Hello everyone,
I am doing single cell two-photon Calcium imaging using whole-cell patch clamp. Briefly, I am patching CA1 cells with an extracellular electrode positioned on the schaffer collaterals to stimulate the CA3-CA1 pathway. I use Fluo-5F and line scan a ROI to detect the calcium transients along the different dendritic positions to see the corresponding change in Ca fluorescence with stimulation. I see a clear corresponding change in fluorescence when I do a line scan along proximal dendrites but fail to see any signal when I move towards distal dendrites. So my questions are the following.
1) Do Ca kinetics differ from proximal to distal dendrites?
2) Is there any other dye suitable for imaging the Ca fluorescence in the distal dendrites?
Any suggestions are welcome and thanks in advance.
P.S. I also tried using Fluo-4ff but it didn't work as well.
Hi Prudhvi,
Check out these papers from the Svoboda lab:
https://doi.org/10.1016/S0006-3495(00)76809-3
https://doi.org/10.1016/S0896-6273(02)00573-1
The first showed how to convert fluorescence signals obtained in situ to [Ca2+] values. In the second, they used fluo-4 or OGB1 co-loaded with Alexa 594. Across both papers, they show [Ca2+] change due to a single AP is largest in spines, then proximal apical dendrites, and smallest in non-spine distal dendritic shaft.
Best of luck with your experiments!
Hey Gerard,
Thank you for the papers and your briefing. I will go over them.
Best,
Prudhvi
Hi Prudhvi,
1) The number of Ca channel is different between proximal and distal dendrite so it wouldn't be suprising if the kinetic of Ca differs too.
2) I'm not focus on dendrites in my imaging but with my dye, I can clearly see them after a stimulation. I currently use the Calbryte 520 AM dye but I'm not patching the cells, maybe the non-AM homolog exist ?
Hi Lora L Martucci ,
Thank you for your input. Yes, there is a cell impermeant homolog of the Calbryte 520 AM dye. Do you have any experience with OGB-5N/6F dye? I have seen many papers using either of these dyes for imaging Ca dynamics in distal dendrites. It is also interesting to know that Ca channel density is different along different locations. Do you have any specific paper in mind regarding the channel expression along the neuron?
Thanks for your time.
Best,
Prudhvi
Hi Prudhvi,
I didn't check this litterature for a while, I can remember only this article : http://www.jneurosci.org/content/17/15/5936
For the dye, I don't know this one, I used its AM homolog (OGB1-AM) wich was good but not that good compared to Calbryte.
Best
Lora
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