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!
Jose.
On 2008-11-30, Bill <connelly.bill 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!
>
