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 µm 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.

The pre-Bötzinger complex is necessary for the expression of post-inspiration

“…The present study shows for the first time that unilateral pre-BötC inhibition silences both inspiratory and post-inspiratory output in the ipsilateral vagal nerve, which mediates the laryngeal valving of respiratory airflow (see Paton & Dutschmann, 2002). This observation is functionally consistent with recent experiments in which optogenetic activation of pre-BötC neurons during expiration prolongs expiration (Hülsmann et al ., 2021). The observation that unilateral inhibition of the pre-BötC causes suppression of inspiratory hypoglossal motor output is in line with previous reports that pre-BötC activity specifically drives the inspiratory activity of the hypoglossal nerve (Smith et al ., 1991, Del Negro et al ., 2002, 2005).…”

“…Finally, optogenetic activation of a subset of pre-BötC neurons in vivo showed phasedependent effects such that activation of pre-BötC neurons during inspiration increases respiratory frequency, whereas activation during post-inspiration depresses respiratory frequency (Hülsmann et al, 2021). Taken together, these data suggest a novel role for the pre-BötC in determining the timing of the inspiratory/post-inspiratory phase transition.…”

“…Further, recent measurements of respiratory local field potentials in the pre-BötC in situ demonstrated the robust expression of LFPs at the transition from expiration to inspiration and from inspiration to post-inspiration (Dhingra et al ., 2020). Finally, optogenetic activation of a subset of pre-BötC neurons in vivo showed phase-dependent effects such that activation of pre-BötC neurons during inspiration increases respiratory frequency, whereas activation during post-inspiration depresses respiratory frequency (Hülsmann et al ., 2021). Taken together, these data suggest a novel role for the pre-BötC in determining the timing of the inspiratory/post-inspiratory phase transition.…”

Neural mechanisms underlying respiratory regulation within the preBötzinger complex of the rabbit

“…Although the blockade of both GABA A and glycine receptors does not stop the breathing rhythm, these inhibitory amino acids provide important contributions to the respiratory pattern formation. Inhibitory transmission during both inspiratory and expiratory phases of respiration may play a critical role in the control of both amplitude and frequency of the respiratory output (Menuet et al, 2020;Hülsmann et al, 2021). In fact, optogenetic studies performed in in vivo and in situ rodent preparations demonstrate that photoinhibition of preBötC neurons applied during inspiration stops the phrenic burst and triggers postinspiration, while during expiration prolongs the expiratory duration (Menuet et al, 2020).…”

“…Data and recordings adapted from Mutolo et al (2005). rhythmogenic neurons in in vivo mice (Hülsmann et al, 2021). At variance with previous experiments in the rodent preBötC (e.g.…”