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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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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.
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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.

Mentions: A critical role of inhibition in the preBötC in controlling respiratory pattern is likely in the production of apneas10, such as during swallowing or breathholding. To determine whether preBötC GlyT2 neurons participate in generating apneas, we photoinhibited these neurons during Breuer-Hering lung inflation reflex (BHIR)-induced apneas10. The lungs were inflated with sufficient continuous positive airway pressure (CPAP) to produce apneas that lasted ~11 s (~4 cm H2O) during control periods (Fig. 5a, top). Photoinhibiting Arch-transfected preBötC GlyT2 neurons (1 s pulse) broke the apnea with one or two breaths (Fig. 5a, bottom; n = 3). Laser onset at 5.5 ± 0.5 s after onset of CPAP (CPAP + Laser) induced a first breath at 5.8 ± 0.5 s, i.e., a 300 ms delay from the laser onset versus an expected first breath at 11.8 ± 1.4 s during control (CPAP only) periods (ANOVA; P = 3×10−6; Fig. 5b; n = 3).


Optogenetic perturbation of preBötzinger complex inhibitory neurons modulates respiratory pattern.

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

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.
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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.
Mentions: A critical role of inhibition in the preBötC in controlling respiratory pattern is likely in the production of apneas10, such as during swallowing or breathholding. To determine whether preBötC GlyT2 neurons participate in generating apneas, we photoinhibited these neurons during Breuer-Hering lung inflation reflex (BHIR)-induced apneas10. The lungs were inflated with sufficient continuous positive airway pressure (CPAP) to produce apneas that lasted ~11 s (~4 cm H2O) during control periods (Fig. 5a, top). Photoinhibiting Arch-transfected preBötC GlyT2 neurons (1 s pulse) broke the apnea with one or two breaths (Fig. 5a, bottom; n = 3). Laser onset at 5.5 ± 0.5 s after onset of CPAP (CPAP + Laser) induced a first breath at 5.8 ± 0.5 s, i.e., a 300 ms delay from the laser onset versus an expected first breath at 11.8 ± 1.4 s during control (CPAP only) periods (ANOVA; P = 3×10−6; Fig. 5b; 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