Optogenetic perturbation of preBötzinger complex inhibitory neurons modulates respiratory pattern

Abstract

Inhibitory neurons make up a substantial fraction of the neurons in the preBötzinger complex (preBötC), a site that is critical for mammalian eupneic breathing. We investigated the role of glycinergic preBötC neurons in respiratory rhythmogenesis in mice using optogenetically targeted excitation and inhibition. Channelrhodopsin-2 (ChR2) or Archaerhodopsin (Arch) were expressed in glycinergic preBötC neurons of glycine transporter 2 (Glyt2, also known as Slc6a5)-Cre mice. In ChR2-transfected mice, brief inspiratory-phase bilateral photostimulation targeting the preBötC prematurely terminated inspiration, whereas expiratory-phase photostimulation delayed the onset of the next inspiration. Prolonged photostimulation produced apneas lasting as long as the light pulse. Inspiratory-phase photoinhibition in Arch-transfected mice during inspiration increased tidal volume without altering inspiratory duration, whereas expiratory-phase photoinhibition shortened the latency until the next inspiration. During persistent apneas, prolonged photoinhibition restored rhythmic breathing. We conclude that glycinergic preBötC neurons modulate inspiratory pattern and are important for reflex apneas, but that the rhythm can persist after substantial dampening of their activity.

Figure 1. Cre-dependent ChR2 or Arch expression targeted to preBötC GlyT2 neurons

(a) Representative confocal mosaic micrograph of sagittal brainstem section of GlyT2-cre⁺ mouse showing the extent of eYFP⁺ and Sst⁺ neurons after injection of AAV2/1-Ef1α-DIO-ChR2-eYFP into preBötC (n = 5). Vertical white lines at bottom mark approximate rostral-caudal boundaries of Bötzinger Complex (BötC) and preBötzinger Complex (preBötC). Outline of cannula tip placement at top right. No labeled somas were found in the brainstem outside the boundaries of this micrograph. Abbreviations: VII: facial nucleus; NA: nucleus ambiguus; LRN: lateral reticular nucleus. (b) High magnification micrograph of bracketed segment in (a) showing eYFP⁺ preBötC neurons (white arrows) intermingled with Sst⁺ neurons (blue arrows). (c, d) Distribution of eYFP/GFP⁺ (blue line), marking location of ChR2- (c; n = 3) or Arch- (d; n = 3) expressing neurons and Sst⁺ (red line) neurons relative to caudal boundary of facial nucleus. Error bars: mean ± s.e.m. (e) Representative single channel and overlay confocal micrographs showing eYFP⁺ preBötC neurons (green) after AAV2/1-Ef1α-DIO-ChR2-eYFP injection. Section also labeled for Sst (red) and NeuN (blue) immunoreactivity. (f) Single channel and overlay confocal micrographs (n = 3) showing eYFP⁺ preBötC neurons (green) with glycine immunoreactivity (red).

Figure 2. Photostimulation of preBötC GlyT2 neurons depresses breathing

(a) Left: Schematic depicting bilateral placement of optical cannulae targeting preBötC. Right: schematic airflow trace depicting definitions of stimulus phase (ϕ_stim), expected phase (ϕ_e), induced phase (ϕ_i), and phase shift (ϕ_shift), demarcated with horizontal arrows, relative to the reference cycle that spans 0° – 360°; the control cycle preceding reference cycle spans phase 360° – 0°. Black bar below trace is laser-on period that defines the start of ϕ_stim. ϕ_e is the period of the previous control respiratory cycle, and ϕ_shift is the difference between ϕ_e and ϕ_i. (b) Shift in respiratory phase (ϕ_shift) resulting from bilateral photostimulation (100 ms pulse) of preBötC GlyT2 neurons in GlyT2-cre⁺ (Cre⁺, red; n = 5) and GlyT2-cre⁻ (Cre⁻, blue; n = 5) mice. Stimulus phase (ϕ_stim) is depicted on x-axis (b, e, f) with inspiration (black bar) defined from 0° – 72°, with gray bar: (b) the subsequent expiration defined from 72° – 360° or (e, f) the preceding expiration defined from −288° – 0°. P values: 150 – 180°: P = 0.007; 180 – 210°: P = 2×10⁻⁵; 210 – 240°: P = 4×10⁻⁶; 240 – 270°: P = 4×10⁻⁷; 270 – 300°: P = 1×10⁻⁹; 300 – 330°: P = 5×10⁻¹⁰; 330 – 360°: P = 4×10⁻¹⁰. (c, d) Representative airflow and tidal volume (airflow) traces illustrating effect of photostimulation (100 ms pulse; black bar beneath trace) during inspiration (c) and expiration (d). (e, f) Comparison of ratio of peak inspiratory airflow (e; 0 – 30°: P = 4×10⁻¹⁰; 30 – 60°: P = 0.02) or inspiratory duration (f; 0 – 30°: P = 4×10⁻¹⁰) in GlyT2-cre⁺ (Cre⁺, red) and GlyT2-cre⁻ (Cre⁻, blue) anesthetized mice. Dotted vertical line (e, f) at 0° indicates onset of inspiration. Error bars, mean ± s.e.m. Statistical significance was determined with a one-way ANOVA and pair-wise comparisons were made with Tukey’s HSD test. * P < 0.05; ** P < 0.001; ^† P < 10⁻⁸.

Figure 3. Prolonged photostimulation of preBötC GlyT2 neurons results in apnea

(a) Representative airflow trace illustrating effect of 1 s pulse train of photostimulation (black bars beneath trace; 7× 100ms pulses with a 50 ms interpulse interval; n = 5). (b) Overlay of airflow traces aligned to the laser onset at various phases of respiratory cycle (7× 100 ms pulses with a 50 ms interpulse interval) reveals that the next breath occurred at a fairly constant delay after the laser shuts off. (c) Representative graph of latency to next breath as a function of initial phase of photostimulation. Dotted line indicates mean latency. (d, e, f) Photostimulation with a 1 s pulse train (7× 100 ms pulses with 50 ms interpulse interval) of awake, behaving mice in a plethysmograph under eupneic (d; n = 5), hypoxic (e; n = 5), or hypercapnic (f; n = 5) states. (g) Airflow during bilateral 20 s pulse train of light (100 ms pulses with 50 ms interpulse interval; n = 3) in anesthetized mouse.

Figure 4. Photoinhibition of preBötC GlyT2 neurons augments breathing

(a) Schematic depicting bilateral placement of optical cannulae targeting preBötC. (b) Representative airflow and tidal volume (∫airflow) traces (3 pairs) illustrating effect of brief photoinhibition (100 ms pulse; black bar) during inspiration. (c, d) Comparison of photoinhibited cycle to prior control cycle as a function of stimulus phase (ϕ_stim), for ratio of peak inspiratory airflow (c; n = 7; 0 – 30°: P = 2×10⁻⁷; 300 – 330°: P = 0.01; 330 – 360°: P = 5×10⁷) or inspiratory duration (d; n = 7; all P > 0.05) in Cre⁺ (red) and Cre⁻ (blue) anesthetized mice. Respiratory stimulus phase (ϕ_stim) on x-axis (c, d, e) with inspiration (black) defined from 0° to 78° (−360° to −282°) and expiration (gray) defined from 78° to 360° (-282° to 0°). The dotted vertical line (c, d) at 0° indicates onset of inspiration. (e) Shift in respiratory phase (ϕ_shift) resulting from preBötC-targeted laser pulses (100 ms pulse) in GlyT2-cre⁺ (Cre⁺, red; n = 7) and GlyT2-cre⁻ (Cre⁻, blue; n = 5) mice. P values: 150 – 180°: P = 0.002; 180 – 210°: P = 2×10⁻⁵; 210 – 240°: P = 8×10⁻⁶; 240 – 270°: P = 0.01. (f) Representative light response in response to photoinhibition (black bar) compared to endogenous sigh (arrow). (g) Representative airflow and tidal volume (∫airflow) traces illustrating effect of brief photoinhibition (100 ms pulse; black bar) during expiration. (h) Representative airflow and tidal volume traces illustrating that longer photostimulation (5 s continuous pulse) increased peak inspiratory airflow and frequency (n = 3). Error bars, mean ± s.e.m. Statistical significance was determined with a one-way ANOVA and pair-wise comparisons were made with Tukey’s HSD test. * P < 0.05; ** P < 0.001; ^† P < 10⁻⁸.

Figure 5. Photoinhibition of preBötC GlyT2 neurons during a reflex apnea rescues breathing

(a) Breuer-Hering lung inflation reflex (BHIR) triggered by continuous positive airway pressure (CPAP; ~4 cm H₂O; dotted black line) induced apnea in a tracheotomized, anesthetized Arch-transfected mouse in control (top) and bilateral photoinhibition (bottom, black bar) conditions. Top: After onset of BHIR, first breathe was at ~11 sec (black arrow). Bottom: 1s pulse applied 5.5 ± 0.5 s from onset of BHIR produced a first breath (indicated with a black arrow) 5.8 ± 0.5 s after the start of reflex, i.e., a 300 ms delay from the laser onset, versus an expected first breath at 11.8 ± 1.4 s, as seen during control (CPAP only) periods. (b) Duration of induced apnea, measured from onset of BHIR to onset of the next inspiration, in CPAP only and CPAP + laser conditions. Means are indicated with black horizontal lines (n = 3; P = 3×10⁻⁶). Statistical significance was determined with an unpaired t-test. * P < 10⁻⁵.

Figure 6. Unit recording with concurrent ChR2 or Arch activation

(a) Schematic showing cannulae implantation at a 27° angle and vertical electrode placement to record from units within the cone of light (473 nm or 593 nm) in opsin-transfected mice. (b) Representative airflow traces (red) and respiratory-modulated units (blue; i) expiratory, ii) inspiratory, iii) pre-inspiratory, iv) pre-inspiratory, v) inspiratory) showing strong inhibition during 1s continuous laser pulse (black bar). Black arrows indicate the expected onset of inspiration following photostimulation. In total, 18 neurons were recorded from 3 mice. (c) Representative recording of multiple units following photostimulation of ChR2-expressing preBötC GlyT2 neurons with airflow trace. Inset: Shorter time-scale at onset of continuous laser pulse (black line) showing large field potential within ~2 msec. Vertical black line shows alignment of laser onset and initial artifact on trace. (d) Response of preBötC neurons to photoinhibition of Arch-expressing preBötC GlyT2 neurons with 1 s continuous laser pulse (black line). Black arrows indicate the expected onset of inspiration following photoinhibition as measured on the airflow trace. In total, 12 neurons were recorded from 2 mice.