Hi all,
I am conducting 10 minute gap free voltage clamp recordings in hippocampal slice cultures, and would like to be able to monitor the Cm, Rm and Ra at regular intervals (possibly every two minutes) (i am using clampex 11.1). Is it possible to periodically integrate an episodic stimulation event into a gap free recording, so that i can look at and calculate these values from the capacitive transients?
You may apply a linear ramp pulse any time to evaluate any change in access resistance.
You probably can't do that in gap-free mode, but you can set up a long repeating episodic stimulation and add your test pulse in the Waveform.
Short pulses for series (access) resistance monitoring will not affect your gap-free recording too much. Looking at membrane properties (input resistance etc) will require longer pulses, so it might be more useful to interrupt the recording and run a full pulse/step/ramp battery if you expect membrane changes.
Just change your holding voltage by a small amount (say -10 mV) for a second or two and then adjust it back to where ever you were holding it before.
One option might be if you superimpose your voltage clamp protocol with 10 Hz train of 50 ms +1mV pulses which alternate with 50 ms -1mV pulses (period = 100 ms). Thus, the effect on your voltage protocol and the measured current would be minimal.
Then, you need to connect your additional voltage train signal to one line of oscilloscope, which will trigger it, while the current output should connected to 10 Hz notch filter and then applied to a second input of the same oscilloscope. Then you will be able to see the current response to 2 mV voltage steps, which will show to you Ra (somewhat close to the amplitude of this filtered current step), while Cmem and Rmem might be estimated from the decay rate between the steps and the phase shift between the current and the voltage pulses.
However, you need to calibrate your system with equivalent electric circuit of a cell, with different parameters, that are close to your cell parameters.
You could record in Gap Free protocol and set it for 10 minutes, than have it load an episodic stimulation protocol to record series resistance for a minute and then set that to load a the 10 minute gap free protocol. They would load each other until the user broke the cycle.
This option is in clampex under configure-> sequencing keys.
Add the first key as the (record) gap free protocol; on the sequencing tab under next key select the protocol for series resistance. Then make another key for series resistance that loads the gap free protocol.
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