K-Cl Cotransporter 2 Research Articles

The α-7 Nicotinic Receptor Positive Allosteric Modulator Alleviates Lipopolysaccharide Induced Depressive-like Behavior by Regulating Microglial Function, Trophic Factor, and Chloride Transporters in Mice.

Neuroinflammation contributes to the pathophysiology of major depressive disorder (MDD) by inducing neuronal excitability via dysregulation of microglial brain-derived neurotrophic factor (BDNF), Na-K-Cl cotransporter-1 (NKCC1), and K-Cl cotransporter-2 (KCC2) due to activation of BDNF-tropomyosin receptor kinase B (TrkB) signaling. Allosteric modulation of α7 nAChRs has not been investigated on BDNF, KCC2, and NKCC1 during LPS-induced depressive-like behavior. Therefore, we examined the effects of PNU120596, an α7 nAChR positive allosteric modulator, on the expression of BDNF, KCC2, and NKCC1 in the hippocampus and prefrontal cortex using Western blot analysis, immunofluorescence assay, and real-time polymerase chain reaction. The effects of ANA12, a TrkB receptor antagonist, on LPS-induced cognitive deficit and depressive-like behaviors were determined using the Y-maze, tail suspension test (TST), and forced swim test (FST). Pharmacological interactions between PNU120596 and ANA12 were also examined. Experiments were conducted in male C57BL/6J mice. LPS administration (1 mg/kg) resulted in increased expression of BDNF and the NKCC1/KCC2 ratio and decreased expression of KCC2 in the hippocampus and prefrontal cortex. PNU120596 pretreatment (4 mg/kg) attenuated the LPS-induced increase in the expression of BDNF and NKCC1/KCC2 ratio and the reduction in KCC2 expression in these brain regions. In addition, ANA12 (0.25 or 0.50 mg/kg) reduced the LPS-induced cognitive deficit and depressive-like behaviors measured by a reduced spontaneous alternation in the Y-maze and increased immobility duration in TST and FST. Coadministration of PNU120596 (1 mg/kg) and ANA12 (0.25 mg/kg) prevented the LPS-induced cognitive deficit and depressive-like behaviors. Overall, PNU120596 prevented the LPS-induced depressive-like behavior by likely decreasing neuronal excitability via targeting microglial α7 nAChR in the hippocampus and prefrontal cortex.

NKCC1 and KCC2 Chloride Transporters Have Different Membrane Dynamics on the Surface of Hippocampal Neurons.

Na-K-2Cl cotransporter 1 (NKCC1) regulates chloride influx in neurons and thereby GABAA receptor activity in normal and pathological conditions. Here, we characterized in hippocampal neurons the membrane expression, distribution and dynamics of exogenous NKCC1a and NKCC1b isoforms and compared them to those of the chloride extruder K-Cl cotransporter 2 (KCC2). We found that NKCC1a and NKCC1b behave quite similarly. NKCC1a/1b but not KCC2 are present along the axon initial segment where they are confined. Moreover, NKCC1a/1b are detected in the somato-dendritic compartment at a lower level than KCC2, where they form fewer, smaller and less compact clusters at perisynaptic and extrasynaptic sites. Interestingly, ~60% of dendritic clusters of NKCC1a/1b are colocalized with KCC2. They are larger and brighter than those devoid of KCC2, suggesting a particular NKCC1a/1b-KCC2 relationship. In agreement with the reduced dendritic clustering of NKCC1a/1b compared with that of KCC2, NKCC1a/1b are more mobile on the dendrite than KCC2, suggesting weaker cytoskeletal interaction. NKCC1a/b are confined to endocytic zones, where they spend more time than KCC2. However, they spend less time in these compartments than at the synapses, suggesting that they can rapidly leave endocytic zones to increase the membrane pool, which can happen in pathological conditions. Thus, NKCC1a/b have different membrane dynamics and clustering from KCC2, which helps to explain their low level in the neuronal membrane, while allowing a rapid increase in the membrane pool under pathological conditions.

Waggle needling Yanglingquan (GB34) enhances cerebral blood flow and ameliorates condition of spastic paresis in rats with middle cerebral artery occlusion induced by surgical nylon suture.

To evaluate the therapeutic efficacy and the influence on cerebral blood supply of waggle needling Yanglingquan (GB34) on spastic paresis (SP) rats after middle cerebral artery occlusion (MCAO) induced and investigate its mechanism of relieving neurobehavior deficiency. SP rat model was produced by permanent MCAO. Rats were divided into five groups: blank control group (Control), sham operation group (Sham), model group (Model), waggle needling group (WN) and perpendicular needling group (PN). SP rats were treated with acupuncture from day 3 after MCAO, once a day for 6 d. The modified neurological severity score (mNSS) and modified Ashworth scale (MAS) were conducted on days 0, 1, 3, 5, 7 and 9. Cerebral blood flow (CBF) in ischemic cortex was measured by laser speckle imaging 5 min pre ischemia, 5 min post ischemia, and after intervention on day 9. All rats were sacrificed at day 9 and the protein and mRNA expressions of γ2 subunit of the γ-aminobutyric acid receptor A (GABAAγ2) and K+-Cl?cotransporter 2 (KCC2) in the ischemic cortex and lumber enlargement was measured by Western blotting and real-time quantitative polymerase chain reaction. Both Control and Sham groups showed no changes in mNSS and MAS scores and in the regional CBF. Compared with Model group, both WN and PN treatments significantly ameliorated neurological deficit ( 0.01), decreased muscle tone ( 0.05), and enhanced CBF ( 0.001) in SP rats; moreover, WN showed superior effects than PN ( 0.001). In line with the improvement in neurobehavior, acupuncture interventions up-regulated the expressions of GABAAγ2 and KCC2 in the ischemic cortex as well as lumber enlargement ( 0.01) in SP rats, and those changes were more obvious in WN ( 0.05). Acupuncture at Yanglingquan (GB34) enhanced cerebral blood flow and ameliorated SP in permanent MCAO rats, while waggle needling was superior to regular perpendicular needling. Waggle needling Yanglingquan (GB34) would be a potential complementary therapy for SP.

Naloxone could limit morphine hypersensitivity: Considering the molecular mechanisms

BackgroundNaloxone has been used as an opioid antagonist to prevent multiple adverse side effects of opioid-like tolerance and hyperalgesia. This study has investigated naloxone combined with morphine to limit pain hypersensitivity. In addition, the expression of brain-derived neurotrophic factor (BDNF) and K+ Cl− cotransporter2 (KCC2) were also studied. MethodsForty-eight adult male Wistar rats (180–220 g) were divided into eight groups, with six rats in each group. Rats were divided into two tolerance and hyperalgesia groups; the sham group, the morphine group, the treatment group (naloxone along with morphine), and the sham group (naloxone along with saline) for eight consecutive days. Tail-flick test was performed on days 1, 5, and 8, and the plantar test on days 1 and 10. On days 8 and 10, the lumbar segments of the spinal cord were collected, and BDNF and KCC2 expression were analyzed using western blotting and immunohistochemistry, respectively. ResultsResults showed that tolerance and hyperalgesia developed following eight days of repeated morphine injection. BDNF expression significantly increased, but KCC2 was downregulated. Co-administration of naloxone and morphine decreased tolerance and hyperalgesia by decreasing BDNF and increasing KCC2 expression, respectively. ConclusionThis study suggests that BDNF and KCC2 may be candidate molecules for decreased morphine tolerance and hyperalgesia.

PD23-05 THERAPEUTIC EFFECTS OF KCC2 CHLORIDE ION TRANSPORTER ACTIVATION ON DETRUSOR OVERACTIVITY IN MICE WITH SPINAL CORD INJURY

You have accessJournal of UrologyCME1 Apr 2023PD23-05 THERAPEUTIC EFFECTS OF KCC2 CHLORIDE ION TRANSPORTER ACTIVATION ON DETRUSOR OVERACTIVITY IN MICE WITH SPINAL CORD INJURY Kyohei Watanabe, Masaru Ishibashi, Takahisa Suzuki, Atsushi Otsuka, Naoki Yoshimura, Hideaki Miyake, and Atsuo Fukuda Kyohei WatanabeKyohei Watanabe More articles by this author , Masaru IshibashiMasaru Ishibashi More articles by this author , Takahisa SuzukiTakahisa Suzuki More articles by this author , Atsushi OtsukaAtsushi Otsuka More articles by this author , Naoki YoshimuraNaoki Yoshimura More articles by this author , Hideaki MiyakeHideaki Miyake More articles by this author , and Atsuo FukudaAtsuo Fukuda More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003296.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: GABA generally exerts inhibitory effects in the central nervous system, including the spinal cord. However, it may also act as an excitatory transmitter under pathological conditions, such as injury. This paradoxical signaling of GABA is dependent on the intracellular Cl- concentration in neurons. K+-Cl-co-transporter 2 (KCC2) maintains a low intracellular Cl- concentration by pumping K+ and Cl- ions out of the cell. We aimed to clarify whether the down regulation of KCC2 in the sacral parasympathetic nucleus (SPN) of the lumbosacral spinal cord, from which the efferent pathway innervating the bladder originates, causes cellular hyperexcitability and triggers detrusor overactivity (DO) in spinal cord injury (SCI). METHODS: SCI was produced by the Th8-9 spinal cord transection in female C57BL/6 mice. We randomly divided mice into four groups: a spinal cord intact (SI)-vehicle, SI-CLP290, SCI-vehicle, and SCI-CLP290 groups. At 4 weeks after SCI, CLP290, a KCC2 activator was administered, and cystometry was performed. Thereafter, neuronal activity with c-fos staining and KCC expression in the cholinergic preganglionic parasympathetic neurons in the SPN were examined using immunohistochemistry. Firing properties of neurons in the SPN region were evaluated by extracellular recordings in spinal cord slice preparations. RESULTS: DO evident as non-voiding contractions (NVC) was significantly reduced by CLP290 treatment in SCI mice (Figure 1A-D). The number of c-fos-positive cells and co-expression of c-fos in choline acetyltransferase (ChAT)-positive cells were decreased in the SPN region of SCI-CLP290 group vs. SCI-vehicle group. KCC2 immunoreactivity was present on the cell membrane of SPN neurons, and the normalized fluorescence intensity of KCC2 in ChAT-positive SPN neurons was decreased in SCI-vehicle group, vs. SI-vehicle group, but recovered in SCI-CLP290 group. Extracellular recordings showed that CLP290 suppressed the high frequency firing activity of SPN neurons in SCI mice (Figure 1E-F). CONCLUSIONS: This study is the first report to suggest that activation of KCC2 chloride ion transporter may be a therapeutic modality for the treatment of SCI-induced DO by targeting bladder efferent pathways. Source of Funding: This work supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and a HUSM Grant-in-Aid © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e672 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kyohei Watanabe More articles by this author Masaru Ishibashi More articles by this author Takahisa Suzuki More articles by this author Atsushi Otsuka More articles by this author Naoki Yoshimura More articles by this author Hideaki Miyake More articles by this author Atsuo Fukuda More articles by this author Expand All Advertisement PDF downloadLoading ...

Preclinical Development of the Na-K-2Cl Co-transporter-1 (NKCC1) Inhibitor ARN23746 for the Treatment of Neurodevelopmental Disorders.

Alterations in the expression of the Cl- importer Na-K-2Cl co-transporter-1 (NKCC1) and the exporter K-Cl co-transporter 2 (KCC2) lead to impaired intracellular chloride concentration in neurons and imbalanced excitation/inhibition in the brain. These alterations have been observed in several neurological disorders (e.g., Down syndrome and autism). Recently, we have reported the discovery of the selective NKCC1 inhibitor "compound ARN23746" for the treatment of Down syndrome and autism in mouse models. Here, we report on an extensive preclinical characterization of ARN23746 toward its development as a clinical candidate. ARN23746 shows an overall excellent metabolism profile and good brain penetration. Moreover, ARN23746 is effective in rescuing cognitive impairment in Down syndrome mice upon per os administration, in line with oral treatment of neurodevelopmental disorders. Notably, ARN23746 does not present signs of toxicity or diuresis even if administered up to 50 times the effective dose. These results further support ARN23746 as a solid candidate for clinical trial-enabling studies.

The effects of postnatal erythropoietin and nano-erythropoietin on behavioral alterations by mediating K-Cl co-transporter 2 in the valproic acid-induced rat model of autism.

In this study, based on the excitatory/inhibitory imbalance theory of autism, the time window of GABA switch, the role of K-Cl co-transporter 2 (KCC2) in adjustment GABA switch, and brain permeability to erythropoietin (EPO), the effects of postnatal -EPO and- nano- erythropoietin (NEPO) have been evaluated in the valproic acid (VPA) rat model of autism. The VPA was administered for animal modeling of autism at gestational day (GD) 12.5 (600 mg/kg). Male offsprings were injected with EPO and NEPO in a clinically proper postnatal dosing regimen on postnatal days (PND) 1-5, and autistic-like behaviors were tested at the end of the first month. Then animals were sacrificed, and neuron morphology and KCC2 expression were examined by Nissl staining and Western blot. According to our findings, high-dose NEPO improved autism-associated phenotypes. Neuroprotective effects of EPO and NEPO have been shown in the hippocampus. Postnatal NEPO treatment reversed KCC2 expression abnormalities induced by prenatal VPA. Our results might support the role of KCC2 in ASD and the excitatory/inhibitory imbalance hypothesis. We suggested Nano- erythropoietin and other KCC2 interventions as a new approach to the early treatment and prevention of autism.

The developmental changes in intrinsic and synaptic properties of prefrontal neurons enhance local network activity from the second to the third postnatal weeks in mice

The prefrontal cortex (PFC) is characterized by protracted maturation. The cellular mechanisms controlling the early development of prefrontal circuits are still largely unknown. Our study delineates the developmental cellular processes in the mouse medial PFC (mPFC) during the second and the third postnatal weeks and characterizes their contribution to the changes in network activity. We show that spontaneous inhibitory postsynaptic currents (sIPSC) are increased, whereas spontaneous excitatory postsynaptic currents (sEPSC) are reduced from the second to the third postnatal week. Drug application suggested that the increased sEPSC frequency in mPFC at postnatal day 10 (P10) is due to depolarizing γ-aminobutyric acid (GABA) type A receptor function. To further validate this, perforated patch-clamp recordings were obtained and the expression levels of K-Cl cotransporter 2 (KCC2) protein were examined. The reversal potential of IPSCs in response to current stimulation was significantly more depolarized at P10 than P20 while KCC2 expression is decreased. Moreover, the number of parvalbumin-expressing GABAergic interneurons increases and their intrinsic electrophysiological properties significantly mature in the mPFC from P10 to P20. Using computational modeling, we show that the developmental changes in synaptic and intrinsic properties of mPFC neurons contribute to the enhanced network activity in the juvenile compared with neonatal mPFC.

Analgesic Effect of Bumetanide on Neuropathic Pain in Patients With Spinal Cord Injury.

Introduction:The current study evaluated the analgesic effects of bumetanide as an adjunctive in the management of neuropathic pain following Spinal Cord Injury (SCI). The peripheral expression of Na-K-Cl Cotransporter-1 (NKCC1) and K-Cl Cotransporter-2 (KCC2) genes in polymorphonuclear lymphocytes (PMLs) was assessed as a possible biomarker indicating central mechanisms underlying the observed response.Methods:Through an open-label, single-arm, pilot trial of bumetanide (2 mg/d), an add-on treatment was conducted on 14 SCI patients for 19 weeks. This study consisted of 3 phases: pre-treatment (1 month), titration (3 weeks), and active treatment (4 months). Ultimately, 9 patients completed the study. The primary outcome variables were the endpoint pain score using the Numeric Rating Scale (NRS), and also the short-form of the McGill pain questionnaire. Secondary endpoints included the short-form of the health survey that assesses the quality of life. Blood samples were collected and used for determining the expression of NKCC1 and KCC2 genes in transcription and translation levels.Results:Bumetanide treatment significantly decreased average pain intensity according to the NRS and the short-form of the McGill pain questionnaire scores. Baseline expression of KCC2 protein was low between groups and increased significantly following treatment (P<0.05). In the current study, pain improvement was accompanied by the greater mean change from the baseline (improvement) for the overall quality of life.Conclusion:These data highlighted the analgesic effect of bumetanide on neuropathic pain and indicated the potential role of the upregulation of KCC2 protein and involvement of GABAergic disinhibition in producing neuropathic pain.

Analgesic Effect of Bumetanide on Neuropathic Pain in Patients with Spinal Cord Injury

Objectives: The current study evaluated the analgesic effects of bumetanide as an adjunctive treatment in the management of neuropathic pain following spinal cord injury. The peripheral expression of Na-K-Cl-cotransporter-1 1 and K-Cl-cotransporter-2 (KCC2)2 genes in polymorphonuclear lymphocytes (PMLs) assessed as a possible biomarker indicating central mechanisms underlying the observed response. Methods: Through an open-label, single arm, pilot trial of bumetanide (2mg/day), as add-on treatment conducted in 14 SCI patients for 19 weeks. This study consisted of 3 phases: pre-treatment (1month), titration (3 weeks), and active treatment (4 months). Ultimately, 9 patients completed the study. The primary outcome variables were the endpoint pain score using the numeric rating scale (NRS), and also the short-form McGill Pain Questionnaire. Secondary endpoints included the Short-Form Health Survey that assesses the quality of life. Blood samples were collected and used for determining the expression of NKCC1 and KCC2 genes in transcription and translation levels. Results: Bumetanide treatment significantly decreased average pain intensity according to the NRS and the short form of the McGill Pain Questionnaire scores. Baseline expression of KCC2 protein was low between groups and increased significantly following treatment (P&lt;0.05). In the current study, pain improvement accompanied by the greater mean change from the baseline (improvement) for the overall quality of life. Conclusions: These data highlighted the analgesic effect of bumetanide on neuropathic pain and indicated the probable role of upregulation of KCC2 protein and involvement of GABAergic disinhibition in producing neuropathic pain.

Preliminary study of analgesic effect of bumetanide on neuropathic pain in patients with spinal cord injury

Background/objectiveThe current study evaluated the analgesic effects of bumetanide as an adjunctive treatment in managing neuropathic pain following spinal cord injury. The peripheral expression level of Na-K-Cl-cotransporter-1 (NKCC1) and K-Cl-cotransporter-2 (KCC2) genes in polymorphonuclear lymphocytes (PMLs) assessed as a possible biomarker indicating central underlying mechanisms. MethodsThis open-label, single-arm, pilot trial of bumetanide (2 mg/day) is an add-on treatment conducted in 14 SCI patients for 19 weeks. The whole duration consisted of three phases: pre-treatment (1 month), titration (3 weeks), and active treatment (4 months). Ultimately, nine patients completed the study. The primary outcome variables were the endpoint pain score measured by the numeric rating scale (NRS), and the short-form McGill Pain Questionnaire. Secondary endpoints included the Short-Form Health Survey that measures the quality of life. Blood samples were collected and used for determining the expression of NKCC1 and KCC2 genes in transcription and translation levels. ResultsBumetanide treatment significantly reduced average pain intensity according to the NRS and the short form of the McGill Pain Questionnaire scores. The baseline expression of KCC2 protein was low between groups and increased significantly following treatment (P < 0.05). Through the current study, pain improvement accompanied by the more significant mean change from the baseline for the overall quality of life. ConclusionThese data might be a piece of preliminary evidence for the analgesic effect of bumetanide on neuropathic pain and could support the potential role of the upregulation of KCC2 protein and involvement of GABAergic disinhibition in producing neuropathic pain.

Novel Treatment Targets Based on Insights in the Etiology of Depression: Role of IL-6 Trans-Signaling and Stress-Induced Elevation of Glutamate and ATP.

Inflammation and psychological stress are risk factors for major depression and suicide. Both increase central glutamate levels and activate the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system. Both factors also affect the function of the chloride transporters, Na-K-Cl-cotransporter-1 (NKCC1) and K-Cl-cotransporter-2 (KCC2), and provoke interleukin-6 (IL-6) trans-signaling. This leads to measurable increases in circulating corticosteroids, catecholamines, anxiety, somatic and psychological symptoms, and a decline in cognitive functions. Recognition of the sequence of pathological events allows the prediction of novel targets for therapeutic intervention. Amongst others, these include blockade of the big-K potassium channel, blockade of the P2X4 channel, TYK2-kinase inhibition, noradrenaline α2B-receptor antagonism, nicotinic α7-receptor stimulation, and the Sgp130Fc antibody. A better understanding of downstream processes evoked by inflammation and stress also allows suggestions for tentatively better biomarkers (e.g., SERPINA3N, MARCKS, or 13C-tryptophan metabolism).

Crossing the Chloride Channel: The Current and Potential Therapeutic Value of the Neuronal K+-Cl-Cotransporter KCC2

Chloride (Cl−) homeostasis is an essential process involved in neuronal signalling and cell survival. Inadequate regulation of intracellular Cl− interferes with synaptic signalling and is implicated in several neurological diseases. The main inhibitory neurotransmitter of the central nervous system is γ-aminobutyric acid (GABA). GABA hyperpolarises the membrane potential by activating Cl− permeable GABAA receptor channels (GABAAR). This process is reliant on Cl− extruder K+-Cl− cotransporter 2 (KCC2), which generates the neuron's inward, hyperpolarising Cl− gradient. KCC2 is encoded by the fifth member of the solute carrier 12 family (SLC12A5) and has remained a poorly understood component in the development and severity of many neurological diseases for many years. Recent advancements in next-generation sequencing and specific gene targeting, however, have indicated that loss of KCC2 activity is involved in a number of diseases including epilepsy and schizophrenia. It has also been implicated in neuropathic pain following spinal cord injury. Any variant of SLC12A5 that negatively regulates the transporter's expression may, therefore, be implicated in neurological disease. A recent whole exome study has discovered several causative mutations in patients with epilepsy. Here, we discuss the implications of KCC2 in neurological disease and consider the evolving evidence for KCC2's potential as a therapeutic target.

Effects of α7 nicotinic receptor positive allosteric modulator, PNU120596, on BDNF, NKCC1, and KCC2 expressions following LPS‐induced depressive‐like behavior in mice

Previous evidence indicates that neuroinflammation plays a critical role in the pathophysiology of major depressive disorder (MDD) by inducing neuronal excitability via dysregulation of microglial brain‐derived neurotrophic factor (BDNF), Na‐K‐Cl cotransporter‐1 (NKCC1), and K‐Cl cotransporter‐2 (KCC2). However, the role of microglial α7 nicotinic acetylcholine receptors (nAChRs), exhibiting anti‐inflammatory function during the activation, on these mediators is unknown. The objective of the present study was to determine the effects of PNU120596 on BDNF, NKCC1, and KCC2 expressions in lipopolysaccharide (LPS)‐induced mouse model of MDD using Western blot analysis, immunofluorescence staining, and real‐time polymerase chain reaction. We also examined the pharmacological interaction between PNU120596 and ANA12, an antagonist of TrkB which is the primary receptor for BDNF, on LPS‐induced depressive‐like behavior using forced swim test (FST), tail suspension test (TST), and Y‐maze test. Systemic administration of LPS (1 mg/kg, i.p.) significantly increased BDNF protein expression and mRNA ratio of NKCC1 to KCC2, and decreased KCC2 mRNA expression in the hippocampus and prefrontal cortex, the brain regions implicated in MDD. The PNU120596 (1 or 4 mg/kg, i.p.) dose‐dependently prevented the LPS‐induced dysregulated BDNF, NKCC1, and KCC2 expressions in these brain regions. Combination treatment with PNU120596 (1 mg/kg, i.p.) and ANA12 (0.25 mg/kg, i.p.) prevented LPS‐induced increased immobility time during FST and TST and LPS‐induced decreased spontaneous alternations (%) during Y‐maze test. Overall, our results suggest that PNU120596 prevented LPS‐induced depressive‐like behavior by decreasing neuronal excitability by targeting microglial α7 nAChR in the hippocampus and prefrontal cortex. (Supported in part by grant from Saudi Arabian Cultural Mission, USA).Support or Funding InformationSupported in part by grant from Saudi Arabian Cultural Mission, USA.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pharmaco-resistant Neonatal Seizures: Critical Mechanistic Insights from a Chemoconvulsant Model.

Neonatal seizures are harmful to the developing brain and are associated with mortality and long-term neurological comorbidities. Hypoxic-ischemic encephalopathy (HIE) seizures represent a significant proportion of such seizures. Phenobarbital (PB) remains the first line anti-seizure drug (ASD) treatment but fails ~50% of the time. Translational models of neonatal seizures are crucial to investigating mechanisms underlying PB-resistance. A model of PB-resistant ischemic seizures in postnatal day 7 (P7) CD-1 mice reported K-Cl cotransporter 2 (KCC2) degradation that has been shown to be due to activation of the TrkB pathway. We investigated PB-efficacy in a pentylenetetrazole (PTZ) model of neonatal seizures in the same strain and age using identical treatment protocols to gain insights into mechanisms underlying PB-resistance. A single dose of PTZ (80mg/kg; IP) consistently induced repetitive seizures that did not progress to status epilepticus (SE). PB (25 mg/kg; IP, single dose) significantly suppressed the PTZ-induced seizures. This was associated with significant KCC2 upregulation and stable Na-K-Cl cotransporter 1 (NKCC1) expression at 24h. The TrkB pathway was not activated. PTZ seizure burdens were significantly higher than those reported for ischemic seizures, indicating seizure severity did not dictate the differences in PB-efficacy. Bumetanide (BTN) (0.1-0.2 mg/kg; IP) did not work as an anti-seizure agent, similar to the ischemic model. When investigating mechanisms underlying the emergence of PB-resistance in translational models, the method by which seizures are induced may dictate mechanisms underlying emergence of PB-resistance.

Dose-dependent reversal of KCC2 hypofunction and phenobarbital-resistant neonatal seizures by ANA12

Neonatal seizures have an incidence of 3.5 per 1000 newborns; while hypoxic-ischemic encephalopathy (HIE) accounts for 50–60% of cases, half are resistant to 1st-line anti-seizure drugs such as phenobarbital (PB). Tyrosine receptor kinase B (TrkB) activation following ischemic injury is known to increase neuronal excitability by downregulation of K-Cl co-transporter 2 (KCC2); a neuronal chloride (Cl−) co-transporter. In this study, three graded doses of ANA12, a small-molecule selective TrkB antagonist, were tested in CD1 mice at P7 and P10 following induction of neonatal ischemia by a unilateral carotid ligation. The PB loading dose remained the same in all treatment groups at both ages. Evaluation criteria for the anti-seizure efficacy of ANA12 were: (1) quantitative electroencephalographic (EEG) seizure burden and power, (2) rescue of post-ischemic KCC2 and pKCC2-S940 downregulation and (3) reversal of TrkB pathway activation following ischemia. ANA12 significantly rescued PB resistant seizures in a dose-dependent manner at P7 and improved PB efficacy at P10. Additionally, female pups responded better to lower doses of ANA12 compared to males. ANA12 significantly reversed post-ischemic KCC2 downregulation and TrkB pathway activation at P7 when PB alone was inefficacious. Rescuing KCC2 hypofunction may be critical for preventing emergence of refractory seizures.

K-Cl Cotransporter 2-mediated Cl- Extrusion Determines Developmental Stage-dependent Impact of Propofol Anesthesia on Dendritic Spines.

General anesthetics potentiating γ-aminobutyric acid (GABA)-mediated signaling are known to induce a persistent decrement in excitatory synapse number in the cerebral cortex when applied during early postnatal development, while an opposite action is produced at later stages. Here, the authors test the hypothesis that the effect of general anesthetics on synaptogenesis depends upon the efficacy of GABA receptor type A (GABAA)-mediated inhibition controlled by the developmental up-regulation of the potassium-chloride (K-Cl) cotransporter 2 (KCC2). In utero electroporation of KCC2 was used to prematurely increase the efficacy of (GABAA)-mediated inhibition in layer 2/3 pyramidal neurons in the immature rat somatosensory cortex. Parallel experiments with expression of the inward-rectifier potassium channel Kir2.1 were done to reduce intrinsic neuronal excitability. The effects of these genetic manipulations (n = 3 to 4 animals per experimental group) were evaluated using iontophoretic injection of Lucifer Yellow (n = 8 to 12 cells per animal). The total number of spines analyzed per group ranged between 907 and 3,371. The authors found a robust effect of the developmental up-regulation of KCC2-mediated Cl transport on the age-dependent action of propofol on dendritic spines. Premature expression of KCC2, unlike expression of a transport-inactive KCC2 variant, prevented a propofol-induced decrease in spine density. In line with a reduction in neuronal excitability, the above result was qualitatively replicated by overexpression of Kir2.1. The KCC2-dependent developmental increase in the efficacy of GABAA-mediated inhibition is a major determinant of the age-dependent actions of propofol on dendritic spinogenesis.

Cortical GABAergic excitation contributes to epileptic activities around human glioma.

Brain gliomas are highly epileptogenic. Excitatory glutamatergic mechanisms are involved in the generation of epileptic activities in the neocortex surrounding gliomas. However, chloride homeostasis is known to be perturbed in glioma cells. Thus, the contribution of γ-aminobutyric acidergic (GABAergic) mechanisms that depend on intracellular chloride merits closer study. We studied the occurrence, networks, cells, and signaling basis of epileptic activities in neocortical slices from the peritumoral surgical margin resected around human brain gliomas. Postoperative glioma tissue from 69% of patients spontaneously generated interictal-like discharges, synchronized, with a high-frequency oscillation signature, in superficial layers of neocortex around areas of glioma infiltration. Interictal-like events depended both on glutamatergic AMPA receptor-mediated transmission and on depolarizing GABAergic signaling. GABA released by interneurons depolarized 65% of pyramidal cells, in which chloride homeostasis was perturbed because of changes in expression of neuronal chloride cotransporters: KCC2 (K-Cl cotransporter 2) was reduced by 42% and expression of NKCC1 (Na-K-2Cl cotransporter 1) increased by 144%. Ictal-like activities were initiated by convulsant stimuli exclusively in these epileptogenic areas. This study shows that epileptic activities are sustained by excitatory effects of GABA in human peritumoral neocortex, as reported in temporal lobe epilepsies, suggesting that both glutamate and GABA signaling and cellular chloride regulation processes, all also involved in oncogenesis as already shown, induce an imbalance between synaptic excitation and inhibition underlying epileptic discharges in glioma patients. Thus, the control of chloride in neurons and glioma cells may provide a therapeutic target for patients with epileptogenic gliomas.

Changes in spinal inhibitory networks induced by furosemide in humans.

During neural development in animals, GABAergic and glycinergic neurons are first excitatory, and then become inhibitory in the mature state. This developmental shift is due mainly to strong expression of the cation-chloride K-Cl cotransporter 2 (KCC2) and down-regulation of Na-K-Cl cotransporter 1 (NKCC1) during maturation. The down-regulation of co-transporter KCC2 after spinal cord transection in animals leads to the depolarising (excitatory) action of GABA and glycine and thus results in a reduction of inhibitory synaptic efficiency. Furosemide, a loop diuretic, has been shown to selectively and reversibly block inhibitory postsynaptic potentials without affecting excitatory postsynaptic potentials in animal spinal neurons. Moreover, this diuretic has been also demonstrated to block the cation-chloride co-transporters. Here, we used furosemide to demonstrate changes in spinal inhibitory networks in healthy human subjects. Non-invasive electrophysiological techniques were used to assess presynaptic inhibition, postsynaptic inhibition and the efficacy of synaptic transmission between muscle afferent terminals and soleus motoneurons in the spinal cord. Orally administered furosemide, at doses commonly used in the clinic (40 mg), significantly reduced spinal inhibitory interneuronal activity for at least 70 min from intake compared to control experiments in the same subjects while no changes were observed in the efficacy of synaptic transmission between muscle afferent terminals and soleus motoneurons. The reduction of inhibition was dose-dependent. Our results provide indirect evidence that reversible changes in the cation-chloride transport system induce modulations of inhibitory neuronal activity at spinal cord level in humans.

K‐CL COTRANSPORTER‐2 (KCC2) EXPRESSION IN CHICKEN CARDIOMYOCYTES

K‐Cl cotransporter‐2 (KCC2) is widely considered to be expressed only in mature central neurons. Here, we report the novel finding that KCC2 is expressed in chicken cardiomyocytes. Previously, we prepared antibodies against a fusion protein encoded by exons 22–24 of rat KCC2 that broadly recognized KCC2 in vertebrate brains. While these antibodies have never shown reactivity with protein outside the CNS of therian mammals, they have never been tested in other tissues of lower vertebrates. We confirmed in rat that KCC2 was exclusively expressed in brain, but in chicken, we observed expression in both brain and heart. In silico analysis showed that while exon 22 is unique to the KCC2 isoform in therian mammals, it is retained in KCC2's closest paralog, KCC4, of lower vertebrates, including chicken. A blocking peptide was used to eliminate cross‐reactivity with chicken KCC4 protein, but it did not prevent reactivity with chicken heart. Furthermore, real time PCR revealed robust KCC2 transcript expression in chicken brain and heart, but nearly 10 times less KCC4 transcript in both tissues. These data confirm that our KCC2 antibodies are indeed recognizing KCC2 in chicken heart. Immunofluorescence microscopy revealed prominent plasma membrane KCC2 labeling in chicken ventricular myocytes. We hypothesize that KCC is critical for cardiomyocyte Cl− homeostasis, especially under conditions of elevated heart rates.