I am currently using a high K+ model (8.5 mM K+ with 1mM CaCl2 and 1.3mM MgSO4) for inducing seizures in acute rat hippocampal slices (4-8wks old). Recently, we see a lot of spreading depression's (SDs) in our slices which makes it almost impossible to study any drug effects. Most of our slices start to show SDs within a few "ictal-like bursts" and then occur regularly after every ictal burst or once every 2-3 bursts. We also find the incidence of SDs to increase whenever the recorded potential (field) is of higher amplitude, usually stable until they exceed ~4-5mV.
The slices are maintained at 34-35 C in an interface chamber (flowing) with a flow rate of 1.5ml/min and bubbled at a high rate. Potentials are recorded with a ~1mOhm glass electrode filled with high K+ acsf.
Reducing temperature to 34 C and increasing flow rates doesn't seem to help much. Is there any other way to reduce SDs?
Sometimes spreading depression occurs due to the
hypoxic condition especially in high potassium seizure model. It may be avoided by preparing slices with continuous provision of enough oxygen. You must also provide contin. oxygen at the time of recording in interface chamber, increase the oxygen level if you are already providing during both procedure. When you transfer the slices into the interface chamber, wait for at least one hour before recording.
it may also depend on the quality of slices, try to be as fast as possible.
You can also reduce the concentration of both KCL and MgSO4. I guess you already know that high K is a standard model for SD. Therefore reducing KCl may help. In compensation you can reduce the conc. of MgSO4 which increase the excitability.
Elevated K+ concentration to around 8.5 mM would be expected to increase K(+) conductance, particularly at Kir-channel conductance. That might influence SD.
Hi,
This is a normal phenomenon, in fact it is known as a good way to induce SD for those studying it.
Your ACSF contains already a lot of K+. During a seizure, external K+ raises to more than 8 mM in normal ACSF. In your conditions, you should be around 12-16 mM during the seizure, above the threshold to trigger SD according to U. Heinemann's work (check his numerous papers on this). Using an interface chamber is more problematic because there is a strong accumulation of ions etc., as compared to fast perfusion systems in submerged chambers.
You could do the same experiments in submerged slices, or use the low Mg model instead of high K, or a mixture of high K and low Mg.
hope that helps
Thank you all for your responses.
@Abdul Wahab: Reducing both KCl and MgSO4 concentrations sounds like a quick solution (and interesting). I can try this out. Thank you!.
@Christophe Bernard: We have had issues (quality and repeatability) with inducing seizures under submerged conditions. We find the slices to respond better and stronger to high K+ with the flowing type interface chamber we are currently using. (Haas-top slice holder from Warner instruments). Since I will also be looking at oscillations, I think submerged chambers might affect my recordings. Can I have your opinion on this? Thank you!
You can use a submerged chamber with double flow, which allows studying oscillations:
http://www.ncbi.nlm.nih.gov/pubmed/19200237
This works very well
the secret:
very fast perfusion >10 ml/min
both sides of the slice perfused
physiological temperature
There is a hungarian company selling the chamber and perfusion/heating system - cheaper than Warner
Hi
There are several issues to take into account when working with the high K model of epilepsy. First the seizures discharges are among other things dependent on ephaptic interactions and potassium accumulation in the extra cellular space. Seizures can therefore be evoked reliably in an interface preparation whereas using a submerged slices preparation will not work. A Hass interface chamber is the perfect experimental chamber for the model. SD occur usually during the ictal type of seizure discharge and caused by to low oxygenation. There are 3 ways to improve oxygenation. Make the supply reliable by sufficient flow of humidified gas with 95 % oxygen and 5% CO2. Decrease slice thickness. Slices of between 400 to 450 micrometer should be optimal making them to thin hampers seizure discharges. Lowering the temperature slightly (one degree) to reduce oxygen demands of the tissue. To low temperature will stop seizure discharges. The ion composition of ACSF is critical to induce seizures too. In your case i would try lowering potassium a little rather than lowering the concentration of divalent cations. We successfully used ACSF composed of : 124 NaCl, 3.5 KCl, 1.25 Na2PO4, 1.2 MgSO4, 1.2 CaCl2, 26 NaHCO3, and D-glucose, 10. High-K+ ACSF has 7.5 mM KCl. Placement of a stimulation electrode in a slice exposed to high K is also prone to evoke a spreading depression. Age of your experimental animal might be an issue too. I would suggest you use animal more than 8 weeks old.
Besides all good suggestion already made, I would additionally suggest trying increase the calcium level a little. May be it helps.
@Morten Jenson: Thank you very much for your suggestions! As you have mentioned, SD precisely occurs during early Ictal -Clonic phase, especially in slices which produce high amplitude activity. We have infact tried lowering the temperature down to 34C, which seems to do the trick sometimes but in some slices we also end up with more interictal-like bursts. We make 450uM slices and we oxygenate quite heavily.
I will try reducing the KCl to 7.5mM and increasing CaCl2 to 1.2. Thank you!
@Rubem Guedes: Thank you for you suggestion. I will try increasing calcium levels.
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