Figure 2. Activation of intermediate BA-NAc neurons by social reward and/or social aversion using TRAPing and c-Fos.
(A) Schematic of TRAP system. In the presence of a salient stimulus, Fos promoter-Fos-iCreERT2 recombinase knock-in allele is activated/expressed. Endogenous c-Fos is expressed with a short half-life. iCreERT2 migrates to the cytosol, where it remains and degrades in the absence of 4-OHT. After 4-OHT injection, it binds to iCreERT2, the latter translocates to the nucleus, Cre-Lox recombination occurs at the tdTomato reporter knock-in allele, resulting in permanent expression of tdTomato. (B) Experimental design with Fos-TRAP2 male mice. Handling; CTB infusion & recovery/tracing: stereotactic surgery for bilateral CTB-Alexa Fluor 647 injection in NAc; Single-housing; i.p. hab: habituation to intraperitoneal injection (of vehicle) on 3 days; Social stimulus 1: exposure to either social reward (SR), social aversion (SA) or left undisturbed (No S), followed by 4-OHT at 50 mg/kg i.p.; TRAPing: 7 days for recombination and expression of tdTomato; Social stimulus 2: exposure to either SR, SA or No S; Ex-vivo: 90 min after onset of social stimulus 2, mice perfused-fixed. (C) Schematic of experimental procedures. For details, see legend Fig. 1B. Brains were sectioned coronally (40 µm) and 1 section per 0.1 mm from bregma -1.6 to -2.0 mm was used for c-Fos immunostaining, confocal imaging and quantification of CTB+, tdTom+ and c-Fos+ BA-NAc neurons. (D-F) tdTom and/or c-Fos expression by CTB+ int-BA-NAc neurons: data are individual values calculated as the sum of [mean of both hemispheres per section] 5 sections (-1.6, -1.7, -1.8, -1.9, -2.0), as well as group mean ± SEM. (D) Absolute number of tdTom+/c-Fos- and tdTom+/c-Fos+ BA-NAc neurons. There were more activated (tdTom+ and/or c-Fos+) neurons in each of SR-SA, SA-SR, SR-SR and SA-SA mice than in No S mice (stimulus group main effect: F(4, 39)=6.01, p=0.0007; a vs b, p<0.05, Dunnett’s multiple comparison test). There was a stimulus group x activity marker interaction effect (F(4,39)=16.41, p<0.0001): there were more tdTom+/c-Fos- neurons than tdTom+/c-Fos+ in No S, SR-SA and SA-SR mice, and an equable number of such single- and double-labelled neurons in SR-SR and SA-SA mice (Sidak’s multiple comparison). (E) Percentage of tdTom+ BA-NAc neurons that were also c-Fos+. There was a stimulus group main effect (F(4,39)=39.01, p<0.0001): percentage of tdTom+ neurons that were also c-Fos+was lower in experimental-social mice (SR-SA and SA-SR) and No S mice than in control-social mice (SR-SR and SA-SA) (Tukey’s multiple comparison test). (F) Absolute number of tdTom-/c-Fos+ BA-NAc neurons. There was a stimulus group main effect (Welch’s ANOVA, W(4.00, 16.15)=41.82, p<0.0001): there were more such neurons in SR-SA and SA-SR mice than in No S mice, and there were more such neurons in SR-SR and SA-SA mice than in No S, SR-SA, SA-SR mice (Dunnett’s T3 multiple comparison). Note the different Y-axis scales in D and F. (G) Representative confocal micrographs for left-hemisphere BA at bregma -1.8 from an SR-SA mouse (upper images) and an SA-SR mouse (lower images): Left to Right: CTB, tdTom, c-Fos, Merged. In merged: open arrows indicate CTB+/tdTom+/c-Fos-(monovalent) BA-NAc neurons and filled arrows CTB+/tdTom+/c-Fos+(dual-valent) BA-NAc neurons. The merged images also indicate the approximate medial and lateral subdivisions of the BA. Scale bar = 100 μm. Brightness and contrast have been adjusted for display purposes.