Does anyone have a good uncaging protocol for neurons in culture? I'm experiencing cell death after the addition of 30uM RuBi Glutamate to my DIV14 dissociated cortical neuron cultures. I am protecting the plate (Mattek dish) from ambient light, except when imaging fluorescently-tagged actin with 543nm light on a laser scanning confocal. The entire plate is toast in a little over an hour, even in areas not yet visited during imaging, so I'm wondering if it's a pH or toxicity problem? Or if perhaps RuBi glutamate is so easy to uncage, that the light of the computer monitor is enough to set it off? I am a merely a cell biologist, so any guidance or advice from the Neuro community would be appreciated ;)
Dear Sara, while I have experience uncaging (MNI-)glutamate in acute brain slices, I have not personally used the RuBi compound, nor have I worked with cultured neurons. That said, some thoughts:
The original publication characterizing RuBi includes in its title the phrase "with visible or IR light", and shows an action spectrum in Figure 1 (see reference below). Caveat: I don't fully understand the method used to obtain this spectrum. This spectrum shows a tail extending beyond 550 nm. Therefore I think it is certainly possible that the intense 543 nm illumination during confocal imaging is uncaging the RuBi - and this would include the solution between the focal plane and the light source. So perhaps the laser-uncaged Glu is diffusing beyond the illuminated region?
Salierno, M., E. Marceca, D. S. Peterka, R. Yuste, and R. Etchenique (2010, April). A fast ruthenium polypyridine cage complex photoreleases glutamate with visible or IR light in one and two photon regimes. Journal of Inorganic Biochemistry 104 (4), 418-422.
What happens if you add the RuBi and wait 1-2 hours without performing confocal imaging, are the cells healthier?
Personally I would avoid any intense illumination (in the UV-visible range) of the sample once the RuBi has been added. During imaging, the sample will likely be illuminated for a very long time (minutes) compared to the duration of typical uncaging illumination (on the order of 1 ms). Perhaps you can find an imaging illumination intensity low enough to avoid uncaging RuBi, but it seems risky.
Alternatively, you could try a different caged glutamate compound (e.g. MNI-glutamate) with a more advantageous spectrum (in the UV)? You can find the spectrum for MNI-glutamate in this paper (as well as a thorough treatment of the subject):
Trigo, F. F., J. E. T. Corrie, and D. Ogden (2009, May). Laser photolysis of caged compounds at 405nm: Photochemical advantages, localisation, phototoxicity and methods for calibration. Journal of Neuroscience Methods 180 (1), 9-21.
Hopefully this approach would allow you to perform fluorescence imaging without uncaging the glutamate.
Hi Benjamin,
Thanks for the thoughtful response! I tried your suggestion of culturing the neurons in RuBI glutamate for a bit and they looked perfectly healthy, so yes, I think I'm in a situation where my imaging is doing the uncaging! At least I've learned what glutamate toxicity looks like. I was able to do an uncaging experiment today with iRFP-actin and the cells stayed healthy throughout the experiment. Sadly iRFP is much dimmer than mRuby but it will have to do! I'd love to use MNI-glutamate, but 405 uncaging is not compatible with the nearest laser scanning confocal, so I'll need another microscope to pursue that. Now to tackle the next problem - how to translate slice uncaging protocols to cultured neurons!
Sara
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