Following established protocols(Article Adenylyl cyclase-dependent form of chemical long-term potent...
) for forskolin-induced late-phase LTP in acute hippocampal slices (400 µm), slices are initially treated with 50 µM forskolin in standard ACSF for 5 minutes. Subsequently, they are perfused for 5 minutes with a high-potassium/high-calcium magnesium-free ACSF solution (10 mM CaCl₂, 30 mM KCl, 0 mM MgSO₄; other components unchanged: 124 mM NaCl, 25 mM NaHCO₃, 1 mM NaH₂PO₄, and 10 mM glucose). However, slices consistently deteriorate and die during or immediately after this 5-minute perfusion in the high-K⁺/high-Ca²⁺ solution. This is evidenced by the complete disappearance of field excitatory postsynaptic potentials (fEPSPs) and confirmed indicators of cell death.Could anyone kindly suggest where the issue might lie? I would greatly appreciate any insights or suggestions for modifying the protocol to maintain slice viability.
Hi,
you are simply adding ~26mM KCl, ~8mM CaCl2 and remove ~1mM MgSO4? This will increase your osmolarity by about 60mOsm/L (a rough guess).
An increase like this will kill your cells.
Brain slices are very sensitive to small changes in osmolarity. If you change anything in your ACSF, you should carefully monitor the osmolarity and adapt it. At best you don´t have any change.
Hope this is helpfull.
Best,
Stefan
Stefan may be right. The original JNeurosci 1999 paper is a little vague about this solution. We treated our slices with 52 mM K for two hours, and we decreased Na by the same amount to keep the osmolarity close to normal. They survived okay. The original paper may have done this too and just left it unstated. Hope that will work for you!
-Matthew
Simply add the K by 50 mM for 2 hours and decrease the Na level by same concentration to maitain the near normal osmolarity
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