No expression when delivering AAV1.syn.CaMP6f to ~P180 rat thalamic neurons. Anyone had success with viral GCaMP6 delivery in adult rats?

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William M Connelly

Hey Stuart,

You must be desperate to be posting in here, so I'm just going to chuck some random ideas at you.

I'm sure you're not diluting it, but if you are, try pure saline rather than PBS/HEPES Buffer.

Make sure the virus is good: Put it in the cortex of some 4-5 week old mice. If you don't see expression in two weeks the virus must be TOTALLY busted.

Buy a new batch of the same Virus. Or get the CAG promoter. Or get the CamKII promoter (should work for TC, not RTN, obv).

Are you SURE you imaging system is working? The first day I was doing GCaMP imaging, I thought my expression had failed. It turned out the laser wasn't aligned properly, and I was getting low power (

Stuart M Cain

Hey Bill,

Thanks for tips and yes, i'm out of ideas. I got the virus from UPenn but don't have previous experience to compare to, except using genetically-encoded GCaMP7. I'll order the virus you recommend from Stanford.

You're right, I'm not diluting it.

We've tried using both laser-2P and LED-camera stimulation-visualization and had no luck. Not tested the power though so I'll look into that.

I do wonder whether it is just really difficult to express in adult neurons but with the ChR2 working so well even at P250 I can't be sure.

Anyway, thanks again for the tips.More to try yet.

So looks like the GCaMP6f is expressing. Issue was that we were getting very strong expression at the injection site but not so good further away. Do you have any suggestions on concentration to inject?

Nadine Katrin Gut

Hi Stuart,

I got beautiful expression with the same virus in the thalamus (better than with CamKII btw). I would stick with the undiluted virus. I tested 1:4 and it will still give you a decent expression, but less than with undiluted.

Hi Nadine,

Thanks for the information.Good to know you have seen nice expression. I have a couple of questions if you don't mind?:

1.Which neurons are you looking at in the thalamus?

2. Do you see spontaneous calcium activity or do you have stimulate?

3. If you stimulate what do you stimulate with?

4. What are using using to image? Camera vs 2P?

4. What are you using to excite, LED or laser?

5. What time resolution are you acquiring at?