I was worried that anyone might not respond because my questions were too basic...

Thank you so much for the response.

>Firstly, you do not need 13 pulses for leak subtraction. Especially for such a large slow current. 4 pulses will be adequate.

I may have misunderstood about the P/N subtraction... I just thought that, for the leak subtraction, I have to sum the thirteen -10mV pre-pulse traces, invert the polarity and subtract it from the main trace. Neurons are depolarized for 130 mV max (from -110 to +20 mV) in my plan, and therefore, -32.5mV step (=130/4) is required to subtract the leak with the 4 pre-pulses. But such hyperpolarization is too large. Hyperpolarization-activated inward current will be induced. (I'm thinking about adding low Cesium to ACSF for this problem.)

Instead of the summation above, is it OK to average several (4 or 6) 10mV-height pre-pulses and multiply the averaged trace (13 times for example)?

>Cell capacitance should LARGELY be subtracted by adjusting for the whole-cell parameters on the preamplifier.

I did not compensate cell capacitance in synaptic current experiments (only the pipette capacitance was compensated), and I checked the series resistance with the capacitive current. (So I did not touch the capacitance dials on the amp after break-in.) How do you monitor the change in series resistance when the cell capacitance is subtracted by the amp? Calculate from the residual currents? or keep adjusting/reading the Rs dial?

>If your only "blocker" is TTX, you're going to have significant calcium currents that will muddy the situation. I suggest Cadmium to block them.

I tried the recording two days ago, but the traces were terrible. It is as you pointed out. I would rather not use cadmium if possible, because it needs a strict emission control... Is it acceptable to use Manganese instead of Cadmium?

>Have you considered using nucleate patches or on-cell recording?

I didn't think of it. That sounds good. I will try the cell-attached mode too.

Thank you again for helpful suggestions.

Regards,

Fukushima

Thank you again for a lot of helpful information.

>I wrote a little blog on the subject, that might interest you. 

I checked your blog just now. I was impressed with the phrase "The important thing is to know when the approximation is good, and when it is bad". This is a quite simple and a very very important thing, but this is also easily forgotten...

>I've never come across people using Manganese to block calcium currents.

I found a paper in which Manganese is added to the ACSF (http://www.jneurosci.org/content/20/11/4145.long).

But, as you said, Cd is the most popular. I will stock Cd.

Thanks to you, I have a feeling things will be all right!

Regards,

Fukushima

Hi, Akihiro!

I've been involved in a side project regarding A-type potassium current in the CA1, and I have to say it is... challenging ;) As William noticed when performing whole-cell patch clamp you have to use cadmium or cobalt. We were using cobalt, but it was difficult to get a stable recording, and our whole system was pink. So I would also suggest trying attached mode instead of whole-cell, which will also solve the problem of space clamp.  

If you are still working on this project, it would also be interesting to check the latency of the first spike, as A-type channels are strongly involved in this phenomenon ( it was mentioned for example in Kim et al., 2005 in Journal of Physiology).  I would strongly recommend it as a separate type of experiments. Recordings in current clamp are less frustrating, and you will have another lead that something is going on with the A-type channels :)  

Good luck with your experiments! ;)