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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.

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

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Detection of Dbx1 preBötC neurons.(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.DOI:http://dx.doi.org/10.7554/eLife.03427.004
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fig1s1: Detection of Dbx1 preBötC neurons.(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.DOI:http://dx.doi.org/10.7554/eLife.03427.004

Mentions: Dbx1 neurons were detected and mapped within the preBötC, and then laser ablated individually, in sequence, while monitoring respiratory network functionality via XII motor nerve output. Experiments began with an ‘initialization phase’ that defined the domain for detection and ablation, which was bilateral. We used a Dbx1 Cre-driver line (Dbx1CreERT2) coupled with floxed reporter mice (Gt(ROSA)26Sorflox-stop-tdTomato) to locate Dbx1 neurons via fluorescence. Viewed in the transverse plane of slices that expose the preBötC at their surface (i.e., preBötC-surface slices), Dbx1 neurons form a bilaterally symmetrical V-shape starting dorsally at the border of the XII motor nuclei and continuing ventrolaterally to the preBötC (Figure 1A and Figure 1—figure supplement 1A). Dorsally, the preBötC adjoins the semi-compact division of the nucleus ambiguus (scNA); the ventral border of the preBötC is orthogonal to the dorsal boundary of the principal sub-nucleus of the inferior olive (IOPloop) (Ruangkittisakul et al., 2011, 2014). These spatial relationships visible in bright field or epifluorescence allow us to pinpoint the preBötC (Figure 1A). At the cellular level, identifying putative rhythmogenic neurons on the basis of fluorescent protein expression alone is acceptable because the overwhelming majority of Dbx1 preBötC neurons are inspiratory (e.g., Figure 1B) (Picardo et al., 2013).10.7554/eLife.03427.003Figure 1.Dbx1 preBötC neurons.


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 preBötC neurons.(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.DOI:http://dx.doi.org/10.7554/eLife.03427.004
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fig1s1: Detection of Dbx1 preBötC neurons.(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.DOI:http://dx.doi.org/10.7554/eLife.03427.004
Mentions: Dbx1 neurons were detected and mapped within the preBötC, and then laser ablated individually, in sequence, while monitoring respiratory network functionality via XII motor nerve output. Experiments began with an ‘initialization phase’ that defined the domain for detection and ablation, which was bilateral. We used a Dbx1 Cre-driver line (Dbx1CreERT2) coupled with floxed reporter mice (Gt(ROSA)26Sorflox-stop-tdTomato) to locate Dbx1 neurons via fluorescence. Viewed in the transverse plane of slices that expose the preBötC at their surface (i.e., preBötC-surface slices), Dbx1 neurons form a bilaterally symmetrical V-shape starting dorsally at the border of the XII motor nuclei and continuing ventrolaterally to the preBötC (Figure 1A and Figure 1—figure supplement 1A). Dorsally, the preBötC adjoins the semi-compact division of the nucleus ambiguus (scNA); the ventral border of the preBötC is orthogonal to the dorsal boundary of the principal sub-nucleus of the inferior olive (IOPloop) (Ruangkittisakul et al., 2011, 2014). These spatial relationships visible in bright field or epifluorescence allow us to pinpoint the preBötC (Figure 1A). At the cellular level, identifying putative rhythmogenic neurons on the basis of fluorescent protein expression alone is acceptable because the overwhelming majority of Dbx1 preBötC neurons are inspiratory (e.g., Figure 1B) (Picardo et al., 2013).10.7554/eLife.03427.003Figure 1.Dbx1 preBötC neurons.

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