Problems getting good signals

I have been trying to learn how to use the MEA-64 system for several weeks and consistantly have a few problems:

1. The slice often does not sit firmly enough on the electroedes so the signal is really weak. Adding more weight on top or decreasing ACSF level often does not help.

AND

2. When I use a new electroede, the slice sticks to the electrode (Even after incubating the new electrode with slices), and when I take off the brain slice, the electrodes come off with it.

AND

3. On occassions I finally get decent fiber volleys, latency, and fields in the Schaffer Collaterals. But when I stimululate with classic theta bursts, I rarely if ever get LTP. From the responses, I can tell that the slices are healthy.

I would appreciate any advice on the following matters.

Thank You
Ammar Hawasli

Dear Ammar,

Do you use MCS MEA-60 or Panasonic MED-64 arrays? I heard the latter are a bit fragile.

In terms of the slice attachement stability, I also have these problems occasionally. I think using 3D Ayanda MEAs instead of usual MEAs could solve the problem partially. Also, the grid is essential, unless you culture the slice on an MEA. However, both kinds of arrays are rather robust and should not be damaged easily.

In terms of LTP, my values are usually slightly lower than the ones described in the literature. I use 2X100 Hz @ 50s with the tetanic stimulation being 150% of the baseline and get 20-60% potentiation depending on the mouse strain I use. Do you use rats or mice? I believe the lower LTP levels could have something to do with the monopolar stimulation of the slice (in contrast to bipolar stimulation, used in most classical studies).

Max

Dear Ammar Hawasli

I would like to comment the problems from the manufacturer's point of view, and hope I can help you:

>1. The slice often does not sit firmly enough on the
>electroedes so the signal is really weak. Adding more weight
>on top or decreasing ACSF level often does not help.

Do you coat the MEAs and if so, which method do you use? Coating often improves the attachment of the slice.

>2. When I use a new electroede, the slice sticks to the
>electrode (Even after incubating the new electrode with
>slices), and when I take off the brain slice, the electrodes
>come off with it.

This is not a known issue; as Max pointed out, the electrodes are quite robust. How do you remove the slice? In most cases, it is sufficient to rinse the MEAs with distilled water. You should not touch the electrode field with any device in any way.

Best regards, Christine (MCS)

Thanks for your responses.

I am using the MED-64 system. After I open the new electrode,I fill the well with ACSF and 3-4 slices of mouse brain tissue. I let it rock slowly at Room temp for 2 hours. At this stage the slices are floating. I was thinking of using 1 mg/mL albumin in PBS instead of ACSF to have better coating.


Can either of you make suggestions on grids or ways to weigh the slice down? Which brand of platinum ring is optimal for you to weigh a 350 micron slice onto the electrode?

Any help and suggestions would be much appreciated.
Thank you so much,
Ammar

>
>Can either of you make suggestions on grids or ways to weigh
>the slice down? Which brand of platinum ring is optimal for
>you to weigh a 350 micron slice onto the electrode?
>

Dear Ammar,

We use poly-D-lysine coated MCS MEAs or 3D Ayanda MEAs. We cut the brain in either sagittal or sagittal-horisontal plane and trim the hippocampal slices of the surrounding tissue. I worked mainly with two types of grids. The first grid was just a usual U-shaped flattened piece of platinum wire (@ 80-120 mg) glued onto wedding veil. The other grid was sort of a double version of the previous one. Another platinum wire was glued from the other side of the veil symmetrically to the first piece of platinum. In this case, the veil is "lifted" from the surface on the distance equal to the thickness of the flattenned wire. Therefore, if you use 350 um slices, the thickness of the wire should be around 300 uM and not more than 350 uM, otherwise the veil will not hold the slice sitting still on MEAs. This kind of grid is less damaging because you can vary the pressure on the slice by changing the thickness of the wire.

In some manuals it is advised to nearly dry the hippocampal slice on the array with filter paper wedges, however my observation is that at least with 3D arrays it is definitely not necessary. Moreover, it is easier to change the position of the slice if the whole well is filled with ACSF.

I cannot comment on MED-64 system, as I never used it. However, I used to touch the electrodes on MCS MEAs with a brush sometimes and it did not cause any harm, I worked with that array for several months...

I hope that helps. Good luck!

Max

Thanks for the suggestions. I have inquired about getting a 3d electrode. Now i can get consistant .4-.7 mV signals but my issue now is that the signal starts to fade (i.e. the response dies off) during my baseline. I am thinking my slice is dying in the middle of the experiment. This has happened to me often.

Any suggestions from your past experiences.

Thanks.
Ammar
Novice

>my issue now is that the signal starts to fade (i.e. the
>response dies off) during my baseline. I am thinking my
>slice is dying in the middle of the experiment. This has
>happened to me often.

Dear Ammar,

The loss of contact between the slice and electrodes could also lead to fading of the EPSPs. In such a case, the decrease is rather rapid (@ 2-5 min). Make sure the grid really holds the slice tightly. Good luck!

Thanks for your advice. Things have significantly improved in the last few weeks. I am getting good signals these days and can predict when I have a healthy pathway in which I can see PPF and LTP.

However, yesterday I encountered a new problem. All was going well two days ago. I have been using the same electrode for about 2 weeks. I am very careful with it; the dots looks find under a scope; I store it in dH2o between uses. My responses went from normal spike, fiber volley field, to an odd response. I looks kind of like a exponential decay graph.

My question is How do you tell the difference between a dirty electrode (which I could clean with EDTA-collagenase) and a bad electrode? Is the visibility of the individual probes on the grid the best way to know if the electrode is good?


Thanks,
Ammar