It is said that dendritic spines can naturally occur among primary mouse neurons and their density or morphology can be thoroughly assayed. So can anybody provide any information?
At the moment we want to evaluate effect of some chemicals at synaptic level, but we wonder how valid such effect on cultured spines is, given that synapses need extra modulation in vivo.
Thanks a lot if you could help!
Any experiment performed in vitro will only give hints on the true mechanisms of in vivo brain function and physiology. However, the statistical power, reproducibility and feasibility to answer a particular question may be enhanced in vitro by offering a reduced number of system variables. I believe for your question it is perfectly fine to start in vitro. You could measure synaptic strength by measuring frequency and amplitude of miniature postsynaptic currents in cultured neurons or better in acute brain slices, that are less likely to suffer from 'unnatural' plasticity. mEPSCs or mIPSCs are measured in the presence of TTX, so network activity should not effect your measurement. This is in contrast to culture medium and bath solutions that you should carefully think of, but will depend on the particular drug you aim to test. You could then verify your results in vivo. May be look at Gina Turrigianos work on homeostatic plasticity that she first described in vitro and later verfied in vivo. For example: http://www.ncbi.nlm.nih.gov/pubmed/23678123
I agree with Cornelia in terms of how in vitro data shed light on in vivo functions. However, what are the methods used to test in vivo synaptic strength and plasticity?
If you have access to a good microscope (confocal, 2-photon) you can try to measure the number of spines over some period of time. From this you can extract data such as spine density (spines per micrometer), fractions of dynamic spines (new spines, lost spines, transient spines) and their evolution over time etc.
You could also measure intensities which are found to correlate with actual spine volume, as validated with electron microscopy (see link).
One step further is to construct models based on these measured spine parameters in order to make predictions and extract fundamental characteristics of your system.
As you alluded to, it is known that primary cultures do make an excess of connections as compared to the in vivo situation, so ultimately any results will have to be validated in vivo. It stills makes sense to start with cultures if your lab is adjusted to those methods, as setting up in vivo studies can take a considerable amount of time (years).
I hope this helps!
Vassilis
http://ac.els-cdn.com/S0896627305000048/1-s2.0-S0896627305000048-main.pdf?_tid=5eb972d6-a21b-11e4-80fa-00000aacb361&acdnat=1421920096_0b5b0c7a6210b14bfbe5ef6e14493a3e
One could also induce plasticity in vivo and then measure parameters of synaptic strength ex vivo e.g. using electron microscopy or patch-clamp in acute brain slices. But first you would need to get an idea on which time scales and at which concentrations your drug is working. It could be that you need to incubate cells for days to see an effect or you can throw it on and it changes synaptic properties immediately.
This is a difficult question because synaptic properties are heterogeneous (they vary from cell to cell and from synapse to synapse). You should first identify a group to cells you want to study (say in the V1 area) and then you could try two-photon imaging, and/or acute brain slices and/or in vivo patching.
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