[Neuroscience] Re: question of inferior olive slice

Bill via neur-sci%40net.bio.net (by connelly.bill from gmail.com)
Mon Jun 23 18:41:29 EST 2008


The principle behind the sucrose solution is simple. During strong
insults to the brain, depolarization causes Cl- influx which
eventually bursts the cell due to osmotic pressure. Further down the
track, the calcium influx causes by the depolarization has activated
cell death pathways. By removing Na+ and Cl- from the solution, you
stop any depolarization and you stop and Cl- influx respectively. You
can actually use a low sodium solution (Like Choline Cl-) because
without the Na+ you can't get the depolarization in the first place.
But weighing out sucrose is more pleasant than choline chloride.

See the original paper on Sucrose by George Aghajanian, where it was
developed for making slices of the facial nuclei (not too far from
where you are working)

Aghajanian GK, Rasmussen K.
Intracellular studies in the facial nucleus illustrating a simple new
method for obtaining viable motoneurons in adult rat brain slices.
Synapse. 1989;3(4):331-8

I think who ever said they used 124mM sucrose made a mistake (I hope
they did at least), you need to double the sucrose relative to
NaCl...  The solution I use is 248 sucrose, 3 KCl, 2 MgCl2, 1 CaCl2,
1.25 NaH2PO4, 26 NaHCO3, and 10 D-glucose. Some people use 10mM Mg2+
and 0.1mM Ca2+ but I never found it made a difference.

I strongly recommend the sucrose solution, I expect you will find a
vast improvement in your slices (where ever you cut your slices from
in fact).

I also recommend incubating your slices at 35 degrees for half an hour
to an hour after slicing. Another thing which I have found enhances
the viability of some neurons is adding 1mM Na Ascorbate and 3mM
Pyruvate (makes CA3 pyramidal, dentate granule and cerebellar granule
neurons appear more healthy, but seems to have no effect on cortical
neurons).

Finally, 20 days seems to be getting a little on the old side for such
a mylin rich area. When I have worked in those kind of places, 21 days
old was my absolute limit for finding viable cells. If at all possible
I would work from 17 days old or younger.

All of that wont help too much with the problem of minigies. My only
advise would be to take your time. With animals of this age, the
decapitated brain seems to survive for a long time before you need to
cut slices. Get you boss to buy you some ultra fine forceps, some
moria spring scissors and a dissecting scope, and make sure you don't
have any coffee before the dissection!


On Jun 19, 12:47 pm, wwt <wwt0... from gmail.com> wrote:
> Hi! everyone. Recently, I was cutting inferior olive sagittal slice in
> rats (about 20d, female and male) and doing patch on these.
> Unfortunately, the health of slice always was too bad. As you know,
> the inferior olive is at very superficial position of ventral brain
> stem. When I extract the brain stem, I feel it is too difficult to
> avoid hurting the inferior olive. And there is a layer of meninge
> adhering on the surface of inferior olive. It is almost impossible to
> cut off meninges clearly in 20d rats during my operation. So the brain
> stem is always extruded during cutting process. But if I clean the
> meninges before cutting, it is ease to drag the tissue, even operation
> carefully. How can I do for avoiding these?
> Yosi Yarom's Research Lab use sucrose ACSF to cut inferior olive
> slice. The concentration of sucrose is about 124mM in their cutting
> solution( for example: J Neurophysiol 85:1686-1696, 2001.http://jn.physiology.org/cgi/content/full/85/4/1686#BIBL). And there
> are no NaCl in cutting solution. But this solution is a kind of low
> osmotic pressure, about 220 mOsm. I feel it is strange. Do you know
> the principle or reason of it?



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