Hi collegues,
I'm trying to study the glutamate release probability in hippocampal cultured neurons. I started using hyperosmotic extracellular solution (1M sucrose) but this method evaluates the RRP inducing Calcium-independent release of glutamate. Do you know any methods to study Calcium-dependent release?
Thanks in advance for helping me
Have you checked to see if your cultures have spontaneously developed autapses? I found that inhibitory cells from neocortical cultures regularly had autapses. Though I can't remember finding many excitatory autapses.
You also don't need to do dual whole-cell recordings. You could perform loose seal recordings on the second cell. This will only allow you to investigate the connections in one direction, but you can use the same pipette, which makes it a lot faster.
Hi,
For studying calcium dependent glutamate release, you could use two-photon Ca2+ imaging-derived optical quantal analysis and correlated electron microscopic reconstructions. Check this article: (doi: 10.1038/nn.3137)
Hi guys, thanks for your answers. I'm searching for a pharmacological method to study by patch-clamp recordings the release probability. I'm searching on pubmed about this but at the moment i did not found nothing useful for my experiments. Maybe the only possibility is to use double patch-clamp recordings :)
Maybe you should be more specific about what you actually want to do. It is impossible to measure the exact release probability at a synapse using only "pharmacology". If you want to to put a number on the release probability (that is, a number between 0 and 1) you must have some kind of read out of whether a vesicle has been released or not. I gave you two only electrophysiological methods that do not require dual whole-cell recordings. Daniel gave you an optical method (which, in culture, can just be done with one-photon fluorescence). There ARE other possibilities beside double whole-cell recording.
Hi William,
you are right, but in my project i have to evaluate the release probability in hippocampal cultured neurons silenced for a protein. The silencing for this protein in my opinion is causing some alterations in the glutamate release probability through some transynaptic effects. I tried to study the RRP using hypertonic solution and the pool of vesicles seems similar between 2 conditions, but sucrose-induced release it's not calcium mediated. For this reason i would to study the calcium dependent release and to do this i can't use autaptic cells and i was searching for an easier as possible electrophysiological approach to perform this experiment.
Thanks for the contribution.
Luca
Well if all you are interested in is a *relative* change, then you can use field stimulation. You can place a large bipolar electrode somewhere on your cultured neurons, and evoke an EPSP, then look at the paired pulse ratio. If the paired pulse ratio is higher in your "altered" cells, then you probability has decreased.
You could also look at the rate of miniature EPSCs, assuming similar plating density, you could get alterations in the rate of mEPSCs if the proability of release goes down.
The first approach could be useful in my experimental situation. For the second one i have the data about the frequency of mEPSCs but this parameter changes if the release probability changes and also if the number of post-synaptic contacts changes, so if we hypothesize that the number of post-synaptic contacts decreases and the release probability increases maybe we do not see a net effect on the frequency of mEPSCs, do you agree?
You can measure glutamate, aspartate, glicine, GABA release using HPLC
If you are interested in the exactly method I can give it to you.
Hi M. Pilar,
i'm searching for an electrophysiological method to study glutamate release. Thanks for your input anyway.
M.Pilar Conzález
Hello M. Pilar,
It was quite a while since your answer, but I am very interested in measuring glutamate release! Could you send me the method, please?
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