We have homogenized Taenia crassiceps larvae and are puffing the homogenate onto pyramidal neurons during whole-cell patch-clamp recordings in organotypic hippocampal slice cultures. We need to put the puffer pipette very close to the neurons - ie the neurons move during the puffing - we see obvious depolarization ie 10 - 20 mV worth, that is not blocked by glutamate receptor blockers (kynurenic acid, AP5, CNQX). The pH is roughly between 7 and 8. Osmolarity of the homogenate is 300 ish. Also the K+ concentration within the homogenate is 4 mM and the effect is there when using a caesium internal. Is what we are seeing an artefact? What substances cause neurons to depolarize, what should we be thinking of as causative agents that might be in the homogenate?
Hello Joseph,
Did you try blocking Calcium channels?
I am not sure what you are looking for. Looking at Calcium currents as a possible source of depolarization may be helpful. Especially when rise in internal calcium through cascading activation of calcium currents is known to activate several cellular messenger pathways several of which have been implicated in quite a few pathophysiological conditions.
Some candidates that come to mind are
1) N-type Calcium channels, for instance, have been shown to be mechanosensitive (since you mentioned puffing very near the neuronal membrane)
2) Another possibility could be the activation of currents though non-specific cation channels (like the ones activated by maitotoxin) and subsequent activation of calcium currents (L-type channels). Maitotoxin is a toxin produced by a dinoflagellate. It is known to cause Ciguatera a sickness caused by eating certain types of fish.
Hope you find this helpful
Shreya
Hi Shreya, that is very useful - mechanosensitive channels are something to think about seriously as well as the non-specific cation channels. Thanks!
Have you thought of ATP receptors?
As suggested in the previous comment, calcium channels may be involved, and they may be mechanosenstive. Perhaps it's also worth looking into the cationic TRP channels? (https://www.nature.com/nature/journal/v432/n7018/full/nature03066.html)
But if it turns out to not be mechanosensitive, I wonder if P2X channels are involved? Ca2+ entry through P2X channels could lead to enough depolarization to activate other voltage gated calcium channels like the L-type. Cells of any sort I imagine are full of ATP!
I realize that it is a rather obvious comment, but what happens when you puff on control media/saline that does not include the homogenate, from the same distance?
I second Jonathan, first you need to figure out if it is an agent in your homogenate or simply mechanically evoked.
Another alternative is to (volume permitting) bath your slice in the homogenate.
The timecourse and evolution of the depolarization can also help to limit your search to mechanisms of comparable timecourse.
Try to do an I/V in voltage clamp (basically hold the cell at different Vm during subsequent puffs), that can tell you about the ionic composition of your depolarizing current through the reversal potential.
I would expect multiple agents, though, as a homogenate will contain lot's of possible candidates (especially these worms do secrete neurotransmitters to fool the gut nervous system, right?). Another idea is to puff different homogenates (like brain, but that likely will have high K) and compare.
Let us know how is it going, sounds like a fun experiment.
Most likely the homogenate contains glutamate as it is near millimolar concentrations in cytoplasm which will activate glutamatergic receptors in the pyramidal neurons. Glycine concentration is also likely sufficient to activate GluN1 subunits of heteromeric NMDA receptors. I suggest coapplication of ionotropic glutamate receptor antagonists, eg. CNQX and APV.