Rhythmogenic states of the respiratory network reveal differential opioid sensitivity

Abstract

Opioid induced respiratory depression (OIRD) is the major cause of death associated with opioid use. Although mortality increases dose-dependently, variability in the opioid response makes opioids particularly dangerous. The variability may be partly explained by rhythmogenic properties of the preBötzinger Complex (preBötC), a region that constitutes the minimal circuitry necessary for breathing. At the core of rhythmogenesis are periodic inspiratory bursts that emerge from synchronized activity within the preBötC. Between each burst, excitation builds among a growing number of neurons that synchronize to form a burst. Implicated in this are network-based mechanisms between recurrently connected excitatory and inhibitory neurons, as well as intrinsic-based mechanisms involving intrinsically bursting neurons that express persistently active inward cation currents, such as the persistent sodium current (INaP). Importantly, these mechanisms are sensitive to neuromodulation and depend on network excitability. Here, we test the hypothesis that shifts in the relative rhythmogenic role of network- and intrinsic- based mechanisms allow the preBötC to operate along a continuum of states, and consequently modulates sensitivity to OIRD. Horizontal brainstem slices were exposed to varying concentrations of aCSF [K+] or pharmacological manipulation of INaP prior to bath-application of the µ-opioid receptor agonist DAMGO. The aCSF [K+] required to induce bursting varied amongst slices. 48% required 8mM [K+], 17% required 6.5mM [K+], 10% required 5mM [K+], and 24% were rhythmic at 3mM [K+]. Unexpectedly we found upon initiating bursting, further increases in aCSF [K+] either increased or decreased rhythm stability, following a bell curve. Along this curve, increasing [K+] initially increased rhythm stability from 5% to 30% stabilitymax. After stabilitymax was reached, further 2mM increases in [K+] decreased stability by -40%, -55%, -52%, and -71%. The trajectory of stability to increasing aCSF [K+] predicted OIRD sensitivity. Slices that did not reach stabilitymax by 8mM [K+] displayed high sensitivity to OIRD and were silenced prior to 200nM DAMGO. In contrast, slices that reached stabilitymax at aCSF [K+] lower than 8mM and destabilized in 8mM [K+], were only modestly suppressed (-32% change in burst frequency) by 200nM DAMGO. To test the role of INaP in OIRD sensitivity, INaP was suppressed with 400nM ATTX or 20µM Riluzole prior to DAMGO. Both treatments increased OIRD sensitivity and silenced all slices prior to 200nM DAMGO. Lastly, to test for a protective role of INaP against OIRD INaP was potentiated with 400nM veratridine before or after DAMGO. Veratridine prior to DAMGO prevented OIRD, whereas veratridine following OIRD rescued bursting. Our results suggest the primary mechanisms of rhythm generation are flexible, and shift based upon the amount of network excitability and intrinsic bursting. Further, changing these dynamic properties creates states of rhythmogenesis that become highly sensitive, or insensitive to OIRD.