Viral Vectors for Optogenetics of Hypothalamic Neuropeptides - Viral Vector Approaches in Neurobiology and Brain Diseases - page 308

in myelination in older slices that leads to increased scattering of light
at deeper levels in the slice. For this reason, the use of a slicing
method that allows a maximal survival of neurons at the surface of
the slice during the preparation can be an important advantage.
In this regard, we have obtained in our lab excellent results
with a new cutting approach that uses a relatively simple and low-
cost vibratome developed by Precisionary Instruments. The
approach is based on two innovations: the fi rst one consists of sta-
bilizing the brain tissue inside an agar column and the second of
compressing the edge of the brain tissue slightly during the cutting
process. In brief, the process works as follows: after the rat brain has
been removed from the skull and split over the midine into two,
one hemisphere is glued onto a plastic piston. This piston is inserted
into a closely fi tting metal cylinder to a level that leaves the brain
just below the upper edge of the cylinder. Agar solution at 37 °C is
subsequently poured around the brain and rapidly solidifi ed by
immediately gripping the metal cylinder into a freezing-cold metal
holder that tightly fi ts around the cylinder. The metal cylinder (with
piston, brain, and agar inside it) is then horizontally inserted
through a hole into a cutting chamber that is subsequently fi lled
with 4 °C ACSF. A microprecision drive is placed against the back
end of the piston in order to advance it in controlled steps (typically
between 400 to 500 m), thereby causing at the front end the

colon of agar and brain tissue to protrude in similar steps from the
cylinder. At this front end, a razor blade is used to cut the protrud-
ing edge of the agar/brain colon. This razor blade is mounted onto
a unit that can vibrate horizontally and travel vertically at controlled
speeds to move through the agar/brain colon resulting in 400-500
microm thick slices. The compression of the tissue is obtained by a
slight inward curvature of the edges of the metal cylinder, which
thereby exerts a uniform pressure on the tissue and agar column
just at the point where it protrudes from the cylinder before being
cut by the razor blade. For the preparation of slices that are express-
ing channelrhodopsin, the whole procedure should be performed
in a dark compartment/room in order to limit the possible activa-
tion of ChR2 by ambient light.

μ

1. Decapitate the animal.
2. Rapidly remove the brain and submerge it in cooled (0-3 °C)
and oxygenated (95 % O 2 and 5 % CO 2 ) artifi cial cerebrospinal
fl uid (ACSF; in mM: 118 NaCl, 25 NaHCO 3 , 10 D -Glucose,
2 KCl, 2 MgCl 2 , 1.2 NaH 2 PO 4 , and 2 CaCl 2 with 300 mOsm;
pH 7.3) with a MgCl 2 concentration increased to 10 mM in
order to block NMDA excitotoxicity.
3. Use a vibroslicer (see above) to make slices of 400-500

Step by Step

m
thickness of the region of interest. This particular thickness
allows a reliable manipulation of the slice and a suffi cient num-

μ