Quantitative analysis of neuronal firing patterns was used to study the effects of chronic suppression of bioelectric activity (BEA) on functional development in primary cultures of fetal rat cerebral cortex. BEA was monitored with extracellular electrodes in active control cultures or, after return to control medium, in cultures chronically silenced with tetrodotoxin (TTX) at around 7, 14, 21 and 42 days in vitro. Spike trains of single neurons lasting up to 25 min duration were analyzed using a previously published set of computer programs. In control cultures, the main developmental trends seen in a previous study could be replicated. After development in the presence of TTX, activity levels were increased at all ages, and a high incidence was found of a single firing pattern characterized by stereotyped burst firing, while showing a low minute order variability in firing rate and low dependencies between successive intervals; conversely, the incidence of variable/non-burst firing was decreased relative to untreated cultures. The former firing pattern (i.e. non-variable bursting) could also be produced through acute addition of the GABA (A)-antagonist picrotoxin to control cultures, and resembled interictal burst firing observed in models of chronic epilepsy in vivo. These similarities suggest that chronic silencing of the cultures may have resulted in a functional disinhibition of the neuronal network; such disinhibition might be related to the increased cell death which we observed with chronic TTX-treatment in the same cultures.

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The development of spontaneous bioelectric activity (SBA) was studied in dissociated occipital cortex cultures prepared from 19 day old rat fetuses. All cultures, recorded one per diem from 5 to 33 days in vitro (div), showed SBA. Computer analysis of 76 extracellularly recorded single unit spike trains was carried out after selection on the basis of stationarity criteria. Statistically significant developmental trends were found in (i) interspike interval dependencies and (ii) fluctuations in mean firing rate, on the order of a minute or longer. The highly dependent firing patterns, including stereotyped bursting, were present mostly in the 9-12 div group, whereas minute-to-minute fluctuations in the intensity of firing were considerably more pronounced in the oldest group (22-33 div) than in the younger cultures. In addition, firing categories defined on the basis of factor-analysis revealed that such fluctuations were almost exclusively to be found in neurons which fired in a pronounced 'burst', rather than a relatively continuous fashion. Only a few mature appearing synaptic structures were observed electron microscopically prior to 12 div, but increased steadily in number thereafter. No cultures prior to 14 div, but all cultures older than this, stained positively for the presence of glutamic acid decarboxylase. An extensive immunoreactive, putative GABAergic, network was present by three weeks in vitro.

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SUMMARYPreviously we have presented evidence for a direct relationship between post-mitotic new membrane formation and changes in the electrical membrane characteristics during cytokinesis of Xenopus eggs [l, 21. In the present study the phenomena underlying the hyperpolarization of the electrical membrane potential during cytokinesis were investigated. Total Na+ and K+ contents at the onset of the first and second cleavage were measured independently by flame spectophotometry and by means of ion-selective electrodes. Total Cl-content was measured by the latter method only. The water content was determined from the difference between wet weight and dry weight. 3H,0-influx experiments yielded an independent estimate of the water content (0.737 ,ul/egg), a rate constant for the influx of 1.412 x 10e3 set,-r and a low water permeability of 1.87 x 1O-5 cm set-I. They furthermore revealed the absence of intracellular water compartmentation. The Naf, K+ and Cl-concentrations remained constant during first cleavage at 58.6, 87.3 and 62.6 mM/l cell water, respectively. The membrane potential (E,), the membrane resistance (R,), and the intracellular ion activities of Na+, K+ and Cl-(a;,, ak and a$ were measured simultaneously and continuously during cleavage, using conventional glass microelectrodes and ion-selective microelectrodes. a' na showed an increase from 19.4 to 22.4 mM concomitant with the hyperpolarization of Em and the decline of R,. a; and a& remained constant at 51.4 and 53.1 mM, respectively. From the calculated activity coefficients it was concluded that all Cl-ions were free, whereas 30 % of the Kf ions and 60 % of the Naf ions were bound. The influence of changes in the ionic composition of the medium on E,,, was analysed in the uncleaved egg, in normally cleaving eggs, and in eggs cleaving outside the vitelline membrane. The latter conditions leads to exposure of the whole area of newly formed membrane to the medium. The cell membrane of the uncleaved egg exhibits no permselectivity. In normally cleaving eggs the relative permeability P&Px was 0.73, while in eggs cleaving outside the vitelline membrane it was 0.19. It was concluded that the changes of E,,, during cytokinesis are due to the insertion of a part of the newly formed membrane into the cell surface. The K+ permeability of this new membrane is at least five times greater that that of the pre-existing membrane. The possible role of the hyperpolarization of E,,, in the regulation of the cell cycle is briefly discussed.In previous reports [2, 31 the characteristic changes of the electrical membrane potential (E,) and the electrical membrane resistance (R,) during the first cytokinetic cycle of Xenopus laevis eggs were described. A temporal and causal relationship was found between these changes and new membrane formation. Ihe electrical membrane characteristics start to change at 6-8 min from the onset of the cleavage. From that time on morphological signs of the incorporation of new membrane material in the bottom of the cleavage furrow are ...