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[Neuroscience] Re: Neur-sci Digest, Vol 89, Issue 9

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Wed Nov 28 17:17:20 EST 2012


Dear Jeffery,

I would like to reply to your question about series resistance and EPSPs.

The series resistance is a major source of errors in  recordings on a 
cellular level.  "Series resistance" not only a simple "resistor", it is 
composed of  complex networks. For better understanding one simplifies 
by subdividing this complex system into two subsystems: the access or 
microelectrode resistance and an intrinsic resistance inside the cell, 
This part is often neglected but in many cases it has significant 
impacts on the recordings. Both parts of the series resistance   again 
are not only  simple "resistors" but a complex distributed networks.

Series resistance has a twofold impact on electrophysiological 
recordings, especially in single electrode recording configurations. 
First of all, the resistance of the microelectrode or patch electrode 
(which in case of a single electrode recording system is a significant 
part of the series resistance) forms a   lowpass filter with the stray 
capacitance around  the electrode. This problem is solved to a 
satisfactory degree  by the use of "capacity compensation" circuits in 
most of the recording amplifiers. Secondly, any current flow through the 
electrode will cause a voltage deflection both on the access resistance 
as well as on the intrinsic series resistance inside the cell.  This is 
a significant source of error both in current clamp and in voltage clamp 
recordings. The series resistance and the membrane resistance form a 
voltage divider, therefore the correct measurement of the membrane 
potential requires special efforts. Also, the combination of series 
resistance and membrane resistance together with the membrane capacity 
form a lowpass filter which also has a serious impact o the recordings, 
especially in voltage clamp mode. See e.g. C.M. Armstromg and W.F. 
Gilly,  Access Resistance and Space Clamp Problems associated with 
Whole-Cell Patch Clamping, Methods in Enzymology 207, 1992).

At this point we need to distinguish between current clamp (CC) and 
voltage clamp (VC) recording. "Series resistance" although present also 
in CC recordings is a term used generally in conjunction with VC 
recordings. In case of two electrode voltage clamping it defines the 
resistance between the current injection electrode tip and the membrane. 
In case of a continuous single electrode clamp (e.g. patch clamp 
amplifier) the series resistance is both the access resistance and the 
intrinsic series resistance (same as in two electrode clamping). Both 
two electrode clamp amplifiers as well as patch clamps have a unit for 
series resistance compensation, this is an important part of the 
amplifier tuning procedure.

About series resistance in conjunction with voltage clamp and patch 
clamp amplifiers there is a vast literature. The origin of this 
resistance and the impact on the recording has been analyzed since a 
long time, see e.g. the important papers published by Bob Eisenberg and 
colleagues in  the seventies of last century.

    * R. S. EISENBERG and E. ENGEL The Spatial Variation of Membrane
      Potential Near a Small Source of Current in a Spherical Cell, THE
      JOURNAL OF GENERAL PHYSIOLOGY VOLUME 55 1, 736-57,  1970
    * EISENBERG, R. S., and E. A. JOHNSON., Three Dimensional Electrical
      Field Problems in Physiology, in: JAV Butler and D Noble (eds)
      Progress in Molecular Biology and Biophysics, Vol. 20, Pergamon
      Press, Oxford& New York, 1970
    * Engel E, Barcilon V, Eisenberg RS. The interpretation of
      current-voltage relations recorded from a spherical cell with a
      single microelectrode. Biophys J. 1972 Apr;12(4):384-403

There is of course more literature e.g. textbooks like

    * Jack, J.J.B., Noble, D. and Tsien, R.W. (1975) "Electric Current
      Flow in Excitable Cells". Claredon Press, Oxford., 
    * Ferreira, H.G, and Marshall, M.W. (1985) "The biophysical basis of
      excitability", Cambridge University Press, Cambridge
    * B. Hille "Ion Channels of Excitable Membranes", Sinauer
      Associates, 1992 .....


The reason why I mentioned the Eisenberg papers is that they give an 
insight into the problems of current clamp measurements as well. In the 
CC terminology "series resistance" is not used.  CC amplifiers usually 
have a "BRIDGE BALANCE knob which allows to compensate the voltage drop 
across the microelectrode or access resistance. See e.g. D. Ogden "The 
Plymouth Workshop Handbook" 
(http://www.utdallas.edu/~tres/microelectrode/me.html), first chapter 
http://www.utdallas.edu/~tres/microelectrode/microelectrodes_ch01.pdf.

In this chapter both the capacity compensation as well as bridge balance 
is described in detail, including examples of how to set it up. These 
means you need an amplifier with these features. The situation is 
different if you use a patch clamp amplifier in current clamp mode. This 
amplifier is much different from an "BRIDGE" amplifier and can cause  
problems, especially due to the lack of bridge balance. See papers  

    * Magistretti, J., M. Mantegazza, E. Guatteo, and E. Wanke. 1996.
      Action potentials recorded with patch-clamp amplifiers: are they
      genuine? Trends Neurosci. 19:530 -534.,
    * Jacopo Magistretti, Massimo Mantegazza, Marco de Curtis, and Enzo
      Wanke Modalities of Distortion of Physiological Voltage Signals by
      Patch-Clamp Amplifiers: A Modeling Study, Biophysical Journal
      Volume 74 February 1998 831-842

So far the ideal amplifier for CC investigations will a BRIDGE amplifier 
with good capacity compensation.

You asked also about the discontinuous amplifier. These amplifiers 
sample the potential in the gap between the discontinuous current 
injection, thus eliminate the electrode resistance (or access 
resistance) completely. Such an amplifier need a very good capacity 
compensation and must be capable of high switching frequencies. I 
developed such an amplifier for my engineer thesis, please see the 
following papers:

    * Richter, D.W., Pierrefiche, O., Lalley, P.M. and Polder, H.R.
      (1996) Voltage-clamp analysis of neurons within deep layers of the
      brain. Journal of Neuroscience Methods 67: 121-131
    * Draguhn, A., M. Pfeiffer, U. Heinemann and H.R. Polder (1997) A
      simple hardware model for the direct observation of voltage-clamp
      performance under realistic conditions, J. Neurosci. Meth. 78:105-113
    * Polder, H.R.  and D.  Swandulla (2001) The use of control theory
      for the design of voltage clamp systems: A Simple and standardized
      procedure for evaluating system parameters, J. Neurosci. Meth.,
      109: 97-109
    * Sutor, B., Ch. Grimm and H.R. Polder (2003) Voltage-Clamp
      controlled Current-Clamp Recordings From Neurons: An
      Electrophysiological Technique Enabling the Detection of Fast
      Potentials Changes at Preset Holding Potentials, Pflugers Arch.
      446, 133-141.

This amplifier is commercially available and used in hundreds of 
publications both with sharp and patch electrodes. It has both a bridge 
mode and the SWITCHED CC/VC modes, and is the "ideal" amplifier for 
intracellular or whole cell current and voltage clamp investigations.

Please contact me again if you have further questions.

Best regards,

Hans Reiner Polder
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neur-sci-request from oat.bio.indiana.edu wrote:

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>Today's Topics:
>
>   1. Re: Help with series resistance and EPSP variations	(Jeff II)
>      (Jeffrey Lopez)
>   2. Re: Re: Help with series resistance and EPSP	variations (Jeff
>      II) (Dr. Michael Ferber)
>  
>
>
> ------------------------------------------------------------------------
>
> Subject:
> [Neuroscience] Re: Help with series resistance and EPSP variations 
> (Jeff II)
> From:
> Jeffrey Lopez <jeffrey.yaime from googlemail.com>
> Date:
> Mon, 26 Nov 2012 13:28:00 +0100
> To:
> neur-sci from oat.bio.indiana.edu
>
> To:
> neur-sci from oat.bio.indiana.edu
> CC:
> neur-sci from magpie.bio.indiana.edu
>
>
>Hi Michael, thanks a lot for your comments and the link.
>Yes, I am doing current clamp recordings from soma as you mentioned.
>Still I dont get the point how do you conclude that RS is equivalent
>to the resistance of the cytoplasm (Ri).
>On the other side, how would affect changes in RS the time constant or
>the slope of my EPSP?
>And how will solve the problem the use of a discontinuous mode amplifier?
>Thanks again, best wishes
>
>
>
>2012/11/25, neur-sci-request from oat.bio.indiana.edu
><neur-sci-request from oat.bio.indiana.edu>:
>  
>
>>Send Neur-sci mailing list submissions to
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>>or, via email, send a message with subject or body 'help' to
>>	neur-sci-request from net.bio.net
>>
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>>than "Re: Contents of Neur-sci digest..."
>>
>>
>>Today's Topics:
>>
>>   1. Re: Help with series resistance and EPSP variations
>>      (Dr. Michael Ferber)
>>
>>
>>----------------------------------------------------------------------
>>
>>Message: 1
>>Date: Sat, 24 Nov 2012 11:43:18 +0100
>>From: "Dr. Michael Ferber" <Michael.Ferber from gmx.de>
>>Subject: Re: [Neuroscience] Help with series resistance and EPSP
>>	variations
>>To: neur-sci from magpie.bio.indiana.edu
>>Message-ID: <2234704.ku0AEN0ukF from ragnarok>
>>Content-Type: text/plain; charset="us-ascii"
>>
>>Hmm.... hwta type of experiment are you doing? I assume your patchj
>>electrode
>>is lovcated at the soma and you are in current clamp mode. Your EPSPs are
>>eliceted somewhere distant in the dendrites of a neuron. Right? Uner these
>>circumstances I would say that your RS is equivalent to the resistance if
>>the
>>cytoplasm. To solve your question you may have a look to the length constant
>>
>>of axons (or dendrites).
>>
>>See for example here: http://neuroscience.uth.tmc.edu/s1/chapter03.html
>>
>>
>>Best wishes
>>Michael
>>
>>    
>>
>>>Hi everybody, there is a question I have since some time but still
>>>dont have a convinvcing answer.
>>>When your series resistance changes during a patch clamp experiment,
>>>1- which influence does it have on my EPSP slope and amplitude?
>>>2-why?
>>>3-how would it help or not to use a discontinuous (switching) amplifier?
>>> If anyone could help me to understand I will appreciate it, please
>>>try to be explicit in answering. Thanks a lot in advance!
>>>
>>>_______________________________________________
>>>Neur-sci mailing list
>>>Neur-sci from net.bio.net
>>>http://www.bio.net/biomail/listinfo/neur-sci
>>>      
>>>
>>--
>>Dr. Michael Ferber
>>Gottstreustr. 3
>>D-34127 Kassel
>>Tel:(+49) 0561 8165785
>>mobil: (+49) 01577 3965785
>>email: Michael.Ferber from gmx.de
>>
>>
>>
>>------------------------------
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>>End of Neur-sci Digest, Vol 89, Issue 7
>>***************************************
>>
>>    
>>
>
>
>  
>
>
> ------------------------------------------------------------------------
>
> Subject:
> Re: [Neuroscience] Re: Help with series resistance and EPSP variations 
> (Jeff II)
> From:
> "Dr. Michael Ferber" <Michael.Ferber from gmx.de>
> Date:
> Mon, 26 Nov 2012 23:21:16 +0100
> To:
> neur-sci from magpie.bio.indiana.edu
>
> To:
> neur-sci from magpie.bio.indiana.edu
>
>
>Hi Jeffrey,
>  
>
>>Yes, I am doing current clamp recordings from soma as you mentioned.
>>Still I dont get the point how do you conclude that RS is equivalent
>>to the resistance of the cytoplasm (Ri).
>>    
>>
>if I remember correct RS is the sum of all resistances between tip of 
>electrode and membrane. This means it is mainly the resistance of the 
>cytoplasm + the resistances between the membrane and the ground. (structures 
>around the nerve cell, resistance of the bathing solutions and more) Under 
>normal conditions these should be constant. In patch clamp experiments the 
>cytoplasm may change its resistance due to exchange with the pipette solution.  
>So this is the only value that may change. 
>  
>
>>On the other side, how would affect changes in RS the time constant or
>>the slope of my EPSP?
>>    
>>
>In current clamp you record potential changes. If an EPSP is elicited distant 
>from your electrode it spreads passively and is damped according to the values 
>of membrane resitance membrane capacitance and resistance of the cytoplasm. 
>This means the more distant the origin of your EPSP the smaller the amplitude 
>and the lower the slope of your EPSP. If you reduce the cytoplasmatic 
>resistance  without affecting RM Amplitude and slope of the EPSP  will 
>increase compared to the initinal conditions.
>  
>
>>And how will solve the problem the use of a discontinuous mode amplifier?
>>    
>>
>I do not see any benefit. As far as I know switching modes mainly apply to 
>voltage clamp recordings. 
>  
>
>>Thanks again, best wishes
>>    
>>
>There is also an old thread dealing with these things 
>http://www.bio.net/bionet/mm/neur-sci/2007-February/thread.html#61664 
>([Neuroscience] Series resistance and capacitance compensation in current 
>clamp)
>
>
>Best wishes
>Michael
>
>  
>
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