Article Figures & Data
Figures
- Figure 1.
IB4 intensity versus cell size. A–D, Staining intensity of IB4 binding versus cell size in control (non-dye-injected) (A, B) and dye-injected, identified (C, D) DRG neurons. A, B, All neuronal profiles with visible nuclei in sections of three L5 DRGs (one from each of three rats) were measured. Relative intensity of IB4 binding is plotted against soma size (A; cross-sectional area) and as size distribution histograms (B). B, Cross-hatched histograms of all neurons are superimposed by gray histograms of all IB4+ neurons (intensity ≥20%), and superimposed in black are histograms of strongly IB4+ neurons (intensity ≥40%). The inset histogram a shows all IB4− (intensity <20%), and histogram b replicates the gray histogram in B to show all IB4+ (intensity ≥20%) neurons for ease of comparison with a. C, D, Relative intensity of IB4 binding versus cell size in dye-injected, physiologically identified neurons is shown, with overall layout similar to A and B. C, Nociceptors are indicated with filled circles and LTMs with open circles. NOC, Nociceptive neurons; CUNR, C-fiber unresponsive neurons; +ve, positive; -ve, negative. D, Neurons are subdivided according to their dorsal root CVs. Histogram shading in D is as for B. C, There was a significant correlation between the cross-sectional area and IB4-binding intensity in all dye-injected neurons studied (n = 64, Aα/β unresponsive excluded; p < 0.0001; r2 = 0.37) as well as in all nociceptor-type units (n = 38; p = 0.0005; r2 = 0.29) and all LTMs (n = 26, p < 0.01, r2 = 0.27). Vertical dotted lines indicate boundaries between small, medium, and large neurons. Horizontal dotted lines in A and C indicate 20 and 40% borderlines between negative (<20%), weakly positive (20–40%), and strongly positive (>40%) neurons.
- Figure 2.
A, Intensity of IB4 binding in relation to sensory properties and CV. Medians are shown with fine horizontal lines. A-fiber unresponsive units are excluded. NOC, Nociceptive neurons; UNR, C-fiber unresponsive neurons; LTM, low-threshold mechanoreceptive neurons; F/G, field or guard hair neurons; RA, rapidly adapting LTM neurons; SA, slowly adapting LTMs; MS, muscle spindle afferents; +ve, positive; -ve, negative. The dotted lines from the y-axis are as described for Figure 1. Horizontal lines above the columns indicate statistical tests (dotted, Wilcoxon ranking test; solid, Kruskal–Wallis test) between column medians. There was no significant difference between the C-nociceptive and C-unresponsive groups or between the four Aα/β-LTM groups (Kruskal–Wallis test). These were therefore combined to create C-nociceptor-type and Aα/β-LTM groups, respectively. C-nociceptor-type units were compared using a Kruskal–Wallis test with all other groups except C-LTM (too few data). Levels of significance are shown above the lines linking appropriate groups: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001. B, The inset shows the data for C-fiber nociceptors divided into subgroups defined by receptive properties and receptive field depth in the tissues. MC, C-mechano-cold; PM, C-polymodal; MH, C-mechano-heat; Sup, superficial; Derm, dermal; Sub, subcutaneous. Open circles around data points indicate which units showed spontaneous (Spont)/ongoing firing. C, An example of a dye-injected C-fiber unresponsive nociceptor-type neuron (top) and the same neuron after immunocytochemistry show strong IB4-binding intensity.
- Figure 3.
TrkA and IB4 colocalization in identified DRG neurons. Immunoreactivity for trkA and IB4 binding measured on different sections through the same dye-injected neurons. A, B, The relationship between relative intensity of IB4-binding (y-axis) and trkA (x-axis) immunoreactivity on different sections of the same C-fiber neurons (A) and Aδ-LTM units (B). From each axis, the solid lines indicate the 20% (positive/negative) borderline and the dotted lines indicate the 40% (weakly positive/strongly positive) borderline. For all C-fiber neurons positive (>20%) for both (5 nociceptive and 3 unresponsive units), there was a significant negative linear correlation between the relative intensity of the two markers (p < 0.05; r2 = 0.62; n = 8). +ve, positive; -ve, negative. C, Photomicrographs of three representative neurons to show IB4 binding and trkA staining on two different sections. Arrows indicate dye-injected profiles before immunocytochemistry (columns 1 and 3) and after immunocytochemistry (columns 2 and 4). Sensory receptive properties and conduction velocity are given on column 1 image; percentage relative staining intensity is also shown (columns 2 and 4). The scale bar (top left image) applies to all photomicrographs. MC, C-mechano-cold; NOC, nociceptive; HTM, high threshold mechanoreceptor.
- Figure 4.
IB4 intensity versus CV. Relationship between IB4-binding immunointensity and CV in identified DRG neurons. IB4 staining for C- (<0.8 m/s) and A- (>1.5 m/s) fiber neurons are shown separately. A-fiber unresponsive units are excluded. The vertical dotted lines from the x-axis indicate the upper border of CV for C-fibers (0.8 ms), C/Aδ- (1.5 m/s), and Aδ-fibers (6.5 m/s). Regression lines, p values, and r2 values are given for significant linear correlations. The dotted lines from the y-axis and symbols are as in Figure 2. NOC, Nociceptive neurons; LTM, low-threshold mechanoreceptive; C UNR, C-fiber unresponsive neurons; +ve, positive; -ve, negative.
- Figure 5.
Electrophysiology and Na+ channel expression in IB4+ and IB4− C-fiber neurons. A, B, Examples of typical somatic APs evoked by dorsal root stimulation in an IB4− C-nociceptor (A), and an IB4+ C-nociceptor (B). derm, Dermal. C–H, Comparison of electrophysiological properties (C–F) and relative intensities of two TTX-resistant Na+ channels (G, H) between IB4− and IB4+ C-fiber neurons (CV, <0.8 m/s). C, AP duration at base; D, AP rise time; E, CV; F, Em; G, Nav1.9 relative intensity; H, Nav1.8 relative intensity. AP duration at base (AP durn. base; C) and AP rise time (D) are plotted only in neurons with overshooting somatic AP and membrane potential more negative than, or equal to, −40 mV. Mann–Whitney U tests were performed to compare the median values of each plotted variable between all of the IB4+ (C-fiber-nociceptor type, nociceptive and unresponsive) units and all of the IB4− units together (nociceptor type plus C-LTM) or all C-fiber IB4− nociceptor-type units. Where a significance was found, asterisks indicate significance levels: *p < 0.05, **p < 0.01. Open symbols indicate C-LTMs; solid symbols indicate C-fiber nociceptor-type units. LTM, Low-threshold mechanoreceptors; NOC, C-nociceptor-type neurons.
- Figure 6.
IB4 intensity versus electrophysiology and Na+ channel expression in C-neurons. A–D, IB4-binding intensity in C-fiber DRG neurons in relation to membrane properties. A, AP duration; B, AP rise time (RT); C, AP height; D, Em. E, F, IB4-binding intensity plotted against Na+ channel expression. E, Nav1.9 intensity; F, Nav1.8 intensity. The criteria for accepting neurons for AP variable analysis are as in Figure 5. NOC/NOCI, nociceptors; UNR, unresponsive; NOC-TYPE, nociceptors and unresponsive units altogether. Where a significant linear correlation exists, regression lines, p values, and r2 values are given for significant linear correlations. The dotted lines from the y-axis and symbols are as in Figure 2.
Tables
CV Sensory receptor type All cells Relative immunointensity IB4+ weak (20–40%) IB4+ strong (≥ 40%) Total IB4+ (≥ 20%) N Median % N % cells n % cells N % cells C NOCI 18 45 3 17 10 55 13 72 UNR 14 55 3 21 7 50 10 71 LTM 2 7 0 0 0 0 Total 34 42 6 18 17 50 23 68 Aδ NOCI 12 5.7 0 0 0 0 0 0 LTM 6 28 3 50 0 0 3 50 Total 18 9 3 17 0 0 3 17 Aα/β NOCI 21 4 0 0 0 0 0 0 LTM 44 0.4 0 0 0 0 0 0 Total 65 1.6 0 0 0 0 0 0 -
Median immunointensity in all neurons is shown regardless of whether they were IB4+ or IB4− (column 3). Numbers and percentages of neurons in different groups, defined by CV and sensory properties (columns 1 and 2), which showed weak positive (20–40%) or strongly positive (≥40%) or all positive (≥20%) immunostaining for IB4 binding are shown.
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Types IB4 CV Em Action potential Nav1.9 Nav1.8 n m/s n mV n Base (ms) RT (ms) FT (ms) Ht (mV) Over (mV) n % rel intens n % rel intens All + 24 0.39 13 57.9 12 6.5 2.1 4.01 77 21 13 71 12 46 − 11 0.52* 10 47.6* 5 3* 1.04** 1.92† 61 6.4 7 17* 7 49 LTMs − 2 0.48 2 47.6 2 1.4 0.67 0.72 52 4.5 1 19 1 31 Noci type + 24 0.39 13 57.9 12 6.5 2.1 4.01 77 21 13 71 12 46 − 9 0.59* 8 47.9† 3 3.5 1.35* 2.18 79 29 6 17* 6 50 -
Median values for IB4+ and IB4− C-fiber neurons for CV, Em, and AP variables. AP variables included base (AP duration at base), RT (rise time), FT (fall time), Ht (AP height), and Over (AP overshoot). The values for All IB4+ and for C-nociceptor-type IB4+ neurons are the same, because all IB4+ C-fiber neurons were nociceptor type. Ems were included only if they were more negative than or equal to −40 mV. AP variable values were from neurons with Em of at least −40 mV and an overshooting AP. Statistical tests were Mann–Whitney U tests between all IB4+ and all IB4− neurons (asterisks in All IB4− row) and between IB4+ and IB4− C-nociceptor-type neurons (asterisks in C-nociceptor type IB4− row).
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↵*p < 0.05;
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↵**p < 0.01;
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↵†p = 0.05–0.1. rel intens, Relative intensity. For distribution of values, see Fig. 5.
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