Search

The K+-Cl–cotransporter KCC2, encoded by theSlc12a5gene, is a neuron-specific chloride extruder that tunes the strength and polarity of GABAAreceptor-mediated transmission. In addition to its canonical ion transport function, KCC2 also regulates spinogenesis and excitatory synaptic function through interaction with a variety of molecular partners. KCC2 is enriched in the vicinity of both glutamatergic and GABAergic synapses, the activity of which in turn regulates its membrane stability and function. KCC2 interaction with the submembrane actin cytoskeleton via 4.1N is known to control its anchoring near glutamatergic synapses on dendritic spines. However, the molecular determinants of KCC2 clustering near GABAergic synapses remain unknown. Here, we used proteomics to identify novel KCC2 interacting proteins in the adult rat neocortex. We identified both known and novel candidate KCC2 partners, including some involved in neuronal development and synaptic transmission. These include gephyrin, the main scaffolding molecule at GABAergic synapses. Gephyrin interaction with endogenous KCC2 was confirmed by immunoprecipitation from rat neocortical extracts. We showed that gephyrin stabilizes plasmalemmal KCC2 and promotes its clustering in hippocampal neurons, mostly but not exclusively near GABAergic synapses, thereby controlling KCC2-mediated chloride extrusion. This study identifies gephyrin as a novel KCC2 anchoring molecule that regulates its membrane expression and function in cortical neurons.SIGNIFICANCE STATEMENTFast synaptic inhibition in the brain is mediated by chloride-permeable GABAAreceptors (GABAARs) and therefore relies on transmembrane chloride gradients. In neurons, these gradients are primarily maintained by the K/Cl cotransporter KCC2. Therefore, understanding the mechanisms controlling KCC2 expression and function is crucial to understand its physiological regulation and rescue its function in the pathology. KCC2 function depends on its membrane expression and clustering, but the underlying mechanisms remain unknown. We describe the interaction between KCC2 and gephyrin, the main scaffolding protein at inhibitory synapses. We show that gephyrin controls plasmalemmal KCC2 clustering and that loss of gephyrin compromises KCC2 function. Our data suggest functional units comprising GABAARs, gephyrin, and KCC2 act to regulate synaptic GABA signaling.

An upregulation of the Na+-K+-2Cl-co-transporter NKCC1, the main chloride importer in mature neurons, can lead to depolarizing/excitatory responses mediated by GABAAreceptors and thus to hyperactivity. Understanding the regulatory mechanisms of NKCC1 would help prevent intra-neuronal chloride accumulation that occurs in pathologies with defective inhibition. The cellular and molecular regulatory mechanisms of NKCC1 are poorly understood. Here, we report in mature hippocampal neurons that GABAergic activity controls the membrane diffusion and clustering of NKCC1 via the chloride-sensitive WNK1 kinase and the downstream SPAK kinase that directly phosphorylates NKCC1 on key threonine residues. At rest, this signaling pathway has little effect on intracellular Cl-concentration but it participates to the elevation of intraneuronal Cl-concentration in hyperactivity condition associated with an up-regulation of NKCC1. The fact that the chloride exporter KCC2 is also regulated in mature neurons by the WNK1 pathway indicates that this pathway will be a target of choice in the pathology.

SummaryNKCC1 and KCC2 transporters regulate neuronal chloride homeostasis and thus synaptic inhibition. KCC2 activity is tuned by diffusion-capture in an activity-dependent manner. The mechanisms controlling NKCC1 in neurons are unknown. We found using super-resolution imaging that NKCC1 like KCC2 form nanodomains in the somato-dendritic membrane at extrasynaptic sites and at the periphery of excitatory and inhibitory synapses. NKCC1 nanoclusters are half the size and density in molecules than KCC2 clusters. This is accompanied by a higher mobility of NKCC1 compared to KCC2 in the dendritic membrane, suggesting a weaker NKCC1 anchoring to the cytoskeleton. In contrast, NKCC1, but not KCC2, is confined to endocytic zones, which would explain its controlled surface expression and the fact that endocytic zones would provide a reservoir from which NKCC1 could be released into the membrane. Finally, we show an increased confinement of NKCC1 in axons but not in dendrites upon glutamatergic activity blockade, indicating a selective mechanism of regulation in the axon. We propose that a rapid regulation of NKCC1 by lateral diffusion in the axon would control presynaptic glutamate release and the firing of action potentials.

Many psychiatric diseases including depression, schizophrenia and anxiety have been associated with serotonin (5-HT) neuron dysfunction. Pacemaker-like firing of raphe 5-HT neurons was proposed to be under unique 5-HT1Areceptor-mediated autoinhibition. We previously showed that 5-HT2Breceptors were expressed by 5-HT neurons together with 5-HT1Areceptors. However, functional consequences on 5-HT neurons of putative interaction between these receptors are unknown. Using co-immunoprecipitation, BRET, confocal and super-resolution microscopy in hippocampal and 5-HT neurons, we present converging evidence that 5-HT1Aand 5-HT2Breceptors can form heterodimers and co-cluster at the surface of dendrites. 5-HT2Breceptor clusters were redistributed upon 5-HT1Areceptor expression supporting functional interactions between the two receptors. Furthermore, 5-HT2Breceptor expression prevented agonist-induced internalization of 5-HT1Areceptors, whereas 5-HT1Areceptors mimicked the clustering effect of 5-HT2Breceptor stimulation on its surface expression. The functional impact of this interactionin-vivowas assessed by recording 5-HT neuron excitability from mice lacking 5-HT2Breceptors in 5-HT neurons. Upon 5-HT1Areceptor stimulation, the firing activity of 5-HT neurons was increased in the absence of 5-HT2Breceptors and decreased in their presence through regulation of SK channels, thus demonstrating functional output of this interaction in controlling 5-HT neuron firing activity.

ATP signaling and surface P2X4 receptors are upregulated selectively in neurons and/or glia in various CNS disorders including anxiety, chronic pain, epilepsy, ischemia, and neurodegenerative diseases. However, the cell-specific functions of P2X4 in pathological contexts remain elusive. To elucidate P2X4 functions, we created a conditional transgenic knock-in P2X4 mouse line (Floxed P2X4mCherryIN) allowing the Cre activity-dependent genetic swapping of the internalization motif of P2X4 by the fluorescent mCherry protein to prevent constitutive endocytosis of P2X4. By combining molecular, cellular, electrophysiological, and behavioral approaches, we characterized two distinct knock-in mouse lines expressing noninternalized P2X4mCherryIN either exclusively in excitatory forebrain neurons or in all cells natively expressing P2X4. The genetic substitution of wild-type P2X4 by noninternalized P2X4mCherryIN in both knock-in mouse models did not alter the sparse distribution and subcellular localization of P2X4 but increased the number of P2X4 receptors at the surface of the targeted cells mimicking the pathological increased surface P2X4 state. Increased surface P2X4 density in the hippocampus of knock-in mice altered LTP and LTD plasticity phenomena at CA1 synapses without affecting basal excitatory transmission. Moreover, these cellular events translated into anxiolytic effects and deficits in spatial memory. Our results show that increased surface density of neuronal P2X4 contributes to synaptic deficits and alterations in anxiety and memory functions consistent with the implication of P2X4 in neuropsychiatric and neurodegenerative disorders. Furthermore, these conditional P2X4mCherryIN knock-in mice will allow exploring the cell-specific roles of P2X4 in various physiological and pathological contexts.

Synapse stabilization Early in brain development, neurons connect to each other enthusiastically. With development, an overabundance of synapses is winnowed down to refine efficiently connected circuits. Inactive synapses are prime targets for elimination, whereas active synapses tend to be retained. Gomez-Castro et al . took a closer look at how those choices are made (see the Perspective by Blum and Lopes). When postsynaptic adenosine receptors are muted or do not find enough extracellular adenosine, synapses get eliminated. Neurotransmitter-dependent signaling pathways drive protein kinase A to phosphorylate the postsynaptic scaffolding molecule gephyrin. Together with a partner synaptogenic membrane protein, gephyrin is required for the stabilization of γ-aminobutyric acid receptors. Adenosine receptors thus detect synaptic activity and in turn drive the stabilization of synapses that produce such activity. —PJH

International audience; The serotonin 2B (5-HT2B) receptor coupled to Gq-protein contributes to the control of neuronal excitability and is implicated in various psychiatric disorders. The mechanisms underlying its brain function are not fully described. Using peptide affinity chromatography combined with mass spectrometry, we found that the PDZ binding motif of the 5-HT2B receptor located at its C-terminal end interacts with the scaffolding protein channel interacting PDZ protein (CIPP). We then showed, in COS-7 cells, that the association of the 5-HT2B receptor to CIPP enhanced receptor-operated inositol phosphate (IP) production without affecting its cell surface and intracellular levels. Co-immunoprecipitation experiments revealed that CIPP, the 5-HT2B receptor, and the NR1 subunit of the NMDA receptor form a macromolecular complex. CIPP increased 5-HT2B receptor clustering at the surface of primary cultured hippocampal neurons and prevented receptor dispersion following agonist stimulation, thus potentiating IP production and intracellular calcium mobilization in dendrites. CIPP or 5-HT2B receptor stimulation in turn dispersed NR1 clusters colocalized with 5-HT2B receptors and increased the density and maturation of dendritic spines. Collectively, our results suggest that the 5-HT2B receptor, the NMDA receptor, and CIPP may form a signaling platform by which serotonin can influence structural plasticity of excitatory glutamatergic synapses.

International audience; This chapter explores non-canonical functions of the neuronal chloride/potassium KCC2 transporter at glutamatergic, excitatory synapses. The first section describes KCC2 expression and membrane dynamics in cortical neurons to show that KCC2 is enriched in dendritic spines that host excitatory synapses. Then, it reviews KCC2 protein binding partners, with a specific focus on those that may contribute to the specific confinement and role of KCC2 in dendritic spines. With this background, the next section describes how KCC2 contributes to both dendritic spine morphology and excitatory synaptic function and plasticity. The chapter ends with a discussion on the implications of the multiple functions of KCC2, in particular with respect to the pathology.

In biological membranes many factors such as cytoskeleton, lipid composition, crowding and molecular interactions deviate lateral diffusion from the expected random walks. These factors have different effects on diffusion but act simultaneously so the observed diffusion is a complex mixture of diffusive behaviors (directed, >Brownian, anomalous or confined). Therefore commonly used approaches to quantify diffusion based on averaging of the displacements, such as the mean square displacement, are not adapted to the analysis of this heterogeneity. We introduce a new parameter, the packing coefficient Pc, which gives an estimate of the degree of free movement that a molecule displays in a period of time independently of its global diffusivity. Applying this approach to two different situations (diffusion of a lipid probe and trapping of receptors at synapses), we show that Pc detected and localized temporary changes of diffusive behavior both in time and in space. More importantly, it allowed the detection of periods with very high confinement (~immobility), their frequency and duration, and thus it can be used to calculate the effective kon and koff of scaffolding interactions such those that immobilize receptors at synapses.

ATP signaling and surface P2X4 receptors are upregulated selectively in neurons and/or glia in various CNS disorders including anxiety, chronic pain, epilepsy, ischemia, and neurodegenerative diseases. However, the cell-specific functions of P2X4 in pathological contexts remain elusive. To elucidate P2X4 functions, we created a conditional transgenic knock-in P2X4 mouse line (Floxed P2X4mCherryIN) allowing the Cre activity-dependent genetic swapping of the internalization motif of P2X4 by the fluorescent mCherry protein to prevent constitutive endocytosis of P2X4. By combining molecular, cellular, electrophysiological, and behavioral approaches, we characterized two distinct knock-in mouse lines expressing noninternalized P2X4mCherryIN either exclusively in excitatory forebrain neurons or in all cells natively expressing P2X4. The genetic substitution of wild-type P2X4 by noninternalized P2X4mCherryIN in both knock-in mouse models did not alter the sparse distribution and subcellular localization of P2X4 but increased the number of P2X4 receptors at the surface of the targeted cells mimicking the pathological increased surface P2X4 state. Increased surface P2X4 density in the hippocampus of knock-in mice altered LTP and LTD plasticity phenomena at CA1 synapses without affecting basal excitatory transmission. Moreover, these cellular events translated into anxiolytic effects and deficits in spatial memory. Our results show that increased surface density of neuronal P2X4 contributes to synaptic deficits and alterations in anxiety and memory functions consistent with the implication of P2X4 in neuropsychiatric and neurodegenerative disorders. Furthermore, these conditional P2X4mCherryIN knock-in mice will allow exploring the cell-specific roles of P2X4 in various physiological and pathological contexts.

Neuronal Cl- homeostasis is regulated by the activity of two cation chloride co-transporters (CCCs), the K+-Cl- cotransporter KCC2 and the Na+-K+-Cl- cotransporter NKCC1, which are primarily extruding and importing chloride in neurons, respectively. Several neurological and psychiatric disorders including epilepsy, neuropathic pain, schizophrenia and autism are associated with altered neuronal chloride (Cl-) homeostasis. A current view is that the accumulation of intracellular Cl- in neurons as a result of KCC2 down-regulation and/or NKCC1 up-regulation may weaken inhibitory GABA signaling and thereby promote the development of pathological activities. CCC activity is determined mainly by their level of expression in the plasma membrane. Furthermore, CCCs undergo "diffusion-trapping" in the membrane, a mechanism that is rapidly adjusted by activity-dependent post-translational modifications i.e. phosphorylation/dephosphorylation of key serine and threonine residues. This represents probably the most rapid cellular mechanism for adapting CCC function to changes in neuronal activity. Therefore, interfering with these mechanisms may help restoring Cl- homeostasis and inhibition under pathological conditions. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'.

The fundamental role of the brain-specific myelin transcription factor 1-like (MYT1L) gene in cases of intellectual disability and in the aetiology of neurodevelopmental disorders is increasingly recognised. Yet, its function remains under-investigated. Here, we identify a network of helix-loop-helix (HLH) transcriptional regulators controlled by MYT1L, as indicated by our analyses in human neural stem cells and in the human brain. Using cell-based knockdown approaches and microarray analyses we found that (i) MYT1L is required for neuronal differentiation and identified ID1, a HLH inhibitor of premature neurogenesis, as a target. (2) While MYT1L prevented expression of ID1, it induced expression of a large number of terminal differentiation genes. (3) Consistently, expression of MYT1L in the human brain coincided with neuronal maturation and inversely correlated with that of ID1 and ID3 throughout the lifespan. (4) Genetic polymorphisms that reduced expression of MYT1L in the hippocampus resulted in increased expression of ID1 and ID3, decreased levels of the proneural basic HLH (bHLH) transcriptional regulators TCF4 and NEUROD6 and decreased expression of genes involved in long-term potentiation and synaptic transmission, cancer and neurodegeneration. Furthermore, our neuroimaging analyses indicated that MYT1L expression associated with hippocampal volume and activation during episodic memory recall, as measured by blood-oxygen-level dependent (BOLD) signals. Overall, our findings suggest that MYT1L influences memory-related processes by controlling a neuronal proliferation/differentiation switch of ID-bHLH factors.Neuropsychopharmacology accepted article preview online, 04 May 2017. doi:10.1038/npp.2017.91.

Synaptic plasticity relies on the rapid changes in neurotransmitter receptor number at postsynaptic sites. Using superresolution photoactivatable localization microscopy imaging and quantum dot–based single-particle tracking in rat hippocampal cultured neurons, we investigated whether the phosphorylation status of the main scaffolding protein gephyrin influenced the organization of the gephyrin scaffold and GABAA receptor (GABAAR) membrane dynamics. We found that gephyrin phosphorylation regulates gephyrin microdomain compaction. Extracellular signal–regulated kinase 1/2 and glycogen synthase kinase 3β (GSK3β) signaling alter the gephyrin scaffold mesh differentially. Differences in scaffold organization similarly affected the diffusion of synaptic GABAARs, suggesting reduced gephyrin receptor–binding properties. In the context of synaptic scaling, our results identify a novel role of the GSK3β signaling pathway in the activity-dependent regulation of extrasynaptic receptor surface trafficking and GSK3β, protein kinase A, and calcium/calmodulin-dependent protein kinase IIα pathways in facilitating adaptations of synaptic receptors.

The K-Cl cotransporter KCC2 plays an essential role in neuronal chloride homeostasis, and thereby influences the efficacy and polarity of GABA signaling. Although KCC2 is expressed throughout the somatodendritic membrane, it is remarkably enriched in dendritic spines, which host most glutamatergic synapses in cortical neurons. KCC2 has been shown to influence spine morphogenesis and functional maturation in developing neurons, but its function in mature dendritic spines remains unknown. Here, we report that suppressing KCC2 expression decreases the efficacy of excitatory synapses in mature hippocampal neurons. This effect correlates with a reduced postsynaptic aggregation of GluR1-containing AMPA receptors and is mimicked by a dominant negative mutant of KCC2 interaction with cytoskeleton but not by pharmacological suppression of KCC2 function. Single-particle tracking experiments reveal that suppressing KCC2 increases lateral diffusion of the mobile fraction of AMPA receptor subunit GluR1 in spines but not in adjacent dendritic shafts. Increased diffusion was also observed for transmembrane but not membrane-anchored recombinant neuronal cell adhesion molecules. We suggest that KCC2, likely through interactions with the actin cytoskeleton, hinders transmembrane protein diffusion, and thereby contributes to their confinement within dendritic spines.

Expression of the neuronal K/Cl transporter KCC2 is tightly regulated throughout development and by both normal and pathological neuronal activity. Changes in KCC2 expression have often been associated with altered chloride homeostasis and GABA signaling. However, recent evidence supports a role of KCC2 in the development and function of glutamatergic synapses through mechanisms that remain poorly understood. Here we show that suppressing KCC2 expression in rat hippocampal neurons precludes long-term potentiation of glutamatergic synapses specifically by preventing activity-driven membrane delivery of AMPA receptors. This effect is independent of KCC2 transporter function and can be accounted for by increased Rac1/PAK- and LIMK-dependent cofilin phosphorylation and actin polymerization in dendritic spines. Our results demonstrate that KCC2 plays a critical role in the regulation of spine actin cytoskeleton and gates long-term plasticity at excitatory synapses in cortical neurons.SIGNIFICANCE STATEMENTChanges in the expression of neuronal chloride transporters, such as KCC2, occur during postnatal development and are induced in a variety of neurological and psychiatric conditions. Such changes are expected to primarily impact GABA signaling because GABAA receptors are predominantly permeable to chloride ions. However, the KCC2 transporter forms clusters near glutamatergic synapses and interacts with several actin-related proteins. We show that KCC2 is strictly required for LTP expression at hippocampal excitatory synapses. This effect is due to KCC2 interaction with the Rac1/PAK signaling pathway that controls actin polymerization. Suppressing this interaction promotes actin polymerization thereby hindering AMPA receptor traffic upon KCC2 suppression. Alterations of KCC2 expression therefore impact not only GABAergic signaling but also glutamatergic synaptic function and long term plasticity.

The role of the scaffolding protein gephyrin at hippocampal inhibitory synapses is not well understood. A previous study (Kneussel et al., 1999) reported a complete loss of synaptic clusters of the major GABAAR subunits α2 and γ2 in hippocampal neurons lacking gephyrin. In contrast, we show here that GABAAR α2 and γ2 subunits do cluster at pyramidal synapses in hippocampal cultures from gephyrin-/- mice, albeit at reduced levels compared with control neurons. Synaptic aggregation of GABAAR α1 on interneurons was identical between the culture types. Furthermore, we recorded miniature IPSCs (mIPSCs) from gephyrin-/- neurons. Although the mean mIPSC amplitude was reduced (by 23%) compared with control, the frequency of these events was unchanged. Cell surface labeling experiments indicated that gephyrin contributes, in part, to aggregation but not to insertion or stabilization of GABAAR α2 and γ2 in the plasma membrane. Thus, a major gephyrin-independent component of hippocampal inhibitory synapse development must exist. We also report that glycine receptors cluster at GABAergic synapses in a subset of hippocampal interneurons and pyramidal neurons. Unlike GABAARs, synaptic clustering of glycine receptors was completely abolished in gephyrin-/- neurons. Finally, artificial extrasynaptic aggregation of GABAAR was able to redistribute and cocluster gephyrin by a mechanism requiring a neuron-specific modification or intermediary protein. We propose a model of hippocampal inhibitory synapse development in which some GABAARs cluster at synapses by a gephyrin-independent mechanism and recruit gephyrin. This clustered gephyrin may then recruit glycine receptors, additional GABAARs, and other signal-transducing components.

The murine genome contains approximately 70 protocadherin (Pcdh) genes. Many are expressed in the nervous system, suggesting that Pcdhs may specify neuronal connectivity. Here, we analyze the 22 contiguous genes of the Pcdh-gamma cluster. Individual neurons express subsets of Pcdh-gamma genes. Pcdh-gamma proteins are present in most neurons and associated with, but not confined to, synapses. Early steps in neuronal migration, axon outgrowth, and synapse formation proceed in mutant mice lacking all 22 Pcdh-gamma genes. At late embryonic stages, however, dramatic neurodegeneration leads to neonatal death. In mutant spinal cord, many interneurons are lost, but sensory and motor neurons are relatively spared. In cultures from mutant spinal cord, neurons differentiate and form synapses but then die. Thus, Pcdh-gamma genes are dispensable for at least some aspects of connectivity but required for survival of specific neuronal types.

The dystrophin glycoprotein complex (DGC) is a multimolecular complex that links the extracellular matrix to the cytoskeleton. The DGC is present at the skeletal neuromuscular junction and required for its maturation and maintenance. Members of the DGC are also expressed in brain. We used cultured hippocampal neurons to analyze the distribution, regulation, and role in synaptogenesis of the major transmembrane component of the DGC, dystroglycan; one of its extracellular ligands, agrin; and one of its cytoskeletal binding partners, dystrophin. alpha-Dystroglycan, beta-dystroglycan, and dystrophin clustered at a subset of inhibitory synapses containing GABA(A)R subunits alpha1, alpha2, and gamma2, and the inhibitory receptor anchoring protein gephyrin. DGC components were not detected at excitatory glutamatergic synapses. Dystroglycan is the first identified adhesive macromolecule at mature GABA synapses. Developmentally, dystroglycan clustered at synaptic loci after synaptic vesicles, GABA(A)R, and gephyrin, the latter being closely associated with GABA(A)R at all stages of synaptogenesis analyzed. Analysis of gephyrin -/-, agrin -/-, and mdx mouse hippocampal neurons in culture indicated that synaptic clustering of dystroglycan occurs independently of gephyrin, agrin, and dystrophin. In dystroglycan-deficient neurons, cultured from a conditional mutant strain, GABAergic synapses differentiated with clusters of gephyrin and GABA(A)R apposed to synaptic terminals, but these synapses did not contain detectable dystrophin. Thus the DGC is not essential for GABAergic synaptogenesis but is likely to function in modulating inhibitory synapses or conferring specialized properties on a subset of them.

In the spinal cord, GABA and glycine mediate inhibition at separate or mixed synapses containing glycine and/or GABA(A) receptors (GlyR and GABA(A)R, respectively). We have analysed here the sequence of events leading to inhibitory synapse formation during synaptogenesis of embryonic spinal cord neurons between 1 and 11 days in vitro (DIV). We used immunocytochemical methods to detect simultaneously an antigen specific to inhibitory terminals, the vesicular inhibitory amino acid transporter (VIAAT), and one of the following postsynaptic elements: GlyR, GABA(A)R or gephyrin, the anchoring protein of GlyR, which is also associated with GABA(A)R. Quantitative analysis revealed that until 5 DIV most gephyrin clusters were not adjacent to VIAAT-positive profiles, but became associated with them at later stages. In contrast, GlyR and GABAAR clustered predominantly in front of VIAAT-containing terminals at all stages. However, about 10% of receptor aggregates were detected at nonsynaptic loci. The two receptors colocalized in 66.2+/-2.5% of the inhibitory postsynaptic domains after 11 DIV, while 30.3+/-2.6% and 3.4+/-0.8% of them contained only GlyR and GABA(A)R, respectively. Interestingly, at 3 DIV GABA(A)R clustered at a postsynaptic location prior to gephyrin and GlyR; GABA(A)R could thus be the initiating element in the construction of mixed glycine and GABAergic synapses. The late colocalization of gephyrin with GABA(A)R, and the demonstration by other groups that, in the absence of gephyrin, postsynaptic GABA(A)R is not detected, suggest that gephyrin is involved in the stabilization of GABA(A)R rather than in its initial accumulation at synaptic sites.

Benzodiazepines (BZDs) are widely used in the treatment of a variety of neurological and psychiatric conditions including anxiety, insomnia and epilepsy. BZDs are thought to act predominantly by affecting the gating of GABAA receptor channels, resulting in enhanced GABA-mediated currents in neurons. However, mutations mimicking the effect of BZDs on GABAAR channel gating have been shown to also impact the membrane dynamics and synaptic anchoring of the receptors. Here, using single molecule tracking combined with electrophysiological recordings, we show that BZD ligands rapidly influence the dynamic behavior of GABAARs in hippocampal neurons. Application of the inverse BZD agonist DMCM rapidly increased the diffusion and reduced the clustering of GABAARs at synapses, resulting in reduced postsynaptic currents. Conversely, the BZD full agonist diazepam had little effect at rest but reduced lateral diffusion and increased synaptic stabilization and clustering of GABAARs upon sustained neuronal activity, resulting in enhanced potency of inhibitory synapses. These effects occurred in the absence of detectable changes in gephyrin clusters, suggesting they did not reflect a rapid dispersion of the synaptic scaffold. Thus, alterations of the diffusion and synaptic anchoring of GABAARs represent a novel, unsuspected mechanism through which BZDs rapidly modulate GABA signaling in central neurons.

The neuronal K/Cl transporter KCC2 exports chloride ions and thereby influences the efficacy and polarity of GABA signaling in the brain. KCC2 is also critical for dendritic spine morphogenesis and the maintenance of glutamatergic transmission in cortical neurons. Because KCC2 plays a pivotal role in the function of central synapses, it is of particular importance to understand the cellular and molecular mechanisms underlying its regulation. Here, we studied the impact of membrane diffusion and clustering on KCC2 function. KCC2 forms clusters in the vicinity of both excitatory and inhibitory synapses. Using quantum-dot-based single-particle tracking on rat primary hippocampal neurons, we show that KCC2 is slowed down and confined at excitatory and inhibitory synapses compared with extrasynaptic regions. However, KCC2 escapes inhibitory synapses faster than excitatory synapses, reflecting stronger molecular constraints at the latter. Interfering with KCC2–actin interactions or inhibiting F-actin polymerization releases diffusion constraints on KCC2 at excitatory but not inhibitory synapses. Thus, F-actin constrains KCC2 diffusion at excitatory synapses, whereas KCC2 is confined at inhibitory synapses by a distinct mechanism. Finally, increased neuronal activity rapidly increases the diffusion coefficient and decreases the dwell time of KCC2 at excitatory synapses. This effect involves NMDAR activation, Ca2+influx, KCC2 S940 dephosphorylation and calpain protease cleavage of KCC2 and is accompanied by reduced KCC2 clustering and ion transport function. Thus, activity-dependent regulation of KCC2 lateral diffusion and clustering allows for a rapid regulation of chloride homeostasis in neurons.

The K-Cl co-transporter KCC2 plays multiple roles in the physiology of central neurons and alterations of its function and/or expression are associated with several neurological conditions. By regulating intraneuronal chloride homeostasis, KCC2 strongly influences the efficacy and polarity of the chloride-permeable -aminobutyric acid (GABA) type A and glycine receptor (GlyR) mediated synaptic transmission. This appears particularly critical for the development of neuronal circuits as well as for the dynamic control of GABA and glycine signaling in mature networks. The activity of the transporter is also associated with transmembrane water fluxes which compensate solute fluxes associated with synaptic activity. Finally, KCC2 interaction with the actin cytoskeleton appears critical both for dendritic spine morphogenesis and the maintenance of glutamatergic synapses. In light of the pivotal role of KCC2 in the maturation and function of central synapses, it is of particular importance to understand the cellular and molecular mechanisms underlying its regulation. These include development- and activity-dependent modifications both at the transcriptional and post-translational levels. We emphasize the importance of post-translational mechanisms such as phosphorylation and dephosphorylation, oligomerization, cell surface stability, clustering and membrane diffusion for the rapid and dynamic regulation of KCC2 function.

Neuronal activity modulates the membrane diffusion of postsynaptic γ-aminobutyric acid (GABA)(A) receptors (GABA(A)Rs), thereby regulating the efficacy of GABAergic synapses. The K289M mutation in GABA(A)Rs subunit γ2 has been associated with the generalized epilepsy with febrile seizures plus (GEFS+) syndrome. This mutation accelerates receptor deactivation and therefore reduces inhibitory synaptic transmission. Yet, it is not clear why this mutation specifically promotes febrile seizures. We show that upon raising temperature both the number of GABA(A)Rs clusters and the frequency of miniature inhibitory postsynaptic currents decreased in neurons expressing the K289M mutant but not wild-type (WT) recombinant γ2. Single-particle tracking experiments revealed that raising temperature increases the membrane diffusion of synaptic GABA(A)Rs containing the K289M mutant but not WT recombinant γ2. This effect was mediated by enhanced neuronal activity as it was blocked by glutamate receptor antagonists and was mimicked by the convulsant 4-aminopyridine. Our data suggest the K289M mutation in γ2 confers GABA(A)Rs with enhanced sensitivity of their membrane diffusion to neuronal activity. Enhanced activity during hyperthermia may then trigger the escape of receptors from synapses and thereby further reduce the efficacy of GABAergic inhibition. Alteration of the membrane diffusion of neurotransmitter receptors therefore represents a new mechanism in human epilepsy.

Summary An activity-dependent change in synaptic efficacy is a central tenet in learning, memory, and pathological states of neuronal excitability. The lateral diffusion dynamics of neurotransmitter receptors are one of the important parameters regulating synaptic efficacy. We report here that neuronal activity modifies diffusion properties of type-A GABA receptors (GABA A R) in cultured hippocampal neurons: enhanced excitatory synaptic activity decreases the cluster size of GABA A Rs and reduces GABAergic mIPSC. Single-particle tracking of the GABA A R γ2 subunit labeled with quantum dots reveals that the diffusion coefficient and the synaptic confinement domain size of GABA A R increases in parallel with neuronal activity, depending on Ca 2+ influx and calcineurin activity. These results indicate that GABA A R diffusion dynamics are directly linked to rapid and plastic modifications of inhibitory synaptic transmission in response to changes in intracellular Ca 2+ concentration. This transient activity-dependent reduction of inhibition would favor the onset of LTP during conditioning.

SummaryIn the spinal cord, most inhibitory synapses have a mixed glycine-GABA phenotype. Using a pharmacological approach, we report an NMDAR activity-dependent regulation of the mobility of GlyRs but not GABAARs at inhibitory synapses in cultured rat spinal cord neurons. The NMDAR-induced decrease in GlyR lateral diffusion was correlated with an increase in receptor cluster number and glycinergic mIPSC amplitude. Changes in GlyR diffusion properties occurred rapidly and before the changes in the number of synaptic receptors. Regulation of synaptic GlyR content occurred without change in the amount of gephyrin. Moreover, NMDAR-dependent regulation of GlyR lateral diffusion required calcium influx and calcium release from stores. Therefore, excitation may increase GlyR levels at synapses by a calcium-mediated increase in postsynaptic GlyR trapping involving regulation of receptor-scaffold interactions. This provides a mechanism for a rapid homeostatic regulation of the inhibitory glycinergic component at mixed glycine-GABA synapses in response to increased NMDA excitatory transmission.

The dynamics of membrane proteins in living cells has become a major issue to understand important biological questions such as chemotaxis, synaptic regulation, or signal transduction. The advent of semi-conductor quantum dots (QDs) has opened new perspectives for the study of membrane properties because these new nanomaterials enable measurements at the single molecule level with high signal-to-noise ratio. Probes used until now indeed encounter significant limitations: organic fluorophores and fluorescent proteins rapidly photobleach, whereas gold particles and latex beads, although more stable, are bulky and usually stain only one protein per experiment. In comparison, QDs are bright and photostable fluorescent probes with a size on the order of 10 nm can be used with standard immunochemical methods. We present the experimental protocols and methods of analysis which we used to investigate the dynamics of individual GABAA receptors in the axonal growth cone of spinal neurons in culture. Single QD tracking is nevertheless a general method, suitable to study many transmembrane proteins.

The dynamics of membrane proteins in living cells has become a major issue to understand important biological questions such as chemotaxis, synaptic regulation, or signal transduction. The advent of semi-conductor quantum dots (QDs) has opened new perspectives for the study of membrane properties because these new nanomaterials enable measurements at the single molecule level with high signal-to-noise ratio. Probes used until now indeed encounter significant limitations: organic fluorophores and fluorescent proteins rapidly photobleach, whereas gold particles and latex beads, although more stable, are bulky and usually stain only one protein per experiment. In comparison, QDs are bright and photostable fluorescent probes with a size on the order of 10 nm can be used with standard immunochemical methods. We present the experimental protocols and methods of analysis which we used to investigate the dynamics of individual GABAA receptors in the axonal growth cone of spinal neurons in culture. Single QD tracking is nevertheless a general method, suitable to study many transmembrane proteins.

Lateral diffusion of neurotransmitter receptors in and out of synapses has been postulated as a core mechanism for rapid changes in receptor number at synapses during plastic processes. In this study, we have used single particle tracking to investigate how changes in glycine receptor (GlyR) lateral diffusion properties might account for changes in receptor number at synapses after disruption of the cytoskeleton in dissociated spinal cord neurons. We found that pharmacological disruption of F-actin and microtubules decreased the amount of GlyR and gephyrin, the backbone of the inhibitory postsynaptic scaffold, at synapses. F-actin and microtubule disruption increased GlyR exchanges between the synaptic and extrasynaptic membranes and decreased receptor dwell time at synapses. GlyR lateral diffusion was predominantly controlled by microtubules in the extrasynaptic membrane and by actin at synapses. Both diffusion coefficients and confinement at synapses were affected after F-actin disruption. Our results indicate that receptor exchanges between the synaptic and extrasynaptic compartments depend on the properties of both the postsynaptic differentiation and the extrasynaptic membrane. Consequently, GlyR number at synapses may be rapidly modulated by the cytoskeleton through the regulation of lateral diffusion in the plasma membrane and of receptor stabilization at synapses.

Semiconductor quantum dots (QDs) are nanometer-sized fluorescent probes suitable for advanced biological imaging. We used QDs to track individual glycine receptors (GlyRs) and analyze their lateral dynamics in the neuronal membrane of living cells for periods ranging from milliseconds to minutes. We characterized multiple diffusion domains in relation to the synaptic, perisynaptic, or extrasynaptic GlyR localization. The entry of GlyRs into the synapse by diffusion was observed and further confirmed by electron microscopy imaging of QD-tagged receptors.

The characterization of the Caenorhabditis elegans unc-47 gene recently allowed the identification of a mammalian (gamma)-amino butyric acid (GABA) transporter, presumed to be located in the synaptic vesicle membrane. In situ hybridization data in rat brain suggested that it might also take up glycine and thus represent a general Vesicular Inhibitory Amino Acid Transporter (VIAAT). In the present study, we have investigated the localization of VIAAT in neurons by using a polyclonal antibody raised against the hydrophilic N-terminal domain of the protein. Light microscopy and immunocytochemistry in primary cultures or tissue sections of the rat spinal cord revealed that VIAAT was localized in a subset (63-65%) of synaptophysin-immunoreactive terminal boutons; among the VIAAT-positive terminals around motoneuronal somata, 32.9% of them were also immunoreactive for GAD65, a marker of GABAergic presynaptic endings. Labelling was also found apposed to clusters positive for the glycine receptor or for its associated protein gephyrin. At the ultrastructural level, VIAAT immunoreactivity was restricted to presynaptic boutons exhibiting classical inhibitory features and, within the boutons, concentrated over synaptic vesicle clusters. Pre-embedding detection of VIAAT followed by post-embedding detection of GABA or glycine on serial sections of the spinal cord or cerebellar cortex indicated that VIAAT was present in glycine-, GABA- or GABA- and glycine-containing boutons. Taken together, these data further support the view of a common vesicular transporter for these two inhibitory transmitters, which would be responsible for their costorage in the same synaptic vesicle and subsequent corelease at mixed GABA-and-glycine synapses.

Listeria monocytogenes is an intracellular pathogen that causes severe central nervous system infection in humans and animals. The ability of this bacterium to penetrate nerve cells was investigated by using rat spinal cell cultures. Entry into distinct cell types, i.e., glial cells and neurons, was monitored by a differential immunofluorescence technique with antibodies against cell type-specific markers and the bacterial pathogen. L. monocytogenes was detected predominantly within macrophages constituting the microglia. Astrocytes and oligodendrocytes, the major components of macroglia, were infected to a lesser extent. Surprisingly, Listeria innocua , a noninvasive and nonpathogenic species, also has the capacity to enter into these three types of glial cells. Entry into neurons was a very rare event. In contrast, we found that L. monocytogenes could efficiently invade neurons when these latter cells were cocultivated with Listeria -infected mouse macrophages. In this case, infection of neurons occurs by cell-to-cell spread via an actA -dependent mechanism. These data support the notion that infected phagocytes can be vectors by which L. monocytogenes gains access to privileged niches such as the central nervous system.

Agrin, a synaptic basal lamina protein synthesized by motoneurons is involved in the aggregation of nicotinic acetylcholine receptors (nAchRs) at the neuromuscular junction. Agrin transcripts are broadly expressed in the central nervous system (CNS) including non-cholinergic regions. This wide distribution of agrin mRNAs raises the question of its function in these areas. To approach this question, we analysed the expression and cellular distribution of agrin in primary cultures of rat embryonic dorsal horn neurons. Polymerase chain reaction analysis demonstrated that the four agrin isoform (B0, B8, B11, B19) mRNAs are expressed as early as 4 days in vitro, before the formation of functional synaptic contacts. Western blots also showed that agrin-like proteins are secreted in conditioned medium from 7 days cultures. We analysed the subcellular distribution of agrin by double immunolabeling and fluorescence microscopy. We found that agrin is synthesized by almost all neurons and was present in the somata and in the axons but not in dendrites within the sensitivity of the detection. This intra-axonal localisation of agrin could only be seen after permeabilization. Furthermore, agrin immunoreactive axons were found adjacent to gephyrin, the postsynaptic glycine receptor-associated protein. Altogether, our results suggest that, as established at the neuromuscular junction, agrin may be involved in pre- to postsynaptic interactions in the central nervous system.

The flux of neurotransmitter receptors in and out of synapses depends on receptor interaction with scaffolding molecules. However, the crowd of transmembrane proteins and the rich cytoskeletal environment may constitute obstacles to the diffusion of receptors within the synapse. To address this question, we studied the membrane diffusion of the γ-aminobutyric acid type A receptor (GABA(A)R) subunits clustered (γ2) or not (α5) at inhibitory synapses in rat hippocampal dissociated neurons. Relative to the extrasynaptic region, γ2 and α5 showed reduced diffusion and increased confinement at both inhibitory and excitatory synapses but they dwelled for a short time at excitatory synapses. In contrast, γ2 was ~3-fold more confined and dwelled ~3-fold longer in inhibitory synapses than α5, indicating faster synaptic escape of α5. Furthermore, using a gephyrin dominant-negative approach, we showed that the increased residency time of γ2 at inhibitory synapses was due to receptor-scaffold interactions. As shown for GABA(A)R, the excitatory glutamate receptor 2 subunit (GluA2) of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) had lower mobility in both excitatory and inhibitory synapses but a higher residency time at excitatory synapses. Therefore barriers impose significant diffusion constraints onto receptors at synapses where they accumulate or not. Our data further reveal that the confinement and the dwell time but not the diffusion coefficient report on the synapse specific sorting, trapping and accumulation of receptors.

INTRODUCTIONThis protocol describes a highly sensitive approach for tracking the motion of membrane molecules over extended time periods with single-molecule resolution. This technique uses nanometer-sized quantum dots (QDs) linked to the extracellular part of the proteins to be followed. Single-fluorophore epifluorescence imaging then reveals the membrane diffusion of the particle of interest. Two methods are presented for labeling neurons with the primary antibody of choice along with a secondary anti-Fab antibody that is either biotinylated or directly coupled to the desired QD. The behavior of QD-labeled molecules can then be followed within the cell using epifluorescence imaging.

We have evaluated the influence of the secretory phenotype of presynaptic boutons on the accumulation of postsynaptic glycine receptors (GlyRs), type A GABA receptors (GABA(A)Rs), and gephyrin clusters. The cellular distribution of these components was analyzed on motoneurons cultured either alone or with glycinergic and/or GABAergic neurons. In motoneurons cultured alone, we observed gephyrin clusters at nonsynaptic sites and in front of cholinergic boutons, whereas glycine and GABA(A) receptors formed nonsynaptic clusters. These receptors are functionally and pharmacologically similar to those found in cultures of all spinal neurons. Motoneurons receiving GABAergic innervation from dorsal root ganglia neurons displayed postsynaptic clusters of gephyrin and GABA(A)Rbeta but not of GlyRalpha/beta subunits. In motoneurons receiving glycinergic and GABAergic innervation from spinal interneurons, gephyrin, GlyRalpha/beta, and GABA(A)Rbeta formed mosaics at synaptic loci. These results indicate that (1) the transmitter phenotype of the presynaptic element determines the postsynaptic accumulation of specific receptors but not of gephyrin and (2) the postsynaptic accumulation of gephyrin alone cannot account for the formation of GlyR-rich microdomains.