I plan to record the synaptic transmission at the Calyx of held for the first time. But I find it quite difficult to get a right transverse slice. I dissected out the brain stem, but could rarely get it to stand (mouse is about P21) when glued onto the platform of vibrotome (Leica VT1200s). How could it be done?
If you dissect out the brainstem with a large chunk of cortex and cerebellum, you can then make a roughly coronal cut at some point right through the cerebellum and use this flat surface to glue the brainstem/cerebellum to the vibratome stage. You then end up cutting transverse/horizontal (depending on the angle of the cerebellar cut) from the spinal cord end down to the auditory brainstem in the pons (visible as a white bulge due to the myelinated fibres running across the surface).
Thank you Kiri! I will try it. I am just thinking about cutting slice with an angle (about 30 degree) tilting towards the cerebellum as the red dash line shown in the following picture. What do you think about it?
I guess the transverse cut would work best for evoked EPSC of Calyx synapse, as you can see in J. G. G. Borst's paper (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1156851/). Probably it will preserve most of the connection from anteroventral cochlear nucleus this way.
I was a masters student in Borst's lab back in 2006-2007. I've performed the procedure myself quite often, but it has been several years.
Of what I remember (but I used P7 rats), Kiri is quite right in keeping a part of the cerebellum intact. You have an extra flat surface that you can use to glue, and in addition it supports the sectioning (when you mount it so that you first slice though the brainstem, and then to the cerebellum. You do not have to actually make sections of the cerebellum, just stop, but it gives some structural support). Alternatively, you could put some agar behind your brainstem section.
Another tip: If I remember correctly, if you cut from the caudal side (which you probably do), you are almost reaching the MNTB when you see the facial nerves appearing (both on the left and right sight, the white cables). The next consecutive sections should contain the mntb around the midline!
Good luck!
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