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. 2018 Mar 21;7:e32036. doi: 10.7554/eLife.32036

Figure 5. Imiquimod’s effects in the mouse.

(A) Proportions of IMQ+/AITC+ and AITC+ only DRG neurons observed in calcium imaging experiments (n = 83 IMQ+/AITC+ neurons, n = 781 AITC+ only DRG neurons). (B, C) Representative traces from calcium imaging experiments on dissociated mouse DRG neurons from WT (B) (n = 15 neurons) and Trpa1-/- (C) (n = 23 neurons) mice. (A–C), 100 μM IMQ and 100 μM AITC used. (D) Quantification of scratching bouts in WT and Trpa1-/- mice following IMQ (10 μg) injections. (E, F) Representative traces from calcium imaging experiments in which 100 μM IMQ, 100 μM CQ, 100 μM DC, and 100 μM AITC were applied. IMQ+/CQ+DCA+/AITC+ (e) and AITC+ only (f) neurons are shown. (G) Quantification of the IMQ+ neuronal populations from the experiments shown in (E, F) (n = 27 IMQ+/CQ+DCA+/AITC+ neurons, 7 IMQ+/AITC+ neurons). (H) Comparison of maximum ΔF/F between IMQ+/AITC+ and AITC+ only. 100 μM IMQ, 10 μM AITC used. All calcium imaging experiments performed on dissociated mouse DRG neurons. (I) Quantification of scratching bouts in WT mice following injections of varying AITC concentrations. *p<0.05, **p<0.01, ***p<0.001, Student’s t-test. Bars are expressed as mean + s.e.m.

Figure 5.

Figure 5—figure supplement 1. Calcium imaging of discrete neuronal subpopulations in the mouse.

Figure 5—figure supplement 1.

(A, B) Representative traces from calcium imaging experiments featuring dissociated mouse DRG neurons sequentially bathed in 100 μM IMQ and 10 μM AITC. IMQ+/AITC+ (A) and AITC+ only (B) neurons were both identified. As shown in (B), many of the AITC+ only neurons peaked at lower ΔF/F values than the IMQ+/AITC+ neurons. (C) Within the IMQ+/AITC+ population (n = 37 neurons), 100 μM IMQ was demonstrated to be a weaker stimulus than 10 μM AITC. (D, E) Representative traces of calcium imaging experiments where DRG neurons were stimulated with 100 μM IMQ and 100 nM CAPS. Two main functional subpopulations were identified--one that responded to both IMQ and CAPS (D), and another that responded only to 100 nM CAPS (E). Interestingly, many of the CAPS+ only cells peaked at lower ΔF/F values during the CAPS stimulus than the neurons that responded to both IMQ and CAPS. (F) Comparison of maximum ΔF/F between IMQ+/CAPS+ and CAPS+ only neurons. (G) Quantification of scratching bouts in WT and Trpa1-/- mice following IMQ injections. (H) Representative traces from from one calcium imaging experiment in which 100 μM loxoribine was applied to dissociated DRG neurons. As shown, loxoribine did not elicit any fluorescence changes in DRG neurons (n = 51), whereas 100 μM IMQ and 100 μM AITC elicited robust intracellular calcium flux. (I) Increasing the concentration of AITC activates greater proportions of DRG neurons (71/752, 379/1107, and 864/1671 neurons for the 5 μM, 10 μM, and μM AITC experiments). (J) IMQ+ neuron populations are enriched within populations that respond to lower concentrations of AITC (26/71 5 μM AITC+ neurons, 37/220 10 μM AITC+ neurons, 83/864 100 μM AITC+ neurons). Fluorescence intensity is expressed as ΔF/F using the 340/380 ratio values. Bars represented as means + s.e.m., Student’s t-test. *p<0.05, **p<0.01, ***p<0.001.