Chloride Concentration In Neurons Research Articles

Regulation of Neuronal Chloride Homeostasis by Pro- and Mature Brain-Derived Neurotrophic Factor (BDNF) via KCC2 Cation-Chloride Cotransporters in Rat Cortical Neurons.

The strength of inhibitory neurotransmission depends on intracellular neuronal chloride concentration, primarily regulated by the activity of cation-chloride cotransporters NKCC1 (Sodium-Potassium-Chloride Cotransporter 1) and KCC2 (Potassium-Chloride Cotransporter 2). Brain-derived neurotrophic factor (BDNF) influences the functioning of these co-transporters. BDNF is synthesized from precursor proteins (proBDNF), which undergo proteolytic cleavage to yield mature BDNF (mBDNF). While previous studies have indicated the involvement of BDNF signaling in the activity of KCC2, its specific mechanisms are unclear. We investigated the interplay between both forms of BDNF and chloride homeostasis in rat hippocampal neurons and in utero electroporated cortices of rat pups, spanning the behavioral, cellular, and molecular levels. We found that both pro- and mBDNF play a comparable role in immature neurons by inhibiting the capacity of neurons to extrude chloride. Additionally, proBDNF increases the endocytosis of KCC2 while maintaining a depolarizing shift of EGABA in maturing neurons. Behaviorally, proBDNF-electroporated rat pups in the somatosensory cortex exhibit sensory deficits, delayed huddling, and cliff avoidance. These findings emphasize the role of BDNF signaling in regulating chloride transport through the modulation of KCC2. In summary, this study provides valuable insights into the intricate interplay between BDNF, chloride homeostasis, and inhibitory synaptic transmission, shedding light on the underlying cellular mechanisms involved.

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.

Repurposing cancer drugs identifies kenpaullone which ameliorates pathologic pain in preclinical models via normalization of inhibitory neurotransmission

Inhibitory GABA-ergic neurotransmission is fundamental for the adult vertebrate central nervous system and requires low chloride concentration in neurons, maintained by KCC2, a neuroprotective ion transporter that extrudes intracellular neuronal chloride. To identify Kcc2 gene expression‑enhancing compounds, we screened 1057 cell growth-regulating compounds in cultured primary cortical neurons. We identified kenpaullone (KP), which enhanced Kcc2/KCC2 expression and function in cultured rodent and human neurons by inhibiting GSK3ß. KP effectively reduced pathologic pain-like behavior in mouse models of nerve injury and bone cancer. In a nerve-injury pain model, KP restored Kcc2 expression and GABA-evoked chloride reversal potential in the spinal cord dorsal horn. Delta-catenin, a phosphorylation-target of GSK3ß in neurons, activated the Kcc2 promoter via KAISO transcription factor. Transient spinal over-expression of delta-catenin mimicked KP analgesia. Our findings of a newly repurposed compound and a novel, genetically-encoded mechanism that each enhance Kcc2 gene expression enable us to re-normalize disrupted inhibitory neurotransmission through genetic re-programming.

Chloride dynamics alter the input-output properties of neurons.

Fast synaptic inhibition is a critical determinant of neuronal output, with subcellular targeting of synaptic inhibition able to exert different transformations of the neuronal input-output function. At the receptor level, synaptic inhibition is primarily mediated by chloride-permeable Type A GABA receptors. Consequently, dynamics in the neuronal chloride concentration can alter the functional properties of inhibitory synapses. How differences in the spatial targeting of inhibitory synapses interact with intracellular chloride dynamics to modulate the input-output function of neurons is not well understood. To address this, we developed computational models of multi-compartment neurons that incorporate experimentally parametrised mechanisms to account for neuronal chloride influx, diffusion, and extrusion. We found that synaptic input (either excitatory, inhibitory, or both) can lead to subcellular variations in chloride concentration, despite a uniform distribution of chloride extrusion mechanisms. Accounting for chloride changes resulted in substantial alterations in the neuronal input-output function. This was particularly the case for peripherally targeted dendritic inhibition where dynamic chloride compromised the ability of inhibition to offset neuronal input-output curves. Our simulations revealed that progressive changes in chloride concentration mean that the neuronal input-output function is not static but varies significantly as a function of the duration of synaptic drive. Finally, we found that the observed effects of dynamic chloride on neuronal output were mediated by changes in the dendritic reversal potential for GABA. Our findings provide a framework for understanding the computational effects of chloride dynamics on dendritically targeted synaptic inhibition.

Transient ischemia facilitates neuronal chloride accumulation and severity of seizures.

ObjectivePreceding oxygen glucose deprivation (OGD) and ongoing seizures have both been reported to increase neuronal chloride concentration ([Cl−]i), which may contribute to anticonvulsant failure by reversing the direction of chloride currents at inhibitory GABAA synapses.MethodsThe effects of OGD on [Cl−]i, seizure activity, and anticonvulsant efficacy were studied in a chronically epileptic in vitro preparation.ResultsSeizures initially increased during OGD, followed by suppression. On reperfusion, seizure frequency and [Cl−]i progressively increased, and phenobarbital efficacy was reduced. Bumetanide (10 μmol/L) and furosemide (1 mmol/L) prevented or reduced the OGD induced [Cl−]i increase. Phenobarbital efficacy was enhanced by bumetanide (10 μmol/L). Furosemide (1 mmol/L) suppressed recurrent seizures.Interpretation[Cl−]i increases after OGD and is associated with worsened seizure activity, reduced efficacy of GABAergic anticonvulsants, and amelioration by antagonists of secondary chloride transport.

Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo

Intracellular chloride ([Cl-]i) and pH (pHi) are fundamental regulators of neuronal excitability. They exert wide-ranging effects on synaptic signaling and plasticity and on development and disorders of the brain. The ideal technique to elucidate the underlying ionic mechanisms is quantitative and combined two-photon imaging of [Cl-]i and pHi, but this has never been performed at the cellular level in vivo. Here, by using a genetically encoded fluorescent sensor that includes a spectroscopic reference (an element insensitive to Cl- and pH), we show that ratiometric imaging is strongly affected by the optical properties of the brain. We have designed a method that fully corrects for this source of error. Parallel measurements of [Cl-]i and pHi at the single-cell level in the mouse cortex showed the in vivo presence of the widely discussed developmental fall in [Cl-]i and the role of the K-Cl cotransporter KCC2 in this process. Then, we introduce a dynamic two-photon excitation protocol to simultaneously determine the changes of pHi and [Cl-]i in response to hypercapnia and seizure activity.

Role of Vasopressin in Rat Models of Salt-Dependent Hypertension

Dietary salt intake increases both plasma sodium and osmolality and therefore increases vasopressin (VP) release from the neurohypophysis. Although this effect could increase blood pressure by inducing fluid reabsorption and vasoconstriction, acute activation of arterial baroreceptors inhibits VP neurons via GABAA receptors to oppose high blood pressure. Here we review recent findings demonstrating that this protective mechanism fails during chronic high salt intake in rats. Two recent studies showed that chronic high sodium intake causes an increase in intracellular chloride concentration in VP neurons. This effect causes GABAA receptors to become excitatory and leads to the emergence of VP-dependent hypertension. One study showed that the increase in intracellular chloride was provoked by a decrease in the expression of the chloride exporter KCC2 mediated by local secretion of brain-derived neurotrophic factor and activation of TrkB receptors. Prolonged high dietary salt intake can cause pathological plasticity in a central homeostatic circuit that controls VP secretion and thereby contribute to peripheral vasoconstriction and hypertension.

Developmental Decrease of Neuronal Chloride Concentration Is Independent of Trauma in Thalamocortical Brain Slices.

The intraneuronal chloride concentration ([Cl-]i) is paramount for determining the polarity of signaling at GABAA synapses in the central nervous system. Sectioning hippocampal brain slices increases [Cl-]i in the superficial layers. It is not known whether cutting trauma also increases [Cl-]i in the neocortex and thalamus, and whether the effects of trauma change during development. We used Cl- imaging to study the [Cl-]i vs. the distance from the cut surface in acute thalamocortical slices from mice at developmental ages ranging from post-natal day 5 (P5) to P20. We demonstrate: 1) [Cl-]i is higher in the most superficial areas in both neocortical and thalamic brain slices at all ages tested and, 2) there is a developmental decrease in [Cl-]i that is independent of acute trauma caused by brain slicing. We conclude that [Cl-]i has a developmental progression during P5-20 in both the neocortex and thalamus. However, in both brain regions and during development the neurons closest to the slicing trauma have an elevated [Cl-]i.

Syndrome of severe pain associated with a continuous bumetanide infusion

Syndrome of severe pain associated with a continuous bumetanide infusion

Comment on "Local impermeant anions establish the neuronal chloride concentration".

Glykys et al. (Reports, 7 February 2014, p. 670) conclude that, rather than ion transporters, "local impermeant anions establish the neuronal chloride concentration" and thereby determine "the magnitude and direction of GABAAR currents at individual synapses." If this were possible, perpetual ion-motion machines could be constructed. The authors' conclusions conflict with basic thermodynamic principles.

Response to comments on "Local impermeant anions establish the neuronal chloride concentration".

We appreciate the interest in our paper and the opportunity to clarify theoretical and technical aspects describing the influence of Donnan equilibria on neuronal chloride ion (Cl(-)) distributions.

Comment on "Local impermeant anions establish the neuronal chloride concentration".

Glykys et al. (Reports, 7 February 2014, p. 670) proposed that cytoplasmic impermeant anions and polyanionic extracellular matrix glycoproteins establish the local neuronal intracellular chloride concentration, [Cl(-)]i, and thereby the polarity of γ-aminobutyric acid type A (GABAA) receptor signaling. The experimental procedures and results in this study are insufficient to support these conclusions. Contradictory results previously published by these authors and other laboratories are not referred to.

Local Impermeant Anions Establish the Neuronal Chloride Concentration

Neuronal intracellular chloride concentration [Cl(-)](i) is an important determinant of γ-aminobutyric acid type A (GABA(A)) receptor (GABA(A)R)-mediated inhibition and cytoplasmic volume regulation. Equilibrative cation-chloride cotransporters (CCCs) move Cl(-) across the membrane, but accumulating evidence suggests factors other than the bulk concentrations of transported ions determine [Cl(-)](i). Measurement of [Cl(-)](i) in murine brain slice preparations expressing the transgenic fluorophore Clomeleon demonstrated that cytoplasmic impermeant anions ([A](i)) and polyanionic extracellular matrix glycoproteins ([A](o)) constrain the local [Cl(-)]. CCC inhibition had modest effects on [Cl(-)](i) and neuronal volume, but substantial changes were produced by alterations of the balance between [A](i) and [A](o). Therefore, CCCs are important elements of Cl(-) homeostasis, but local impermeant anions determine the homeostatic set point for [Cl(-)], and hence, neuronal volume and the polarity of local GABA(A)R signaling.

Abnormal chloride homeostasis in the substancia nigra pars reticulata contributes to locomotor deficiency in a model of acute liver injury.

BackgroundAltered chloride homeostasis has been thought to be a risk factor for several brain disorders, while less attention has been paid to its role in liver disease. We aimed to analyze the involvement and possible mechanisms of altered chloride homeostasis of GABAergic neurons within the substantia nigra pars reticulata (SNr) in the motor deficit observed in a model of encephalopathy caused by acute liver failure, by using glutamic acid decarboxylase 67 - green fluorescent protein knock-in transgenic mice.MethodsAlterations in intracellular chloride concentration in GABAergic neurons within the SNr and changes in the expression of two dominant chloride homeostasis-regulating genes, KCC2 and NKCC1, were evaluated in mice with hypolocomotion due to hepatic encephalopathy (HE). The effects of pharmacological blockade and/or activation of KCC2 and NKCC1 functions with their specific inhibitors and/or activators on the motor activity were assessed.ResultsIn our mouse model of acute liver injury, chloride imaging indicated an increase in local intracellular chloride concentration in SNr GABAergic neurons. In addition, the mRNA and protein levels of KCC2 were reduced, particularly on neuronal cell membranes; in contrast, NKCC1 expression remained unaffected. Furthermore, blockage of KCC2 reduced motor activity in the normal mice and led to a further deteriorated hypolocomotion in HE mice. Blockade of NKCC1 was not able to normalize motor activity in mice with liver failure.ConclusionOur data suggest that altered chloride homeostasis is likely involved in the pathophysiology of hypolocomotion following HE. Drugs aimed at restoring normal chloride homeostasis would be a potential treatment for hepatic failure.

Regional differences in the expression of K+–Cl− 2 cotransporter in the developing rat cortex

The type 2 potassium–chloride cotransporter (KCC2) is the main regulator of intracellular chloride concentration in CNS neurons, and plays a crucial role in spine development that is independent of its ion cotransport function. The expression pattern of KCC2 is upregulated during postnatal development showing area and layer-specific differences in distinct brain areas. We examined the regional and ultrastructural localisation of KCC2 in various areas of developing neocortex and paleocortex during the first two postnatal weeks. Light-microscopy examination revealed diffuse neuropil and discrete funnel-shaped dendritic labelling in the piriform and entorhinal cortices at birth. Subsequently, during the beginning of the first postnatal week, diffuse KCC2 labelling gradually started to appear in the superficial layers of the neocortex while the punctate-like labelling of dendrites in the piriform, entorhinal and perirhinal cortices become more pronounced. By the end of the first postnatal week, discrete dendritic expression of KCC2 was visible in all neocortical and paleocortical areas. The expression level did not change during the second postnatal week suggesting that, in contrast to hippocampus, adult pattern of KCC2 in the cortical cells is already established by the end of the first postnatal week. Quantitative electron microscopy examination revealed that in superficial layers of both neo- and paleocortex, the majority of KCC2 signal was plasma membrane associated but the number of transport vesicle-associated immunosignal increased with development. In deep layers, KCC2 immunolabeling was evenly distributed in plasma membrane and transport vesicles showing no obvious change with maturation. The number of KCC2 immunogold particles increased in dendritic spines with no association with synapses. This observation points to the dual role of KCC2 in spine genesis and ion cotransport.

Immunolocalization of cation-chloride cotransporters in the developing and mature spinal cord of opossums, Monodelphis domestica.

Spinal inhibition is required to generate coordinated outputs between antagonistic muscles during locomotion. It relies on low neuronal chloride concentration set by two cation-chloride cotransporters, NKCC1 and KCC2 which, respectively, pumps Cl− in or out of neurons. It is generally accepted that NKCC1 is gradually inactivated during development, while KCC2 is upregulated and activated, resulting in low intracellular [Cl−]. Newborn opossums are very immature but perform rhythmic and alternate movements of the forelimbs to crawl on the mother's belly and attach to a teat. Their hindlimbs are immobile. The alternation of the forelimbs suggests that mechanisms allowing spinal inhibition are present at birth. We studied the anatomical basis of inhibition in the spinal enlargements of postnatal opossums by immunolocalizing NKCC1 and KCC2. In some specimens, motoneurons and sensory afferents were labeled with TRDA prior to immunolabeling. At birth, both NKCC1 and KCC2 are detected in the presumptive gray and white matter of the ventral and the intermediolateral cord of both enlargements, but are sparse in the dorsal horn, where KCC2 is mostly seen on a small bundle of dendrites along primary afferents. KCC2 labeling is bright and has a mesh-like appearance in the gray matter and a radial appearance in the white matter, whereas NKCC1 is pale and diffuse. The subsequent expression of the cotransporters follows general ventrodorsal and mediolateral gradients, with the lumbar segments slightly lagging the cervical segments, until the mature pattern is observed around the 5th week. At all ages studied, KCC2 labeling is strong in the periphery of neurons. NKCC1 labeling decreases and becomes more uniformly distributed in the cells with age. Despite the significant anatomical and motor differences between the forelimbs and the hindlimbs of neonatal opossums, the maturation of KCC2 and NKCC1 is quite similar in both enlargements.

Lack of the Sodium-Driven Chloride Bicarbonate Exchanger NCBE Impairs Visual Function in the Mouse Retina

Regulation of ion and pH homeostasis is essential for normal neuronal function. The sodium-driven chloride bicarbonate exchanger NCBE (Slc4a10), a member of the SLC4 family of bicarbonate transporters, uses the transmembrane gradient of sodium to drive cellular net uptake of bicarbonate and to extrude chloride, thereby modulating both intracellular pH (pHi) and chloride concentration ([Cl−]i) in neurons. Here we show that NCBE is strongly expressed in the retina. As GABAA receptors conduct both chloride and bicarbonate, we hypothesized that NCBE may be relevant for GABAergic transmission in the retina. Importantly, we found a differential expression of NCBE in bipolar cells: whereas NCBE was expressed on ON and OFF bipolar cell axon terminals, it only localized to dendrites of OFF bipolar cells. On these compartments, NCBE colocalized with the main neuronal chloride extruder KCC2, which renders GABA hyperpolarizing. NCBE was also expressed in starburst amacrine cells, but was absent from neurons known to depolarize in response to GABA, like horizontal cells. Mice lacking NCBE showed decreased visual acuity and contrast sensitivity in behavioral experiments and smaller b-wave amplitudes and longer latencies in electroretinograms. Ganglion cells from NCBE-deficient mice also showed altered temporal response properties. In summary, our data suggest that NCBE may serve to maintain intracellular chloride and bicarbonate concentration in retinal neurons. Consequently, lack of NCBE in the retina may result in changes in pHi regulation and chloride-dependent inhibition, leading to altered signal transmission and impaired visual function.

Transient increase in neuronal chloride concentration by neuroactive aminoacids released from glioma cells

Neuronal chloride concentration ([Cl−]i) is known to be dynamically modulated and alterations in Cl− homeostasis may occur in the brain at physiological and pathological conditions, being also likely involved in glioma-related seizures. However, the mechanism leading to changes in neuronal [Cl−]i during glioma invasion are still unclear. To characterize the potential effect of glioma released soluble factors on neuronal [Cl−]i, we used genetically encoded CFP/YFP-based ratiometric Cl-(apical) Sensor transiently expressed in cultured hippocampal neurons. Exposition of neurons to glioma conditioned medium (GCM) caused rapid and transient elevation of [Cl−]i, resulting in the increase of fluorescence ratio, which was strongly reduced by blockers of ionotropic glutamate receptors APV and NBQX. Furthermore, in HEK cells expressing GluR1-AMPA receptors, GCM activated ionic currents with efficacy similar to those caused by glutamate, supporting the notion that GCM contains glutamate or glutamatergic agonists, which cause neuronal depolarization, activation of NMDA and AMPA/KA receptors leading to elevation of [Cl−]i. Chromatographic analysis of the GCM showed that it contained several aminoacids, including glutamate, whose release from glioma cells did not occur via the most common glial mechanisms of transport, or in response to hypoosmotic stress. GCM also contained glycine, whose action contrasted the glutamate effect. Indeed, strychnine application significantly increased GCM-induced depolarization and [Cl−]i rise. GCM-evoked [Cl−]i elevation was not inhibited by antagonists of Cl− transporters and significantly reduced in the presence of anion channels blocker NPPB, suggesting that Cl− selective channels are a major route for GCM-induced Cl− influx. Altogether, these data show that glioma released aminoacids may dynamically alter Cl− equilibrium in surrounding neurons, deeply interfering with their inhibitory balance, likely leading to physiological and pathological consequences.

Opposite temperature effect on transport activity of KCC2/KCC4 and N(K)CCs in HEK-293 cells

BackgroundCation chloride cotransporters play essential roles in many physiological processes such as volume regulation, transepithelial salt transport and setting the intracellular chloride concentration in neurons. They consist mainly of the inward transporters NCC, NKCC1, and NKCC2, and the outward transporters KCC1 to KCC4. To gain insight into regulatory and structure-function relationships, precise determination of their activity is required. Frequently, these analyses are performed in HEK-293 cells. Recently the activity of the inward transporters NKCC1 and NCC was shown to increase with temperature in these cells. However, the temperature effect on KCCs remains largely unknown.FindingsHere, we determined the temperature effect on KCC2 and KCC4 transport activity in HEK-293 cells. Both transporters demonstrated significantly higher transport activity (2.5 fold for KCC2 and 3.3 fold for KCC4) after pre-incubation at room temperature compared to 37°C.ConclusionsThese data identify a reciprocal temperature dependence of cation chloride inward and outward cotransporters in HEK-293 cells. Thus, lower temperature should be used for functional characterization of KCC2 and KCC4 and higher temperatures for N(K)CCs in heterologous mammalian expression systems. Furthermore, if this reciprocal effect also applies to neurons, the action of inhibitory neurotransmitters might be more affected by changes in temperature than previously thought.

Novel Repression ofKcc2Transcription by REST–RE-1 Controls Developmental Switch in Neuronal Chloride

Transcriptional upregulation of Kcc2b, the gene variant encoding the major isoform of the KCC2 chloride transporter, underlies a rapid perinatal decrease in intraneuronal chloride concentration (chloride shift), which is necessary for GABA to act inhibitory. Here we identify a novel repressor element-1 (RE-1) site in the 5' regulatory region of Kcc2b. In primary cortical neurons, which recapitulate the chloride shift in culture, the novel upstream RE-1 together with a known intronic RE-1 site function in concerted interaction to suppress Kcc2b transcription. With critical relevance for the chloride shift, only in the presence of the dual RE-1 site could inhibition of REST upregulate Kcc2b transcription. For this, we confirmed increased KCC2 protein expression and decreased intraneuronal chloride. Kcc2b developmental upregulation was potentiated by BDNF application, which was fully dependent on the presence of dual RE-1. In addition, the developmental chloride shift and GABA switch, from excitatory to inhibitory action, was accelerated by REST inhibition and slowed by REST overexpression. These results identify the REST-dual RE-1 interaction as a novel mechanism of transcriptional Kcc2b upregulation that significantly contributes to the ontogenetic shift in chloride concentration and GABA action in cortical neurons, which is fundamental for brain function in health and disease. Thus, we present here a new logic for the perinatal chloride shift, which is critical for establishment of GABAergic cortical inhibitory neurotransmission.