Recovery of Serotonergic Innervation in Spinal Motor Nuclei below Cervical Spinal Injury: Effects of Intermittent Hypoxia

Abstract

Cervical spinal cord injury (cSCI) disrupts serotonergic innervation below the injury. Since serotonin underlies motor functional recovery following cSCI, it is important to know the extent of serotonergic reinnervation after incomplete cSCI and to understand factors that accelerate that process. Repetitive acute intermittent hypoxia (rAIH) is one treatment known to elicit serotonin‐dependent respiratory and limb motor plasticity, partially restoring breathing and limb functions after chronic cSCI. Chronic intermittent hypoxia (CIH) similar to that experienced during sleep apnea elicits neuropathology, and potentially undermines the capacity for AIH‐induced motor plasticity. Although rAIH was previously reported to increase serotonergic innervation of the phrenic motor nucleus in intact rats, the impact of different intermittent hypoxia (IH) protocols on serotonergic innervation of respiratory motor nuclei in rats with or without chronic cSCI has not been investigated. Further, the impact of IH on serotonergic innervation of non‐respiratory motor nuclei has never been investigated. Thus, we tested the hypotheses that rAIH and CIH exert differential effects on serotonergic innervation of phrenic, intercostal, forelimb and axial motor nuclei motor nuclei post‐cSCI. Serotonergic innervation was assessed via immunofluorescence in male Sprague Dawley rats with and without C2 spinal hemisection (C2Hx; 12 wks post‐injury) exposed to 28 days of: 1) normoxia; 2) daily AIH (10, 5min 10.5% O2 episodes per day; 5min normoxic intervals); 3) mild CIH (5‐min 10.5% O2 episodes; 5min intervals; 8 hrs/day); and 4) moderate CIH (2min 10.5% O2 episodes; 2min intervals; 8 hrs/day). Rats received intrapleural injections of Cholera toxin B subunit 14 days before C2Hx to label phrenic and intercostal motor neurons. Forelimb and axial motor nuclei regions were determined using anatomical landmarks. In all regions investigated, no IH protocol impacted serotonergic innervation in intact or injured rats, contrary to previous reports. All areas had significant reductions in the number of serotonergic structures ipsilateral to C2Hx (p<0.001). Serotonergic structures innervating ipsilateral phrenic nuclei were larger (p<0.001), resulting in an overall increase in total innervation (p<0.001). Serotonergic structures innervating ipsilateral intercostal nuclei were also larger (p<0.001), but total innervation was reduced (p<0.001) due to larger reductions in number. In forelimb and axial nuclei, the size of serotonergic structures was unaffected by C2Hx (p<0.001), resulting in less total innervation ipsilateral to C2Hx (p<0.0001). Thus, C2Hx deferentially impacts serotonin reinnervation in spinal motor nuclei ipsilateral to C2Hx. The functional impact of increased serotonergic structure size in phrenic and intercostal motor nuclei is unclear. Our results indicate substantial serotonergic reinnervation below cSCI at this time point, potentially enabling serotonin‐dependent, AIH‐induced plasticity with chronic cSCI.Support or Funding InformationSPARC OT2OD023854 NIH T32 HD043730 NIH K12 HD055929 and UF McKnight Brain Institute