Using simultaneous voltage and calcium imaging to study fast Ca 2+ channels

International audienceThe combination of fluorescence measurements of membrane potential and intracellular Ca2+ concentration allows correlating the electrical and calcium activity of a cell with spatial precision. The technical advances allowing this type of measurement were achieved only recently and represent an important step in the progress of the voltage imaging approach pioneered over 40 years ago by Lawrence B. Cohen. Here, we show how this approach can be used to investigate the function of Ca2+ channels using the foreseen possibility to extract Ca2+ currents from imaging experiments. The kinetics of the Ca2+ current, mediated by voltage-gated Ca2+ channels, can be accurately derived from the Ca2+ fluorescence measurement using Ca2+ indicators with KD>10  μM that equilibrate in <1  ms. In this respect, the imaging apparatus dedicated to this application is described in detail. Next, we illustrate the mathematical procedure to extract the current from the Ca2+ fluorescence change, including a method to calibrate the signal to charge flux density. Finally, we show an example of simultaneous membrane potential and Ca2+ optical measurement associated with an action potential at a CA1 hippocampal pyramidal neuron from a mouse brain slice. The advantages and limitations of this approach are discussed

Oxygen/Glucose deprivation induces a reduction in synaptic AMPA receptors on hippocampal CA3 neurons mediated by mGluR1 and adenosine A3 receptors

Hippocampal CA1 pyramidal neurons are highly sensitive to ischemic damage, whereas neighboring CA3 pyramidal neurons are less susceptible. It is proposed that switching of AMPA receptor (AMPAR) subunits on CA1 neurons during an in vitro model of ischemia, oxygen/glucose deprivation (OGD), leads to an enhanced permeability of AMPARs to Ca2+, resulting in delayed cell death. However, it is unclear whether the same mechanisms exist in CA3 neurons and whether this underlies the differential sensitivity to ischemia. Here, we investigated the consequences of OGD for AMPAR function in CA3 neurons using electrophysiological recordings in rat hippocampal slices. Following a 15 min OGD protocol, a substantial depression of AMPAR-mediated synaptic transmission was observed at CA3 associational/commissural and mossy fiber synapses but not CA1 Schaffer collateral synapses. The depression of synaptic transmission following OGD was prevented by metabotropic glutamate receptor 1 (mGluR1) or A3 receptor antagonists, indicating a role for both glutamate and adenosine release. Inhibition of PLC, PKC, or chelation of intracellular Ca2+ also prevented the depression of synaptic transmission. Inclusion of peptides to interrupt the interaction between GluA2 and PICK1 or dynamin and amphiphysin prevented the depression of transmission, suggesting a dynamin and PICK1-dependent internalization of AMPARs after OGD. We also show that a reduction in surface and total AMPAR protein levels after OGD was prevented by mGluR1 or A3 receptor antagonists, indicating that AMPARs are degraded following internalization. Thus, we describe a novel mechanism for the removal of AMPARs in CA3 pyramidal neurons following OGD that has the potential to reduce excitotoxicity and promote neuroprotectio

Enhanced SUMOylation and SENP-1 protein levels following oxygen and glucose deprivation in neurones

Here, we show that oxygen and glucose deprivation (OGD) causes increased small ubiquitin-like modifier (SUMO)-1 and SUMO-2/3 conjugation to substrate proteins in cultured hippocampal neurones. Surprisingly, the SUMO protease SENP-1, which removes SUMO from conjugated proteins, was also increased by OGD, suggesting that the neuronal response to OGD involves a complex interplay between SUMOylation and deSUMOylation. Importantly, decreasing global SUMOylation in cultured hippocampal neurones by overexpression of the catalytic domain of SENP-1 increased neuronal vulnerability to OGD-induced cell death. Taken together, these results suggest a neuroprotective role for neuronal SUMOylation after OGD

Cortactin regulates endo-lysosomal sorting of AMPARs via direct interaction with GluA2 subunit

Abstract AMPA receptor (AMPAR) trafficking is a key determinant of synaptic strength and synaptic plasticity. Under basal conditions, constitutive trafficking maintains surface AMPARs by internalization into the endosomal system, where the majority are sorted and targeted for recycling back to the plasma membrane. NMDA receptor (NMDAR)-dependent Long-Term Depression (LTD) is characterised by a reduction in synaptic strength, and involves endosomal sorting of AMPARs away from recycling pathways to lysosomes. The mechanisms that determine whether AMPARs are trafficked to lysosomes or to recycling endosomes, especially in response to NMDAR stimulation, are unclear. Here, we define a role for the actin-regulatory protein cortactin as a mediator of AMPAR endosomal sorting by direct interaction with the GluA2 subunit. Disrupting GluA2-cortactin binding in neurons causes the targeting of GluA2/A3-containing receptors to lysosomes and their consequent degradation, resulting in a loss of surface and synaptic GluA2 under basal conditions and an occlusion of subsequent LTD expression. Furthermore, we show that NMDAR stimulation causes a dissociation of endogenous cortactin from GluA2 via tyrosine phosphorylation of cortactin. These results demonstrate that cortactin maintains GluA2/A3 levels by directing receptors away from lysosomes, and that disrupting GluA2-cortactin interactions to target GluA2/A3 to lysosomes is an essential component of LTD expression

The Small GTPase Arf1 Modulates Arp2/3-Mediated Actin Polymerization via PICK1 to Regulate Synaptic Plasticity

SummaryInhibition of Arp2/3-mediated actin polymerization by PICK1 is a central mechanism to AMPA receptor (AMPAR) internalization and long-term depression (LTD), although the signaling pathways that modulate this process in response to NMDA receptor (NMDAR) activation are unknown. Here, we define a function for the GTPase Arf1 in this process. We show that Arf1-GTP binds PICK1 to limit PICK1-mediated inhibition of Arp2/3 activity. Expression of mutant Arf1 that does not bind PICK1 leads to reduced surface levels of GluA2-containing AMPARs and smaller spines in hippocampal neurons, which occludes subsequent NMDA-induced AMPAR internalization and spine shrinkage. In organotypic slices, NMDAR-dependent LTD of AMPAR excitatory postsynaptic currents is abolished in neurons expressing mutant Arf1. Furthermore, NMDAR stimulation downregulates Arf1 activation and binding to PICK1 via the Arf-GAP GIT1. This study defines Arf1 as a critical regulator of actin dynamics and synaptic function via modulation of PICK1

Cortactin regulates endo-lysosomal sorting of AMPARs via direct interaction with GluA2 subunit

Abstract AMPA receptor (AMPAR) trafficking is a key determinant of synaptic strength and synaptic plasticity. Under basal conditions, constitutive trafficking maintains surface AMPARs by internalization into the endosomal system, where the majority are sorted and targeted for recycling back to the plasma membrane. NMDA receptor (NMDAR)-dependent Long-Term Depression (LTD) is characterised by a reduction in synaptic strength, and involves endosomal sorting of AMPARs away from recycling pathways to lysosomes. The mechanisms that determine whether AMPARs are trafficked to lysosomes or to recycling endosomes, especially in response to NMDAR stimulation, are unclear. Here, we define a role for the actin-regulatory protein cortactin as a mediator of AMPAR endosomal sorting by direct interaction with the GluA2 subunit. Disrupting GluA2-cortactin binding in neurons causes the targeting of GluA2/A3-containing receptors to lysosomes and their consequent degradation, resulting in a loss of surface and synaptic GluA2 under basal conditions and an occlusion of subsequent LTD expression. Furthermore, we show that NMDAR stimulation causes a dissociation of endogenous cortactin from GluA2 via tyrosine phosphorylation of cortactin. These results demonstrate that cortactin maintains GluA2/A3 levels by directing receptors away from lysosomes, and that disrupting GluA2-cortactin interactions to target GluA2/A3 to lysosomes is an essential component of LTD expression

Differential Regulation of GABABReceptor Trafficking by Different Modes ofN-methyl-d-aspartate (NMDA) Receptor Signaling

Inhibitory GABAB receptors (GABABRs) can down-regulate most excitatory synapses in the CNS by reducing postsynaptic excitability. Functional GABABRs are heterodimers of GABAB1 and GABAB2 subunits and here we show that the trafficking and surface expression of GABABRs is differentially regulated by synaptic or pathophysiological activation of NMDA receptors (NMDARs). Activation of synaptic NMDARs using a chemLTP protocol increases GABABR recycling and surface expression. In contrast, excitotoxic global activation of synaptic and extrasynaptic NMDARs by bath application of NMDA causes the loss of surface GABABRs. Intriguingly, exposing neurons to extreme metabolic stress using oxygen/glucose deprivation (OGD) increases GABAB1 but decreases GABAB2 surface expression. The increase in surface GABAB1 involves enhanced recycling and is blocked by the NMDAR antagonist AP5. The decrease in surface GABAB2 is also blocked by AP5 and by inhibiting degradation pathways. These results indicate that NMDAR activity is critical in GABABR trafficking and function and that the individual subunits can be separately controlled to regulate neuronal responsiveness and survival

PICK1 inhibition of the Arp2/3 complex controls dendritic spine size and synaptic plasticity

Dendritic spine remodelling involves regulated actin dynamics and is essential for synaptic plasticity. The Arp2/3 inhibitor PICK1 is here shown to regulate spine size: inducing shrinkage during long-term depression

Contribution à l étude du rôle des récepteurs tachykinines de type 2 dans la neurorégulation de l activité motrice du côlon chez l Homme

La présente étude a été développée pour analyser l expression des récepteurs tachykinines de type 2 (NK2r) au niveau du côlon chez l Homme. Nos investigations ont été réalisées sur des spécimens ex vivo et maintenus en survie in vitro. L analyse des NK2r a été réalisée au niveau des plexus nerveux myentériques et des musculeuses longitudinale et circulaire. Des méthodes de biologie moléculaire, et d immunofluorescence couplées à une analyse informatique d images obtenues en microscopie confocale ont été utilisées. Par RT-qPCR, nous avons caractérisé l ARNm du gène codant pour le NK2r fonctionnel (a-TACR2) et déterminé son niveau d expression. Les NK2r sont exprimés dans les neurones myentériques, qui sur la base de leur contenu en médiateurs peuvent être rattachés aux neurones sensitifs afférents primaires intrinsèques, aux interneurones et aux neurones moteurs. Ces données dans leur ensemble montrent qu au niveau des plexus myentériques, les tachykinines peuvent intervenir, via les NK2r, dans la régulation pré- et post-synaptique de la transmission des messages nerveux. Dans les musculeuses, les NK2r sont exprimés au niveau pré-jonctionnel au sein des varicosités nerveuses et au niveau post-jonctionnel, sur les cellules musculaires. Au niveau des cellules musculaires, l activation des NK2r est suivie de leur internalisation puis de leur recyclage dont nous détaillons pour la première fois la cinétique. Nos travaux apportent une nouvelle contribution au rôle des NK2r dans la neurorégulation de l activité motrice du côlon humain et ouvrent des perspectives nouvelles de recherche sur leur implication dans les troubles moteurs intestinaux liés à certaines maladies inflammatoires du tractus digestif.AIX-MARSEILLE3-BU Sc.St Jérô (130552102) / SudocSudocFranceF

Functional coupling of diverse voltage-gated Ca 2+ channels underlies high- fidelity of fast dendritic Ca 2+ signals during burst firing

International audienceIn CA1 hippocampal pyramidal neurons, the dendritic Ca2+ signal associated with somatic firing represents a fundamental activation code for several proteins. This signal, mediated by voltage-gated Ca2+ channels (VGCCs), varies along the dendrites. In this study, using a recent optical technique based on the low-affinity indicator Oregon Green 488 BAPTA-5N, we analysed how activation and deactivation of VGCCs produced by back-propagating action potentials (bAPs) along the apical dendrite shape the Ca2+ signal at different locations in CA1 hippocampal pyramidal neurons of the mouse. We measured, at multiple dendritic sites, the Ca2+ transients and the changes in membrane potential associated with bAPs at 50 μs temporal resolution and we estimated the kinetics of the Ca2+ current. We found that during somatic bursts, the bAPs decrease in amplitude along the apical dendrite but the amplitude of the associated Ca2+ signal in the initial 200 μm dendritic segment does not change. Using a detailed pharmacological analysis, we demonstrate that this effect is due to the perfect compensation of the loss of Ca2+ via high-voltage-activated (HVA) VGCCs by a larger Ca2+ component via low-voltage-activated (LVA) VGCCs, revealing a mechanism coupling the two VGCC families of K+ channels. More distally, where the bAP does not activate HVA-VGCCs, the Ca2+ signal is variable during the burst. Thus, we demonstrate that HVA- and LVA-VGCCs operate synergistically to stabilise Ca2+ signals associated with bAPs in the most proximal 200 μm dendritic segment