How did you perform the in vivo injections (injection method, volume, etc)? How long did you wait post-injection?

Does your virus express a fluorescent protein fused to the ReaChR? That is helpful for confirming expression, e.g. in fixed slices ex vivo.

How did you stimulate in vivo? How did you stimulate the organotypic slices (through objective, with fiber, what light source, etc)?

Hi Benjamin, sorry my question wasn´t really detailed:

For organotypic slices with use a pressure-based injection system and inject few nanolitres in different locations in CA3. We wait 10 days minimum before doing ephys recording in CA1. For brain-injections, we use a nanoject or a picospitzer and inject 500nL to 1uL and wait at least 3 weeks before the recordings.

Our virus didn´t express any fluoresecent tag but carried a HA for post-staining.

The in-vitro optogenetic experiment are done using a LED light source (coolLED) coming from the objective with a 2ms pulse of light (I tried different durations) every minute at different wavelength (the best one should be 595 nm). For in-vivo, the probe is combined to an optical fiber and again we tried different pulse-length and wavelength.

The trafficking of the channel seems to be good in organotypic slices but I don´t know if it is the case when we inject it directly in brain.

Hi Noemie,

more question first.

-what speed did you inject with in vivo? (100nl/min?)

-what virus do you use?

-did you infect CA3 or LA in vivo?

-what is the light intensity at the fiber tip in vivo?

-what is the diameter and numerical aperture of the optic fiber?

First thing to do is to check the expression in vivo. If there is not an abundance of HA tag all other experiments are a waste of time.

Second, in your organotypic slice you recorded in CA1 while you injected in CA3. In vivo I would record an stimulate as closely together as you can and preferably in the area you injected the virus. This will increase your chances of recording spikes. You will need to record near cell bodies of infected neurons.

LED light sources couple less well than laser light sources. you will loose more light going from patch cord to implanted fiber. you can calculate the light intensity at different distances with this tool from the Deisseroth lab

https://web.stanford.edu/group/dlab/cgi-bin/graph/chart.php

another great online resource is https://www.openoptogenetics.org/index.php?title=Main_Page

I hope this helps, good luck

Nils

Dear Niels, thanks for your answer:

- We injected 30nl/sec with the Nanoject, with the picospitzer it might be almost the same (it is hard to control the speed as it is manual injections).

-the virus was AAV with hSyn as a promoter

-In-vivo we injected in MGM and tried to record from LA or from MGM directly

-For the light intensity in-vitro I don´t really know, I can measure it, but in both organotypic or acute slices we used the same parameters

We will do the staining asap to confirm the virus expression, and we will repeat the experiment with another virus carrying a color tag.

Let´s see.

Dear Noemie

for in vivo injections rates of 30nl/minute are more common. The slower the better as it does less damage.

In slices I do not think the light intensity should be a problem. It worked for the organotypics. I was more concerned about the in vivo experiments. Bad coupling, scatter in the tissue and small diameter of the fiber makes that the amount of light is quite small and intensity drops of a lot with distance.

good luck!

Thanks for your answer and advice, we will try again by changing parameters one by one and see.

All the best,