Using Viral Vectors to Study the Memory Trace in Mice - Viral Vector Approaches in Neurobiology and Brain Diseases - page 144

Fig. 3 Imaging of the memory trace. ( a ) A low magnifi cation image of the right half of a coronal section from
the brain stained with Hoechst 33258. The location of the LA is indicated, as is the basolateral amygdala (BL)
and the hippocampus (hpc). The LA can be located under low magnifi cation by following the external capsule
(ec) down from the hpc to where it bifurcates at the tip of the LA. ( b ) Example of an image obtained from a
catFISH experiment. Left panel : overlay of nuclei stained with Hoechst 33258 ( blue ) and GFP mRNA ( green ).
Right panel : the same image but displaying nuclear Arc mRNA expression ( red ) instead of GFP. Neurons
expressing both GFP and Arc, GFP alone, or Arc alone are indicated. Inset of right panel : example of a neuron
with cytosolic arc expression that would be excluded from the analysis. ( c ) Example of the quantifi cation from
a typical catFISH experiment. Animals infused with CREB (GFP-CREB) or GFP show similar Arc mRNA expres-
sion in the LA. However, CREB-positive neurons have a greater probability of also being Arc-positive when
compared to overall Arc, while noninfused neurons show a decreased probability of expressing Arc. By com-
parison, in animals infused with GFP alone, there is an equal probability of Arc expression in GFP-positive and
GFP-negative neurons
hippocampus, follow the external capsule ventrally past the
rhinal fi ssure where it bifurcates at the tip of LA. The LA
should appear as a triangular structure (Fig. 3a ). The back-
ground in the “green” (FITC) channel may also help locating
the correct position of LA.
2. Locate an area that has a group of GFP-positive cells within
the LA ( see Note 15) and then set up acquisition parameters
to capture the nuclear counterstain, Alexa 488 (GFP), and
Alexa 647 (arc) signals at 40× magnifi cation (Fig. 3b ). It is
advisable to add a control channel that lies between the spec-
trum of GFP and arc signal, for example rhodamine signal ( see
Note 16).