Electrophysiology and ultrastructure of mouse hypothalamic neurons in culture: a correlative analysis during...

The development of the electrical activity of hypothalamic neurons in dissociated cell cultures obtained from 14 day old mice foetuses was studied using patch extracellular and intracellular recording techniques. Electrophysiological data were compared with morphological observations obtained by electron microscopy. During patch recording, excitability of the cells was tested by the application of a 40 mM KCl solution. Tetrodotoxin (TTX, 10(-6) M in the delivery pipette) and Co2+ (10(-2) M in the delivery pipette) were applied to the recorded cell by pressure in order to study the involvement of sodium and calcium currents in the electrical activity during the in vitro development. From the first day of incubation, TTX and Co2+ were able to block reversibly the spontaneous electrical activity. However, only TTX application inhibited action potentials which suggests that calcium currents could be poorly involved in the action potential generation at the beginning of neuronal differentiation. Three different phases were found in the electrophysiological development of hypothalamic neurons in culture. The first phase (between the 1st and the 5th day of incubation) was characterized by an increase in the ratio of the spontaneously active cells (15% at day 1 and 90% at day 5). This increase paralleled the increase of the ratio of excitable cells. During this period no post-synaptic activity was detected. Morphologically, at 36 h, no synaptic contact was observed and growth cones were found to be very primitive. The second phase, between the 6th and the 9th day of culture, was characterized by a decrease in the ratio of spontaneously active cells and by the appearance, in a few cases, of a postsynaptic potential activity. During this phase the majority of the silent cells were excitable. At this stage neurons formed well differentiated neurites and growth cones. Synaptogenesis had already started and several stages of synapse formation could be seen. The third phase of the development, from 10 days of incubation, was characterized by an increase in post synaptic potential activity. During this period, numerous mature synapses could be observed although most of the synaptic contacts were located on neurites. In addition, some synapses were apposed onto degenerated structures. In conclusion, hypothalamic neurons in culture appear to differentiate in 3 steps: a primitive stage during which spontaneous electrical activity and excitability develop without any synaptic contact; a 2nd stage during which synaptic contacts develop, followed by a third stage of synapse maturation where mature synapses are formed whereas transient synapses degenerate.