I've never worked in Fish, so it's hard for me to comment. Can you actually notice the cells dying? That is to say, if directly after you've cut the slices, and you put them under the scope, can you see living cells, and then an hour later, when you come back, are they bloated/gone? If they are already bloated/not visible directly after you cut the slices, then it's likely you are doing something terribly wrong; either you solutions are hugely wrong (which it doesn't sound like) or your dissection technique needs a lot of work.

Maybe you should give us more details, like: Dissection time, aCSF make up, Vibrotime make, slice thickness. Also, what kind of holding chamber are you using. I know a group who had dead slices for 6 months after buying an off the shelf holding chamber that didn't work.

There is an alternative idea, that rests on the notion that your slices are actually fine, and your problem lies elsewhere. How certain are you that your optics are correct, and you electrodes are good? Have you tried to visualize slices produced by a group that produces good brain slices? Maybe your optics are out, and hence you can only see dead cells (often dead cells have a very black membrane), and hence the seals are bad. 

Thank you for taking the time. I'll try to answer all your questions...

dissection: 3 to 5 minutes to remove the forebrain block from the head; 3 to 5 minutes to embed it in agarose, trim the rostral par to have a flat surface to allow siting in the vibratome chamber; and 3-5 minutes to cut 350 micron sections. All done in ice-cold aCSF. This is a similar technique as I was doing before for a sensory area (electric lateral line lobe, ELL).

aCSF (in mM): 124 NaCl, 3 KCl, 0.75 KH2PO4, 2 CaCl2, 1.5 MgSO4, 24 NaHCO3, 10 D-glucose

Recording chamber: P1 from Warner Inst ha I'm using since a while.

Visualisation: DIC. Works fine. Yet, I wouldn't never say great wih DIC! But it works similarly as other people around. I recenly re-calibrated it.

Other group for his prep: it's the first time somebody does fish telencephalic slice for recording. Sad!

I's not only the seal that are bad, it's also the fact that they "disappear" or are very fragile.

The sad/funny part is that I usually prepare two extra slices that I give to my collegue for him to record DL neurons (hippocampus-like region). These are small neurons and his neurons and recordings are fine. I record areas, DD an DC, with big neurons.

Ah, well then I wonder, how do your slices compare to the arborization of these neurons? For instance, if I were to cut a neocortical rodent brain slice parallel to the surface of the brain the neurons would be much less healthy than if I cut the slice perpendicular to the surface. This is because the parallel slice will cut through all the thick apical dendrites of the pyramidal cells.

Also, different areas of the (mammalian) brain have different physical properties when it comes to slices. e.g. Cortical rodent slices are beautiful to look at when cut from animals of almost any age (certainly less than 2 months old). However, the substantia nigra produces very damaged slices and/or slices with very hard to visualize cells when the animals get above 3 weeks old (largely due to increased Myelination I believe). So perhaps this area just isn't good to work with?

How old are the fish? Perhaps, you should pay more attention to brain surgery. More quickly and gentlely.

Sorry to be another mammalian scientist unable to comment specifically on fish but perhaps following this algorithm I usually use to troubleshoot may help:

1. Check dissection: are you taking too long or too little time with the dissection? Check your dissection time is comparable with those who have no problems with getting slice recordings from fish preparations at similar ages.

2. Check slicer. are you slices compressing? If so, then they will have a distorted shape and have little life left in them. Since you have DIC optics, you can also check for more subtle slicing problems - if the most superficial surface is a LOT unhealthier then deeper layers, then you have a slicing issue. It may be an embedding issue so check your preparation is firmly anchored and not oscillating during slicing. Other causes are: your solutions isn't cold enough during slicing (i.e. not

I'm not doing fish,

In mouse we noticed a great increase in the quality of our slices if we partially freeze down the ACSF to an ice slush before the sections (250 ml ACSF, 60 min oxigenated in ice + 15 min -80°C + crusch the ice with a pimmer). Clearly the temperature is below 0. Furthermore the osmolarity of the resulting medium is high (>400 mOsm). I think this helps the cells to survive. This agrees with the "high sucrose" used by many authors for slice preparation. So, regardless of which solutions you use, I'd try increasing the osmolarity during sections (unless you are doing this already); possibly cooling down the ACSF in the -80°C freezer for a short.

A second matter is: for some neurons (dopaminergic, substantia nigra) we need to cut and keep the preparation in 25 mM glucose-ACSF until the recordings (@ 10 mM glucose-ACSF). This is because the particular type of neuron is sensible to low glucose and dies quickly eventhough other neurons in the same slice are alive and well.

I also notice a very big difference according to which blades one uses for sections ( better > worse: Gillette supersilver > Korma > Derby > Wilkinsons > Dorko > Shark). Generally better blades are thinner.

For adult animals and very tricky preps, it is also used to cool down the anestetized animal (cardiac perfusion) with chilled ACSF before the dissection. For mammals one needs an animal application. You might have an easy life trying this out and cooling down your living fishes before extracting their brains (?).

Quick and careful handling of the slices is of course mandatory.

Good luck!

I assume that your cells look fine after 30 minutes? If so I doubt it is the way your are cutting your tissue. Are you holding your slices in normal ACSF, as it appears from your initial statement? If your are maintaining them in kynurenic acid for instance, that could cause problems. Reduced calcium will also cause problems. And choline or sucrose for extended periods of time will also cause your cells to die. They need to sit as normal as possible once the sectioning is complete.

I found that my cells (spinal motoneurons in mice) had improved when I reduced the time the tissue spends in the agar block. I prepare an agar cube ahead of time as a support for the tissue. The size of it is about 1cm3 to fit the tissue sample and the vibratome chmber.  Then I use low gelling point agarose (~4% in ACSF) and "paint" the tissue placed on the agar cube, and immerse it immediately in cold ACSF (Vibratome chamber). This process attaches well the tissue to the agar cube for the sectioning and reduces the time the tissue spends in the agar prior to sectioning from 3-5minutes to 10 seconds. Also if you prepare the agar cube ahead of time you can better control the angles for the type of slices you want to achieve: transverse or oblique etc. I find this process way easier and faster than sculpting the agar with the tissue in it.

May be you can also check your patch pipettes according to the size of the cells, the parameters of the pipettes change, so it could explain why yours cells die and don't seal whereas for the other they work fine.

I find that having everything (blades, tubing etc) very clean or aseptic helps. Also. has anyone any experience of using antibiotics in aCSF, if so could they maybe give us some ideas?

thanks

What about usingbcultured slice preparation? For a recent paper see: Gen Comp Endocrinol. 2014 May 20. pii: S0016-6480(14)00192-0. doi: 10.1016/j.ygcen.2014.05.017. [Epub ahead of print] Long-term GnRH-induced gonadotropin secretion in a novel hypothalamo-pituitary slice culture from tilapia brain. Bloch CL, Kedar N, Golan M, Gutnick MJ, Fleidervish IA, Levavi-Sivan B5.

Is the osmolarity of fish CSF close to the one of mammalian? If not, the standard ACSF might cause trouble to a fish brain.

osmolarity is around 300mOsm.

I am using a different antioxidant called N-acetyl-cysteine (NAC) (12 mM) added to the same cutting solution you are using to prepare slices.  Only caveat is that you need to adjust pH to 7.3 when you supplement with NAC, but this strategy has improved my slices. 

We use an equimolar concentration of sucrose instead of NaCl for our mouse slices. This increases cell viability over NaCl, however have never used choline so not sure how this compares