Functionally intact in vitro preparation generating respiratory activity in neonatal and mature mammals

The present report describes a novel rhythmically active brainstem slice preparation that generates respiratory activity spontaneously in both mice and rats of varying maturational states. The brainstems of neonatal (0-4 days) and mature (3-8 weeks) mice and rats were isolated and a 600- to 750-microns thick slice cut to include the dorsomedial and the ventrolateral regions of the complete rostro-caudal extent of the medulla. This plane of section we have termed "tilted-sagittal". Rhythmically discharging neurones were recorded extracellularly from both the dorsal and ventral regions of the slice. The recording sites of these neurones were found in the hypoglossal motonucleus (XII) and in areas of the ventrolateral medulla that includes the ventral respiratory group (VRG) region. Histological examination revealed the preservation of neuronal structures important for cardiorespiratory regulation and reflex control including the nucleus of the solitary tract as well as the nucleus ambiguus. In addition, pontine structures including the A5 region were also preserved. Rhythmic activity was found only in slices where the ambiguual column was preserved in its entirety. The mean frequency of discharge of XII neurones was 20 and 10 bursts per minute in neonates and mature rodents respectively. In preparations of mature animals we demonstrate that this frequency increased significantly (P < 0.05) by either raising temperature from 29 degrees C to 38 degrees C (54%), elevating extracellular potassium concentration from 4 to 7.5 mM (52%), blocking potassium channels (20%) or decreasing pH from 7.4 to 7.0 (18%). The burst duration to frequency ratio of XII and VRG rhythmic neurones was similar and therefore indicative of a common brainstem oscillator. Consistent with this finding was that rhythmic activity in the VRG persisted despite removal of the dorsomedial region of the slice. In contrast, rhythmic XII neurones became tonic following mechanical disconnection of the VRG.