Variable sensitivity to noxious heat is mediated by differential expression of the CGRP gene

Abstract

Heat sensitivity shows considerable functional variability in humans and laboratory animals, and is fundamental to inflammatory and possibly neuropathic pain. In the mouse, at least, much of this variability is genetic because inbred strains differ robustly in their behavioral sensitivity to noxious heat. These strain differences are shown here to reflect differential responsiveness of primary afferent thermal nociceptors to heat stimuli. We further present convergent behavioral and electrophysiological evidence that the variable responses to noxious heat are due to strain-dependence of CGRP expression and sensitivity. Strain differences in behavioral response to noxious heat could be abolished by peripheral injection of CGRP, blockade of cutaneous and spinal CGRP receptors, or long-term inactivation of CGRP with a CGRP-binding Spiegelmer. Linkage mapping supports the contention that the genetic variant determining variable heat pain sensitivity across mouse strains affects the expression of the Calca gene that codes for CGRPalpha.

Fig. 1.

Differential sensory, neurosecretory, and transcriptional functions in primary afferent neurons of AKR and C57BL/6 mice. (A) Averaged heat responses of mechano-heat-sensitive C-fibers in the isolated skin-saphenous nerve preparation stimulated by radiant heat to the epidermis (lower trace shows stimulus profile). AKR: 8.1 spikes per stimulus ± 1.8 (SEM), threshold 44.3 ± 0.6°C; C57BL/6: 34.2 ± 0.8 spikes per stimulus, threshold 40.1 ± 0.54°C; both strain differences P < 0.01, Mann-Whitney U test. Detailed methods, further statistics, and data on mechanical sensitivity of the C-fibers are given in supporting information. (B) Stimulus-response curves of heat-induced (5 min) iCGRP release from isolated dorsal hindpaw skin. Baseline release (dotted line) was the same in both strains (152 ± 4 pg per g of skin in 5 min); all stimulated increases over baseline were significant (P < 0.01, Wilcoxon test). ^**, P < 0.01 compared to other strain, Mann-Whitney U test. (C) Responses induced by KCl (60 mM) and capsaicin (1 μM), baseline release is indicated by a dotted line; ^*, P < 0.05; ^**, P < 0.01. (D) Immunocytochemical strain comparison in lumbar DRGs. Percentage of iCGRP-staining neurons in four randomly chosen sections from each of five animals of either strain (n = 20 sections). Detailed methods and a representative photomicrograph can be found in supporting information. (E) Total iCGRP content of hairy skin; “n” refers to skin flaps from both hindpaws of 5/4 animals (^***, P < 0.001; Mann-Whitney U test). For a table relating stimulated iCGRP release to total skin content and comparing between the mouse strains, see supporting information. (F) Quantitative gene expression of Calca, coding for αCGRP (Left), and Tac1, coding for preprotachykinin-A (substance P; SP) (Right). Amounts of mRNA in lumbar DRGs of four to five animals of either strain, determined by real-time PCR quantification (TaqMan) and expressed in arbitrary units (AU) relative to the housekeeping genes cyclophilin or GAPDH. ^*, P < 0.01, Mann-Whitney U test.

Fig. 2.

Differential noxious heat sensitivity in AKR and C57BL/6 mice and its abolition by pharmacological manipulations at CGRP receptors and by CGRP inactivation. (A) Complete and reversible abolition of the AKR vs. C57BL/6 strain difference by injection of 5 μg of CGRPα into the right hindpaw; 1 μg of CGRPα produced a partial effect. Symbols represent mean ± SEM latency (s) to withdraw the paw from the noxious stimulus; n = 12-21 per strain per condition. ^*, P < 0.05 compared to vehicle-injected mice of same strain (Student's t test) and compared to PWL at time = 0; only the AKR-CGRP groups displayed a significant repeated measures effect. The left (noninjected) hindpaw was tested simultaneously; contralateral CGRP injection had no effect in either strain (data not shown). Injection of CGRPβ (5 and 10 μg) also produced no effect (data not shown). No CGRPα dose up to 20 μg produced any significant PWL changes in C57BL/6 mice (data not shown). (B) Reversible attenuation of the AKR vs. C57BL/6 strain difference in heat nociception by injection of 0.1 μg but not 0.01 μg of BIBN4096BS (BIBN) into the spinal cord. Symbols represent mean ± SEM withdrawal latency (s) from both hindpaws averaged; n = 5-8 per strain per condition. ^*, P < 0.05 compared to vehicle-treated mice of the same strain (Student's t test) and compared to PWL at time = 0; only the C57BL/6-BIBN (0.1 μg) group displayed a significant repeated measures effect. No BIBN dose up to 1.0 μg produced any significant PWL changes in AKR mice. (C) Reduced sensitivity to noxious heat in C57BL/6 mice after chronic inactivation of endogenous CGRP, and its complete reversal by exogenous CGRP administration. The radiant heat source was attenuated to adjust PWLs to be ≈8 s. The PEGylated anti-CGRP Spiegelmer NOX-504P was systemically injected (25 μg per mouse, i.p.) once daily over 6 days and caused a bilateral reduction on day 7 in PWLs as compared to vehicle injections (bar graph, means ± SEM). The subsequent CGRPα injection (5 μg, s.c.) into the left hindpaw caused a profound sensitization only in the CGRP-deprived but not vehicle-treated animals (line graph, means ± SEM). ^*, P < 0.01 (ANOVA followed by LSD test).

Fig. 3.

Reversible abolition of multiple strain differences in heat nociception by exogenous administration of CGRP into the hindpaw, and related Calca gene expressions. (A) Bars represent mean ± SEM latency (s) to withdraw the paw from the noxious stimulus before (Top; average of four baseline measurements), 5 min (Middle), and 20 min (Bottom) after injection of 5 μg CGRP. F values provided are from one-way ANOVAs performed at each time point. (B) Basal Calca gene expression in CGRP “responders” (AKR, A, DBA/2) and “nonresponders” (C57BL/6, C57BL/10, CD-1). Bars represent mean ± SEM Calca expression in lumbar (L4-L6) DRGs relative to GAPDH; n = 3 per strain. Status regarding response to CGRP injection and range of basal latencies are provided above each group of strains. Calca expression in all low-latency “responder” strains significantly exceeded that of all high-latency “nonresponder” strains (ANOVA followed by LSD test).

Fig. 4.

Location of a major gene affecting thermal nociception on the paw-withdrawal test on mid-chromosome 7. (A) The green curve shows logarithm of the odds (LOD) scores (on a Kosambi-corrected map) for linkage at successive locations (in cM) along chromosome 7 in (AKR × C57BL/6)F₂ mice, from a full-genome search for linkage. No other strongly suggestive QTLs or strong epistatic interactions were observed. The purple curve shows LOD scores in a subsequent (BALB/c × C57BL/6)F₂ intercross, genotyped only at five markers on chromosome 7. In both cases, a recessive model, yielding the best fit, is shown. Horizontal dotted lines represent “highly significant” (99.9th percentile) genome-wide LOD score thresholds for each experiment as determined by permutation analysis. Horizontal solid lines represent the 1-LOD drop-off confidence interval for each experiment. Calca (arrowhead) falls within confidence intervals in both experiments. (B) Congenic mice (A.B6-Tyr⁺/J) featuring a C57BL/6-derived genomic segment on mid-chromosome 7 placed on an A strain genetic background display reduced basal PWLs and no CGRP-induced hyperalgesia. Symbols represent mean ± SEM. (s) PWLs before and after CGRP injection at time = 0, n = 6-10 per genotype. Vehicle injection produced no changes in any genotype (data not shown). Shown are data from female mice; male congenic mice displayed a smaller (but still statistically significant) reduction in PWLs.