Hi all,
I am interested in AMPAR mediated mini EPSCs in hippocampal neurons. My current recording configuration (hibernate E as bath solution and Cs gluconate as internal) seem to allow me to record from them up to day in vitro (DIV) 25 for about 30 min with fairly stable access. The sad story is that I do not see a lot of mini events (1 - 2 events every 3 - 5 seconds --> much less than 1 Hz). I can see quite a lot spontaneous events starting at DIV 11 already (1 - 2 event every second or so). Does it sound like something you experience before? How would you recommend troubleshooting it? Maybe, like, changing the recording condition or culture condition to have more mini AMPAR EPSCs? Thanks a lot!!!
Best,
Huong
Below are some more information if you would like to know....
When the neurons are younger (Div 12 - 15), there are a lot of action potential driven EPSCs [huge events, > 100 pA]. And when they get to Div 25, there are mostly very small events (20 pA, more or less). The small events decay time is approximately from 4 - 30 ms.
Regarding the culture, Coverslips are coated with 1 mg/ml Poly D lysine. I plate the neurons from E17 - E 18 hippocampi at 1.4 millions neurons per 100 mm dish containing 6 coverslips. The coverslips are submerged in serum containing media. The coverslips have wax feet so I can flip them up side down into 60 mm dishes with neurobasal + B27 + glutamax + 20 % media conditioned by astrocytes [which facilitates the growth of a lot of astrocytes underneath the neurons].
Sometimes I also co-culture the neuron coverslips in dishes with astrocyte feeder layers [in which the cells are not touching each other i.e. Banker culture]. The viability and development look fairly good. I can see a fairly dense network of dendrites already at DIv 11 and it just gets denser over time. Cells are evenly distributing across the coverslip, not much fasciculation or any sign of substrate problem. I feed them twice a week after the first week.
hi, you can use latrotoxin to pharmacollogically increase the minis frequency.Also you can increase the KCl concentration (couple of mM, no more) of your culture medium. You are right, the spontaneous activity start around DIV 10 but I don't see any strong decrease @ DIV 25.
Hi Christophe,
Thank you for the response! How far can you go with the KCl? I have 5.4 mM in my bath solution at the moment. Could you go up to 10 mM or maybe that is too much? I would prefer to try that before trying the latrotoxin, though it sounds like an interesting solution.
Regarding the spontaneous activities, I actually see less big events at DIV 25 in comparison to DIV 11. Looks like you are seeing a different trend? :)
Huong, what temperature are you working at? If room temp, you could try physiological and see if that speeds things up.
I am indeed recording at room temperature. Yes increasing to 30oC or 32oC could be a good option. I need to secure a heater for that first. :) Did you try patching on culture neurons at physiological temperature? Did you just heat up the solution before it is going to the chamber or how you did that? Is there anything tricky about it at all? Would love to hear a bit more about this.
Working at 28 degrees will help. As will increasing the density of you cultures? I mostly use cortical cells to begin with and the mEPSC frequency is really high (Becann-Kelly 2014 FCN); we tried several things to try increase the frequency in a couple of genotypes, such as nicotine. Variable effects, I think due to the fact our frequency is so high already. I attribute this to the 2mM CaCl in our ACSF, which I think icreased Pr to Ca-saturated release levels...
I am sure that higher temperatures will increase mEPSC rates. Also higher external calcium. You can use a Warner Systems heater.
Hello Huong, I would suggest to go with temperature first. Minis is a spontaneous fusion event that depend on membrane fluidity and thus temperature. If not enough, I would increase KCl (no more than 2-3mM) then Ca if you have to but I would stay clear of LTX or Nic (cost and potential side effects on your experiments). For warming up your chamber on a budget, get a a small water bath/heating block from the unused equipment of the molecular biologists in your lab. Coil your perfusion line in it . If you can also steal (I mean borrow) a peristaltic pump from your colleagues then make a heat exchanger as close a possible to the chamber to warm up the perfusion line, with a short length of stainless steel tubing (a 12 gauge needle is OK). If you use water-immersion objective, ou will also have to warm it up. I hope this helps
Thank you all for the suggestion!!!
@Austen: Yes, increasing the density would also do it!
@Thomas: Did you mean the KCl should not exceed 3 mM? Or that I should not add more than another 3 mM into the solution? I have 5.4 mM at the moment. Regarding the heating system, so you think both the objective and the solution need to be warmed up to create the effect? This makes some sense although I have seen either of these strategies on separate systems but have not seen both on the same rig before.
If you have a ceramic covered objective you may not need to heat it up. We do not heat up the objective. The Warner system bath heater has a temperature sensor that monitors the temperature of the bath in a feedback loop. This make is relatively stable. We keep our brain slices during recordings at 33-34 degrees Celsius without any problems.
@Henrique P. von Gersdorff _ I am curious does the Warner system contribute any noise?
Yes. You must be careful to ground things well and use a smooth bath perfusion system (Shield equipment well to avoid ground loops). We use a pump to get a uniform flow rate.
Getting rid of noise is an ancient art form for electrophysiologists and the perennial bane of our existence...
I don't have ceramic shield on the objective. Sounds like this could act as a heat sink but your heating system might overcome that problem right? Thanks Henrique! May I get a bit more information about the system that your lab is using? It sounds like a really good one! Is it the one in this link?
https://www.warneronline.com/product_info.cfm?id=702
Yes, this is what we use for our brain slice patch clamp recordings.
Good luck with your recordings!
Huong, my gut reaction is that something is wrong with your primary neurons. Cultured neurons are typically ridiculously active, pumping out minis at 10-15 hz, which just increases over time. In fact most neurobiologists studying activity-dependent processes in cultures have to "quiet" neurons down by inhibiting activity with APV or TTX for hours to days prior to stimulation. That you should be looking for ways to increase mini frequency and amplitude suggests to me that your cultures are not as healthy as they should be.
Huong,
I do not have personal experience recording minis in hippocampus nor in hippocampal cultures, but there are a lot of collaborators around me doing so. As far as I recall, the frequencies they report are rarely over 1 Hz (most commonly around 0.5 Hz). The first time I saw these frequencies were puzzling to me because I was recording mEPSCs in cortex where frequencies could easily go above 10 Hz in some cells.
I do not know what the hibernate E solution is but one possibility is that its composition is exactly what is keeping your cell cultures so quiet. Check the K+ concentration in that solution, if it is below 3 mM, that may be culprit in part. In any case, increasing the K+ concentration should help with increasing mini frequency.
Hope this helps!!
Hi Bryen and Felipe, thank you so much for your inputs. I saw your responses earlier but waited until I did more recording before answering you. It looks like I am having a bit more activity of AMPAR on our new prep of neurons. These are the ones we culture with astrocytes feeder layers. I have ~ 1 Hz of mini frequency, similar to what Felipe recalled. :)
@Bryen: Thanks for the thoughtful concern. We are following the protocol of Banker. Are you using other protocol? Would you mind sharing a bit more information with me. Also, I checked on the literature and did not find any report of 10 - 15 Hz mini AMPA frequency on hippocampal neurons. I am a bit puzzled when you said using APV and TTX to inhibit mini activity. As far as I know, APV together with TTX might induce scaling up of the AMPAR activity (studies from Lu Chen lab at Stanford). Anyhow, cell health is always an issue for culture. I do see that when the neurons are not happy-looking (grainy membrane, fasiculating processess, etc), their activity is not great... I am trying all kinds of things to keep them as healthy as I could but there are always more to learn. :) Would love to hear more from you.
@Felipe: Yup, from my limited experience with both cultures, the cortical neurons seem to have much more activity in comparison to the hippocampal. I am just shocked if they are heaven-and-hell different like that (0.5 Hz and 10 Hz, yikes!). The hibernate E is already having 5.4 mM of K+, so it might not need more potassium, I think. But I will keep this suggestion in mind!
Hi Huong. Just to clarify my earlier post. You could increase KCl by a few mM but I would keep final [ ] below 10mM. I also have a question about amplitude. Is it possible that you have a lot of events below your detection limit (in the noise)? You mentioned that initially the spont activity is higher and that EPSPs often trigger a spike. Would that trigger LTD (smaller event) or even synapse elimination ? You may test this by adding a 50-200nM of TTX contiuously to your culture medium to reduce activity. Another possibility already mentioned to get more mEPSC is to increase density (again, unless the network self-regulate through LTD). I am also surprised by the difference between cortical and Hipp culture activity. Is this at similar cell density (final density, not seeding density since survival might be different). Hope this helps
Dear Houng. A way around could be using Cs based internal solution. It will improve the space clamp and will remove the leak conductance from dendrites. As a result you will be able to detect inputs which are more distal. This should make some difference. Because of the uncontrolled expansion of neurons in cultures and more distal location of excitatory inputs, most of them do not reach the somata. While it is true that the rate of synaptic activity in cultures is typically higher, the majority of them are GABAergic and are blockade of GABAa antagonists. Hope you will see some improvements and good luck - Saak
@Thomas: As always, many ideas! Thank you! :) It is very helpful indeed!
- Regarding your concern for LTD, I did not see any reduction of spon events within once cells. About 2 weeks ago, I recored from ~ 4 week old neurons (who were not co-cultured with astrocytes) and they did not have much of the big EPSCs. This week we started to have Banker cultured neurons at ~ 3 week old and these all seem to have very robust spontaneous EPSCs and look very nice (3D-looking, smooth membrane, nice processes).
- I have not analyzed the data but looks like these new neurons have higher mini frequency already (still lower than 1 Hz though). I might still need to heat them up or try your suggestion with the KCl to get more response and reduce recording time. Thank you for this!
- The density is about as high as we can do considering other factors. I tried higher density before but the media just got acidic so quickly and the neurons were not healthy...
-For your question about cortical and hippocampal neurons, no, they did not look like they had the same final density. The cortical neurons usually looked slightly denser than the hippocampal neurons even if we plated them at same density. I don't have any quantification on this yet though. This was mostly from my quick glance at the traces
@Saak: That is super insightful! I do use Cs gluconate as internal with all of the voltage clamp experiment but did not really think about the geography of culture processes that hard. :) From my staining experiments, culture neurons do have synapses everywhere, and their dendrites stretch quite far. I have not measured and compared these numbers to in vivo neurons. Would be interesting to learn more.
I did not know that culture neurons have mainly GABAergic synaptic activity... I saw quite a lot of mini-like activity (events
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