Calcium-dependent responses in neurons of the isolated respiratory network of newborn rats.

1. Membrane potentials (Em) and currents (Im) were measured using whole-cell patch clamp techniques in inspiratory (Insp, types I-III), preinspiratory (Pre-I) and tonic expiratory (Exp) neurons of the ventral respiratory group (VRG) in the isolated brainstem-spinal cord preparation of 0- to 4-day-old rats. 2. After blocking on-going synaptic activity with 0.2-0.5 microM tetrodotoxin (TTX), Ca(2+)-dependent responses were analysed using patch pipettes containing 120 mM Cs+ and 20 mM tetraethylammonium (TEA) to block K+ conductances. 3. In all cells studied, all-or-none high voltage-activated (HVA) Ca2+ spikes with an activation threshold of -33 +/- 6.9 mV (n = 37) were evoked by depolarizing current pulses. 4. In less than 15% of Insp and Pre-I cells and in 20% of Exp neurons, termination of hyperpolarizing pulses led to low voltage-activated (LVA) Ca2+ spikes with a threshold potential of between -70 and -60 mV (n = 7). 5. In more than 50% of Insp III and Pre-I neurons, depolarizing pulses evoked graded 'plateau' potentials with an amplitude of 5-20 mV. Slow voltage ramp commands revealed that this type of Ca2+ response was due to an inward current with a mean activation threshold of -42 +/- 2.1 mV (n = 5). These intermediate voltage-activated (IVA) plateau potentials persisted for several seconds after termination of depolarizing current pulses and decreased in amplitude at more negative holding potentials. 6. The HVA and LVA Ca2+ spikes as well as the IVA plateau potentials and the underlying inward current were potentiated after extracellular addition of 2 mM Ba2+ whereas 1-2 mM Co2+ led to blockade of these responses. 7. Nifedipine (10 microM) selectively suppressed HVA Ca2+ potentials whereas 0.2-0.4 microM omega-agatoxin-IVA reduced the IVA response without major effects on HVA Ca2+ spikes. omega-Conotoxin-GVIA (2 microM) led to a partial blockade of both IVA and HVA potentials. 8. After extracellular application of TTX, Ba2+ and/or TEA, HVA and LVA Ca2+ spikes as well as IVA plateau potentials were also revealed using patch pipettes containing K+ instead of Cs+ and TEA. 9. The results indicate that neonatal respiratory neurons have a complex set of Ca(2+)-dependent membrane conductances. The relevance of these conductances for initiation and maintenance of respiratory bursts is discussed.