My boss wants me to look at holding current during whole-cell recording to check if the cell is good but she doesn't know how. Can anybody help me? Thanks.
you can look at holding current two ways very easily.
As long as you are not applying online P/N leak subtraction, the basline current of your traces before you step to a different voltage will = holding current for that Vm.
So if you are holding at -80mV and the baseline current is -50pA that is the holding current.
Of course, even simpler is to have the setting for the digital readout of your amplifier (if you have one) set to display the Im (membrane current) while you are holding your cells at whatever voltage you choose (e.g. -80mV or -60mV).
All the conventional axon instruments amplifers have a LCD or LED display for just such a purpose. The Multiclamps do not, but it is instead displayed in the Mutliclamp software on your computer screen
Thank you for the quick reply. I need to look at it during spontaneous event recording. How can I determine the baseline current? Can I use membrane test?
The amplitude of the holding current is the value of the basline current during spontaneous recordings.
See figure 1Aa in the paper below
Nicotinic receptors mediate increased GABA release in brain through a tetrodotoxin-insensitive mechanism during prolonged exposure to nicotineP. J. Zhu and V. A. ChiappinelliNeuroscience Volume 115, Issue 1, 15 November 2002, Pages 137-144
The effects of nicotine on the spontaneous release of GABA from nerve terminals in the chick lateral spiriform nucleus were examined using whole cell patch-clamp recording in brain slices. Exposure to 1 μM nicotine produced an early immediate increase in the frequency of spontaneous postsynaptic GABAergic currents. This effect was blocked in the presence of 0.5 μM tetrodotoxin. However, a prolonged application of 0.1–1 μM nicotine (>3 min) caused a tetrodotoxin-insensitive increase in the frequency of spontaneous GABAergic currents. This late tetrodotoxin-insensitive effect was blocked by the nicotinic antagonists dihydro-β-erythroidine (30 μM) and mecamylamine (10 μM), but not by methyllycaconitine (50–100 nM), indicating that activation of high affinity nicotine receptors was mainly responsible for this effect. This enhancement was inhibited by the high threshold Ca2+ channel blocker Cd2+ (100 μM), but not by dantrolene or ryanodine. The tetrodotoxin-insensitive enhancement of the frequency of GABA currents by nicotine was reduced by inhibition of cAMP-dependent protein kinase with HA1004 (30 μM), but not by inhibition of protein kinase C with staurosporine (1 μM), and was facilitated by forskolin (10 μM) or bromo-cAMP (50 μM).
The results indicate that nicotine-enhanced GABA release can operate through both tetrodotoxin-sensitive and -insensitive mechanisms in a single brain region and that a second messenger cascade may be involved in the tetrodotoxin-insensitive enhancement by nicotine.Author Keywords: chick lateral spiriform nucleus; cadmium; second messenger cascadePMID: 12401328
In figure 1Aa, look at how the basline (holding current) increases with the application of 1μM Nicotine by −116±6 pA from the control level of −77±9 pA, and also how the frequncy of spontaneous firing increases.
All this information should be recorded in your traces if you have set up the scale factors on your amplifer correctly, and as I said before, the amplifier will give you a constant readout of the holding current that you can write down in you lab note book.
When people say that 'leak subtraction was done off line' would they be referring to the baseline shown in the figure you posted?
Does it relate to serial resistance in anyway?