The precise and efficient transport, targeting and surface expression of AMPA receptors (AMPARs; GluR1-4) and kainate receptors (KARs; GluR5-7 and KA1-2) is fundamental to neuronal function. These processes are facilitated and regulated by spatially and temporally coordinated protein-protein interactions and considerable progress has been achieved in defining the exact roles of some of these interactions but much remains to be determined. An area of intense interest is assessment of the dynamic aspects of AMPAR and KAR trafficking in living neurones. In our lab we are using immunocytochemical techniques together with GFP variants to study receptor translocation, surface expression, lateral diffusion in the plasma membrane, endocytosis,recycling and exocytosis in live cells with high spatial and temporal resolution (Ashby et al. 2004b). We use virus-mediated transduction to express fluorophore-tagged AMPAR and KAR subunits in cultured hippocampal neurones and hippocampal slices to directly visualize receptors. We then determine the rates, directions and extent of movement using photobleach procedures and confocal microscopy. Although conventional GFP (and its spectral variants) provide insight into subunit and receptor transport inside neurones (Perestenko & Henley, 2003), these GFP-tagged receptors are limited for study of real time changes in surface expression. To overcome this we use a GFP derivative called super ecliptic pHluorin (SEP) that alters in fluorescence according to pH. SEP does not fluoresce at pH <6.0 but the fluorescence intensity increases with pH up to a maximum at 8.5 (Ashby et al. 2004b). Since intracellular organelles are luminally acidified, a SEP tag placed on the extracellular side of a transmembrane protein will have very little fluorescence when the protein is inside the cell. This means that SEP fluorescence only comes from proteins on the cell surface. These pHluorin-receptor constructs have many uses, for example, when expressed in hippocampal neurones pHluorin-GluR2 fluorescence is particularly enriched in the heads of dendritic spines and we have tracked the movement of synaptic and extrasynaptic AMPAR receptors in response to NMDAR stimulation (chem-LTD) (Ashby et al. 2004a). We have also shown that in cultured hippocampal neurones the surface expression of GluR6-containing KARs is dynamically regulated. Intriguingly, internalised KARs are sorted into recycling or degradative pathways depending on the endocytotic stimulus. Sustained kainate activation results in lysosomal targeting and degradation of the receptor whereas NMDAR activation evokes internalisation to early endosomes with subsequent recycling back into the plasma membrane. These processes provide mechanisms for both rapid and chronic changes in the number of functional receptors (Martin & Henley, 2004). Some of our current work is aimed at monitoring these trafficking events in near real-time using SEP-tagged KAR subunits. In addition, we are also actively investigating the parameters for surface diffusion of SEP-GluRs and how surface receptor movement is influenced by membrane topology. It has been shown that individual AMPARs diffuse within the plasma membrane using single particle tracking (Borgdorff & Choquet, 2002) but it remains unclear how these movements affect the overall distribution of synaptic proteins. Furthermore, lateral diffusion in dendritic spines has not previously been directly assessed but our recent experiments suggest that lateral diffusion can account for a rapid exchange of AMPARs at spines. Dendritic spines compartmentalize cytoplasmic molecules, a process suggested to underlie the synapse-specificity of plasticity. However, many of the proteins targeted for modification during plasticity are transmembrane (e.g. glutamate receptors) or membrane-associated (e.g. PSD95). The synapse-specificity of signalling would be compromised if these proteins laterally diffuse between synapses. Our data indicate that plasma membrane 3-D shape can control the redistribution of such proteins by physically limiting their movements at dendritic spines. This suggests that the spine neck acts to retain membrane proteins close to individual synapses. In summary, recent and on-going work in our lab is directed at understanding the cellular processes that regulate constitutive and activity-dependent glutamate receptor trafficking in neurones. A key approach is to visualise, in near real-time, receptor movement both inside the cell and at the plasma membrane and to determine how the dynamics and the trafficking are altered by physiological, pharmacological and biochemical manipulations.