Astrocyte-derived lactate in stress disorders.

Stress disorders are psychiatric disorders arising following stressful or traumatic events. They could deleteriously affect an individual's health because they often co-occur with mental illnesses. Considerable attention has been focused on neurons when considering the neurobiology of stress disorders. However, like other mental health conditions, recent studies have highlighted the importance of astrocytes in the pathophysiology of stress-related disorders. In addition to their structural and homeostatic support role, astrocytes actively serve several functions in regulating synaptic transmission and plasticity, protecting neurons from toxic compounds, and providing metabolic support for neurons. The astrocyte-neuron lactate shuttle model sets forth the importance of astrocytes in providing lactate for the metabolic supply of neurons under intense activity. Lactate also plays a role as a signaling molecule and has been recently studied regarding its antidepressant activity. This review discusses the involvement of astrocytes and brain energy metabolism in stress and further reflects on the importance of lactate as an energy supply in the brain and its emerging antidepressant role in stress-related disorders.

Photo-Chemical Stimulation of Neurons with Organic Semiconductors.

Recent advances in light-responsive materials enabled the development of devices that can wirelessly activate tissue with light. Here it is shown that solution-processed organic heterojunctions can stimulate the activity of primary neurons at low intensities of light via photochemical reactions. The p-type semiconducting polymer PDCBT and the n-type semiconducting small molecule ITIC (a non-fullerene acceptor) are coated on glass supports, forming a p-n junction with high photosensitivity. Patch clamp measurements show that low-intensity white light is converted into a cue that triggers action potentials in primary cortical neurons. The study shows that neat organic semiconducting p-n bilayers can exchange photogenerated charges with oxygen and other chemical compounds in cell culture conditions. Through several controlled experimental conditions, photo-capacitive, photo-thermal, and direct hydrogen peroxide effects on neural function are excluded, with photochemical delivery being the possible mechanism. The profound advantages of low-intensity photo-chemical intervention with neuron electrophysiology pave the way for developing wireless light-based therapy based on emerging organic semiconductors.

Gut microbiota modulates expression of genes involved in the astrocyte-neuron lactate shuttle in the hippocampus.

The gut microbiota modulates brain physiology, development, and behavior and has been implicated as a key regulator in several central nervous system disorders. Its effect on the metabolic coupling between neurons and astrocytes has not been studied to date, even though this is an important component of brain energy metabolism and physiology and it is perturbed in neurodegenerative and cognitive disorders. In this study, we have investigated the mRNA expression of 6 genes encoding proteins implicated in the astrocyte-neuron lactate shuttle (Atp1a2, Ldha, Ldhb, Mct1, Gys1, Pfkfb3), in relation to different gut microbiota manipulations, in the mouse brain hippocampus, a region with critical functions in cognition and behavior. We have discovered that Atp1a2 and Pfkfb3, encoding the ATPase, Na+/K+ transporting, alpha 2 sub-unit, respectively and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, two genes predominantly expressed in astrocytes, were upregulated in the hippocampus after microbial colonization of germ-free mice for 24 h, compared with conventionally raised mice. Pfkfb3 was also upregulated in germ-free mice compared with conventionally raised mice, while an increase in Atp1a2 expression in germ-free mice was confirmed only at the protein level by Western blot. In a separate cohort of mice, Atp1a2 and Pfkfb3 mRNA expression was upregulated in the hippocampus following 6-week dietary supplementation with prebiotics (fructo- and galacto-oligosaccharides) in an animal model of chronic psychosocial stress. To our knowledge, these findings are the first to report an influence of the gut microbiota and prebiotics on mRNA expression of genes implicated in the metabolic coupling between neurons and astrocytes.

Benchmarking the Performance of Electropolymerized Poly(3,4-ethylenedioxythiophene) Electrodes for Neural Interfacing.

The development of electronics adept at interfacing with the nervous system is an ever-growing effort, leading to discoveries in fundamental neuroscience applied in clinical setting. Highly capacitive and electrochemically stable electronic materials are paramount for these advances. A systematic study is presented where copolymers based on 3,4-ethylenedioxythiophene (EDOT) and its hydroxyl-terminated counterpart (EDOTOH) are electropolymerized in an aqueous solution in the presence of various counter anions and additives. Amongst the conducting materials developed, the copolymer p(EDOT-ran-EDOTOH) doped with perchlorate in the presence of ethylene glycol shows high specific capacitance (105 F g-1 ), and capacitance retention (85%) over 1000 galvanostatic charge-discharge cycles. A microelectrode array-based on this material is fabricated and primary cortical neurons are cultured therein for several days. The microelectrodes electrically stimulate targeted neuronal networks and record their activity with high signal-to-noise ratio. The stability of charge injection capacity of the material is validated via long-term pulsing experiments. While providing insights on the effect of additives and dopants on the electrochemical performance and operational stability of electropolymerized conducting polymers, this study highlights the importance of high capacitance accompanied with stability to achieve high performance electrodes for biological interfacing.

Lactate and pyruvate promote oxidative stress resistance through hormetic ROS signaling.

L-lactate was long considered a glycolytic by-product but is now being recognized as a signaling molecule involved in cell survival. In this manuscript, we report the role of L-lactate in stress resistance and cell survival mechanisms using neuroblastoma cells (SH-SY5Y) as well as the C. elegans model. We observed that L-lactate promotes cellular defense mechanisms, including Unfolded Protein Response (UPR) and activation of nuclear factor erythroid 2-related factor 2 (NRF2), by promoting a mild Reactive Oxygen Species (ROS) burst. This increase in ROS triggers antioxidant defenses and pro-survival pathways, such as PI3K/AKT and Endoplasmic Reticulum (ER) chaperones. These results contribute to the understanding of the molecular mechanisms involved in beneficial effects of L-lactate, involving mild ROS burst, leading to activation of unfolded protein responses and detoxification mechanisms. We present evidence that this hormetic mechanism induced by L-lactate protects against oxidative stress in vitro and in vivo. This work contributes to the identification of molecular mechanisms, which could serve as targets for future therapeutic approaches for cell protection and aging-related disorders.

Gangliosides: Treatment Avenues in Neurodegenerative Disease.

Gangliosides are cell membrane components, most abundantly in the central nervous system (CNS) where they exert among others neuro-protective and -restorative functions. Clinical development of ganglioside replacement therapy for several neurodegenerative diseases was impeded by the BSE crisis in Europe during the 1990s. Nowadays, gangliosides are produced bovine-free and new pre-clinical and clinical data justify a reevaluation of their therapeutic potential in neurodegenerative diseases. Clinical experience is greatest with monosialo-tetrahexosyl-ganglioside (GM1) in the treatment of stroke. Fourteen randomized controlled trials (RCTs) in overall >2,000 patients revealed no difference in survival, but consistently superior neurological outcomes vs. placebo. GM1 was shown to attenuate ischemic neuronal injuries in diabetes patients by suppression of ERK1/2 phosphorylation and reduction of stress to the endoplasmic reticulum. There is level-I evidence from 5 RCTs of a significantly faster recovery with GM1 vs. placebo in patients with acute and chronic spinal cord injury (SCI), disturbance of consciousness after subarachnoid hemorrhage, or craniocerebral injuries due to closed head trauma. In Parkinson's disease (PD), two RCTs provided evidence of GM1 to be superior to placebo in improving motor symptoms and long-term to result in a slower than expected symptom progression, suggesting disease-modifying potential. In Alzheimer's disease (AD), the role of gangliosides has been controversial, with some studies suggesting a "seeding" role for GM1 in amyloid ß polymerization into toxic forms, and others more recently suggesting a rather protective role in vivo. In Huntington's disease (HD), no clinical trials have been conducted yet. However, low GM1 levels observed in HD cells were shown to increase cell susceptibility to apoptosis. Accordingly, treatment with GM1 increased survival of HD cells in vitro and consistently ameliorated pathological phenotypes in several murine HD models, with effects seen at molecular, cellular, and behavioral level. Given that in none of the clinical trials using GM1 any clinically relevant safety issues have occurred to date, current data supports expanding GM1 clinical research, particularly to conditions with high, unmet medical need.

L-Lactate Regulates the Expression of Synaptic Plasticity and Neuroprotection Genes in Cortical Neurons: A Transcriptome Analysis.

Lactate, a product of aerobic glycolysis in astrocytes, is required for memory formation and consolidation, and has recently emerged as a signaling molecule for neurons and various cell types in peripheral tissues. In particular lactate stimulates mRNA expression of a few plasticity-related genes. Here, we describe a RNA-seq study that unravels genome-wide transcriptomic responses to this energy metabolite in cortical neurons. Our results show that mRNA expression of 20 immediate-early genes involved in the MAPK signaling pathway and in synaptic plasticity were increased by more than twofold following 1 h of lactate stimulation. This effect was dependent on NMDA receptor (NMDAR) activity since it was prevented by pre-treatment with MK-801. Comparison with published datasets showed that a significant proportion of genes modulated by lactate were similarly regulated by a stimulation protocol activating specifically synaptic NMDARs known to result in upregulation of pro-survival and downregulation of pro-death genes. Remarkably, transcriptional responses to lactate were reproduced by NADH (for 74 of the 113 genes, FDR < 0.05), suggesting a redox-dependent mechanism of action. Longer-term gene expression changes observed after 6 h of lactate treatment affected genes involved in regulating neuronal excitability and genes coding for proteins localized at synapses. Gene set enrichment analyses performed with ranked lists of expressed genes revealed effects on molecular functions involved in epigenetic modulation, and on processes relevant to sleep physiology and behavioral phenotypes such as anxiety and hyperactivity. Overall, these results strengthen the notion that lactate effectively regulates activity-dependent and synaptic genes, and highlight new signaling effects of lactate in plasticity and neuroprotection.

Development of inhibitory synaptic inputs on layer 2/3 pyramidal neurons in the rat medial prefrontal cortex.

Inhibitory control of pyramidal neurons plays a major role in governing the excitability in the brain. While spatial mapping of inhibitory inputs onto pyramidal neurons would provide important structural data on neuronal signaling, studying their distribution at the single cell level is difficult due to the lack of easily identifiable anatomical proxies. Here, we describe an approach where in utero electroporation of a plasmid encoding for fluorescently tagged gephyrin into the precursors of pyramidal cells along with ionotophoretic injection of Lucifer Yellow can reliably and specifically detect GABAergic synapses on the dendritic arbour of single pyramidal neurons. Using this technique and focusing on the basal dendritic arbour of layer 2/3 pyramidal cells of the medial prefrontal cortex, we demonstrate an intense development of GABAergic inputs onto these cells between postnatal days 10 and 20. While the spatial distribution of gephyrin clusters was not affected by the distance from the cell body at postnatal day 10, we found that distal dendritic segments appeared to have a higher gephyrin density at later developmental stages. We also show a transient increase around postnatal day 20 in the percentage of spines that are carrying a gephyrin cluster, indicative of innervation by a GABAergic terminal. Since the precise spatial arrangement of synaptic inputs is an important determinant of neuronal responses, we believe that the method described in this work may allow a better understanding of how inhibition settles together with excitation, and serve as basics for further modelling studies focusing on the geometry of dendritic inhibition during development.

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

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Nkx2.1-derived astrocytes and neurons together with Slit2 are indispensable for anterior commissure formation.

Guidepost cells present at and surrounding the midline provide guidance cues that orient the growing axons through commissures. Here we show that the transcription factor Nkx2.1 known to control the specification of GABAergic interneurons also regulates the differentiation of astroglia and polydendrocytes within the mouse anterior commissure (AC). Nkx2.1-positive glia were found to originate from three germinal regions of the ventral telencephalon. Nkx2.1-derived glia were observed in and around the AC region by E14.5. Thereafter, a selective cell ablation strategy showed a synergistic role of Nkx2.1-derived cells, both GABAergic interneurons and astroglia, towards the proper formation of the AC. Finally, our results reveal that the Nkx2.1-regulated cells mediate AC axon guidance through the expression of the repellent cue, Slit2. These results bring forth interesting insights about the spatial and temporal origin of midline telencephalic glia, and highlight the importance of neurons and astroglia towards the formation of midline commissures.

Genes involved in the astrocyte-neuron lactate shuttle (ANLS) are specifically regulated in cortical astrocytes following sleep deprivation in mice.

STUDY OBJECTIVES: There is growing evidence indicating that in order to meet the neuronal energy demands, astrocytes provide lactate as an energy substrate for neurons through a mechanism called "astrocyte-neuron lactate shuttle" (ANLS). Since neuronal activity changes dramatically during vigilance states, we hypothesized that the ANLS may be regulated during the sleep-wake cycle. To test this hypothesis we investigated the expression of genes associated with the ANLS specifically in astrocytes following sleep deprivation. Astrocytes were purified by fluorescence-activated cell sorting from transgenic mice expressing the green fluorescent protein (GFP) under the control of the human astrocytic GFAP-promoter. DESIGN: 6-hour instrumental sleep deprivation (TSD). SETTING: Animal sleep research laboratory. PARTICIPANTS: Young (P23-P27) FVB/N-Tg (GFAP-GFP) 14Mes/J (Tg) mice of both sexes and 7-8 week male Tg and FVB/Nj mice. INTERVENTIONS: Basal sleep recordings and sleep deprivation achieved using a modified cage where animals were gently forced to move. MEASUREMENTS AND RESULTS: Since Tg and FVB/Nj mice displayed a similar sleep-wake pattern, we performed a TSD in young Tg mice. Total RNA was extracted from the GFP-positive and GFP-negative cells sorted from cerebral cortex. Quantitative RT-PCR analysis showed that levels of Glut1, α-2-Na/K pump, Glt1, and Ldha mRNAs were significantly increased following TSD in GFP-positive cells. In GFP-negative cells, a tendency to increase, although not significant, was observed for Ldha, Mct2, and α-3-Na/K pump mRNAs. CONCLUSIONS: This study shows that TSD induces the expression of genes associated with ANLS specifically in astrocytes, underlying the important role of astrocytes in the maintenance of the neuro-metabolic coupling across the sleep-wake cycle.

An ion transport-independent role for the cation-chloride cotransporter KCC2 in dendritic spinogenesis in vivo.

The neuron-specific K-Cl cotransporter, KCC2, is highly expressed in the vicinity of excitatory synapses in pyramidal neurons, and recent in vitro data suggest that this protein plays a role in the development of dendritic spines. The in vivo relevance of these observations is, however, unknown. Using in utero electroporation combined with post hoc iontophoretic injection of Lucifer Yellow, we show that premature expression of KCC2 induces a highly significant and permanent increase in dendritic spine density of layer 2/3 pyramidal neurons in the somatosensory cortex. Whole-cell recordings revealed that this increased spine density is correlated with an enhanced spontaneous excitatory activity in KCC2-transfected neurons. Precocious expression of the N-terminal deleted form of KCC2, which lacks the chloride transporter function, also increased spine density. In contrast, no effect on spine density was observed following in utero electroporation of a point mutant of KCC2 (KCC2-C568A) where both the cotransporter function and the interaction with the cytoskeleton are disrupted. Transfection of the C-terminal domain of KCC2, a region involved in the interaction with the dendritic cytoskeleton, also increased spine density. Collectively, these results demonstrate a role for KCC2 in excitatory synaptogenesis in vivo through a mechanism that is independent of its ion transport function.

Intracellular chloride concentration influences the GABAA receptor subunit composition.

GABA(A) receptors (GABA(A)Rs) exist as different subtype variants showing unique functional properties and defined spatio-temporal expression pattern. The molecular mechanisms underlying the developmental expression of different GABA(A)R are largely unknown. The intracellular concentration of chloride ([Cl(-)](i)), the main ion permeating through GABA(A)Rs, also undergoes considerable changes during maturation, being higher at early neuronal stages with respect to adult neurons. Here we investigate the possibility that [Cl(-)](i) could modulate the sequential expression of specific GABA(A)Rs subtypes in primary cerebellar neurons. We show that [Cl(-)](i) regulates the expression of α3-1 and δ-containing GABA(A) receptors, responsible for phasic and tonic inhibition, respectively. Our findings highlight the role of [Cl(-)](i) in tuning the strength of GABAergic responses by acting as an intracellular messenger.

Fluoxetine regulates the expression of neurotrophic/growth factors and glucose metabolism in astrocytes.

RATIONALE: The pharmacological actions of most antidepressants are ascribed to the modulation of serotonergic and/or noradrenergic transmission in the brain. During therapeutic treatment for major depression, fluoxetine, one of the most commonly prescribed selective serotonin reuptake inhibitor (SSRI) antidepressants, accumulates in the brain, suggesting that fluoxetine may interact with additional targets. In this context, there is increasing evidence that astrocytes are involved in the pathophysiology of major depression. OBJECTIVES: The aim of this study was to examine the effects of fluoxetine on the expression of neurotrophic/growth factors that have antidepressant properties and on glucose metabolism in cultured cortical astrocytes. RESULTS: Treatment of astrocytes with fluoxetine and paroxetine, another SSRI antidepressant, upregulated brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), and VGF mRNA expression. In contrast, the tricyclic antidepressants desipramine and imipramine did not affect the expression of these neurotrophic/growth factors. Analysis of the effects of fluoxetine on glucose metabolism revealed that fluoxetine reduces glycogen levels and increases glucose utilization and lactate release by astrocytes. Similar data were obtained with paroxetine, whereas imipramine and desipramine did not regulate glucose metabolism in this glial cell population. Our results also indicate that the effects of fluoxetine and paroxetine on glucose utilization, lactate release, and expression of BDNF, VEGF, and VGF are not mediated by serotonin-dependent mechanisms. CONCLUSIONS: These data suggest that, by increasing the expression of specific astrocyte-derived neurotrophic factors and lactate release from astrocytes, fluoxetine may contribute to normalize the trophic and metabolic support to neurons in major depression.

Regulation of dendritic development by BDNF requires activation of CRTC1 by glutamate.

Dendritic growth is essential for the establishment of a functional nervous system. Among extrinsic signals that control dendritic development, substantial evidence indicates that BDNF regulates dendritic morphology. However, little is known about the underlying mechanisms by which BDNF controls dendritic growth. In this study, we show that the MAPK signaling pathway and the transcription factor cAMP response element-binding protein (CREB) mediate the effects of BDNF on dendritic length and complexity. However, phosphorylation of CREB alone is not sufficient for the stimulation of dendritic growth by BDNF. Thus, using a mutant form of CREB unable to bind CREB-regulated transcription coactivator (CRTC1), we demonstrate that this effect also requires a functional interaction between CREB and CRTC1. Moreover, inhibition of CRTC1 expression by shRNA-mediated knockdown abolished BDNF-induced dendritic growth of cortical neurons. Interestingly, we found that nuclear translocation of CRTC1 results from activation of NMDA receptors by glutamate, a process that is essential for the effects of BDNF on dendritic development. Together, these data identify a previously unrecognized mechanism by which CREB and the coactivator CRTC1 mediate the effects of BDNF on dendritic growth.

Expression of brain-derived neurotrophic factor is not modulated by chronic mild stress in the rat hippocampus and amygdala.

Accumulating evidence supports a role for brain-derived neurotrophic factor (BDNF) in depression. However, most of these studies have been performed in animal models that have a low face validity with regard to the human disease. Here, we examined the regulation of BDNF expression in the hippocampus and amygdala of rats subjected to the chronic mild stress (CMS) model of depression, a paradigm that induces anhedonia, a core symptom of depression. We found that exposure of rats to the CMS paradigm did not modulate BDNF mRNA expression in the hippocampus and amygdala. In addition, chronic administration of imipramine, which reversed CMS-induced anhedonia, did not alter BDNF mRNA expression in these limbic structures.

Excitatory GABA action is essential for morphological maturation of cortical neurons in vivo.

GABA exerts excitatory actions on embryonic and neonatal cortical neurons, but the in vivo function of this GABA excitation is essentially unknown. Using in utero electroporation, we eliminated the excitatory action of GABA in a subpopulation of rat ventricular progenitors and cortical neurons derived from these progenitors by premature expression of the Cl- transporter KCC2, as confirmed by the changes in the reversal potential of GABA-induced currents and the resting membrane potential after GABA(A) receptor blockade. We found that radial migration to layer II/III of the somatosensory cortex of neurons derived from the transfected progenitors was not significantly affected, but their morphological maturation was markedly impaired. Furthermore, reducing neuronal excitability of cortical neurons in vivo by overexpressing an inward-rectifying K+ channel, which lowered the resting membrane potential, mimicked the effect of premature KCC2 expression. Thus, membrane depolarization caused by early GABA excitation is critical for morphological maturation of neonatal cortical neurons in vivo.

Role of activity-dependent regulation of neuronal chloride homeostasis in development.

The polarity of neurotransmission mediated by the gamma-amino butyric acid (GABA) type A receptor depends crucially on intracellular chloride concentration, which is largely determined by the expression and function of cation/chloride co-transporters. Recent evidence shows how both activity and neurotrophic factors can affect GABAergic transmission in the mammalian central nervous system through their effects on the neuron-specific chloride-extruding transporter KCC2. In particular, GABAergic neurotransmission early in development, sustained neuronal activity in mature networks and brain-derived neurotrophic factor each modulate the expression or function of KCC2. The resulting changes in intracellular chloride concentration alter the nature or strength of fast GABAergic neurotransmission, profoundly affecting the development and function of neuronal networks.

A critical role for system A amino acid transport in the regulation of dendritic development by brain-derived neurotrophic factor (BDNF).

Dendritic development is essential for the establishment of a functional nervous system. Among factors that control dendritic development, brain-derived neurotrophic factor (BDNF) has been shown to regulate dendritic length and complexity of cortical neurons. However, the cellular and molecular mechanisms that underlie these effects remain poorly understood. In this study, we examined the role of amino acid transport in mediating the effects of BDNF on dendritic development. We show that BDNF increases System A amino acid transport in cortical neurons by selective up-regulation of the sodium-coupled neutral amino acid transporter (SNAT)1. Up-regulation of SNAT1 expression and System A activity is required for the effects of BDNF on dendritic growth and branching of cortical neurons. Further analysis revealed that induction of SNAT1 expression and System A activity by BDNF is necessary in particular to enhance synthesis of tissue-type plasminogen activator, a protein that we demonstrate to be essential for the effects of BDNF on cortical dendritic morphology. Together, these data reveal that stimulation of neuronal differentiation by BDNF requires the up-regulation of SNAT1 expression and System A amino acid transport to meet the increased metabolic demand associated with the enhancement of dendritic growth and branching.

Modulation of GABAergic transmission by activity via postsynaptic Ca2+-dependent regulation of KCC2 function.

Activity-induced modification of GABAergic transmission contributes to the plasticity of neural circuits. In the present work we found that prolonged postsynaptic spiking of hippocampal neurons led to a shift in the reversal potential of GABA-induced Cl- currents (E(Cl)) toward positive levels in a duration- and frequency-dependent manner. This effect was abolished by blocking cytosolic Ca2+ elevation and mimicked by releasing Ca2+ from internal stores. Activity- and Ca2+-induced E(Cl) shifts were larger in mature neurons, which express the K-Cl cotransporter KCC2 at high levels, and inhibition of KCC2 occluded the shifts. Overexpression of KCC2 in young cultured neurons, which express lower levels of KCC2 and have a more positive E(Cl), resulted in hyperpolarized E(Cl) similar to that of mature cells. Importantly, these young KCC2-expressing neurons became responsive to neuronal spiking and Ca2+ elevation by showing positive E(Cl) shifts. Thus, repetitive postsynaptic spiking reduces the inhibitory action of GABA through a Ca2+-dependent downregulation of KCC2 function.