Opioid‐Induced Respiratory Depression (OIRD) in C57BL/6J (B6) Mice is Mitigated by Prefrontal Cortex Neostigmine Administration
Authors: Sun Qiwei W. , Glovak Zachary T. , Baghdoyan Helen A. , Lydic Ralph 
Published: May 2022

Opioids diminish the wakefulness stimulus for breathing in humans (10.1152/physiol.00043.2020) and mice (10.1152/jn.00017.2021). The neuronal networks underlying the wakefulness stimulus remain poorly understood. Cholinergic transmission in the prefrontal cortex (PFC) promotes cortical EEG activation (10.1152/jn.2002.87.6.2817), increases wakefulness (10.3389/fnins.2020.567849), and stimulates breathing (10.1096/fasebj.2021.35.S1.01735). This study is using whole‐body plethysmography (WBP) to test the hypothesis that microinjection of the acetylcholinesterase inhibitor neostigmine (Neo) into the PFC attenuates OIRD caused by systemic fentanyl administration in male B6 mice. All procedures followed the FASEB Statement of Principles for the use of Animals in Research and Education. Previous studies of the dose‐dependent effects of fentanyl in B6 mice used CO2 to stimulate breathing (10.1038/npp.2015.201). Therefore, we first quantified the dose‐dependent effects of systemic fentanyl administration on B6 mice breathing room air. Mice (n=12) received a 0.3 mL intraperitoneal (IP) injection of saline (control) and 8 increasing doses of fentanyl (0.001 to 3 mg/kg), each separated by one half‐log unit. Repeated measures one‐way ANOVA showed that fentanyl caused a dose‐dependent decrease (F(2.47; 27.12)= 20.30, p<0.0001) in minute ventilation (V̇E) quantified as mL/min/g body weight. Additional mice (n=4) were implanted with a 26‐gauge guide cannula aimed for the PFC at coordinates (mm) A/P = 1.94; M/L = 0.3; D/V = 2.5 relative to bregma (histology pending). Seven to ten days after surgery, WBP was used to evaluate the respiratory effects of 0.1 mg/kg IP fentanyl in combination with unilateral PFC microinjection of saline (control) or 0.016 nmol/50nL Neo (4.8 ng). Breathing was compared among three conditions: 1=(IP saline+PFC saline), 2=(IP fentanyl+PFC saline), and 3=(IP fentanyl+PFC Neo). Centrally administered drugs rapidly diffuse away from a microinjection site. Accordingly, V̇E was quantified during the initial 10 min after PFC microinjection. Comparison between conditions 1 and 2 showed that IP fentanyl caused a 19.7% decrease in V̇E. Comparing conditions 2 and 3 showed that IP fentanyl+PFC Neo caused a 44.3% increase in V̇E. Regression analyses revealed that PFC Neo delayed the onset of OIRD. During the initial 5 min after PFC Neo, time accounted for 84% of the variance in V̇E compared to 60% of the variance after PFC saline. During the 6 to 10 min after PFC injection, time explained only 27% of the variance in V̇E, likely due to Neo diffusing away from the PFC. The slope of the minute ventilation line (V̇E=(0.03)(min)+3.2) increased after IP fentanyl + PFC Neo and decreased (V̇E=(‐0.12)(min)+3.8) after IP fentanyl + PFC saline. The slopes of these two functions were significantly different (p<0.01). For up to 8 min, 4.8 ng of Neo delivered into the PFC diminished OIRD caused by 0.1 mg/kg IP fentanyl. These findings support our working hypothesis that enhancing cholinergic transmission in the PFC promotes to the wakefulness stimulus for breathing and mitigates OIRD.