Immunohistochemistry and confocal microscopy
For c-Fos immunofluorescence, series of sections were collected from
-1.6 to -2.0 mm i.e. the AP intermediate BA. Using the same
custom-written Fiji macro as described for the BL/6 mouse experiment,
the numbers and threshold gray values for CTB+,
tdTom+, c-Fos+,
CTB+/tdTom+,
CTB+/c-Fos+ and
CTB+/tdTom+/c-Fos+cells/neurons were determined. The numbers and densities of
cells/neurons were calculated as the sum of [mean of both hemispheres
per section] for 5 sections (-1.6, -1.7, -1.8, -1.9, -2.0). In
addition, for c-Fos, the integrated density of each
c-Fos+ BA-NAc neuron was calculated as area x mean
gray value.
The TRAPing parameters used were optimized by pilot study, in which
reporter expression by BA cells (not specifically BA-NAc neurons ) was
quantified. A maximal efficacy of about 30% tdTom labeling of c-Fos
labeling was realized, i.e. if a stimulus (e.g. SR, SA, footshock)
resulted in an average of 100 c-Fos+ cells, it
resulted in an average of 30 tdTom+ cells. Protocol
changes, including higher 4-OHT dose, signal amplification with tdTomato
immunostaining, did not increase TRAPing efficacy.