Studies on neuronal network activity of olfactory bulb, spinal cord and frontal cortex grown on microelectrode arrays in vitro : the role of gap junctions in network integration
This project focused on understanding the mechanisms involved in CNS integration. The anatomy and physiology of mammalian olfactory system was investigated in order to develop an organotypic in vitro sensory system to increase our understanding of sensory processing at a neural network level. The olfactory network cultures grown on multielectrode arrays (MEAs) were found to only rarely exhibit electrical activity and it was decided this was an unsuitable preparation for the purposes of this study. The spinal cord was chosen as a secondary sensory system, initially in co-culture with dorsal root ganglia and then alone, with special interest in gap junction function. Gap junctions have received increasing attention as contributors to pattern generation in neuronal ensembles, including the generation or modification of highly coordinated, intense bursting states. The main result section of this study explored the effects of four gap junction blockers (carbenoxolone (CBX), halothane, I-octanol and oleamide) on the spontaneous activity of mouse and rat frontal cortex and spinal cord cultures grown on microelectrode arrays (MEAs). It was our hypothesis that the characteristic coordinated bursting seen in most frontal cortex and in some higher density spinal cord cultures would be influenced via gap junction communication. The four compounds tested generated interesting, and in one case paradoxical effects. Frontal cortex cultures were all inhibited in a dose-dependent manner, which included total cessation of activity by halothane, CBX, I-octanol, or oleamide (at concentrations 250 muM, 100 muM, 20 muM, 20 muM, respectively). All cultures showed spontaneous recovery at lower concentrations and reversibility after culture medium changes at higher concentrations. In addition, measurements of network burst rates and coefficients of variation of burst period indicate that burst coordination among channels was reduced by these compounds. These responses were generally mirrored in the spinal cord, except for CBX, which produced a paradoxical transient intense increase in network spike and burst production. The results of this study show the effect of the gap junction blockers to be not only tissue specific, but also to differ from species to species. It is still unclear whether these differences seen really are through the blockade of gap junctions, or due to the secondary effects of the blockers used. Further studies showed that strychnine (1 muM) prevented this transient activity increase in spinal cord networks, implying that CBX may temporarily block glycine inhibition. Blocking intracellular calcium mobility with thapsigargin (up to 5 muM) did not affect the effects of gap junction blockers used.