The goals of Project 3 are two-fold. The first goal is to develop in vitrolex vivo models of acute intoxication by seizurogenic chemical threat agents by applying high content, rapid throughput technology to quantitatively detect disruption of electrical and Ca^* signaling events that are known to contribute to seizure induction, severity, and promote neuronal damage. The second goal is to apply these methods to investigate existing and new therapeutic strategies for mitigating onset, progression, and severity of seizures elicited by acute intoxication with the GABA antagonist tetramethylenedisulfotetramine (TETS) and the organophosphorus (OP) cholinesterase inhibitor diisopropylfluorophosphate (DFP). The Axion multi-well multichannel electrode arrays permit unparalleled sensitivity in detecting patterns of electrical burst-spike activity in both dissociated neurons and brain slices, whereas the 96-well Tetra High Throughput Cellular Screening System (FLIPR) provides real-time assessments of spontaneous and evoked Ca oscillations. Relevant in vitro systems, including cultures enriched for primary neurons, mixed astrocyte-neuronal co-cultures, and hippocampal slices are chosen for model development because of their predictive value to Projects 1 and 2. A major outcome of the proposed studies will be to screen existing therapeutic leads and discover new therapeutic targets (with Cores A and B) that delay or mitigate triggered seizure activity and subsequent neuropathology produced by TETS and DFP. These in vitro approaches will identify the most promising therapeutic candidates and drug combinations for in vivo testing in Projects 1 and 2. Further validation and optimization of therapeutic strategies, including timing of administration, dosages, and drug combinations, will be assessed ex vivo in a reiterative process with Projects 1 and 2. The receptor based rapid-throughput approaches proposed in Project 3 will expedite more efficient formulation of optimal therapeutic strategies by Cores A and B. Moreover, the molecular and cellular mechanisms by which TETS and DFP alter the fidelity of Ca2+ signaling events to promote injurious neuronal excitability will improve our understanding of how these threat agents promote seizures and impair neural networks, providing insight into novel drug targets. Additional major milestones of the in vitro/ex vivo approach proposed in Project 3 are to accelerate: (1) Implementation of existing therapeutic agents approved for use in humans or currently undergoing clinical trials (referred to in this proposal as Tier 1), and (2) identify new receptor-targeted compounds and mixtures that improve our ability to treat acute (convulsant) and delayed (neuropathology) harm caused by exposures to seizurogenic threat agents (referred to as Tier 2 candidates).
Aim 1 : Develop rapid throughput multielectrode array (MEA) and high-resolution imaging methods to efficiently detect and discriminate threat agents that trigger electrical burst-spike activity, those that dysregulate neuronal Ca2+ signaling, and those that have both activities. 1 .a. Investigate if TETS produces electrical burst-spike activity and abnormal Ca2+ oscillations in neuronal cell cultures and/or neuron/glia co-cultures. I.b. Determine whether TETS and DFP produce similar patterns of burst-spike (epileptiform) activity in acutely dissociated hippocampal slices.
Aim 2 : Determine whether Tier 1 and novel Tier 2 therapeutic candidates, singly or in combination, ameliorate electrical and Ca2+ signaling disturbances triggered by TETS and/or DFP.
Aim 3 : Investigate if therapeutic candidates that target Ca2+ -dependent signaling events provide added neuroprotection against TETS- and DFP-triggered chronic neuropathology. 3.a. Determine whether exposures to TETS or DFP, in the presence or absence of therapeutic intervention, in vivo persistently alters the electrophysiological and Ca2+ signaling properties of hippocampal neurons and slices prepared ex vivo. 3.b. Determine whether exposures to TETS or DFP, in the presence or absence of therapeutic intervention, in vivo persistently alters the biomarkers of oxidative stress and chronic neuropathology in hippocampal neurons and slices prepared ex vivo.
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