Feasibility of electrical recordings from unconnected vertebrate CNS neurons cultured in a three-dimensional extracellular matrix

Abstract

Single, i.e. unconnected spinal or cerebral neurons from chick embryos rapidly regenerate morphologically identifiable axons and dendrites when cultured in a three-dimensional (3-D) extracellular matrix (ECM) consisting of a hydrated native collagen lattice. We now show that it is possible to study the intrinsic electrophysiological properties of such neurons as early as 2 days in culture. Cells were plated at 10 5 cells/dish using Medium 199 containing 10% fetal calf serum but no other supplements or antibiotics-antimycotics. A patch-clamp/whole-cell voltage clamp system was used to record (1) single-channel currents from cell-attached patches, (2) transmembrane potentials during the injection of rectangular currents, and (3) whole-cell currents during voltage clamp. After 2–4 days in culture, isolated cerebral and spinal neurons exhibited single-channel currents. Within 7 days, rectangular currents injected through the recording electrode evoked action potentials. These results demonstrate that unconnected CNS neurons quickly display at least some properties of excitability when cultured in a 3-D ECM. This culture system should facilitate investigation of intrinsic electrical properties of single CNS neurons, and how extrinsic factors including neurotransmitters, hormones, pharmacological agents and contacts with other cells influence electrical activity.