Contribution of astrocytes to familial risk and clinical manifestation of schizophrenia.

Previous studies have implicated several brain cell types in schizophrenia (SCZ), but the genetic impact of astrocytes is unknown. Considering their high complexity in humans, astrocytes are likely key determinants of neurodevelopmental diseases, such as SCZ. Human induced pluripotent stem cell (hiPSC)-derived astrocytes differentiated from five monozygotic twin pairs discordant for SCZ and five healthy subjects were studied for alterations related to high genetic risk and clinical manifestation of SCZ in astrocyte transcriptomics, neuron-astrocyte co-cultures, and in humanized mice. We found gene expression and signaling pathway alterations related to synaptic dysfunction, inflammation, and extracellular matrix components in SCZ astrocytes, and demyelination in SCZ astrocyte transplanted mice. While Ingenuity Pathway Analysis identified SCZ disease and synaptic transmission pathway changes in SCZ astrocytes, the most consistent findings were related to collagen and cell adhesion associated pathways. Neuronal responses to glutamate and GABA differed between astrocytes from control persons, affected twins, and their unaffected co-twins and were normalized by clozapine treatment. SCZ astrocyte cell transplantation to the mouse forebrain caused gene expression changes in synaptic dysfunction and inflammation pathways of mouse brain cells and resulted in behavioral changes in cognitive and olfactory functions. Differentially expressed transcriptomes and signaling pathways related to synaptic functions, inflammation, and especially collagen and glycoprotein 6 pathways indicate abnormal extracellular matrix composition in the brain as one of the key characteristics in the etiology of SCZ.

Protective Role of Low Ethanol Administration Following Ischemic Stroke via Recovery of KCC2 and p75NTR Expression.

A striking result from epidemiological studies show a correlation between low alcohol intake and lower incidence for ischemic stroke and severity of derived brain injury. Although reduced apoptosis and inflammation has been suggested to be involved, little is known about the mechanism mediating this effect in vivo. Increase in intracellular chloride concentration and derived depolarizing GABAAR-mediated transmission are common consequences following various brain injuries and are caused by the abnormal expression levels of the chloride cotransporters NKCC1 and KCC2. Downstream pro-apoptotic signaling through p75NTR may link GABAA depolarization with post-injury neuronal apoptosis. Here, we show that changes in GABAergic signaling, Cl- homeostasis, and expression of chloride cotransporters in the post-traumatic mouse brain can be significantly reduced by administration of 3% ethanol to the drinking water. Ethanol-induced upregulation of KCC2 has a positive impact on neuronal survival, preserving a large part of the cortical peri-infarct zone, as well as preventing the massive post-ischemic upregulation of the pro-apoptotic protein p75NTR. Importantly, intracortical multisite in vivo recordings showed that ethanol treatment could significantly ameliorate stroke-induced reduction in cortical activity. This surprising finding discloses a pathway triggered by low concentration of ethanol as a novel therapeutically relevant target.

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.

Dominant mutations in ITPR3 cause Charcot-Marie-Tooth disease.

OBJECTIVE: ITPR3, encoding inositol 1,4,5-trisphosphate receptor type 3, was previously reported as a potential candidate disease gene for Charcot-Marie-Tooth neuropathy. Here, we present genetic and functional evidence that ITPR3 is a Charcot-Marie-Tooth disease gene. METHODS: Whole-exome sequencing of four affected individuals in an autosomal dominant family and one individual who was the only affected individual in his family was used to identify disease-causing variants. Skin fibroblasts from two individuals of the autosomal dominant family were analyzed functionally by western blotting, quantitative reverse transcription PCR, and Ca2+ imaging. RESULTS: Affected individuals in the autosomal dominant family had onset of symmetrical neuropathy with demyelinating and secondary axonal features at around age 30, showing signs of gradual progression with severe distal leg weakness and hand involvement in the proband at age 64. Exome sequencing identified a heterozygous ITPR3 p.Val615Met variant segregating with the disease. The individual who was the only affected in his family had disease onset at age 4 with demyelinating neuropathy. His condition was progressive, leading to severe muscle atrophy below knees and atrophy of proximal leg and hand muscles by age 16. Trio exome sequencing identified a de novo ITPR3 variant p.Arg2524Cys. Altered Ca2+ -transients in p.Val615Met patient fibroblasts suggested that the variant has a dominant-negative effect on inositol 1,4,5-trisphosphate receptor type 3 function. INTERPRETATION: Together with two previously identified variants, our report adds further evidence that ITPR3 is a disease-causing gene for CMT and indicates altered Ca2+ homeostasis in disease pathogenesis.

Rapid Voltage Sensing with Single Nanorods via the Quantum Confined Stark Effect.

Properly designed colloidal semiconductor quantum dots (QDs) have already been shown to exhibit high sensitivity to external electric fields via the quantum confined Stark effect (QCSE). Yet, detection of the characteristic spectral shifts associated with the effect of the QCSE has traditionally been painstakingly slow, dramatically limiting the sensitivity of these QD sensors to fast transients. We experimentally demonstrate a new detection scheme designed to achieve shot-noise-limited sensitivity to emission wavelength shifts in QDs, showing feasibility for their use as local electric field sensors on the millisecond time scale. This regime of operation is already potentially suitable for detection of single action potentials in neurons at a high spatial resolution.

Implications of the N-terminal heterogeneity for the neuronal K-Cl cotransporter KCC2 function.

The neuron-specific K-Cl cotransporter KCC2 maintains the low intracellular chloride concentration required for the fast hyperpolarizing responses of the inhibitory neurotransmitters γ-aminobutyric acid (GABA) and glycine. The two KCC2 isoforms, KCC2a and KCC2b differ by their N-termini as a result of alternative promoter usage. Whereas the role of KCC2b in mediating the chloride transport is unequivocal, the physiological role of KCC2a in neurons has remained obscure. We show that KCC2a isoform can decrease the intracellular chloride concentration in cultured neurons and attenuate calcium responses evoked by application of the GABAA receptor agonist muscimol. While the biotinylation assay detected both KCC2 isoforms at the cell surface of cultured neurons, KCC2a was not detected at the plasma membrane in immunostainings, suggesting that the N-terminal KCC2a epitope is masked. Confirming this hypothesis, KCC2a surface expression was detected by the C-terminal KCC2 pan antibody but not by the N-terminal KCC2a antibody in KCC2b-deficient neurons. One possible cause for the epitope masking is the binding site of Ste20-related proline-alanine-rich kinase (SPAK) in the KCC2a N-terminus. SPAK, a known regulator of K-Cl cotransporters, was co-immunoprecipitated in a complex with KCC2a but not KCC2b isoform. Moreover, SPAK overexpression decreased the transport activity of KCC2a but not that of KCC2b, as revealed by rubidium flux assay in HEK293 cells. Thus, our data indicate that both KCC2 isoforms perform as chloride cotransporters in neuronal cells, while their N-terminal heterogeneity could play an important role in fine-tuning of the K-Cl transport activity.

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.

A noninvasive optical approach for assessing chloride extrusion activity of the K-Cl cotransporter KCC2 in neuronal cells.

BACKGROUND: Cation-chloride cotransporters (CCCs) are indispensable for maintaining chloride homeostasis in multiple cell types, but K-Cl cotransporter KCC2 is the only CCC member with an exclusively neuronal expression in mammals. KCC2 is critical for rendering fast hyperpolarizing responses of ionotropic γ-aminobutyric acid and glycine receptors in adult neurons, for neuronal migration in the developing central nervous system, and for the formation and maintenance of small dendritic protrusions-dendritic spines. Deficit in KCC2 expression and/or activity is associated with epilepsy and neuropathic pain, and effective strategies are required to search for novel drugs augmenting KCC2 function. RESULTS: We revised current methods to develop a noninvasive optical approach for assessing KCC2 transport activity using a previously characterized genetically encoded chloride sensor. Our protocol directly assesses dynamics of KCC2-mediated chloride efflux and allows measuring genuine KCC2 activity with good spatial and temporal resolution. As a proof of concept, we used this approach to compare transport activities of the two known KCC2 splice isoforms, KCC2a and KCC2b, in mouse neuronal Neuro-2a cells. CONCLUSIONS: Our noninvasive optical protocol proved to be efficient for assessment of furosemide-sensitive chloride fluxes. Transport activities of the N-terminal splice isoforms KCC2a and KCC2b obtained by the novel approach matched to those reported previously using standard methods for measuring chloride fluxes.

KCC2 regulates actin dynamics in dendritic spines via interaction with ß-PIX.

Chloride extrusion in mature neurons is largely mediated by the neuron-specific potassium-chloride cotransporter KCC2. In addition, independently of its chloride transport function, KCC2 regulates the development and morphology of dendritic spines through structural interactions with the actin cytoskeleton. The mechanism of this effect remains largely unknown. In this paper, we show a novel pathway for KCC2-mediated regulation of the actin cytoskeleton in neurons. We found that KCC2, through interaction with the b isoform of Rac/Cdc42 guanine nucleotide exchange factor ß-PIX, regulates the activity of Rac1 GTPase and the phosphorylation of one of the major actin-regulating proteins, cofilin-1. KCC2-deficient neurons had abnormally high levels of phosphorylated cofilin-1. Consistently, dendritic spines of these neurons exhibited a large pool of stable actin, resulting in reduced spine motility and diminished density of functional synapses. In conclusion, we describe a novel signaling pathway that couples KCC2 to the cytoskeleton and regulates the formation of glutamatergic synapses.

Distribution of neuronal KCC2a and KCC2b isoforms in mouse CNS.

The neuronal K-Cl cotransporter KCC2 maintains the low intracellular chloride concentration required for the fast hyperpolarizing actions of inhibitory neurotransmitters in mature central nervous system (CNS). The KCC2 gene produces two isoforms, KCC2a and KCC2b, that differ in their N-termini. Increase of KCC2b in the cortex underlies the developmental shift in γ-aminobutyric acid (GABA)ergic responses, whereas the physiological role of KCC2a is still poorly characterized. The two KCC2 isoforms show equal distribution in mouse brainstem neurons at birth; however their postnatal expression patterns, and the subcellular localization of KCC2a, have not yet been described. Here, we compared the pattern of KCC2a and KCC2b expression in different regions of postnatal mouse CNS by immunohistochemistry by using isoform-specific antibodies. Tissue from KCC2a isoform-specific knockout mice was used as a negative control. KCC2b expression increased postnatally and was widely expressed in adult brain. KCC2a immunoreactivity was low or absent in most parts of the adult cortex, hippocampus, thalamus, and cerebellar cortex. Both isoforms were widely present in the developing and mature hypothalamus, a large part of the brainstem, and the spinal cord. A notable exception was the lack of KCC2a staining in the brainstem auditory system. At the subcellular level, the isoforms were only partially colocalized. In neuronal somas, KCC2b immunoreactivity was concentrated at the plasma membrane, whereas KCC2a signal was not. Moreover, although both isoforms were expressed in microtubule-associated protein (MAP)2-positive dendrites, they appeared in non-overlapping dendritic compartments. The results, together with those of previous studies, suggest that KCC2a and KCC2b have overlapping roles in neonatal neurons but presumably different roles in mature neurons.

Methods for three-dimensional analysis of dendritic spine dynamics.

Dendritic spines are small bulbous expansions that receive input from a single excitatory synapse. Although spines are often characterized by a mushroom-like morphology, they come in a wide range of sizes and shapes, even within the same dendrite. In a developing brain, spines exhibit a high degree of structural and functional plasticity, reflecting the formation and elimination of synapses during the maturation of neuronal circuits. The morphology of spines in developing neurons is affected by synaptic activity, hence contributing to the experience-dependent refinement of neuronal circuits, learning, and memory. Thus, understanding spine dynamics and its regulation is of central importance to studies of synaptic plasticity in the brain. The challenge has been to develop a computer-based assay that will quantitatively assess the three-dimensional change in spine movements caused by various stimuli and experimental conditions. Here, we provide detailed protocols for cell plating, transient transfections, and time-lapse imaging of dendritic spines. For the analysis of dendritic spine dynamics, we present two methods based on quantitative three-dimensional measurements.

Neurturin evokes MAPK-dependent upregulation of Egr4 and KCC2 in developing neurons.

The K-Cl cotransporter KCC2 plays a crucial role in the functional development of GABA(A)-mediated responses rendering GABA hyperpolarizing in adult neurons. We have previously shown that BDNF upregulates KCC2 in immature neurons through the transcription factor Egr4. The effect of BDNF on Egr4 and KCC2 was shown to be dependent on the activation of ERK1/2. Here we demonstrate that the trophic factor neurturin can also trigger Egr4 expression and upregulate KCC2 in an ERK1/2-dependent manner. These results show that Egr4 is an important component in the mechanism for trophic factor-mediated upregulation of KCC2 in immature neurons involving the activation of specific intracellular pathways common to BDNF and Neurturin.

Knocking down of the KCC2 in rat hippocampal neurons increases intracellular chloride concentration and compromises neuronal survival.

KCC2 is a neuron-specific potassium-chloride co-transporter controlling intracellular chloride homeostasis in mature and developing neurons. It is implicated in the regulation of neuronal migration, dendrites outgrowth and formation of the excitatory and inhibitory synaptic connections. The function of KCC2 is suppressed under several pathological conditions including neuronal trauma, different types of epilepsies, axotomy of motoneurons, neuronal inflammations and ischaemic insults. However, it remains unclear how down-regulation of the KCC2 contributes to neuronal survival during and after toxic stress. Here we show that in primary hippocampal neuronal cultures the suppression of the KCC2 function using two different shRNAs, dominant-negative KCC2 mutant C568A or DIOA inhibitor, increased the intracellular chloride concentration [Cl⁻]i and enhanced the toxicity induced by lipofectamine-dependent oxidative stress or activation of the NMDA receptors. The rescuing of the KCC2 activity using over-expression of the active form of the KCC2, but not its non-active mutant Y1087D, effectively restored [Cl⁻]i and enhanced neuronal resistance to excitotoxicity. The reparative effects of KCC2 were mimicked by over-expression of the KCC3, a homologue transporter. These data suggest an important role of KCC2-dependent potassium/chloride homeostasis under neurototoxic conditions and reveal a novel role of endogenous KCC2 as a neuroprotective molecule.

Early growth response 4 mediates BDNF induction of potassium chloride cotransporter 2 transcription.

A major event in the maturation of CNS GABAergic transmission is the qualitative change in GABA(A)-mediated responses from depolarizing to hyperpolarizing. In cortical regions, this is attributed to the increased expression of potassium chloride cotransporter 2b (KCC2b), the main isoform of the neuron-specific K-Cl cotransporter KCC2. We have previously shown that transcription factor early growth response 4 (Egr4) can activate the KCC2b promoter. Here we demonstrate that in immature hippocampal neurons BDNF robustly induces ERK1/2 (extracellular signal-regulated kinase 1/2)-dependent Egr4 expression and rapid Egr4-dependent activation of the KCC2b promoter. The subsequent increase in KCC2b mRNA contributes to the expression of total KCC2 protein levels. These results indicate that Egr4 is an important component in the mechanism of BDNF-dependent KCC2 gene regulation via the ERK1/2 pathway in immature neurons.

Coexpression and heteromerization of two neuronal K-Cl cotransporter isoforms in neonatal brain.

The neuron-specific K-Cl cotransporter KCC2 maintains the low intracellular chloride concentration required for the fast hyperpolarizing actions of inhibitory neurotransmitters. The KCC2 gene codes for two isoforms, KCC2a and KCC2b, which differ in their N termini. The relative expression and cellular distribution of the two KCC2 protein isoforms are unknown. Here, we characterize an antibody against the KCC2a isoform and show that a previously described antibody against KCC2 is specific for the KCC2b isoform (Hubner, C. A., Stein, V., Hermans-Borgmeyer, I., Meyer, T., Ballanyi, K., and Jentsch, T. J. (2001) Neuron 30, 515-524). Immunostaining of dissociated hippocampal cultures confirms that both KCC2 isoforms are neuron-specific. Immunoblot analysis indicates that KCC2b is the major KCC2 isoform in the adult brain, whereas in the neonatal mouse central nervous system, half of total KCC2 protein is KCC2a. At this stage, the two KCC2 isoforms are largely colocalized and show similar patterns of distribution in the brain. When coexpressed in HEK293 cells, KCC2a and KCC2b proteins form heteromeric complexes. Moreover, the two isoforms can be coimmunoprecipitated from the neonatal brain, suggesting the presence of endogenous KCC2a-KCC2b heteromers. Consistent with this, native gel analysis shows that a substantial part of endogenous KCC2 isoforms in the neonatal brain constitute dimers.

KCC2 interacts with the dendritic cytoskeleton to promote spine development.

The neuron-specific K-Cl cotransporter, KCC2, induces a developmental shift to render GABAergic transmission from depolarizing to hyperpolarizing. Now we demonstrate that KCC2, independently of its Cl(-) transport function, is a key factor in the maturation of dendritic spines. This morphogenic role of KCC2 in the development of excitatory synapses is mediated by structural interactions between KCC2 and the spine cytoskeleton. Here, the binding of KCC2 C-terminal domain to the cytoskeleton-associated protein 4.1N may play an important role. A more general conclusion based on our data is that KCC2 acts as a synchronizing factor in the functional development of glutamatergic and GABAergic synapses in cortical neurons and networks.

A novel N-terminal isoform of the neuron-specific K-Cl cotransporter KCC2.

The neuronal K-Cl cotransporter KCC2 maintains the low intracellular chloride concentration required for the hyperpolarizing actions of inhibitory neurotransmitters gamma-aminobutyric acid and glycine in the central nervous system. This study shows that the mammalian KCC2 gene (alias Slc12a5) generates two neuron-specific isoforms by using alternative promoters and first exons. The novel KCC2a isoform differs from the only previously known KCC2 isoform (now termed KCC2b) by 40 unique N-terminal amino acid residues, including a putative Ste20-related proline alanine-rich kinase-binding site. Ribonuclease protection and quantitative PCR assays indicated that KCC2a contributes 20-50% of total KCC2 mRNA expression in the neonatal mouse brain stem and spinal cord. In contrast to the marked increase in KCC2b mRNA levels in the cortex during postnatal development, the overall expression of KCC2a remains relatively constant and makes up only 5-10% of total KCC2 mRNA in the mature cortex. A rubidium uptake assay in human embryonic kidney 293 cells showed that the KCC2a isoform mediates furosemide-sensitive ion transport activity comparable with that of KCC2b. Mice that lack both KCC2 isoforms die at birth due to severe motor defects, including disrupted respiratory rhythm, whereas mice with a targeted disruption of the first exon of KCC2b survive for up to 2 weeks but eventually die due to spontaneous seizures. We show that these mice lack KCC2b but retain KCC2a mRNA. Thus, distinct populations of neurons show a differential dependence on the expression of the two isoforms: KCC2a expression in the absence of KCC2b is presumably sufficient to support vital neuronal functions in the brain stem and spinal cord but not in the cortex.

Upregulation of the neuron-specific K+/Cl- cotransporter expression by transcription factor early growth response 4.

The expression of the neuron-specific K+/Cl- cotransporter (KCC2) is restricted to the CNS and is strongly upregulated during neuronal maturation, yielding a low intracellular chloride concentration that is required for fast synaptic inhibition in adult neurons. To elucidate the mechanisms of KCC2 gene regulation, we analyzed the KCC2 (alias Slc12a5) promoter and proximal intron-1 regions and revealed 10 candidate transcription factor binding sites that are highly conserved in mammalian KCC2 genes. Here we focus on one of these factors, early growth response 4 (Egr4), which shows a similar developmental upregulation in CNS neurons as KCC2. KCC2 luciferase reporter constructs containing the Egr4 site (Egr4(KCC2)) were strongly induced by Egr4 overexpression in neuro-2a neuroblastoma cells and in cultured neurons. Egr4-mediated induction was decreased significantly by point-mutating the Egr4(KCC2). Insertion of Egr4(KCC2) into the KCC2 basal promoter in the endogenous reverse, but not in the opposite, orientation reestablished Egr4-mediated induction. Electrophoretic mobility shift assay confirmed specific Egr4 binding to Egr4(KCC2). Interference RNA-mediated knock-down of Egr4 and a dominant-negative isoform of Egr4 significantly inhibited KCC2 reporter induction and endogenous KCC2 expression in cultured neurons. Together, the results indicate an important role for Egr4 in the developmental upregulation of KCC2 gene expression.

Distinct properties of functional KCC2 expression in immature mouse hippocampal neurons in culture and in acute slices.

A hallmark in the development of GABAergic neurotransmission is the switch in GABA(A)-mediated responses from depolarizing to hyperpolarizing. This occurs due to a gradual decrease in the intracellular concentration of chloride caused by the functional expression of the neuron-specific K-Cl cotransporter KCC2. Whether a mere increase in the amount of KCC2 protein is the rate-limiting step in vivo, or a further activation of the otherwise nonfunctional cotransporter is required, is not clear. Imposing a fixed Cl(-) load via patch pipette we measured the resultant somato-dendritic gradients in reversal potential of GABAergic currents to determine the time course of functional maturation of KCC2-mediated Cl(-) extrusion in two preparations: cultured mouse hippocampal neurons plated at embryonic day 17 and CA1 pyramidal cells in acute slices. We found that in immature neurons in both preparations the gradient is initially small or not detectable. It undergoes an abrupt increase at around days 13-14 in culture, while a more gradual increase occurs between postnatal days 5-14 in slices. Consistent with the presence of a nonfunctional form of KCC2 in immature hippocampal neurons grown in culture, application of the broad-spectrum kinase inhibitor staurosporine produces a rapid and potent up-regulation of KCC2 function in these cultured neurons, but not in neonatal slices. Taken together with our previously published data, these results indicate that the functional activity of KCC2 in vivo parallels the developmental expression of the protein, whereas cultured neurons require an additional activation step (mimicked by staurosporine) for KCC2 to become functional.

Developmental up-regulation of KCC2 in the absence of GABAergic and glutamatergic transmission.

Postsynaptic gamma-aminobutyric acid (GABA)A-mediated responses switch from depolarizing to hyperpolarizing during postnatal development of the rodent hippocampus. This is attributable to a decrease in the concentration of intracellular chloride set by the expression of the neuron-specific K+-Cl- co-transporter, KCC2. A recent in vitro study [Ganguly et al. (2001) Cell, 105, 521-532] showed that KCC2 expression may be under the trophic control of GABAA receptor-mediated transmission. Here we have studied the developmental expression of KCC2 protein in mouse hippocampal dissociated cultures as well as organotypic cultures. A low somatic expression level was found in neurons prior to the formation of the first synapses, as detected by synaptophysin immunoreactivity. Thereafter, KCC2 expression was strongly up-regulated during neuronal maturation. The developmental up-regulation of KCC2 expression was not altered by a chronic application (throughout the culturing period; 2-15 days in vitro) of the action-potential blocker TTX or the N-methyl-d-aspartate (NMDA) and non-NMDA antagonists APV and NBQX. Blockade of GABAA-mediated transmission with picrotoxin did not affect the expression levels of KCC2 protein either. These data show that neither neuronal spiking nor ionotropic glutamatergic and GABAergic transmission are required for the developmental expression of KCC2 in mouse hippocampal neurons in vitro.