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Elucidation of the neuronal mechanisms underlying respiratory rhythmogenesis is a major focal point in respiratory physiology. An area of the ventrolateral medulla, the pre-Bötzinger complex (preBotC), is a critical site. Attention is now focused on understanding the cellular and network properties within the preBotC that underlie this critical function. The inability to clearly identify key "rhythm-generating" neurons within the heterogeneous population of preBotC neurons has been a significant limitation. Here we report an advancement allowing precise targeting of neurons expressing neurokinin-1 receptors (NK1Rs), which are hypothesized to be essential for respiratory rhythmogenesis. The internalization of tetramethylrhodamine conjugated substance P in rhythmically active medullary slice preparations provided clear visualization of NK1R-expressing neurons for subsequent whole-cell patch-clamp recordings. Among labeled neurons, 82% were inspiratory modulated, and 25% had pacemaker properties. We propose that this approach can be used to greatly expedite progress toward understanding the neuronal processes underlying the control of breathing.

Fluorescent Tagging of Rhythmically Active Respiratory Neurons within the Pre-Bötzinger Complex of Rat Medullary Slice Preparations