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 c entral neurons. We used surface chemistry and laser-lithography to produce geometrical patterns of neuron-compatible substrate spaced by less neuron-c ompatible surfaces. The patterned substrates were composed of a laminin-der ived synthetic peptide, PA22-2, and the spacing surfaces of either decyldim ethylsilane (DDMS) or trimethylsilane (TMS). Dissociated rat hippocampal ne urons 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 axon al differentiation of the hippocampal neurons, but less favourable for the development of the resting conductance than the DDMS spacing surfaces. Furt hermore, neurons grown on the patterned substrates frequently made synaptop hysin-positive contacts with one another. Spontaneous post-synaptic current s recorded from such neurons suggest that these contacts were indeed functi onal synapses. When hippocampal neurons were plated at a very low density, they often formed circuitries consisting of only two neurons on the pattern ed substrate. Such a simple circuitry allowed us to analyse synaptic transm ission in a single neuronal pair without the influence of the third neurons . With the clarity of analysis and the readiness of manipulation, our cultu re system would offer a powerful tool for studying development and function s of mammalian central synapses.