NYMC Faculty Publications

Exposing Immature Hippocampal Neurons to Excitotoxins Reveals Distinct Transcriptome and Protein Regulation with Induction of Common Survival Signaling Pathways

Journal Title

Molecular and Cellular Neurosciences

First Page

54

Last Page

69

Document Type

Article

Publication Date

July 2019

Department

Cell Biology and Anatomy

Abstract

Early life traumas lead to neuroprotection by preconditioning mechanisms. To determine which genes and pathways are most likely involved in specific adaptive effects, immature hippocampal cultures were exposed to a single high dose of glutamate (250muM), NMDA (100muM), or KA (300muM) for 48h (5-7 DIV) based on our prior "two hit" in vitro model of preconditioning. Transcriptome profiling and immunocytochemistry of gene candidates were performed 7days later when cultured neurons mature (14 DIV). Many genes were up- and down- regulated involving distinct Ca(2+)-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. Immunohistochemistry showed a marked decrease in the number of Calb1 and Calm2 positive neurons following NMDA but not after glutamate exposure whereas ryanodine and Cav1.2 voltage gated channel expression was less affected. Survivors had marked increases in Calm2 immunostaining; however, high-density neural clusters observed in controls, were depleted after NMDA and partly diminished after glutamate. While NR1 mRNA expression was decreased in the microarray, specific antibodies revealed selective loss of the NR1 C1 splice variant. Calm2 which can inactivate NMDA receptors by binding to C1 but not C2 regions of its NR1 subunit suggests that loss of the C1 splice variant will reduce co-regulation with Calm2 and alter NR1 trafficking, phosphorylation, and NMDA currents following early life NMDA exposure. A dramatic reduction in the density of GABAAalpha5 and GABAB receptor expressing neurons was observed after NMDA exposure but immunodensity measurements were unchanged as was the expression of the GABA synthesizing enzyme, GAD, suggesting that fast inhibitory neurotransmission and response to benzodiazepines and GABAB-mediated IPSPs may be preserved in matured survivors. 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 CB1 interacting protein function. This decrease likely contributes to memory and attention tasks deficits that follow a single early neurological insult. Diverse changes that follow overactivation of excitatory networks of immature neurons appear long-lasting or permanent and are expected to have profound effects on network function and adaptive responses to further insult.

Link to Full Text

Share

COinS