Following behavioral testing, rats were deeply anesthetized with an overdose of sodium pentobarbital. Rats in which Fos plumes were measured were perfused and brains treated as described previously (Reynolds and Berridge, 2008 (link)). These included rats behaviorally tested in the environmental shift group (n=10; which therefore received a 7th final drug or vehicle microinjection and behavioral test 90 minutes prior to perfusion) and a separate dedicated Fos group (n = 36; which were histologically assessed after just a single drug or vehicle microinjection into locations staggered throughout medial shell, administered under conditions identical to the first day of testing for behavioral rats). The purpose of the dedicated Fos group was to assess maximal local impact radius, and avoid danger of under-estimating plume size due to progressive necrosis/gliosis over a series of microinjections that might shrink a final plume. If shrinkage occurred in the behaviorally tested group, that in turn could give rise to overly precise estimates of localization of function in brain maps. This potential distortion of impact estimates by plume shrinkage was prevented in the dedicated group that received only one microinjection.
All rats used for Fos analysis were anesthetized and transcardially perfused 90 minutes after their final or sole bilateral microinjection of vehicle (n=10), DNQX alone (n=13), DNQX plus SCH23390 (n=6), DNQX plus raclopride (n=10), DNQX plus raclopride and SCH23390 (n=3) or no solution (normal, n=3). Brain slices were processed for Fos-like immunoreactivity using NDS, goat anti-cfos (Santa Cruz Biotechnology, Santa Cruz, CA) and donkey anti-goat Alexa Fluor 488 (Invitrogen, Carlsbad, CA) (Faure et al., 2008 (link); Reynolds and Berridge, 2008 (link)). Sections were mounted, air-dried and coverslipped with ProLong Gold antifade reagent (Invitrogen). Zones where the expression of fluorescent Fos was elevated in neurons surrounding microinjection sites (“Fos plumes”) were assessed via microscope as described previously (Reynolds and Berridge, 2008 (link)).
Other brains were removed and fixed in 10% paraformaldehyde for 1–2 days and in 25% sucrose solution (0.1 M NaPB) for 3 days. For assessment of microinjection site locations in behaviorally tested rats, brains were sliced at 60 microns on a freezing microtome, mounted, air-dried and stained with cresyl violet for verification of microinjection sites. Bilateral microinjection sites for each rats were placed on coronal slices from a rat brain atlas (Paxinos and Watson, 2007 ), which were used to extrapolate the position of each site on one sagittal slice. Mapping in the sagittal view allows for the presentation on the same map of the entire rostrocaudal and dorsoventral extents of NAc medial shell. Functional effects on appetitive and fearful behaviors were mapped using color-coding to express the intensity of changes in motivated behaviors for individual behaviorally-tested rats. Symbols were sized to match the maximal diameter of Fos plumes measured as described below. Sites were classified as rostral shell if their NAc placements were located +1.4 to +2.6 mm ahead of bregma, and as caudal shell if their placements were located +0.4 to +1.4 mm ahead of bregma.
All rats used for Fos analysis were anesthetized and transcardially perfused 90 minutes after their final or sole bilateral microinjection of vehicle (n=10), DNQX alone (n=13), DNQX plus SCH23390 (n=6), DNQX plus raclopride (n=10), DNQX plus raclopride and SCH23390 (n=3) or no solution (normal, n=3). Brain slices were processed for Fos-like immunoreactivity using NDS, goat anti-cfos (Santa Cruz Biotechnology, Santa Cruz, CA) and donkey anti-goat Alexa Fluor 488 (Invitrogen, Carlsbad, CA) (Faure et al., 2008 (link); Reynolds and Berridge, 2008 (link)). Sections were mounted, air-dried and coverslipped with ProLong Gold antifade reagent (Invitrogen). Zones where the expression of fluorescent Fos was elevated in neurons surrounding microinjection sites (“Fos plumes”) were assessed via microscope as described previously (Reynolds and Berridge, 2008 (link)).
Other brains were removed and fixed in 10% paraformaldehyde for 1–2 days and in 25% sucrose solution (0.1 M NaPB) for 3 days. For assessment of microinjection site locations in behaviorally tested rats, brains were sliced at 60 microns on a freezing microtome, mounted, air-dried and stained with cresyl violet for verification of microinjection sites. Bilateral microinjection sites for each rats were placed on coronal slices from a rat brain atlas (Paxinos and Watson, 2007 ), which were used to extrapolate the position of each site on one sagittal slice. Mapping in the sagittal view allows for the presentation on the same map of the entire rostrocaudal and dorsoventral extents of NAc medial shell. Functional effects on appetitive and fearful behaviors were mapped using color-coding to express the intensity of changes in motivated behaviors for individual behaviorally-tested rats. Symbols were sized to match the maximal diameter of Fos plumes measured as described below. Sites were classified as rostral shell if their NAc placements were located +1.4 to +2.6 mm ahead of bregma, and as caudal shell if their placements were located +0.4 to +1.4 mm ahead of bregma.
