The respiratory rhythm is generated by a network in the ventral medulla. Recent studies concerning the spatio-temporal activity pattern of its elements used voltage sensitive dyes, having poor response amplitudes. We succeeded using Ca²⁺ indicators to image rhythmic population activity in a conventional PBC slice with single cell resolution, in spite of their questioned tissue penetration and cell selectivity. Of the tested dyes fluo-3 AM was suited best. Increased osmolarity improved labeling. Imaging was performed using a sensitive, high speed CCD-camera and a high NA 20x objective. To correlate optical signals with rhythmic mass activity an extracellular electrode was placed in the contralateral PBC. In 54% of slices optical and electrical activity correlated; 10% of labeled neurons showed obviously correlated rhythmic Ca²⁺ signals. Deeper located neurons became detectable upon off-line image processing using the rhythmic mass activity as a time stamp. Neurons were classified as inspiratory-like (61%), expiratory-like (22%), and pre-inspiratory like (16%). Up to 6 rhythmically active neurons were observed in the field of vision of a given slice. We conclude that CCD imaging is superior to confocal methods in terms of speed and applicability and that Ca²⁺ dyes are reliable and powerful tools to study in vitro rhythmogenesis.

Supported by the DFG (CMPB) and Göttingen University