Electronics miniaturization techniques have been used to fabricate substrata to study contact guidance of neuronal cells, (eg leech segmental ganglion P and R cells, chick embryo cerebellar and cerebral cells, neonatal rat nodose ganglion cells). Three main types of microfabrication were carried out: one or more parallel grooves of varying width and depth; radiating grooves with branches; parallel strips of adhesive and nonadhesive substrate. Some were fabricated with adhesive and nonadhesive silicones, some with adhesive proteins such as laminin, concanavalin A and polylysine. The first and third type of structure often induced orientation into monopolar or bipolar cells. The second type has been less effective in controlling cell shape, possibly because the best dimensions of groove has not yet been determined. The neurites were visualised using fluorescent markers (eg fluorescein diacetate, Fluo-3 or Calcein).
Electrophysiological experiments have been carried out on isolated neurons. Intracellular recordings have been taken in the cell body and whole cell patch clamping has been used to measure passive and active membrane properties and the action of neurotransmitters. Voltage sensitive dyes have also been used for recording electrical activity. A new system was also developed to measure transmembrane potential changes by monitoring changes in fluorescence from potentiometric dyes. Another approach being used developed in extracellular recording using very small electrodes which can be incorporated into the substratum at the bottom of grooves. These electrodes have been used to stimulate cells and electrical activity seems to be unaffected by the shape change from multipolar cells to unipolar or biopolar in the grooved substrates.
The project is aimed at understanding the mechanisms of information processing by neurons in the brain of mammals. In real neuronal networks, signal processing appears to take place within each neuron as well as in the pattern of connections between neurons unlike artificial neural networks in which the computing power relies mainly in the connectivity. Signal processing will be studied using optical and electrical methods to obtain multiple sites recording of electrical and ionic activity of single neurons. The neurons will be cultured on special substrates that will impose the shape of the neurite arborisation, control the pattern of connections and allow local stimulation with micro-etched electrode arrays.
Funding SchemeCSC - Cost-sharing contracts