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Comparative Study
. 2007 Mar;8(3):263-72.
doi: 10.1016/j.jpain.2006.09.005. Epub 2006 Nov 16.

Critical evaluation of the colocalization between calcitonin gene-related peptide, substance P, transient receptor potential vanilloid subfamily type 1 immunoreactivities, and isolectin B4 binding in primary afferent neurons of the rat and mouse

Theodore J Price  1 , Christopher M Flores
Affiliations
Comparative Study

Critical evaluation of the colocalization between calcitonin gene-related peptide, substance P, transient receptor potential vanilloid subfamily type 1 immunoreactivities, and isolectin B4 binding in primary afferent neurons of the rat and mouse

Theodore J Price et al. J Pain. 2007 Mar.

Abstract

Calcitonin gene-related peptide (CGRP) and/or substance P (SP) immunoreactivity as well as isolectin B(4) (IB(4)) binding are commonly used to define peptidergic and non-peptidergic nociceptor populations, respectively. Although this demarcation is well supported in the mouse, there is accumulating evidence to suggest it is not so in the rat. Hence, this investigation was undertaken to evaluate and quantify the colocalization of the neuropeptides CGRP and SP with IB(4) binding sites and the transient receptor potential vanilloid subfamily type 1 (TRPV1) channel and to compare this colocalization between trigeminal (TG) and dorsal root ganglia (DRG) in adult rats. These findings illustrate that there is a substantial overlap ( approximately 45% in the DRG and approximately 30% in the TG) between peptidergic neurons (ie, CGRP- and SP-expressing) and neurons that bind IB(4) in rat sensory ganglia. However, there were also significant differences in the colocalization of these markers between the DRG and TG. For instance, in the DRG, significantly more CGRP-immunoreactive neurons also expressed IB(4) binding sites (44.5%) compared with the TG (27.5%). In contrast, significantly fewer CGRP-immunoreactive neurons in the DRG colocalized TRPV1 immunoreactivity (49.2%) compared with the TG (70%). Moreover, we directly assessed the colocalization of CGRP and IB(4) in the TG of rats and mice using a CGRP antibody that recognizes this peptide in both species. Thus, whereas only an approximately 10% overlap was observed in TG neurons of mouse, significantly greater overlap (approximately 35%) was observed in those of rat.

Perspective: These data indicate that in adult rat sensory ganglia, there is not a clear distinction between the peptidergic and non-peptidergic nociceptor subclasses as a function of IB(4) binding. Furthermore, there are significant differences between the TG and DRG in the degree to which commonly utilized nociceptive neuronal markers are co-expressed. Taken together, the present findings dictate prudence when extrapolating experimental conclusions about the neurochemical classification of neurons between sensory ganglia or between species, including humans.

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Figures

Figure 1
Figure 1. Representative photomicgraphs of double labeling histochemistry
Images illustrate colocalization of analytes in DRG (left panels) and TG (right panels) neurons. The immunolabel is shown in the appropriate color on each panel. For example, in the top-left panel, CGRP-immunoreactive neurons are in green and TRPV1-immunoreactive neurons are in red. Co-labeled neurons appear yellow following the overlay of green and red single-labeled images. Upward arrows demonstrate single-labeled neurons, and downward arrows demonstrate double-labeled neurons.
Figure 2
Figure 2. Percent colocalization of neuronal markers in DRG and TG
The percent colocalization between neurons that were immunoreactive for the first parameter which also were immunoreactive for the second parameter are shown (i.e., column 1 depicts the percentage of CGRP-immunoreactive neurons that also contained IB4 labeling). DRG vs. TG: ★ p<0.05, ★★ p<0.01, two-way ANOVA with Bonferroni post-test).
Figure 3
Figure 3. Comparison of diameters for CGRP-, TRPV1- and SP-immunoreactive and IB4 –binding neurons in DRG and TG
Neuron diameter profiles are shown for each of the analytes used in this study in both DRG (open squares) and TG (closed squares), with neuron diameter plotted on the y-axis in μm. Lines illustrate the mean for each profile. Profiles for CGRP, TRPV1, IB4 and SP from DRG and TG were compared by two-way ANOVA with Bonferroni post-test between the two sensory ganglia. DRG vs. TG: ★★★ p<0.001. At least 200 neurons were measured per condition.
Figure 4
Figure 4. Comparison of neuron diameter profiles between single and double-labeled neurons for each analyte in DRG and TG
Neuron diameter profiles for single-labeled neurons for each analyte are shown with neuron diameter profiles for double-labeled neurons that contained the same analyte in both TG and DRG. Neuron diameter is plotted on the y-axis in μm. Lines illustrate the mean for each profile. A) CGRP alone and CGRP + IB4 or CGRP + TRPV1; B) SP alone and SP + IB4 or SP + TRPV1; C) IB4 alone and IB4 + CGRP, IB4 + SP or IB4 + TRPV1; D) TRPV1 alone and TRPV1 + CGRP, TRPV1 + SP or TRPV1 + IB4. Comparisons were made by one-way ANOVA with Dunnet’s post-test between co-expressing populations and overall populations (left-most data set in each panel): * p<0.5, ** p<0.01.
Figure 5
Figure 5. Comparison of the colocalization of CGRP immunoreactivity and IB4 binding in the TG of rat and mouse
Upper panels show representative photomicrographs of colocalization between CGRP immunoreactivity and IB4 binding sites in the rat and mouse. Vertical arrows show colocalization while horizontal arrows show single-labeled neurons. Scale bar = 20 μm. A) The percent colocalization between CGRP and IB4-binding sites is shown for rat and mouse TG neurons, *** p<0.001, two-way ANOVA, Bonferroni post-test. Total percentage of neurons exhibiting IB4-binding (B) and CGRP immunoreactivity (C) in mouse vs. rat TG, ** p<0.01, unpaired t-test.
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