Hi, I new started patch clamp electrophysiology techniques and I m working in the cohlear nucleus neurons but I cant distinguish neurons and neuroglia in this area. I realize that most of cells which I did seal is glial cells. What could be reason for it?HowHow do I overcome it. Also would you give me some references /resources for current clamp analysis. thanks.
Merhaba arkadaşım.
Neurogliya da hiç action potential yok ve heç neye cavab vermez ama neuron stimulus-a cavab verer. Çok rahat biri birinden seçmak olar
This may be difficult a priori. After patching you can see the resting potential in current clamp: very hyper polarized means its probably glia. With experience you will learn to avoid the glia. Try putting a fluorescent dye in the patch pipette and seeing the morphology of your cells. You will learn to distinguish this after a while and with experience...
I don't have any experience in cochlear nucleus, but have patched many neurons and astrocytes in cortex. As mentioned by Henrique, the hyperpolarized membrane potential might give an indication. Moreover, importantly, when injecting depolarizing currents glial cells will not show the typical fast action potentials that are seen in neurons. So when you are injecting currents that depolarize the cell to 0 mV or higher and still get a linear IV relation without fast action potentials you most probably have patched a glial cell. A third characteristic will be the very low membrane resistance (due to extensive gap junction coupling with other glia). As said before, it mainly takes practice. After some time you will be able to distinguish them based on your microscope image, and manage to distinguish neurons before patching.
I want to thank you for your valuable comments. I hope that happens as your says, and I can do distinction with experience gain. Would you give me some references /resources for current and voltage clamp analysis. Thanks again and best regards .
Dear Caner, I agree with Henrique and Rogier as long as we consider astrocytes as the only glial cell you may encounter when patching in the CNS... Unfortunatelly for neurocentric oriented patch-clampers, but not for the health of your brain, you have also other types of glial cells that have quite different properties than astrocytes in terms of electrophysiological behavior. An easy one to distinghish is the microglia. In physiological conditions (even in slices) the input resistance of these cells is usually in the order of 1gigaohm. They express, no voltage activated currents and thus no action potential. The resting membrane potential is somewhere in between -20 and -70 mV but not so easy to measure due to the comparable membrane and seal resistances. They are not coupled through gap junctions. If you are in pathological conditions or during normal postal development, things may change (voltage activated potassium channels, chloride channels etc...) but still no action potential!. You can check the papers of Helmutt Kettenmann, Claudia Eder, or mine, (although none of us recorded these cells in current clamp I think). The most difficult ones are the NG2 cells of oligodendrocyte progenitors (OPCs). Those cells express voltage activated sodium channels and can fire small action portentials. David Attwell published a paper some years ago showing that some of these cells can fire repetitive action potentials just like neurons but I am not aware of any other report of this kind. What is sure though is that they can fire one or two-three small action potentials. And they also have actual synaptic inputs, so you will detect IPSPs and EPSPs from these cells, but usually at lower frequency than in most neurons. Also, the properties of NG2 cells change during postnatal development. You should check the papers of Dwight Bergles, Christian Steinhauser, Maria-Cecilia Angulo, David Attwell, Maria Kukley for instance to get a better idea of the behavior of these cells. Finally, when NG2/OPCs differentiate into oligodendrocytes, they loose their ability to fire action potential, they don't show anymore synaptic currents and they express different potassium channels. Much easier to distinguish from neurons than NG2 cells.
I know this seems to be an awfull situation for beginers with an interest only in neurons but you will learn rapidly to distinguish neurons from glia. Using good optics (DIC or Dodt contrast) also helps to recognize them in slices where they usually have not the same aspect as neurons.
Dear professor; Thank you very much for your interest in also for taking your time for me .. in fact I'm trying to patch clamp is approximately 1 year and I can receive recordings in current clamp, but I'm talking about the subject it scared me and I thought I made a huge mistake somewhere. I hope that I develop myself.. I took note that author and important points that you mentioned. I will search them.Thank you so much. Best regards.
Sevgili Caner,
I agree with above comments. It is actually quite rare that you would be recording from glial cells in brain slices. In my experince the chances are abot 2-3%, therefore you should not worry about it too much. In brief glial cells would not have action potential or fast inactivating Na currents. You may be having problem in using some of your equipment properly. Do you have a copy of the Axon guide for Electrophysiology and Biophysics, by Alan S Finkel? if not You should get one, because it is very useful for beginners. You can also download my 2013 methods paper describing brain slice preparation and recording methods, methods chapter from the resrach gate.
best wishes, hoscakal, Refik
Bir sey degil. Good luck with your experiments.
best wishes, hoscakal, Refik
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