Spinal cord injury produces not only the initial damage to neurons and glia, but also secondary tissue damage due to a cascade of events. One of the secondary mechanisms involves excitotoxicity due to marked increases in release of excitatory amino acids. The glutamate transporters are hypothesized to contribute to regulation of the extracellular glutamate concentration in the synaptic cleft, providing important function both in normal physiology and in pathological conditions, such as excitotoxicity. Increased release of excitatory amino acids occurs in response to acute trauma to spinal cord. The focus of this grant is to determine the effect of acute spinal cord crush injury in rats on the high affinity glutamate transporters. Pharmacologically distinct sodium (Na)-dependent and chloride (Cl)- dependent glutamate transporters are present throughout the brain. Subtypes of Na-dependent transporters have been cloned from rat, rabbit and humans. While their distribution with in brain has been determined, their distribution within the spinal cord is limited. Prior to determining the effects of spinal cord injury of the glutamate transporters, their distribution in normal rats must be delineated. We hypothesize the Na-dependent and Cl-dependent glutamate transporters will show distinct distributions in the spinal cord and that these transporters will be differentially altered by acute spinal cord crush injury.
The Specific Aims of this grant application are to: 1) Determine the distribution of the Na-dependent and Cl-dependent glutamate transporters and subtypes in cross-sections of rat spinal cord. 1a) Quantify the functional anatomical presence and distribution within the spinal cord of both the pharmacologically distinct Na- dependent and Cl-dependent transporters by autoradiography with [3H]-D-aspartate, [3H]-L-glutamate and [35S]-cystine. 1b) Quantify the subtype distribution of the Na-dependent transporters by the ability of pharmacologically characterized inhibitors of Na-dependent transport to displace [3H]-D-aspartate binding. 1c) The anatomical specificity of the Na-dependent subtypes EAAC1,GLT-1 and GLAST will be determined by immunohistochemistry with primary antibodies specific to each subtype and compared to the results in Aim1b. 2) Determine the effect of acute spinal cord injury on the transporters by crush injury at the T-10 spinal column level. Tissue will be evaluated as in the above three Specific Aims at 1,4 and 12 hours after injury. Transporter alterations will also be correlated to glial responses by GFAP immunohistochemistry. The background information obtained in these above studies will lead to future experiments to elucidate the underlying mechanisms which alter the glutamate transporters and to potential interventions in spinal cord trauma.
