Article Figures & Data
Figures
- Figure 1.
Protruder and retractor hypoglossal MNs in neonatal brainstem slices. A, Sagittal MRI of a neonatal mouse pup (courtesy G. A. Johnson, with permission) showing the tongue (blue hatching) and sites of fluorescent-conjugated cholera toxin injections to retrogradely label protruder (CTB555: red) and retractor (CTB488: green) MNs. B, Neonatal mouse tongue showing the location of protruder and retractor muscles. The base of the tongue is toward the top, the tip toward the bottom of the panel, and the dotted line marks the midline of the dorsal surface. The red shading near the tongue base indicates the general location of protruder muscles, and the green shading on the midlateral aspect indicates the general location of retractor muscles. C, Confocal image of a transverse coronal brainstem section with retrogradely labeled protruder CNXII MNs (red) and retractor (green) CNXII MNs. The ventral CNXII has a higher density of protruder MNs; the dorsal subdivision has a higher density of retractor MNs. Scale bar: 70 μm. D: dorsal, L: lateral.
- Figure 2.
Differences in LgDel protruder and retractor MN spontaneous activity. A, Representative traces of spontaneous AP activity of protruder CNXII MNs (pMNs, red) from neonatal WT (left) and LgDel (right) pups. B, Spontaneous AP activity of retractor CNXII MNs (rMNs, green) from neonatal WT (left) and LgDel (right) pups. C, AP frequency was significantly higher in LgDel rMNs compared with both LgDel pMNs and WT rMNs as shown in C (LgDel rMN (3.34 ± 0.7 Hz, n = 10) compared with LgDel pMN (0.84 ± 0.2 Hz, n = 9, p = 0.011 unpaired Student’s t test and WT rMN (1.34 ± 0.4 Hz, n = 9, p = 0.0398 unpaired Student’s t test). The pMN AP firing was statistically higher from WT (2.56 ± 0.7 Hz, n = 10) than LgDel (0.84 ± 0.2 Hz, n = 10, p = 0.0434, unpaired Student’s t test). D, Typical GABAergic IPSCs events in WT (top) and LgDel (bottom) neonatal rMNs. E, Scatter plots show IPSC frequency was significantly greater in WT (5.82 ± 0.8 Hz) versus LgDel rMNs (2.70 ± 0.7 Hz, unpaired Students’ t test, df = 28, p = 0.0099). For all graphs, data expressed as mean ± SEM; statistical comparisons are noted as *p < 0.05, **p < 0.01.
- Figure 3.
Spontaneous fictive respiratory bursts in WT and LgDel CNXII slices. A, A schematic of a lower medulla section taken at the level of CNXII showing the nucleus (CNXII, box) ventral to the fourth ventricle (IV), segregated populations of pMNs (red) and rMNs (green), CNXII axons extending ventrally exit as CNXII rootlets (asterisks), and adjacent cranial nuclei including the nucleus of the solitary tract (nST), the spinal trigeminal nucleus (spCNV), and nucleus ambiguous (nA). A’, A confocal image of CNXII showing the segregation of the pMNs (red), rMNs (green), and CNXII axons extending ventrally (asterisks). Scale bar: 50 μm. D: dorsal, M: medial. A’’, Spontaneous inspiratory bursting activity recorded from hypoglossal nerve rootlets using a suction electrode (top), and represented as an integrated electrophysiological signal (right). B, Scatterplot of spontaneous inspiratory related bursting frequency recorded from WT and LgDel slices. C, Scatter plot of burst durations from WT and LgDel slices. Burst frequency and duration were not significantly different in the two genotypes; WT (n = 25) and LgDel (n = 10) animals, p > 0.05, unpaired two-tailed Student’s t test.
- Figure 4.
Delayed inspiratory burst-related LgDel protruder MN activity. A, A confocal image of a pMN labeled by intracellular injection with biocytin, after identification using retrograde tracing with CTB-Alexa Fluor 555 (red). The asterisk indicates the single axon extending from this pMN to exit in the ventromedial region of the posterior medulla. Scale bar: 100 μm. V: ventral, L: lateral, IV: fourth ventricle. B, Representative traces showing the relationship between pMN AP activity (top traces) and fictive respiratory bursts recorded at CNXII rootlets (bottom traces) in WT versus LgDel neonatal mice. C, Histograms of AP firing in WT (white bars) and LgDel (black bars) pMNs before, during, and after inspiratory bursts. WT protruder MN AP firing increased in the first 1-s postburst interval (prior: 1.52 ± 0.5 Hz, first second 3.22 ± 0.7 Hz, one-way repeated measures ANOVA with Dunnett’s multiple comparison post hoc test, p = 0.0014, DFn = 4, DFd = 49, F = 5.538), whereas LgDel protruder MNs AP firing increased in the second, but not first, 1-s postburst interval (prior: 0.7 ± 0.1 Hz, first second 0.8 ± 0.1 Hz, second second 2.9 ± 0.7 Hz, one-way repeated measures ANOVA with Dunnett’s multiple comparison post hoc test, p = 0.0009, DFn = 4, DFd = 39, F = 6.408). The AP firing was higher in WT (3.22 ± 0.7 Hz) than in LgDel (0.8 ± 0.1 Hz) at the first 1-s postburst interval (two-way ANOVA with Bonferroni post hoc test, p < 0.05). D, Histograms of EPSC frequency in WT and LgDel pMNs before, during, and after inspiratory bursts. EPSC frequency from WT and LgDel pups increases significantly during the first 1-s postinspiratory burst interval (WT: prior: 3.1 ± 0.5 Hz, first second 5.9 ± 0.8 Hz, second second 3.3 ± 0.5 Hz, one-way repeated measures ANOVA with Dunnett’s multiple comparison post hoc test, DFn = 4, DFd = 59, F = 15.76, p < 0.0001; LgDel protruder MNs: prior: 3.97 ± 0.5 Hz, first second 7.37 ± 1.3 Hz, second second 6.7 ± 1.3 Hz, one-way repeated measures ANOVA with Dunnett’s multiple comparison post hoc test, DFn = 4, DFd = 44, F = 6.909, p = 0.0004); however, LgDel pMN EPSC frequency continued at significantly elevated levels through the second 1-s postburst interval compared with WT (6.7 ± 1.3 Hz in LgDel vs 3.3 ± 0.5 Hz in WT, p < 0.05, two-way ANOVA with Bonferroni post hoc test). E, GABAergic IPSC frequency histograms of WT and LgDel protruder MNs. IPSC frequency increased significantly during the first 1-s postburst interval in WT (prior: 3.2 ± 0.5 Hz, first second 5.3 ± 0.8 Hz, second second 4.0 ± 0.7 Hz, one-way repeated measures ANOVA with Dunnett’s multiple comparison post hoc test, DFn = 4, DFd = 44, F = 13.55, p < 0.0001) and LgDel pups (prior: 3.7 ± 0.5 Hz, first second 5.8 ± 0.6 Hz, second second 4.7 ± 0.6 Hz, one-way repeated measures ANOVA with Dunnett’s multiple comparison post hoc test, DFn = 4, DFd = 49, F = 16.96, p < 0.0001). Data expressed as mean ± SEM; Comparison between WT and LgDel group; *p < 0.05. Comparison among different time points within same group; +p < 0.05, ++p < 0.01, +++p < 0.001.
- Figure 5.
Increased inspiratory burst-related activity in LgDel retractor CNXII MNs. A, A confocal image of a rMN labeled by intracellular injection with a biocytin (fluorophore wavelength), after identification using retrograde tracing with CTB-Alexa Fluor 488 (green). The asterisk indicates the single axon extending from this rMN to exit in the ventromedial region of the posterior medulla. Scale bar: 50 μm. V: ventral, L: lateral, IV: fourth ventricle. B, Representative traces of rMN AP activity (top traces) from WT and LgDel mouse pups in response to fictive inspiratory bursts (bottom traces). C, Histograms of rMN AP frequency in WT (white bars) and LgDel (black bars). rMN AP frequency differs through five distinct epochs before, during, and after inspiratory bursts. In LgDel rMNs AP activity increased from 3.0 ± 0.9 Hz before bursts to 7.0 ± 1.8 Hz in the first 1-s interval following burst initiation (one-way repeated measures ANOVA with Dunnett’s multiple comparison post hoc test, DFn = 4, DFd = 34, F = 3.17, p = 0.0317). This increase was significantly greater than the increased AP frequency in WT rMNs: (WT 1.8 ± 0.2 Hz; LgDel 7.0 ± 1.8 Hz, two-tailed unpaired Student’s t test p = 0.036. DFn = 6, DFd = 7). D, Histograms of EPSC frequency in WT and LgDel pMNs before, during, and after inspiratory bursts. LgDel retractor EPSC frequency increased significantly during the first (11.6 ± 1.5 Hz) and second 1-s postburst intervals (8.8 ± 1.3 Hz) compared with levels before the burst (6.1 ± 0.8 Hz, one-way RM ANOVA DFn = 4, DFd = 44, F = 8.392, p < 0.0001, with Dunnett’s multiple comparison test). LgDel rMN EPSC frequency was greater than that in WT for each of the three 1-s postburst intervals (two-way ANOVA, DFn = 4, DFd = 100, F = 6.3 with Bonferroni multiple comparisons: first second 6.9 ± 1.2 Hz in WT, 11.6 ± 1.5 Hz in LgDel; p = 0.04; second second 3.7 ± 0.8 Hz in WT, 8.8 ± 1.3 Hz in LgDel; p = 0.006; third second 2.8 ± 0.7 Hz, WT, 7.1 ± 1.1 Hz, LgDel; p = 0.007). E, Histograms of GABAergic IPSC frequency in WT and LgDel pMNs before, during, and after inspiratory bursts. No significant change in GABAergic IPSC frequency in LgDel rMNs over the entire duration of the burst (DFn = 4, DFd = 39, F = 2.328, p = 0.0807, one-way ANOVA). WT rMNs GABAergic IPSC frequency was increased significantly from 5.6 ± 1.0 Hz (before the burst) to 12.4 ± 2.5 Hz (during the first 1-s postburst interval, one-way RM ANOVA with Dunnett’s multiple comparison test, DFn = 4, DFd = 39, F = 8.596, p = 0.0001). The rMN IPSC frequency was greater in WT than LgDEl (12.4 ± 2.5 Hz in WT, 5.5 ± 1.9 Hz, two-way ANOVA with Bonferroni multiple comparisons, p = 0.0270, F = 2.92, DFn = 4, DFd = 70). Data expressed as mean ± s.e.m; Comparison between WT and LgDel group *p < 0.05 and **p < 0.01. Comparison among different time points within same group +p < 0.05, +++p < 0.001.
- Figure 6.
Changes in retractor CNXII MNs cell soma and dendritic arborization in the hypoglossal nucleus of LgDel mouse pups. A, B, Representative confocal image renderings of a CNXII pMN (left) and rMN (right) after patch clamp recording followed by intracellular-labeled cell dendritic structure and soma volume in WT (pMN, left) or LgDel (rMN, right). The color coding illustrating the size of cell body volumes; V, ventral, L, lateral. Scale bar: 30 μm. C, Scholl analyses of CNXII pMN (left) and rMN (right) dendritic branching showing statistic difference only in rMN but not in pMN, the total numbers of dendritic intersections at 50- and 90-μm radii were significantly greater in LgDel than WT retractor MNs; at 50 μm: 5.57 ± 0.4 in WT, 10.29 ± 1.4 in LgDel, t = 3.339, p < 0.05; at 90 μm: WT 5.42 ± 1.9, LgDel 10.58 ± 2.1, t = 3.642, p < 0.05, two-way RM ANOVA with Bonferroni post hoc tests. Data presented as Mean ± SED; *p < 0.05.
Movies
- Movie 1.
This video shows a detailed 3-D rendering of retrogradely labeled hypoglossal protruder motorneurons (in red) and retractor motorneurons (in green). Protruders were mainly in the rostral (anterior) and ventral parts of the hypoglossal nucleus, whereas retractors were localized more to the posterior (caudal) and dorsal areas within the XII nucleus.
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