For anybody who has done GCaMP imaging in acute brain slices, I have some questions. We injected AAV expressing GCaMP6f into a particular brain region, waited >2-3 weeks, cut slices, and are using two-photon imaging combined with pressure ejection of drug to try to evoke a response. We get good increases in Ca++ signals with a high K+ concentration puffed onto our cells as a positive control, but we are trying to see an inhibitory effect with GABA. Has anyone done this? What kind of spontaneous activity do you see, if any? Is there a good way to increase activity so that we may be able to turn it down? Any tips and recommendations would be most welcome.
Thanks for your answer! That's the sense I have as well after looking at a lot of papers. Our problem is that we have seen almost no spontaneous activity to try to turn down. What K+ concentration do you find to work best to turn up spontaneous activity to a reasonable degree? We will also try thicker slices.
Additionally, I found that Jonathan Ting used 20 uM NMDA to turn up spontaneous activity (https://www.youtube.com/watch?v=QXA0-MzsSWc). In one of his papers (attached) he also said "we have also used bath application of low concentrations of glutamate receptor agonists such as NMDA and glutamate, which induce more stochastic patterns of firing with slower onset and lasting for sustained periods." I haven't been able to find specific info on what exactly he used though, aside from that youtube video.
Have you used glutamate receptor agonists to turn up spontaneous activity? If so, what drugs and concentrations?
Thanks for the tips, I appreciate it.
https://link.springer.com/protocol/10.1007%2F978-1-4939-1096-0_14
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