Control of Breathing by Raphe Obscurus Serotonergic Neurons in Mice

We used optogenetics to determine the global respiratory effects produced by selectively stimulating raphe obscurus (RO) serotonergic neurons in anesthetized mice and to test whether these neurons detect changes in the partial pressure of CO2, and hence function as central respiratory chemoreceptors. Channelrhodopsin-2 (ChR2) was selectively (similar to 97%) incorporated into similar to 50% of RO serotonergic neurons by injecting AAV2 DIO ChR2-mCherry (adeno-associated viral vector double-foxed inverse open reading frame of ChR2-mCherry) into the RO of ePet-Cre mice. The transfected neurons heavily innervated lower brainstem and spinal cord regions involved in autonomic and somatic motor control plus breathing but eschewed sensory related regions. Pulsed laser photostimulation of ChR2-transfected serotonergic neurons increased respiratory frequency (fR) and diaphragmatic EMG (dEMG) amplitude in relation to the duration and frequency of the light pulses (half saturation, 1 ms; 5-10 Hz). dEMG amplitude and fR increased slowly (half saturation after 10-15 s) and relaxed monoexponentially (tau, 13-15 s). The breathing stimulation was reduced similar to 55% by methysergide (broad spectrum serotonin antagonist) and potentiated (similar to 16%) at elevated levels of inspired CO2 (8%). RO serotonergic neurons, identified by their entrainment to short light pulses (threshold, 0.1-1 ms) were silent (nine cells) or had a low and regular level of activity (2.1 +/- 0.4 Hz; 11 cells) that was not synchronized with respiration. These and nine surrounding neurons with similar characteristics were unaffected by adding up to 10% CO2 to the breathing mixture. In conclusion, RO serotonergic neurons activate breathing frequency and amplitude and potentiate the central respiratory chemoreflex but do not appear to have a central respiratory chemoreceptor function.