RESUMO
Neuronal assemblies within the nervous system produce electrical activity that can be recorded in terms of action potential patterns. Such patterns provide a sensitive endpoint to detect effects of a variety of chemical and physical perturbations. They are a function of synaptic changes and do not necessarily involve structural alterations. In vitro neuronal networks (NNs) grown on micro-electrode arrays (MEAs) respond to neuroactive substances as well as the in vivo brain. As such, they constitute a valuable tool for investigating changes in the electrophysiological activity of the neurons in response to chemical exposures. However, the reproducibility of NN responses to chemical exposure has not been systematically documented. To this purpose six independent laboratories (in Europe and in USA) evaluated the response to the same pharmacological compounds (Fluoxetine, Muscimol, and Verapamil) in primary neuronal cultures. Common standardization principles and acceptance criteria for the quality of the cultures have been established to compare the obtained results. These studies involved more than 100 experiments before the final conclusions have been drawn that MEA technology has a potential for standard in vitro neurotoxicity/neuropharmacology evaluation. The obtained results show good intra- and inter-laboratory reproducibility of the responses. The consistent inhibitory effects of the compounds were observed in all the laboratories with the 50% Inhibiting Concentrations (IC(50)s) ranging from: (mean ± SEM, in µM) 1.53 ± 0.17 to 5.4 ± 0.7 (n = 35) for Fluoxetine, 0.16 ± 0.03 to 0.38 ± 0.16 µM (n = 35) for Muscimol, and 2.68 ± 0.32 to 5.23 ± 1.7 (n = 32) for Verapamil. The outcome of this study indicates that the MEA approach is a robust tool leading to reproducible results. The future direction will be to extend the set of testing compounds and to propose the MEA approach as a standard screen for identification and prioritization of chemicals with neurotoxicity potential.
RESUMO
The release of transmitter at neuromuscular junctions (NMJ) of the opener muscle in crayfish is quantal in nature. This NMJ offers the advantage of being able to record quantal events at specific visually identified release sites, thus allowing measurement of the physiological parameters of vesicle release and its response to be directly correlated with synaptic structure. These experiments take advantage of areas between the varicosities on the nerve terminal that we define as "stems." Stems were chosen as the region to study because of their low synaptic output due to fewer synaptic sites. Through 3D reconstruction from hundreds of serial sections, obtained by transmission electron microscopy (TEM), at a site in which focal macropatch recordings were obtained, the number of synapses and AZs are revealed. Thus, physiological profiles with various stimulation conditions can be assessed in regards to direct synaptic structure. Here, we used the properties of the quantal shape to determine if distinct subsets of quantal signatures existed and if differences in the distributions are present depending on the frequency of stimulation. Such a quantal signature could come about by parameters of area, rise time, peak amplitude, latency, and tau decay. In this study, it is shown that even at defined sites on the stem, with few active zones, synaptic transmission is still complex and the quantal responses appear to be variable even for a given synapse over time. In this study, we could not identify a quantal signature for the conditions utilized.