The Sagittally Sectioned Rat Hindbrain Preparation: Improved Access to the Brainstem Respiratory Network

Neurons that control breathing are distributed along the ventral respiratory column (VRC) in the ventrolateral brain stem, extending from the pons to the spinomedullary junction. These networks can be isolated in the en bloc brainstem-spinal cord preparation, which remains spontaneously active for hours under physiological conditions, but which only permits blind single-unit recordings from respiratory network constituents. The transverse slice preparation isolates the minimal neural substrate sufficient for respiratory rhythmogenesis, but does not permit the study of neuronal signaling along the column. Here, the sagittally sectioned rat hindbrain preparation is described. This preparation exposes the VRC transected along its long axis, at its surface, permitting optical recording of network activity using membrane-permeant Ca²⁺ indicators, as well as single-unit recording under visual control using differential interference contrast microscopy to patch onto cells identified using Ca²⁺ indicators. Because the VRC does not run parallel with the midline or the dorsal brainstem surface, exposing respiratory networks at the preparation’s surface requires that the sagittal surface be cut at the appropriate ventrodorsal (18.6�, relative to the dorsal surface) and rostrocaudal tilt (8.5�, relative to the midline), and mediolateral level. These empirically derived parameters conform with anatomical features of the brainstem: the rostrocaudal tilt aligns with the nucleus ambiguus; the ventrodorsal tilt lies parallel with reticular nuclei striations; the mediolateral level of section abuts a branch of the basilar artery. Detailed methods for isolating the SSRH, stabilizing it in the recording chamber, and recording respiratory networks using a membrane-permeant Ca²⁺ indicator are provided here.