Characterization of acute neurotoxic effects of trimethylolpropane phosphate via neuronal network biosensors

Abstract

We have utilized cultured neuronal networks grown on microelectrode arrays to demonstrate rapid, reliable detection of a toxic compound, trimethylolpropane phosphate (TMPP). Initial experiments, which were performed blind, demonstrated rapid classification of the compound as a convulsant, a finding consistent with previous whole animal neurobehavioral studies. TMPP (2-200 microM) reorganized network spike activity into synchronous, quasi-periodic burst episodes. Integrated burst amplitudes invariably increased, reflecting higher spike frequencies within each burst. The variability of network burst parameters, quantified as coefficients of variation (CVs), was decreased. Mean CVs for burst duration, interburst interval, and burst rate were lowered by 42+/-13, 58+/-5.5, and 62+/-1.8%, respectively (mean+/-SEM, n=8 cultures, 197 channels). These changes in network activity paralleled the effects induced by bicuculline, a known disinhibitory and seizure-inducing drug, and confirmed classification of TMPP as a potential epileptogenic compound. Simple pharmacological tests permit exploration of mechanisms underlying observed activity shifts. The EC(50) for GABA inhibition of network activity was increased from 2.8 to 7.0 microM by 20 microM TMPP and to 20.5 microM by 200 microM TMPP. Parallel dose-response curves suggest that TMPP acts by a competitive antagonism of GABA inhibition, and are consistent with reported patch-clamp analysis of TMPP-induced reduction of inhibitory postsynaptic current amplitudes. The potency of TMPP in generating epileptiform activity in vitro was comparable to concentrations reported for in vivo studies. TMPP and bicuculline produced both increases and decreases in burst rate depending on native spontaneous bursting levels. These results demonstrate a need for multivariate analysis of network activity changes to yield accurate predictions of compound effects.