I am looking for methods which can be used to investigate whether the synapses which are being formed after some drug's treatment are actually forming functional synapses. Is the activity restored back into synapses.
So what are the targets and important aspects to be looked at when studying this?
Dear Apurva
One first approach could be by tasking achievement of experimental subjects (mice, rats, etc.). You could have different groups (control, treated, and maybe placebo) and you would design different tasks that allow you measure the fails and successes of the groups.
Additionally, you can prepare a primary neuron culture that allows the neurons to form a network. In this way you can assess changes of neuronal network in presence and absence of a testing drug.
These are ideas only.
Best Regards
Esri
If we are working on hippocampus, and let's say after the drug treatment the synaptogenesis and spinogenesis increases, can we record LTP from CA3- CA1 region to show that the new synapses are able to form functional synapses. I am trying to think all this in terms of a disease model.
You can use immunohistochemistry to look at various proteins that would indicate functionality, such as vGlut or VIAAT which are neurotransmitter transporters. Or synaptophysin which is a vesicle docking protein though that would mainly tell you if the terminal bouton is functional. You don't give much information about how you plan to know if the synapses are new - is this in vivo or in vitro? Knowing more about your experimental design would narrow down your choices as to how to measure functionality.
This would be on histological slices from hippocampus of the transgenic mice treated with saline or the drug in question. So, I was interested to check
1. If the spine morphology has improved after drug treatment in the transgenic mic in comparison to the control. (this might be investigated in fixed histological sections)
2. Does it form increases synapses as it has been previously studied that there is synaptic loss in the transgenic mice. (this might also be investigated in fixed histological sections)
3. So if this drug is able to improve spinogenesis and synaptogeneis at some time point post drug treatment, does the synapses formed after the drug treatment functional too? I wanted to look at that aspect too. (can be investigated in non fixed live acute hippocampal slices from the transgenic mice or in fixed sections, depending on the methods used to show functionalisation of the synapses formed)
Please suggest me in this direction.
Apurva, there are multiple ways to achieve the goals
1. recording mEPSCs is a very good starting point- in-case there is an increase in the number of synapses following your treatment, there should be corresponding increase in the frequency of mEPSCs. then follow it up with input-output curves, where you make field recordings (CA3-CA1), at different, fixed stimulation intensities. if there is a change in the synapse numbers, there will be a marked difference in the slope of the input-output curve between different groups.
2. The other way to do it is immunocyto chemistry where you could stain for PSD95 (structural), GluA1/2 (functional) components of the postsynptic components and a corresponding presynaptic protein like Bassoon, and count the number of 'functional synapses' in a gven dendritic length, which contain PSD95 & GluA1/2 in apposition to the pre-synaptic marker. Compare these across treatments
@Jai: Thanks a lot Jai. I was too thinking for these investigative methodology for answering the functionalisation of synapses post drug treatment. Your suggestions strengthened my thoughts regarding this.
Just one more key question. Won't recording of LTP from such drug treated animals give better understanding of functionalisation of synapses. Because LTP is reported to be impaired in these animals. If post- treatment LTP at CA3- CA1 synapses improves, then I think it does suggest the synapse functionalisation where it will be first shown that drug leads to increased synaptogenesis using immunohistochemical methods as mentioned by you above.
Thanks for the suggestions. Please do try to comment on this.
Interpretation of LTP in this scenario, where your drug is mediating the formation of new synapses could be trickier than we think. It depends on what information you want to get- if you want to show that administrating the drug improves cognitive abilities, which go hand in hand with the resurgence of LTP in these animals, that is fine.
But if your question is about synaptogenesis, then it is completely independent of the classical LTP, which is brought about by addition of new receptors to existing synapses or unsilencing of silent synapses.
Well, thanks for clearing up this difference. They mean two different thins , each of them important for defining two important processes.
Ofcourse I would like to check both the processes i.e. whether mEPSCs frequency is increasing besides LTP improvement in such animal hippocampal slices.
Thanks a lot. That adds one more important facet in the study. That's the advantage of discussion. Thanks...
This is, actually, not a trivial thing to accomplish. Unless you can isolate down to the specific synapse and actually show that it is transmitting, then the best you can generally do it to look for signs that there is a synapse and to hope that there actually is one there. For example: the suggestion of looking for proteins that serve as synapse markers is a very common and good procedure, but no real guarantee that there is actually a synapse there (it is very likely that there is, but no proof). The 'gold standard', if you will is to use electrophysiological means to show that transmission is actually occurring. The problem with doing this is generally technical, i.e. ensuring that the transmission recorded is from your 'new' synapses, as opposed to formerly existing synapses.
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