Downregulation of KCC2 following LTP contributes to EPSP-spike potentiation in rat hippocampus

GABAergic synaptic inhibition plays a critical role in regulating long-term potentiation (LTP) of glutamatergic synaptic transmission and circuit output. The K(+)-Cl(-) cotransporter 2 (KCC2) is an important factor in determining inhibitory GABAergic synaptic strength besides the contribution of GABA(A) receptor. Although much knowledge has been gained regarding activity-dependent downregulation of KCC2 in many pathological conditions, the potential change and contribution of KCC2 in LTP expression is still unknown. In this study, we found that downregulation of KCC2 was accompanied with the occurrence of LTP but not that of long-term depression in hippocampal CA1 region. Meanwhile, KCC2 level in CA3/DG and adjacent cortex was stable in the process of LTP expression in Schaffer collateral synapses. Blockade of NMDA receptor with APV not only prevented LTP induction also abolished the reduction of KCC2. Furthermore, the inhibition of KCC2 function with furosemide directly induced EPSP-spike (E-S) potentiation, an important component of LTP in hippocampus. The present data suggest a novel mechanism that LTP formation is accompanied by the downregulation of KCC2, which is underlying GABAergic strength and most likely contributes to the E-S potentiation following LTP.     

A shared vesicular carrier allows synaptic corelease of GABA and glycine

The type of vesicular transporter expressed by a neuron is thought to determine its neurotransmitter phenotype. We show that inactivation of the vesicular inhibitory amino acid transporter (Viaat, VGAT) leads to embryonic lethality, an abdominal defect known as omphalocele, and a cleft palate. Loss of Viaat causes a drastic reduction of neurotransmitter release in both GABAergic and glycinergic neurons, indicating that glycinergic neurons do not express a separate vesicular glycine transporter. This loss of GABAergic and glycinergic synaptic transmission does not impair the development of inhibitory synapses or the expression of KCC2, the K+ -Cl- cotransporter known to be essential for the establishment of inhibitory neurotransmission. In the absence of Viaat, GABA-synthesizing enzymes are partially lost from presynaptic terminals. Since GABA and glycine compete for vesicular uptake, these data point to a close association of Viaat with GABA-synthesizing enzymes as a key factor in specifying GABAergic neuronal phenotypes.     

Pumping up the synapse

Synchronized control of excitatory and inhibitory synapse maturation is crucial for normal brain wiring, while its dysfunction leads to neurodevelopmental disorders, including autism. A paper in this issue of Neuron identified a novel role for the KCC2 pump, also responsible for the GABAergic synapse developmental switch, in regulating spiny excitatory synapse maturation, implicating it in the coordinated maturation of inhibitory and excitatory synapses.     

A tale of timing and transport

Excitatory and inhibitory synapses show long-term plasticity, but spike timing-dependent plasticity was seen only at excitatory connections. No longer. In this issue of Neuron, Woodin et al. demonstrate that coincident pre- and postsynaptic activity acts on the neuronal K+/Cl- cotransporter KCC2 to shift the reversal potential for Cl- and thus alters the effectiveness of GABAergic synapses.     

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

Abnormal Expression of Cerebrospinal Fluid Cation Chloride Cotransporters in Patients with Rett Syndrome

Objective

Rett Syndrome is a progressive neurodevelopmental disorder caused mainly by mutations in the gene encoding methyl-CpG-binding protein 2. The relevance of MeCP2 for GABAergic function was previously documented in animal models. In these models, animals show deficits in brain-derived neurotrophic factor, which is thought to contribute to the pathogenesis of this disease. Neuronal Cation Chloride Cotransporters (CCCs) play a key role in GABAergic neuronal maturation, and brain-derived neurotrophic factor is implicated in the regulation of CCCs expression during development. Our aim was to analyse the expression of two relevant CCCs, NKCC1 and KCC2, in the cerebrospinal fluid of Rett syndrome patients and compare it with a normal control group.

Methods

The presence of bumetanide sensitive NKCC1 and KCC2 was analysed in cerebrospinal fluid samples from a control pediatric population (1 day to 14 years of life) and from Rett syndrome patients (2 to 19 years of life), by immunoblot analysis.

Results

Both proteins were detected in the cerebrospinal fluid and their levels are higher in the early postnatal period. However, Rett syndrome patients showed significantly reduced levels of KCC2 and KCC2/NKCC1 ratio when compared to the control group.

Conclusions

Reduced KCC2/NKCC1 ratio in the cerebrospinal fluid of Rett Syndrome patients suggests a disturbed process of GABAergic neuronal maturation and open up a new therapeutic perspective.     

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.     

BDNF-induced TrkB activation down-regulates the K+-Cl- cotransporter KCC2 and impairs neuronal Cl- extrusion

Pathophysiological activity and various kinds of traumatic insults are known to have deleterious long-term effects on neuronal Cl- regulation, which can lead to a suppression of fast postsynaptic GABAergic responses. Brain-derived neurotrophic factor (BDNF) increases neuronal excitability through a conjunction of mechanisms that include regulation of the efficacy of GABAergic transmission. Here, we show that exposure of rat hippocampal slice cultures and acute slices to exogenous BDNF or neurotrophin-4 produces a TrkB-mediated fall in the neuron-specific K+-Cl- cotransporter KCC2 mRNA and protein, as well as a consequent impairment in neuronal Cl- extrusion capacity. After kindling-induced seizures in vivo, the expression of KCC2 is down-regulated in the mouse hippocampus with a spatiotemporal profile complementary to the up-regulation of TrkB and BDNF. The present data demonstrate a novel mechanism whereby BDNF/TrkB signaling suppresses chloride-dependent fast GABAergic inhibition, which most likely contributes to the well-known role of TrkB-activated signaling cascades in the induction and establishment of epileptic activity.     

The Role of K<Superscript>+</Superscript>-Cl<Superscript>−</Superscript>-Cotransporter-2 in Neuropathic Pain

The pain sensory system normally functions under a fine balance between excitation and inhibition. When this balance is perturbed for some reason, it leads to neuropathic pain. There is accumulating evidence that attributes this pain generation to specific dysfunctions of the inhibitory system in the spinal cord. One possible mechanism leading to the induction of these dysfunctions is the down-regulation of K⁺-Cl^?-cotransporter-2 (KCC2) expression. In fact, various neuropathic pain models indicate a decrease of KCC2 expression in the spinal cord. The alteration of KCC2 expression affects GABAergic and glycinergic neurotransmissions, because KCC2 is a potassium-chloride exporter and serves to maintain intracellular chloride concentration. When there is a low level of KCC2 expression, GABAergic and glycinergic neurotransmissions transform from inhibitory signals to excitatory signals. In this review, the hypothesis that an alteration of KCC2 expression has a crucial influence on the initiation/development or maintenance of neuropathic pain is discussed. In addition, it is suggested that the alteration of inhibitory signals is dependent on the time after peripheral nerve injury.     

The neuron-specific K-Cl cotransporter, KCC2. Antibody development and initial characterization of the protein

The neuron-specific K-Cl cotransporter (KCC2) is hypothesized to function as an active Cl- extrusion pathway important in postsynaptic inhibition mediated by ligand-gated anion channels, like gamma-aminobutyric acid type A (GABAA) and glycine receptors. To understand better the functional role of KCC2 in the nervous system, we developed polyclonal antibodies to a KCC2 fusion protein and used these antibodies to characterize and localize KCC2 in the rat cerebellum. The antibodies specifically recognized the KCC2 protein which is an approximately 140-kDa glycoprotein detectable only within the central nervous system. The KCC2 protein displayed a robust and punctate distribution in primary cultured retinal amacrine cells known to form exclusively GABAAergic synapses in culture. In immunolocalization studies, KCC2 was absent from axons and glia but was highly expressed at neuronal somata and dendrites, indicating a specific postsynaptic distribution of the protein. In the granule cell layer, KCC2 exhibited a distinct colocalization with the beta2/beta3-subunits of the GABAA receptor at the plasma membrane of granule cell somata and at cerebellar glomeruli. KCC2 lightly labeled the plasma membrane of Purkinje cell somata. Within the molecular layer, KCC2 exhibited a distinctly punctate distribution along dendrites, indicating it may be highly localized at inhibitory synapses along these processes. The distinct postsynaptic localization of KCC2 and its colocalization with GABAA receptor in the cerebellum are consistent with the putative role of KCC2 in neuronal Cl- extrusion and postsynaptic inhibition.     

GABA itself promotes the developmental switch of neuronal GABAergic responses from excitation to inhibition

GABA is the main inhibitory neurotransmitter in the adult brain. Early in development, however, GABAergic synaptic transmission is excitatory and can exert widespread trophic effects. During the postnatal period, GABAergic responses undergo a switch from being excitatory to inhibitory. Here, we show that the switch is delayed by chronic blockade of GABA(A) receptors, and accelerated by increased GABA(A) receptor activation. In contrast, blockade of glutamatergic transmission or action potentials has no effect. Furthermore, GABAergic activity modulated the mRNA levels of KCC2, a K(+)-Cl(-) cotransporter whose expression correlates with the switch. Finally, we report that GABA can alter the properties of depolarization-induced Ca(2+) influx. Thus, GABA acts as a self-limiting trophic factor during neural development.     

BDNF regulates spontaneous correlated activity at early developmental stages by increasing synaptogenesis and expression of the K+/Cl- co-transporter KCC2

Spontaneous neural activity is a basic property of the developing brain, which regulates key developmental processes, including migration, neural differentiation and formation and refinement of connections. The mechanisms regulating spontaneous activity are not known. By using transgenic embryos that overexpress BDNF under the control of the nestin promoter, we show here that BDNF controls the emergence and robustness of spontaneous activity in embryonic hippocampal slices. Further, BDNF dramatically increases spontaneous co-active network activity, which is believed to synchronize gene expression and synaptogenesis in vast numbers of neurons. In fact, BDNF raises the spontaneous activity of E18 hippocampal neurons to levels that are typical of postnatal slices. We also show that BDNF overexpression increases the number of synapses at much earlier stages (E18) than those reported previously. Most of these synapses were GABAergic, and GABAergic interneurons showed hypertrophy and a 3-fold increase in GAD expression. Interestingly, whereas BDNF does not alter the expression of GABA and glutamate ionotropic receptors, it does raise the expression of the recently cloned K(+)/Cl(-) KCC2 co-transporter, which is responsible for the conversion of GABA responses from depolarizing to inhibitory, through the control of the Cl(-) potential. Together, results indicate that both the presynaptic and postsynaptic machineries of GABAergic circuits may be essential targets of BDNF actions to control spontaneous activity. The data indicate that BDNF is a potent regulator of spontaneous activity and co-active networks, which is a new level of regulation of neurotrophins. Given that BDNF itself is regulated by neuronal activity, we suggest that BDNF acts as a homeostatic factor controlling the emergence, complexity and networking properties of spontaneous networks.     

NMDA receptors in GABAergic synapses during postnatal development

GABA (gamma-aminobutyric-acid), the main inhibitory neurotransmitter in the adult brain, exerts depolarizing (excitatory) actions during development and this GABAergic depolarization cooperates with NMDARs (N-methyl-D-aspartate receptors) to drive spontaneous synchronous activity (SSA) that is fundamentally important for developing neuronal networks. Although GABAergic depolarization is known to assist in the activation of NMDARs during development, the subcellular localization of NMDARs relative to GABAergic synapses is still unknown. Here, we investigated the subcellular distribution of NMDARs in association with GABAergic synapses at the developmental stage when SSA is most prominent in mice. Using multiple immunofluorescent labeling and confocal laser-scanning microscopy in the developing mouse hippocampus, we found that NMDARs were associated with both glutamatergic and GABAergic synapses at postnatal day 6-7 and we observed a direct colocalization of GABA(A)- and NMDA-receptor labeling in GABAergic synapses. Electron microscopy of pre-embedding immunogold-immunoperoxidase reactions confirmed that GluN1, GluN2A and GluN2B NMDAR subunits were all expressed in glutamatergic and GABAergic synapses postsynaptically. Finally, quantitative post-embedding immunogold labeling revealed that the density of NMDARs was 3 times higher in glutamatergic than in GABAergic synapses. Since GABAergic synapses were larger, there was little difference in the total number of NMDA receptors in the two types of synapses. In addition, receptor density in synapses was substantially higher than extrasynaptically. These data can provide the neuroanatomical basis of a new interpretation of previous physiological data regarding the GABA(A)R-NMDAR cooperation during early development. We suggest that during SSA, synaptic GABA(A)R-mediated depolarization assists NMDAR activation right inside GABAergic synapses and this effective spatial cooperation of receptors and local change of membrane potential will reach developing glutamatergic synapses with a higher probability and efficiency even further away on the dendrites. This additional level of cooperation that operates within the depolarizing GABAergic synapse, may also allow its own modification triggered by Ca(2+)-influx through the NMDA receptors.     

Disruption of KCC2 reveals an essential role of K-Cl cotransport already in early synaptic inhibition

Synaptic inhibition by GABA(A) and glycine receptors, which are ligand-gated anion channels, depends on the electrochemical potential for chloride. Several potassium-chloride cotransporters can lower the intracellular chloride concentration [Cl(-)](i), including the neuronal isoform KCC2. We show that KCC2 knockout mice died immediately after birth due to severe motor deficits that also abolished respiration. Sciatic nerve recordings revealed abnormal spontaneous electrical activity and altered spinal cord responses to peripheral electrical stimuli. In the spinal cord of wild-type animals, the KCC2 protein was found at inhibitory synapses. Patch-clamp measurements of embryonic day 18.5 spinal cord motoneurons demonstrated an excitatory GABA and glycine action in the absence, but not in the presence, of KCC2, revealing a crucial role of KCC2 for synaptic inhibition.     

K+-Cl− co-transporter 2 (KCC2) – a membrane trafficking perspective

K⁺-Cl^? co-transporter 2 (KCC2/SLC12A5) is a neuronal specific cation chloride co-transporter which is active under isotonic conditions, and thus a key regulator of intracellular Cl^? levels. It also has an ion transporter-independent structural role in modulating the maturation and regulation of excitatory glutamatergic synapses. KCC2 levels are developmentally regulated, and a postnatal upregulation of KCC2 generates a low intracellular chloride concentration that allows the neurotransmitters γ-aminobutyric acid (GABA) and glycine to exert inhibitory neurotransmission through its Cl^? permeating channel. Functional expression of KCC2 at the neuronal cell surface is necessary for its activity, and impairment in KCC2 cell surface transport and/or internalization may underlie a range of neuropathological conditions. Although recent advances have shed light on a range of cellular mechanisms regulating KCC2 activity, little is known about its membrane trafficking itinerary and regulatory proteins. In this review, known membrane trafficking signals, pathways and mechanisms pertaining to KCC2’s functional surface expression are discussed.     

Differential expression pattern of chloride transporters<Emphasis Type="Italic"> NCC,NKCC2, KCC1, KCC3, KCC4,</Emphasis> and<Emphasis Type="Italic"> AE3</Emphasis> in the developing rat auditory brainstem

During development of inhibitory synapses, the action of the two neurotransmitters GABA and glycine shifts from depolarizing to hyperpolarizing. The shift is due to an age-dependent regulation of the intracellular free chloride concentration ([Cl(-)](i)) in postsynaptic neurons. A model system to study this maturation process is a glycinergic projection in the mammalian auditory brainstem. It is formed in the superior olivary complex (SOC) by neurons of the medial nucleus of the trapezoid body, whose axons terminate in the lateral superior olive (LSO). LSO neurons of perinatal rats and mice are depolarized upon glycine application, whereas older cells (>postnatal day (P) 8) are hyperpolarized. Here we examined the expression of six secondary active chloride transporter genes ( NCC, NKCC2, KCC1, KCC3, KCC4, and AE3) in the rat SOC to unravel the molecular mechanisms underlying this change. RT-PCR analysis demonstrated brainstem expression of KCC1, KCC3, KCC4, and AE3, but not of NCC and NKCC2. RNA in situ hybridization showed that only AE3 is highly expressed both at P3 (high [Cl(-)](i)) and P12 (low [Cl(-)](i)) in LSO neurons. KCC1 and KCC4 are weakly expressed in LSO neurons at P3 and P12, respectively. This study completes the expression analysis of all known chloride transporters sensitive to loop diuretic drugs in the SOC and demonstrates differences in the maturation between hippocampal and brainstem inhibitory synapses.     

Inhibitory synaptic transmission tuned by Ca2+ and glutamate through the control of GABAAR lateral diffusion dynamics

The GABAergic synapses, a primary inhibitory synapse in the mammalian brain, is important for the normal development of brain circuits, and for the regulation of the excitation-inhibition balance critical for brain function from the developmental stage throughout life. However, the molecular mechanism underlying the formation, maintenance, and modulation of GABAergic synapses is less understood compared to that of excitatory synapses. Quantum dot-single particle tracking (QD-SPT), a super-resolution imaging technique that enables the analysis of membrane molecule dynamics at single-molecule resolution, is a powerful tool to analyze the behavior of proteins and lipids on the plasma membrane. In this review, we summarize the recent application of QD-SPT in understanding of GABAergic synaptic transmission. Here we introduce QD-SPT experiments that provide further insights into the molecular mechanism supporting GABAergic synapses. QD-SPT studies revealed that glutamate and Ca²⁺ signaling is involved in (a) the maintenance of GABAergic synapses, (b) GABAergic long-term depression, and GABAergic long-term potentiation, by specifically activating signaling pathways unique to each phenomenon. We also introduce a novel Ca²⁺ imaging technique to describe the diversity of Ca²⁺ signals that may activate the downstream signaling pathways that induce specific biological output.     

Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex

Calcium-activated chloride channels of the anoctamin (alias TMEM16) protein family fulfill critical functions in epithelial fluid transport, smooth muscle contraction and sensory signal processing. Little is known, however, about their contribution to information processing in the central nervous system. Here we examined the recent finding that a calcium-dependent chloride conductance impacts on GABAergic synaptic inhibition in Purkinje cells of the cerebellum. We asked whether anoctamin channels may underlie this chloride conductance. We identified two anoctamin channel proteins, ANO1 and ANO2, in the cerebellar cortex. ANO1 was expressed in inhibitory interneurons of the molecular layer and the granule cell layer. Both channels were expressed in Purkinje cells but, while ANO1 appeared to be retained in the cell body, ANO2 was targeted to the dendritic tree. Functional studies confirmed that ANO2 was involved in a calcium-dependent mode of ionic plasticity that reduces the efficacy of GABAergic synapses. ANO2 channels attenuated GABAergic transmission by increasing the postsynaptic chloride concentration, hence reducing the driving force for chloride influx. Our data suggest that ANO2 channels are involved in a Ca²⁺-dependent regulation of synaptic weight in GABAergic inhibition. Thus, in balance with the chloride extrusion mechanism via the co-transporter KCC2, ANO2 appears to regulate ionic plasticity in the cerebellum.     

Balanced GABAergic and glutamatergic synapse development in hippocampal neurons

Coordinated development of excitatory and inhibitory synapses is crucial for normal function of neuronal circuits. Using homo- and heterochronic cultures of hippocampal neurons, we compared the formation of glutamatergic and GABAergic synapses at different stages and asked whether the age of dendrites affects their ability to accept new glutamatergic and GABAergic synapses. Neurons were transfected with either CFP-actin as a dendritic marker or GFP-synaptophysin as a presynaptic marker. We found that GFP-synaptophysin clusters formed on CFP-actin-labeled dendrites at similar density regardless of pre- and postsynaptic cell type or the age of dendrites (0-2 weeks) upon co-culturing. Therefore, the age of mature dendrites does not affect their ability to accept new synapses. Because GABAergic transmission switches from depolarizing to hyperpolarizing during 1-2 weeks in these cultures, our observations also suggest that this developmental switch does not alter the formation of GABAergic synapses.     

A variant of KCC2 from patients with febrile seizures impairs neuronal Cl− extrusion and dendritic spine formation

Genetic variation in SLC12A5 which encodes KCC2, the neuron-specific cation-chloride cotransporter that is essential for hyperpolarizing GABAergic signaling and formation of cortical dendritic spines, has not been reported in human disease. Screening of SLC12A5 revealed a co-segregating variant (KCC2-R952H) in an Australian family with febrile seizures. We show that KCC2-R952H reduces neuronal Cl⁻ extrusion and has a compromised ability to induce dendritic spines in vivo and in vitro. Biochemical analyses indicate a reduced surface expression of KCC2-R952H which likely contributes to the functional deficits. Our data suggest that KCC2-R952H is a bona fide susceptibility variant for febrile seizures.