Breathing movements in mammals arise from a rhythmic pattern of neural activity, thought to originate in the pre-Bötzinger complex in the lower brainstem. The mechanisms generating the neural rhythm in this region are unknown. The central question is whether the rhythm is generated by a network of bursting pacemaker neurons coupled by excitatory synapses that synchronize pacemaker activity. Here we visualized the activity of inspiratory pacemaker neurons at single-cell and population levels with calcium-sensitive dye. We developed methods to label these neurons retrogradely with the dye in neonatal rodent brainstem slices that retain the rhythmically active respiratory network. We simultaneously used infrared structural imaging to allow patch-clamp recording from the identified neurons. After we pharmacologically blocked glutamatergic synaptic transmission, a subpopulation of inspiratory neurons continued to burst rhythmically but asynchronously. The intrinsic bursting frequency of these pacemaker neurons depended on the baseline membrane potential, providing a cellular mechanism for respiratory frequency control. These results provide evidence that the neuronal kernel for rhythm generation consists of a network of synaptically-coupled pacemaker neurons.

We measured persistent Na(+) current and membrane properties of bursting-pacemaker and nonbursting inspiratory neurons of the neonatal rat pre-Bötzinger complex (pre-BötC) in brain stem slice preparations with a rhythmically active respiratory network in vitro. In whole-cell recordings, slow voltage ramps ( g(NaP)/g(Leak) in nonpacemaker cells (P < 0.0002). We conclude that I(NaP) is ubiquitously expressed by pre-BötC inspiratory neurons and that bursting pacemaker behavior within the heterogeneous population of inspiratory neurons is achieved with specific ratios of these two conductances, g(NaP) and g(Leak).