Connectivity statistics and their implications on activity dynamics in cortical cell cultures

Abstract

Event Abstract Back to Event Connectivity statistics and their implications on activity dynamics in cortical cell cultures Samora Okujeni1, 2*, Steffen Kandler1, 2 and Ulrich Egert2, 3 1 BCCN Freiburg, Neurobiology and Biophysics, Germany 2 Albert-Ludwigs-University Freiburg , Germany 3 BCCN Freiburg, Department of Microsystems Engineering , Germany In the absence of complex architecture, central parameters of connectivity in neuronal networks are the size of dendrites and axons in conjunction with the spatial distribution of neurons. The homeostatic regulation of neurite fields is crucial for the maintenance of stability in the activity dynamics. We analyze these fundamental properties of neuronal networks in dissociated cortical cell cultures grown on micro-electrode arrays (MEAs). These generic random networks display a self-regulated maturation process that is characterized by neurite outgrowth, synapse over-expression and pruning similar to the critical period in the developing cortex. Within this period of network formation we manipulated neuronal connectivity by pharmacological intervention with structural differentiation processes. Previous studies showed that pharmacological inhibition of protein kinase C (PKC) activity enhances neurite outgrowth [1] and impairs cell migration [2] and network pruning [3] in early neural development. Following these findings, we chronically treated cortical cell cultures with PKC inhibitors and characterized them morphologically and functionally. To capture connectivity statistics we applied a modified Scholl analysis [4] to describe the radial dendritic field density of neurons embedded into the network. The method involves the grouping of neurons into classes of defined local neuron density to account for local differences in the overlap of dendritic fields of spatially non-uniformly distributed neurons. Dendrite profiles across conditions were subsequently compared based on these classes. Blocking PKC activity significantly enhanced radial arborization by +15% at intermediate (DIV14) and about +50% at later stages (DIV40) of development. The evolution of dendritic fields furthermore revealed an ongoing extension in cultures exposed to PKC inhibitors in contrast to an incipient reduction found under control conditions. This suggests an impaired downregulation of neurite outgrowth and pruning with the ending of the initial wiring phase. To further confirm these findings, dendrites of neurons in sparse cultures with negligible dendritic field overlap were traced manually and likewise showed significantly increased arborization and enhanced segment extension. By correlating local and global neuron density [5] we moreover found reduced clustering in the networks developing under inhibited PKC activity, indicating impaired cell migration. Electrophysiological recordings were performed during the development to assess functional consequences of the altered network differentiation. In cultures exposed to PKC inhibitors we had a higher yield of electrodes displaying neuronal activity, which accounts for a higher probability of finding neurons in the vicinity of an electrode in absence of neuronal clustering. The number of active electrodes furthermore remained stable in the course of development in contrast to a decrease found under control conditions, which provides electrophysiological evidence for the non-initiation of pruning processes. The activity stayed organised in network-wide bursting events that characteristically emerge in neuronal cell cultures. Bursts however tended to be more compact, occurred at a higher frequency and were stronger synchronized over the MEA. This suggests a faster propagation of activity through the networks with enhanced inter-neuronal connectivity. In summary we show that inhibition of PKC activity in developing cortical cell cultures increases connectivity and relate the structural changes to functional differences in the activity dynamics.