Fibre photometry target validation
After completion of the experiment, mice were deeply anaesthetised and
underwent brain perfusion-fixation for histological assessment in terms
of BA probe placement and BA GCaMP6/EGFP and NAc mCherry expression. As
described in detail elsewhere , the optic fibre implant was removed, and
the brain sectioned coronally at 100 μm using a vibratome (Leica).
Sections underwent Nissl staining (NeuroTrace 640/660 Deep-Red
Fluorescent Nissl Stain, Thermo Fisher), followed by washing in PBS,
mounting on microscope slides, addition of Dako/DAPI fluorescence
mounting medium (Sigma Aldrich), and cover-slipping. Using an
epifluorescence microscope (Axio Observer.Z.1, Zeiss), mounting medium
allowed for localization of GCaMP6/EGFP expression, and Nissl staining
allowed for localization of the probe placement and tracing of the
BA-NAc pathway using mCherry reporter. Using a mouse brain atlas
(Franklin and Paxinos, 2019), the bregma levels of the BA section that
included the most ventral position of the fibre tip in the BA and of the
NAc section with the highest mCherry expression, were identified. Two
GCaMP6 mice were excluded due to a misplaced BA AAV vector injection and
optic fibre placement, resulting in final sample sizes of n=10 for
GCaMP6 and n=4 for EGFP.