Neuron only exists under standard whole cell!?!?!

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frustrated student
frustrated student's picture
Neuron only exists under standard whole cell!?!?!

Hi-

Forgive me in advance for not giving the type or neuron or really specific details.  My PI is paranoid about this issue!!!

Background:
About 6 years worth of data from my lab (using standard whole cell current clamp recordings, fura2 calcium imaging and FLIPR membrane potential dye imaging) support that a specific type of neuron exists.  I have repeated these experiments to ensure that I am able to seal/record the cells in brain slices (or dye/image the cultured neurons) and am confident that what I am looking at are the same neurons we have published on. Basically, in this brain region, approximately 50% of the neurons should be what I am looking for. Moreover, a collaborator has tagged these neurons with GFP (it is tagged to something that is specific to these neurons in this area of the brain) and when I patch the glowing neuron, I get the desired effect 100% of the time. 

Issue with standard whole cell:
That we need to put ATP or GTP in the patch pipette has always been an issue given what we claim is metabolically going on in these neurons.

Reviewer solution:
perf-patch or cell attach these neurons without ATP or GTP in the pipette

Problem:
When I try to perf-patch these neurons using amphotericin or nystatin in the patch pipette  or use loose cell attach techniques I cannot find the neurons!!!  I have patched 104 neurons using perf patch techniques and none respond as they should.

I have no ideas other than the potential for amphotericin or nystatin-dependent pertubations in membrane cholesterols to cause this issue.  I'm lost and my PI has no idea where/how to help me with this issue.  Any ideas?

FYI, before you say that the neuron doesn't exist (that has been a common comment because we can only find it when there is ATP or GTP in the pipette), please note that when imaging (FLIPR or Fura2) there is no ATP or GTP in the bath and the neurons show up as expected.

Bluejay
Bluejay's picture
You might want to look at the

You might want to look at the role of ATP in priming or altering the cell via activation of purinergic receptors, calcium increase and subsequent protein kinase c activaton and phosphorylation.  Most cells contain P2Y receptors.  See Samways & Henderson Cell Signal 2996;18:151-161 for ATP effects on cell receptors..  Suramin is a specific inhibitor of P2Y but you can check out the Sigma or Tocris catalog for others.  Perhaps you would see the effect with ATPgammaS which is a P2Y agonist but would not be hydrolyzed.

frustrated student
frustrated student's picture
thanks.  i'll have a look at

thanks.  i'll have a look at that.  i got one response from a colleague that doesn't seem plausible which is to look at the role of stretch receptors/osmotic pressure receptors because that might play a role in the difference between the types of seals.  that seems like a stretch (no pun intended) no?  i can't see how a simple stretch receptor can change currents over a 2 hour recording.