Synaptic connectivity in micropatterned networks of neuronal cells

The presented thesis describes the formation of functional neuronal networks on an underlying micropattern. Small circuits of interconnected neurons defined by the geometry of the patterned substrate could be observed and were utilised as a model system of reduced complexity for the behaviour of neuronal network formation and activity. The first set of experiments was conducted to investigate aspects of the substrate preparation. Micropatterned substrates were created by microcontact printing of physiological proteins onto polystyrene culture dishes. The substrates displayed a high contrast between the repellant background and the cell attracting pattern, such that neurons seeded onto these surfaces aligned with the stamped structure. Both the patterning process and the cell culture were optimised, yielding highly compliant low-density networks of living neuronal cells. In the second step, cellular physiology of the cells grown on these substrates was investigated by patch-clamp measurements and compared to cells cultivated under control conditions. It could be shown that the growth on a patterned substrate did not result in an impairment of cellular integrity nor that it had an impact on synapse formation or synaptic efficacy. Due to the extremely low-density cell culture that was applied, cellular connectivity through chemical synapses could be observed at the single cell level. Having established that single cells were not negatively affected by the growth on patterned substrates, aspects of network formation were investigated. The formation of physical contact between two cells was analysed through microinjection studies and related to the rate at which functional synaptic contacts formed between two neighbouring cells. Surprisingly, the rate of synapse formation between physically contacting cells was shown to be unaltered in spite of the drastic reduction of potential interaction partners on the micropattern. Additional features of network formation were investigated and found consistent with results reported by other groups: A different rate of synapse formation by excitatory and inhibitory neurons could be reproduced as well as a different rate of frequency-dependent depression at excitatory and inhibitory synapses. Furthermore, regarding simple feedback loops, a significant enrichment of reciprocal connectivity between mixed pairs of excitatory and inhibitory neurons relative to uniform pairs could be demonstrated. This phenomenon has also been described by others in unpatterned cultures [Muller, 1997] and may therefore be a feature underlying neuronal network formation in general. Based on these findings, it can be assumed that inherent features of neuronal behaviour and cellular recognition mechanisms were found in the cultured networks and appear to be undisturbed by patterned growth. At the same time, it was possible to reduce the complexity of the forming networks dramatically in a cell culture on a patterned surface. Thus, features of network architecture and synaptic connectivity could be investigated on the single cell level under highly defined conditions.