Thanks for the preLight and your comments! To answer your questions:
1. Generally speaking yes, a rise in cytosolic calcium was followed by a rise in ER lumenal calcium. There were instances where, at stimulation, the ER lumenal calcium showed a decrease in calcium, that sometime was not proceeded by an increase in calcium (Fig. S1), but this only occurred around 5% of the time. There were instances where I got a more extended cytosolic response, and I could often see a somewhat corresponding ER lumenal response.
2 and 4. I tried to test with ionomycin, but even with l-glutamate in the solution I got too much movement to get any meaningful data. I would like to try again though.
3. The resting fluorescence is similar between Ib and Is neurons at the NMJ, it is only in the axons that the fluorescence is more dim.
5. I would really like to look at changes through development, particularly in the context of the mutants. I would have to think about how to do this technically on my setup though!
6. Ib expression is only lower than Is in the axon not the NMJs. We reasoned that this must be due to the smaller dimeter of the Ib neurons, not a difference in the expression from the drivers.
Thanks again! Let me know if you have any more questions!
Megan Oliva (first author)