Using Viral Vectors to Study the Memory Trace in Mice - Viral Vector Approaches in Neurobiology and Brain Diseases - page 132

the lateral nucleus of the amygdala (LA) as a critical region for the
acquisition and storage of this associative memory [ 1 , 2 ]. Within
the LA, it has been estimated that approximately 20-25 % of
neurons are involved in encoding of a typical CS-US association
[ 3 , 4 ], but the ability to reliably identify and subsequently manipu-
late this subpopulation has proved challenging. Thus, the ability to
identify the proportion of LA neurons critically involved in the
memory trace would facilitate further characterization of the
molecular, cellular, and circuit mechanisms that underlie memory
formation.
In our laboratory, we have delineated a population of neurons
in the LA that are critical for the encoding and storage of condi-
tioned fear memories. By using viral-mediated gene delivery to
manipulate levels of the transcription factor CREB (cAMP/Ca 2+
responsive element binding protein) in individual neurons in the
LA, we found that neurons with relatively high CREB levels have
an increased probability (>70 %) of becoming part of a fear memory
trace compared to their neighbors with relatively lower CREB lev-
els [ 3 ]. Furthermore, by specifi cally deleting neurons with high lev-
els of CREB after memory formation, we showed that these neurons
are indeed critical components of the fear memory trace [ 5 ].
In this chapter, we describe the methods to (1) manipulate
CREB levels in individual LA neuron in vivo using a replication-
defi cient Herpes simplex virus (HSV) gene delivery system and (2)
identify neurons involved in a memory trace using a technique
known as catFISH ( c ompartmental a nalysis of t ranscripts by fl uo-
rescence i n s itu h ybridization). In this latter technique, nuclear
expression of the mRNA for the immediate early gene Arc ( a ctivity-
r egulated c ytoskeletal associated protein) is used as a measure of
recent neuronal activity (e.g., after fear conditioning or fear mem-
ory testing). As the protocol for generating HSV delivery particles
have been described in detail elsewhere [ 6 , 7 ], only a brief over-
view will be provided for this section along with any critical factors
to consider when using this approach.

2 Materials

The catalogue numbers and/or suppliers indicated for the equip-
ment below are those which we have found to be reliable. However,
equivalent items from other suppliers may be used.

2.1 Surgery and
Virus Injection

Suitable anesthetics and analgesics
3 % H 2 O 2
0.9 % saline
dH 2 O
70 % ethanol

2.1.1 Solutions