The breathing motor pattern in mammals originates in brainstem networks. Whether pacemaker neurons play an obligatory role remains a key unanswered question. We performed whole-cell recordings in the preBötzinger Complex in slice preparations from neonatal rodents and tested for pacemaker activity. We observed persistent Na ϩ current (I NaP )-mediated bursting in ϳ5% of inspiratory neurons in postnatal day 0 (P0)-P5 and in P8 -P10 slices. I NaP -mediated bursting was voltage dependent and blocked by 20 M riluzole (RIL). We found Ca 2ϩ current (I Ca )-dependent bursting in 7.5% of inspiratory neurons in P8 -P10 slices, but in P0 -P5 slices these cells were exceedingly rare (0.6%). This bursting was voltage independent and blocked by 100 M Cd 2ϩ or flufenamic acid (FFA) (10 -200 M), which suggests that a Ca 2ϩ -activated inward cationic current (I CAN ) underlies burst generation. These data substantiate our observation that P0 -P5 slices exposed to RIL contain few (if any) pacemaker neurons, yet maintain respiratory rhythm. We also show that 20 nM TTX or coapplication of 20 M RIL ϩ FFA (100 -200 M) stops the respiratory rhythm, but that adding 2 M substance P restarts it. We conclude that I NaP and I CAN enhance neuronal excitability and promote rhythmogenesis, even if their magnitude is insufficient to support bursting-pacemaker activity in individual neurons. When I NaP and I CAN are removed pharmacologically, the rhythm can be maintained by boosting neural excitability, which is inconsistent with a pacemaker-essential mechanism of respiratory rhythmogenesis by the preBötzinger complex.

Inspiratory neurons of the preBötzinger complex (preBötC) form local excitatory networks and display 10-30 mV transient depolarizations, dubbed inspiratory drive potentials, with superimposed spiking. AMPA receptors are critical for rhythmogenesis under normal conditions in vitro but whether other postsynaptic mechanisms contribute to drive potential generation remains unknown. We examined synaptic and intrinsic membrane properties that generate inspiratory drive potentials in preBötC neurons using neonatal mouse medullary slice preparations that generate respiratory rhythm. We found that NMDA receptors, group I metabotropic glutamate receptors (mGluRs), but not group II mGluRs, contributed to inspiratory drive potentials. Subtype 1 of the group I mGluR family (mGluR1) probably regulates a K + channel, whereas mGluR5 operates via an inositol 1,4,5-trisphosphate (IP 3 ) receptor-dependent mechanism to augment drive potential generation. We tested for and verified the presence of a Ca 2+ -activated non-specific cation current (I CAN ) in preBötC neurons. We also found that high concentrations of intracellular BAPTA, a high-affinity Ca 2+ chelator, and the I CAN antagonist flufenamic acid (FFA) decreased the magnitude of drive potentials. We conclude that I CAN underlies robust inspiratory drive potentials in preBötC neurons, and is only fully evoked by ionotropic and metabotropic glutamatergic synaptic inputs, i.e. by network activity.

Breathing movements in mammals depend on respiratory neurons in the preBötzinger Complex (preBötC), which comprise a rhythmic network and generate robust bursts that form the basis for inspiration. Persistent Na + current (I NaP ) is widespread in the preBötC and is hypothesized to play a critical role in rhythm generation because of its subthreshold activation and slow inactivation properties that putatively promote long-lasting burst depolarizations. In neonatal mouse slice preparations that retain the preBötC and generate a respiratory-related rhythm, we tested the role of I NaP with multiple Na + channel antagonists: tetrodotoxin (TTX; 20 nM), riluzole (RIL; 10 µM), and the intracellular Na + channel antagonist QX-314 (2 mM). Here we show that I NaP promotes intraburst spiking in preBötC neurons but surprisingly does not contribute to the depolarization that underlies inspiratory bursts, i.e. the inspiratory drive potential. Local microinjection in the preBötC of 10 µM RIL or 20 nM TTX does not perturb respiratory frequency, even in the presence of bath-applied 100 µM flufenamic acid (FFA), which attenuates a Ca 2+ -activated non-specific cation current (I CAN ) that may also have burst-generating functionality. These data contradict the hypothesis that I NaP in preBötC neurons is obligatory for rhythmogenesis. However, in the presence of FFA, local microinjection of 10 µM RIL in the raphe obscurus causes rhythm cessation, which suggests that I NaP regulates the excitability of neurons outside the preBötC, including serotonergic raphe neurons that project to, and help maintain, rhythmic preBötC function.