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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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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).
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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).

Mentions: We utilized optogenetic tools to probe the functional contribution of glycinergic preBötC neurons to respiratory rhythm and pattern. We used viral transfection to express Channelrhodopsin-2 (ChR2) or Archaerhodopsin (Arch) in glycinergic preBötC neurons by locally injecting cre recombinase-dependent viruses into the preBötC of transgenic mice expressing Cre driven by the glycine transporter 2 (GlyT2) promoter12–14. Since the GlyT2 promoter is specific to glycinergic neurons15, this intersectional strategy allowed us to selectively activate and silence these inhibitory neurons in intact, awake or anesthetized, spontaneously breathing mice. We found that perturbing glycinergic neuronal activity in the preBötC profoundly modulated the amplitude and timing of inspiratory motor output and expiratory period with no indications that they were critical for rhythmogenesis.


Optogenetic perturbation of preBötzinger complex inhibitory neurons modulates respiratory pattern.

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

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).
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Related In: Results  -  Collection

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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).
Mentions: We utilized optogenetic tools to probe the functional contribution of glycinergic preBötC neurons to respiratory rhythm and pattern. We used viral transfection to express Channelrhodopsin-2 (ChR2) or Archaerhodopsin (Arch) in glycinergic preBötC neurons by locally injecting cre recombinase-dependent viruses into the preBötC of transgenic mice expressing Cre driven by the glycine transporter 2 (GlyT2) promoter12–14. Since the GlyT2 promoter is specific to glycinergic neurons15, this intersectional strategy allowed us to selectively activate and silence these inhibitory neurons in intact, awake or anesthetized, spontaneously breathing mice. We found that perturbing glycinergic neuronal activity in the preBötC profoundly modulated the amplitude and timing of inspiratory motor output and expiratory period with no indications that they were critical for rhythmogenesis.

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