Figure 6. Calcium dynamics in PG cells.

The CNS–RG complexes (a–c and f–h) and isolated RG (d) were cultured from tim-gal4/UAS-cameleon (YC2.1-82) flies. Representative data from two different cells were plotted for each experiment. (a) The frequency of spontaneous Ca²⁺ spikes depended on the extracellular Ca²⁺ concentrations. In contrast, exposure to the Ca²⁺-ATPase inhibitor thapsigargin (TG; 1 min, 1 μM) had no effect on the frequency of Ca²⁺ spikes. (b) Effects of TTX (0.3 μM) and the L-type Ca²⁺ channel activator BayK8644 (100 μM) on Ca²⁺ spikes in PG cells. TTX reduced the frequency but did not abolish Ca²⁺ spikes. Exposure to BayK8644 increased the Ca²⁺ spiking frequency again, even in the presence of TTX. (c) PG cells in CNS–RG complexes cultured in medium containing TTX exhibited Ca²⁺ spikes similar to control culture conditions. Nimodipine (2 μM) reduced the Ca²⁺ spiking frequency in PG cells cultured with TTX. (d) The effect of excitation light on [Ca²⁺]_c was tested in PG cells in isolated RG cultures using an LED light source. The low-fluorescent signals were amplified using an EM–CCD gain. The relatively noisy traces at 3×10⁴ photons cm⁻² s⁻¹ (invisible to eye) were caused by the electrical amplification of the signals. Neither the frequency of Ca²⁺ spikes nor baseline [Ca²⁺]_c levels were affected by excitation light in PG cells in isolated RG cultures. (e) To examine the effects of photic stimulation on Ca²⁺ spikes (f–h), a micro-light was located near the brain hemisphere. An excitation light for Ca²⁺ imaging (435–445 nm) was tightly focussed on the RG. (f) Exposure of the brain to blue (455–505 nm; blue trace) but not red (595–645 nm; red trace) light temporarily blocked Ca²⁺ spikes in PG cells in CNS–RG complex cultures. The effects of blue light exposure (in two representative cells as solid and dashed lines) were abolished in PG cells cultured with TTX (g) or in PG cells cultured from cry^b background flies (h). All the above results were consistently observed in 44–119 cells in 3–5 organs.