Breathing movements in mammals arise from a rhythmic pattern of neural activity, thought to originate in the pre-Bötzinger complex1 in the lower brainstem. The mechanisms generating the neural rhythm in this region are unknown2,3,4,5. 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.
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We thank M. J. O'Donovan and K. R. Spring for advice on optical recording; W.S.Rasband for NIH Image programs; G. P. Compo for the CWT denoising algorithm; and R. E. Burke and A. Lev-Tov for critical comments on the manuscript.
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Koshiya, N., Smith, J. Neuronal pacemaker for breathing visualized in vitro. Nature 400, 360–363 (1999). https://doi.org/10.1038/22540
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