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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 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 (ϕstim), 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 (ϕstim) 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 (ϕshift) 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.
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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 (ϕstim), 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 (ϕstim) 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 (ϕshift) 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.

Mentions: In anesthetized ChR2-transfected mice, a 1 s photostimulus (7 × 100 ms pulses, 50 ms interpulse interval) at any time during the respiratory cycle stopped breathing, i.e., produced an apnea, which continued until after the light shut off (Fig. 3a; n = 5). The subsequent respiratory cycle began at a fairly constant delay after the laser turned off regardless of stimulus phase (Fig. 3b, c). The duration of this delay was unique to each mouse (300 – 860 ms, min-max; n = 3), presumably due to differences in the precise position of the virus injection and optical fiber placement. When tested in awake mice, a 1 s pulse train consistently produced apneas (Fig. 3d; n = 5). We further tested photostimulus responses in anesthetized mice when ventilation was increased during 5 min of either hypoxic (8% O2, 92% N2; n = 5) or hypercapnic (5% CO2 in room air; n = 5) inspired gas mixtures, finding that ChR2 activation still consistently produced apnea in either of these conditions of increased respiratory drive (Fig. 3e, f). Prolonged stimulation (up to 20 s pulse trains; n = 3) produced an apnea that persisted until the laser turned off (Fig. 3g).


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 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 (ϕstim), 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 (ϕstim) 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 (ϕshift) 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.
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Related In: Results  -  Collection

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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 (ϕstim), 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 (ϕstim) 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 (ϕshift) 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.
Mentions: In anesthetized ChR2-transfected mice, a 1 s photostimulus (7 × 100 ms pulses, 50 ms interpulse interval) at any time during the respiratory cycle stopped breathing, i.e., produced an apnea, which continued until after the light shut off (Fig. 3a; n = 5). The subsequent respiratory cycle began at a fairly constant delay after the laser turned off regardless of stimulus phase (Fig. 3b, c). The duration of this delay was unique to each mouse (300 – 860 ms, min-max; n = 3), presumably due to differences in the precise position of the virus injection and optical fiber placement. When tested in awake mice, a 1 s pulse train consistently produced apneas (Fig. 3d; n = 5). We further tested photostimulus responses in anesthetized mice when ventilation was increased during 5 min of either hypoxic (8% O2, 92% N2; n = 5) or hypercapnic (5% CO2 in room air; n = 5) inspired gas mixtures, finding that ChR2 activation still consistently produced apnea in either of these conditions of increased respiratory drive (Fig. 3e, f). Prolonged stimulation (up to 20 s pulse trains; n = 3) produced an apnea that persisted until the laser turned off (Fig. 3g).

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