Inspiratory Off-Switch Mediated by Optogenetic Activation of Inhibitory Neurons in the preBötzinger Complex In Vivo

Abstract: The role of inhibitory neurons in the respiratory network is a matter of ongoing debate.

Conflicting and contradicting results are manifold and the question whether inhibitory neurons are

essential for the generation of the respiratory rhythm as such is controversial. Inhibitory neurons

are required in pulmonary reflexes for adapting the activity of the central respiratory network to the

status of the lung and it is hypothesized that glycinergic neurons mediate the inspiratory off-switch.

Over the years, optogenetic tools have been developed that allow for cell-specific activation of

subsets of neurons in vitro and in vivo. In this study, we aimed to identify the effect of activation

of inhibitory neurons in vivo. Here, we used a conditional transgenic mouse line that expresses

Channelrhodopsin 2 in inhibitory neurons. A 200  m multimode optical fiber ferrule was implanted

in adult mice using stereotaxic surgery, allowing us to stimulate inhibitory, respiratory neurons within

the core excitatory network in the preBötzinger complex of the ventrolateral medulla. We show that,

in anesthetized mice, activation of inhibitory neurons by blue light (470 nm) continuously or with

stimulation frequencies above 10 Hz results in a significant reduction of the respiratory rate, in some

cases leading to complete cessation of breathing. However, a lower stimulation frequency (4–5 Hz)

could induce a significant increase in the respiratory rate. This phenomenon can be explained by

the resetting of the respiratory cycle, since stimulation during inspiration shortened the associated

breath and thereby increased the respiratory rate, while stimulation during the expiratory interval

reduced the respiratory rate. Taken together, these results support the concept that activation of

inhibitory neurons mediates phase-switching by inhibiting excitatory rhythmogenic neurons in the

preBötzinger complex.