Sodium and calcium current-mediated pacemaker neurons and respiratory rhythm generation

Abstract

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+ current (INaP)-mediated bursting in ∼5% of inspiratory neurons in postnatal day 0 (P0) -P5 and in P8-P10 slices. INap-mediated bursting was voltage dependent and blocked by 20 μM riluzole (RIL). We found Ca2+ current (ICa)- 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 μM Cd2+ or flufenamic acid (FFA) (10-200 μM), which suggests that a Ca2+-activated inward cationic current (ICAN) 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 20nM TTX or coapplication of 20 μM RIL + FFA (100-200 μM) stops the respiratory rhythm, but that adding 2 μM substance P restarts it. We conclude that I NaP and ICAN enhance neuronal excitability and promote rhythmogenesis, even if their magnitude is insufficient to support bursting-pacemaker activity in individual neurons. When INaP and ICAN 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.