DETERMINANTS OF VULNERABILITY TO EXCITOTOXIC DEATH

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Glutamate-mediated excitotoxic injury has been implicated in ischemic neuronal death and a variety of neurodegenerative disorders. Sensitivity to excitotoxic injury is known to vary between different types of CNS neurons, to change during development, and to be influenced by various neurotrophic factors. This proposal will use two well-characterized model systems, primary cultures of cerebellar granule cell neurons and spinal motor neurons, to determine whether and to what degree alterations in the expression, subunit composition, or phenotypic properties of glutamate receptors contribute to differences we have found in the sensitivity of these two types of neurons to glutamate-induced cell death. The proposal has three specific aims. 1.) Mature cerebellar granule cells are resistant to excitotoxic injury. Immature granule cells express the NMDA- receptor subunit NR2B, whereas mature granule cells express the NR2C subunit. To test directly whether changes in NR2 expression influence the sensitivity of granule cells to excitotoxic death, we will compare the sensitivity of granule cells cultured under conditions where NR2B or NR2C expression predominates and use replication-deficient viral vectors to alter the expression of these two subunits. 2.) We have found that exposure of purified cultures of spinal motor neurons to brain-derived neurotrophic factor (BDNF) greatly increases the vulnerability of these cells to excitotoxic death. To test whether this effect is the result of changes in glutamate-receptor (GluR) phenotype, we Will use electrophysiological methods to compare motor neurons that have been grown in the presence or absense of BDNF. We will also compare the GluR phenotype of motor neurons from BDNF-treated cultures that have or have not been exposed to glutamate. 3.) The effect of BDNF on the GluR expression and sensitivity of motor neurons to excitotoxic injury will be studied in primary cultures of spinal cord from transgenic animals in which motor neurons can be identified by cell-type specific expression of the Green Fluorescent Protein (GFP). The results will be compared with those from experiments on spinal cord cultures from mutant mice we will generate by crossbreeding the GFP-transgenic animals with BDNF knockout mice.