Figure 5. ERα activation is required for potentiation of calcium response in IB4+ small female DRG neurons by low concentration of ryanodine.

(a) Recordings of [Ca²⁺]_i transients in IB4+ and IB4− small DRG neurons, incubated without (left panel) or with (middle and right panels) β-estradiol (100 nM). Ryanodine (2 nM) was applied for 10 min when [Ca²⁺]_i returned to baseline after the first application of caffeine (5 mM). After that, caffeine administration was repeated. Whereas no response was produced by ryanodine itself, a significant potentiation of the response to caffeine after ryanodine application occurred, exclusively in IB4+ neurons and predominantly in cultures incubated with β-estradiol; (b) Recordings of calcium transients induced by two subsequent applications of caffeine (5 mM) in the absence of ryanodine in IB4+ small DRG neurons incubated without (left panel) or with (right panel) β-estradiol; (c) Pooled relative changes in amplitude of the second response to caffeine without ryanodine application, compared as percentage of the first response, in cultures incubated with β-estradiol or vehicle (*p < 0.05, two-tailed unpaired Student’s t-test with Welch’s correction: t₁₄ = 2.56, p = 0.02); (d) Pooled relative changes in amplitude of the response to caffeine after ryanodine application, compared as percentage of the response before ryanodine application, as described in (a). Potentiated (above the 8% cut-off) and non-potentiated neuron activation are plotted as white and grey symbols, respectively; (e) Percentage of potentiated neurons in different groups of cultured neurons (IB4+ without estradiol; IB4+ with estradiol; IB4− with estradiol), compared by Exact Fisher’s test (**p < 0.01, ***p < 0.001, when the IB4+ with estradiol is compared to the other groups: p < 0.0001 vs IB4+ without β-estradiol; p = 0.0018 vs IB4−). Of note, there is a remarkably higher number of potentiated cells among the IB4+ neurons incubated with β-estradiol; (f) Percentage of potentiated IB4+ neurons in culture incubated with β-Estradiol (100 nM) or PPT (100 nM). No significant difference was observed between two groups (p = 0.61 > 0.05, exact Fisher’s test). For panels (a) and (b), the horizontal scale bars correspond to 100 s and, the vertical ones correspond to 0.1 a.u. (arbitrary units of the fluorescence ratio F340/F380).