Dear colleagues, I’ve started working with cultured primary neurons and came across a problem.
I need to depolarize neurons for different time intervals (up to 1 hour) and then use them for an assay 2 days after. The problem is that most of them are dying after depolarization.
I culture neurons in complete neurobasal medium (Neurobasal + 2% B27 supplement + 1%Glutamax +1% penstrep) with 1/3 media change every 3 days. I depolarize them on DIV11-14 by swapping media on Tyrode’s solution (45mM KCl) for up to an hour. Then I wash the cells with Tyrode’s solution (5mM KCl) twice and swap the collected media back.
45mM K+ Tyrode’s: 100 mM* NaCl; 45 mM KCl; 1 mM MgCl2; 1.8 mM CaCl2; 1.04 mM Na2HPO4; 26.2 mM NaHCO3; 10.9 mM HEPES; 10 mM D-glucose
* NaCl is used to adjust osmolarity of the solution, so concentration varies.
Since in the current setup I depolarize cells in 5%CO2 incubator I used buffering formula of Neurobasal media. I adjust the pH to Neurobasal’s pH=7.7 and checked that in CO2 incubator it equilibrates to pH=7.4. And I adjust solution’s osmolarity to match the current neuron’s medium too.
Also, I depolarized neurons in live cell imaging using GCaMP6s to monitor calcium elevation and after minutes I can see that some neurites are destroyed (Fig.1, attached) and after 0.5-1 hour cells don’t look good and most of them die afterwards (Fig.2).
At the same time, I keep seeing papers with no explicit details on solution and osmolarity where cultured primary neurons are stimulated with KCl for hours. For example, here 6 hours of 55mM KCl (https://www.nature.com/articles/nature09033).
I guess there are a lot of people routinely working with primary neuronal culture. Could you please help me, what am I missing?
1-hour incubation is too long and effects in non-reversible. It completely depolarises the cell and possibility of reversal in none. We use for short acute exposure of KCl (30s-1min). See the paper here.
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Thank you Manoj kumar.
I agree with you, I did timelaps imaging with only 5 minutes depolarization, these effects started to occur but they were reversible in contrast to 30 min depolarization.
Well, I just wonder if longer depolarization is possible without neuron morbidity.
Hi Marc,
Thank you for the advice!
I need this setup to develop a genetically encoded biosensor. I'd like to use the simplest method of pharmacological neuronal activation for this. So far, indeed, it seems that toxicity is caused by high potassium concentration. I try to use lower concentrations now and BIC/4-AP stimulation.
What wonders me is evidence in the literature that prolonged depolarization with high potassium concentration is not toxic to neurons. For example, in this paper (https://doi.org/10.1016/0006-8993(95)00552-2) authors specifically assessed viability 24h after treatment. 1hour of 50mM KCl was almost non-toxic.
Hi Max,
Have you done a control experiment using 5 mM K Tyrodes rather than the 45 mM one? It might be worth checking that the process of changing the media back and forth isn't damaging the neurons.
Hi Damian,
Yes, I've observing neurons in 5mM K+ Tyrode's for 1h and they looked fine.
I'm not sure about media change. I know that substituting the significant part of media with fresh one is damaging to the neurons. Also, I recently found an interesting paper (doi.org/10.1371/journal.pone.0025633) where authors observed neuronal toxicity caused by neurobasal media itself. They claimed that this is excitotoxicity caused by L-cysteine which concentration in the commercially available media is, for some reason, 26x higher than in the originally described recipe.
I think the changing of conditioned neuronal media back and forth should inflict minimal damage. I can’t see any other option when using chemicals for stimulation.
Also, I think I’m making some progress. Lower potassium concentrations (25, 35mM) significantly increase calcium with much less toxicity. So probably that will do. I need to do more tests to be sure.
It's never easy is it :) Glad you are making some progress. I use 30 mM KCl when stimulating neurons for Ca imaging, and with even a 2 s exposure get a robust Ca transient. I guess you could estimate the required concentration by using GHK equation and activation potential of the Ca channels (if you are aiming for Ca influx). Good luck!
As also suggested before by others I would definitely consider using Bicuculline (40uM), it's more physiological, way less toxic and reversible if you wash it out. It's also normally used to induce homeostatic downscaling, where it is left for 48h in the medium. A lot of people used KCl so you will find many protocols, but many good scientists advised me against it because of its toxicity. For depolarizing neurons people use also media change (see FLARE form Alice Ting's lab). Good luck!
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