Hi Wiilliam,

Thank you. I work on culture neurons, and there is no Phosphate in my ACSF. I will try Cadmium. BTW, which one do you think is the best, Cadmium or cocktail of VGCCs antagonists? 

Thanks a lot. @William M Connelly

Yu

That is a good suggestion. Let's try cadmium, during waiting for those drugs.

Thanks a lot.

Yu

If you wish to measure the possible Ca2+entry though NMDA receptor you need block the Ca2+ entry through voltage dependent Ca2+ channels. If you use TTX you block de voltage dependent Na+ channel and in this case the cell dost not depolarized? and the voltage dependent Ca2+ channels are not functional. However, it is very difficult to achieve at total blockade of the any voltage dependent channels. If you measurement the intracellular Ca2+ as follow:

Cells + XTT + high K + 0 Mg  you measure the Ca2+ entry through the voltage dependent Ca2+ channels which have not been blockade (remaining voltage dependdent Ca2+ channels) ----- A entry

Cells + XTT + 0 Mg + NMDA   you measure the Ca2+ entry through NMDA receptor and remaining voltage dependent Ca2+ channel--------  B

                B-A =  perhaps this Ca2+ entry will be through NMDA receptor

Hi M.Pilar Conzález,

Thanks for the suggestions. I agree that VGCCs can not be simply abolished by blocking voltage changes with TTX. I think maybe cocktail of VGCCs antagonists.

You impair me, how about use Cells+TTX+0Mg+APV for B? Then A-B should be the contribution of NMDAR? How about [email protected] Conzález

Yu

When you measure cells + TTX (sorry I tell you before XTT, forgot this)  + high K + 0 Mg , the TTX has blocked the most voltage dependent Na+ channels but not all. Under this condition, if you added high K the remaining voltage dependent Na+ channels may induces depolarization and may open voltage dependent Ca2+ channels promoting Ca2+ entry which will be due to a Ca2+ entry via voltage dependent Ca2+channel. This will be the basal Ca2 entry.

 If you now added NMDA the Ca2+ entry will be due to the NMDA receptor. I think

 This way is the same but better that the before that I indicated you.

 If you use cocktail of voltage dependent Ca2+ channels blockers you can not assume that has blocked all these channels. Then, if you added NMDA you can not are sue that the Ca2+ entry was via NMDAR.

 Sorry my mistake with XTT in spite to TTX

That is a good concern. I can not guarantee that antagonists block them all. Maybe I can try both cocktail+NMDA and the paradigm you suggested. If both ways shows similar results, I can confirm there is some changes with NMDAR in my culture.

Thanks a lot. @M.Pilar Conzález

 I think that you can measure Ca2+ entry mediate by NMDAR as I say you before, but you may add the following control to guarantee that this Ca2+ entry is via NMDAR.

 Cell + TTX + 0 Mg + high K+------------- you measure Basal Ca2+

to the same tray added NMDA------------- Ca2+ entry mediated by NMDAR [1]

 Beside that:

 Cell + TTX + 0 Mg + high K+------------- you measure Basal Ca2+

to the same tray added + a NMDAR  antagonist  + Mg + NMDA------------- No Ca2+ entry

 This may confirm that the Ca2+ entry in [1] is mediated NMDAR

Hi,

I would like to share some primary data. I found that signals fluctruated a lot when I using Mg2+ free ACSF for Ca2+ imaging. My data looks quite noisy. Is it normal? @M.Pilar Conzález 

Best,

Yu

Hi William,

They are attached here. Sorry I did not explain carefully. 

I added TTX and cocktail of VGCCs antagonists, and applied NMDA (100uM) in Mg2+ free ACSF. They looks wired. 

BTW, how long do I need to wash if I want to remove APV in cultured neurons? Maybe, I can do something like no APV_APV_no APV?

Best,

Yu

Hi William,

I use Cal520-AM (1 uM for final concentration). Yes, I washed those antagonists ~12 min before imaging. 

About APV, I actually want to put all paradigm into the same image, no APV_with APV_no APV. Is it fine if I introduce a 5min washing to remove APV? Perfusion speed is 2.12 ml/min as I calibrated recently. 

It is a good suggestion about baseline. I should wait longer. Maybe 60s?

Thanks.

Yu