Selective optogenetic stimulation of RTN neurons drives ventilation and active expiration in unanesthetized rats

Abstract

BackgroundThe retrotrapezoid nucleus (RTN) consists of neurons that are chemosensitive (i.e., CO2/pH sensitive), glutamatergic, Phox2b+ and Neuromedin B+ (Nmb+). Lentiviral vectors currently used to transduce RTN neurons with various opto‐ or pharmacogenetic actuators contain a Phox2b‐activated promoter, PRSx8. When injected into the RTN, these vectors also transduce substantial numbers of nearby C1 and catecholaminergic neurons. The purpose of the present study was to determine the effects on breathing elicited by selectively activating RTN neurons.MethodsTo selectively transduce RTN neurons we microinjected a mixture of Lenti‐PRSx8‐ChR2‐mCherry and AAV5‐FLEX‐taCasP3‐TEVp into the RTN of adult TH‐Cre rats. One month after the surgery, RTN neurons were stimulated with blue light (473 nm, 10 ms pulse, 20 Hz, for 20 s). All the measurements were conducted inside a plethysmography chamber. Diaphragm (DIA) and abdominal (ABD) muscle activity were recorded in conscious conditions. Results (mean ± SD), were analyzed using one‐ or two‐way ANOVA as required and differences were considered significant at P<0.05.ResultsRTN neurons (Nmb+, TH−) were selectively transduced (96.8 % ± 4) with the PRSx8 vector, the FLEX‐caspase AAV5 having caused the apoptotic death of C1 neurons (TH+) in the area where neurons were transduced with ChR2. Selective stimulation of RTN (NMB+ neurons; n=6) increased respiratory rate during wake and NREM sleep but not during REM sleep (Δ 30 ± 7 vs. 22.7 ± 6 vs. 4 ± 4 bpm from baseline), increased tidal volume also during quiet waking and non‐REM but not REM sleep (Δ 0.15 ± 0.07 vs. 0.13 ± 0.04 vs. 0.02 ± 0.007 mL/100g) and VE also during wake and non‐REM sleep but not REM (27.8 ± 11 vs. 21.4 ± 8 vs. 3.1 ±1 mL/100g/min). Selective RTN stimulation in rats breathing room air elicited active expiration (from 8.4 ± 7 to 15.9 ± 8 % of normalized abdominal EMG recorded at 9% FiCO2, n=3). RTN stimulation produced greater active expiration in rats exposed to 6% FiCO2 (from 56.3 ± 32 to 125.3 ± 104 % of normalized abdominal EMG).ConclusionsSelective RTN neuron stimulation drives breathing amplitude and frequency during wake, NREM but not in REM sleep, and elicits active expiration, especially at elevated levels of FiCO2.Support or Funding InformationSupported by grants from the National Institutes of Health (HL28785, 18 HL074011) and the Congenital Central Hypoventilation Syndrome Foundation to PGG.