Download microscopy lab - (canvas.brown.edu).

MICROSCOPY LAB
NISSL, CHOLINESTERASE AND HUMAN SPINAL CORD
Goals - The goals of today's lab are: a) to introduce you to the examination of nervous
tissue in the compound light microscope; b) to acquaint you with the use of anatomical
atlases to identify structures of interest in tissue sections; c) to review the basic structure
of the nervous system; and d) to get a little exposure to comparative (inter-species)
differences in brain anatomy. There are three sets of slides to view: 1) a set of sections
through the human spinal cord; 2) Nissl-stained sections of the rat brain; and 3) AChE
sections of the rat brain. Try to budget your time so that you get to all of these slide sets
before the class is over. We have only until 1:00 PM. If your set of slides is deficient in
some way, or if you are just curious, we encourage groups to look at each others' slides,
but please don't intermix slides from different slide sets; keep slides in each set
together so the sets remain complete.
Technical Notes - If one or more of the slides is smeared with Permount, you can clean
it up using a Kimwipe wet with HemoDe, which the TA can provide. If there is lots of
Permount to remove, you might have to use several Kimwipes. Concentrate on the area
of the slide over or under your sections - this is where the optics count.
If you have never used a microscope before and are lost, ask for help from a TA or
instructor. Remember: microscope optics invert your image so that the tissue section
you see through the eyepieces is rotated 180 degrees from what you see if you look
directly down onto the slide on the microscope stage. For this exercise, it is easiest if the
section appears "right-side-up" in the eyepieces, so that the dorsal surface of the section is
up. When viewing the tissue, use a microscope objective lens that is appropriate for your
current needs. If you are trying to get an overview of the tissue section, use a low power
objective; if you want to see details of cellular organization, go to a higher power lens.
Human Spinal Cord Sections - Each of you should have a small slide box containing
transverse sections at various levels through the human spinal cord, some stained for cell
bodies (Nissl stain) and others for myelin (Weil stain). There is also a pair of sections
containing cross-sections of the cauda equina. Using these, address the following:
1) Explore how the amount (thickness) of white matter varies with the level of the
spinal cord. What accounts for this?
2) Try to identify the following features of the spinal cord in one or more sections:
a) Large motorneurons of the ventral horn
b) Expanded ventral horn in cervical or lumbar enlargements
c) Zone (or tract) of Lissauer
d) Substantia gelatinosa (and if possible the cells of the marginal zone or Rexed's
lamina I).
e) Intermediolateral cell column of the thoracic cord.
f) Ventral white commissure
g) Central canal
h) Entry of dorsal rootlets into spinal cord and exit of ventral roots
f) In the cauda equina, fibers differing in caliber and/or myelination as seen in
the cross-sections of the spinal roots.
Rat Nissl Sections - Go through the following steps for each Nissl section in your
collection.
1) Identify what level of the neuraxis it comes from using the rat brain atlases
distributed around the room.
2) Identify at least one distinctive feature (e.g., a nucleus, cell layer, or white-matter
region) in the microscope. Then go to the atlas and learn that structure's identity.
Have we already talked about this structure in class? If so, is it where you expect it
to be? If the structure is not familiar, you may want to ask the TA's or instructor if
they can tell you anything about it. This game of Trivial NeuroPursuit is a great
way to show us up!!
3) Study the atlas plate corresponding to your section and identify at least one
distinctive feature as above. Now find it in your section if you can. If you can't,
the problem may not be yours but because the plane of section or level may be
slightly different from that shown in the atlas.
Rat AChE Sections - As you know, acetylcholinesterase is the degradative enzyme in
the cholinergic system. It's best characterized role is in truncating the effects of
synaptically released acetylcholine. Early work with AChE histochemistry showed that
the enzyme was present in the membranes of certain neurons (e.g., motorneurons) that
were known or very likely to use ACh as their transmitter. This raised the hope that
staining for the enzyme might provide a simple way to map cholinergic neurons in the
brain. However, it is now known that the enzyme can be found in regions that are
exclusively postsynaptic to cholinergic inputs and even in regions where there are few if
any cholinergic elements, either pre-or postsynaptic (e.g., the cerebellar cortex). This
may be related to non-cholinergic and even non-synaptic roles for the enzyme that have
been recently been proposed (see the review by Greenfield cited below). The preferred
method for identification of cholinergic neurons is immunohistochemistry for
demonstrating the presence of choline acetyltransferase (ChAT), the synthetic enzyme for
acetylcholine. In any case, the AChE staining retains its utility as a means of
differentiating certain nuclei (or compartments therein) from surrounding tissue.
Because preparing high-quality histological material takes practice, you may have
generated relatively few sections that are recognizable. Still, by sharing slides among lab
groups, you should be able to come up with several workable ones. Using the rat atlases
(which include plates of AChE-stained sections), first identify which levels of the brain
these sections correspond to. For sections of marginal quality, you may need help from a
TA or the instructor. Now, within each of these sections, identify a few regions that
stand out by virtue of their relatively high levels of enzyme staining. Find the
corresponding nuclei in the rat atlas to determine their identities. Can you locate a cranial
nerve motor nucleus such as the nucleus of cranial nerves III? Are the large motor
neurons there darkly staining? Why might that be?
FOR ADDITIONAL READING:
Silver, A. (1974) The Biology of Cholinesterases. North Holland, Amsterdam.
Greenfield, S. (1984) Acetylcholinesterase may have novel functions in the brain.
Trends Neurosci. 7:364-368.