Formation of hippocampal synapses on patterned substrates of a laminin-derived synthetic peptide

We created a new culture system which provides simple, stereotyped neuronal circuitries suitable for investigating synaptic events between mammalian central neurons. We used surface chemistry and laser-lithography to produce geometrical patterns of neuron-compatible substrate spaced by less neuron-compatible surfaces. The patterned substrates were composed of a laminin-derived synthetic peptide, PA22-2, and the spacing surfaces of either decyldimethylsilane (DDMS) or trimethylsilane (TMS). Dissociated rat hippocampal neurons survived on the patterned substrates for several days without the aid of glia and extended their neurites along the substrates. The TMS spacing surfaces appeared more favourable for the excitability development and axonal differentiation of the hippocampal neurons, but less favourable for the development of the resting conductance than the DDMS spacing surfaces. Furthermore, neurons grown on the patterned substrates frequently made synaptophysin-positive contacts with one another. Spontaneous post-synaptic currents recorded from such neurons suggest that these contacts were indeed functional synapses. When hippocampal neurons were plated at a very low density, they often formed circuitries consisting of only two neurons on the patterned substrate. Such a simple circuitry allowed us to analyse synaptic transmission in a single neuronal pair without the influence of the third neurons. With the clarity of analysis and the readiness of manipulation, our culture system would offer a powerful tool for studying development and functions of mammalian central synapses.