Figure 7.
Sema3A-induced cGMP causes recruitment and expression of CaV2.3 and PKG is required as a co-activator for acquisition of the dendrite identity. (a) Representative traces of Ca2+ currents (left), summary of the current (I)/ voltage (V) relationship (centre) of CaV2.3 currents and cumulative distribution of peak CaV2.3 currents (right) monitored in cultured xSCIN growth cones. Either ODQ (1 μM), Rp-8-pCPT-cGMPS (2.5 μM) or cycloheximide (CHX, 25 μM) was applied in the bath 30 min before experiments were performed. Sema3A (2,000 U·ml–1 in micropipettes) was applied to the growth cone as a gradient. (b–g, i–n) lmmunofluorescence images of Xenopus spinal cords immunostained for xCaV2.3 α1E (b–d, i–k) or MAP2 (e–g, l–n), without MO injection (b–g) or with injection of A-MO specific for either xSema3A (i–k) or xCaV2.3 α1E (l–n). Signals in red (b’–d’, i’–k’) and traces in black (e'–g', l'–n') indicate the location of the xCaV2.3 α1E and MAP2 IRs, respectively, which are overlaid with signals from the GFP injection marker (green, i’–k’, l’–n’). ODQ (1 mM), Rp-8-pCPT-cGMPS (0.25 mM) and 8-Br-cGMP (1 mM) were injected into the hindbrain and spinal cord of stage 30–32 tailbud animals (see Fig. 5g). Scale bar, 20 μm. (h, o) Summaries of normalized xCaV2.3 α1E IR (b–d, i–k) and MAP2-positive volumes (e–g, l–n). xCaV2.3 α1E IR and MAP2-positive volumes in bilateral sides of the spinal cord treated with ODQ or cGMP analogues were normalized against the average IR and positive volumes in control animals (h). Injected sides of spinal cords were normalized to the average IRs and positive volumes in control animals (o). Data are mean ± s.e.m. (n) = number of growth cones and spinal cords examined. Significant differences from control are indicated (*p < 0.05; **p< 0.01).