Development of spontaneous electrical activity by rat hypothalamic neurons in dissociated culture

The development of spontaneous electrical activity by embryonic rat hypothalamic neurons in dissociated culture was monitored using an extracellular, patch electrode recording method. Embryonic day 17 hypothalamic neurons were plated onto a feeder layer of astrocytes obtained from neonatal rat cerebral cortex, and extracellular electrical activity was monitored beginning the day after plating. The rates and patterns of spontaneous discharge were examined using interpike interval histograms. The percentage of spontaneously active neurons increased steadily with time in culture, from 13% in the first week to 56% during the sixth week in vitro. Although the percentage of spontaneously active cells increased, average firing rates did not change with time in culture. The pattern of electrical discharge was primarily random at all time points, with a small number of cells displaying regular activity while 4 cells were classified as phasic/bursty. In general, spontaneous action potential discharge was not dependent on synaptic transmission, as activity persisted after perfusion with bath solution containing either low Ca2+/high Mg2+ or kynurenic acid. Tetrodotoxin consistently and reversibly abolished spontaneous firing, regardless of culture age. We conclude that spontaneous activity in low density hypothalamic culture develops gradually though steadily, and is generated through an endogenous mechanism, independent of synaptic excitation.