Hi David,
You're never going to get as good slices from 2-3 month old animals as
you do from >21 day old animals. So we always just need to remember
that.
My biggest question is how are you cutting the slices? The best CA1
slices I have ever seen are made by dissecting out the hippocampus,
gluing it on the traverse plane, the vibrotoming it up against a
silicone block. One of the main reasons I think this is so good, is
that it makes sure you get perfectly transverse slices, and so you
don't slice off the apical dendrite. That's what I think you should
check, are all the dendrites in the plane of the slice.
I don't think you'll find cardiac perfusion to be very helpful. In my
experience, the biggest difference that makes is in the visibility of
cells, so long as you are capable at the dissection. (if you're heavy
handed, it can afford some protection).
But in the lab I'm currently in they routinely work on 6-8 week
animals, and the get beautiful CA1 slices (not every cell is
patchable, but certainly enough to work with).
You transfer the slice to 35 degrees for half an hour after slicing,
then transfer to room temp, right?
Also, do you have ascorbate and pyruvate in your holding solution. I
recommend it. You might also want to give myo-inositol a go.
On Sep 16, 5:07 pm, Neuro Sun <sungreat... from gmail.com> wrote:
> Hi everyone,
>> I've been patching mice CA1 pyramidal neurons for synaptic transmission
> study and have some shrinking cell problems, I cut 300um brain slices with
> vibratome and store the slice in partial sucrose cutting solution (Jonas P,
> osm ~320), and do recording in normal ACSF (osm 300-310) at 32 C. However,
> in older mice ( > 3 weeks), after cutting the slice, the cells seem to be
> shrinking (fold inward, not smooth round cells), and using the same
> solutions, slices from younger mice looked very good. I don't know why this
> happens, could it be pure age issue? I heard people can patch on really old
> animals(2-3 months) without any problem. I think the next step i want to try
> cardiac perfusion before slicing. It will be so helpful to hear from you
> guys that what do you think of the potential causes for shrinking cells?
>> Yours sincerely,
>> David
