Figure 1. Anterior-posterior distribution of BA-NAc projection neurons and of their c-Fos activation by social reward or social aversion.
(A) Experimental design. Handling; CTB infusion & recovery/tracing: stereotactic surgery for bilateral CTB-Alexa Fluor 555 injection in NAc; Single-housing; Social stimulus: exposure to social reward (SR), social aversion (SA) or left undisturbed (no stimulus, No S); Ex-vivo: 90 min after onset of SR or SA, social mice and No S were perfused-fixed. (B) Representative image (10x, scale bar = 2 mm) at bregma +1.1 showing NAc CTB injection. Schemas for stimulus exposures: SR: 90-min exposure to a (pro-)estrous female in subject’s home cage; SA: subject transferred to cage of an aggressive-dominant male CD-1 mouse, 20 min distal exposure via a divider, followed by proximal exposure for 60 sec attack or 10 min maximum, followed by distal exposure for remainder of the 90 min. Perfused-fixed brains sectioned coronally (40 µm) and 1 section per 0.2 mm from bregma -1.0 to -2.6 mm used for c-Fos immunostaining, confocal imaging and quantification of CTB+ and c-Fos+ BA-NAc neurons. (C-E): BA-NAc c-Fos expression, data are group mean ± SEM, with value for each mouse being the mean of both hemispheres for each bregma level. (C) Total CTB+BA-NAc neurons versus BA bregma level. Neuronal number depended on bregma level (bregma main effect: F(2.44, 85.44)=109.1, p<0.0001) with highest number of neurons at -2.0. There was no main or interaction effect involving group (p≥0.84). (D) Total c-Fos+ BA cells versus BA bregma level. SR and SA led to increased c-Fos+ cells compared with No S mice, with highest number at -1.6; there were more c-Fos+cells in SA versus SR mice at -1.6 and -2.6 (stimulus group x bregma interaction effect: F(16, 280)=7.71, p<0.0001). Different letters denote significant pairwise post hoc stimulus-group differences using Tukey’s multiple comparison. (E) c-Fos+/CTB+ BA-NAc neurons versus BA bregma level. SR and SA led to increased c-Fos+ BA-NAc neurons compared with No S mice, with highest number at -1.6 to -2.0 (intermediate BA); there were more c-Fos+ neurons in SA versus SR mice at -2.4 and -2.6 (posterior BA) (stimulus group x bregma interaction effect: F(16, 280)=6.52, p<0.0001). Different letters denote significant or trend-level pairwise post hoc stimulus-group differences using Tukey’s multiple comparison. (F-H): The same data as in C-E now collapsed into BA subregions, anterior (aBA, -1.0 to -1.4), intermediate (intBA, -1.6 to -2.0) and posterior (pBA, -2.2 to -2.6); data are individual values with mean ± SEM, with values for each mouse being the mean of both hemispheres x 3 bregma levels . (F) Total of CTB+ BA-NAc neurons versus BA subregion. Neuronal number was similar in intBA and pBA and higher than in aBA (subregion main effect: F(1.41, 49.42)=168.60, p<0.0001). There was no main or interaction effect involving group (p≥0.67). (G) Total c-Fos+ BA cells versus BA subregion. In aBA and intBA, SR and SA led to increased c-Fos+ cells compared with No S mice, and SA led to increased c-Fos+ cells compared with SR, whilst in pBA SR and SA led to a similar increase compared with No S (stimulus group x BA subregion interaction effect: F(4, 70)=6.67, p=0.0001). Different letters denote significant pairwise post hoc differences using Tukey’s multiple comparison. (H) c-Fos+/CTB+BA-NAc neurons versus BA subregion. In aBA and intBA, SR and SA led to a similar increase in c-Fos+ BA-NAc neurons compared with No S, whilst in pBA SR and SA led to an increase in c-Fos+ BA-NAc neurons compared with No S and SA to an increase compared with SR (stimulus group x subregion interaction effect: F(4, 70)=10.78, p<0.0001). Different letters denote significant pairwise post hoc differences using Tukey’s multiple comparison. (I) Representative confocal micrographs for left-hemisphere BA at bregma -1.8 from an SR mouse (upper images) and an SA mouse (lower images): Left: CTB. Middle: c-Fos. Right: Merged; the dashed line depicts the approximate medial and lateral subdivisions of the BA. Open arrows indicate examples of BA-NAc CTB+/c-Fos+ neurons. Scale bar = 100 μm. Brightness and contrast have been adjusted for display purposes.