cortex

Project number:

503

Project title:

The dynamics of extrasynaptic glutamate receptors during excitotoxicity investigated by single particle tracking

Project supervisor:

Project description:

The amino acid glutamate mediates the majority of excitatory transmission in the CNS by activating AMPA- and NMDA-type receptors. AMPA receptors mainly mediate ongoing transmission, while NMDA receptor activation initiates both long term potentiation and depression. AMPA receptors inserted in the plasma membrane are highly mobile, and are recruited to synaptic locations by lateral diffusion (Triller and Choquet, 2005). In fact, it has been estimated that the total number of surface receptors may be greater than synaptic receptors (Rusakov et al., 1998). Recently it has been shown that a stargazin-related protein, TARP γ -8, regulates the extrasynaptic expression of AMPA receptors (Rouach et al, 2005). It is still reasonable to think, however, that synapses constitute the main route for interneuronal communication in the brain. The functional roles of spillover (diffusion of neurotransmitter in the extracellular space to activate receptors at other locations than their local synapse) or ectopic release (release of transmitter from non-synaptic sites) are not, as for now, evident. Under pathological situations, however, the role of extrasynaptic glutamate receptors could conceivably change. During excitotoxicity (excessive glutamate release which occurs in several pathological diseases like stroke, seizures and brain trauma) proportionally more released glutamate will be present at extrasynaptic sites, increasing the receptor stimulation here possibly to an even higher degree than in the synapses themselves. To further our understanding of these processes, we plan to elucidate two unavoidable questions:

1a) How big is the extrasynaptic pool of plasma membrane inserted AMPA receptors compared to the synaptic pool? 1b) Is there a differential localization of extrasynaptic receptors in such a manner that certain locations (e.g. dendritic shafts) contain significantly higher concentrations of AMPA receptors than other sites (e.g. cell bodies)?

2a) Is there a dynamic regulation of AMPA receptor lateral diffusion to synapses and out of them under excitotoxic conditions? 2b) Is there a net diffusion of AMPA receptors into or out of the synapses during excitotoxicity?

To answer these questions, we will combine quantitative postembedding immunogold labeling and single particle tracking. The first approach is well established in our lab, and we already have significant amounts of data (intaxt brain tissue) from control situations that we would like to quantitate. We need to supplement these results, however, with similar labeling data from excitotoxic brains. Basically, we will label brain sections embedded in methacrylate resins, after freeze substitution of chemically fixed tissue. When optimized for the target epitope, this approach offers a high labeling efficiency and allows simultaneous visualization of several antigens by use of different gold particle sizes. When used in conjunction with computer programs for automated acquisition and analysis of gold particles, the postembedding immunogold procedure provides an accurate representation of the cellular and subcellular distribution of proteins like glutamate receptors.

Immunogold analysis, however, yields only static information; they are snapshots of the brain. We aim to go one step further, to visualize the movements of AMPA receptors inserted into the plasma membrain into and out of synaptic locations. The single particle tracking protocol, as developed by Daniel Choquet and Antoine Triller (Triller and Choquet, 2005), is a potent tool for studies of the dynamic movement of receptors, and possibly the regulation of this diffusion, under e.g. excitotoxic conditions. Basically, we will label (with quantum dots) extracellular epitopes of AMPA receptors in dissociated hippocampal cultures, and track their movements in real time using a fast and sensitive digital camera (Photometrics Cascade 512B). Such an approach may give us information about the movements, and changes in these movements, during excitotoxic conditions (e.g. ischemia or glutamate stimulation).

Possible cortex partners for rotation:

Denise Manahan-Vaughan, Bochum