Figure 5.

Overexpression of PSCA at E8 blocks nicotine-induced calcium influx via α7-nAChRs. Ciliary ganglia from embryos infected with RCASBP(A)-PSCA or RCASBP(A) open (without an insert) at St. 8–9 were isolated at E8, dissociated, and loaded with Fura-2, 2 h after plating. Nicotine (10 μm) was used to stimulate nAChRs in the presence TTX and cobalt, which block voltage activated influx of calcium. A, Brief application of nicotine induces rapid increase in [Ca²⁺]_i. The α7-specific antagonist MLA blocks 50% of this nicotine-induced response. The third response is the total calcium signal observed in response to perfusion with 25 mm KCl in the absence of TTX and Co²⁺. The mean peak Ca²⁺ responses due to depolarization with 25 mm KCl between open and PSCA infected ciliary ganglion neurons are not significantly different, indicating the all neurons are equally loaded with Fura-2. B, Plot of all the peak nicotine-induced calcium responses observed in PSCA versus open virus infected neurons. Each gray spot represents the response of a single neuron. Graph represents the sum of data from 3 independent experiments using viral injections together with calcium imaging of E8 neurons. PSCA infected neurons exhibit a significantly smaller mean nicotine-induced increase in [Ca²⁺]_i compared with open-infected neurons. Bars indicate mean plus or minus SD (p < 0.001; open: 0.3 ± 0.02, n = 69; PSCA: 0.17 ± 0.01, n = 86). Addition of exogenous αbtx (50 nm) to PSCA infected neurons does not cause additional reduction in the mean nicotine-induced Ca²⁺ whereas addition of αbtx on open infected neurons significantly reduces increases in [Ca²⁺]_i (p < 0.001; open: 0.3 ± 0.02, n = 69; open+btx: 0.14 ± 0.02 n = 31), whereas open with PSCA is not significantly different from open +btx or PSCA +btx; one-way ANOVA with Bonferroni post hoc test.