title: Organotypic slice cultures containing the preBötzinger complex generate respiratory-like rhythms.
abstract: Study of acute brain stem slice preparations in vitro has advanced our understanding of the cellular and synaptic mechanisms of respiratory rhythm generation, but their inherent limitations preclude long-term manipulation and recording experiments. In the current study, we have developed an organotypic slice culture preparation containing the preBötzinger complex (preBötC), the core inspiratory rhythm generator of the ventrolateral brain stem. We measured bilateral synchronous network oscillations, using calcium-sensitive fluorescent dyes, in both ventrolateral (presumably the preBötC) and dorsomedial regions of slice cultures at 7-43 days in vitro. These calcium oscillations appear to be driven by periodic bursts of inspiratory neuronal activity, because whole cell recordings from ventrolateral neurons in culture revealed inspiratory-like drive potentials, and no oscillatory activity was detected from glial fibrillary associated protein-expressing astrocytes in cultures. Acute slices showed a burst frequency of 10.9 ± 4.2 bursts/min, which was not different from that of brain stem slice cultures (13.7 ± 10.6 bursts/min). However, slice cocultures that include two cerebellar explants placed along the dorsolateral border of the brainstem displayed up to 193% faster burst frequency (22.4 ± 8.3 bursts/min) and higher signal amplitude (340%) compared with acute slices. We conclude that preBötC-containing slice cultures retain inspiratory-like rhythmic function and therefore may facilitate lines of experimentation that involve extended incubation (e.g., genetic transfection or chronic drug exposure) while simultaneously being amenable to imaging and electrophysiology at cellular, synaptic, and network levels.
authors.affiliation: Department of Neuroscience and Pharmacology, University of Copenhagen, Copenhagen, Denmark; and Department of Applied Science, The College of William and Mary, Williamsburg, Virginia.
authors.forename: Wiktor S
authors.initials: WS
authors.lastname: Phillips
authors.affiliation: Department of Neuroscience and Pharmacology, University of Copenhagen, Copenhagen, Denmark; and.
authors.forename: Mikkel
authors.initials: M
authors.lastname: Herly
authors.affiliation: Department of Applied Science, The College of William and Mary, Williamsburg, Virginia.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Del Negro
authors.affiliation: Department of Neuroscience and Pharmacology, University of Copenhagen, Copenhagen, Denmark; and jrekling@sund.ku.dk.
authors.forename: Jens C
authors.identifier: http://orcid.org/0000-0002-8568-2332
authors.initials: JC
authors.lastname: Rekling
mesh_terms: D000200:Action Potentials; D000818:Animals; D001253:Astrocytes / Q000378:metabolism / Q000502:physiology; D001933:Brain Stem / Q000166:cytology*; D020013:Calcium Signaling; D019069:Cell Respiration; D063226:Central Pattern Generators / Q000166:cytology*; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009474:Neurons / Q000378:metabolism / Q000502:physiology; D046509:Tissue Culture Techniques / Q000379:methods*
chemical_list: D005904:Glial Fibrillary Acidic Protein
keywords: breathing; central pattern generator; organotypic slice cultures; preBötzinger complex; respiration; rhythm generation; rhythmogenesis