Sealing cultured invertebrate neurons to embedded dish electrodes facilitates long-term stimulation and recording

Recently it has become possible to form small networks of synaptically connected identified invertebrate neurons in culture. Using conventional saline-filled glass electrodes, it is difficult to simultaneously stimulate and record from more than 2 or 3 cultured neurons and to perform experiments lasting longer than several hours. We demonstrate that it is possible to overcome these limitations by using planar arrays of electrodes embedded in the bottom of a culture dish. The arrays employ conductive leads and insulation that are transparent, making the dishes compatible with voltage-sensitive dyes and inverted microscopy. Identified neurons from leech Hirudo medicinalis, slug Aplysia californica, and snail Helisoma trivolvis, have been grown on these arrays. Due to their large size (soma diameter 40-200 μm) these neurons form seals over the dish electrodes. Individual electrodes can then be used to stimulate and to record action potentials in the associated neuron. With sealing, action potentials have been recorded simultaneously from many neurons for up to two weeks, with signal-to-noise ratios as large as 500:1. We developed and tested a simple model that describes the voltage waveforms measured with array electrodes. Potentials measured from electrodes under cell bodies were primarily derivatives of the intracellular potential, while those measured from electrodes under axon stumps were primarily proportional to local inward Na⁺ currents. While it is relatively easy to record action potentials, it is difficult to record postsynaptic potentials because of their small size and slow rate of rise.