Abstracts

Rationale: Immature neurons can resist neurotoxicity associated with overactivation of glutamate receptors and are instead capable of generating substantial neuroprotective/ preconditioning effects in response to seizures or certain excitatory amino acids (EAAs). Our recent in vivo and in vitro microarray transcriptome profiling studies of hippocampal neurons revealed many genes were up- and down- regulated involving distinct Ca2+-binding protein families, G-coupled proteins, various growth factors, synaptic vesicle docking factors, certain neurotransmitter receptors, heat shock, oxidative stress, and certain anti-apoptotic Bcl-2 gene members that influence neuronal survival. Methods: In order to verify the translation of certain candidate genes that most likely are involved in neuroprotection, we used our early life in vitro pre-conditioning protocol to expose immature hippocampal neurons to high doses of glutamate (250 M) or NMDA (100 M) for 48 hr (5-7DIV). Cultured hippocampal neurons were processed for immunohistochemistry with 15 specific antibodies applied 7 days after the exposure, when cultured neurons mature (14DIV). Cultured neurons were dehydrated on coverslips mounted onto slides and analyzed under bright field microscopy. Results: A marked decrease in the number of Calb1 and Calm2 positive neurons was observed following NMDA but not after glutamate pre-treatment whereas ryanodine and Cav1.2 voltage gated channel expression was steady under both conditions. However, surviving neurons had marked increases in their expression of Calm2 in single neurons but clustered neurons observed in controls were depleted of Calm2 after NMDA and highly reduced after glutamate treatment. A dramatic reduction in the density of GABAA alpha 5 and GABAB receptor expressing neurons was also observed by prior overactivation of NMDA type glutamate receptors but immunodensity measurements of most of the survivors was unchanged as was the expression of the GABA synthesizing enzyme, GAD. While NR1 mRNA expression was decreased in prior microarrays, protein expression with specific antibodies revealed selective loss of the NR1 C1 splice variant. Conclusions: In conclusion, fast inhibitory neurotransmission and response to benzodiazapines and GABAB-mediated IPSPs may not be reduced in mature survivors after an early life insult. Calm2 which can induce inactivation of NMDA receptors by binding tightly to C1 but not C2 regions of its NR1 subunit suggests that the C1 splice variant is co-regulated with Calm2 and that simultaneous loss of these proteins would reduce subsequent NR1 trafficking, phosphorylation, and NMDA currents following early life NMDA exposure. Moreover, selective upregulation of Chat and CNRIP was detected after glutamate treatment suggesting this condition would decrease cholinergic and excitatory neurotransmission by decreasing Ach content and the CB1 cannabinoid interacting protein function which likely contribute to memory and attention task deficits that follow a single early life neurological insult (such as a seizure). Hence, diverse translated gene changes that follow overactivation of excitatory networks of immature neurons appear permanent and are expected to have profound effects on network function and adaptive responses to subsequent insults Funding: Private Donor