I am a student who major in neuroscience specializing in the function of calyx.I wanna get a nice mouse slices with MNTB. if it is necessary to get around 30 degree to cut the front part to get a better EPSCs? My slice looks active now, but I seldom get ~10nA EPSC (maybe once a week),I don't know if the electrode placed too superficial or not.Or the slice is too thick(my slice is 270um).By the way, my mouse age is P16-21.
I can't help with the MNTB prep, but if you are sure your currents are too small, can you tell me more about your patching conditions? Specifically:
* What kind of electrode solution are you using? I would recommend Cs-based. It is much harder to clamp with K-based solution.
* Are you using series resistance compensation? You should if you can, like up to 70%. Compensation has a huge effect on amplitude and time-course of EPSCs.
* What size electrode are you using? In the AVCN, we aim for 1-2 MOhm. MNTB is also big cells, so you can use low-resistance pipettes. This improves recording quality and stability.
* What temperature are you using? I'm not sure how that might affect EPSC amplitude, honestly.
* In the AVCN, the amplitude of EPSCs depends on the strain of the mouse. Maybe this is also true in MNTB?
* I doubt slice thickness is an issue.
-Matthew
I used to make the slices using a slight angle (as in this paper Article An organotypic slice culture to Study the formation of calyx...
) to keep the axons intact. That worked well for me.And, as Matthew already indicates, your series resistance becomes critical if you want to record 10 nA. Even if your Rs is 3 MOhm, your driving force will reduce during the current by Ir x Rs. A simple correction can be found in Traynelis‘ paper (Article Software-based correction of single compartment series resis...
), namely (Vc - Vrev)/(Vc - IRs - Vrev). This correction does not include the current that is lost through the capacitance (see Traynelis’ paper).At higher temperatures the EPSC will be faster and therefore more difficult to record without filtering. (That is what I expect, but I have not checked this.)
Lastly, synaptic release depends on the presynaptic AP and the calcium influx. You are using a standard Ringer solution? Or have you decreased the calcium concentration? I highly recommend that you first follow the pioneering papers from Borst, from Forsythe and from Von Gersdorff (and many others) to see if you get the same results.
Good luck!
Slices should be viable up to 350-400 uM even from aged animals for many hours
Joseph J Munier Martijn Sierksma Matthew Xu-Friedman I'm sorry not to check your answers in time.Thanks a lot for your kind reply, in fact, it is really useful especially changing the slices to up to 300um. Now I can get a nice brain slices basically. To clamping them easier, I'll also check my electrode solution and Rs parameter later. By the way, solution is indeed the key details for patching!
All good advice. Don’t be too wedded to large currents Zoey. They produce the largest errors. When you’ve minimised pipette series resistance, you can also reduce voltage error by reducing the current magnitude. First you can clamp at more positive voltages, such as -40 mV, which will reduce driving force AND will also inactivate postsynaptic NaV, so reducing ‘clamp escape’. Second, consider using a low affinity AMPAR antagonist such as kynurenate (see papers from Henrique Von Gersdorff and ours 2002 and 2003). I strongly advise that you work at physiological temperatures, if you are not already doing so. Good luck.
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