergic neurotransmission. KCC2 also plays a pivotal role in the development of excitatory glutamatergic neurotransmission by promoting dendritic spine maturation. The cytoplasmic C-terminal domain (KCC2-CTD) plays a critical regulatory role in the molecular mechanisms controlling the cotransporter activity through dimerization, phosphorylation, and protein interaction.MethodsTo identify novel CTD regulatory motifs, we used the Mu transposon-based mutagenesis system to generate a library of KCC2 mutants with 5 amino acid insertions randomly distributed within the KCC2-CTD. We determined the insertion positions in 288 mutants by restriction analysis and selected clones with a single insertion site outside known KCC2 regulatory motifs. We analyzed the subcellular distribution of KCC2-CTD mutants in cultured cortical neurons using immunocytochemistry and selected ten mutants with ectopic expression patterns for detailed characterization.ResultsA fluorescent Cl−-transport assay in HEK293 cells revealed mutants with both reduced and enhanced Cl−-extrusion activity, which overall correlated with their glycosylation patterns. Live-cell immunostaining analysis of plasma membrane expression of KCC2-CTD mutants in cultured cortical neurons corroborated the glycosylation data. Furthermore, the somatodendritic chloride gradient in neurons transfected with the KCC2-CTD mutants correlated with their Cl−-extrusion activity in HEK293 cells. Gain- and loss-of-function mutant positions were analyzed using available KCC2 cryo-EM structures.DiscussionTwo groups of mutants were identified based on 3D structural analysis. The first group, located near the interface of transmembrane and cytoplasmic domains, may affect interactions with the N-terminal inhibitory peptide regulating KCC2 activity. The second group, situated on the external surface of the cytoplasmic domain, may disrupt interactions with regulatory proteins. Analyzing CTD mutations that modulate KCC2 activity enhances our understanding of its function and is essential for developing novel anti-seizure therapies.

Aim: We aimed to explore whether the combination of CLP290 and bumetanide maximally improves neuropathic pain following spinal cord injury (SCI) and its possible molecular mechanism. Methods: Rats were randomly divided into five groups: Sham, SCI + vehicle, SCI + CLP290, SCI + bumetanide, and SCI + combination (CLP290 + bumetanide). Drug administration commenced on the 7th day post‐injury (7 dpi) and continued for 14 days. All rats underwent behavioral assessments for 56 days to comprehensively evaluate the effects of interventions on mechanical pain, thermal pain, cold pain, motor function, and other relevant parameters. Electrophysiological assessments, immunoblotting, and immunofluorescence detection were performed at different timepoints post‐injury, with a specific focus on the expression and changes of KCC2 and NKCC1 proteins in the lumbar enlargement of the spinal cord. Results: CLP290 and bumetanide alleviated SCI‐associated hypersensitivity and locomotor function, with the combination providing enhanced recovery. The combined treatment group exhibited the most significant improvement in restoring Rate‐Dependent Depression (RDD) levels. In the combined treatment group and the two individual drug administration groups, the upregulation of potassium chloride cotransporter 2 (K+‐Cl−cotransporter 2, KCC2) expression and downregulation of sodium potassium chloride cotransporter 1 (Na+‐K+‐Cl−cotransporter 1, NKCC1) expression in the lumbar enlargement area resulted in a significant increase in the KCC2/NKCC1 ratio compared to the SCI + vehicle group, with the most pronounced improvement seen in the combined treatment group. Compared to the SCI + vehicle group, the SCI + bumetanide group showed no significant paw withdrawal thermal latency (PWTL) improvement at 21 and 35 dpi, but a notable enhancement at 56 dpi. In contrast, the SCI + CLP290 group significantly improved PWTL at 21 days, with non‐significant changes at 35 and 56 days. At 21 dpi, KCC2 expression was marginally higher in monotherapy groups versus SCI + vehicle, but not significantly. At 56 dpi, only the SCI + bumetanide group showed a significant difference in KCC2 expression compared to the control group. Conclusion: Combined application of CLP290 and bumetanide effectively increases the ratio of KCC2/NKCC1, restores RDD levels, enhances GABAA receptor‐mediated inhibitory function in the spinal cord, and relieves neuropathic pain in SCI; Bumetanide significantly improves neuropathic pain in the long term, whereas CLP290 demonstrates a notable short‐term effect. [ABSTRACT FROM AUTHOR]

The elimination of ground reaction force (support withdrawal) vastly affects slow postural muscles in terms of their regulation and structure. One of the effects of support withdrawal in this study was an immediate postural muscle inactivation, followed by the daily gradual development of spontaneous activity of the slow postural soleus muscle in response to rat hindlimb suspension to mimic space flight. The origin of this activity is somewhat akin to muscle spasticity after spinal cord injuries and is the result of KCC2 content decline in the spinal cord's motor neurons. However, the physiological consequences of unloading-induced spontaneous activity remain unexplored. We have conducted an experiment with the administration of a highly specific KCC2 activator during 7-day unloading. For this experiment, 32 male Wistar rats were divided into 4 groups: C+placebo, C+CLP-290 (100 mg/kg b w), 7HS+placebo, and 7HS+CLP—hindlimb-suspended group with CLP-290 administration (100 mg/kg b w). The soleus muscles of the animals were dissected and analyzed for several proteostasis- and metabolism-related parameters. CLP-290 administration to the unloaded animals led to the upregulation of AMPK downstream (p-ACC) and mTOR targets (p-p70S6k and p-4E-BP) and an enhanced PGC1alpha decrease vs. the 7HS group, but neither prevented nor enhanced atrophy of the soleus muscle or myofiber CSA. [ABSTRACT FROM AUTHOR]

Bipolar disorder (BP) is a recurring psychiatric condition characterized by alternating episodes of low energy (depressions) followed by manias (high energy). Cortical network activity produced by GABAergic interneurons may be critical in maintaining the balance in excitatory/inhibitory activity in the brain during development. Initially, GABAergic signaling is excitatory; with maturation, these cells undergo a functional switch that converts GABAA channels from depolarizing (excitatory) to hyperpolarizing (inhibitory), which is controlled by the intracellular concentration of two chloride transporters. The earliest, NKCC1, promotes chloride entry into the cell and depolarization, while the second (KCC2) stimulates movement of chloride from the neuron, hyperpolarizing it. Perturbations in the timing or expression of NKCC1/KCC2 may affect essential morphogenetic events including cell proliferation, migration, synaptogenesis and plasticity, and thereby the structure and function of the cortex. We derived induced pluripotent stem cells (iPSC) from BP patients and undiagnosed control (C) individuals, then modified a differentiation protocol to form GABAergic interneurons, harvesting cells at sequential stages of differentiation. qRT-PCR and RNA sequencing indicated that after six weeks of differentiation, controls transiently expressed high levels of NKCC1. Using multi-electrode array (MEA) analysis, we observed that BP neurons exhibit increased firing, network bursting and decreased synchrony compared to C. Understanding GABA signaling in differentiation may identify novel approaches and new targets for treatment of neuropsychiatric disorders such as BP. [ABSTRACT FROM AUTHOR]

The electromyographic activity of the soleus muscle is a reliable indicator of its functional status. Support unloading causes an immediate cessation of electrical activity of the soleus muscle, which resumes upon restoration of the support load. Prolonged support unloading, however, results in the emergence of spontaneous electrical activity of the soleus muscle. Previous research has established a correlation between this activity and the presence of the potassium-chloride cotransporter (KCC2) on the membranes of spinal cord motor neurons. It has also been demonstrated that the administration of the KCC2 activator prochlorperazine can eliminate spontaneous soleus muscle activity. Here, we aimed to investigate the effect of CLP290, an alternative KCC2 activator, on the spontaneous tonic activity of the rat soleus muscle. The results indicated that daily administration of CLP290 to rats during a 14-day period of hindlimb suspension prevented the reduction in KCC2 levels in lumbar spinal cord motor neurons and the increase in soleus muscle spontaneous tonic activity. Notably, there were no significant differences in the cross-sectional area of slow-type fibers between the antiorthostatic suspension groups with and without CLP290 administration. [ABSTRACT FROM AUTHOR]

Valproic acid (VPA) has anticancer, anti-inflammatory, and epigenetic effects. The study aimed to determine the expression of carcinogenesis-related SLC5A8, SLC12A2, SLC12A5, CDH1, and CDH2 in adult glioblastoma U87 MG and T98G cells and the effects of 0.5 mM, 0.75 mM, and 1.5 mM doses of VPA. RNA gene expression was determined by RT-PCR. GAPDH was used as a control. U87 and T98G control cells do not express SLC5A8 or CDH1. SLC12A5 was expressed in U87 control but not in T98G control cells. The SLC12A2 expression in the U87 control was significantly lower than in the T98G control. T98G control cells showed significantly higher CDH2 expression than U87 control cells. VPA treatment did not affect SLC12A2 expression in U87 cells, whereas treatment dose-dependently increased SLC12A2 expression in T98G cells. Treatment with 1.5 mM VPA induced SLC5A8 expression in U87 cells, while treatment of T98G cells with VPA did not affect SLC5A8 expression. Treatment of U87 cells with VPA significantly increased SLC12A5 expression. VPA increases CDH1 expression depending on the VPA dose. CDH2 expression was significantly increased only in the U87 1.5 mM VPA group. Tested VPA doses significantly increased CDH2 expression in T98G cells. When approaching treatment tactics, assessing the cell's sensitivity to the agent is essential. [ABSTRACT FROM AUTHOR]

BackgroundVolatile anesthetic exposure during development leads to long-term cognitive deficits in rats which are dependent on age and sex. Female rats are protected relative to male rats for the same exposure on postnatal day 7. Here we test our hypothesis that androgens can modulate chloride cotransporter expression to alter the susceptibility to neurotoxicity from GABAergic drugs using female rats with exogenous testosterone exposure.MethodsFemale rats were injected with testosterone (100 μg/animal) or vehicle on postnatal days 1 to 6. On postnatal day 7, the animals were randomized to either isoflurane exposure or sham. Spatial memory was assessed with the Barnes maze starting on postnatal day 41. Western blots were run from testosterone treated postnatal day 7 animals to measure levels of chloride cotransporters sodium-potassium-chloride symporter (NKCC1) and chloride-potassium symporter 5 (KCC2).ResultsExogenous testosterone modulated isoflurane anesthetic neurotoxicity in female rats based on poor performance in the probe trial of the Barnes Maze. By contrast, females with vehicle and isoflurane exposure were able to differentiate the goal position. These behavioral differences corresponded to differences in the protein levels of NKCC1 and KCC2 after exogenous testosterone exposure, with NKCC1 increasing ( P

Spinal cerebrospinal fluid-contacting neurons (CSF-cNs) form an evolutionary conserved bipolar cell population localized around the central canal of all vertebrates. CSF-cNs were shown to express molecular markers of neuronal immaturity into adulthood; however, the impact of their incomplete maturation on the chloride (Cl-) homeostasis as well as GABAergic signaling remains unknown. Using adult mice from both sexes, in situ hybridization revealed that a proportion of spinal CSF-cNs (18.3%) express the Na+-K+-Cl- cotransporter 1 (NKCC1) allowing intracellular Cl- accumulation. However, we did not find expression of the K+-Cl- cotransporter 2 (KCC2) responsible for Cl- efflux in any CSF-cNs. The lack of KCC2 expression results in low Cl- extrusion capacity in CSF-cNs under high Cl- load in whole-cell patch clamp. Using cell-attached patch clamp allowing recordings with intact intracellular Cl- concentration, we found that the activation of ionotropic GABAA receptors (GABAA-Rs) induced both depolarizing and hyperpolarizing responses in CSF-cNs. Moreover, depolarizing GABA responses can drive action potentials as well as intracellular calcium elevations by activating voltage-gated calcium channels. Blocking NKCC1 with bumetanide inhibited the GABAinduced calcium transients in CSF-cNs. Finally, we show that metabotropic GABAB receptors have no hyperpolarizing action on spinal CSF-cNs as their activation with baclofen did not mediate outward K+ currents, presumably due to the lack of expression of G-protein-coupled inwardly rectifying potassium (GIRK) channels. Together, these findings outline subpopulations of spinal CSF-cNs expressing inhibitory or excitatory GABAA-R signaling. Excitatory GABA may promote the maturation and integration of young CSF-cNs into the existing spinal circuit. [ABSTRACT FROM AUTHOR]

Anesthesia is proposed to have different underlying mechanisms in terms of molecular targets and neural circuits. Emergence from anesthesia is a dynamic process that is driven by the brain's inherent abilities and can be partially controlled, in addition to being dependent on anesthetic elimination. Animal-model studies indicate that the recovery of consciousness from the minimally responsive state during anesthesia is a molecularly distinct process from the behavioral recovery observed after anesthesia is discontinued. There are similarities and differences in the nuclei, neural circuits, neurotransmitters, and arousal systems involved in emergence from anesthesia and awakening from sleep. 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. [ABSTRACT FROM AUTHOR]

Background: 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. Objective: The present study aimed to investigate the potential enhancement of NEM on the sleep-inducing properties of alprazolam (Alp). Methods: 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. Results: 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. Conclusions: 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. [ABSTRACT FROM AUTHOR]

Neurodevelopment is a highly organized and complex process involving lasting and often irreversible changes in the central nervous system. Inherited disorders of neurotransmission (IDNT) are a group of genetic disorders where neurotransmission is primarily affected, resulting in abnormal brain development from early life, manifest as neurodevelopmental disorders and other chronic conditions. In principle, IDNT (particularly those of monogenic causes) are amenable to gene replacement therapy via precise genetic correction. However, practical challenges for gene replacement therapy remain major hurdles for its translation from bench to bedside. We discuss key considerations for the development of gene replacement therapies for IDNT. As an example, we describe our ongoing work on gene replacement therapy for succinic semialdehyde dehydrogenase deficiency, a GABA catabolic disorder. [ABSTRACT FROM AUTHOR]

Zinc is essential for many physiological functions, with a major role in digestive system, skin health, and learning and memory. On the cellular level, zinc is involved in cell proliferation and cell death. A selective zinc sensing receptor, ZnR/GPR39 is a Gq‐coupled receptor that acts via the inositol trisphosphate pathway to release intracellular Ca2+. The ZnR/GPR39 serves as a mediator between extracellular changes in Zn2+ concentration and cellular Ca2+ signalling. This signalling pathway regulates ion transporters activity and thereby controls the formation of transepithelial gradients or neuronal membrane potential, which play a fundamental role in the physiological function of these tissues. This review focuses on the role of Ca2+ signalling, and specifically ZnR/GPR39, with respect to the regulation of the Na+/H+ exchanger, NHE1, and of the K+/Cl− cotransporters, KCC1‐3, and also describes the physiological implications of this regulation. [ABSTRACT FROM AUTHOR]

Corticotropin-releasing hormone (CRH) neurons play an important role in the regulation of neuroendocrine responses to stress. The excitability of CRH neurons is regulated by inhibitory GABAergic inputs. However, it is unclear when GABAergic regulation of CRH neurons is established during fetal brain development. Furthermore, the exact progression of the developmental shift of GABA action from depolarization to hyperpolarization remains unelucidated. Considering the importance of CRH neuron function in subsequent hypothalamic-pituitary-adrenal (HPA) axis regulation during this critical phase of development, we investigated the ontogeny of GABAergic inputs to CRH neurons and consequent development of chloride homeostasis. Both CRH neuron soma in the paraventricular nucleus (PVN) and axons projecting to the median eminence could be identified at embryonic day 15 (E15). Using acute slices containing the PVN of CRF-VenusΔNeo mice, gramicidin perforated-patch clamp-recordings of CRH neurons at E15, postnatal day 0 (P0), and P7 were performed to evaluate the developmental shift of GABA action. The equilibrium potential of GABA (EGABA) was similar between E15 and P0 and showed a further hyperpolarizing shift between P0 and P7 that was comparable to EGABA values in adult CRH neurons. GABA primarily acted as an inhibitory signal at E15 and KCC2 expression was detected in CRH neurons at this age. Activation of the HPA axis has been proposed as the primary mechanism through which prenatal maternal stress shapes fetal development and subsequent long-term disease risk. We therefore examined the impact of maternal food restriction stress on the development of chloride homeostasis in CRH neurons. We observed a depolarization shift of EGABA in CRH neurons of pups exposed to maternal food restriction stress. These results suggest that Cl- homeostasis in early developmental CRH neurons attains mature intracellular Cl- levels, GABA acts primarily as inhibitory, and CRH neurons mature and function early compared with neurons in other brain regions, such as the cortex and hippocampus. Maternal food restriction stress alters chloride homeostasis in CRH neurons of pups, reducing their inhibitory control by GABA. This may contribute to increased CRH neuron activity and cause activation of the HPA axis in pups. [ABSTRACT FROM AUTHOR]

Abstract Developmental neurogenesis is a tightly regulated spatiotemporal process with its dysregulation implicated in neurodevelopmental disorders. NMDA receptors are glutamate-gated ion channels that are widely expressed in the early nervous system, yet their contribution to neurogenesis is poorly understood. Notably, a variety of mutations in genes encoding NMDA receptor subunits are associated with neurodevelopmental disorders. To rigorously define the role of NMDA receptors in developmental neurogenesis, we used a mutant zebrafish line (grin1 −/−) that lacks all NMDA receptors yet survives to 10 days post-fertilization, offering the opportunity to study post-embryonic neurodevelopment in the absence of NMDA receptors. Focusing on the forebrain, we find that these fish have a progressive supernumerary neuron phenotype confined to the telencephalon at the end of embryonic neurogenesis, but which extends to all forebrain regions during postembryonic neurogenesis. This enhanced neuron population does not arise directly from increased numbers or mitotic activity of radial glia cells, the principal neural stem cells. Rather, it stems from a lack of timely maturation of transit-amplifying neuroblasts into post-mitotic neurons, as indicated by a decrease in expression of the ontogenetically-expressed chloride transporter, KCC2. Pharmacological blockade with MK-801 recapitulates the grin1 −/− supernumerary neuron phenotype, indicating a requirement for ionotropic signaling. Thus, NMDA receptors are required for suppression of indirect, transit amplifying cell-driven neurogenesis by promoting maturational termination of mitosis. Loss of suppression results in neuronal overpopulation that can fundamentally change brain circuitry and may be a key factor in pathogenesis of neurodevelopmental disorders caused by NMDA receptor dysfunction.

Patients with neurological diseases, such as schizophrenia, tend to show low K+-Cl- co-transporter 2 (KCC2) levels in the brain. The cause of these diseases has been associated with stress and neuroinflammation. However, since the pathogenesis of these diseases is not yet fully investigated, drug therapy is still limited to symptomatic therapy. Targeting KCC2, which is mainly expressed in the brain, seems to be an appropriate approach in the treatment of these diseases. In this review, we aimed to discuss about stress and inflammation, KCC2 and Gamma-aminobutyric acid (GABA) function, diseases which decrease the KCC2 levels in the brain, factors that regulate KCC2 activity, and the possibility to overcome neuronal dysfunction targeting KCC2. We also aimed to discuss the relationships between neurological diseases and LPS caused by Porphyromonas gingivalis (P. g), which is a type of oral bacterium. Clinical trials on oxytocin, sirtuin 1 (SIRT1) activator, and transient receptor potential cation channel subfamily V Member 1 activator have been conducted to develop effective treatment methods. We believe that KCC2 modulators that regulate mitochondria, such as oxytocin, glycogen synthase kinase 3β (GSK3β), and SIRT1, can be potential targets for neurological diseases.

Background 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. Objective The present study aimed to investigate the potential enhancement of NEM on the sleep-inducing properties of alprazolam (Alp). Methods 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. Results 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. Conclusions 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.

Developmental neurogenesis is a tightly regulated spatiotemporal process with its dysregulation implicated in neurodevelopmental disorders. NMDA receptors are glutamate-gated ion channels that are widely expressed in the early nervous system, yet their contribution to neurogenesis is poorly understood. Notably, a variety of mutations in genes encoding NMDA receptor subunits are associated with neurodevelopmental disorders. To rigorously define the role of NMDA receptors in developmental neurogenesis, we used a mutant zebrafish line (grin1−/−) that lacks all NMDA receptors yet survives to 10 days post-fertilization, offering the opportunity to study post-embryonic neurodevelopment in the absence of NMDA receptors. Focusing on the forebrain, we find that these fish have a progressive supernumerary neuron phenotype confined to the telencephalon at the end of embryonic neurogenesis, but which extends to all forebrain regions during postembryonic neurogenesis. This enhanced neuron population does not arise directly from increased numbers or mitotic activity of radial glia cells, the principal neural stem cells. Rather, it stems from a lack of timely maturation of transit-amplifying neuroblasts into post-mitotic neurons, as indicated by a decrease in expression of the ontogenetically-expressed chloride transporter, KCC2. Pharmacological blockade with MK-801 recapitulates the grin1−/− supernumerary neuron phenotype, indicating a requirement for ionotropic signaling. Thus, NMDA receptors are required for suppression of indirect, transit amplifying cell-driven neurogenesis by promoting maturational termination of mitosis. Loss of suppression results in neuronal overpopulation that can fundamentally change brain circuitry and may be a key factor in pathogenesis of neurodevelopmental disorders caused by NMDA receptor dysfunction. [ABSTRACT FROM AUTHOR]

Phenobarbital (PB) remains the first-line medication for neonatal seizures. Yet, seizures in many newborns, particularly those associated with perinatal ischemia, are resistant to PB. Previous animal studies have shown that in postnatal day P7 mice pups with ischemic stroke induced by unilateral carotid ligation, the tyrosine receptor kinase B (TrkB) antagonist ANA12 (N-[2-[[(hexahydro-2-oxo-1H-azepin-3-yl)amino]carbonyl]phenyl]-benzo[b]thiophene-2-carboxamide, 5 mg/kg) improved the efficacy of PB in reducing seizure occurrence. To meet optimal standards of effectiveness, a wider range of ANA12 doses must be tested. Here, using the unilateral carotid ligation model, we tested the effectiveness of higher doses of ANA12 (10 and 20 mg/kg) on the ability of PB to reduce seizure burden, ameliorate cell death (assessed by Fluoro-Jade staining), and affect neurodevelopment (righting reflex, negative geotaxis test, open field test). We found that a single dose of ANA12 (10 or 20 mg/kg) given 1 h after unilateral carotid ligation in P7 pups reduced seizure burden and neocortical and striatal neuron death without impairing developmental reflexes. In conclusion, ANA12 at a range of doses (10–20 mg/kg) enhanced PB effectiveness for the treatment of perinatal ischemia-related seizures, suggesting that this agent might be a clinically safe and effective adjunctive agent for the treatment of pharmacoresistant neonatal seizures. [ABSTRACT FROM AUTHOR]

Impaired rate-dependent depression (RDD) of the spinal H-reflex occurs in diabetic rodents and a sub-set of patients with painful diabetic neuropathy. RDD is unaffected in animal models of painful neuropathy associated with peripheral pain mechanisms and diabetic patients with painless neuropathy, suggesting RDD could serve as a biomarker for individuals in whom spinal disinhibition contributes to painful neuropathy and help identify therapies that target impaired spinal inhibitory function. The spinal pharmacology of RDD was investigated in normal rats and rats after 4 and 8 weeks of streptozotocin-induced diabetes. In normal rats, dependence of RDD on spinal GABAergic inhibitory function encompassed both GABAA and GABAB receptor sub-types. The time-dependent emergence of impaired RDD in diabetic rats was preceded by depletion of potassium-chloride co-transporter 2 (KCC2) protein in the dorsal, but not ventral, spinal cord and by dysfunction of GABAA receptor-mediated inhibition. GABAB receptor-mediated spinal inhibition remained functional and initially compensated for loss of GABAA receptor-mediated inhibition. Administration of the GABAB receptor agonist baclofen restored RDD and alleviated indices of neuropathic pain in diabetic rats, as did spinal delivery of the carbonic anhydrase inhibitor acetazolamide. Pharmacological manipulation of RDD can be used to identify potential therapies that act against neuropathic pain arising from spinal disinhibition.

Disinhibition during early stages of Alzheimer's disease is postulated to cause network dysfunction and hyperexcitability leading to cognitive deficits. However, the underlying molecular mechanism remains unknown. Here we show that, in mouse lines carrying Alzheimer's disease-related mutations, a loss of neuronal membrane potassium-chloride cotransporter KCC2, responsible for maintaining the robustness of GABAA-mediated inhibition, occurs pre-symptomatically in the hippocampus and prefrontal cortex. KCC2 downregulation was inversely correlated with the age-dependent increase in amyloid-β 42 (Aβ42). Acute administration of Aβ42 caused a downregulation of membrane KCC2. Loss of KCC2 resulted in impaired chloride homeostasis. Preventing the decrease in KCC2 using long term treatment with CLP290 protected against deterioration of learning and cortical hyperactivity. In addition, restoring KCC2, using short term CLP290 treatment, following the transporter reduction effectively reversed spatial memory deficits and social dysfunction, linking chloride dysregulation with Alzheimer's disease-related cognitive decline. These results reveal KCC2 hypofunction as a viable target for treatment of Alzheimer's disease-related cognitive decline; they confirm target engagement, where the therapeutic intervention takes place, and its effectiveness. [ABSTRACT FROM AUTHOR]

Neonatal hypoxia–ischemia (HI) is one of the main causes of tissue damage, cell death, and imbalance between neuronal excitation and inhibition and synaptic loss in newborns. GABA, the major inhibitory neurotransmitter of the central nervous system (CNS) in adults, is excitatory at the onset of neurodevelopment and its action depends on the chloride (Cl−) cotransporters NKCC1 (imports Cl−) and KCC2 (exports Cl−) expression. Under basal conditions, the NKCC1/KCC2 ratio decreases over neurodevelopment. Thus, changes in this ratio caused by HI may be related to neurological disorders. The present study evaluated the effects of bumetanide (NKCC cotransporters inhibitor) on HI impairments in two neurodevelopmental periods. Male Wistar rat pups, 3 (PND3) and 11 (PND11) days old, were submitted to the Rice-Vannucci model. Animals were divided into 3 groups: SHAM, HI-SAL, and HI-BUM, considering each age. Bumetanide was administered intraperitoneally at 1, 24, 48, and 72 h after HI. NKCC1, KCC2, PSD-95, and synaptophysin proteins were analyzed after the last injection by western blot. Negative geotaxis, righting reflex, open field, object recognition test, and Morris water maze task were performed to assess neurological reflexes, locomotion, and memory function. Tissue atrophy and cell death were evaluated by histology. Bumetanide prevented neurodevelopmental delay, hyperactivity, and declarative and spatial memory deficits. Furthermore, bumetanide reversed HI-induced brain tissue damage, reduced neuronal death and controlled GABAergic tone, maintained the NKCC1/KCC2 ratio, and synaptogenesis close to normality. Thereby, bumetanide appears to play an important therapeutic role in the CNS, protecting the animals against HI damage and improving functional performance. [ABSTRACT FROM AUTHOR]

Patients with neurological diseases, such as schizophrenia, tend to show low K+-Cl- co-transporter 2 (KCC2) levels in the brain. The cause of these diseases has been associated with stress and neuroinflammation. However, since the pathogenesis of these diseases is not yet fully investigated, drug therapy is still limited to symptomatic therapy. Targeting KCC2, which is mainly expressed in the brain, seems to be an appropriate approach in the treatment of these diseases. In this review, we aimed to discuss about stress and inflammation, KCC2 and Gamma-aminobutyric acid (GABA) function, diseases which decrease the KCC2 levels in the brain, factors that regulate KCC2 activity, and the possibility to overcome neuronal dysfunction targeting KCC2. We also aimed to discuss the relationships between neurological diseases and LPS caused by Porphyromonas gingivalis (P. g), which is a type of oral bacterium. Clinical trials on oxytocin, sirtuin 1 (SIRT1) activator, and transient receptor potential cation channel subfamily V Member 1 activator have been conducted to develop effective treatment methods. We believe that KCC2 modulators that regulate mitochondria, such as oxytocin, glycogen synthase kinase 3β (GSK3β), and SIRT1, can be potential targets for neurological diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A recent study published in the journal Anesthesiology & Perioperative Science explores the role of KCC2 in the recovery of consciousness from anesthesia. The study suggests that the downregulation of KCC2 in the ventral posteromedial nucleus (VPM) of the thalamus leads to disinhibition and accelerated recovery of VPM neuron excitability, enabling the emergence of consciousness from anesthetic inhibition. The authors propose that the VPM is a key nucleus in thalamocortical signal transmission and cortical activation, and that KCC2 downregulation-mediated disinhibition may be a crucial mechanism for neural network reconstruction after anesthesia. The study also suggests that the ubiquitin degradation of KCC2 may serve as a common molecular mechanism for the active reboot of consciousness, independent of the specific anesthetic used. Additionally, the study reports on the occurrence of seizure-like tremors during the period of exiting from the minimally responsive state (MRS) and before the recovery of consciousness, suggesting a potential link between KCC2 degradation and anesthetic epilepsy. [Extracted from the article]

The hypothalamus contains a number of nuclei that subserve a variety of functions, including generation of circadian rhythms, regulation of hormone secretion and maintenance of homeostatic levels for a variety of physiological parameters. Within the hypothalamus, γ-amino-butyric acid (GABA) is one of the major neurotransmitters responsible for cellular communication. Although GABA most commonly serves as an inhibitory neurotransmitter, a growing body of evidence indicates that it can evoke post-synaptic excitation as a result of the active regulation of intracellular chloride concentration. In this review, we consider the evidence for this ionic plasticity of GABAergic synaptic transmission in five distinct cases in hypothalamic cell populations. We argue that this plasticity serves as part of the functional response to or is at least associated with dehydration, lactation, hypertension and stress. As such, GABA excitation should be considered as part of the core homeostatic mechanisms of the hypothalamus.

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. Conclusion: SLC12A5 shows promise to function as a novel effective biomarker for GBM and deserves further systematic research. [ABSTRACT FROM AUTHOR]

Synaptic inhibition plays a crucial role in regulating neuronal excitability, which is the foundation of nervous system function. This inhibition is largely mediated by the neurotransmitters GABA and glycine that activate Cl-- permeable ion channels, which means that the strength of inhibition depends on the Cl- gradient across the membrane. In neurons, the Cl- gradient is primarily mediated by two secondarily active cation-chloride cotransporters (CCCs), NKCC1 and KCC2. CCC-mediated regulation of the neuronal Cl- gradient is critical for healthy brain function, as dysregulation of CCCs has emerged as a key mechanism underlying neurological disorders including epilepsy, neuropathic pain, and autism spectrum disorder. This review begins with an overview of neuronal chloride transporters before explaining the dependent relationship between these CCCs, Cl- regulation, and inhibitory synaptic transmission. We then discuss the evidence for how CCCs can be regulated, including by activity and their protein interactions, which underlie inhibitory synaptic plasticity. For readers who may be interested in conducting experiments on CCCs and neuronal excitability, we have included a section on techniques for estimating and recording intracellular Cl-, including their advantages and limitations. Although the focus of this review is on neurons, we also examine how Cl- is regulated in glial cells, which in turn regulate neuronal excitability through the tight relationship between this nonneuronal cell type and synapses. Finally, we discuss the relatively extensive and growing literature on how CCC-mediated neuronal excitability contributes to neurological disorders. [ABSTRACT FROM AUTHOR]

Abstract Background 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. Methods 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. Results 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. Conclusions Sustained elevation of IL-1β in the hippocampus may induce cognitive impairment by upregulation of KCC2 during early development.

In adults, γ-aminobutyric acid (GABA) type A receptor (GABAAR)-mediated inhibition depends on the maintenance of low intracellular chloride anion concentration through neuron-specific potassium-chloride cotransporter-2 (KCC2). KCC2 has been widely reported to have a plasticity change during the course of epilepsy development, with an early downregulation and late recovery in neuronal cell membranes after epileptic stimulation, which facilitates epileptiform burst activity. Furosemide is a clinical loop diuretic that inhibits KCC2. Here, we first confirmed that furosemide pretreatment could effectively prevented convulsant stimulation-induced neuronal membrane KCC2 downregulation in the hippocampus in both in vivo and in vitro cyclothiazide-induced seizure model. Second, we verified that furosemide pretreatment rescued KCC2 function deficits, as indicated by EGABA depolarizing shift and GABAAR inhibitory function impairment induced via cyclothiazide treatment. Further, we demonstrated that furosemide also suppressed cyclothiazide-induced epileptiform burst activity in cultured hippocampal neurons and lowered the mortality rate during acute seizure induction. Overall, furosemide prevents membrane KCC2 downregulation during acute seizure induction, restores KCC2-mediated GABA inhibition, and interrupts the progression from acute seizure to epileptogenesis.

Avulsion injury results in motoneuron death due to the increased excitotoxicity developing in the affected spinal segments. This study focused on possible short and long term molecular and receptor expression alterations which are thought to be linked to the excitotoxic events in the ventral horn with or without the anti-excitotoxic riluzole treatment. In our experimental model the left lumbar 4 and 5 (L4, 5) ventral roots of the spinal cord were avulsed. Treated animals received riluzole for 2 weeks. Riluzole is a compound that acts to block voltage-activated Na+ and Ca2+ channels. In control animals the L4, 5 ventral roots were avulsed without riluzole treatment. Expression of astrocytic EAAT-2 and that of KCC2 in motoneurons on the affected side of the L4 spinal segment were detected after the injury by confocal and dSTORM imaging, intracellular Ca2+ levels in motoneurons were quantified by electron microscopy. The KCC2 labeling in the lateral and ventrolateral parts of the L4 ventral horn was weaker compared with the medial part of L4 ventral horn in both groups. Riluzole treatment dramatically enhanced motoneuron survival but was not able to prevent the down-regulation of KCC2 expression in injured motoneurons. In contrast, riluzole successfully obviated the increase of intracellular calcium level and the decrease of EAAT-2 expression in astrocytes compared with untreated injured animals. We conclude that KCC2 may not be an essential component for survival of injured motoneurons and riluzole is able to modulate the intracellular level of calcium and expression of EAAT-2.

Simple Summary: Naked mole-rats live in crowded underground burrows where concentrations of oxygen can be low and concentrations of carbon dioxide can be high. Accordingly, this species is tolerant of low oxygen levels and high carbon dioxide levels, which would be deadly to most surface dwellers. The current article reviews what we know about these unusual tolerances and their underlying mechanisms. Understanding these mechanisms could lead to new strategies for treating human disorders related to low oxygen and high carbon dioxide, as experienced, for example, during a heart attack. Naked mole-rats (Heterocephalus glaber) are very unusual among subterranean mammals in that they live in large colonies and are extremely social, spending large amounts of time gathered together in underground nests more than a meter below the surface. Many respiring individuals resting in deep, poorly ventilated nests deplete the oxygen supply and increase the concentration of carbon dioxide. Consistent with living in that atmosphere, naked mole-rats tolerate levels of low oxygen and high carbon dioxide that are deadly to most surface-dwelling mammals. Naked mole-rats appear to have evolved a number of remarkable adaptations to be able to thrive in this harsh atmosphere. In order to successfully survive low oxygen atmospheres, they conserve energy utilization by reducing the physiological activity of all organs, manifest by reduced heart rate and brain activity. Amazingly, they resort to the anaerobic metabolism of fructose rather than glucose as a fuel to generate energy when challenged by anoxia. Similarly, high carbon dioxide atmospheres normally cause tissue acidosis, while naked mole-rats have a genetic mutation preventing both acid-induced pain and pulmonary edema. Together, these putative adaptations and the tolerances they provide make the naked mole-rat an important model for studying a host of biomedical challenges. [ABSTRACT FROM AUTHOR]

Neurological diseases including Alzheimer's, Huntington's disease, Parkinson's disease, Down syndrome and epilepsy, and neuropsychiatric disorders such as schizophrenia, are conditions that affect not only individuals but societies on a global scale. Current therapies offer a means for small symptomatic relief, but recently there has been increasing demand for therapeutic alternatives. The γ-aminobutyric acid (GABA)ergic signaling system has been investigated for developing new therapies as it has been noted that any dysfunction or changes to this system can contribute to disease progression. Expression of the K-Cl-2 (KCC2) and N-K-C1-1 (NKCC1) cation-chloride cotransporters (CCCs) has recently been linked to the disruption of GABAergic activity by affecting the polarity of GABAA receptor signaling. KCC2 and NKCC1 play a part in multiple neurological and neuropsychiatric disorders, making them a target of interest for potential therapies. This review explores current research suggesting the pathophysiological role and therapeutic importance of KCC2 and NKCC1 in neuropsychiatric and neurological disorders. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Bone cancer pain is a global medical concern with limited treatment options that significantly reduce the quality of life for cancer patients. Therefore, identifying a promising therapeutic target for bone cancer pain is urgently needed., Objective: Our previous research indicated that KCC2 may be associated with the modulation of HDAC2 in a rat model of bone cancer pain. The current study aimed to investigate whether KCC2 in the lumbar spinal cord is a key downstream molecule in the modulation of HDAC2 related to bone cancer pain., Methods: In this study, we assessed the expression levels of KCC2 and HDAC2 in the lumbar spinal cord of rats with bone cancer pain using Western blotting and RT-PCR. Mechanical hyperalgesia was evaluated using Von Frey hairs, and immunofluorescence was employed to localize KCC2 in central nervous system cells., Results: The expression of KCC2 was down-regulated in a time-dependent manner in the lumbar spinal cord of rats with bone cancer pain. Furthermore, the use of an RNA-interfering lentivirus targeting HDAC2 restored KCC2 expression and alleviated mechanical hyperalgesia in these rats. Notably, the analgesic effect of the HDAC2-targeting lentivirus was completely reversed by the KCC2 inhibitor VU0240551., Conclusion: KCC2 in the lumbar spinal cord mediated the modulation of HDAC2 in rat models of bone cancer pain, suggesting that KCC2 could be a promising therapeutic target for treating bone cancer pain., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The with-no-lysine (WNK) family of serine-threonine kinases and its downstream kinases of STE20/SPS1-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase-1 (OSR1) may regulate intracellular Cl- homeostasis through phosphorylation of cation-Cl- co-transporters. WNK3 is expressed in fetal and postnatal brains, and its expression level increases during development. Its roles in neurons, however, remain uncertain. Using WNK3 knockout (KO) mice, we investigated the role of WNK3 in the regulation of the intracellular Cl- concentration ([Cl-]i) and the excitability of layer V pyramidal neurons in the medial prefrontal cortex (mPFC). Gramicidin-perforated patch-clamp recordings in neurons from acute slice preparation at the postnatal day 21 indicated a significantly depolarized reversal potential for GABAA receptor-mediated currents by 6mV, corresponding to the higher [Cl-]i level by ~4mM in KO mice than in wild-type littermates. However, phosphorylation levels of SPAK and OSR1 and those of neuronal Na+-K+-2Cl- co-transporter NKCC1 and K+-Cl- co-transporter KCC2 did not significantly differ between KO and wild-type mice. Meanwhile, the resting membrane potential of neurons was more hyperpolarized by 7mV, and theminimumstimulus current necessary for firing induction was increased in KO mice. These were due to an increased inwardly rectifying K+ (IRK) conductance, mediated by classical inwardly rectifying (Kir) channels, in KO neurons. The introduction of an active form of WNK3 into the recording neurons reversed these changes. The potential role of KCC2 function in the observed changes of KO neurons was investigated by applying a selective KCC2 activator, CLP290. This reversed the enhanced IRK conductance in KO neurons, indicating that both WNK3 and KCC2 are intimately linked in the regulation of resting K+ conductance. Evaluation of synaptic properties revealed that the frequency of miniature excitatory postsynaptic currents (mEPSCs) was reduced, whereas that of inhibitory currents (mIPSCs) was slightly increased in KO neurons. Together, the impact of these developmental changes on the membrane and synaptic properties was manifested as behavioral deficits in pre-pulse inhibition, a measure of sensorimotor gating involving multiple brain regions including the mPFC, in KO mice. Thus, the basal function of WNK3 would be the maintenance and/or development of both intrinsic and synaptic excitabilities. [ABSTRACT FROM AUTHOR]