Hi Giacomo, in my experience, maintaining intact dura mater favors the register quality by reducing mechanical artefacts. Therefore delete it or remove tissues formed on the brain will not help to improve the quality of the VSD signal.

Maybe your dye is going bad? If it's been around for awhile the VSD could have bleached or otherwise denatured. You might test it by imaging it on some membrane that you can control the voltage across. If it's possible you could try to position an extracellular electrode near a cell you know is stained to compare its activity w/ fluorescence - test whether it's the dye or the cell. 

I'm not sure about those dyes in particular, but VSD's are also commonly toxic - you might be killing cells even if the brain looks fine. If nothing else, repeatedly exposing the brain to light would result in phototoxicity. As far as I know much of the volume from lost neurons will be replaced with glia, which would dilute/degrade your signal. 

Also, who knows! Depending on your task, the decrease in signal might just be a decrease in activity. If the monkey is mastering the task, one would expect to see less cortical activity.

 Hi Mariluz and Jonny, thank you for answering!

@Mariluz: I am not sure I understand what you mean. I need to open the dura to stain the brain with the dye and image the cortical response. I stain the brain applying the solution with the dye on the cortex (I fill up the recording chamber, see method in the paper I added above) for two hours. New layers that grow on the cortex don't allow the dye to reach the cortex (they form a sort of barrier). For a certain time, removing these layers I am actually able to restore a correct staining. I would be interested in understanding better your point :)

 @Jonny: 1) I don't think there is a problem with the dye. When I stain with the same dye a cortex more "fresh" in a different monkey the staining is successful. 2) I am only able to record the neural response across several millimeters of cortex, unfortunately I don't have the single neuron resolution.  3) Yes, I also think that one of the most important reason the signal is degraded is the photo-toxicity and the toxicity of the dye. Do you know more about that? This is exactly that kind of info I hope to obtain asking the question on RG.  However, looking at the behavior of the monkey it seems the piece of cortex I am using keep doing its job very well.. (but I see this is not a strong point :) ) 4) I am in a primary sensory cortex and I am using many different stimuli therefore I  would be surprise this big effect of decrease in response was just due to a sort of adaptation.

Hi Giacomo, it is also possible to stain the cortex without removing the dura (Lippert et al J Neurophysiol 2007; Devoshire et al., EJN 2010; Morales-Botello et al., 2012).

I also think that phototoxicity can be a cause of your problem. But other possibility I can think of is that  some residual dye accumulates after each staining and contributes to increased background fluorescence, this would result in a decrease if signal-to-noise ratio. I try to clean the cortex longer to remove residual dye and see if it improves the signal-to-noise ratio.

Grinvald and Hildesheim, Nature 2004 (reference about phototoxicity...)

I hope I've helped you...

MªLuz

Thank you very much Mariluz!  I am checking out the papers you suggested :)

Hi Giacomo, My guess is that the cortical tissue is undergoing chronic inflammation. We only have experience in rat but if the cortex is "irritated" e.g., touched, the staining becomes poor. Somehow inflammation reduces inter cellular spaces and prevent diffusion of dye molecules.  

Hi Jian-young, thank you for your answer! 

In my opinion, chronic inflammation could  indeed explain the phenomena we are discussing here.

However, in this regard there is something that sounds a little odd to me. From time to time, the cortical area we are working on can undergo a (not severe) acute inflammation. The signs of inflammation  are quite clear: capillary vessels become very evident in dense network patches and the whole cortical area looks reddish. After 3 days of antibiotics these signs disappear and the staining works again. Instead, in this case when the staining start to become chronically impaired, the cortex looks OK and evident signs of inflammation can not be observed.

Do you think that chronic inflammation could still be present even without any evident sign?

Do you think the use of anti-inflammatory drugs before the staining could restore the dye penetration?

Thank you!

We have tried cortiso which works well in rats. you might want to try locally application before staining. How long do you stain? We circulate the dye solution for 2 hours. circulation seems improving.  

Dear Jian-young I am very grateful for your suggestions. I stain for 2h as well but without circulation.

I will try both things and I will report here if it solved my problem. Thank you!

Hey Giac! Well I agree with Jian-young that this has something to do with chronic inflammation or other mechanisms relative to chronic interaction with the brain. I would try not doing experiment for an entire week or two just to see if that may be the cause. Have you noticed any behavioral changes from the monkey at the same time?

Hi , maybe the body vaccinate itself against your dye. Try another dye after you  fail to record any good signal and also you can check there if there is any electrical activity in the brain to record. If electrical activity exists, it has got to be the vaccinate thing.

Thank you Sandrine and Hosein for your answers. 

@Jian-young: I have tested both systemic and local inflammation treatment with Dexamethasone in the same animal in which I could not record VSD signals anymore from one month. One day I injected the dexa. 2h before the staining. Another day I applied dexa. directly on the cortex for 10 minutes just before the staining. The day of the local application I was able to record a small VSD signal. Although the staining quality is in itself quite variable across days and it is just a single observation, it seems promising to me, at least to understand the cause of the problem with the staining. Unfortunately I have not found a system to circulate the dye yet.

@Sandrine: I have tried to wait for one week and it did not seem to help for my problem. The behavior of the monkey seems fine.

@Hosein: I am already alternating two different dyes. Your point seems to me consistent with the chronic inflammation idea.  I have already planned to use a single electrode to test the neural activity next month.