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 (I_NaP)-mediated bursting in ∼5% 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 μmriluzole (RIL). We found Ca²⁺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 μmCd²⁺or flufenamic acid (FFA) (10-200 μm), which suggests that a Ca²⁺-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 nmTTX or coapplication of 20 μmRIL + FFA (100-200 μm) stops the respiratory rhythm, but that adding 2 μmsubstance P restarts it. We conclude thatI_NaPandI_CANenhance neuronal excitability and promote rhythmogenesis, even if their magnitude is insufficient to support bursting-pacemaker activity in individual neurons. WhenI_NaPandI_CANare 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.

The preBötzinger complex (preBötC) is essential for normal respiratory rhythm generation in rodents, for which the underlying mechanisms remain unknown. Excitatory preBötC pacemaker neurons are proposed to be necessary for rhythm generation. Here we report the presence of a population of preBötC glycinergic pacemaker neurons. We used rhythmicin vitrotransverse slice preparations from transgenic mice where neurons expressing the glycine transporter 2 (GlyT2) gene coexpress enhanced green fluorescent protein (EGFP). We combined epifluorescence and whole-cell patch-clamp recording to study preBötC EGFP-labeled, i.e., glycinergic, inspiratory-modulated neurons with pacemaker properties. We defined glycinergic pacemaker neurons as those preBötC EGFP neurons that exhibited the following: (1) ectopic bursting in rhythmic slices when depolarized during their normally silent period and (2) bursting when depolarized in nonrhythmic slices (following AMPA receptor blockade). Forty-two percent of EGFP-labeled neurons were inspiratory (n= 48 of 115), of which 23% (n= 11 of 48 inspiratory; 10% of the total recorded) were pacemakers. We conclude that there is a population of preBötC inspiratory-modulated glycinergic, presumably inhibitory, pacemaker neurons that constitute a substantial fraction of all preBötC pacemaker neurons. These findings challenge contemporary models for respiratory rhythmogenesis that assume the excitatory nature of preBötC pacemaker neurons. Testable and nontrivial predictions of the functional role of excitatory and inhibitory pacemaker neurons need to be proposed and the necessary experiments performed.