Search

BackgroundExaggerated responses to sensory stimuli, a hallmark of Fragile X syndrome (FXS), contribute to anxiety and learning challenges. Sensory hypersensitivity is recapitulated in the Fmr1 knockout (KO) mouse model of FXS. Recent studies in Fmr1 KO mice have demonstrated differences in activity of cortical interneurons and a delayed switch in the polarity of GABA signaling during development. Previously, we reported that blocking the chloride transporter NKCC1 with the diuretic bumetanide, could rescue synaptic circuit phenotypes in primary somatosensory cortex (S1) of Fmr1 KO mice. However, it remains unknown whether bumetanide can rescue earlier circuit phenotypes or sensory hypersensitivity in Fmr1 KO mice.MethodsWe used acute and chronic systemic administration of bumetanide in Fmr1 KO mice and performed in vivo 2-photon calcium imaging to record neuronal activity, while tracking mouse behavior with high-resolution videos.ResultsWe demonstrate that layer (L) 2/3 pyramidal neurons in S1 of Fmr1 KO mice show a higher frequency of synchronous events at postnatal day (P) 6 compared to wild-type controls. This was reversed by acute administration of bumetanide. Furthermore, chronic bumetanide treatment (P5-P14) restored S1 circuit differences in Fmr1 KO mice, including reduced neuronal adaptation to repetitive whisker stimulation, and ameliorated tactile defensiveness. Bumetanide treatment also rectified the reduced feedforward inhibition of L2/3 neurons in S1 and boosted the circuit participation of parvalbumin interneurons.ConclusionsThis further supports the notion that synaptic, circuit, and sensory behavioral phenotypes in Fmr1 KO can be mitigated by inhibitors of NKCC1, such as the FDA-approved diuretic bumetanide.

Objective: Following hypoxic–ischemic (HI) brain injury, neuronal cytoplasmic chloride concentration ([Cl−]i) increases, potentially contributing to depolarizing γ‐aminobutyric acid (GABA) responses, onset of seizures, and the failure of antiepileptic drugs that target inhibitory chloride‐permeable GABAA receptors. Post‐HI seizures characteristically begin hours after injury, by which time substantial accumulation of [Cl−]i may have already occurred. In immature neurons, a major pathway for Cl− influx is the reversible Na+‐K+‐2Cl− cotransporter NKCC1. Methods: Spontaneous neuronal network, neuronal [Cl−]i, and GABA activity were determined in hippocampal preparations from neonatal Clomeleon and SuperClomeleon/DLX‐cre mice to test whether blocking NKCC1 earlier after oxygen–glucose deprivation (OGD) injury would more effectively ameliorate the increase in [Cl−]i, ictallike epileptiform discharges (ILDs), and the failure of the GABAergic anticonvulsant phenobarbital. Results: In vitro, murine intact hippocampi were free of ILDs for 12 h after preparation. Transient OGD resulted in a gradual increase in [Cl−]i, depolarizing action of GABA, and facilitation of neuronal network activity. Spontaneous ILDs began 3–5 h after injury. Blocking NKCC1 with 2–10 μmol·L−1 bumetanide reduced [Cl−]i equally well when applied up to 10 h after injury. Whereas phenobarbital or bumetanide applied separately were less effective when applied later after injury, ILDs were successfully suppressed by the combination of phenobarbital and bumetanide regardless of the number of prior ILDs or delay in application. Significance: The present age‐specific group studies demonstrate that after OGD, NKCC1 transport activity significantly contributes to progressive [Cl−]i accumulation, depolarizing action of GABA, and delayed onset of ILDs. In this neonatal model of neuronal injury and ILDs, earlier treatment with bumetanide alone more efficiently recovered control baseline [Cl−]i and depressed epileptiform discharges. However, there was no time dependency to the anti‐ictal efficacy of the combination of phenobarbital and bumetanide. These in vitro results suggest that after perinatal injury, early pre‐emptive treatment with phenobarbital plus bumetanide would be as efficacious as late treatment after seizures are manifest. [ABSTRACT FROM AUTHOR]

Background: Hydrocephalus is characterized by secretion, circulation, and absorption disorder of cerebrospinal fluid (CSF) with high morbidity and complication rate. The relationship between inflammation and abnormal secretion of CSF by choroid plexus epithelium (CPE) had received more attention. In this study, we aim to detect the role of Toll‐like receptor 4/nuclear factor‐kappa B/Na+/K+/2Cl‐cotransporter 1(TLR4/NF‐κB/NKCC1) signal pathway in the development of hydrocephalus. Method: Hydrocephalus was induced in adult rats (8 weeks) by intracisternal kaolin injection, then pyrrolidinedithiocarbamate (PDTC) and bumetanide were administrated to the rats mode. Then the rat model was evaluated, and ventricular volume was calculated at different time points. Then CPE, cortex, preventricular tissue, and CSF were obtained. Protein expressions of TLR‐4, NKCC/serine–threonine STE20/SPS1‐related, proline‐alanine‐rich kinase (SPAK), pNKCC1, pSPAK, GFAP, AQP1, and AQP4 were measured by RT‐PCR, western blot, and immunofluorescence (IF) stains in CPE, respectively. Result: Our data showed that inflammation factors tumor necrosis factor‐(TNF‐α), interleukin 18(IL‐18), and glial fibrillary acidic protein (GFAP) concentrations were significantly higher in the model group than in controls. The TLR4/NF‐κB/NKCC1 signal pathway were actived by NF‐κB‐p65, NKCC1, pNKCC1‐ pSPAK complex, and Aquaporin1 (AQP1) high expression. PDTC and bumetanide use can help regular TLR4/NF‐κB/NKCC1 expression and reduced AQP1 expression by down‐regulate NF‐B‐p65 and inhibiting NKCC1, respectively. As a result, the treatment groups alleviated CPE abnormal secretion and ventricle enlargement. Conclusion: These results confirmed that the inflammatory reaction contributes TLR4/NF‐κB/NKCC1 mediated CPE abnormal secretion and consequent hydrocephalus. Regulation of TLR4/NF‐κB/NKCC1 and AQP1 can prevent this process. Our study provides a strong rationale for further exploring alleviating CPE abnormal secretion as a therapeutic perspective of hydrocephalus. [ABSTRACT FROM AUTHOR]

The potassium–chloride cotransporter KCC2 is the main extruder of Cl- in neurons. It plays a fundamental role in the activity of the inhibitory neurotransmitters (GABA and glycine) since low levels of KCC2 promote intracellular Cl- accumulation, leading to the depolarizing activity of GABA and glycine. The downregulation of this cotransporter occurs in neurological disorders characterized by hyperexcitability, such as epilepsy, neuropathic pain, and spasticity. KCC2 is also downregulated after axotomy. If muscle reinnervation is allowed, the KCC2 levels recover in motoneurons. Therefore, we argued that target-derived neurotrophic factors might be involved in the regulation of KCC2 expression. For this purpose, we performed the axotomy of extraocular motoneurons via the monocular enucleation of adult rats, and a pellet containing either VEGF or BDNF was chronically implanted in the orbit. Double confocal immunofluorescence of choline acetyl-transferase (ChAT) and KCC2 was carried out in the brainstem sections. Axotomy led to a KCC2 decrease in the neuropil and somata of extraocular motoneurons, peaking at 15 days post-lesion, with the exception of the abducens motoneuron somata. VEGF administration prevented the axotomy-induced KCC2 downregulation. By contrast, BDNF either maintained or reduced the KCC2 levels following axotomy, suggesting that BDNF is involved in the axotomy-induced KCC2 downregulation in extraocular motoneurons. The finding that VEGF prevents KCC2 decrease opens up new possibilities for the treatment of neurological disorders coursing with neuronal hyperactivity due to KCC2 downregulation. [ABSTRACT FROM AUTHOR]

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]

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]

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]

Bumetanide is used widely as a tool and off‐label treatment to inhibit the Na‐K‐2Cl cotransporter NKCC1 in the brain and thereby to normalize intra‐neuronal chloride levels in several brain disorders. However, following systemic administration, bumetanide only poorly penetrates into the brain parenchyma and does not reach levels sufficient to inhibit NKCC1. The low brain penetration is a consequence of both the high ionization rate and plasma protein binding, which restrict brain entry by passive diffusion, and of brain efflux transport. In previous studies, bumetanide was determined in the whole brain or a few brain regions, such as the hippocampus. However, the blood–brain barrier and its efflux transporters are heterogeneous across brain regions, so it cannot be excluded that bumetanide reaches sufficiently high brain levels for NKCC1 inhibition in some discrete brain areas. Here, bumetanide was determined in 14 brain regions following i.v. administration of 10 mg/kg in rats. Because bumetanide is much more rapidly eliminated by rats than humans, its metabolism was reduced by pretreatment with piperonyl butoxide. Significant, up to 5‐fold differences in regional bumetanide levels were determined with the highest levels in the midbrain and olfactory bulb and the lowest levels in the striatum and amygdala. Brain:plasma ratios ranged between 0.004 (amygdala) and 0.022 (olfactory bulb). Regional brain levels were significantly correlated with local cerebral blood flow. However, regional bumetanide levels were far below the IC50 (2.4 μM) determined previously for rat NKCC1. Thus, these data further substantiate that the reported effects of bumetanide in rodent models of brain disorders are not related to NKCC1 inhibition in the brain. [ABSTRACT FROM AUTHOR]

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]

Cerebrovascular diseases and their sequalae, such as ischemic stroke, chronic cerebral hypoperfusion, and vascular dementia are significant contributors to adult disability and cognitive impairment in the modern world. Astrocytes are an integral part of the neurovascular unit in the CNS and play a pivotal role in CNS homeostasis, including ionic and pH balance, neurotransmission, cerebral blood flow, and metabolism. Astrocytes respond to cerebral insults, inflammation, and diseases through unique molecular, morphological, and functional changes, collectively known as reactive astrogliosis. The function of reactive astrocytes has been a subject of debate. Initially, astrocytes were thought to primarily play a supportive role in maintaining the structure and function of the nervous system. However, recent studies suggest that reactive astrocytes may have both beneficial and detrimental effects. For example, in chronic cerebral hypoperfusion, reactive astrocytes can cause oligodendrocyte death and demyelination. In this review, we will summarize the (1) roles of ion transporter cascade in reactive astrogliosis, (2) role of reactive astrocytes in vascular dementia and related dementias, and (3) potential therapeutic approaches for dementing disorders targeting reactive astrocytes. Understanding the relationship between ion transporter cascade, reactive astrogliosis, and cerebrovascular diseases may reveal mechanisms and targets for the development of therapies for brain diseases associated with reactive astrogliosis. [ABSTRACT FROM AUTHOR]

Background: Astrogliosis and white matter lesions (WML) are key characteristics of vascular contributions to cognitive impairment and dementia (VCID). However, the molecular mechanisms underlying VCID remain poorly understood. Stimulation of Na‐K‐Cl cotransport 1 (NKCC1) and its upstream kinases WNK (with no lysine) and SPAK (the STE20/SPS1‐related proline/alanine‐rich kinase) play a role in astrocytic intracellular Na+ overload, hypertrophy, and swelling. Therefore, in this study, we assessed the effect of SPAK inhibitor ZT‐1a on pathogenesis and cognitive function in a mouse model of VCID induced by bilateral carotid artery stenosis (BCAS). Methods: Following sham or BCAS surgery, mice were randomly assigned to receive either vehicle (DMSO) or SPAK inhibitor ZT‐1a treatment regimen (days 14–35 post‐surgery). Mice were then evaluated for cognitive functions by Morris water maze, WML by ex vivo MRI‐DTI analysis, and astrogliosis/demyelination by immunofluorescence and immunoblotting. Results: Compared to sham control mice, BCAS‐Veh mice exhibited chronic cerebral hypoperfusion and memory impairments, accompanied by significant MRI DTI‐detected WML and oligodendrocyte (OL) death. Increased activation of WNK‐SPAK‐NKCC1‐signaling proteins was detected in white matter tissues and in C3d+GFAP+ cytotoxic astrocytes but not in S100A10+GFAP+ homeostatic astrocytes in BCAS‐Veh mice. In contrast, ZT‐1a‐treated BCAS mice displayed reduced expression and phosphorylation of NKCC1, decreased astrogliosis, OL death, and WML, along with improved memory functions. Conclusion: BCAS‐induced upregulation of WNK‐SPAK‐NKCC1 signaling contributes to white matter‐reactive astrogliosis, OL death, and memory impairment. Pharmacological inhibition of the SPAK activity has therapeutic potential for alleviating pathogenesis and memory impairment in VCID. [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

Autism spectrum disorder (ASD) is thought to result from susceptibility genotypes and environmental risk factors. The offspring of women who experience pregnancy infection have an increased risk for autism. Maternal immune activation (MIA) in pregnant animals produces offspring with autistic behaviors, making MIA a useful model for autism. However, how MIA causes autistic behaviors in offspring is not fully understood. Here, we show that NKCC1 is critical for mediating autistic behaviors in MIA offspring. We confirmed that MIA induced by poly(I:C) infection during pregnancy leads to autistic behaviors in offspring. We further demonstrated that MIA offspring showed significant microglia activation, excessive dendritic spines, and narrow postsynaptic density (PSD) in their prefrontal cortex (PFC). Then, we discovered that these abnormalities may be caused by overexpression of NKCC1 in MIA offspring’s PFCs. Finally, we ameliorated the autistic behaviors using PFC microinjection of NKCC1 inhibitor bumetanide (BTN) in MIA offspring. Our findings may shed new light on the pathological mechanisms for autism caused by pregnancy infection.

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]

Cerebrovascular diseases and their sequalae, such as ischemic stroke, chronic cerebral hypoperfusion, and vascular dementia are significant contributors to adult disability and cognitive impairment in the modern world. Astrocytes are an integral part of the neurovascular unit in the CNS and play a pivotal role in CNS homeostasis, including ionic and pH balance, neurotransmission, cerebral blood flow, and metabolism. Astrocytes respond to cerebral insults, inflammation, and diseases through unique molecular, morphological, and functional changes, collectively known as reactive astrogliosis. The function of reactive astrocytes has been a subject of debate. Initially, astrocytes were thought to primarily play a supportive role in maintaining the structure and function of the nervous system. However, recent studies suggest that reactive astrocytes may have both beneficial and detrimental effects. For example, in chronic cerebral hypoperfusion, reactive astrocytes can cause oligodendrocyte death and demyelination. In this review, we will summarize the (1) roles of ion transporter cascade in reactive astrogliosis, (2) role of reactive astrocytes in vascular dementia and related dementias, and (3) potential therapeutic approaches for dementing disorders targeting reactive astrocytes. Understanding the relationship between ion transporter cascade, reactive astrogliosis, and cerebrovascular diseases may reveal mechanisms and targets for the development of therapies for brain diseases associated with reactive astrogliosis.

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.

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 Regulation of the volume and electrolyte composition of the cerebrospinal fluid (CSF) is vital for brain development and function. The Na-K-Cl co-transporter NKCC1 in the choroid plexus (ChP) plays key roles in regulating CSF volume by co-transporting ions and mediating same-direction water movements. Our previous study showed ChP NKCC1 is highly phosphorylated in neonatal mice as the CSF K+ level drastically decreases and that overexpression of NKCC1 in the ChP accelerates CSF K+ clearance and reduces ventricle size [1]. These data suggest that NKCC1 mediates CSF K+ clearance following birth in mice. In this current study, we used CRISPR technology to create a conditional NKCC1 knockout mouse line and evaluated CSF K+ by Inductively Coupled Plasma Optical Emission spectroscopy (ICP-OES). We demonstrated ChP-specific reduction of total and phosphorylated NKCC1 in neonatal mice following embryonic intraventricular delivery of Cre recombinase using AAV2/5. ChP-NKCC1 knockdown was accompanied by a delayed perinatal clearance of CSF K+. No gross morphological disruptions were observed in the cerebral cortex. We extended our previous results by showing embryonic and perinatal rats shared key characteristics with mice, including decreased ChP NKCC1 expression level, increased ChP NKCC1 phosphorylation state, and increased CSF K+ levels compared to adult. Collectively, these follow up data support ChP NKCC1’s role in age-appropriate CSF K+ clearance during neonatal development.

Abstract Background Idiopathic intracranial hypertension (IIH) is a condition characterized by increased intracranial pressure (ICP), impaired vision, and headache. Most cases of IIH occur in obese women of childbearing age, though age, BMI, and female sex do not encompass all aspects of IIH pathophysiology. Systemic metabolic dysregulation has been identified in IIH with a profile of androgen excess. However, the mechanistic coupling between obesity/hormonal perturbations and cerebrospinal fluid dynamics remains unresolved. Methods Female Wistar rats were either fed a high fat diet (HFD) for 21 weeks or exposed to adjuvant testosterone treatment for 28 days to recapitulate IIH causal drivers. Cerebrospinal fluid (CSF) and blood testosterone levels were determined with mass spectrometry, ICP and CSF dynamics with in vivo experimentation, and the choroid plexus function revealed with transcriptomics and ex vivo isotope-based flux assays. Results HFD-fed rats presented with increased ICP (65%), which was accompanied by increased CSF outflow resistance (50%) without altered CSF secretion rate or choroid plexus gene expression. Chronic adjuvant testosterone treatment of lean rats caused elevated ICP (55%) and CSF secretion rate (85%), in association with increased activity of the choroid plexus Na+,K+,2Cl− cotransporter, NKCC1. Conclusions HFD-induced ICP elevation in experimental rats occurred with decreased CSF drainage capacity. Adjuvant testosterone, mimicking the androgen excess observed in female IIH patients, elevated the CSF secretion rate and thus ICP. Obesity-induced androgen dysregulation may thus contribute to the disease mechanism of IIH.

• This systematic review included 26 rodent studies describing 38 experiments (28 in-vivo and ten in-vitro) and two small human studies evaluating the role of bumetanide to treat neonatal seizures. • Among 38 animal experiments, bumetanide was reported to have antiseizure effects in 21, pro-seizure in six and ineffective in 11. • Both human studies did not show benefits of bumetanide their primary analyses, but one of them found benefit on post-hoc analysis. • Hearing impairment was detected in 5/37 surviving infants in the bumetanide group vs 0/13 in controls. • Four of the five infants with hearing impairment had received aminoglycosides concurrently. Bumetanide, an inhibitor of the sodium-potassium-chloride cotransporter-1, has been suggested as an adjunct to phenobarbital for treating neonatal seizures. A systematic review of animal and human studies was conducted to evaluate the efficacy and safety of bumetanide for neonatal seizures. PubMed, Embase, CINAHL and Cochrane databases were searched in March 2023. 26 animal (rat or mice) studies describing 38 experiments (28 in-vivo and ten in-vitro) and two human studies (one RCT and one open-label dose-finding) were included. The study designs, methods to induce seizures, bumetanide dose, and outcome measures were heterogeneous, with only 4/38 experiments being in animal hypoxia/ischaemia models. Among 38 animal experiments, bumetanide was reported to have antiseizure effects in 21, pro-seizure in six and ineffective in 11. The two human studies (n = 57) did not show the benefits of bumetanide as an add-on agent to phenobarbital in their primary analyses, but one study reported benefit on post-hoc analysis. Overall, hearing impairment was detected in 5/37 surviving infants in the bumetanide group vs. 0/13 in controls. Four of the five infants with hearing impairment had received aminoglycosides concurrently. Other adverse effects reported were diuresis, mild-to-moderate dehydration, hypotension, and electrolyte disturbances. The studies did not report on long-term neurodevelopment. The certainty of the evidence was very low. Animal data suggest that bumetanide has inconsistent effects as an antiseizure medication in neonates. Data from human studies are scarce and raise some concerns regarding ototoxicity when given with aminoglycosides. Well conducted studies in animal models of hypoxic-ischaemic encephalopathy are urgently needed. Future RCTs, if conducted in human neonates, should have an adequate sample size, assess neurodevelopment, minimize using aminoglycosides, be transparent about the potential ototoxicity in the parent information sheet, conduct early hearing tests and have trial-stopping rules that include hearing impairment as an outcome. [ABSTRACT FROM AUTHOR]

Mutations in the SLC12A2 gene, which encodes the Na-K-2Cl cotransporter-1 (NKCC1), are linked to various conditions such as neurodevelopmental deficits, deafness, and fluid secretion in different epithelia. Cases of complete NKCC1 deficiency in young patients are straightforward, leading to clinical presentations that overlap with the phenotypes observed in NKCC1 knockout mouse models. However, cases involving deleterious variants in one allele are more difficult, as the clinical presentation is variable, and the cause-effect relationship is not always clear. For instance, we worked on a single patient's case from multiple angles and published six related papers to convince ourselves of the cause-and-effect relationship between her NKCC1 mutation and her clinical presentations. The cluster of mutations in a small portion of the carboxyl terminus and its association with deafness point to a cause-and-effect relationship, even if the molecular mechanism is unknown. Overall, the preponderance of evidence suggests that the SLC12A2 gene is a human disease-causing and likely haploinsufficient gene that requires further investigation. [ABSTRACT FROM AUTHOR]

Background: The balance between the activity of the Na+/K+/Cl- cotransporter (NKCC1) that introduces Cl- into the cell and the K+/Cl- cotransporter (KCC2) that transports Cl- outside the cell is critical in determining the inhibitory or excitatory outcome of GABA release. Mounting evidence suggests that the impairment of GABAergic inhibitory neurotransmission plays a crucial role in the pathophysiology of epilepsy, both in patients and animal models. Previous studies indicate that decreased KCC2 expression is linked to audiogenic seizures in GASH/Sal hamsters, highlighting that Cl- imbalance can cause neuronal hyperexcitability. In this study, we aimed to investigate whether the Na+/K+/Cl- cotransporter NKCC1 is also affected by audiogenic seizures and could, therefore, play a role in neuronal hyperexcitability within the GASH/Sal epilepsy model. Methods: NKCC1 protein expression in both the GASH/Sal strain and wild type hamsters was analyzed by immunohistochemistry and Western blotting techniques. Brain regions examined included cortex, hippocampus, hypothalamus, inferior colliculus and pons-medulla oblongata, which were evaluated both at rest and after sound-inducing seizures in GASH/Sal hamsters. A complementary analysis of NKCC1 gene slc12a2 expression was conducted by real-time PCR. Finally, protein and mRNA levels of glutamate decarboxylase GAD67 were measured as an indicator of GABA release. Results: The induction of seizures caused significant changes in NKCC1 expression in epileptic GASH/Sal hamsters, despite the similar brain expression pattern of NKCC1 in GASH/Sal and wild type hamsters in the absence of seizures. Interestingly, the regulation of brain NKCC1 by seizures demonstrated regional specificity, as protein levels exclusively increased in the hippocampus and hypothalamus. Complementary real-time PCR analysis revealed that NKCC1 regulation was post-transcriptional only in the hypothalamus. In addition, seizures also modulated GAD67 mRNA levels in a brain region-specific manner. The increased GAD67 expression in the hippocampus and hypothalamus of the epileptic hamster brain suggests that NKCC1 upregulation overlaps with GABA release in these regions during seizures. Conclusions: Our results indicate that seizure induction causes dysregulation of NKCC1 expression in GASH/Sal animals, which overlaps with changes in GABA release. These observations provide evidence for the critical role of NKCC1 in how seizures affect neuronal excitability, and support NKCC1 contribution to the development of secondary foci of epileptogenic activity. [ABSTRACT FROM AUTHOR]

Background: Idiopathic intracranial hypertension (IIH) is a condition characterized by increased intracranial pressure (ICP), impaired vision, and headache. Most cases of IIH occur in obese women of childbearing age, though age, BMI, and female sex do not encompass all aspects of IIH pathophysiology. Systemic metabolic dysregulation has been identified in IIH with a profile of androgen excess. However, the mechanistic coupling between obesity/hormonal perturbations and cerebrospinal fluid dynamics remains unresolved. Methods: Female Wistar rats were either fed a high fat diet (HFD) for 21 weeks or exposed to adjuvant testosterone treatment for 28 days to recapitulate IIH causal drivers. Cerebrospinal fluid (CSF) and blood testosterone levels were determined with mass spectrometry, ICP and CSF dynamics with in vivo experimentation, and the choroid plexus function revealed with transcriptomics and ex vivo isotope-based flux assays. Results: HFD-fed rats presented with increased ICP (65%), which was accompanied by increased CSF outflow resistance (50%) without altered CSF secretion rate or choroid plexus gene expression. Chronic adjuvant testosterone treatment of lean rats caused elevated ICP (55%) and CSF secretion rate (85%), in association with increased activity of the choroid plexus Na+,K+,2Cl− cotransporter, NKCC1. Conclusions: HFD-induced ICP elevation in experimental rats occurred with decreased CSF drainage capacity. Adjuvant testosterone, mimicking the androgen excess observed in female IIH patients, elevated the CSF secretion rate and thus ICP. Obesity-induced androgen dysregulation may thus contribute to the disease mechanism of IIH. [ABSTRACT FROM AUTHOR]

Regulation of the volume and electrolyte composition of the cerebrospinal fluid (CSF) is vital for brain development and function. The Na-K-Cl co-transporter NKCC1 in the choroid plexus (ChP) plays key roles in regulating CSF volume by co-transporting ions and mediating same-direction water movements. Our previous study showed ChP NKCC1 is highly phosphorylated in neonatal mice as the CSF K+ level drastically decreases and that overexpression of NKCC1 in the ChP accelerates CSF K+ clearance and reduces ventricle size [1]. These data suggest that NKCC1 mediates CSF K+ clearance following birth in mice. In this current study, we used CRISPR technology to create a conditional NKCC1 knockout mouse line and evaluated CSF K+ by Inductively Coupled Plasma Optical Emission spectroscopy (ICP-OES). We demonstrated ChP-specific reduction of total and phosphorylated NKCC1 in neonatal mice following embryonic intraventricular delivery of Cre recombinase using AAV2/5. ChP-NKCC1 knockdown was accompanied by a delayed perinatal clearance of CSF K+. No gross morphological disruptions were observed in the cerebral cortex. We extended our previous results by showing embryonic and perinatal rats shared key characteristics with mice, including decreased ChP NKCC1 expression level, increased ChP NKCC1 phosphorylation state, and increased CSF K+ levels compared to adult. Collectively, these follow up data support ChP NKCC1's role in age-appropriate CSF K+ clearance during neonatal development. [ABSTRACT FROM AUTHOR]

Fluid homeostasis is fundamental for brain function with cerebral edema and hydrocephalus both being major neurological conditions. Fluid movement from blood into brain is one crucial element in cerebral fluid homeostasis. Traditionally it has been thought to occur primarily at the choroid plexus (CP) as cerebrospinal fluid (CSF) secretion due to polarized distribution of ion transporters at the CP epithelium. However, there are currently controversies as to the importance of the CP in fluid secretion, just how fluid transport occurs at that epithelium versus other sites, as well as the direction of fluid flow in the cerebral ventricles. The purpose of this review is to evaluate evidence on the movement of fluid from blood to CSF at the CP and the cerebral vasculature and how this differs from other tissues, e.g., how ion transport at the blood-brain barrier as well as the CP may drive fluid flow. It also addresses recent promising data on two potential targets for modulating CP fluid secretion, the Na+/K+/Cl− cotransporter, NKCC1, and the non-selective cation channel, transient receptor potential vanilloid 4 (TRPV4). Finally, it raises the issue that fluid secretion from blood is not constant, changing with disease and during the day. The apparent importance of NKCC1 phosphorylation and TRPV4 activity at the CP in determining fluid movement suggests that such secretion may also vary over short time frames. Such dynamic changes in CP (and potentially blood-brain barrier) function may contribute to some of the controversies over its role in brain fluid secretion.

BackgroundThe balance between the activity of the Na+/K+/Cl− cotransporter (NKCC1) that introduces Cl− into the cell and the K+/Cl− cotransporter (KCC2) that transports Cl− outside the cell is critical in determining the inhibitory or excitatory outcome of GABA release. Mounting evidence suggests that the impairment of GABAergic inhibitory neurotransmission plays a crucial role in the pathophysiology of epilepsy, both in patients and animal models. Previous studies indicate that decreased KCC2 expression is linked to audiogenic seizures in GASH/Sal hamsters, highlighting that Cl− imbalance can cause neuronal hyperexcitability. In this study, we aimed to investigate whether the Na+/K+/Cl− cotransporter NKCC1 is also affected by audiogenic seizures and could, therefore, play a role in neuronal hyperexcitability within the GASH/Sal epilepsy model.MethodsNKCC1 protein expression in both the GASH/Sal strain and wild type hamsters was analyzed by immunohistochemistry and Western blotting techniques. Brain regions examined included cortex, hippocampus, hypothalamus, inferior colliculus and pons-medulla oblongata, which were evaluated both at rest and after sound-inducing seizures in GASH/Sal hamsters. A complementary analysis of NKCC1 gene slc12a2 expression was conducted by real-time PCR. Finally, protein and mRNA levels of glutamate decarboxylase GAD67 were measured as an indicator of GABA release.ResultsThe induction of seizures caused significant changes in NKCC1 expression in epileptic GASH/Sal hamsters, despite the similar brain expression pattern of NKCC1 in GASH/Sal and wild type hamsters in the absence of seizures. Interestingly, the regulation of brain NKCC1 by seizures demonstrated regional specificity, as protein levels exclusively increased in the hippocampus and hypothalamus. Complementary real-time PCR analysis revealed that NKCC1 regulation was post-transcriptional only in the hypothalamus. In addition, seizures also modulated GAD67 mRNA levels in a brain region-specific manner. The increased GAD67 expression in the hippocampus and hypothalamus of the epileptic hamster brain suggests that NKCC1 upregulation overlaps with GABA release in these regions during seizures.ConclusionsOur results indicate that seizure induction causes dysregulation of NKCC1 expression in GASH/Sal animals, which overlaps with changes in GABA release. These observations provide evidence for the critical role of NKCC1 in how seizures affect neuronal excitability, and support NKCC1 contribution to the development of secondary foci of epileptogenic activity.

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.

Syndromic sensorineural hearing loss is multigenic and associated with malformations of the ear and other organ systems. Herein we describe a child admitted to the NIH Undiagnosed Diseases Program with global developmental delay, sensorineural hearing loss, gastrointestinal abnormalities, and absent salivation. Next-generation sequencing revealed a uniparental isodisomy in chromosome 5, and a 22 kb homozygous deletion in SLC12A2, which encodes for sodium, potassium, and chloride transporter in the basolateral membrane of secretory epithelia. Functional studies using patient-derived fibroblasts showed truncated SLC12A2 transcripts and markedly reduced protein abundance when compared with control. Loss of Slc12a2 in mice has been shown to lead to deafness, abnormal neuronal growth and migration, severe gastrointestinal abnormalities, and absent salivation. Together with the described phenotype of the Slc12a2-knockout mouse model, our results suggest that the absence of functional SLC12A2 causes a new genetic syndrome and is crucial for the development of auditory, neurologic, and gastrointestinal tissues.

Abstract Background Hydrocephalus is a severe complication of intracerebral hemorrhage with ventricular extension (ICH-IVH) and causes cerebrospinal fluid (CSF) accumulation. The choroid plexus epithelium plays an important role in CSF secretion and constitutes the blood–CSF barrier within the brain–immune system interface. Although the NLRP3 inflammasome, as a key component of the innate immune system, promotes neuroinflammation, its role in the pathogenesis of hydrocephalus after hemorrhage has not been investigated. Therefore, this study aimed to investigate the potential mechanism of NLRP3 in hydrocephalus to discover a potential marker for targeted therapy. Methods A rat model of hydrocephalus after ICH-IVH was developed through autologous blood infusion in wild-type and Nlrp3−/− rats. By studying the features and processes of the model, we investigated the relationship between the NLRP3 inflammasome and CSF hypersecretion in the choroid plexus. Results The ICH-IVH model rats showed ventricular dilation accompanied by CSF hypersecretion for 3 days. Based on the choroid plexus RNA-seq and proteomics results, we found that an inflammatory response was activated. The NLRP3 inflammasome was investigated, and the expression levels of NLRP3 inflammasome components reached a peak at 3 days after ICH-IVH. Inhibition of NLRP3 by an MCC950 inflammasome inhibitor or Nlrp3 knockout decreased CSF secretion and ventricular dilation and attenuated neurological deficits after ICH-IVH. The mechanism underlying the neuroprotective effects of NLRP3 inhibition involved decreased phosphorylation of NKCC1, which is a major protein that regulates CSF secretion by altering Na+- and K+-coupled water transport, via MCC950 or Nlrp3 knockout. In combination with the in vitro experiments, this experiment confirmed the involvement of the NLRP3/p-NKCC1 pathway and Na+ and K+ flux. Conclusions This study demonstrates that NKCC1 phosphorylation in the choroid plexus epithelium promotes NLRP3 inflammasome-mediated CSF hypersecretion and that NLRP3 plays an important role in the pathogenesis of hydrocephalus after hemorrhage. These findings provide a new therapeutic strategy for treating hydrocephalus.

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]

The sodium–potassium–chloride transporter NKCC1 of the SLC12 family performs Na+‐dependent Cl−‐ and K+‐ion uptake across plasma membranes. NKCC1 is important for regulating cell volume, hearing, blood pressure, and regulation of hyperpolarizing GABAergic and glycinergic signaling in the central nervous system. Here, we present a 2.6 Å resolution cryo‐electron microscopy structure of human NKCC1 in the substrate‐loaded (Na+, K+, and 2 Cl−) and occluded, inward‐facing state that has also been observed for the SLC6‐type transporters MhsT and LeuT. Cl− binding at the Cl1 site together with the nearby K+ ion provides a crucial bridge between the LeuT‐fold scaffold and bundle domains. Cl−‐ion binding at the Cl2 site seems to undertake a structural role similar to conserved glutamate of SLC6 transporters and may allow for Cl−‐sensitive regulation of transport. Supported by functional studies in mammalian cells and computational simulations, we describe a putative Na+ release pathway along transmembrane helix 5 coupled to the Cl2 site. The results provide insight into the structure–function relationship of NKCC1 with broader implications for other SLC12 family members. Synopsis: The Na+‐K+‐2Cl− cotransporter NKCC1 (SLC12A2) performs Na+‐dependent uptake of Cl− and K+ ions and is an important factor in ion homeostasis and osmotic control. Its mechanisms of ion recognition and release are addressed here by cryo‐EM microscopy, ion uptake studies, and molecular dynamics simulations. A 2.6 Å resolution cryo‐EM map reveals lipid molecules at a dimeric interface and three cholesterol molecules per protomer that regulate its transport activity.Analysis of the revealed intracellular water networks and bound ions indicates that the Cl– ion at a Cl2 site is released reversibly prior to Na+ release.Intracellular Na+ ion release is modeled and indicates guidance along transmembrane helix 5 by two negatively charged glutamate residues.The Cl2 site, which overlaps with a conserved glutamate residue in the SLC6 transporter, may sense chloride to regulate transport. [ABSTRACT FROM AUTHOR]

Ultrastructural studies of contusive spinal cord injury (SCI) in mammals have shown that the most prominent acute changes in white matter are periaxonal swelling and separation of myelin away from their axon, axonal swelling, and axonal spheroid formation. However, the underlying cellular and molecular mechanisms that cause periaxonal swelling and the functional consequences are poorly understood. We hypothesized that periaxonal swelling and loss of connectivity between the axo-myelinic interface impedes neurological recovery by disrupting conduction velocity, and glial to axonal trophic support resulting in axonal swelling and spheroid formation. Utilizing in vivo longitudinal imaging of Thy1 YFP+ axons and myelin labeled with Nile red, we reveal that periaxonal swelling significantly increases acutely following a contusive SCI (T13, 30 kdyn, IH Impactor) versus baseline recordings (laminectomy only) and often precedes axonal spheroid formation. In addition, using longitudinal imaging to determine the fate of myelinated fibers acutely after SCI, we show that ∼73% of myelinated fibers present with periaxonal swelling at 1 h post SCI and ∼ 51% of those fibers transition to axonal spheroids by 4 h post SCI. Next, we assessed whether cation-chloride cotransporters present within the internode contributed to periaxonal swelling and whether their modulation would increase white matter sparing and improve neurological recovery following a moderate contusive SCI (T9, 50 kdyn). Mechanistically, activation of the cation-chloride cotransporter KCC2 did not improve neurological recovery and acute axonal survival, but did improve chronic tissue sparing. In distinction, the NKKC1 antagonist bumetanide improved neurological recovery, tissue sparing, and axonal survival, in part through preventing periaxonal swelling and disruption of the axo-myelinic interface. Collectively, these data reveal a novel neuroprotective target to prevent periaxonal swelling and improve neurological recovery after SCI. [Display omitted] • Periaxonal swelling precedes axon degeneration post-contusive SCI in vivo. • ∼73% of dorsal column axons develop periaxonal swelling by 1 h post-SCI. • Bumetanide promotes acute and chronic tissue sparing after SCI. • Bumetanide significantly improves components of locomotor recovery. [ABSTRACT FROM AUTHOR]

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.

Ion disturbances are among the most remarkable deficits in spinal cord injury (SCI). GABA is an integral part of neural interaction. Action of the GABAA receptor depends on the amount of intracellular chloride. Homeostasis of chloride is controlled by two co-transporters, NKCC1 and KCC2. Previous studies revealed that NKCC1 are disturbed in SCI. In this study, NKCC1 is highly expressed in the epicenter of the lesioned spinal cord at 3 hours after induction of the lesion and reached the peak around 6 hours after SCI. Bumetanide (2 and 4 mg/day), as a specificNKCC1inhibitor, was used at 3 hours post SCI for 28 days. The functional recovery outcomes were measured by the Basso-Beattie-Bresnahan (BBB) locomotor rating scale, ladder walking test, and hot plate test. The rats that received bumetanide 4 mg/day exhibited improved recovery of locomotor function, reduction of NKCC1 gene expression, and upregulation of GAP protein levels 28 days post SCI. Histological tissue evaluations confirmed bumetanide's neuroprotective and regenerative effects. This study provides novel evidence for the benefits of bumetanide in early administration after SCI. [ABSTRACT FROM AUTHOR]

The excitatory action of gamma-aminobutyric-acid (GABA) in the median-eminence (ME) led to the steady-state release of corticotropinreleasing hormone (CRH) from CRH axon terminals, which modulates the hypothalamic-pituitary-adrenal (HPA) axis. However, in ME, the source of excitatory GABAergic input is unknown. We examined agouti-related peptide (AgRP) expressing neurons in the arcuate nucleus as a possible source for excitatory GABAergic input. Here, we show that a subpopulation of activated AgRP neurons directly project to the CRH axon terminals in ME elevates serum corticosterone levels in 60% food-restricted mice. This increase in serum corticosterone is not dependent on activation of CRH neuronal soma in the paraventricular nucleus. Furthermore, conditional deletion of Na+-K+-2Cl- cotransporter-1 (NKCC1), which promotes depolarizing GABA action, from the CRH axon terminals results in significantly lower corticosterone levels in response to food restriction. These findings highlight the important role of a subset of AgRP neurons in HPA axis modulation via NKCC1-dependent GABAergic excitation in ME. [ABSTRACT FROM AUTHOR]

Background and Objectives: Sevoflurane (Sev) exposure may provoke deleterious effects on cognitive function. This study explores the mechanism of long non-coding RNA growth arrest specific transcript 5 (LncRNA GAS5) in Sev-induced cognitive dysfunction in neonatal rats., Methods: Cognitive dysfunction was induced by Sev anesthesia in 7-day-old Sprague-Dawley rats, followed by open field test, novel object recognition, radial arm maze, and Morris water maze to evaluate cognitive function of rats. The subcellular localization of LncRNA GAS5 was detected by nucleocytoplasmic isolation assay, and the binding of miR-137 to LncRNA GAS5 and NKCC1 was detected by RNA pull down and dual-luciferase reporter assay, respectively. Adenovirus-packaged sh-LncRNA GAS5 was injected into the hippocampus of Sev rats. qRT-PCR and Western blot were performed to detect the expressions of LncRNA GAS5, miR-137 and NKCC1 in the hippocampus of rats., Results: Sev anesthesia led to cognitive dysfunction in neonatal rats. LncRNA GAS5 was highly expressed in Sev rats, and inhibition of LncRNA GAS5 alleviated Sev-induced cognitive dysfunction in rats. LncRNA GAS5 targeted miR-137, and miR-137 inhibited NKCC1 expression. Knockdown of miR-137 or overexpression of NKCC1 reversed the effect of LncRNA GAS5 inhibition on cognitive dysfunction in sev rats., Conclusion: LncRNA GAS5 promotes Sev-induced cognitive dysfunction in neonatal rats via the miR-137/NKCC1 axis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Interleukin 18 (IL-18), a proinflammatory cytokine, has been implicated in various neurological disorders, including cerebrovascular disease and psychiatric disorders. In a previous study, IL-18 was observed to activate microglia and enhance the inflammatory response following intracranial hemorrhage (ICH). However, the underlying mechanism remains unclear. In the present study, we found that IL-18 and IL-18 receptor (IL-18 R) are primarily secreted by neurons during the early stages after ICH, with microglia becoming the predominant source at 12-24 h after ICH. Meanwhile, the expression level of IL-18 R increased following ICH, along with an augmentation in the binding affinity of IL-18 R to IL-18. Subsequently, the deficiency of IL-18 R mitigated neurological impairment and subsequent activation of inflammatory pathways in mice post-ICH. Moreover, our findings suggest that IL-18-induced neurological injury after ICH may be mediated by the interaction between IL18R and NKCC1. Significantly, the NKCC1 inhibitor rescued the neurologic injury after ICH. In conclusion, our study suggests that targeting the IL-18/IL-18R/NKCC1 pathway could be an effective therapeutic strategy to attenuate secondary brain injury after ICH., Competing Interests: On behalf of all authors, the corresponding author states that there is no conflict of interest., (© 2024 The Authors. Published by Elsevier Inc.)

Ion disturbances are among the most remarkable deficits in spinal cord injury (SCI). GABA is an integral part of neural interaction. Action of the GABAA receptor depends on the amount of intracellular chloride. Homeostasis of chloride is controlled by two co-transporters, NKCC1 and KCC2. Previous studies revealed that NKCC1 are disturbed in SCI. In this study, NKCC1 is highly expressed in the epicenter of the lesioned spinal cord at 3 hours after induction of the lesion and reached the peak around 6 hours after SCI. Bumetanide (2 and 4 mg/day), as a specific NKCC1 inhibitor, was used at 3 hours post SCI for 28 days. The functional recovery outcomes were measured by the Basso–Beattie–Bresnahan (BBB) locomotor rating scale, ladder walking test, and hot plate test. The rats that received bumetanide 4 mg/day exhibited improved recovery of locomotor function, reduction of NKCC1 gene expression, and upregulation of GAP protein levels 28 days post SCI. Histological tissue evaluations confirmed bumetanide’s neuroprotective and regenerative effects. This study provides novel evidence for the benefits of bumetanide in early administration after SCI.

The specific NKCC1 cotransporter antagonist, bumetanide, attenuates the severity of Autism Spectrum Disorders (ASD), and many neurodevelopmental or neurodegenerative disorders in animal models and clinical trials. However, the pervasive expression of NKCC1 in many cell types throughout the body is thought to challenge the therapeutic efficacy of bumetanide. However, many peripheral functions, including intestinal, metabolic, or vascular, etc., are perturbed in brain disorders contributing to the neurological sequels. Alterations of these functions also increase the incidence of the disorder suggesting complex bidirectional links with the clinical manifestations. We suggest that a more holistic view of ASD and other disorders is warranted to account for the multiple sites impacted by the original intra-uterine insult. From this perspective, large-spectrum active repositioned drugs that act centrally and peripherally might constitute a useful approach to treating these disorders. [ABSTRACT FROM AUTHOR]

Schizophrenia (SCZ) is a debilitating, destructive, and chronic mental disorder and affects approximately one percent of the human population. Diagnosis in psychiatry is based on the patient's descriptions of his/her symptoms, interviewer's observations, history of disorder over time, and response to treatment. All of these data measure phenotype-based functions. But it appears that accurate diagnosis of such a complex disorder must be based on valid and reliable factors. In the present study, gene selection was based on the possible role of γ-aminobutyric acid (GABA) in psychopathology of SCZ and expression in blood. We evaluated the association of Na+-K+-Cl− co-transporter 1 (NKCC1) and K+-Cl− co-transporter 2 (KCC2) genes' messenger ribonucleic acid (mRNA) levels, and also the NKCC1/KCC2 ratio with positive and negative syndrome scale (PANSS) and brief psychiatric rating scale (BPRS) scores in an SCZ group. By using real-time PCR (RT-PCR), the present study is the first attempt to explore levels of NKCC1 and KCC2 expression at mRNA level and their relative expression in human peripheral blood of patients with SCZ. Our results showed that the NKCC1 to KCC2 mRNA ratio is significantly increased (but based on the delta cycle of threshold [∆Ct] is significantly lower) in the total sample of cases rather than controls (p = 0.045) and also higher in male sample cases rather than male controls (p = 0.016). In female samples, we found a trend toward a significant effect between the case and control participants (p = 0.075). We also found statistically significant association between mRNA of NKCC1 and KCC2 genes and NKCC1/KCC2 mRNA ratio with the positive and negative syndrome scale (PANSS) and brief psychiatric rating scale (BPRS) scores. [ABSTRACT FROM AUTHOR]

In his editorial, Kevin Staley criticizes our recent work demonstrating the lack of effect of bumetanide in a novel model of neonatal seizures. The main points in our response are that (1) our work is on an asphyxia model, not one on "hypercarbia only"; (2) clinically relevant parenteral doses of bumetanide applied in vivo lead to concentrations in the brain parenchyma that are at least an order of magnitude lower than what would be sufficient to exert any direct effect—even a transient one—on neuronal functions, including neonatal seizures; and (3) moreover, bumetanide's molecular target in the brain is the Na‐K‐2Cl cotransporter NKCC1, which has vital functions in neurons, astrocytes, and oligodendrocytes as well as microglia. This would make it impossible even for highly brain‐permeant NKCC1 blockers to specifically target depolarizing and excitatory actions of γ‐aminobutyric acid in principal neurons of the brain, which is postulated as the rationale of clinical trials on neonatal seizures. [ABSTRACT FROM AUTHOR]

Background: Hydrocephalus is a severe complication of intracerebral hemorrhage with ventricular extension (ICH-IVH) and causes cerebrospinal fluid (CSF) accumulation. The choroid plexus epithelium plays an important role in CSF secretion and constitutes the blood-CSF barrier within the brain-immune system interface. Although the NLRP3 inflammasome, as a key component of the innate immune system, promotes neuroinflammation, its role in the pathogenesis of hydrocephalus after hemorrhage has not been investigated. Therefore, this study aimed to investigate the potential mechanism of NLRP3 in hydrocephalus to discover a potential marker for targeted therapy.Methods: A rat model of hydrocephalus after ICH-IVH was developed through autologous blood infusion in wild-type and Nlrp3-/- rats. By studying the features and processes of the model, we investigated the relationship between the NLRP3 inflammasome and CSF hypersecretion in the choroid plexus.Results: The ICH-IVH model rats showed ventricular dilation accompanied by CSF hypersecretion for 3 days. Based on the choroid plexus RNA-seq and proteomics results, we found that an inflammatory response was activated. The NLRP3 inflammasome was investigated, and the expression levels of NLRP3 inflammasome components reached a peak at 3 days after ICH-IVH. Inhibition of NLRP3 by an MCC950 inflammasome inhibitor or Nlrp3 knockout decreased CSF secretion and ventricular dilation and attenuated neurological deficits after ICH-IVH. The mechanism underlying the neuroprotective effects of NLRP3 inhibition involved decreased phosphorylation of NKCC1, which is a major protein that regulates CSF secretion by altering Na+- and K+-coupled water transport, via MCC950 or Nlrp3 knockout. In combination with the in vitro experiments, this experiment confirmed the involvement of the NLRP3/p-NKCC1 pathway and Na+ and K+ flux.Conclusions: This study demonstrates that NKCC1 phosphorylation in the choroid plexus epithelium promotes NLRP3 inflammasome-mediated CSF hypersecretion and that NLRP3 plays an important role in the pathogenesis of hydrocephalus after hemorrhage. These findings provide a new therapeutic strategy for treating hydrocephalus. [ABSTRACT FROM AUTHOR]

Traumatic brain injury (TBI) is one of the leading causes of death, long-term disability, and neurological impairments in children and young adults. TBI is associated with cognitive deficits and memory impairment. The Na+-K+-2Cl (NKCC) cotransporter contributes to the pathophysiology of common neurological disorders following TBI. The present study was designed to explore the potential therapeutic effect of bumetanide (a relatively specific inhibitor of NKCC1) on TBI-induced passive avoidance learning deficit in a rat model of controlled cortical impact (CCI) injury. Male rats were randomly divided into the following groups: (i) control animals that have undergone a sham surgical procedure, except for CCI delivery, (ii) TBI; received CCI delivery, (iii) Bumetanide plus TBI; received bumetanide 30 min before CCI delivery, (iv) TBI plus bumetanide; received bumetanide 30 min after CCI delivery. Passive avoidance learning (PAL) was assessed using a step-through apparatus on day 8 after TBI induction. CCI resulted in PAL deficits. The step-through latency in the acquisition trial (STLa) was significantly higher in the injured animals, and both pre-CCI and post-CCI bumetanide treatment decreased STLa. A decrease in step-through latency in the retention trial (STLr) and an increase in time spent in the dark compartment (TDC) during the retention test was observed in the injured animals. Both pre-CCI and post-CCI bumetanide treatment increased STLr and decreased TDC in injured animals. These findings suggest that bumetanide has a neuroprotective effect on TBI-induced memory impairment in rats and pharmacological inhibition of NKCC1 is protective against brain injury. [ABSTRACT FROM AUTHOR]

The function of Litopenaeus vannamei Na+/K+/2Cl− cotransporter 1 (NKCC1) under nitrite stress was investigated. The full-length cDNA sequence of the L. vannamei NKCC1 gene was cloned using the rapid amplification of cDNA ends (RACE) technique, and the sequence was analysed using bioinformatics tools. Expression and localisation of NKCC1 in tissues were assessed using real-time quantitative PCR and in situ hybridisation, respectively. The impact of nitrite stress on the survival, physiology, biochemistry and tissue structure of L. vannamei was investigated following silencing of NKCC1 by RNA interference. The 3143 bp cDNA sequence of L. vannamei NKCC1 encodes a polypeptide of 918 amino acids. It is evolutionarily conserved. NKCC1 expression was highest in gill tissue, particularly within cuticle and gill epithelial cells. After silencing NKCC1 , an increase in shrimp survival was observed, accompanied by a significant reduction in nitrite entry into the body (P < 0.05). Moreover, the oxidative stress enzyme system remained unaffected and damage to gill tissue was alleviated. The results suggest that NKCC1 is involved in regulating nitrite uptake, and plays a crucial role in facilitating nitrite entry into the organism through gill tissue. The findings provide a vital experimental basis for addressing concerns related to nitrite toxicity. [Display omitted] • Molecular characterization of NKCC1 in Litopenaeus vannamei. • NKCC1 gene was mainly expressed in the cuticle and gill epithelial cells in gill tissue. • Interference with NKCC1 can improve the survival rate of prawn, reduce the accumulation of nitrite, and damage to gill tissue. [ABSTRACT FROM AUTHOR]

BackgroundRecent evidence indicates that histamine, acting on histamine 1 receptor (H1R), resets the circadian clock in the mouse suprachiasmatic nucleus (SCN) by increasing intracellular Ca(2+) concentration ([Ca(2+)]i) through the activation of CaV1.3 L-type Ca(2+) channels and Ca(2+)-induced Ca(2+) release from ryanodine receptor-mediated internal stores.ResultsIn the current study, we explored the underlying mechanisms with various techniques including Ca(2+)- and Cl(-)-imaging and extracellular single-unit recording. Our hypothesis was that histamine causes Cl(-) efflux through cystic fibrosis transmembrane conductance regulator (CFTR) to elicit membrane depolarization needed for the activation of CaV1.3 Ca(2+) channels in SCN neurons. We found that histamine elicited Cl(-) efflux and increased [Ca(2+)]i in dissociated mouse SCN cells. Both of these events were suppressed by bumetanide [Na(+)-K(+)-2Cl(-) cotransporter isotype 1 (NKCC1) blocker], CFTRinh-172 (CFTR inhibitor), gallein (Gβγ protein inhibitor) and H89 [protein kinase A (PKA) inhibitor]. By itself, H1R activation with 2-pyridylethylamine increased the level of cAMP in the SCN and this regulation was prevented by gallein. Finally, histamine-evoked phase shifts of the circadian neural activity rhythm in the mouse SCN slice were blocked by bumetanide, CFTRinh-172, gallein or H89 and were not observed in NKCC1 or CFTR KO mice.ConclusionsTaken together, these results indicate that histamine recruits the H1R-Gβγ-cAMP/PKA pathway in the SCN neurons to activate CaV1.3 channels through CFTR-mediated Cl(-) efflux and ultimately to phase-shift the circadian clock. This pathway and NKCC1 may well be potential targets for agents designed to treat problems resulting from the disturbance of the circadian system.

Valproic acid (VPA) is a histone deacetylase inhibitor with sex-specific immunomodulatory and anticancer effects. This study aimed to investigate the effect of 0.5 and 0.75 mM VPA on NKCC1 (SLC12A2), KCC2 (SLC12A5) and SLC5A8 (SLC5A8) co-transporter gene expressions in pediatric PBT24 (boy's) and SF8628 (girl's) glioblastoma cells. The SLC12A2, SLC12A5 and SLC5A8 RNA expressions were determined by the RT-PCR method. The SLC12A2 and SLC5A8 expressions did not differ between the PBT24 and SF8628 controls. The SLC12A5 expression in the PBT24 control was significantly higher than in the SF8628 control. VPA treatment significantly increased the expression of SLC12A2 in PBT24 but did not affect SF8628 cells. VPA increased the SLC12A5 expression in PBT24 and SF8628 cells. The SLC12A5 expression of the PBT24-treated cells was significantly higher than in corresponding SF8628 groups. Both VPA doses increased the SLC5A8 expression in PBT24 and SF8628 cells, but the expression was significantly higher in the PBT24-treated, compared to the respective SF8628 groups. The SLC5A8 expression in PBT24-treated cells was 10-fold higher than in SF8628 cells. The distinct effects of VPA on the expression of SLC12A2, SLC12A5 and SLC5A8 in PBT24 and SF8628 glioblastoma cells suggest differences in tumor cell biology that may be gender-related. [ABSTRACT FROM AUTHOR]

Autism spectrum disorder (ASD), as a common neurodevelopmental disorder that encompasses impairments in social communication and interaction, as well as repetitive and restrictive behavior, still awaits an effective treatment strategy. The involvement of GABAergic neurotransmission, and especially a deficit of GABAA receptors that contain the α5 subunits, were implicated in pathogenesis of ASD. Therefore, we tested MP‐III‐022, a positive allosteric modulator (PAM) selective for α5GABAA receptors, in Wistar rats prenatally exposed to valproic acid, as an animal model useful for studying ASD. Postweaning rats of both sexes were treated for 7 days with vehicle or MP‐III‐022 at two doses pharmacokinetically determined as selective, and thereafter tested in a behavioral battery (social interaction test, elevated plus maze, spontaneous locomotor activity, and standard and reverse Morris water maze). Additional rats were used for establishing a primary neuronal culture and performing calcium imaging, and determination of hippocampal mRNA levels of GABRA5, NKCC1, and KCC2. MP‐III‐022 prevented impairments in many parameters connected with social, repetitive and restrictive behavioral domains. The lower and higher dose was more effective in males and females, respectively. Intriguingly, MP‐III‐022 elicited certain changes in control animals similar to those manifested in valproate animals themselves. Behavioral results were mirrored in GABA switch and spontaneous neuronal activity, assessed with calcium imaging, and also in expression changes of three genes analyzed. Our data support a role of α5GABAA receptors in pathophysiology of ASD, and suggest a potential application of selective PAMs in its treatment, that needs to be researched in a sex‐specific manner. Lay Summary: In rats prenatally exposed to valproate as a model of autism, a modulator of α5GABAA receptors ameliorated social, repetitive and restrictive impairments, and, intriguingly, elicited certain autism‐like changes in control rats. Behavioral results were mirrored in GABA switch and spontaneous neuronal activity, and partly in gene expression changes. This shows a role of α5GABAA receptors in pathophysiology of ASD, and a potential application of their selective modulators in its treatment. [ABSTRACT FROM AUTHOR]