ABSTRACT
Title
NO-cGMP pathway controls network activity in dissociated cortical neurons coupled to Micro-Electrode Arrays
Authors
C. Cervetto 1, M. Tedesco 2, M. Chiappalone 3, M. Marcoli 1, G. Maura (1,4,5) & S. Martinoia (2,3)
(1) DIMES, Pharmacology and Toxicology, University of Genoa, Genoa, Italy; (2) NBT-DIBE, University of Genoa, Genoa, Italy; (3) NeuroTech-NBT, Italian Institute of Technology (IIT), Genoa, Italy; (4) Center of Excellence Biomed Res., University of Genoa, Genoa, Italy; (5) National Institute of Neuroscience (INN), Turin, Italy
(1) DIMES, Pharmacology and Toxicology, University of Genoa, Genoa, Italy; (2) NBT-DIBE, University of Genoa, Genoa, Italy; (3) NeuroTech-NBT, Italian Institute of Technology (IIT), Genoa, Italy; (4) Center of Excellence Biomed Res., University of Genoa, Genoa, Italy; (5) National Institute of Neuroscience (INN), Turin, Italy
Abstract
Neuronal networks coupled to microelectrode arrays (MEAs) allowing long-term, non-invasive, multielectrode detection of spontaneous and evoked firing activity and neuronal network synchronization are a valuable experimental model for studying neuronal dynamics. We previously found that in cerebrocortical neuron networks exhibiting a mature behavior, synaptic connectivity was mostly related to glutamate pathway activation: activation of NMDA receptors evoked changes of firing and bursting rate highly correlated to the effects on glutamatergic transmission, as assessed by measurement of glutamate release. Although activation of the NMDA-dependent nitric oxide (NO)-cyclic GMP (cGMP) pathway is hypothesized to play roles in the control of neuron network plasticity at glutamatergic synapses, roles for NO signalling in the control of the integrated network activity had scarcely been addressed. Here we report on the effects of pharmacological manipulation of the NO-cGMP pathway on the dynamics of rat cerebrocortical neuron networks. A detailed analysis of drug effects on the network electrophysiological behavior, in terms of spike and burst activity is presented, and a novel approach related to changes in the synchronization level among neuronal units in the network under chemical stimulation is addressed. Determination of glutamate released by cortical neuronal networks in combination with recording of integrated activity allowed to establish correlation between emerging patterns of network activity and functioning of glutamatergic excitatory synapses.
Briefly, primary cultures of cortical neurons prepared from E18 rats were plated over poly-d-lysine and laminin coated microelectrode arrays of 60 planar TiN/SiN microelectrodes and kept alive in healthy conditions for several weeks. Electrophysiological signals were recorded simultaneously from 60 electrodesstarting from the second week in vitro, to allow for synaptic maturation of the network;the experimental sessions started with a 20 min recording of the network spontaneous activity, in physiological solution as control condition; a 20 min recording was applied for each experimental condition. Signals were recorded and monitored by using a home made developed software for realtime spike detection and burst analysis, and a commercial software for on line visualizations and raw data storage. Endogenous glutamate was measured by HPLC in Aliquots of the bath solution; the analytical method involved automatic precolumn derivatization with o-phthalaldehyde followed by separation on C18 reverse phase chromatography column and fluorimetric detection.
Our findings confirm dependence of complex network behavior from the functioning of glutamate transmission in cerebrocortical cultures. We find that NO synthase inhibition by NARG inhibited both firing activity and glutamate release, while the NO donor SNAP transiently increased firing rate and glutamate release, indicating that glutamatergic transmission and network activity are controlled by the gaseous messenger NO. As NO has been reported to possess both cGMP-dependent and cGMP independent pathways, we investigated on the effects of interference with cGMP production. We found that inhibition of the guanylyl cyclase activity by ODQ inhibited both firing activity and glutamate release; addition of the membrane permeable analogue of cGMP, 8Br-cGMP, increased both electrophysiological activity and transnmitter release. The finding indicatesthat the cGMP dependent patwhays is involved in the NO role in sustaining network activity and glutamatergic transmission activation. We conclude that collective behavior of mature cerebrocortical neuronal network is controlled by activation of the NO-cGMP pathway and that manipulation of the pathway affects electrophysiological network behavior and glutamate release.
Briefly, primary cultures of cortical neurons prepared from E18 rats were plated over poly-d-lysine and laminin coated microelectrode arrays of 60 planar TiN/SiN microelectrodes and kept alive in healthy conditions for several weeks. Electrophysiological signals were recorded simultaneously from 60 electrodesstarting from the second week in vitro, to allow for synaptic maturation of the network;the experimental sessions started with a 20 min recording of the network spontaneous activity, in physiological solution as control condition; a 20 min recording was applied for each experimental condition. Signals were recorded and monitored by using a home made developed software for realtime spike detection and burst analysis, and a commercial software for on line visualizations and raw data storage. Endogenous glutamate was measured by HPLC in Aliquots of the bath solution; the analytical method involved automatic precolumn derivatization with o-phthalaldehyde followed by separation on C18 reverse phase chromatography column and fluorimetric detection.
Our findings confirm dependence of complex network behavior from the functioning of glutamate transmission in cerebrocortical cultures. We find that NO synthase inhibition by NARG inhibited both firing activity and glutamate release, while the NO donor SNAP transiently increased firing rate and glutamate release, indicating that glutamatergic transmission and network activity are controlled by the gaseous messenger NO. As NO has been reported to possess both cGMP-dependent and cGMP independent pathways, we investigated on the effects of interference with cGMP production. We found that inhibition of the guanylyl cyclase activity by ODQ inhibited both firing activity and glutamate release; addition of the membrane permeable analogue of cGMP, 8Br-cGMP, increased both electrophysiological activity and transnmitter release. The finding indicatesthat the cGMP dependent patwhays is involved in the NO role in sustaining network activity and glutamatergic transmission activation. We conclude that collective behavior of mature cerebrocortical neuronal network is controlled by activation of the NO-cGMP pathway and that manipulation of the pathway affects electrophysiological network behavior and glutamate release.