A brainstem preparation allowing simultaneous access to respiratory motor output and cellular properties of motoneurons in American bullfrogs.

Breathing is generated by a complex neural circuit, and the ability to monitor the activity of multiple network components simultaneously is required to uncover the cellular basis of breathing. In neonatal rodents, a single brainstem slice can be obtained to record respiratory-related motor nerve discharge along with individual rhythm-generating cells or motoneurons because of the close proximity of these neurons in the brainstem. However, most ex vivo preparations in other vertebrates can only capture respiratory motor outflow or electrophysiological properties of putative respiratory neurons in slices without relevant synaptic inputs. Here, we detail a method to horizontally slice away the dorsal portion of the brainstem to expose fluorescently labeled motoneurons for patch-clamp recordings in American bullfrogs. This 'semi-intact' preparation allows tandem recordings of motor output and single motoneurons during respiratory-related synaptic inputs. The rhythmic motor patterns are comparable to those from intact preparations and operate at physiological temperature and [K+]. Thus, this preparation provides the ability to record network and cellular outputs simultaneously and may lead to new mechanistic insights into breathing control across vertebrates.