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Optogenetic perturbation of preBötzinger complex inhibitory neurons modulates respiratory pattern.

Sherman D, Worrell JW, Cui Y, Feldman JL - Nat. Neurosci. (2015)

Bottom Line: Inhibitory neurons make up a substantial fraction of the neurons in the preBötzinger complex (preBötC), a site that is critical for mammalian eupneic breathing.Channelrhodopsin-2 (ChR2) or Archaerhodopsin (Arch) were expressed in glycinergic preBötC neurons of glycine transporter 2 (Glyt2, also known as Slc6a5)-Cre mice.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, California, USA.

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.

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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.
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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.

Mentions: Preinspiratory or early inspiratory bilateral photoinhibition (ϕstim: −120° – 15°) of Arch-transfected preBötC GlyT2 neurons (100 ms pulse; 593 nm; Fig 4a; n=7) increased peak inspiratory airflow (137.0 ± 3.8% of control; P = 2×10−9; n=7) and tidal volume (Fig. 4b, c) without a significant change in inspiratory duration (103.0 ± 0.9% of control; P=1; n=7; Fig. 4b, d) or respiratory phase (P = 1; n = 7; Fig. 4e). Compared to these photoinhibition-augmented breaths, sighs, i.e., larger breaths endogenously generated periodically to hyperinflate the lungs, had greater amplitudes (Fig. 4f). Sighs had peak inspiratory airflows that were 173 ± 15% of control (P = 10−5; n = 7) that was also statistically different from photoinhibition-induced augmented breaths (P = 0.02; n = 7). Bilateral photoinhibition during midexpiration (ϕstim: −210° – −120°) produced a shift in respiratory phase manifested by an earlier onset of the subsequent inspiration (52.3° ± 6.2° phase shift; P = 8×10−6; n = 7; Fig. 4e, g), with the strongest effect at 180° – −150°. Prolonged photoinhibition (5 s pulse) increased respiratory frequency but the effect was transient and did not last for the full duration of the laser pulse (Fig. 4h; n = 3).


Optogenetic perturbation of preBötzinger complex inhibitory neurons modulates respiratory pattern.

Sherman D, Worrell JW, Cui Y, Feldman JL - Nat. Neurosci. (2015)

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.
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Related In: Results  -  Collection

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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.
Mentions: Preinspiratory or early inspiratory bilateral photoinhibition (ϕstim: −120° – 15°) of Arch-transfected preBötC GlyT2 neurons (100 ms pulse; 593 nm; Fig 4a; n=7) increased peak inspiratory airflow (137.0 ± 3.8% of control; P = 2×10−9; n=7) and tidal volume (Fig. 4b, c) without a significant change in inspiratory duration (103.0 ± 0.9% of control; P=1; n=7; Fig. 4b, d) or respiratory phase (P = 1; n = 7; Fig. 4e). Compared to these photoinhibition-augmented breaths, sighs, i.e., larger breaths endogenously generated periodically to hyperinflate the lungs, had greater amplitudes (Fig. 4f). Sighs had peak inspiratory airflows that were 173 ± 15% of control (P = 10−5; n = 7) that was also statistically different from photoinhibition-induced augmented breaths (P = 0.02; n = 7). Bilateral photoinhibition during midexpiration (ϕstim: −210° – −120°) produced a shift in respiratory phase manifested by an earlier onset of the subsequent inspiration (52.3° ± 6.2° phase shift; P = 8×10−6; n = 7; Fig. 4e, g), with the strongest effect at 180° – −150°. Prolonged photoinhibition (5 s pulse) increased respiratory frequency but the effect was transient and did not last for the full duration of the laser pulse (Fig. 4h; n = 3).

Bottom Line: Inhibitory neurons make up a substantial fraction of the neurons in the preBötzinger complex (preBötC), a site that is critical for mammalian eupneic breathing.Channelrhodopsin-2 (ChR2) or Archaerhodopsin (Arch) were expressed in glycinergic preBötC neurons of glycine transporter 2 (Glyt2, also known as Slc6a5)-Cre mice.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, California, USA.

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.

Show MeSH
Related in: MedlinePlus