Chemogenetic activation of phrenic motoneurons
Authors: Benevides Ethan , Rana Sabhya , Sunshine Michael , Jensen Victoria , Fuller David 
Published: May 2023

Disruption of bulbospinal synaptic pathways after cervical spinal cord injury (SCI) can impair or prevent the activation of spinal respiratory motor neurons. We aimed to determine if DREADD technology (i.e., designer receptors exclusively activated by designer drugs) could be used to produce sustained activation of phrenic motoneurons as reflected by increased diaphragm electromyogram (EMG) output. In C57/bl6 mice (wild-type, WT), an AAV9-hSyn-HA-hM3D(Gq)-mCherry vector was bilaterally injected into the mid-cervical (C4) spinal ventral horn. This was expected to drive expression of the excitatory DREADD receptor (hM3Dq) in a non-specific manner, throughout the ventral horn. In ChAT-Cre mice, a cre-dependent construct (AAV9-hSyn-DIO-hM3D(Gq)-mCherry) was injected at C4, and this was expected to drive hM3Dq primarily in phrenic motoneurons, which are ChAT-positive. We hypothesized that 6-8 weeks following spinal AAV injection, the highly selective DREADD ligand JHU37160 (J60) would result in an increase in phasic (inspiratory) diaphragm EMG bursting, and this response would be stronger in the ChAT-Cre mice (due to the more selective DREADD expression). Post-mortem histology confirmed widespread ventral horn neuron transduction in the WT spinal cord. In contrast, neuronal transduction in ChAT-Cre mice was primarily restricted to C3-5 putative motoneurons, following the known distribution of phrenic motoneurons. Diaphragm EMG recordings were made during spontaneous breathing under isoflurane anesthesia. After >20 min of baseline recording, intraperitoneal delivery of J60 (0.1 mg/kg) resulted in a rapid increase in the peak inspiratory diaphragm EMG burst amplitude in all ChAT-Cre mice, but the response was highly variable in WT mice. Analysis of peak EMG using a two-way repeated measures analysis of variance (RM ANOVA) showed an interaction between strain (WT vs. ChAT-Cre) and time post-J60 (F(5,117)=9.58, p<0.001). Tukey post-hoc test revealed peak diaphragm EMG was increased vs. baseline in ChAT-Cre mice at 5-, 15-, 30-, 60-, and 90-min post-injection (p<0.05), and EMG was greater in ChAT-Cre vs. WT at each time point (p<0.05). Respiratory rate progressively declined in ChAT-Cre but not WT mice (2-way RM ANOVA, F(6,138)=3.98, p<0.001). Additional experiments using a ChAT-Cre rat model (n=2) confirmed efficacy after cervical SCI. Specifically, at 7 days following C2 spinal cord hemilesion, rats expressing the hM3Dq receptor in ChAT-positive, mid-cervical ventral horn neurons (driven by AAV9-hSyn-DIO-hM3D(Gq)-mCherry) showed an immediate and sustained (>90 min) increase in diaphragm EMG bursting upon J60 administration. These results indicate the mid-cervical DREADD expression targeting primarily motoneurons enables a rapid and sustained increase in diaphragm output upon DREADD ligand administration. R01HL139708-04 (DF), R01HD052682-13 (DF), UF Graduate Fellowship (EB) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.