Dynamics and plasticity in developing neuronal networks in vitro.

When dissociated cortical tissue is brought into culture, neurons readily grow out by forming axonal and dendritic arborizations and synaptic connections. These developing neuronal networks in vitro display spontaneous firing activity from about the end of the first week in vitro. When cultured on multielectrode arrays firing activity can be recorded from many neurons simultaneously over long periods of time. These experimental approaches provide valuable data for studying firing dynamics in neuronal networks in relation to an ongoing development of neurons and synaptic connectivity in the network. This chapter summarizes recent findings on the characteristics and developmental changes in the spontaneous firing dynamics. These changes include long-lasting transient periods of increased firing at individual sites on a time scale of days to weeks, and an age-specific repetitive pattern of synchronous network firing (network bursts) on a time scale of seconds. Especially the spatio-temporal organization of firing within network bursts showed great stability over many hours. In addition, a progressive day-to-day evolution was observed, with an initial broadening of the burst firing rate profile during the 3rd week in vitro (WIV) and a pattern of abrupt onset and precise spike timing from the 5th WIV onwards. These developmental changes are discussed in the light of structural changes in the network and activity-dependent plasticity mechanisms. Preliminary findings are presented on the pattern of spike sequences within network burst, as well as the effect of external stimulation on the spatio-temporal organization within network bursts.

Compensatory physiological responses to chronic blockade of amino acid receptors during early development in spontaneously active organotypic cerebral cortex explants cultured in vitro.

Paired organotypic explants from rat occipital cortex were cultured for up to three weeks in the presence of selective blockers of amino acid receptor blockers, during which period spontaneous action potential generation was monitored electrophysiologically. In contrast to isolated explants (Corner, M.A., van Pelt, J., Wolters, P.S., Baker, R.E.and Nuytinck, R.H. (2002) Physiological e.ects of sustained blockade of excitatory synaptic transmission on spontaneously active developing neuronal networks--an inquiry into the reciprocal linkage between intrinsic biorhythms and neuroplasticity in early ontogeny. Neurosci. Biobehav. Rev., 26: 127-185), which upregulated their initially depressed spontaneous bursting activity only under conditions of N-methyl D-aspartate (NMDA) receptor blockade, cross-innervated co-cultures showed a large degree of functional recovery even when combined NMDA and AMPA receptor blockade was carried out. This compensatory activity could be eliminated by acute addition of a selective kainate receptor blocker to the medium. When kainate along with AMPA and NMDA receptor mediated activity was chronically suppressed, however, considerable functional recovery--in the form of recurrent burst discharges--took place gradually over a period of three weeks in vitro. These spontaneous bursts disappeared rapidly upon treatment with the muscarinic receptor blocker, atropine, but continuous low-level firing emerged at the same time. Similar "tonic" background activity was induced in control cultures as well, but without any noticeable reduction in burst discharges. Co-cultured neocortex explants, in which cyto-morphological maturation proceeds to a far greater degree than in isolated explants (Baker, R.E.and van Pelt, J. (1997) Co-cultured but not isolated cortical explants display normal dendritic development: a longterm quantitative study. Dev. Brain Res., 98: 21-27) are evidently capable of an astonishing degree of functional compensation for loss of excitatory synaptic drive during development. It could be shown, furthermore, that such homeostatic responses are not mediated largely by a weakening of inhibitory mechanisms in the absence of spontaneous firing. Chronic inhibitory synaptic blockade, on the other hand, led to intensified bursting activity which gradually normalized over a 3-week culture period. The cellular basis for this reversal of the disinhibited state, as well as for the residual neuronal firing even after cholinergic mechanisms have been largely eliminated, is at present unknown. The degree to which immature cortical networks attempt to compensate for altered levels of physiological activity, as documented in the present report, is another indication of how important such activity can be for normal development (see Corner, M.A., van Pelt, J., Wolters, P.S., Baker, R.E. and Nuytinck, R.H. (2002) Physiological e.ects of sustained blockade of excitatory synaptic transmission on spontaneously active developing neuronal networks-an inquiry into the reciprocal linkage between intrinsic biorhythms and neuroplasticity in early ontogeny. Neurosci. Biobehav. Rev., 26: 127-185).. At the same time, the large variations in overall firing levels and "macro-scale" temporal patterns from culture to culture within a given series, despite all attempts at identical preparation of the explants, can only mean that the "set-points" for such regulation are themselves subject to unknown ontogenetic factors which, apparently, are nonuniformly distributed even within a restricted region of the neocortex. On the other hand, it was striking to note that, regardless of age or treatment, an unexpected degree of consistency in temporal patterning existed at "mini-" and "micro-" time-scales (viz., EEG delta and beta frequency ranges, respectively) even when network bursting tendencies became greatly reduced in favor of tonic firing.

Long-term characterization of firing dynamics of spontaneous bursts in cultured neural networks.

Extracellular action potentials were recorded from developing dissociated rat neocortical networks continuously for up to 49 days in vitro using planar multielectrode arrays. Spontaneous neuronal activity emerged toward the end of the first week in vitro and from then on exhibited periods of elevated firing rates, lasting for a few days up to weeks, which were largely uncorrelated among different recording sites. On a time scale of seconds to minutes, network activity typically displayed an ongoing repetition of distinctive firing patterns, including short episodes of synchronous firing at many sites (network bursts). Network bursts were highly variable in their individual spatio-temporal firing patterns but showed a remarkably stable underlying probabilistic structure (obtained by summing consecutive bursts) on a time scale of hours. On still longer time scales, network bursts evolved gradually, with a significant broadening (to about 2 s) in the third week in vitro, followed by a drastic shortening after about one month in vitro. Bursts at this age were characterized by highly synchronized onsets reaching peak firing levels within less than ca. 60 ms. This pattern persisted for the rest of the culture period. Throughout the recording period, active sites showed highly persistent temporal relationships within network bursts. These longitudinal recordings of network firing have, thus, brought to light a reproducible pattern of complex changes in spontaneous firing dynamics of bursts during the development of isolated cortical neurons into synaptically interconnected networks.