Biology Reference
In-Depth Information
Fig. 2
Chamber used for fear conditioning and fear memory testing. Conditioning chamber has metal bars on
fl oor and is washed with water before use. The chamber is modifi ed for testing where the fl oor and walls are
changed as shown and the chamber is washed with ethanol to present a different scent from the conditioning
session
#6 below), quickly render the animal unconscious by placing it
in a chamber containing halothane. Remove the mouse from
the chamber, dissect out the brain and immediately quick-
freeze the tissue by burying it under a layer of granular/pow-
dered dry ice. Once the brain is completely frozen, it can be
wrapped in foil and stored at −80 °C.
6. Expression of
Arc
mRNA subsequent to neuronal activation
is readily detectable within the nuclei of neurons approxi-
mately 5-8 min after fear conditioning or fear testing [
8
,
9
]
(
see
Note 6).
3.4 Finding the
Memory Trace:
catFISH Analysis
The catFISH protocol described here is modifi ed from Guzowski
and Worley's [
9
] protocol. Readers are advised to consult the orig-
inal paper for additional information. In this protocol, the activity
state of neurons and viral infection are visualized by double fl uo-
rescent in situ hybridization to detect intranuclear arc (active) and
GFP (infected) mRNA (
see
Note 7 for a description of probes).
Through cell counting, the relative percent of activation in infected
and noninfected neuron population can then be calculated.
1. Equilibrate the brains, blades, brain matrix, mould, mounting
base in the cryostat at the optimal cutting temperature (typi-
cally −20 °C).
2. Place the brain in the matrix and, with a blade, cut the tissue
coronally into ~1-cm-thick chunks containing the LA. The
brain chunks from all mice should be cut approximately in
the same plane.
3. Add a thin layer of embedding medium at the bottom of the
mold and, within the cryostat chamber, quickly mount the
3.4.1 Tissue Processing
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