We measured a low-threshold, inactivating K+ current, i.e., A-current (IA), in respiratory neurons of the preBötzinger Complex (preBötC) in rhythmically active slice preparations from neonatal C57BL/6 mice. The majority of inspiratory neurons (21/34 = 61.8%), but not expiratory neurons (1/8 = 12.5%), expressed IA. In whole cell and somatic outside-out patches IA activated at –60 mV (half-activation voltage measured –16.3 mV) and only fully inactivated above –40 mV (half- inactivation voltage measured –85.6 mV), indicating that IA can influence membrane trajectory at baseline voltages during respiratory rhythm generation in vitro. 4-Aminopyridine (4-AP, 2 mM) attenuated IA in both whole cell and somatic outside-out patches. In the context of rhythmic network activity, 4-AP caused irregular respiratory-related motor output on XII nerves and disrupted rhythmogenesis as detected with whole-cell and field recordings in the preBötC. Whole-cell current-clamp recordings showed that 4-AP changed the envelope of depolarization underlying inspiratory bursts (i.e., inspiratory drive potentials) from an incrementing pattern to a decrementing pattern during rhythm generation and abolished current pulse-induced delayed excitation. These data suggest that IA opposes excitatory synaptic depolarizations at baseline voltages of approximately –60 mV and influences the inspiratory burst pattern. We propose that IA promotes orderly recruitment of constituent rhythmogenic neurons by minimizing the activity of these neurons until they receive massive coincident synaptic input, which reduces the periodic fluctuations of inspiratory activity.
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.AMPAreceptors 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 (IP3) receptor-dependent mechanism to augment drive potential generation. We tested for andverified the presence of a Ca2+-activated non-specific cation current (ICAN) in preBötC neurons. We also found that high concentrations of intracellular BAPTA, a high-affinity Ca2+ chelator, and the ICAN antagonist flufenamic acid (FFA) decreased the magnitude of drive potentials. We conclude that ICAN 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.
Recent experiments in vivo and in vitro have advanced our understanding of the sites and mechanisms involved in mammalian respiratory rhythm generation. Here we evaluate and interpret the new evidence for two separate brainstem respiratory oscillators and for the essential role of emergent network properties in rhythm generation. Lesion studies suggest that respiratory cell death might explain morbidity and mortality associated with neurodegenerative disorders and ageing.
