Hi Huong,

I've not used the protocol you're planning, but have done a fair amount of patch clamp on hippocampal neurons from E18 rats, as well as multi cell recordings. I find that maturity of function is later than 14 days, say 20 to 25 days, and that recording stability at these later time points is better.

I've not published the multi cell recordings yet, but the maturation study is here:

Article Neuronal Electrophysiological Function and Control of Neurit...

Hi,

I did  some NMDAR-mediated mEPSC on hippocampal neurons from E18 rats. You may have a look at it (Ning K et al J Neurosci 2004).

Cheers,

Ke

Thank you Justin and Ke! I will look at your publications. :)

Dear Huong,

We routinely do field stimulation across 6mm of neuron monolayer and we get consistent results with 12-16mA. A few basic questions: I guess that you place the two Pt foils on opposite sides of the culture (?) and that you apply your stimulations through and isolation unit (I do not know one that can do 65mA output). If you connect an Ohmmeter instead of the isolation unit, how much resistance do you get ? If it exceeds a few kOhms, try roughing the surface with sandpaper and also google "platinum black". Regarding the pulse pattern, you may have to measure the capacitance of your Pt electrode pair (after the procedure above) to check charging time constant. If your pulses are too short, your cells may "see" much less voltage than you think.  I hope this helps, Thomas  

Hi Thomas,

Thank you for your response. Yes, I placed the two foils on the two opposite edges of the culture, which is about 22 mm apart (as this is the size of my coverslips - 22x 22 mm). We use a modified version of the Isostim A320, which should be able to generate up to 100 mA. 

By saying connecting an ohmmeter, did you mean to measure the resistance between the two foils? 

For measuring capacitance, do you suggest just use a protocol in with clampex to do this? I guess one thing I have not explored much is the duration of the stim. I have tried changing the stim intensity and polarity extensively but none of them seem to give consistent response yet. 

You are always so helpful! Thank you so much!

Best,

Huong

Dear Huong, yes the resistance between the foils when immersed in the bath. Simply connect your two wires to an Ohmmeter (instead of the A320).  If your Pt foils are somewhat hydrophobic, the resistance/capacitance could be very high. Also do you have to keep the electrodes so far appart? Two parallel Pt wires, 1mm appart on a mount connected to a simple micromanipulator would allow you to place them just above the cell layer and to lower the saline level, maximizing the efficacy. For capacitance, you could simply connect an oscilloscope to the two wires, you will see immediately what your output pulse looks like. You would have to increase pulse duration until voltage reaches steady-state.  Note: if you use the digidata instead of an oscilloscope, you will have to provide 1/10 attenuation. Good luck

Dear Thomas,

I see! I will try what you suggested. 

Regarding the electrode distance, I mounted the foil onto my chamber and usually just put the coverslips in between. It makes sense to shorten the distance between the two foils. I guess in your case they are put together as a totally separate component? Would you mind taking a photo of your set up to share with me? Do you/could you move them in order to patch on different parts of the coverslip? I guess this also depends on the size of your coverslip and chamber. 

Yes, I am using a digidata from Axon Instruments. I am not sure if I understand the rationale behind the 1/10 ratio.  Are you scaling at 10X ratio when reading through the Digidata?

Thanks so much!

Best,

Huong

Dear Huong, The compliance of the A320 is about 100V (ie: it will increase output voltage as much as necessary to reach your target current, only limited by the internal voltage source ~100V). The maximum input voltage for the Digidata is 10V, hence the need for a 1/10 voltage divider. If you want to keep fixed electrode, I would suggest to have a look at Warner's RC-49MFS and RC-27NE2 chambers for some ideas. For the moveable field electrode, something like the two-pronged fork, like the miniature version of the one used to hold the Thanksgiving turkey, :o). If the two prongs/electrodes are 2mm apart, this leaves a huge space for patching in between. Good luck

Hi Thomas,

Oh I see. Thank you for helping me understand the 1/10 voltage divider. 

We actually just got a 3 mm spacing bipolar platinum stim electrode from FHC, which probably functions similar to the two-prolonged electrodes that you mentioned. I am going to try them out tomorrow. I am hoping that with this new electrode, I won't need as much current and get more reliable response. In your experience, how far do you usually put the stim electrodes in comparison to the recorded neurons? Like, will they be on the same field of view when looking with 60 X objective? With these movable electrodes, what is the range of your stim duration?

Best,

Huong

Dear Huong, Yes this is exactly what I would suggest to buy or build. I guess that you got a PBSB3.. from FHC. The exposed tip is quite long on those and I would recommend bending the tip to make the exposed Pt wire parallel to your coverslip, for maximum "area of effect" . With a 60x objective, the fov is about 350um  so you can patch "between" the two bent electrodes (spacing of 2.8mm)  without optical interference. Disclaimer: I assume not responsibility regarding any real or perceived damage to your brand new FHC electrode, but I strongly recommend doing the bending under a binocular macroscope  :-). Good luck