RESUMO
Unfailing respiration depends on neural mechanisms already present in mammals before birth. Experiments were made to determine how inspiratory and expiratory neurons are grouped in the brainstem of fetal mice. A further aim was to assess whether rhythmicity arises from a single pacemaker or is generated by multiple sites in the brainstem. To measure neuronal firing, a fluorescent calcium indicator dye was applied to embryonic central nervous systems isolated from mice. While respiratory commands were monitored electrically from third to fifth cervical ventral roots, activity was measured optically over areas containing groups of respiratory neurones, or single neurones, along the medulla from the facial nucleus to the pre-Bötzinger complex. Large optical signals allowed recordings to be made during individual respiratory cycles. Inspiratory and expiratory neurones were intermingled. A novel finding was that bursts of activity arose in a discrete area intermittently, occurring during some breaths, but failing in others. Raised CO2 partial pressure or lowered pH increased the frequency of respiration; neurons then fired reliably with every cycle. Movies of activity revealed patterns of activation of inspiratory and expiratory neurones during successive respiratory cycles; there was no evidence for waves spreading systematically from region to region. Our results suggest that firing of neurons in immature respiratory circuits is a stochastic process, and that the rhythm does not depend on a single pacemaker. Respiratory circuits in fetal mouse brainstem appear to possess a high safety factor for generating rhythmicity, which may or may not persist as development proceeds.