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

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Abstract
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 (preBötC), is a critical site. Attention is now focused on understanding the cellular and network properties within the preBötC that underlie this critical function. The inability to clearly identify key “rhythm-generating” neurons within the heterogeneous population of preBötC 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.