Bumetanide induces post-traumatic microglia-interneuron contact to promote neurogenesis and recovery.

Although the Na-K-Cl cotransporter (NKCC1) inhibitor bumetanide has prominent positive effects on the pathophysiology of many neurological disorders, the mechanism of action is obscure. Attention paid to elucidating the role of Nkcc1 has mainly been focused on neurons, but recent single cell mRNA sequencing analysis has demonstrated that the major cellular populations expressing NKCC1 in the cortex are non-neuronal. We used a combination of conditional transgenic animals, in vivo electrophysiology, two-photon imaging, cognitive behavioural tests and flow cytometry to investigate the role of Nkcc1 inhibition by bumetanide in a mouse model of controlled cortical impact (CCI). Here, we found that bumetanide rescues parvalbumin-positive interneurons by increasing interneuron-microglia contacts shortly after injury. The longitudinal phenotypic changes in microglia were significantly modified by bumetanide, including an increase in the expression of microglial-derived BDNF. These effects were accompanied by the prevention of CCI-induced decrease in hippocampal neurogenesis. Treatment with bumetanide during the first week post-CCI resulted in significant recovery of working and episodic memory as well as changes in theta band oscillations 1 month later. These results disclose a novel mechanism for the neuroprotective action of bumetanide mediated by an acceleration of microglial activation dynamics that leads to an increase in parvalbumin interneuron survival following CCI, possibly resulting from increased microglial BDNF expression and contact with interneurons. Salvage of interneurons may normalize ambient GABA, resulting in the preservation of adult neurogenesis processes as well as contributing to bumetanide-mediated improvement of cognitive performance.

High-intensity training with short and long intervals regulate cortical neurotrophic factors, apoptosis markers and chloride homeostasis in rats with stroke.

BACKGROUND/PURPOSE: The optimal endurance exercise parameters remain to be defined to potentiate long-term functional recovery after stroke. We aim to assess the effects of individualized high-intensity interval training (HIIT) with either long or short intervals on neurotrophic factors and their receptors, apoptosis markers and the two-main cation-chloride cotransporters in the ipsi- and contralesional cerebral cortices in rats with cerebral ischemia. Endurance performance and sensorimotor functions were also assessed METHODS: Rats with a 2 h transient middle cerebral artery occlusion (tMCAO) performed work-matched HIIT4 (intervals: 4 min) or HIIT1 (intervals: 1 min) on treadmill for 2 weeks. Incremental exercises and sensorimotor tests were performed at day 1 (D1), D8, and D15 after tMCAO. Molecular analyses were achieved in both the paretic and non-paretic triceps brachii muscles and the ipsi- and contralesional cortices at D17 RESULTS: Gains in endurance performance are in a time-dependent manner from the first week of training. This enhancement is supported by the upregulation of metabolic markers in both triceps brachii muscles. Both regimens alter the expression of neurotrophic markers and chloride homeostasis in a specific manner in the ipsi- and contralesional cortices. HIIT acts on apoptosis markers by promoting anti-apoptotic proteins in the ipsilesional cortex CONCLUSION: HIIT regimens seem to be of clinical relevance in the critical period of stroke rehabilitation by strongly improving aerobic performance. Also, the observed cortical changes suggest an influence of HIIT on neuroplasticity in both ipsi- and contralesional hemispheres. Such neurotrophic markers might be considered as biomarkers of functional recovery in individuals with stroke.

Current perspectives on mitochondrial dysfunction in migraine.

Mitochondria are an autonomous organelle that plays a crucial role in the metabolic aspects of a cell. Cortical spreading depression (CSD) and fluctuations in the cerebral blood flow have for long been mechanisms underlying migraine. It is a neurovascular disorder with a unilateral manifestation of disturbing, throbbing and pulsating head pain. Migraine affects 2.6% and 21.7% of the general population and is the major cause of partial disability in the age group 15-49. Higher mutation rates, imbalance in concentration of physiologically relevant molecules and oxidative stress biomarkers have been the main themes of discussion in determining the role of mitochondrial disability in migraine. The correlation of migraine with other disorders like hemiplegic migraine; mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes [MELAS]; tension-type headache (TTH); cyclic vomiting syndrome (CVS), ischaemic stroke; and hypertension has helped in the assessment of the physiological and morphogenetic basis of migraine. Here, we have reviewed the different nuances of mitochondrial dysfunction and migraine. The different mtDNA polymorphisms that can affect the generation and transmission of nerve impulse has been highlighted and supported with research findings. In addition to this, the genetic basis of migraine pathogenesis as a consequence of mutations in nuclear DNA that can, in turn, affect the synthesis of defective mitochondrial proteins is discussed along with a brief overview of epigenetic profile. This review gives an overview of the pathophysiology of migraine and explores mitochondrial dysfunction as a potential underlying mechanism. Also, therapeutic supplements for managing migraine have been discussed at different junctures in this paper.

Time-dependent cortical plasticity during moderate-intensity continuous training versus high-intensity interval training in rats.

The temporal pattern of cortical plasticity induced by high-intensity interval training (HIIT) and moderate-intensity continuous training (MICT) is required to clarify their relative benefits to prevent neurological disorders. The purpose of this study is to define the time-dependent effects of work-matched HIIT and MICT on cortical plasticity, endurance, and sensorimotor performances over an 8-week training period in healthy rats. Adult healthy rats performed incremental exercise tests and sensorimotor tests before and at 2, 4, and 8 weeks of training. In parallel, cortical markers related to neurotrophic, angiogenic, and metabolic activities were assessed. Results indicate that HIIT induced an early and superior endurance improvement compared to MICT. We found significant enhancement of speed associated with lactate threshold (SLT) and maximal speed (Smax) in HIIT animals. MICT promoted an early increase in brain-derived neurotrophic factor and angiogenic/metabolic markers but showed less influence at 8 weeks. HIIT upregulated the insulin-like growth factor-1 (IGF-1) as well as neurotrophic, metabolic/angiogenic markers at 2 and 8 weeks and downregulated the neuronal K-Cl cotransporter KCC2 that regulates GABAA-mediated transmission. HIIT and MICT are effective in a time-dependent manner suggesting a complementary effect that might be useful in physical exercise guidelines for maintaining brain health.

"Janus-Faced" α-Synuclein: Role in Parkinson's Disease.

Parkinson's disease (PD) is a pathological condition characterized by the aggregation and the resultant presence of intraneuronal inclusions termed Lewy bodies (LBs) and Lewy neurites which are mainly composed of fibrillar α-synuclein (α-syn) protein. Pathogenic aggregation of α-syn is identified as the major cause of LBs deposition. Several mutations in α-syn showing varied aggregation kinetics in comparison to the wild type (WT) α-syn are reported in PD (A30P, E46K, H 50Q, G51D, A53E, and A53T). Also, the cell-to-cell spread of pathological α-syn plays a significant role in PD development. Interestingly, it has also been suggested that the pathology of PD may begin in the gastrointestinal tract and spread via the vagus nerve (VN) to brain proposing the gut-brain axis of α-syn pathology in PD. Despite multiple efforts, the behavior and functions of this protein in normal and pathological states (specifically in PD) is far from understood. Furthermore, the etiological factors responsible for triggering aggregation of this protein remain elusive. This review is an attempt to collate and present latest information on α-syn in relation to its structure, biochemistry and biophysics of aggregation in PD. Current advances in therapeutic efforts toward clearing the pathogenic α-syn via autophagy/lysosomal flux are also reviewed and reported.

Is High-Intensity Interval Training Suitable to Promote Neuroplasticity and Cognitive Functions after Stroke?

Stroke-induced cognitive impairments affect the long-term quality of life. High-intensity interval training (HIIT) is now considered a promising strategy to enhance cognitive functions. This review is designed to examine the role of HIIT in promoting neuroplasticity processes and/or cognitive functions after stroke. The various methodological limitations related to the clinical relevance of studies on the exercise recommendations in individuals with stroke are first discussed. Then, the relevance of HIIT in improving neurotrophic factors expression, neurogenesis and synaptic plasticity is debated in both stroke and healthy individuals (humans and rodents). Moreover, HIIT may have a preventive role on stroke severity, as found in rodents. The potential role of HIIT in stroke rehabilitation is reinforced by findings showing its powerful neurogenic effect that might potentiate cognitive benefits induced by cognitive tasks. In addition, the clinical role of neuroplasticity observed in each hemisphere needs to be clarified by coupling more frequently to cellular/molecular measurements and behavioral testing.

Effects of Different High-Intensity Interval Training Regimens on Endurance and Neuroplasticity After Cerebral Ischemia.

BACKGROUND AND PURPOSE: The objective is to compare the effects of high-intensity interval training (HIIT) with long versus short intervals on endurance and motor performance. Their influence on neuroplasticity markers is assessed in the ipsilesional and contralesional cortex and hippocampus since their remodeling could improve functional recovery. METHODS: Rats performed work-matched HIIT4 (long intervals: 4 minutes) or HIIT1 (short intervals: 1 minute) on treadmill for 2 weeks following transient middle cerebral artery occlusion. Forelimb grip strength evaluated motor function while incremental exercise tests measured the endurance performance. Key neuroplasticity markers were assessed by Western blot. RESULTS: Both regimens were effective in enhancing both the speed associated with the lactate threshold and maximal speed at D8 and D15. Neuroplasticity markers were upregulated in the contralesional hemisphere after training contrary to the ipsilesional side. Grip strength completely recovered but is faster with HIIT4. CONCLUSIONS: HIIT with short and long intervals induced early aerobic fitness and grip strength improvements. Our findings revealed that neuroplasticity markers were upregulated in the contralesional cortex and hippocampus to promote functional recovery.

High-intensity interval training is superior to moderate intensity training on aerobic capacity in rats: Impact on hippocampal plasticity markers.

The use of endurance regimens could be improved by defining their respective effectiveness on aerobic fitness and brain health that remains controversial. We aimed at comparing work-matched high-intensity interval training (HIIT) with moderate-intensity continuous training (MICT) on aerobic performance and muscular plasticity markers in healthy rats. Cognitive functions and brain plasticity markers were also investigated following the 8-week training. Rats performed the incremental exercise test and behavioural tests before and after training at day 1 (D1), D15, D29 and D57. Key cerebral markers were assessed by Western blot and quantitative polymerase chain reaction to provide information on brain function related to angiogenesis, aerobic metabolism and neurotrophin activity at D59. Muscular protein levels involved in angiogenesis and aerobic metabolism were measured in both triceps brachii and soleus muscles. HIIT induced superior improvement of aerobic fitness compared to MICT, as indicated by enhancement of speed associated with lactate threshold (SLT) and maximal speed (Smax). In the triceps brachii muscle, markers of angiogenesis and aerobic activity were upregulated as well as myokines involved in neuroplasticity. Moreover, levels of key brain plasticity markers increased in the hippocampus after 8 weeks of HIIT, without improving cognitive functions. These findings might contribute to define physical exercise guidelines for maintaining brain health by highlighting the promising role of HIIT when using SLT for distinguishing low running speed from high running speed. Further studies are required to confirm these brain effects by exploring synaptic plasticity and neurogenesis mechanisms when exercise intensity is standardized and individualized.

Novel carbon film induces precocious calcium oscillation to promote neuronal cell maturation.

Different types of carbon materials are biocompatible with neural cells and can promote maturation. The mechanism of this effect is not clear. Here we have tested the capacity of a carbon material composed of amorphous sp3 carbon backbone, embedded with a percolating network of sp2 carbon domains to sustain neuronal cultures. We found that cortical neurons survive and develop faster on this novel carbon material. After 3 days in culture, there is a precocious increase in the frequency of neuronal activity and in the expression of maturation marker KCC2 on carbon films as compared to a commonly used glass surface. Accelerated development is accompanied by a dramatic increase in neuronal dendrite arborization. The mechanism for the precocious maturation involves the activation of intracellular calcium oscillations by the carbon material already after 1 day in culture. Carbon-induced oscillations are independent of network activity and reflect intrinsic spontaneous activation of developing neurons. Thus, these results reveal a novel mechanism for carbon material-induced neuronal survival and maturation.

Bumetanide Prevents Brain Trauma-Induced Depressive-Like Behavior.

Brain trauma triggers a cascade of deleterious events leading to enhanced incidence of drug resistant epilepsies, depression, and cognitive dysfunctions. The underlying mechanisms leading to these alterations are poorly understood and treatment that attenuates those sequels are not available. Using controlled-cortical impact as an experimental model of brain trauma in adult mice, we found a strong suppressive effect of the sodium-potassium-chloride importer (NKCC1) specific antagonist bumetanide on the appearance of depressive-like behavior. We demonstrate that this alteration in behavior is associated with an impairment of post-traumatic secondary neurogenesis within the dentate gyrus of the hippocampus. The mechanism mediating the effect of bumetanide involves early transient changes in the expression of chloride regulatory proteins and qualitative changes in GABA(A) mediated transmission from hyperpolarizing to depolarizing after brain trauma. This work opens new perspectives in the early treatment of human post-traumatic induced depression. Our results strongly suggest that bumetanide might constitute an efficient prophylactic treatment to reduce neurological and psychiatric consequences of brain trauma.

Altered interplay between endoplasmic reticulum and mitochondria in Charcot-Marie-Tooth type 2A neuropathy.

Mutations in the MFN2 gene encoding Mitofusin 2 lead to the development of Charcot-Marie-Tooth type 2A (CMT2A), a dominant axonal form of peripheral neuropathy. Mitofusin 2 is localized at both the outer membrane of mitochondria and the endoplasmic reticulum and is particularly enriched at specialized contact regions known as mitochondria-associated membranes (MAM). We observed that expression of MFN2R94Q induces distal axonal degeneration in the absence of overt neuronal death. The presence of mutant protein leads to reduction in endoplasmic reticulum and mitochondria contacts in CMT2A patient-derived fibroblasts, in primary neurons and in vivo, in motoneurons of a mouse model of CMT2A. These changes are concomitant with endoplasmic reticulum stress, calcium handling defects, and changes in the geometry and axonal transport of mitochondria. Importantly, pharmacological treatments reinforcing endoplasmic reticulum-mitochondria cross-talk, or reducing endoplasmic reticulum stress, restore the mitochondria morphology and prevent axonal degeneration. These results highlight defects in MAM as a cellular mechanism contributing to CMT2A pathology mediated by mutated MFN2.

Gamma-aminobutyric acidergic transmission underlies interictal epileptogenicity in pediatric focal cortical dysplasia.

OBJECTIVE: Dysregulation of γ-aminobutyric acidergic (GABAergic) transmission has been reported in lesional acquired epilepsies (gliomas, hippocampal sclerosis). We investigated its involvement in a developmental disorder, human focal cortical dysplasia (FCD), focusing on chloride regulation driving GABAergic signals. METHODS: In vitro recordings of 47 human cortical acute slices from 11 pediatric patients who received operations for FCD were performed on multielectrode arrays. GABAergic receptors and chloride regulators were pharmacologically modulated. Immunostaining for chloride cotransporter KCC2 and interneurons were performed on recorded slices to correlate electrophysiology and expression patterns. RESULTS: FCD slices retain intrinsic epileptogenicity. Thirty-six of 47 slices displayed spontaneous interictal discharges, along with a pattern specific to the histological subtypes. Ictal discharges were induced in proepileptic conditions in 6 of 8 slices in the areas generating spontaneous interictal discharges, with a transition to seizure involving the emergence of preictal discharges. Interictal discharges were sustained by GABAergic signaling, as a GABAA receptor blocker stopped them in 2 of 3 slices. Blockade of NKCC1 Cl- cotransporters further controlled interictal discharges in 9 of 12 cases, revealing a Cl- dysregulation affecting actions of GABA. Immunohistochemistry highlighted decreased expression and changes in KCC2 subcellular localization and a decrease in the number of GAD67-positive interneurons in regions generating interictal discharges. INTERPRETATION: Altered chloride cotransporter expression and changes in interneuron density in FCD may lead to paradoxical depolarization of pyramidal cells. Spontaneous interictal discharges are consequently mediated by GABAergic signals, and targeting chloride regulation in neurons may be considered for the development of new antiepileptic drugs. Ann Neurol 2019; 1-14 ANN NEUROL 2019;85:204-217.

Degradable and Injectable Hydrogel for Drug Delivery in Soft Tissues.

Injectable hydrogels are promising platforms for tissue engineering and local drug delivery as they allow minimal invasiveness. We have here developed an injectable and biodegradable hydrogel based on an amphiphilic PNIPAAm- b-PLA- b-PEG- b-PLA- b-PNIPAAm pentablock copolymer synthesized by ring-opening polymerization/nitroxide-mediated polymerization (ROP/NMP) combination. The hydrogel formation at around 30 °C was demonstrated to be mediated by intermicellar bridging through the PEG central block. Such a result was particularly highlighted by the inability of a PEG- b-PLA- b-PNIPAAm triblock analog of the same composition to gelify. The hydrogels degraded through hydrolysis of the PLA esters until complete mass loss due to the diffusion of the recovered PEG and PNIPAAm/micelle based residues in the solution. Interestingly, hydrophobic molecules such as riluzole (neuroprotective drug) or cyanine 5.5 (imaging probe) could be easily loaded in the hydrogels' micelle cores by mixing them with the copolymer solution at room temperature. Drug release was correlated to polymer mass loss. The hydrogel was shown to be cytocompatible (neuronal cells, in vitro) and injectable through a small-gauge needle (in vivo in rats). Thus, this hydrogel platform displays highly attractive features for use in brain/soft tissue engineering as well as in drug delivery.

Cerebral Ischemia Changed the Effect of Metabosensitive Muscle Afferents on Somatic Reflex Without Affecting Thalamic Activity.

The purpose of the present study was to examine the contribution of group III and IV metabosensitive afferents at spinal and supraspinal levels in rats subjected to middle cerebral artery occlusion (MCAO) with reperfusion during the acute phase. Animals were randomized in Control (n = 23), SHAM (n = 18), MCAO-D1 (n = 10), and MCAO-D7 (n = 20) groups. Rats performed the Electrical Von Frey and the Adhesive removal tests before the surgery and at day 1 (D1), D3, and D7 after MCAO. Animals were subjected to electrophysiological recordings including the responses of group III/IV metabosensitive afferents to combinations of chemical activators and the triceps brachii somatic reflex activity at D1 or D7. The response of ventral posterolateral (VPL) thalamic nuclei was also recorded after group III/IV afferent activation. Histological measurements were performed to assess the infarct size and to confirm the location of the recording electrodes into the VPL. Behavioral results indicated that MCAO induced disorders of both mechanical sensibility and motor coordination of paretic forepaw during 7 days. Moreover, injured animals exhibited an absence of somatic reflex inhibition from the group III/IV afferents at D1, without affecting the response of both these afferents and the VPL. Finally, the regulation of the central motor drive by group III/IV afferents was modified at spinal level during the acute phase of cerebral ischemia and it might contribute to the observed behavioral disturbances.

Effects of High- Versus Moderate-Intensity Training on Neuroplasticity and Functional Recovery After Focal Ischemia.

BACKGROUND AND PURPOSE: This study was designed to compare the effects of high-intensity interval training (HIT) and moderate-intensity aerobic training (MOD) on functional recovery and cerebral plasticity during the first 2 weeks after cerebral ischemia. METHODS: Rats were randomized as follows: control (n=15), SHAM (n=9), middle cerebral artery occlusion (n=13), middle cerebral artery occlusion at day 1 (n=7), MOD (n=13), and HIT (n=13). Incremental tests were performed at day 1 (D1) and 14 (D14) to identify the running speed associated with the lactate threshold (SLT) and the maximal speed (Smax). Functional tests were performed at D1, D7, and D14. Microglia form, cytokines, p75NTR (pan-neurotrophin receptor p75), potassium-chloride cotransporter type 2, and sodium-potassium-chloride cotransporter type 1 expression were made at D15. RESULTS: HIT was more effective to improve the endurance performance than MOD and induced a fast recovery of the impaired forelimb grip force. The ionized calcium binding adaptor molecule 1 (Iba-1)-positive cells with amoeboid form and the pro- and anti-inflammatory cytokine expression were lower in HIT group, mainly in the ipsilesional hemisphere. A p75NTR overexpression is observed on the ipsilesional side together with a restored sodium-potassium-chloride cotransporter type 1/potassium-chloride cotransporter type 2 ratio on the contralesional side. CONCLUSIONS: Low-volume HIT based on lactate threshold seems to be more effective after cerebral ischemia than work-matched MOD to improve aerobic fitness and grip strength and might promote cerebral plasticity.

A Novel View on the Role of Intracellular Tails in Surface Delivery of the Potassium-Chloride Cotransporter KCC2.

A plethora of neurological disorders are associated with alterations in the expression and localization of potassium-chloride cotransporter type 2 (KCC2), making KCC2 a critical player in neuronal function and an attractive target for therapeutic treatment. The activity of KCC2 is determined primarily by the rates of its surface insertion and internalization. Currently the domains of KCC2 dictating its trafficking and endocytosis are unknown. Here, using live-cell immunolabeling and biotinylation of KCC2 proteins expressed in murine neuroblastoma N2a cells, human embryonic kidney 293 cells, or primary cultures of rat hippocampal neurons, we identified a novel role for the intracellular N and C termini in differentially regulating KCC2 surface expression. We report that the N terminus is required for KCC2 insertion into the plasma membrane, whereas the C terminus is critical for the membrane stability of KCC2. Our results provide novel insights into the structure-function role of specific KCC2 domains and open perspectives in exploring structural organization of this protein.

Depolarizing γ-aminobutyric acid contributes to glutamatergic network rewiring in epilepsy.

OBJECTIVE: Rewiring of excitatory glutamatergic neuronal circuits is a major abnormality in epilepsy. Besides the rewiring of excitatory circuits, an abnormal depolarizing γ-aminobutyric acidergic (GABAergic) drive has been hypothesized to participate in the epileptogenic processes. However, a remaining clinically relevant question is whether early post-status epilepticus (SE) evoked chloride dysregulation is important for the remodeling of aberrant glutamatergic neuronal circuits. METHODS: Osmotic minipumps were used to infuse intracerebrally a specific inhibitor of depolarizing GABAergic transmission as well as a functionally blocking antibody toward the pan-neurotrophin receptor p75 (p75NTR ). The compounds were infused between 2 and 5 days after pilocarpine-induced SE. Immunohistochemistry for NKCC1, KCC2, and ectopic recurrent mossy fiber (rMF) sprouting as well as telemetric electroencephalographic and electrophysiological recordings were performed at day 5 and 2 months post-SE. RESULTS: Blockade of NKCC1 after SE with the specific inhibitor bumetanide restored NKCC1 and KCC2 expression, normalized chloride homeostasis, and significantly reduced the glutamatergic rMF sprouting within the dentate gyrus. This mechanism partially involves p75NTR signaling, as bumetanide application reduced SE-induced p75NTR expression and functional blockade of p75NTR decreased rMF sprouting. The early transient (3 days) post-SE infusion of bumetanide reduced rMF sprouting and recurrent seizures in the chronic epileptic phase. INTERPRETATION: Our findings show that early post-SE abnormal depolarizing GABA and p75NTR signaling fosters a long-lasting rearrangement of glutamatergic network that contributes to the epileptogenic process. This finding defines promising and novel targets to constrain reactive glutamatergic network rewiring in adult epilepsy. Ann Neurol 2017;81:251-265.

In vivo Calcium Imaging of Evoked Calcium Waves in the Embryonic Cortex.

The dynamics of intracellular calcium fluxes are instrumental in the proliferation, differentiation, and migration of neuronal cells. Knowledge thus far of the relationship between these calcium changes and physiological processes in the developing brain has derived principally from ex vivo and in vitro experiments. Here, we present a new method to image intracellular calcium flux in the cerebral cortex of live rodent embryos, whilst attached to the dam through the umbilical cord. Using this approach we demonstrate induction of calcium waves by laser stimulation. These waves are sensitive to ATP-receptor blockade and are significantly increased by pharmacological facilitation of intracellular-calcium release. This approach is the closest to physiological conditions yet achieved for imaging of calcium in the embryonic brain and as such opens new avenues for the study of prenatal brain development. Furthermore, the developed method could open the possibilities of preclinical translational studies in embryos particularly important for developmentally related diseases such as schizophrenia and autism.

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.

Current view on the functional regulation of the neuronal K(+)-Cl(-) cotransporter KCC2.

In the mammalian central nervous system (CNS), the inhibitory strength of chloride (Cl(-))-permeable GABAA and glycine receptors (GABAAR and GlyR) depends on the intracellular Cl(-) concentration ([Cl(-)]i). Lowering [Cl(-)]i enhances inhibition, whereas raising [Cl(-)]i facilitates neuronal activity. A neuron's basal level of [Cl(-)]i, as well as its Cl(-) extrusion capacity, is critically dependent on the activity of the electroneutral K(+)-Cl(-) cotransporter KCC2, a member of the SLC12 cation-Cl(-) cotransporter (CCC) family. KCC2 deficiency compromises neuronal migration, formation and the maturation of GABAergic and glutamatergic synaptic connections, and results in network hyperexcitability and seizure activity. Several neurological disorders including multiple epilepsy subtypes, neuropathic pain, and schizophrenia, as well as various insults such as trauma and ischemia, are associated with significant decreases in the Cl(-) extrusion capacity of KCC2 that result in increases of [Cl(-)]i and the subsequent hyperexcitability of neuronal networks. Accordingly, identifying the key upstream molecular mediators governing the functional regulation of KCC2, and modifying these signaling pathways with small molecules, might constitute a novel neurotherapeutic strategy for multiple diseases. Here, we discuss recent advances in the understanding of the mechanisms regulating KCC2 activity, and of the role these mechanisms play in neuronal Cl(-) homeostasis and GABAergic neurotransmission. As KCC2 mediates electroneutral transport, the experimental recording of its activity constitutes an important research challenge; we therefore also, provide an overview of the different methodological approaches utilized to monitor function of KCC2 in both physiological and pathological conditions.