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
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!