The Role of Astrocyte Activation in Anticholinesterase-induced Synaptic Changes and Behavioral Deficits

Anticholinesterases like soman or paraoxon can cause seizures, neurodegeneration, memory impairment, and the development of anxiety, the most prevalent behavioral deficit resulting from nerve agent exposure. Activated astrocytes have been linked to brain injury, in part through the generation of harmful radicals and the secretion of glycosaminoglycans (GAGs) that hinder neuronal regeneration. In this proposal by Dr. Ben Bahr (PI), in vitro and in vivo experiments will make up a systematic study for understanding how astrocyte activation contributes to anticholinesterase neurotoxicity. Rat hippocampal slice cultures infused with the OP pesticide paraoxon will provide a model of anticholinesterase exposure to determine those astrocytic responses that colocalize with distinct events of neuronal deterioration. The mechanistic links between astrocyte activation and the neurotoxicity will be extended from the slice model to paraoxon-treated rats. The listed objectives are relevant to the Army Research Office's interests: Objective 1. As noted in previous reports, reactive astrocytes play a role in the brain's responses to toxicity, thus experiments are designed to determine the astroglial activation events that correspond with paraoxon-induced neurodegenerative changes. Across subfields of hippocampal slices, confocal microscopy will test for the colocalization between markers of reactive astrocytes (increased expression of GFAP, CSPGs) and markers of neurodegeneration (cytoskeletal breakdown, free-radical damage, synaptic loss). Tests will also look for links between activated astrocytes and areas exhibiting disruption in plasticity-related myosin dynamics. Objective 2. To understand the mechanistic steps involved, agents known to inhibit astrocytemediated radical production and GAG levels will be tested for reducing the neurodegenerative events in paraoxon-treated slices or the increased vulnerability exhibited post-treatment. The latter is tested since prior anticholinesterase exposure leads to enhanced vulnerability to excitotoxic insults (stroke, TBI). The agents include 1) inhibitors of specific mitogen-activated protein kinases that make up a phospho-relay system (JNK, p38, ERK), and 2) GAGases. Objective 3. Mechanistic findings from the hippocampal slice model will be extended to an in vivo model of rats injected with a range of paraoxon dosages. Following treatment, correlational analyses will test for statistical relationships between astrocyte activation markers vs. behavioral changes and vs. measures of neurodegeneration in several brain regions. Also, paraoxon-treated rats will be tested for enhanced vulnerability to kainic acid-induced seizure damage, and the correlational relationships are expected to be strengthened by the pathogenic synergy.