Dear all,
I am interested in doing patch-clamp recording in dendrites to investigate if one segment of the dendritic tree into the amygdala receives inputs from two different regions. My question is: How can I be sure that the signal I see is because of the portion that I am patching is receiving information from the upstream regions that I am stimulating, or because I am detecting signal coming from other branches activated by the inputs ?
Thanks in advance for your help.
Best,
This is a pretty hard thing to do, and I would recommend a set of experiments to show this. Firstly, anatomical studies would be a great start (e.g. AAVs expressing two different colour markers injected into your two upstream regions, then fill a neuron in the amygdala with a third colour of Alexa. It wont prove that you are getting true synapses (you need EM for that) but it's a great place to start).
Electrophysiologically, you should be able to combine evidence to suggest that an upstream nucleus is not responsible for the passing on the information by looking at the delay between stimulating and the rise of the EPSP... and also the variance in the onset. If your pathway is disynaptic, you should have a much greater delay and a much much greater variance.
Assuming you are on rig with suitable fluorescence, when you patch the dendrite you can insure that there are no dendrites near by. Then you can bring in a puffer pipette filled with CNQX/AP5 and Alexa, and puff the blockers locally. This will show you if the inputs are arriving on the same dendrite. This will be a pretty annoying experiment, as most of the time I assume the input you are recruiting will be on seperate dendrites or mixed across multiple dendrites. Still, if you can even get a single recording showing that you can block both inputs with a single puff, this will prove that the inputs CAN be on the same dendrite.
Good points by William.
One thing I would recommend doing before you get bogged down in control experiments is to do some modelling work. Depending on the structure of the neurons you are looking at and on how distal you are patching, it is very likely that inputs to other branches will be very strongly attenuated by the time the 'reach' your recording site.
There is a very large body of working looking at electrical signaling in dendrites theoretically and experimentally, I would start by looking at the early work by Greg Stuart, Matthew Larkum, Michael Hausser, along with the modelling work done by people like Arnd Roth.
Good luck,
Christian
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