Manuela Marcoli
University of Genova, Italy
Title: Functional neuron-specific endpoints for in vitro neurotoxicity testing
Biography
Biography: Manuela Marcoli
Abstract
Statement of the Problem: In accordance with 3Rs, alternativ models are required to replace standard neurotoxicity testing. Highcontent, high-throughput tools are needed considering specific features of nervous system (NS) functioning to identify neurotoxic vs. cytotoxic effects. By considering intercellular communication through transmitters and transmitter sensors (receptors), and collective behavior of neuron network as relevant NS functional features, the purpose of this study is to develop tools providing neuron-specific endpoints.
Methodology & Theoretical Orientation: A multi-disciplinary electrophysiological, neurochemical and immunocytochemical approach, combining electrical activity recording of neuron network (on engineered micro-electrode arrays (MEAs) equipped with 60 electrodes onto which cerebrocortical neurons were cultured; data analysis through a home-made software and measurement of transmitter release was used to assess network maturation and to detect effectiveness of neuroactive/neurotoxic substances.
Findings: During network development, maturation of glutamatergic/GABAergic neuron networks, target for relevant neurotoxicity mechanisms (excitotoxicity) and drugs classes, was observed. In mature networks, synaptic connectivity was related to activation of glutamatergic pathways, and the system behaved as a sensitive sensor of glutamatergic transmission functioning. Activation or blockade of NMDA/AMPA receptors, or blockade of glutamate transporters, induced firing and bursting activity variations related to the effects on transmitter release. Also, the network sensed the fine transmission variations involved in synapse plasticity: the collective network behavior and glutamate release were controlled by NMDA-dependent NO-cGMP pathway, as indicated by its pharmacological manipulation (NO synthase/guanylyl cyclase inhibitors, NO donors/8Br-cGMP). By presenting examples of network activity modulation by neuroactive substances (glutamate/GABA receptor agonists/antagonists) and by known neurotoxicants (e.g., domoic acid, chlorpyrifos oxon), and ineffectiveness of molecules not exhibiting acute neurotoxic effects, we report evidence that MEAs-coupled neuron networks can represent an integrated approach for neurotoxicity testing based on functional neuronspecific endpoints. They might provide an effective in vitro alternative tool for evaluating substance neurotoxicity, also providing a mechanistic approach.