Hi everyone,
I do whole cell patch clamp recordings from layer V neurons. In current clamp mode (with zero holding current), I check spontaneous action potential firing in vitro in acute coronal rats slices using K-gluconate-based solution (in mM 140 K-Gluconate, 10 KCl, 0.1 CaCl2, 10 HEPES, 10 EGTA, 5 MgATP, 0.5 NaGTP, 10 Phosphocreatine, 280-290 mosmol, pH 7.3) at room temperature. ACSF is 295-300 mosmol, pH 7.3 and no ions omitted or modified. I mostly get quite low firing rates or silent neurons (compared to neuroelectro data - www.neuroelectro.org-though data here is mostly from in vivo experiments) in ~2 h-long recordings. Could you give me some hints about the factors affecting the spontaneous firing rate? Any link or reference would also be highly appreciated. Thank you in advance.
You may try to measure spontaneous action currents in cortical neuron using cell-attached current recordings. Action currents can reflect the firing of action potentials with no change in intracellular milieu.
Some times spontaneous neuronal activity requires oscillation of calcium, which affects other channels (like small conductance potassium channels). I would try just zero pipette calcium and none or 0.1 mM EGTA.
Yes. Sometimes, under current clamp conditions, seal resistance may be unstable. The baseline level of resting potential may fluctuate and hence generate action potential if potential change reaches the threshold level. "Spontaneous" action potentials could be virtually overestimated.
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