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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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Related in: MedlinePlus

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 (ϕstim), expected phase (ϕe), induced phase (ϕi), and phase shift (ϕshift), 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 ϕstim. ϕe is the period of the previous control respiratory cycle, and ϕshift is the difference between ϕe and ϕi. (b) Shift in respiratory phase (ϕshift) 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 (ϕstim) 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.
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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 (ϕstim), expected phase (ϕe), induced phase (ϕi), and phase shift (ϕshift), 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 ϕstim. ϕe is the period of the previous control respiratory cycle, and ϕshift is the difference between ϕe and ϕi. (b) Shift in respiratory phase (ϕshift) 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 (ϕstim) 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.

Mentions: To efficiently deliver light specifically into the preBötC, we implanted optical cannulae bilaterally from a dorsal approach, about 200 – 400 µm dorsal to the preBötC (Fig 1a–b). By placing the cannulae close to the preBötC, we ensured that the pool of light-responsive neuronal somas able to affect breathing were preBötC GlyT2 neurons. As delineated in the DISCUSSION, the most parsimonious explanation of our results is that the light-induced responses were due to activation of preBötC GlyT2 neurons.


Optogenetic perturbation of preBötzinger complex inhibitory neurons modulates respiratory pattern.

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

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 (ϕstim), expected phase (ϕe), induced phase (ϕi), and phase shift (ϕshift), 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 ϕstim. ϕe is the period of the previous control respiratory cycle, and ϕshift is the difference between ϕe and ϕi. (b) Shift in respiratory phase (ϕshift) 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 (ϕstim) 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.
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Related In: Results  -  Collection

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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 (ϕstim), expected phase (ϕe), induced phase (ϕi), and phase shift (ϕshift), 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 ϕstim. ϕe is the period of the previous control respiratory cycle, and ϕshift is the difference between ϕe and ϕi. (b) Shift in respiratory phase (ϕshift) 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 (ϕstim) 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.
Mentions: To efficiently deliver light specifically into the preBötC, we implanted optical cannulae bilaterally from a dorsal approach, about 200 – 400 µm dorsal to the preBötC (Fig 1a–b). By placing the cannulae close to the preBötC, we ensured that the pool of light-responsive neuronal somas able to affect breathing were preBötC GlyT2 neurons. As delineated in the DISCUSSION, the most parsimonious explanation of our results is that the light-induced responses were due to activation of preBötC GlyT2 neurons.

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