Surface Of Hippocampal Neurons Research Articles

NKCC1 and KCC2 Chloride Transporters Have Different Membrane Dynamics on the Surface of Hippocampal Neurons.

Na-K-2Cl cotransporter 1 (NKCC1) regulates chloride influx in neurons and thereby GABAA receptor activity in normal and pathological conditions. Here, we characterized in hippocampal neurons the membrane expression, distribution and dynamics of exogenous NKCC1a and NKCC1b isoforms and compared them to those of the chloride extruder K-Cl cotransporter 2 (KCC2). We found that NKCC1a and NKCC1b behave quite similarly. NKCC1a/1b but not KCC2 are present along the axon initial segment where they are confined. Moreover, NKCC1a/1b are detected in the somato-dendritic compartment at a lower level than KCC2, where they form fewer, smaller and less compact clusters at perisynaptic and extrasynaptic sites. Interestingly, ~60% of dendritic clusters of NKCC1a/1b are colocalized with KCC2. They are larger and brighter than those devoid of KCC2, suggesting a particular NKCC1a/1b-KCC2 relationship. In agreement with the reduced dendritic clustering of NKCC1a/1b compared with that of KCC2, NKCC1a/1b are more mobile on the dendrite than KCC2, suggesting weaker cytoskeletal interaction. NKCC1a/b are confined to endocytic zones, where they spend more time than KCC2. However, they spend less time in these compartments than at the synapses, suggesting that they can rapidly leave endocytic zones to increase the membrane pool, which can happen in pathological conditions. Thus, NKCC1a/b have different membrane dynamics and clustering from KCC2, which helps to explain their low level in the neuronal membrane, while allowing a rapid increase in the membrane pool under pathological conditions.

Neuron Class and Target Variability in the Three-Dimensional Localization of SK2 Channels in Hippocampal Neurons as Detected by Immunogold FIB-SEM.

Small-conductance calcium-activated potassium (SK) channels are crucial for learning and memory. However, many aspects of their spatial organization in neurons are still unknown. In this study, we have taken a novel approach to answering these questions combining a pre-embedding immunogold labeling with an automated dual-beam electron microscope that integrates focused ion beam milling and scanning electron microscopy (FIB/SEM) to gather 3D map ultrastructural and biomolecular information simultaneously. Using this new approach, we evaluated the number and variability in the density of extrasynaptic SK2 channels in 3D reconstructions from six dendritic segments of excitatory neurons and six inhibitory neurons present in the stratum radiatum of the CA1 region of the mouse. SK2 immunoparticles were observed throughout the surface of hippocampal neurons, either scattered or clustered, as well as at intracellular sites. Quantitative volumetric evaluations revealed that the extrasynaptic SK2 channel density in spines was seven times higher than in dendritic shafts and thirty-five times higher than in interneurons. Spines showed a heterogeneous population of SK2 expression, some spines having a high SK2 content, others having a low content and others lacking SK2 channels. SK2 immunonegative spines were significantly smaller than those immunopositive. These results show that SK2 channel density differs between excitatory and inhibitory neurons and demonstrates a large variability in the density of SK2 channels in spines. Furthermore, we demonstrated that SK2 expression was associated with excitatory synapses, but not with inhibitory synapses in CA1 pyramidal cells. Consequently, regulation of excitability and synaptic plasticity by SK2 channels is expected to be neuron class- and target-specific. These data show that immunogold FIB/SEM represent a new powerful EM tool to correlate structure and function of ion channels with nanoscale resolution.

Aging power spectrum of membrane protein transport and other subordinated random walks

Single-particle tracking offers detailed information about the motion of molecules in complex environments such as those encountered in live cells, but the interpretation of experimental data is challenging. One of the most powerful tools in the characterization of random processes is the power spectral density. However, because anomalous diffusion processes in complex systems are usually not stationary, the traditional Wiener-Khinchin theorem for the analysis of power spectral densities is invalid. Here, we employ a recently developed tool named aging Wiener-Khinchin theorem to derive the power spectral density of fractional Brownian motion coexisting with a scale-free continuous time random walk, the two most typical anomalous diffusion processes. Using this analysis, we characterize the motion of voltage-gated sodium channels on the surface of hippocampal neurons. Our results show aging where the power spectral density can either increase or decrease with observation time depending on the specific parameters of both underlying processes.

Human Autoantibodies Against N-Methyl-D-Aspartate Receptor Modestly Alter Dopamine D1 Receptor Surface Dynamics.

Circulating autoantibodies directed against extracellular domains of the glutamatergic N-methyl-D-aspartate receptors (NMDAR-Ab) elicit psychotic symptoms in humans and behavioral deficits in animal models. Recent advances suggest that NMDAR-Ab exert their pathogenic action by altering the trafficking of NMDAR, which results in a synaptic NMDAR hypofunction consistent with the consensual glutamatergic hypothesis of psychotic disorders. Yet, dysfunction in the dopaminergic signaling is also proposed to be at the core of psychotic disorders. Since NMDAR and dopamine D1 receptors (D1R) form membrane signaling complexes, we investigated whether NMDAR-Ab purified from patients with NMDAR-encephalitis or schizophrenia impaired D1R surface dynamics. As previous data demonstrated that NMDAR-Ab, at least from NMDAR-encephalitis patients, mainly bind to hippocampal NMDAR, we used single nanoparticle imaging to track D1R specifically at the surface of hippocampal neurons that were exposed to either purified G type immunoglobulins (IgGs) from NMDAR-Ab seropositive patients suffering from NMDAR-encephalitis or schizophrenia, or control IgGs from healthy NMDAR-Ab seropositive or seronegative subjects. We report that overnight incubation with NMDAR-Ab from patients, but not from healthy carriers, decreased the surface dynamics of D1R compared with NMDAR-Ab seronegative IgGs. This decrease was abolished, and even reversed, in D1R mutant that cannot physically interact with NMDAR. Overall, our data indicate that NMDAR-Ab from patients with psychotic symptoms alter the trafficking of D1R, likely through the surface crosstalk between NMDAR and D1R.

Elucidating distinct ion channel populations on the surface of hippocampal neurons via single-particle tracking recurrence analysis.

Protein and lipid nanodomains are prevalent on the surface of mammalian cells. In particular, it has been recently recognized that ion channels assemble into surface nanoclusters in the soma of cultured neurons. However, the interactions of these molecules with surface nanodomains display a considerable degree of heterogeneity. Here, we investigate this heterogeneity and develop statistical tools based on the recurrence of individual trajectories to identify subpopulations within ion channels in the neuronal surface. We specifically study the dynamics of the K^{+} channel Kv1.4 and the Na^{+} channel Nav1.6 on the surface of cultured hippocampal neurons at the single-molecule level. We find that both these molecules are expressed in two different forms with distinct kinetics with regards to surface interactions, emphasizing the complex proteomic landscape of the neuronal surface. Further, the tools presented in this work provide new methods for the analysis of membrane nanodomains, transient confinement, and identification of populations within single-particle trajectories.

PI4KIIα phosphorylation by GSK3 directs vesicular trafficking to lysosomes.

Glycogen synthase kinase 3 (GSK3) is essential for normal development and function of the central nervous system. It is especially important for regulating neurotransmission, although the downstream substrates mediating this function are not yet clear. In the present paper, we report the lipid kinase phosphatidylinositol 4-kinase II α (PI4KIIα) is a novel substrate of GSK3 that regulates trafficking and cell-surface expression of neurotransmitter receptors in neurons. GSK3 phosphorylates two distinct sites in the N-terminus of PI4KIIα (Ser5 and Ser47), promoting binding to the adaptor protein 3 (AP-3) complex for trafficking to the lysosome to be degraded. Blocking phosphorylation reduces trafficking to the lysosome, stabilizing PI4KIIα and its cargo proteins for redistribution throughout the cell. Importantly, a reduction in PI4KIIα expression or phosphorylation increases α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor expression at the surface of hippocampal neurons. These studies implicate signalling between GSK3 and PI4KIIα as a novel regulator of vesicular trafficking and neurotransmission in the brain.

The N‐terminal fragment of the β‐amyloid precursor protein of Alzheimer's disease (N‐APP) binds to phosphoinositide‐rich domains on the surface of hippocampal neurons

The function of the β-amyloid precursor protein (APP) of Alzheimer's disease is poorly understood. The secreted ectodomain fragment of APP (sAPPα) can be readily cleaved to produce a small N-terminal fragment (N-APP) that contains heparin-binding and metal-binding domains and that has been found to have biological activity. In the present study, we examined whether N-APP can bind to lipids. We found that N-APP binds selectively to phosphoinositides (PIPs) but poorly to most other lipids. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 )-rich microdomains were identified on the extracellular surface of neurons and glia in primary hippocampal cultures. N-APP bound to neurons and colocalized with PIPs on the cell surface. Furthermore, the binding of N-APP to neurons increased the level of cell-surface PI(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate. However, PIPs were not the principal cell-surface binding site for N-APP, because N-APP binding to neurons was not inhibited by a short-acyl-chain PIP analogue, and N-APP did not bind to glial cells which also possessed PI(4,5)P2 on the cell surface. The data are explained by a model in which N-APP binds to two distinct components on neurons, one of which is an unidentified receptor and the second of which is a PIP lipid, which binds more weakly to a distinct site within N-APP. Our data provide further support for the idea that N-APP may be an important mediator of APP's biological activity.

Single-molecule imaging of the functional crosstalk between surface NMDA and dopamine D1 receptors

Dopamine is a powerful modulator of glutamatergic neurotransmission and NMDA receptor-dependent synaptic plasticity. Although several intracellular cascades participating in this functional dialogue have been identified over the last few decades, the molecular crosstalk between surface dopamine and glutamate NMDA receptor (NMDAR) signaling still remains poorly understood. Using a combination of single-molecule detection imaging and electrophysiology in live hippocampal neurons, we demonstrate here that dopamine D1 receptors (D1Rs) and NMDARs form dynamic surface clusters in the vicinity of glutamate synapses. Strikingly, D1R activation or D1R/NMDAR direct interaction disruption decreases the size of these clusters, increases NMDAR synaptic content through a fast lateral redistribution of the receptors, and favors long-term synaptic potentiation. Together, these data demonstrate the presence of dynamic D1R/NMDAR perisynaptic reservoirs favoring a rapid and bidirectional surface crosstalk between receptors and set the plasma membrane as the primary stage of the dopamine-glutamate interplay.

Regulation of STIM1 and SOCE by the Ubiquitin-Proteasome System (UPS)

The ubiquitin proteasome system (UPS) mediates the majority of protein degradation in eukaryotic cells. The UPS has recently emerged as a key degradation pathway involved in synapse development and function. In order to better understand the function of the UPS at synapses we utilized a genetic and proteomic approach to isolate and identify novel candidate UPS substrates from biochemically purified synaptic membrane preparations. Using these methods, we have identified Stromal interacting molecule 1 (STIM1). STIM1 is as an endoplasmic reticulum (ER) calcium sensor that has been shown to regulate store-operated Ca2+ entry (SOCE). We have characterized STIM1 in neurons, finding STIM1 is expressed throughout development with stable, high expression in mature neurons. As in non-excitable cells, STIM1 is distributed in a membranous and punctate fashion in hippocampal neurons. In addition, a population of STIM1 was found to exist at synapses. Furthermore, using surface biotinylation and live-cell labeling methods, we detect a subpopulation of STIM1 on the surface of hippocampal neurons. The role of STIM1 as a regulator of SOCE has typically been examined in non-excitable cell types. Therefore, we examined the role of the UPS in STIM1 and SOCE function in HEK293 cells. While we find that STIM1 is ubiquitinated, its stability is not altered by proteasome inhibitors in cells under basal conditions or conditions that activate SOCE. However, we find that surface STIM1 levels and thapsigargin (TG)-induced SOCE are significantly increased in cells treated with proteasome inhibitors. Additionally, we find that the overexpression of POSH (Plenty of SH3′s), an E3 ubiquitin ligase recently shown to be involved in the regulation of Ca2+ homeostasis, leads to decreased STIM1 surface levels. Together, these results provide evidence for previously undescribed roles of the UPS in the regulation of STIM1 and SOCE function.

Functional Consequences of the Interactions among the Neural Cell Adhesion Molecule NCAM, the Receptor Tyrosine Kinase TrkB, and the Inwardly Rectifying K+ Channel KIR3.3

Cell adhesion molecules and neurotrophin receptors are crucial for the development and the function of the nervous system. Among downstream effectors of neurotrophin receptors and recognition molecules are ion channels. Here, we provide evidence that G protein-coupled inwardly rectifying K(+) channel Kir3.3 directly binds to the neural cell adhesion molecule (NCAM) and neurotrophin receptor TrkB. We identified the binding sites for NCAM and TrkB at the C-terminal intracellular domain of Kir3.3. The interaction between NCAM, TrkB, and Kir3.3 was supported by immunocytochemical co-localization of Kir3.3, NCAM, and/or TrkB at the surface of hippocampal neurons. Co-expression of TrkB and Kir3.1/3.3 in Xenopus oocytes increased the K(+) currents evoked by Kir3.1/3.3 channels. This current enhancement was reduced by the concomitant co-expression with NCAM. Both surface fluorescence measurements of microinjected oocytes and cell surface biotinylation of transfected CHO cells indicated that the cell membrane localization of Kir3.3 is regulated by TrkB and NCAM. Furthermore, the level of Kir3.3, but not of Kir3.2, at the plasma membranes was reduced in TrkB-deficient mice, supporting the notion that TrkB regulates the cell surface expression of Kir3.3. The premature expression of developmentally late appearing Kir3.1/3.3 in hippocampal neurons led to a reduction of NCAM-induced neurite outgrowth. Our observations indicate a decisive role for the neuronal K(+) channel in regulating NCAM-dependent neurite outgrowth and attribute a physiologically meaningful role to the functional interplay of Kir3.3, NCAM, and TrkB in ontogeny.

PICK1-mediated Glutamate Receptor Subunit 2 (GluR2) Trafficking Contributes to Cell Death in Oxygen/Glucose-deprived Hippocampal Neurons

Oxygen and glucose deprivation (OGD) induces delayed cell death in hippocampal CA1 neurons via Ca2+/Zn2+-permeable, GluR2-lacking AMPA receptors (AMPARs). Following OGD, synaptic AMPAR currents in hippocampal neurons show marked inward rectification and increased sensitivity to channel blockers selective for GluR2-lacking AMPARs. This occurs via two mechanisms: a delayed down-regulation of GluR2 mRNA expression and a rapid internalization of GluR2-containing AMPARs during the OGD insult, which are replaced by GluR2-lacking receptors. The mechanisms that underlie this rapid change in subunit composition are unknown. Here, we demonstrate that this trafficking event shares features in common with events that mediate long term depression and long term potentiation and is initiated by the activation of N-methyl-d-aspartic acid receptors. Using biochemical and electrophysiological approaches, we show that peptides that interfere with PICK1 PDZ domain interactions block the OGD-induced switch in subunit composition, implicating PICK1 in restricting GluR2 from synapses during OGD. Furthermore, we show that GluR2-lacking AMPARs that arise at synapses during OGD as a result of PICK1 PDZ interactions are involved in OGD-induced delayed cell death. This work demonstrates that PICK1 plays a crucial role in the response to OGD that results in altered synaptic transmission and neuronal death and has implications for our understanding of the molecular mechanisms that underlie cell death during stroke.

Delivering AMPA Receptors to the Synapse

Incorporation of AMPA-type glutamate receptors containing the GluR1 subunit into the postsynaptic membrane is associated with long-term potentiation (LTP), a form of synaptic plasticity that may underlie some forms of memory. The trafficking of GluR1-containing receptors to the cell surface is regulated by multiple interactions that occur with the C-terminal domain (CTD), including interactions with kinases that phosphorylate the CTD. Phosphorylation of Ser 845 , reportedly mediated by the adenosine 3′,5′-monophosphate (cAMP)-regulated enzyme protein kinase A (PKA), appears to be required for GluR1 synaptic insertion. Serulle et al . provide evidence that Ser 845 can also be phosphorylated by guanosine 3′,5′-monophosphate (cGMP)-dependent protein kinase II (cGKII), thereby providing a mechanism for nitric oxide signaling downstream of NMDA-type glutamate receptors to increase synaptic strength and contribute to LTP. cGKII was identified in a modified yeast two-hybrid screen for proteins that interacted with the CTD of GluR1. In vitro binding studies suggested that activation of cGKII (binding to the cGMP analog 8-Br-cGMP) increased its interaction with the GluR1 CTD. In lysates from rat brain, cGKII and GluR1 were present in the same subcellular fraction and coimmunoprecipitated. In cultured hippocampal neurons, endogenous cGKII and GluR1 colocalized and coimmunoprecipitated. The interaction was enhanced when the cells were (i) exposed to 8-Br-cGMP to stabilize cGKII in the active conformation, (ii) exposed to glycine to induce a chemical form of LTP (chemLTP) that involves activation of NMDA receptors and activation of nitric oxide synthase and soluble guanylate cyclase (the enzyme that produces cGMP), or (iii) exposed to a nitric oxide donor. In vitro kinase assays demonstrated that the GluR1 CTD was phosphorylated by cGKII at Ser 845 , the same site phosphorylated by PKA. In cultured neurons, chemLTP also triggered phosphorylation of GluR1 at Ser 845 , and this was blocked by the presence of an NMDA receptor antagonist or cGKII inhibitor. Both cAMP analogs and cGMP analogs increased the abundance of GluR1 at the surface of hippocampal neurons but did not induce LTP. The chemLTP condition stimulated an increase in synaptic GluR1 and induced LTP, which was measured as a sustained increase in miniature excitatory postsynaptic currents. The interaction of GluRI with PKA is indirect, requiring the PDZ scaffolding protein SAP97. The interaction with cGKII appears to be direct and involves Arg 837 and does not require SAP97 as a mediator. With mutants either lacking the SAP97 binding region or containing a point mutation at Arg 837 , the authors demonstrated that the cAMP- and cGMP-mediated GluR1 trafficking and phosphorylation pathways were independent. In hippocampal mouse slices, pharmacological inhibitors of the NO to cGMP pathway or expression of a dominant-negative fragment of the cGKII that disrupted the interaction between cGKII and GluR1 inhibited LTP in response to tetanic stimulation. Thus, it appears that both cAMP and cGMP pathways converge on Ser 845 to regulate GluR1 trafficking. Y. Serulle, S. Zhang, I. Ninan, D. Puzzo, M. McCarthy, L. Khatri, O. Arancio, E. B. Ziff, A GluR1-cGKII interaction regulates AMPA receptor trafficking. Neuron 56 , 670-688 (2007). [PubMed]

Extracellular Interactions between GluR2 and N-Cadherin in Spine Regulation

Via its extracellular N-terminal domain (NTD), the AMPA receptor subunit GluR2 promotes the formation and growth of dendritic spines in cultured hippocampal neurons. Here we show that the first N-terminal 92 amino acids of the extracellular domain are necessary and sufficient for GluR2's spine-promoting activity. Moreover, overexpression of this extracellular domain increases the frequency of miniature excitatory postsynaptic currents (mEPSCs). Biochemically, the NTD of GluR2 can interact directly with the cell adhesion molecule N-cadherin, in cis or in trans. N-cadherin-coated beads recruit GluR2 on the surface of hippocampal neurons, and N-cadherin immobilization decreases GluR2 lateral diffusion on the neuronal surface. RNAi knockdown of N-cadherin prevents the enhancing effect of GluR2 on spine morphogenesis and mEPSC frequency. Our data indicate that in hippocampal neurons N-cadherin and GluR2 form a synaptic complex that stimulates presynaptic development and function as well as promoting dendritic spine formation.

Brevican isoforms associate with neural membranes.

Brevican is a neural-specific proteoglycan of the brain extracellular matrix, which is particularly abundant in the terminally differentiated CNS. It is expressed by neuronal and glial cells, and as a component of the perineuronal nets it decorates the surface of large neuronal somata and primary dendrites. One brevican isoform harbors a glycosylphosphatidylinositol anchor attachment site and, as shown by ethanolamine incorporation studies, is indeed glypiated in stably transfected HEK293 cells as well as in oligodendrocyte precursor Oli-neu cells. The major isoform is secreted into the extracellular space, although a significant amount appears to be tightly attached to the cell membrane, as it floats up in sucrose gradients. Flotation is sensitive to detergent treatment. Brevican is most prominent in the microsomal, light membrane and synaptosomal fractions of rat brain membrane preparations. The association with the particulate fraction is in part sensitive to chondroitinase ABC and phosphatidylinositol-specific phospholipase C treatment. Furthermore, brevican staining on the surface of hippocampal neurons in culture is diminished after hyaluronidase or chondroitinase ABC treatment. Taken together, this could provide a mechanism by which perineuronal nets are anchored on neuronal surfaces.

Activity- and Ca(2+)-dependent modulation of surface expression of brain-derived neurotrophic factor receptors in hippocampal neurons.

Brain-derived neurotrophic factor (BDNF) has been shown to regulate neuronal survival and synaptic plasticity in the central nervous system (CNS) in an activity-dependent manner, but the underlying mechanisms remain unclear. Here we report that the number of BDNF receptor TrkB on the surface of hippocampal neurons can be enhanced by high frequency neuronal activity and synaptic transmission, and this effect is mediated by Ca(2+) influx. Using membrane protein biotinylation as well as receptor binding assays, we show that field electric stimulation increased the number of TrkB on the surface of cultured hippocampal neurons. Immunofluorescence staining suggests that the electric stimulation facilitated the movement of TrkB from intracellular pool to the cell surface, particularly on neuronal processes. The number of surface TrkB was regulated only by high frequency tetanic stimulation, but not by low frequency stimulation. The activity dependent modulation appears to require Ca(2+) influx, since treatment of the neurons with blockers of voltage-gated Ca(2+) channels or NMDA receptors, or removal of extracellular Ca(2+), severely attenuated the effect of electric stimulation. Moreover, inhibition of Ca(2+)/calmodulin-dependent kinase II (CaMKII) significantly reduced the effectiveness of the tetanic stimulation. These findings may help us to understand the role of neuronal activity in neurotrophin function and the mechanism for receptor tyrosine kinase signaling.

Quantitation of AMPA receptor surface expression in cultured hippocampal neurons

Protein and messenger RNA levels of the AMPA-type glutamate receptor subunits 1–3 are high in many brain regions, but it is not known how much of the glutamate receptor protein is expressed on the surface of neurons in the form of functional receptors. To provide insight into this matter, western blot immunoreactivities for glutamate receptors 1 and 2/3, as well as binding of the specific ligand [ 3H]AMPA, were quantified following three independent treatments modifying surface receptors in intact primary hippocampal cultures: (i) proteolysis of surface receptors by chymotrypsin, (ii) cross-linking of surface receptors with the membrane-impermeant reagent bis(sulfosuccinimidyl)suberate, and (iii) biotinylation of surface receptors with the membrane-impermeant reagent sulfosuccinimidyl-2(biotinamido)ethyl-1,3-dithiopropionate. All three of these methods demonstrated that 60–70% of total glutamate receptor subunit 1 protein and 40–50% of total glutamate receptor 2/3 protein are expressed on the surface of hippocampal neurons. Parallel studies revealed that 52% of total [ 3H]AMPA binding sites could be precipitated with avidin beads following biotinylation of intact cultures, providing an estimate of [ 3H]AMPA binding site surface expression in accord with the estimates of the surface expression of glutamate receptor subunits 1–3. Experiments examining the surface expression of 32P-labeled glutamate receptor subunit 1 demonstrated that approximately 65% of the phosphorylated form of the subunit is located in the plasma membrane, an estimate similar to the that derived via western blot for the entire glutamate receptor subunit 1 population in the same samples. Moreover, no significant change in the surface expression profile of the glutamate receptor subunits 1–3 was observed following stimulatory treatments known to increase glutamate receptor phosphorylation. These data indicate that slightly more than half of the AMPA receptors in cultured hippocampal neurons are located in the plasma membrane, and that AMPA receptor surface expression is not rapidly altered by glutamate receptor phosphorylation.