Glycinergic pacemaker neurons in preBötzinger complex of neonatal mouse

Abstract

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 rhythmic in vitro transverse 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.

Figure 1.

Examples of preBötC GlyT2-EGFP pacemaker neurons. A, Respiratory-modulated discharge in a GlyT2-EGFP pacemaker neuron current clamped at interburst V_m ≈ −60 mV. A1, Bursting activity in 10 μm CNQX bath applied. Network activity is blocked. B, B1, Another example of a respiratory-modulated discharge and bursting in a GlyT2-EGFP neuron. C, Examples of respiratory-modulated discharge and “ectopic bursts” (asterisks) after depolarizing V_m. Action potentials have been truncated.

Figure 2.

NK1R and GlyT2-EGFP do not colocalize in the preBötC. A, EGFP-labeled neurons in transverse medullary slices of neonatal GlyT2-EGFP mice. NK1R-ir (red) was used to identify the preBötC. Arrows indicate dorsal (D) and lateral (L) orientation of slice. B, We observed little to no colocalization between NK1R-ir and EGFP signal. Note the absence of yellow in the merged channels. C1–C4, Whole-cell patch recordings were made under IR-DIC (C1). Patched cells were tested electrophysiologically for pacemaker properties and filled with rhodamine via the patch electrode (C2). We confirmed that the neurons are GlyT2-EGFP by merging the images taken with EGFP and rhodamine filters (C2–C4). Scale bar, 15 μm. IO, Inferior olive; SP5, spinal trigeminal nucleus; XIIN, hypoglossal nucleus.