Inspiratory-timed phrenic nerve stimulation activates phrenic proprioceptive afferents and induces plasticity

Direct electrical stimulation of the phrenic nerve/diaphragm muscle is an alternative to mechanical ventilation to sustain breathing in conditions such as spinal cord injury. Previous studies from our lab suggest that phrenic nerve stimulation induces a long-lasting increase in contralateral phrenic output. This form of respiratory plasticity depends on activation of phrenic afferents and is referred to as phrenic afferent induced plasticity (pAIP). However, the mechanisms necessary for inducing pAIP are unknown. We hypothesize that pAIP represents a form of spinal cord associative plasticity that is contingent upon the activation of proprioceptors timed specifically with the inspiratory phase. To investigate this hypothesis, we evaluated the expression of pAIP after delivering electrical stimulation in-phase or out-of-phase with inspiration and used RNA scope to identify the phenotype of activated afferents in the dorsal root ganglion. For this, both the left and right phrenic nerves were isolated in urethane anesthetized, mechanically ventilated, vagotomized, and paralyzed male rats. After recording baseline output, we delivered phrenic nerve stimulation (5 x 5-minute episodes of 40Hz, 160µA, 0.1ms pulse width, interleaved with 4-minutes of no stimulation) on the left side during inspiration (n=8) or expiration (n=4) and evaluated the expression of pAIP on the right side and compared to animals not receiving stimulation (time controls, n=11). Blood samples were collected during the experiment and PaCO2 was kept within ±1.5 mmHg from baseline values to remove the contribution of chemoreflexes. At the end of experiment, the C4 dorsal root ganglions were stained for c-Fos to identify activated afferents and parvalbumin to labeled proprioceptive neurons. Our results show that stimulation delivered during inspiration increases contralateral output and this response to stimulation progressively increases with repeated episodes of stimulation. After five episodes, a long-lasting increase in contralateral phrenic motor output is observed at 60 minutes (37.80% ± 7.34, Mean ± SEM) compared to baseline (p=0.0046) and time controls (p=0.0010) indicating pAIP. In contrast, stimulation delivered during expiration resulted in a smaller increase in phrenic amplitude (9.70% ± 2.66), which we found is not different from baseline (P= 0.9739) or time-controls (3.30% ± 1.27, p=0.8429). We also observed a substantial population of parvalbumin neurons co-localized with c-Fos within the dorsal root ganglia (DRG) at C4 level, indicating activation of phrenic proprioceptors. On going analysis will quantify the number of c-Fos activated proprioceptors in each group. Taken together, these findings support our hypothesis that inspiratory-timed, but not expiratory-timed stimulation induces pAIP and activates proprioceptors. By understanding the mechanisms necessary for inducing pAIP, our results help refine current approaches to therapies aimed at enhancing respiratory recovery. Funding: National Institute of Health, National Heart Lung and Blood Institute, 1R01HL179209-01 (to KAS). This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.