Article Figures & Data
Figures
- Figure 1.
Transection of infraorbital nerve induced anatomic remodeling in whisker sensory pathway. A, Schematic drawing of genetic visualization of PrV2-origin lemniscal fibers. A1, Alleles of Krox-20Cre and Ai14 mouse lines. These lines were crossed. A2, Selective labeling of PrV2-origin lemniscal fibers with tdTomato. Boxed area over the left Pr5 indicates the region shown in C2. Horizontal dotted lines indicate horizontal planes shown in C1 and D. B, Experimental schedule of IONC. C, tdTomato signals in the PrV2 (C2) and on the ascending tract (C1) did not change after IONC. Scale bar, 200 µm. D, tdTomato signals in the V2 VPM of Krox20-Ai14 mice. Arrowheads indicate the affected V2 VPM. Yellow enclosures indicate automatically detected ROIs for analyses. Scale bar, 500 µm. E, Ratio of contralateral to ipsilateral tdTomato signals of the V2 VPM in fluorescent intensity and area. Values are represented as mean ± SD, 18 images from nine mice were analyzed for each group. Statistical significance was tested by two-way repeated measures ANOVA (main effect of IONC). ***p < 0.001; two-tailed. Pr5, principle trigeminal nucleus; PrV2, maxillary Pr5; V2, maxillary region; V3, mandibular region; VPM, ventral posteromedial thalamic nucleus.
- Figure 2.
IONC increased non-PrV2-origin lemniscal fiber terminals in V2 VPM related to newly recruited lemniscal fibers onto VPM neurons. A, VGluT2-immunostained V2 VPM sections of Krox20-Ai14 mice. VGluT2 is a marker of lemniscal fiber terminals in the VPM. Top, Significant increase of tdT-negative (non-PrV2-origin) VGluT2 puncta in the contralateral V2 VPM after IONC. Scale bar, 20 µm. Bottom, The soma and dendrites of V2 VPM neurons were visualized by fluoro Nissl and MAP2 stainings, respectively, in addition to VGluT2 and tdTomato (top) to examine subcellular distribution of the VGluT2 puncta in their origin-specific manner. * indicates a V2 VPM neuron; arrows indicate somatic puncta; arrowheads indicate dendritic puncta. Scale bar, 5 µm. B, Summary density of VGluT2 puncta in the V2 VPM. tdT(+) = tdTomato-positive VGluT2; tdT(-) = tdTomato-negative ones. C, IONC decreased proportion of tdT(+) VGluT2 puncta in the contralateral V2 VPM. D, Summary numbers of VGluT2 puncta on the soma (left) and proximal dendrites (right) in the contralateral V2 VPM. tdT-positive puncta decreased whereas tdT-negative ones increased both on the soma and dendrites. A marker indicates each V2 VPM neuron examined. tdT(+) = tdTomato-positive VGluT2; tdT(-) = tdTomato-negative ones. E, Correlated postoperative time course of anatomic and functional lemniscal fiber remodeling in the V2 VPM. Top: VGluT2 immunostaining. Scale bar, 20 µm. Bottom: Discrete lemniscal fiber-mediated EPSCs in a V2 VPM neuron in response to increasing electrical stimuli on the medial lemniscal fiber bundle (Takeuchi et al., 2012). F, Correlation between anatomic and functional lemniscal fiber remodeling in the contralateral V2 VPM. r, Pearson’s correlation coefficient. Broken line is the regression line. G, Experimental schedule of whisker deprivation. H, VGluT2-immunostained sections of the V2 VPM in Krox20-Ai14 mice after whisker deprivation. Scale bar, 20µm. I, Summary density of VGluT2 puncta in the contralateral V2 VPM. tdT(+) = tdTomato-positive VGluT2; tdT(-) = tdTomato-negative ones. Values are represented as mean (±SD). Three to nine mice (eight images from each mouse) were analyzed for each group in A–I. In total, 94 neurons from four mice (4-10 neurons from each image) were analyzed for each group in D. In total, 215 neurons from 91 mice and 118 images from 14 mice were analyzed for E and F. Statistical significance was tested by two-way repeated measures ANOVA (main effect of IONC or whisker deprivation; B–D and I) and Pearson’s product-moment correlation test (F). *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant; two-tailed.
- Figure 3.
IONC decreased size of PrV2-origin lemniscal fiber terminals in V2 VPM and amplitude of lemniscal fiber-derived miniature EPSCs. A, VGluT2-immunostained V2 VPM sections of Krox20-Ai14 mice. Scale bar, 5 µm. B, Cumulative probabilities of tdT-positive VGluT2 puncta in the V2 VPM after sham and IONC operations. Nine mice (eight images from each mouse) were analyzed for each group. C, Averaged traces of asynchronously released miniature EPSCs from a lemniscal fiber in the VPM. Traces of 1517 and 2356 events from 12 and 15 neurons of sham- and IONC-operated mice, respectively. D, Cumulative probabilities of lemniscal fiber-derived miniature EPSC amplitude. Statistical significance was tested by two-sample Kolmogorov-Smirnov test (B and D). ***p < 0.001; two-tailed.
- Figure 4.
IONC induced morphologic changes of PrV2-origin lemniscal fibers in V2 VPM. A, BDA, an anterograde neural tracer, was injected in the left PrV2. B, PrV2-origin lemniscal fibers in the contralateral V2 VPM. Scale bar, 10 µm. C–E, Projection images of 3-D reconstructed terminal morphology of single PrV2-origin lemniscal fibers (C1, D1, and E1) and their quantitative analyses (C2, D2, and E2). C, Scale bar, 50 µm. D, Ectopic branches were defined as branches over 50 µm away from the barycenter of boutons. E, Distances between all pairs of boutons and terminals in each PrV2-origin fiber were analyzed. Values are represented as mean ± SD. A marker indicates each fiber (C2, and D2 left). Twenty-one and 44 fibers from three mice were analyzed for sham and IONC groups, respectively. Statistical significance was tested by two-way repeated measures ANOVA (main effect of IONC; C2, and D2 left) and two-sample Kolmogorov-Smirnov test (D2 right, and E2). **p < 0.01; ***p < 0.001; two-tailed.
- Figure 5.
IONC induced lemniscal fiber innervations from non-V2 brainstem nuclei to V2 VPM. A, A solution containing CTB, an efficient neural tracer, was injected in the right V2 VPM of sham (left)- or IONC (right)-operated Krox20-Ai14 mice to retrogradely label projection neurons terminating the V2 VPM. Injection sites of analyzed mice were reconstructed as green enclosures on the brain atlas of Franklin and Paxinos (2008). Injection sites shown as pictures were depicted by thick lines. Scale bars, 400 µm. B, Retrogradely labeled neurons in the V2 and V3 regions of the Pr5 in sham (left) and IONC (right). See Materials and Methods for details regarding identification of V2 and V3 regions. CO-stained adjacent sections are also shown (B1, leftmost). Squared regions in B1 are magnified in B2. Scale bars for low (B1) and high (B2) magnifications, 400 and 100 µm, respectively. C, Retrogradely labeled neurons in the interpolar subnucleus of the spinal trigeminal nuclei (SpI), and the DCNs in sham (left) and IONC (right). CTB-immunofluorescence of squared regions is magnified in the rightmost and CO-stained adjacent sections are also shown (leftmost). Scale bars for low and high magnifications, 400 and 100 µm. Cu, cuneate nucleus; Gr, gracile nucleus. D, Summary bar graph showing numbers of retrogradely labeled neurons from the V2 VPM. Values are represented as mean ± SD. Three and four mice (four ROIs of separate sections from each animal) were examined for sham and IONC groups, respectively. Statistical significance was tested by two-way repeated measures ANOVA (main effect of IONC). ***p < 0.001; n.s., not significant; two-tailed. E, Nissl- and CO-stained adjacent brainstem sections of an IONC-operated mouse showing Cu and the caudal subnucleus of the spinal trigeminal nuclei (SpC) in the ipsilateral side of IONC. Clear cyto-architecture was retained; there is no collapse of boundaries between Cu and SpC. Mx, matrix region of the medulla. Scale bar, 400 µm.
- Figure 6.
IONC induced ectopic receptive fields of V2 VPM neurons and mechanical hypersensitivities on mandibular region of face. A, C, Recording sites located by electrical lesion made after receptive field recordings. Only recordings in the V2 VPM characterized by dense tdTomato-labeled lemniscal fiber terminals were analyzed. A representative image is shown (A), and all recording sites analyzed were reconstructed on the brain atlas of Franklin and Paxinos (2008) (C). Black circles = recording sites of maxillary receptive fields only; blue circles = recording sites of mandibular receptive fields; red circles = recording sites of nontrigeminal receptive fields; Magenta circles = recording sites of both mandibular and nontrigeminal receptive fields; multiplication signs = nonresponsive sites. Magenta brackets indicate the manually traced V2 VPM regions. Roman numerals indicate recording sites of representative recordings shown in B and D. Scale bars, 1 mm. Cx, cerebral cortex; Hip, hippocampus; VPM, ventral posteromedial thalamic nucleus. B, Representative recording of IONC-operated mouse at the recording site (iii). Red vertical line indicates the timing of cue for stimulation. Time scale, 250 ms; Scale for peristimulus time histogram, 10 spikes. D, Representative receptive fields of V2 VPM neurons. Each Roman numeral corresponds to the recording from the site indicated in C. Colored areas on the age-matched mouse traces indicate receptive fields at the recording sites. Magenta = sham; green = IONC. Body surface at which there is stimulus-locked significant increase in firing rate from basal firing is considered as the receptive field. E, Face-escape thresholds assessed by von Frey filament stimuli on whisker pad and lower jaw before and 1–15 d after IONC operation on P21. Each value was normalized to that of sham group. Twelve mice for each group were examined. Values are represented as mean ± SD. Statistical significance was tested by two-way repeated ANOVA and post hoc Tukey HSD test. *p < 0.05; **p < 0.01; n.s., not significant; two-tailed.
- Figure 7.
Long-lasting lemniscal fiber remodeling related to ectopic receptive fields of V2 VPM neurons and mechanical hypersensitivities on ectopic receptive fields. A, Experimental schedule. B, tdTomato signals in the V2 VPM of Krox20-Ai14 mice long after IONC. Arrowheads indicate the affected V2 VPM. Scale bar, 500 µm. C, VGluT2-immunostained sections of the V2 VPM. Significant increases of tdT-negative VGluT2 puncta after IONC. Scale bar, 20 µm. D, Ratios of contralateral to ipsilateral tdTomato signals of the V2 VPM in fluorescent intensity and area. Four mice (four images from each mouse) were examined for each group. E, Summary density of VGluT2 puncta in the contralateral V2 VPM. tdT(+) = tdTomato-positive VGluT2; tdT(-) = tdTomato-negative ones. F, IONC decreased proportion of tdT-positive VGluT2 puncta in the contralateral V2 VPM. Four mice (eight images from each mouse) were examined for each group in E and F. G, Recording sites located by electrical lesion made after receptive field recordings. All recordings made in the V2 VPM were analyzed and reconstructed on the brain atlas of Franklin and Paxinos (2008). Black circles = recording sites of maxillary receptive fields only; blue circles = recording sites of mandibular receptive fields; multiplication signs = nonresponsive sites. Magenta brackets indicate the V2 VPM regions. Roman numerals indicate the recording sites of representative recordings shown in H. Scale bar, 1 mm. H, Representative receptive fields of V2 VPM neurons. Each Roman numeral corresponds to the recording from the site indicated in G. Colored areas on the mouse cartoons indicate receptive fields at the recording sites. Magenta = sham; green = IONC. I, Face-escape thresholds assessed by von Frey filament stimuli on whisker pad and lower jaw before and 0.5–4.5 months after IONC operation on P21. Nerve regeneration was prevented for long-term evaluation (see Materials and Methods). Each value was normalized to that of the sham group. Nine mice were examined for each group. Values are represented as mean ± SD. Statistical significance was tested by two-way repeated measures ANOVA (main effect of IONC; D–F) and two-way repeated measures ANOVA and post hoc Tukey HSD test (I). *p < 0.05; **p < 0.01; ***p < 0.001; two-tailed.
- Figure 8.
No critical window of IONC-induced lemniscal fiber remodeling. A, IONC operation was conducted on adult Krox20-Ai14 mice. B, tdTomato signals in the V2 VPM of Krox20-Ai14 mice IONC operated in adult. Arrowheads indicate the affected thalamus. Scale bar, 500 µm. C, Ratios of contralateral to ipsilateral tdTomato signals of the V2 VPM in fluorescent intensity and area. Sixteen images from five mice were examined for each group. D, VGluT2-immunostained sections of the contralateral V2 VPM in Krox20-Ai14 mice IONC operated in adult. Scale bar, 20 µm. E, Summary density of VGluT2 puncta in the V2 VPM. tdT(+) = tdTomato-positive VGluT2; tdT(-) = tdTomato-negative ones. F, Summary proportion of tdT-positive VGluT2 in the V2 VPM. Three mice (eight images from each mouse) were examined for each group in E and F. Values are represented as mean ± SD. Statistical significance was tested by two-way repeated measures ANOVA (main effect of IONC; C, E, and F). *p < 0.05; ***p < 0.001; n.s., not significant; two-tailed.
Tables
Data structure Type of test Power
(α = 0.05)a (Fig. 1E, intensity) Normal Two-way repeated measures ANOVA 1.0000000 a (Fig. 1E, area) Normal Two-way repeated measures ANOVA 1.0000000 b [Fig. 2B, tdT(+)] Normal Two-way repeated measures ANOVA 1.0000000 c [Fig. 2B, tdT(-)] Unknown Two-way repeated measures ANOVA 1.0000000 d Normal Two-way repeated measures ANOVA 0.9786688 e (Fig. 2C) Normal Two-way repeated measures ANOVA 1.0000000 f [Fig. 2D, soma, tdT(+)] Unknown Two-way repeated measures ANOVA 1.0000000 f [Fig. 2D, dendrites, tdT(+)] Unknown Two-way repeated measures ANOVA 1.0000000 g [Fig. 2D, soma, tdT(-)] Unknown Two-way repeated measures ANOVA 1.0000000 g [Fig. 2D, dendrites, tdT(-)] Unknown Two-way repeated measures ANOVA 1.0000000 h Unknown Pearson’s χ2 test 0.8460473 i (Fig. 2F) Unknown t test for noncorrelation 0.9759325 j [Fig. 2I, tdT(+)] Normal Two-way repeated measures ANOVA 0.2974602 j [Fig. 2I, tdT(-)] Normal Two-way repeated measures ANOVA 0.1588404 j [%tdT(+)] Unknown Two-way repeated measures ANOVA 0.2390769 k Unknown Two-way repeated measures ANOVA 1.0000000 l (Fig. 3B) Unknown Kolmogorov-Smirnov two-sample test n.a.
(p = 5.5 × 10−28)m Normal Two-way repeated measures ANOVA 0.1673505 n (Fig. 3D) Unknown Kolmogorov-Smirnov two-sample test n.a.
(p = 9.0 × 10−83)o (Fig. 4C2, Bouton) Normal Two-way repeated measures ANOVA 1.0000000 o (Fig. 4C2, Terminal) Normal Two-way repeated measures ANOVA 1.0000000 o (Fig. 4C2, Branch) Normal Two-way repeated measures ANOVA 1.0000000 p (Fig. 4D2 right) Unknown Kolmogorov-Smirnov two-sample test n.a.
(p = 1.7 × 10−7)q (Fig. 4D2 left) Unknown Two-way repeated measures ANOVA 0.9999438 r (Fig. 4E2 left) Unknown Kolmogorov-Smirnov two-sample test n.a.
(p = 2.7 × 10−148)r (Fig. 4E2 right) Unknown Kolmogorov-Smirnov two-sample test n.a.
(p = 2.7 × 10−115)s (Fig. 5D, PrV2) Normal Two-way repeated measures ANOVA 1.0000000 s (Fig. 5D, PrV3) Unknown Two-way repeated measures ANOVA 1.0000000 s (Fig. 5D, V2 SpI) Normal Two-way repeated measures ANOVA 0.9465853 s (Fig. 5D, V3 SpI) Normal Two-way repeated measures ANOVA 1.0000000 s (Fig. 5D, DCN) Normal Two-way repeated measures ANOVA 1.0000000 t Unknown Two-way repeated measures ANOVA 1.0000000 u (Fig. 6E right) Normal Two-way repeated measures ANOVA 0.8840900 v (Fig. 7D, 1 m intensity) Normal Two-way repeated measures ANOVA 1.0000000 v (Fig. 7D, 1 m area) Normal Two-way repeated measures ANOVA 1.0000000 v (Fig. 7D, 3 m intensity) Normal Two-way repeated measures ANOVA 1.0000000 v (Fig. 7D, 3 m area) Normal Two-way repeated measures ANOVA 1.0000000 w Normal Two-way repeated measures ANOVA 1.0000000 x Normal Two-way repeated measures ANOVA 0.5385280 y [Fig. 7E, 3 m, tdT(+)] Normal Two-way repeated measures ANOVA 1.0000000 z [Fig. 7E, 3 m, tdT(-)] Normal Two-way repeated measures ANOVA 1.0000000 aa [Fig. 7F, 3 m, %tdT(+)] Normal Two-way repeated measures ANOVA 1.0000000 bb (Fig. 7I bottom) Normal Two-way repeated measures ANOVA 1.0000000 cc (Fig. 8C, intensity) Normal Two-way repeated measures ANOVA 1.0000000 cc (Fig. 8C, area) Normal Two-way repeated measures ANOVA 1.0000000 dd [Fig. 8E, tdT(+)] Normal Two-way repeated measures ANOVA 1.0000000 ee [Fig. 8E, tdT(-)] Normal Two-way repeated measures ANOVA 1.0000000 ff Normal Two-way repeated measures ANOVA 0.1898718 gg [Fig. 8F, contra] Normal Two-way repeated measures ANOVA 1.0000000 Each small alphabetical character indicates statistical tests labeled with a p value and the small alphabetical character in the Results section. Data normality was determined using the Lilliefors test. Nonparametric statistical analysis produced similar results. Post hoc power analysis was performed using G*Power 3 software (Faul et al., 2007). Post hoc powers of two-way repeated measures ANOVA were calculated on main effect of operation (IONC or whisker deprivation) except w and x (main effect of survival period). n.a., not applicable; p values were provided instead.
Operation RF location Corresponding brain stem nuclei Number of recordings Proportion to total mapped sites (%) Sham Whisker(s) or whisker pad PrV2, V2 SpI 39 92.9 Nonresponsive None 3 7.1 IONC Whisker(s) or whisker pad PrV2, V2 SpI 3 4.6 Lower jaw PrV3, V3 SpI 9 13.8 Chest DCN 6 9.2 Fore paw DCN 3 4.6 Hind paw DCN 1 1.5 Trunk DCN 1 1.5 Tail DCN 1 1.5 Nonresponsive None 50 76.9 Recordings were made from the right V2 VPM. All receptive fields (RFs) were found in the contralateral (left) side of the face and/or body. For the sham group, 42 recording sites were mapped from six mice (six penetrations) in total. For the IONC group, 65 recording sites were mapped from five mice (seven penetrations) in total. Note that in several cases each recording site had multiple RFs such as the lower jaw and the chest. PrV2, maxillary region of the principle trigeminal nucleus; PrV3, mandibular region of the principle trigeminal nucleus; V2 SpI, maxillary region of the interpolar subnucleus of the spinal trigeminal nuclei; V3 SpI, mandibular region of the interpolar subnucleus of the spinal trigeminal nuclei.
Operation RF location Corresponding brain stem nuclei Number of recordings Proportion to total mapped sites (%) Sham Whisker(s) or whisker pad PrV2, V2 SpI 20 95.2 Nonresponsive None 1 4.8 IONC Whisker(s) or whisker pad PrV2, V2 SpI 7 13.5 Lower jaw PrV3, V3 SpI 8 15.4 Nonresponsive None 38 73.1 Recordings were made from the right V2 VPM. All receptive fields (RFs) were found in the contralateral (left) side of the face and/or body. For the sham group, 21 recording sites were mapped from three mice (three penetrations) in total. For the IONC group, 52 recording sites were mapped from three mice (five penetrations) in total. Note that in several cases each recording site had multiple RFs such as the lower jaw and the whisker pad. PrV2, maxillary region of the principle trigeminal nucleus; PrV3, mandibular region of the principle trigeminal nucleus; V2 SpI, maxillary region of the interpolar subnucleus of the spinal trigeminal nuclei; V3 SpI, mandibular region of the interpolar subnucleus of the spinal trigeminal nuclei.
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