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Ryuichi Shigemoto "A Novel Approach to the High Resolution Quantitative Localisation of Membrane Molecules in the CNS: Glutamate Receptors"

Abstract :

eurons in the brain have various membrane domains specialized for signal reception, integration of inputs, generation of action potentials, and transmission of output to next targets. Each membrane domain has a specific subset of molecules crucial for its characteristic function for neuronal signal processing. The synaptic domain has the largest repertoire of functional molecules such as neurotransmitter receptors, ion channels, adhesion molecules and their associating proteins embedded in a dense matrix of protein complexes. Activity of individual synapses is reflected in dynamics of these functional molecules in the domain. Conventional immunogold electron microscopy has revealed concentration of some proteins in synaptic sites but insufficient accessibility of immunochemicals often hampers detection of immunoreactivity in the dense matrix and quantitative measurements of the activity-dependent dynamics in the synaptic domain. To investigate precise localization and activity-dependent changes of synaptic molecules, we used SDS-digested freeze-fracture replica labeling, which can expose epitopes on the membrane surface with strong detergents and reveal two-dimensional and quantitative localization of membrane molecules.

Excitatory synapses are identified with clusters of intramembrane particles (IMP) on E-face of the postsynaptic membrane specialization. Immunogold labeling for ionotropic glutamate receptors, AMPA, kainate, and NMDA receptors was concentrated in these synaptic domains in the cerebellum and hippocampus. The efficacy of the AMPA receptor labeling with SDS-FRL was high enough to detect one channel with one immunogold particle as revealed by combination with an electrophysiological measurement. All excitatory synaptic domains (IMP clusters on postsynaptic E-face) in the hippocampal CA1 area and dentate molecular layer were labeled for GluR1-4 APMA and NR1 NMDA receptor subunits indicating that AMPA receptors are present even in "silent" synapses. Inhibitory synapses and presynaptic active zone were also identified with labeling for GABAA receptor subunits and CAZ proteins, respectively.
To investigate activity-dependent dynamics of AMPA receptors in the synaptic domain, we measured density changes for GluR1-4 before and after induction of cerebellar motor learning in the cerebellum. We identified parallel fiber (PF) - Purkinje cell (PC) synapses by labeling for GluR delta2 subunit and analyzed AMPA receptor density before and after adaptation of the horizontal optokinetic response (HOKR) eye movement in the middle one third of the flocculus, where long-term depression occurs with the motor learning. We found a significant reduction of labeling density at PF-PC synapses after one hour of training accompanied with a significant gain increase of HOKR. No such difference in AMPAR density was detected in the flocculus of untrained animals or in the paraflocculus of the trained animals. These results suggest that short-term HOKR adaptation is caused by reduced synaptic AMPAR content in the flocculus. 

Selected publications

Tanaka J, Matsuzaki M, Tarusawa E, Momiyama A, Molnar E, Kasai H, Shigemoto R.
Number and density of AMPA receptors in single synapses in immature cerebellum.
J Neurosci. 2005 Jan 26;25(4):799-807.
Lujan R, Shigemoto R, Lopez-Bendito G.
Glutamate and GABA receptor signalling in the developing brain.
Neuroscience. 2005;130(3):567-80.

Christophe Mulle