Miniscope & TRAP tagging

[P Wilbers]

Hi all,

I am planning on working with miniscopes in the future and would like some input. Hope someone can help me. 

I would like to combine the recording of cortical neurons with the tagging of a task-related ensemble, using the TRAP system. This means that in addition to visualizing the pan-neuronal gCamp signal, I would like to isolate the recordings from a specific neuronal population. This could maybe be done with a different fluorophore that marks this population, or by for example marking these neurons in one way or another so that they can be traced back in the general recording. However, as the current versions of the miniscope are not able to visualize different wavelengths in a stable manner and the V4 dual-channel miniscope is (as far as I know) not yet available, I'm looking for other ways to do this. 

Does anyone have experience with visualizing / highlighting / tagging a specific population of neurons in addition to the pan-neuronal gCamp signal? 

Some ideas I have are:

- Chemo-tagging: insert an hM4Di in the TRAPped neurons, activate / inhibit them and try to determine what neurons were TRAPped based on this. However, as DREADDs act on the timescale of minutes to hours, this is not as clear as it might be with opto-tagging.

- GFP-tagging: insert a GFP in the TRAPped neurons, so that they have a bright signal in the miniscope recording (same wavelength as the gCamp). This would be done at the end of the experiment, as recordings would no longer be possible afterwards. However, I do not know if this GFP is enough to distinguish TRAPped fluorescent neurons from the signal emitted by the gCamps due to baseline calcium presence. 

Making the gCamp dependent on the TRAP is not possible, as I would like to record activity before I TRAP the neurons. 

Many thanks in advance. 

Pelle