Showing papers by "Daniel Choquet published in 2006"

NMDA receptor surface mobility depends on NR2A-2B subunits

Abstract: The NR2 subunit composition of NMDA receptors (NMDARs) varies during development, and this change is important in NMDAR-dependent signaling. In particular, synaptic NMDAR switch from containing mostly NR2B subunit to a mixture of NR2B and NR2A subunits. The pathways by which neurons differentially traffic NR2A- and NR2B-containing NMDARs are poorly understood. Using single-particle and -molecule approaches and specific antibodies directed against NR2A and NR2B extracellular epitopes, we investigated the surface mobility of native NR2A and NR2B subunits at the surface of cultured neurons. The surface mobility of NMDARs depends on the NR2 subunit subtype, with NR2A-containing NMDARs being more stable than NR2B-containing ones, and NR2A subunit overexpression stabilizes surface NR2B-containing NMDARs. The developmental change in the synaptic surface content of NR2A and NR2B subunits was correlated with a developmental change in the time spent by the subunits within synapses. This suggests that the switch in synaptic NMDAR subtypes depends on the regulation of the receptor surface trafficking.

AMPA and NMDA glutamate receptor trafficking: multiple roads for reaching and leaving the synapse.

Abstract: Glutamate receptor trafficking in and out of synapses is one of the core mechanisms for rapid changes in the number of functional receptors during synaptic plasticity. Recent data have shown that the fast gain and loss of receptors from synaptic sites are accounted for by endocytic/exocytic processes and by their lateral diffusion in the plane of the membrane. These events are interdependent and regulated by neuronal activity and interactions with scaffolding proteins. We review here the main cellular steps for AMPA and NMDA receptor synthesis, traffic within intracellular organelles, membrane exocytosis/endocytosis and surface trafficking. We focus on new findings that shed light on the regulation of receptor cycling events and surface trafficking and the way that this might reshape our thinking about the specific regulation of receptor accumulation at synapses.

Multiple routes for glutamate receptor trafficking: surface diffusion and membrane traffic cooperate to bring receptors to synapses.

Abstract: Trafficking of glutamate receptors into and out of synapses is critically involved in the plasticity of excitatory synaptic transmission. Endocytosis and exocytosis of receptors have initially been thought to account alone for this trafficking. However, membrane proteins also traffic through surface lateral diffusion in the plasma membrane. We describe developments in electrophysiological and optical approaches that have allowed for the real-time measurement of glutamate receptor surface trafficking in live neurons. These include (i) specific imaging of surface receptors using a pH-sensitive fluorescent protein; (ii) design of a photoactivable drug to locally inactivate surface receptors and monitor electrophysiologically their recovery; and (iii) application of single-molecule fluorescence microscopy to directly track the movement of individual surface receptors with nanometer resolution inside and outside synapses. Together, these approaches have demonstrated that glutamate receptors diffuse at high rates in the neuronal membrane and suggest a key role for surface diffusion in the regulation of receptor numbers at synapses.

Simultaneous excitation of multiple fluororophores with a compact femtosecond laser

Abstract: We report on a flexible multiphoton imaging system, suitable for simultaneous and efficient excitation of red (DsRed), yellow (YFP), green (GFP) and blue (DAPI) fluorophores. We used a simple, compact laser system, consisting of a 1 µm high energy diode-pumped oscillator and a tunable wavelength extension using a photonics crystal fibre. The combination of a near IR excitation wavelength, high energy per pulse for efficient three photon excitation and spectral extension for GFP excitation allows for high flexibility.We present experimental results of simultaneous and efficient imaging fluorophores couples from the UV to the red (DAPI-RFP, GFP-RFP).

Lateral Diffusion of Excitatory Neurotransmitter Receptors During Synaptogenesis

Abstract: The understanding of how receptors accumulate within the developing synapse has captured a lot of attention. Lateral diffusion of surface neurotransmitter receptors has emerged as a key pathway to regulate receptor trafficking and surface distribution, in addition to the receptor cycling between intracellular and plasma membrane pools. The neurotransmitter receptor lateral diffusion depends on several factors, such as interactions with other proteins highly enriched within the synaptic structure. During synaptogenesis, receptors aggregate and cluster within developing synapses. As described in this chapter, lateral diffusion of receptors is likely to play an important role in such process since receptor lateral diffusion is high during synaptogenesis, providing favourable conditions for the “capture” of receptors within synaptic contacts. Indeed, the “diffusion-trap” model for receptor accumulation in developing synapses has now gained experimental support from excitatory synapses, although direct evidence to test this model is still lacking due to the absence of adequate tools to precisely control extrasynaptic receptor lateral diffusion. Within the developing synapse, it also emerges that neurotransmitter release generates surface instability of the receptors and their stabilization requires additional factors.