Functional imaging reveals respiratory network activity during hypoxic and opioid challenge in the neonate rat tilted sagittal slab preparation Running head: Functional imaging in the tilted sagittal slab

In mammals, respiration-modulated networks are distributed rostrocaudaly in the ventrolateral quadrant of the medulla. Recent studies have established that in neonate rodents, two spatially separate networks along this column, the parafacial respiratory group (pFRG) and the pre-Bötzinger complex (preBötC), are hypothesized to be sufficient for respiratory rhythm generation, but little is known about the connectivity within or between these networks. In order to be able to observe how these networks interact, we have developed a neonate rat medullary tilted sagittal slab, which exposes one column of respiration-modulated neurons on its surface, permitting functional imaging with cellular resolution. Here we examined how respiratory networks responded to hypoxic challenge and opioid-induced depression. At the systems level, the sagittal slab was congruent with more intact preparations: hypoxic challenge led to a significant increase in respiratory period and inspiratory burst amplitude, consistent with gasping. At opioid concentrations sufficient to slow respiration, we observed periods at integer multiples of control, matching quantal slowing. Consistent with single-unit recordings in more intact preparations, respiratory networks were distributed bimodally along the rostrocaudal axis, with respiratory neurons concentrated at the caudal pole of the facial nucleus, and 350 microns caudally, at the level of the pFRG and the preBötC respectively. Within these regions neurons active during hypoxia-and/or opioid-induced depression were ubiquitous, and interdigitated. In particular, contrary to earlier reports, opiate-insensitive neurons were found at the level of the preBötC. 2 Introduction (441 words): In mammals, breathing is a continuous rhythmic behavior that constantly adapts itself to maintain blood-gas homeostasis (Khoo 2000). Because essential circuits for respiratory rhythm generation are localized in ventrolateral medulla, this behavior provides a platform for elucidating how cellular and network properties interact to generate a mammalian adaptive behavior. In a landmark series of experiments (Smith et al. 1991), networks capable of generating respiratory-related rhythm were isolated from rodent rostral medulla in a 300 µm slice. In this preparation, a circumscribed region in the ventrolateral quadrant, the pre-Bötzinger Complex (preBötC), was identified as necessary and sufficient for respiratory rhythm generation in the slice. The preBötC was found to have a high concentration of propriobulbar respiratory neurons (Dobbins and Feldman 1994), a subset of which had endogenous bursting properties (Johnson et al. 1994). Because an earlier study had established that in vitro respiratory rhythm persisted following blockade of Cl