Out of the many possible explanations, I think the one that is most likely (and least satisfactory!) is that it's just a poor health region that you're recording from. This to a large extent depends on the age of animal you're slicing from - the younger, the healthier the slices would typically be. But also fEPSP recordings, can take a lot of "hunting around" for regions in the slice that will produce nice/healthy responses; sometimes you'll find that by moving your recording electrode a few micrometers laterally and/or deeper in the slice will give you dramatically improved post-synaptic response to pre-synaptic fibre volley ratio.

The other thing to consider is you mentioned you're using sagittal slices, which I don't think is the most common configuration (most people use whole brain coronal, or transverse cuts of isolated hippocampus), so it may be that in this configuration, it is less likely to obtain large post-synaptic responses.

There are lots of excellent resources in the literature that may be of use to you, this is an example of one that I found particularly helpful:

Bortolotto, Z.A., Anderson, W.W., Isaac, J.T., and Collingridge, G.L. 2001. Curr. Protocol Neurosci. Chapter 6: Unit 6.13.

Best of luck!

Thanks a lot for your very helpful answer!

I do agree! The fibre volley dies at last. If you would block all synaptic components you will see a big fibre volley which is, at that size, normally covered by the fEPSP. Unhealthy slices are showing the same. Best regards

I totally agree Haider's comment. From the your example trace, it looks like the slice is not healthy enough for the recording. or move around or deeper of the electrodes.  It's better to use coronal slicing. It will give you more synaptic connection. good luck.

Dear Doris ,

I agree with colleagues that your slices are not in good condition. As you know, slices should be left for at least an hour to incubate after cutting, or even longer, and then start measurement. The slices should not be hypoxic . I suggest to start with somatic response from pyramidal cells as first, which is more easy to find than dendritic response. You will see if the slice is good. Then quickly move registration electrode to the stratum radiatum. Good luck.

Thanks a lot for all the answers. It is very confusing, because I have done slice preparations (spinal cord with attached dorsal root/brain stem) for many years and never had any problems with slice viability (have done LTP in the spinal cord).

Now, when looking at CA1 neurnos, they do look really bad. When we patch neurons (a healthy looking one) from the pyramidal layer we see input (eEPSCs), but we've never seen 'nice' field potentials. We do the prep in a sucrose rich solution and then keep it for at least 1hr in oxygenated ACSF for recovery.

Do you have any ideas what else might be critical for slice viability. Is the way you anaestethize the animal before decapitating it crucial? Does anaesthesia with O2/CO2 harm the cells?  What about the pH? Are they very sensititve to temperature?

Has anyone ever experienced a period in which his/her slices were really bad, and then something was changed, anf all of a sudden it was ok again?

Thanks for the input!

Hi Doris,

I've attached our step by step protocols for cutting and some of our ACSF solutions below. We've used both anaestheized and unanaesthetized aniimals and have had good success. The critical features are to cut in chilled ACSF and then to briefly warm your slices again afterwards. 

1. For young animals (

Hello all, I'm also having issues with optimal slice health. How long do you allow a mouse brain to sit in carbogenated ice-cold ACSF before making the proper cuts prior to mounting? Thanks in advance!

It doesn't have to be long. In fact, after removing the brain and letting it sink in cold ACSF for a few seconds, we just move our brain onto a cold surface (upside down petri dish) that has a piece of filter paper immersed in cold ACSF on it and do our cuts. The brain then quickly is glued onto a stage where it is then immersed in ice cold ACSF for cutting. If you're doing adult animals, use the choline solution.

Brian R Christie Thanks so much! I've been hearing/reading about leaving it in ice cold ACSF anywhere from a few seconds to two minutes! I use an upside-down petri dish in ice with ACSF immersed filter paper to do the remaining cuts as well. Do you think that this added time in cold ACSF is just wasting time (and therefore brain)? I've been immersing the brain post-extraction for one minute before I do my cuts!

Hi

The pH of ACSF is a critical point in this case , in low pH fiber volley tend to be larger than synaptic response

I can't say for sure that it's a waste of time, but I can say that we get excellent responses without doing it. Farshad is also correct that making sure the pH (osmolarity) of your solutions is another critical factor. If your cells die bc they are off, you can also get a larger fiber volley than a response.