Thanks Bill, that helps a lot.
I wasn't sure what the difference between Capacitance/Series resistance
compensation and bridge compensation was before.
I don't know if the voltage error caused by the pipette and access
resistance would be constant across different current steps though..
First of all, even though the resistance is fixed per recording, the
current across it is different for different steps.
Secondly, due to the pipette capacitance, the effect of increasing step
size on "membrane potential" probably won't be linear either.
In another words, the error will be increased for increasing step size.
Yes, medium spiny neurons do express strong Kir, which is supposed to be
responsible for their so-called "down-state" membrane potential.
This is cited by many papers and is used as a recognizable feature of
medium spiny neurons (eg. Nisenbaum and Wilson 1995, JNeurosci
15(6):4449-4463; Wang et al. 2006, Neuron 50, 443-452; Kawaguchi et al.
1989, JNeurophysiol 62(5):1052-1068 just to name a couple).
It sucks that our amplifier doesn't seem to have bridge compensation
circuitry (it seems that the next model up, the EPC-7 Plus, does have
it.. whilst ours, the EPC-7, doesn't!). It doesn't have fast
current-clamp either..
Hyunchul
Bill wrote:
> Oh man, been there and done that. The first poster I ever presented,
> the effect of the drug was actually just increasing series resistance.
>> Any way. If you're in current clamp, there is no such thing as series
> resistance compensation. Compensation is for when your in voltage
> clamp, and it compensates for the voltage lost over the series
> resistance.
>> When you're in current clamp series resistance is usually dealt with
> by a bridge balance. Here the problem is that when you step X pA into
> the cell you create a voltage across the series resistance, so the
> potential change you measure is a combination of current interacting
> with Rs and Rm. I.e. the bridge balance subtracts the voltage drop
> across the microelectrode from your trace.
>> As your initial Rs would be low because you're using a patch-pipette,
> if your series resistance didn't change throughout the course of your
> experiment, your lack of bridge balancing would not really matter
> (i.e. the voltage created over Rs would be constant, though I still
> wouldn't publish the data in good faith). But if it changed,
> significantly, the actual voltage step you read will be more than the
> cell is actually experiencing, because a good proportion of that
> voltage could be occurring over the microelectrode.
>> So: the HEKA manual is right, Rs compensation and correction do not
> apply in I-clamp. However, you need to find how to balance the bridge.
>>> Now I'm no expert on Medium Spinys, but it is my understanding that MS
> do not rectify that much, and in fact have an amazingly Ohmic
> behaviour in the negative direction. It's cholingergic neurons that
> have a big Ih current. (Kawaguchi, J Neurophysiol, 1992). Do you see
> the classically hyperpolarized membrane potential of a MS? The lack of
> rectification isn't explainable by any form of series resistance mess
> up (unless you were in voltage clamp).
>>> On Feb 23, 12:26 pm, Hyunchul Lee <aur... from rocketmail.com> wrote:
>>> Thank you, Dr. Ferber- but let me clarify my question-
>>>> We're making current clamp recordings, and injecting square current
>> pulses whilst in current clamp.
>> Our amplifier seems to "turn off" capacitance/series resistance
>> compensation in current clamp mode, though I'm not so sure.
>> In our preparation, a strong inward rectifying current is expected to be
>> seen in medium spiny neurons by applying hyperpolarizing pulses.
>> We've been applying 20 pA steps from 0 to -1nA (the maximum current
>> injection possible for our amplifier), yet we do not see any inward
>> rectification.
>> Inward rectification is a defining characteristic of medium spiny neurons.
>> These cells have been labelled iontophoretically with neurobiotin, and
>> we see that they are indeed medium spiny neurons.
>> We've checked our intracellular and ACSF ionic concentrations against
>> what others are using, and find no great difference.
>> The cell fires what appear to be normal action potentials with
>> depolarizing pulses in current clamp.
>>>> I'm thinking that perhaps- if the access resistance is quite high, the
>> injected current will tend to pass through the walls of the pipette
>> rather than into the cell, thus charging the interior of the pipette and
>> thus affecting the potential difference between the pipette and the
>> ground. This is just my thinking, but the HEKA manual seems to indicate
>> that compensation dials are disabled when switching to current clamp
>> "for the benefit of the user". Is there anything we can do, short of
>> getting a new amplifier?
>>>> Thanks,
>> Hyunchul
>>>> Dr. Michael Ferber wrote:
>>>>>> 1. Will this stuff up our membrane potential values during large current
>>>> steps (<1nA)? In what way?
>>>>>>> In voltage clamp(!!!!) the problem is the ratio between RS and the membrane
>>> resistance. Here you have a voltage-divider. Therefore the membrane potential
>>> does not corespond to your settings and the mistake depends on the ratio of
>>> RM and RS. Due to the fact that the RM depends on the number of open channels
>>> the mistake is not linear and increases with increasing membrane current
>>> (=decreasing membrane resistance). If RS is low and RM is high everything is
>>> fine, but if you have huge membrane currents the real membrane potential may
>>> be far away from the value you set.
>>>>>>> 2. Is there any legitimate way we can adjust our data post-hoc?
>>>>>>> I see no way.
>>>>>>> 3. Will compensating make a difference in current clamp for our case?
>>>>>>> In this case you apply a constant current and the potential is floating
>>> freely.... and if you compensate your electrode properly the very fast
>>> components (RS, pipette resistance ....) should be cancelled anyway.
>>>>>> Capacitance compensation makes your systen faster (simplyfied). This may be of
>>> interest if you are interested in the kinetics of processes (i.e activation
>>> of channels) .
>>>>>> Regards
>>> Michael
>>>>>> _______________________________________________
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--
Hyunchul Lee - PhD student
Systems Neuroscience Laboratory
N401 Anderson-Stuart Building (F13)
The University of Sydney, NSW, 2006 Australia
Ph:9036-7126 Mob:0422-754-597
email:hlee from medsci.usyd.edu.au
