Long-term optical recording of patterns of electrical activity in ensembles of cultured Aplysia neurons

Abstract

1. Left upper quadrant (LUQ) cells isolated from the abdominal ganglion of Aplysia were maintained in culture to study how the cellular and synaptic properties of individual neurons contribute to the generation of patterns of electrical activity by neuronal ensembles. 2. Conventional microelectrodes were used to examine the spiking characteristics of individually cultured LUQ cells in vitro and to characterize their synaptic interactions. 3. In vitro, in contrast to in situ, LUQ neurons innervate other LUQ neurons. Intracellular recordings from pairs of LUQ cells showed that the prevalent type of postsynaptic potential was purely inhibitory. The other type of response was a dual-action postsynaptic potential, with inhibition followed by a delayed, slow excitation. 4. We established a set of criteria for the use of multiple-site optical recording techniques, in combination with impermeant probes of membrane potential, to observe the patterns of electrical activity generated by ensembles of co-cultured LUQ cells. 5. The spiking activity of individual cells within the neuronal ensembles was detected by means of the change in optical absorption of cells that were vitally stained with the dye RH155. The change in absorption was typically delta A congruent to 4 X 10(-4) per spike. We achieved a signal-to-noise (peak-to-peak) ratio of approximately 10 for a 50 X 50-microns photodetector field and an incident intensity of approximately 10 mW/cm2, close to the theoretical limit. 6. These conditions permitted, for the first time, continuous optical recording from cultured neurons for periods of up to 3 h with no discernible photodynamic damage or photobleaching. This long-term optical recording permitted examination of the different patterns of electrical activity generated by individual neuronal ensembles under several different experimental conditions. 7. An elaborate tracery of regenerated neurites present in these cultures resulted in individual photodetectors recording simultaneously the activity of multiple neurons. We reconstructed the temporal firing patterns for individual neurons within ensembles even with all the neurons active simultaneously and determined the functional connections in the ensemble by analyzing the temporal relationships between firing patterns of individual neurons. Excitatory as well as inhibitory functional interactions could be observed within the neuronal ensemble, the latter after the tonic activity of the neurons was increased by reducing the extracellular [Mg2+]. 8. Examination of the optical data from ensembles constructed from identified cells having characteristic physiological responses allowed us to address the question of how cellular and synaptic properties affect the patterns of electrical activity generated by neuronal ensembles.(ABSTRACT TRUNCATED AT 400 WORDS)