As you pointed out, when I used a mixed NaCl/Sucrosse solution with
ascorbic acid (3mM), the quality of the slices were slightly better, but
the cells were much more easy to patch. I am currently thinking to put
ascorbic acid during my recording solution (without Sucrosse).
On the other hand, I used pyruvic acid (1mM). Is there any reasons other
than "neuroprotection" involved in the effect of pyruvic acid on brain
slice quality? I did not find any conclussive report.
Next week I will do the same cutting solution but with glycerol (mixed
NaCl/Glycerol + Ascorbic acid + pyruvate). I will let you know as soon
Best wishes and thank you very much!
On 2008-12-01, Bill <connelly.bill from gmail.com> wrote:
> Mmm, there is something funny about CA3 cells, they've got to be one
> of the most delicate set of cells.
>> The reason I reccomend the ascorbate is because of its important role
> in preventing the edema that occurs in slices. Check out:
>> Ascorbate inhibits edema in brain slices.
> Brahma B, Forman RE, Stewart EE, Nicholson C, Rice ME.
> J Neurochem. 2000 Mar;74(3):1263-70
>> I've really noticed an improvement in the patchability of small cells
> if the slices are constantly incubated in ascorbate.
>> I wonder about 3mM myo-inositol for similar reasons, though I've never
> tried that.
>> Ah well, let me know how things go, I'm curious about the glycerol, I
> saw that paper, and it seemed a bit... amazing.
>> On Dec 1, 4:32 am, Jose Guzman <n... from neurohost.org> wrote:
>> Well, I agree with you, P21-30 are juvenile animals. I want to get
>> recordings of CA3 pyramidal neurons of the hippocampus. So far, I have
>> not much problems untill P18. However, due to the nature of my experiments,
>> I need to study older animals, and to have relative big number of cells
>> alive (only one or two cells per slice is not enough). Due to the extremely
>> high interconnectivity of those neurons, and the high NMDAR expression
>> I was thinking to use APV to avoid neurotoxicity due to Glu, but if
>> you say it's a waste of time I will think first about other solutions.
>>>> I am carefully following Bischofberger et al, 2006. My cutting angle
>> seems to be OK, because after filling the cells with biocytine I can
>> clearly see both axon and dendrites (in animals < P18). My vibratome
>> is a Leica VT1200, so the state-of-the art.
>>>> And YES, my cells look very crinkly (I was looking for that word!)
>>>> I use a cutting solution in which NaCl is only partly replaced by
>> sucrosse (both for storage and cutting). No Glu-antagonists or
>> anti-oxidant agents like pyruvate or ascorbic acid.
>>>> I will try soon the Glycerol-based solution, and additionally the
>> solution containing pyruvate and ascorbic acid (with my normal sucrosse
>> solution), and I will let you know how it was.
>>>> Thank you very much in advance for your wisdom advices!
>>>> On 2008-11-30, Bill <connelly.b... from gmail.com> wrote:
>>>> > Hi Jose,
>>>> > Just off the bat, lets remember that 21-30 days in not 'old'. Indeed,
>> > it is barely mature. Old is 18 month old rats.
>>>> > Secondly, for 21-30 do not 'need' specialized cutting solution. The
>> > biggest change you see between slices from neonatal and ~30 day old
>> > brain slices is nothing to do there being more dead cells, it is
>> > simply that it is harder to see the neurons (though there are some
>> > areas of exception, e.g. highly mylinated areas).
>>>> > The fact that the sucrose based solution didn't make a big difference
>> > informs you that depolarization toxicity is not your problem.
>>>> > It would have been nice to know where your slices are from, so I could
>> > be certain, but assuming your working from some kind of cortical
>> > region:
>> > Sucrose is good. You might as well do it all the time >P14. AP5 is a
>> > waste of time, acute depolarization induced neurotoxicity is NMDA
>> > receptor independent.
>> > Incubate your solution at 35 degrees for half an hour after slicing,
>> > esp good for small cells.
>> > If your neurons are looking a bit desicated/crinkly/hard try 1mM
>> > Pyruvate and 3mM ascorbate in your incubating/holding aCSF
>> > What brand of slicer are you using? As your brain gets older, this
>> > gets far more important. You can cut nice slices from 12 day old brain
>> > with your hand and a razor blade, those neuros don't care. Make sure
>> > you're vibrotome has been serviced some time in the not too distant
>> > past. I've seen old Camden slicers that bounced around on the table
>> > because they needed salt crust chipped out of the motor and some
>> > lubrication.
>>>> > But I stress this. If you're making slices from 21 day old animals,
>> > and your neurons look unhealthy (vs just harder to see) you are doing
>> > something wrong, indepedent of cutting solution. I worked on 8 week
>> > old mice for 2 years, and you can't get the neurons looking as round
>> > and full as 2 week old slices, and you can't get the neurons as
>> > visible (talk to someone who works on young cat thalamus if you want
>> > to hear about invisible neurons). But from a 21 day old animal, the
>> > neurons should still be like lovely round grapes, just harder to see
>> > than in a 14 day old. And if you're working with pyramidal cells,
>> > remember to keep your slice plane in the plane of the dendrites, those
>> > guys really hate having their dendrites cut.
>>>> > Also, if you're working from an ultra mylinated structure, like the
>> > brain stem, it is vaguely impossible to get reasonable slices from 99%
>> > of that area in animals over ~21 days old, give or take a couple days.
>>>> > I hope you haven't found this too didactic, but this is my experience
>> > (I'll probably get told this is completely wrong by someone else).
>>>> > Oh finally, you can try intracardiac perfussion of your
>> > neuroprotective cutting solution. Its a pain, and its fiddley, I don't
>> > think it helps with the neuroprotection, but I think it helps makes
>> > the neurons in the slices more visible.
>>>> > On Nov 28, 5:11 am, Jose Guzman <n... from neurohost.org> wrote:
>> >> Hi everybody,
>>>> >> I am currently working in hippocampal brain slices to do patch-clamp
>> >> electrophysiology. Due to some experimental issues, I will have to
>> >> record to neurons in old animals (P21-30). The problem is that the
>> >> neurons I need are very susceptible to hypoxia , so the preparation
>> >> requires some special cutting solutions.
>>>> >> I collected the 3 major types of cutting solutions, and I would really
>> >> like to know if any of you tried these in his/her experiments . These
>> >> solutions have some minor variations in other publications (may contain
>> >> additionally APV or kyneuric acid to prevent glutamate excitotoxicity),
>> >> but basically are described as follows:
>>>> >> 1.- Sucrose based solution: total or partial replacement of NaCl
>> >> by Sucrose. (Aghajanian and Rasmussen, 1989, Moyer and Brown,1998).
>>>> >> 2.- Glycerol based solution: the same as the previous but with
>> >> Glycerol replacement. (Ye et al., 2006).
>>>> >> 3.- KGluconate based solution: without Ca2+ or Na+:
>> >> specially indicated for patching dendrites (Dugué et al., 2005).
>>>> >> A major problem I found is that these papers are not very
>> >> much cited in other publications (for example, Ye et al.,
>> >> 2006 has only 6 citations). Personally, I found that
>> >> Sucrose-based solutions made an improvement (although not
>> >> very big) but I would really like to hear from your
>> >> experiences.
>>>> >> Thank you very much for your feedback!