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Laser ablation of Dbx1 neurons in the pre-Bötzinger complex stops inspiratory rhythm and impairs output in neonatal mice.

Wang X, Hayes JA, Revill AL, Song H, Kottick A, Vann NC, LaMar MD, Picardo MC, Akins VT, Funk GD, Del Negro CA - Elife (2014)

Bottom Line: To understand the neural origins of rhythmic behavior one must characterize the central pattern generator circuit and quantify the population size needed to sustain functionality.Breathing-related interneurons of the brainstem pre-Bötzinger complex (preBötC) that putatively comprise the core respiratory rhythm generator in mammals are derived from Dbx1-expressing precursors.These results demonstrate that a single canonical interneuron class generates respiratory rhythm and contributes in a premotor capacity, whereas these functions are normally attributed to discrete populations.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Science, The College of William and Mary, Williamsburg, United States.

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Detection of Dbx1 neuron targets via fluorescence and image processing.(A1, B1, C1, D1) Images from the preBötC of Dbx1+/CreERT2; Rosa26tdTomato mice showing tdTomato in neurons derived from Dbx1-expressing precursors (i.e., Dbx1 neurons). Scale bar in A1 is 20 µm and applies to all panels. C1 and D1 show the same field of view at two different depths (−20 and −10 µm, respectively). (A2, B2, C2, D2) Masks of ROIs obtained by analyzing the corresponding images above. Red ROIs are deemed valid targets by the circularity test, which evaluates somatic shape; blue ROIs that fail the circularity test are rejected. Circularity analyses distinguish somata from auto-fluorescent detritus (A1, A2) as well as contiguous soma-dendrite images (B1, B2) and isolated segments (shafts) of dendrites (C1, C2, D1, D2). Non-somatic auto-fluorescence is rejected because it does not accurately indicate underlying neurons. Dendritic segments are not valid targets because they are difficult to target in the ablation phase of the experiments and their cell bodies are detectable in adjacent focal planes. Often, a cell rejected by the circularity test in one focal plane (e.g., C2, graygray double arrowhead) is validated in the adjacent plane (D2, graygray double arrowhead). When ROIs that pass the circularity test are detected in more than one focal plane, they are validated or rejected according to the priority rule. ROIs from a deeper focal plane (−20 µm) are validated by circularity and thus colored red (C2, circled ROIs). Subsequent detection of overlaying ROIs at the superficial focal plane (−10 µm), which also pass the circularity test, are nonetheless rejected by the priority rule and thus colored blue (D2, circled ROIs). These criteria for target detection are more fully described in ‘Materials and methods’ and Wang et al. (2013).DOI:http://dx.doi.org/10.7554/eLife.03427.005
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fig1s2: Detection of Dbx1 neuron targets via fluorescence and image processing.(A1, B1, C1, D1) Images from the preBötC of Dbx1+/CreERT2; Rosa26tdTomato mice showing tdTomato in neurons derived from Dbx1-expressing precursors (i.e., Dbx1 neurons). Scale bar in A1 is 20 µm and applies to all panels. C1 and D1 show the same field of view at two different depths (−20 and −10 µm, respectively). (A2, B2, C2, D2) Masks of ROIs obtained by analyzing the corresponding images above. Red ROIs are deemed valid targets by the circularity test, which evaluates somatic shape; blue ROIs that fail the circularity test are rejected. Circularity analyses distinguish somata from auto-fluorescent detritus (A1, A2) as well as contiguous soma-dendrite images (B1, B2) and isolated segments (shafts) of dendrites (C1, C2, D1, D2). Non-somatic auto-fluorescence is rejected because it does not accurately indicate underlying neurons. Dendritic segments are not valid targets because they are difficult to target in the ablation phase of the experiments and their cell bodies are detectable in adjacent focal planes. Often, a cell rejected by the circularity test in one focal plane (e.g., C2, graygray double arrowhead) is validated in the adjacent plane (D2, graygray double arrowhead). When ROIs that pass the circularity test are detected in more than one focal plane, they are validated or rejected according to the priority rule. ROIs from a deeper focal plane (−20 µm) are validated by circularity and thus colored red (C2, circled ROIs). Subsequent detection of overlaying ROIs at the superficial focal plane (−10 µm), which also pass the circularity test, are nonetheless rejected by the priority rule and thus colored blue (D2, circled ROIs). These criteria for target detection are more fully described in ‘Materials and methods’ and Wang et al. (2013).DOI:http://dx.doi.org/10.7554/eLife.03427.005

Mentions: (A) Fluorescent image of a transverse slice from a Dbx1+/CreERT2; Rosa26tdTomato mouse pup. Anatomical landmarks are illustrated including: XII, the hypoglossal motor nucleus; scNA, semi-compact nucleus ambiguus; and IOP, the principal inferior olive. The domain for detection and ablation is indicated by the white boxes, bilaterally. Scale bar is 500 µm. (B) Mask of targets showing validated Dbx1 (red) and invalidated (blue) cells for all focal planes to a depth of −80 µm. Each image is 412 × 412 µm2 (as in Figure 1C). Image processing routines for detecting and validating Dbx1 neuron targets are detailed in ‘Materials and methods’, Figure 1—figure supplement 2, and a methodological paper (Wang et al., 2013). Note that the highest fraction of validated Dbx1 target cells is found at deeper focal planes, e.g., −80 µm due to the ‘priority rule’, which applies to overlapping ROIs in adjacent focal planes. According to the priority rule, the ROI from the deeper focal is accepted as a ‘bona fide’ target and the redundant ROI at the superficial level is rejected. Also see Figure 1—figure supplement 2C,D.


Laser ablation of Dbx1 neurons in the pre-Bötzinger complex stops inspiratory rhythm and impairs output in neonatal mice.

Wang X, Hayes JA, Revill AL, Song H, Kottick A, Vann NC, LaMar MD, Picardo MC, Akins VT, Funk GD, Del Negro CA - Elife (2014)

Detection of Dbx1 neuron targets via fluorescence and image processing.(A1, B1, C1, D1) Images from the preBötC of Dbx1+/CreERT2; Rosa26tdTomato mice showing tdTomato in neurons derived from Dbx1-expressing precursors (i.e., Dbx1 neurons). Scale bar in A1 is 20 µm and applies to all panels. C1 and D1 show the same field of view at two different depths (−20 and −10 µm, respectively). (A2, B2, C2, D2) Masks of ROIs obtained by analyzing the corresponding images above. Red ROIs are deemed valid targets by the circularity test, which evaluates somatic shape; blue ROIs that fail the circularity test are rejected. Circularity analyses distinguish somata from auto-fluorescent detritus (A1, A2) as well as contiguous soma-dendrite images (B1, B2) and isolated segments (shafts) of dendrites (C1, C2, D1, D2). Non-somatic auto-fluorescence is rejected because it does not accurately indicate underlying neurons. Dendritic segments are not valid targets because they are difficult to target in the ablation phase of the experiments and their cell bodies are detectable in adjacent focal planes. Often, a cell rejected by the circularity test in one focal plane (e.g., C2, graygray double arrowhead) is validated in the adjacent plane (D2, graygray double arrowhead). When ROIs that pass the circularity test are detected in more than one focal plane, they are validated or rejected according to the priority rule. ROIs from a deeper focal plane (−20 µm) are validated by circularity and thus colored red (C2, circled ROIs). Subsequent detection of overlaying ROIs at the superficial focal plane (−10 µm), which also pass the circularity test, are nonetheless rejected by the priority rule and thus colored blue (D2, circled ROIs). These criteria for target detection are more fully described in ‘Materials and methods’ and Wang et al. (2013).DOI:http://dx.doi.org/10.7554/eLife.03427.005
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fig1s2: Detection of Dbx1 neuron targets via fluorescence and image processing.(A1, B1, C1, D1) Images from the preBötC of Dbx1+/CreERT2; Rosa26tdTomato mice showing tdTomato in neurons derived from Dbx1-expressing precursors (i.e., Dbx1 neurons). Scale bar in A1 is 20 µm and applies to all panels. C1 and D1 show the same field of view at two different depths (−20 and −10 µm, respectively). (A2, B2, C2, D2) Masks of ROIs obtained by analyzing the corresponding images above. Red ROIs are deemed valid targets by the circularity test, which evaluates somatic shape; blue ROIs that fail the circularity test are rejected. Circularity analyses distinguish somata from auto-fluorescent detritus (A1, A2) as well as contiguous soma-dendrite images (B1, B2) and isolated segments (shafts) of dendrites (C1, C2, D1, D2). Non-somatic auto-fluorescence is rejected because it does not accurately indicate underlying neurons. Dendritic segments are not valid targets because they are difficult to target in the ablation phase of the experiments and their cell bodies are detectable in adjacent focal planes. Often, a cell rejected by the circularity test in one focal plane (e.g., C2, graygray double arrowhead) is validated in the adjacent plane (D2, graygray double arrowhead). When ROIs that pass the circularity test are detected in more than one focal plane, they are validated or rejected according to the priority rule. ROIs from a deeper focal plane (−20 µm) are validated by circularity and thus colored red (C2, circled ROIs). Subsequent detection of overlaying ROIs at the superficial focal plane (−10 µm), which also pass the circularity test, are nonetheless rejected by the priority rule and thus colored blue (D2, circled ROIs). These criteria for target detection are more fully described in ‘Materials and methods’ and Wang et al. (2013).DOI:http://dx.doi.org/10.7554/eLife.03427.005
Mentions: (A) Fluorescent image of a transverse slice from a Dbx1+/CreERT2; Rosa26tdTomato mouse pup. Anatomical landmarks are illustrated including: XII, the hypoglossal motor nucleus; scNA, semi-compact nucleus ambiguus; and IOP, the principal inferior olive. The domain for detection and ablation is indicated by the white boxes, bilaterally. Scale bar is 500 µm. (B) Mask of targets showing validated Dbx1 (red) and invalidated (blue) cells for all focal planes to a depth of −80 µm. Each image is 412 × 412 µm2 (as in Figure 1C). Image processing routines for detecting and validating Dbx1 neuron targets are detailed in ‘Materials and methods’, Figure 1—figure supplement 2, and a methodological paper (Wang et al., 2013). Note that the highest fraction of validated Dbx1 target cells is found at deeper focal planes, e.g., −80 µm due to the ‘priority rule’, which applies to overlapping ROIs in adjacent focal planes. According to the priority rule, the ROI from the deeper focal is accepted as a ‘bona fide’ target and the redundant ROI at the superficial level is rejected. Also see Figure 1—figure supplement 2C,D.

Bottom Line: To understand the neural origins of rhythmic behavior one must characterize the central pattern generator circuit and quantify the population size needed to sustain functionality.Breathing-related interneurons of the brainstem pre-Bötzinger complex (preBötC) that putatively comprise the core respiratory rhythm generator in mammals are derived from Dbx1-expressing precursors.These results demonstrate that a single canonical interneuron class generates respiratory rhythm and contributes in a premotor capacity, whereas these functions are normally attributed to discrete populations.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Science, The College of William and Mary, Williamsburg, United States.

Show MeSH
Related in: MedlinePlus