Role of NMDA receptor trafficking during synaptic maturation and plasticity

La synapse glutamatergique assure la majeure partie de la transmission excitatrice du cerveau et des changements de sa force constituent un corrélat cellulaire des processus d’apprentissage et de mémoire. Ces processus adaptatifs nécessitent souvent l’activation des récepteurs ionotropiques au glutamate de type NMDA (NMDAR) et l’influx calcique dans le compartiment postsynaptique qui suit leur ouverture. Jusqu’alors, l’activation des voix de signalisations sous-jacentes était considérée comme le seul mécanisme essentiel à la plasticité synaptique. Il est apparu récemment que les NMDAR diffusent à la surface des neurones, assurant un remodelage dynamique de leur distribution. La possibilité que la dynamique de surface des NMDAR joue un rôle déterminant dans les propriétés plastiques des synapses a donc émergé. Au cours de ma thèse, je me suis intéressé à cette problématique à l’aide d’approches d’imagerie dynamique à haute-résolution (ex. suivi de nanoparticules uniques, FRAP) et d’outils moléculaires de haute spécificité (ex. ligand biomimétique, x-link de récepteurs via les anticorps). J’ai dans un premier temps étudié la dynamique de surface des NMDAR endogènes au cours de la plasticité synaptique au sein de réseaux neuronaux hippocampiques in vitro. Mes résultats révèlent que l’induction de la potentialisation à long terme (LTP) des synapses glutamatergiques s’accompagne d’une redistribution latérale des NMDAR de surface dans la région postsynaptique. De façon remarquable, la réduction de la diffusion de surface des NMDAR via des anticorps commerciaux, mais aussi des anticorps purifiés de patients atteints d’encéphalite auto-immune, ciblant des épitopes extracellulaires des NMDAR, bloque la LTP. Dans un second temps, je me suis intéressé à la régulation de cette dynamique des NMDAR. En collaboration avec le groupe de Stéphane Oliet (CRI, INSERM), nous avons découvert qu’une redistribution rapide de surface des NMDAR s’opère différemment sous l’effet des co-agonistes du récepteur, la glycine et la D-sérine, et cela de façon dépendante des sous-unités GluN2A/GluN2B des NMDAR. De plus, j’ai démontré que l’interaction directe entre les NMDAR et les récepteurs dopaminergiques D1 membranaires contrôle la distribution des deux types de récepteurs aux abords de la synapse et module la plasticité synaptique. L’ensemble de ces données indique que la dynamique de surface des NMDAR est régulée par la présence d’un neuromodulateur, la dopamine, et de co-agonistes, contrôlant de façon dynamique la fenêtre plastique des synapses.Glutamate synapse mediates most synaptic excitation in the brain and changes in its strength constitute a cellular basis for learning and memory processes. These adaptive properties often require ionotropic glutamate NMDA receptor (NMDAR) and the calcium influx in the postsynaptic compartment following their opening. So far, the activation of the subsequent signaling pathways was considered as the only mechanism essential for synaptic plasticity. It recently appeared that NMDAR diffuse at the neuronal surface, dynamically shaping their distribution. Whether the NMDAR surface dynamics and its potential regulators play an instrumental role in the plastic properties of synapses emerged thus as a possibility. During my PhD, I tackled this question using a combination of high resolution imaging techniques (e.g. single nanoparticle tracking, FRAP) and high specificity molecular approaches (e.g. biomimetic ligand, antibody based receptor cross-link). First, I studied surface dynamics of endogenous NMDAR during synaptic plasticity on hippocampal neurons in vitro. My results reveal that the induction of glutamate synapse long-term potentiation (LTP) is accompanied by a lateral redistribution of surface NMDAR within the postsynaptic area. Strikingly, reducing the surface diffusion of NMDAR using both commercial and purified antibodies from autoimmune encephalitis patients targeting extracellular epitopes of the NMDAR prevents LTP. Second I investigated whether NMDAR dynamics were regulated. In collaboration with Stephane Oliet’s group (CRI, INSERM), we uncovered that rapid surface redistribution can also be achieved differentially using the NMDAR co-agonists, glycine and D-serine, in a GluN2A/GluN2B NMDAR subunit dependent manner. In addition, I demonstrated that the direct interaction between NMDAR and dopamine D1 receptor at the membrane controls both receptors distribution in the synaptic area and modulates synaptic plasticity. Altogether, these data indicate that the NMDAR surface dynamics is regulated by ambient neuromodulators such as dopamine and co-agonists, dynamically controlling then the plastic range of synapses

Role of NMDA receptor trafficking during synaptic maturation and plasticity

La synapse glutamatergique assure la majeure partie de la transmission excitatrice du cerveau et des changements de sa force constituent un corrélat cellulaire des processus d’apprentissage et de mémoire. Ces processus adaptatifs nécessitent souvent l’activation des récepteurs ionotropiques au glutamate de type NMDA (NMDAR) et l’influx calcique dans le compartiment postsynaptique qui suit leur ouverture. Jusqu’alors, l’activation des voix de signalisations sous-jacentes était considérée comme le seul mécanisme essentiel à la plasticité synaptique. Il est apparu récemment que les NMDAR diffusent à la surface des neurones, assurant un remodelage dynamique de leur distribution. La possibilité que la dynamique de surface des NMDAR joue un rôle déterminant dans les propriétés plastiques des synapses a donc émergé. Au cours de ma thèse, je me suis intéressé à cette problématique à l’aide d’approches d’imagerie dynamique à haute-résolution (ex. suivi de nanoparticules uniques, FRAP) et d’outils moléculaires de haute spécificité (ex. ligand biomimétique, x-link de récepteurs via les anticorps). J’ai dans un premier temps étudié la dynamique de surface des NMDAR endogènes au cours de la plasticité synaptique au sein de réseaux neuronaux hippocampiques in vitro. Mes résultats révèlent que l’induction de la potentialisation à long terme (LTP) des synapses glutamatergiques s’accompagne d’une redistribution latérale des NMDAR de surface dans la région postsynaptique. De façon remarquable, la réduction de la diffusion de surface des NMDAR via des anticorps commerciaux, mais aussi des anticorps purifiés de patients atteints d’encéphalite auto-immune, ciblant des épitopes extracellulaires des NMDAR, bloque la LTP. Dans un second temps, je me suis intéressé à la régulation de cette dynamique des NMDAR. En collaboration avec le groupe de Stéphane Oliet (CRI, INSERM), nous avons découvert qu’une redistribution rapide de surface des NMDAR s’opère différemment sous l’effet des co-agonistes du récepteur, la glycine et la D-sérine, et cela de façon dépendante des sous-unités GluN2A/GluN2B des NMDAR. De plus, j’ai démontré que l’interaction directe entre les NMDAR et les récepteurs dopaminergiques D1 membranaires contrôle la distribution des deux types de récepteurs aux abords de la synapse et module la plasticité synaptique. L’ensemble de ces données indique que la dynamique de surface des NMDAR est régulée par la présence d’un neuromodulateur, la dopamine, et de co-agonistes, contrôlant de façon dynamique la fenêtre plastique des synapses.Glutamate synapse mediates most synaptic excitation in the brain and changes in its strength constitute a cellular basis for learning and memory processes. These adaptive properties often require ionotropic glutamate NMDA receptor (NMDAR) and the calcium influx in the postsynaptic compartment following their opening. So far, the activation of the subsequent signaling pathways was considered as the only mechanism essential for synaptic plasticity. It recently appeared that NMDAR diffuse at the neuronal surface, dynamically shaping their distribution. Whether the NMDAR surface dynamics and its potential regulators play an instrumental role in the plastic properties of synapses emerged thus as a possibility. During my PhD, I tackled this question using a combination of high resolution imaging techniques (e.g. single nanoparticle tracking, FRAP) and high specificity molecular approaches (e.g. biomimetic ligand, antibody based receptor cross-link). First, I studied surface dynamics of endogenous NMDAR during synaptic plasticity on hippocampal neurons in vitro. My results reveal that the induction of glutamate synapse long-term potentiation (LTP) is accompanied by a lateral redistribution of surface NMDAR within the postsynaptic area. Strikingly, reducing the surface diffusion of NMDAR using both commercial and purified antibodies from autoimmune encephalitis patients targeting extracellular epitopes of the NMDAR prevents LTP. Second I investigated whether NMDAR dynamics were regulated. In collaboration with Stephane Oliet’s group (CRI, INSERM), we uncovered that rapid surface redistribution can also be achieved differentially using the NMDAR co-agonists, glycine and D-serine, in a GluN2A/GluN2B NMDAR subunit dependent manner. In addition, I demonstrated that the direct interaction between NMDAR and dopamine D1 receptor at the membrane controls both receptors distribution in the synaptic area and modulates synaptic plasticity. Altogether, these data indicate that the NMDAR surface dynamics is regulated by ambient neuromodulators such as dopamine and co-agonists, dynamically controlling then the plastic range of synapses

Regulation of dopamine D1 receptor dynamics within the postsynaptic density of hippocampal glutamate synapses.

Dopamine receptor potently modulates glutamate signalling, synaptic plasticity and neuronal network adaptations in various pathophysiological processes. Although key intracellular signalling cascades have been identified, the cellular mechanism by which dopamine and glutamate receptor-mediated signalling interplay at glutamate synapse remain poorly understood. Among the cellular mechanisms proposed to aggregate D1R in glutamate synapses, the direct interaction between D1R and the scaffold protein PSD95 or the direct interaction with the glutamate NMDA receptor (NMDAR) have been proposed. To tackle this question we here used high-resolution single nanoparticle imaging since it provides a powerful way to investigate at the sub-micron resolution the dynamic interaction between these partners in live synapses. We demonstrate in hippocampal neuronal networks that dopamine D1 receptors (D1R) laterally diffuse within glutamate synapses, in which their diffusion is reduced. Disrupting the interaction between D1R and PSD95, through genetical manipulation and competing peptide, did not affect D1R dynamics in glutamatergic synapses. However, preventing the physical interaction between D1R and the GluN1 subunit of NMDAR abolished the synaptic stabilization of diffusing D1R. Together, these data provide direct evidence that the interaction between D1R and NMDAR in synapses participate in the building of the dopamine-receptor-mediated signalling, and most likely to the glutamate-dopamine cross-talk

GFP insertion at the N-terminus of PSD-95 prevents the D1R-PSD95 interaction-induced D1R surface delivery.

<p>(<b>A</b>) Schematic representation of the interaction between PSD-95, characterized by its PDZ binding (squares), SH3 and GK domains (rounds), and D1R (<i>upper panel</i>). Note that the interaction occurs at the N-terminus domain of PSD95. Two variants of PSD95 were transfected in HEK cells: PSD95-CT_GFP that contains a GFP at its C-terminus and PSD95-NT_GFP that contain a GFP at its N-terminus (insertion at amino acid position 32). HEK cells were transfected with D1R-CFP and either PSD95-CT_GFP or PSD95-NT_GFP (do not bind to D1R). The surface content of D1R was measured by immuncytochemistry in the transfected cells (<i>lower panel</i>). (<b>B</b>) Line scan (white line in A) of the immunofluorescence of surface D1R from a HEK cell transfected with D1R alone. Note that D1R are enriched at the plasma membrane. (<b>C</b>) Quantification of the surface content of D1R in the various conditions. The co-expression of PSD95-CT_GFP increases the surface content of D1R (P<0.05), whereas the co-expression of PSD95-NT_GFP has no significant effect on the surface content of D1R (P>0.05).</p

Vestibular Lesion-Induced Developmental Plasticity in Spinal Locomotor Networks during Xenopus laevis Metamorphosis

International audienceDuring frog metamorphosis, the vestibular sensory system remains unchanged, while spinal motor networks undergo a massive restructuring associated with the transition from the larval to adult biomechanical system. We investigated in Xenopus laevis the impact of a pre-(tadpole stage) or post-metamorphosis (juvenile stage) unilateral labyrinthectomy (UL) on young adult swimming performance and underlying spinal locomotor circuitry. The acute disruptive effects on locomotion were similar in both tadpoles and juvenile frogs. However, animals that had metamorphosed with a preceding UL expressed restored swimming behavior at the juvenile stage, whereas animals lesioned after metamorphosis never recovered. Whilst kinematic and electrophysiological analyses of the propulsive system showed no significant differences in either juvenile group, a 3D biomechanical simulation suggested that an asymmetry in the dynamic control of posture during swimming could account for the behavioral restoration observed in animals that had been labyrinthectomized before metamorphosis. This hypothesis was subsequently supported by in vivo electromyography during free swimming and in vitro recordings from isolated brainstem/spinal cord preparations. Specifically, animals lesioned prior to metamorphosis at the larval stage exhibited an asymmetrical propulsion/posture coupling as a post-metamorphic young adult. This developmental alteration was accompanied by an ipsilesional decrease in propriospinal coordination that is normally established in strict left-right symmetry during metamorphosis in order to synchronize dorsal trunk muscle contractions with bilateral hindlimb extensions in the swimming adult. Our data thus suggest that a disequilibrium in descending vestibulospinal information during Xenopus metamorphosis leads to an altered assembly of adult spinal locomotor circuitry. This in turn enables an adaptive compensation for the dynamic postural asymmetry induced by the vestibular imbalance and the restoration of functionally-effective behavior

Unilateral labyrinthectomy-induced alterations in static posture of juvenile <i>Xenopus</i>.

<p>Back and limb joint angles on the left (L) and right (R) sides were measured in stationary intact and lesioned juveniles. The number of animals in each group is indicated in parentheses. A unilateral labyrinthectomy performed before metamorphosis caused larger subsequent alterations in static posture than a post-metamorphic UL. Note that large individual variations among the UL54 group for the R hip angle (indicated by high SEM value compared to the two other groups) were responsible for the low ANOVA power (given in parentheses for non-significant ANOVA tests). The number of animals in each group is indicated in brackets. ns: non-significant;</p>***<p>p<0.001;</p>**<p>p<0.01;</p>*<p>p<0.05.</p

A unilateral labyrinthectomy at pre- or post-metamorphosis leads to distinct degrees of locomotor impairment in freely-behaving juvenile frogs.

<p><b>A.</b> Images of the hindlimb extension phase (near its termination) during three consecutive cycles of normal swimming (top) and rolling behavior (bottom) expressed by control and unilateral labyrinthectomy (UL; red dot)-lesioned stage 66 <i>Xenopus</i>. Each cycle consisted of alternate limb flexions (F) and extensions (E). UL-induced rolling behavior consisted of a semi-complete rotation of the animal around its longitudinal body axis during each hindlimb extension. <b>B–D.</b> Swimming behavior of intact control animals (n = 6) before and after metamorphosis (<b>B</b>), and acute and chronic effects of a right-side UL performed at stage 54 before (n = 9, UL54, <b>C</b>) or at stage 66 after (n = 12, UL66, <b>D</b>) metamorphosis. Histograms show the percentage of swim cycles in which normal rectilinear (unfilled), circling (light grey) or rolling (dark grey) trajectories were expressed in each animal group. Error bars indicate SEM.</p

3D model of a UL juvenile with twisted body trunk.

<p>Geometrical model of a lesioned animal’s trunk using the 3D finite element (FE) method, based on anatomical characteristics and the assumption that the main body/water interactive forces occur at the level of the trunk (<b>A</b>). Once the initial FE model body was built, a 37° torsion () was applied in the antero-posterior axis (<b>B</b> right) in order to simulate the mean body twist towards the lesioned side observed in UL juveniles (<b>B</b> left). Red markers n (nose), r (right hip) and l (left hip) indicate model orientation and together with the two dashed lines (see <b>C</b>), illustrate the model’s torsion. <b>C:</b> The two artificial front and rear rigid body components, respectively simulating the scapula and pelvis belts, were linked by an elastic portion to which the torsion was applied. The pink and orange dashed lines indicate the medial plans of the front and rear rigid body components (green plans), respectively, while the l and r red markers correspond to the linear left and right limits of the rear medial plan, and the n marker indicates the front of the anterior plan. <i>Dorsalis</i> muscles were simulated by two actuators (blue dashed lines) placed on both sides of the antero-posterior axis between the two rigid components. <b>D:</b> Lateral (right) view of the twisted FE model corresponding to UL-induced distortion in juvenile frogs. Arrowhead indicates that the left hip marker (l) is on the non-visible side of the model.</p

Summary of changes occurring in spinal locomotor-related networks during metamorphosis and after a right-side labyrinthectomy.

<p><b>A:</b> Normal metamorphic modifications to spinal motor networks responsible for propulsion and dynamic postural adjustments during swimming (see Beyeler et al., 2008). Note the symmetrical left-right organization in the post-metamorphic juvenile frog. <b>B:</b> In already metamorphosed animals, UL causes asymmetry in the activity of descending brainstem commands to the spinal motor networks, producing an over-excitation on the lesioned side that leads to the expression of rolling behavior. This persistent descending imbalance during juvenile-to-adult maturation has no long-term influence on spinal network organization and animals never recover an effective locomotor capability. <b>C:</b> An acute UL in pre-metamorphic tadpoles also produces an asymmetric descending influence that now persists through metamorphosis (see Lambert et al. 2013). In such an unbalanced developmental environment, however, the adult spinal motor networks are built differently from normal through the establishment of a local asymmetry in propriospinal interactions that are somehow able to counterbalance the asymmetry in the descending commands to allow the restoration of swimming in the post-metamorphic frog. Red arrows: post-UL development; Black arrows: normal development; Double arrow: metamorphic development; Simple arrow: post-metamorphic maturation; Red cross: acute UL; Red dot: persistent UL. The widths of vertical arrows, arrowheads and circuit symbols are proportional to levels of activity.</p

Solely a pre-metamorphic UL alters dorsal muscle/limb extensor muscle coordination in the post-metamorphic frog.

<p>Sample left (L) and right (R) electromyographic (EMG) recordings from dorsal back muscle <i>dorsalis trunci</i> (dt) and ankle extensor muscle <i>plantaris longus</i> (pl) in intact (<b>A</b>), UL54 (<b>B</b>) and UL66 (<b>C</b>) juveniles. The sites of the vestibular lesion (UL) and EMG electrode placements are shown at left. The corresponding circular plots (layout equivalent to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071013#pone-0071013-g003" target="_blank">Figure 3</a> except that each dot represents the mean for a single forward rectilinear swim episode) indicate the lack of bilateral <i>dorsalis</i> and right side (ipsilesional) <i>dorsalis</i>/<i>plantaris</i> coordination in the UL54 group only. Scale bars: 1s.</p