Evidence for monovalent intermediate BA-NAc neurons with respect to salient social stimuli
Having identified that in BL/6 mice the int-BA has a relatively high absolute number and density of NAc projectors that are engaged by salient social stimuli, in a next experiment the adult male offspring ofFos-TRAP2  x Ai14 mouse strains , referred to asFos-TRAP2 mice, were studied to investigate whether these int-BA-NAc neurons are mono- or dual-valent with respect to SR and SA. Targeted recombination in active populations (TRAP) allows for the study of activation of an immediate early gene, in this case Fos , in the same neurons by two stimuli presented at different time points (Fig. 2A). Whilst the efficacy of the TRAPing method is somewhat limited , the literature indicates it to be adequate for current purposes given the inclusion of appropriate control groups.
Fos-TRAP2 male mice were injected in the NAc core/shell at bregma +1.0-1.2 mm with CTB-647 (Fig. 2B, Fig. S6). After 10 days, mice were single caged and, after a further 3-4 days, underwent either No S, SR or SA, as described above (Fig. 2C, Table S1). All mice were then injected with 4-hydroxytamoxifen (4-OHT) to induce Fos -dependent iCreERT2-driven recombination, leading to permanent, cell-specific tdTom expression. After 7 days, mice in the experimental-social groups were exposed to the opposite social stimulus, SA or SR, for 90 min (i.e. SR-SA or SA-SR groups), mice in the control-social groups were re-exposed to the same social stimulus for 90 min (i.e. SR-SR or SA-SA groups), and No S mice remained No S (No S-No S group) (Fig. 2C). All 25 SR mice (SR-SA, SA-SR, SR-SR) approached and mounted the female, and 17 of these copulated and penis licked, and in both exposures in the case of SR-SR mice (Table S2). All SA mice were attacked by the aggressive-dominant male for between 30-60 s during the proximate phase, and in both exposures in SA-SA mice (Table S2). All mice then underwent brain perfusion-fixation. Brains were sectioned coronally at int-BA -1.6 to -2.0, and c-Fos immunofluorescence staining, confocal microscopy and image quantification were carried out.
As expected, the total number of CTB+ int-BA-NAc neurons was similar across groups (Fig. S7A). Fot total int-BA cells, the number of tdTom+ int-BA cells was greater in mice exposed to SR or SA than in No S mice, and in mice exposed SA than in mice exposed to SR (Fig. S7B). In line with the limited efficacy of TRAPing, for each social-stimulus group, the total number of tdTom+ int-BA cells was substantially less than that of c-Fos+ int-BA cells (Fig. 2G; Fig. S7B vs C). The number of c-Fos+ int-BA cells was highest in SA-SA mice (Fig. S7C).
Focusing on int-BA-NAc (CTB+) neurons (Fig. 2G), we first identified tdTom+ neurons (Fig. 2G), and then scored whether these were c-Fos- or c-Fos+ (Fig. 2G). The absolute number of tdTom+ neurons was higher in each group of stimulus mice than in No S mice (Fig. 2D). In SR-SA and SA-SR mice, the absolute number of tdTom+/c-Fos- neurons was higher than that of tdTom+/c-Fos+neurons, whereas in control SR-SR and SA-SA mice these numbers were equable (Fig. 2D). This was confirmed by analyzing the percentage of tdTom+ neurons that were c-Fos+(Fig. 2E): In mice exposed to SR-SA or SA-SR, 16.4% ± 2.1% (mean ± SEM, n=17) of tdTom+ neurons were also c-Fos+, whereas in mice exposed to SR-SR or SA-SA, it was 49.8% ± 2.7 % (mean ± SEM, n=16). Given that about 50% of BA-NAc neurons were re-activated by re-exposure to SR or SA, then that only less than 20% of SR- or SA-activated BA-NAc neurons were re-activated by SA or SR, respectively, indicates that a substantial proportion of SR and SA responsive BA-NAc neurons are monovalent, at least with respect to these specific social stimuli. For tdTom- BA-NAc neurons (Fig. 2F), more of these were c-Fos+ in control-social (SR-SR, SA-SA) mice than in experimental-social (SR-SA, SA-SR) mice, suggesting that first exposure to SR or SA leads to sensitization of the neuronal c-Fos response at re-exposure. Interestingly, this observed increase in the number of BA-NAc tdTom-/c-Fos+ neurons co-occurred with a reduced mean BA-NAc neuron c-Fos signal-intensity (Fig. S8), consistent with a combination of sensitization of some and habituation of other neurons.
This experiment demonstrates that a substantial proportion of the intermediate BA neurons projecting to the NAc core/shell at bregma +1.0-1.2 are monovalent with respect to the social stimuli used, being engaged by either a (pro-)estrous female or an aggressive-dominant male, and with only a minority being engaged by both social stimuli. This finding was obtained despite the absolute number of social stimulus-responsive BA-NAc neurons likely being under-estimated due to the known quantitative limits of the TRAPing method.