Independence of synaptic specificity from neuritic guidance.

Neuronal circuits are interconnected with a high degree of specificity. While axonal guidance has been demonstrated to be crucial for the choice of the correct target region, its role in specificity at the level of individual cells remains unclear. Specificity of synapse formation may either result from precise guidance of axonal outgrowth onto the target or depend on a molecular "match" between pre- and postsynapse. To distinguish between these possibilities, an in vitro system was used in which neuritic outgrowth of rat cortical neurons is accurately guided along the narrow pathways of a surface micropattern. The micropattern consisted of a blend of extracellular matrix molecules applied to a cell repellent background of polystyrene by microcontact printing. The system reproduces guidance by attractant and repellent surface cues while no other signals that may influence synapse formation, like gradients of trophic factors or accumulations of signaling molecules, are provided. While the number of contact points between neighboring cells was strongly reduced on patterned substrates due to the geometrical restrictions, frequency of synapse formation was not different from homogeneous cultures. Thus it was unaffected by stringent guidance onto the target cell or by the number of cell-cell contacts. Moreover, a statistically significant enrichment of reciprocal contacts between mixed pairs of excitatory and inhibitory neurons over probabilistic predictions was found, which has similarly been shown by others in dissociated neuronal cultures. Our results indicate that precise axonal guidance is insufficient for target-specific synapse formation and suggest that instead recognition between individual cells is required.

Connectivity patterns in neuronal networks of experimentally defined geometry.

Experimental control over the position and connectivity pattern of neurons on a surface is of central interest for applications in biotechnology, such as cell-based biosensors and tissue engineering. By restricting neuronal networks to a simple grid pattern, a drastic reduction of network complexity can be achieved relative to networks on homogeneous substrates. Therefore, patterned neuronal networks are also a valuable tool in research on neuronal signal transduction. Microcontact printing has emerged as a simple and efficient method for surface patterning to direct cellular attachment. Although the formation of synaptic contacts in networks of rat cortical cells on such surfaces has been demonstrated, evidence of more complex circuits has been lacking. Triple patch-clamp measurements were performed to analyze connectivity in neuronal networks complying with a grid-shaped micropattern. Cells adhered stringently to the pattern and interconnected to a range of different types of circuits: linear connections, feedback loops, as well as branching and converging pathways. We conclude that in spite of the severe geometric restrictions, a complex repertoire of different connectivity patterns can form along the provided pathways. At the same time, network complexity is kept low enough to allow the study of these patterns at the resolution of single cell-cell contacts.

Analysis of electrotonic coupling in patterned neuronal networks.

Microcontact printing of laminin is known as an efficient approach for guiding neuronal cell migration and neurite outgrowth on artificial surfaces. In the present study, ultrathin (approximately 250 microm) brain stem slices of Sprague-Dawley rats (E15-E18) were cultured on laminin-patterned substrates, such that neuronal cells migrating out of the slices formed grid-shaped neuronal networks along the geometry defined by the pattern. The interconnections between neighbouring pairs of neurons within these artificial networks were assessed electrophysiologically by double patch-clamp recordings and optically by microinjection of fluorescent dyes. Both functional and electrotonic synapses were detected. Based on the recorded data and simulations in PSpice, an electrical model for electrotonically coupled cells was derived. In this model the neuritic pathway is described as a cylindric cable, and gap junctions are represented by an ohmic resistor. Applying this model in the data analysis, the average inner radius of neurites could be determined to be approximately 0.1 microm. In addition, evidence was found for a correlation between the path-width of the applied pattern and the diameter of neurites growing along these paths.