Evidence for bursting pacemaker neurones in cultured spinal cord cells

Abstract

Intracellular recordings were made from dissociated mouse spinal cord cells in primary culture. One type of spinal cord neurone, with a large cell body (40–50 μm), 3–5 short neurites, and a mean resting potential of −65 mV, was found to fire rhythmic bursts of action potentials with a phase duration of approximately 1s when the membrane potential was depolarized to −55 mV. These bursts did not arise from spontaneous synaptic input, but appeared to result from endogenous ionic conductance properties of the membrane resembling those observed in molluscan bursting pacemaker neurones. Ionic conductances underlying this bursting activity were studied pharmacologically by local application of ionic conductance blockers. Pacemaker potentials depended on Na + conductance, since tetrodotoxin and Na-free medium were the most potent agents for blocking spontaneous rhythmic activity. However, a Ca 2+ conductance was involved in the depolarizing phase of membrane potential oscillations, since Ba 2+ application increased oscillation amplitude. Action potentials observed during the bursts were Na +- and Ca 2+-dependent. They did not differ significantly from those observed in other spinal cord neurones in culture. Application of tetraethylammonium, CoCl 2, BaCl 2 and 4-aminopyridine revealed at least three different potassium conductances which controlled this bursting pacemaker activity. A delayed potassium conductance controlled spike duration, a Ca-dependent potassium conductance controlled the duration of the burst and underlay the hyperpolarizing phase terminating the burst, and finally, a transient potassium conductance appeared to be involved in the control of phase duration. The demonstration that spinal cord neurones growing in monolayer culture display typical bursting pacemaker activity raises the possibility that bursting pacemaker neurones in the mammalian spinal cord may be involved in a phasic pattern generator that could control such activities as walking and the respiratory rhythm.