I am trying to examine the active and passive firing properties of neurons from brain slices in vitro and need to know which properties are most beneficial to examine and the best protocol for examining these using whole cell patch clamp techniques. Most of what I have been finding in the literature involves using intracellular recordings to obtain the properties or have conducted these analyses in vivo.
Hello,
I may suggest one of my earlier study :
Postnatal changes in electrophysiological properties of rat nucleus tractus solitarii neurons.
Vincent A, Tell F.
Eur J Neurosci. 1997 Aug;9(8):1612-24.
But you have te keempin mind that whole-cell recording may alter firing properties in different ways : 1/ buffering calcium with EGTA would reduce AHP.
2/ Dialysing intracellular milieu may disrupt any G protein regulated mechanisms (e.g Ih current)
Regards
Hi Eric,
There are several parameters to consider when you study the intrinsic properties of a neuron, i.e. in absence of synaptic activity. I always use blockers of fast synaptic transmission: 5 uM gabazine, 50 uM APV, 20 uM DNQX. Blocking synaptic activity will help to reduce trial-to-trial variability.
The pipette solution also matters: K-gluconate based solutions are the most used, will give you reliable results for long recordings. K-methylsulphate solutions seem to preserve better the Ca-activated K-channels responsible for the slow AHP. I prefrer K-gluconate based solutions with 0.1 or 0.2 mM EGTA.
Basic properties to consider are: resting membrane potential, input resistance, membrane time constant and sag. The resting membrane potential is measured in 0 current configuration. To measure the input resistance you can inject 3 or 4 subthreshold 1 s long current steps (hyper- and depolarizing) of small amplitudes and measure the voltage deflection at the end of the voltage response. Then you plot the I-V curve and perform a linear regression (V in mV and I in nA will give you Rinput in MOhm), the input resistance will be the slope of the linear fit. The membrane time constant is measured from an exponential fit to the subthreshold voltage responses. See Spruston & Johnston 1992.
The sag is a property expressed by neurons expressing HCN channels. In response to hyperpolarizing steps the neurons will first passively hyperpolarized and with some delay HCN channels will activate depolarizing the membrane. See Dickson et al 2000. To measure the sag you can inject 1s long hyperpolarizing current steps and measure the ratio between the voltage response at the steady-state and the voltage response at the peak.
The action potential threshold can be measured in many ways. I usually use 10 ms long depolarizing current steps and pick the action potential fired by the minimum injected current amplitude. I measure the threshold from the 1st derivative at the point where the derivative is 20 mV/ms. Other parameters of single action potential to consider are: amplitude, half-width, fast AHP, ADP and medium AHP.
The delay of the first spike is also important and can be measured from 1 s long depolarizing current steps. See Storm 1988.
The frequency-current relationship is also importamt and you just need to inject 1s long depolarizing current steps. You plot the frequency to the current amplitude and from the f-I curve you can calculate the rheobase, gain and the point where the cell enters depolarization block.
The adaptation is a parameter that gives you information about the temporal distribution of spikes during a train. I usually measure it with an index: (1 – (Flast/Finitial)) where Flast is the average frequency of the last two or three ISIs and Finitial is the average frequency of the first two or three ISIs. The colsest the value to 0 the more regular is the firing; the closest the value to 1 the more adapting is the firing.
Thanks Maximiliano for the response and the references. Would you suggest using the passive membrane properties (Cm, Rm, Ra, tau) in the membrane test window of Clampex? Do you think that would be an accurate measurement of the passive properties? Have you conducted these analyses with Cs-based internal solutions? If so, have you noticed much of a difference in the results when compared to K-gluconate based Internal solutions? I know that many people state that Cs based solutions reduce the Ih currents, but I have not had this happen to me with my work thus far. Also, can you please explain the adaptation (your last paragraph). I'm not sure what this is. Thanks again for your help
Hi Eric,
No, it would not be accurate. You have to analyse those properties with Clampfit offline, or if you know how to automate analysis with MatLab it might be possible to do it during the experiment. I never measured those parameter with a Cs-based solution because that would block most of the potassium currents. Moreover you cannot record the firing activity of a neurons with a Cs-based pipette solution.
The adaptation is a phenomenon by which the firing of a cell slows down during a train of action potentials. So in some cases (like CA1 pyramidal cells or stellate cells of the entorhinal cortex) the firing frequency is higher at the beginning of a train of action potentials than at the end. There are different mechanisms that underly adaptation in different cell types. For example, slow activating potassium channels, like Kv7. Also Ca-activated K channels that underly the slow AHP. Also the inactivation of Na channels can have a role in the adaptation.
Different cell types can express different firing patterns some are regula firing (i.e. the interspike interval does not change during a train of spikes) some are adapting (i.e. the interspike interval is bigger at the end of a train than at its onset.
I think it is more accurate to study the temporal distribution of spikes in a train in traces that contain the same number of spikes. In that way you can reduce variance due to differences in input resistance between cells or treatments (control vs drug).
Of course the details of each protocol will depend on the properties of the cell type you want to study.
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