Glutamate is the principal excitatory neurotransmitter throughout the CNS, including the spinal cord. It acts on ionotropic (iGluR) and metabotropic glutamate receptors. Three iGluR families have been identified by the development of more-or-less selective agonists: N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate receptors. Both AMPA (GluR1-4) and NMDA (NR1, NR2A-D) receptors have been detected in the spinal cord and these play a major role in physiological processes such as fast excitatory transmission, synaptic plasticity and neuronal development. In addition, they have also been implicated in pathological conditions including neuropathic pain and neurodegenerative disorders. However, very little is known about the synaptic distribution of these receptors in the spinal gray matter. This is because conventional immunocytochemical techniques, generally used to investigate the location of proteins in the CNS, fail to detect these subunits at synapses due to the presence of an elaborate protein meshwork associated with the postsynaptic membrane, which masks synaptic receptors. Postembedding immunocytochemistry on freeze-substituted, Lowicryl-embedded material is a technique which has been used exclusively for the detection of synaptic AMPA and NMDA receptors. This project initially set out to use this method to examine these receptors on neurons of the adult rat spinal cord, with emphasis on their involvement in the sensory processing of the dorsal horn. With antibodies against the GluR1, GluR2/3, NR1, NR2A and NR2B subunits, heavy labelling was observed at many asymmetrical synapses and where the plane of section was perpendicular to the cleft, most of the immunogold particles were associated with the postsynaptic density. To examine the receptor expression pattern of selected cell populations a new method was developed which involved the combination of postembedding electron microscopy with immunofluorescence and confocal microscopy. However, during the course of this study heavy immunogold labelling of dense-cored vesicles (dcvs) inside axonal boutons was observed with all NMDA antibodies. Several studies have found iGluRs in primary afferent terminals in the spinal gray matter and these are thought to function as presynaptic receptor, in order to determine whether gold particles found over dcvs corresponded to presynaptic receptors in transit, immunogold reactions were carried out on transgenic mice which lacked the NR2A subunit. Surprisingly, not only did the dev labelling remain in these knock-out animals, but there was also a significant synaptic labelling. This suggested that the postembedding immunogold labelling observed with the NR2A antibody was non-specific. Since the labelling patterns were similar with other NMDA antibodies this cast doubts on the validity of the postembedding method for detecting NMDA receptors.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:404285 |
| Date | January 2004 |
| Creators | Nagy, Gergely György |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/30731/ |
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