Re: Neur-sci Digest, Vol 89, Issue 9

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Re: Neur-sci Digest, Vol 89, Issue 9

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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
[hidden email]




[hidden email] 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 <[hidden email]>
> Date:
> Mon, 26 Nov 2012 13:28:00 +0100
> To:
> [hidden email]
>
> To:
> [hidden email]
> CC:
> [hidden email]
>
>
>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, [hidden email]
><[hidden email]>:
>  
>
>>Send Neur-sci mailing list submissions to
>> [hidden email]
>>
>>To subscribe or unsubscribe via the World Wide Web, visit
>> http://www.bio.net/biomail/listinfo/neur-sci
>>or, via email, send a message with subject or body 'help' to
>> [hidden email]
>>
>>You can reach the person managing the list at
>> [hidden email]
>>
>>When replying, please edit your Subject line so it is more specific
>>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" <[hidden email]>
>>Subject: Re: [Neuroscience] Help with series resistance and EPSP
>> variations
>>To: [hidden email]
>>Message-ID: <2234704.ku0AEN0ukF@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
>>>[hidden email]
>>>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: [hidden email]
>>
>>
>>
>>------------------------------
>>
>>_______________________________________________
>>Neur-sci mailing list
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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" <[hidden email]>
> Date:
> Mon, 26 Nov 2012 23:21:16 +0100
> To:
> [hidden email]
>
> To:
> [hidden email]
>
>
>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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>
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>

--

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Phone: +49-(0)7141-97302-30
Fax:   +49-(0)7141-97302-40
Web: www.npielectronic.com; Email: [hidden email]

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