Anteroposterior distributions of BA-NAc neurons activated by salient social stimuli
The first study aim was to determine the anteroposterior (AP) distribution profiles of BA neurons that project to NAc and are activated by salient social stimuli in terms of c-Fos protein expression (Fig. 1A). C57BL/6J male mice were injected in the NAc core and shell at bregma +1.0-1.2 mm with the neuronal retrograde tracer, cholera toxin subunit β, conjugated with Alexa Fluor 555 (CTB-555) (Fig. S1A). After 10 days, mice were single-caged and after a further 3-4 days allocated randomly to stimulus groups (Fig. 1B, Table S1): No stimulus (No S): the mouse remained undisturbed in the home cage to provide basal c-Fos levels for comparison. Social reward (SR): a (pro-)estrous BL/6 female was placed into the home cage of the mouse for 90 min and distal and proximal socio-sexual interactions occurred; all SR mice approached, mounted and copulated with the female and penis licked (Table S2). Social aversion (SA): the mouse was placed in the home cage of an aggressive-dominant CD-1 male for 90 min, comprising periods of physical separation that allowed only distal social interactions, either side of a short period of proximal contact during which agonistic social interactions occurred without wounding, with all SA mice attacked for between 30-60 s during this proximal phase (Table S2). Following exposure to SR or SA (and No S), mice underwent brain perfusion-fixation. Brain coronal sectioning, c-Fos immunofluorescence staining, confocal microscopy and image quantification, were conducted (Fig. 1A, B).
In coronal sections that included the BA, immunostaining for neuronal and glial markers indicated that CTB was localized exclusively in glutamatergic neurons (Fig. S2). In sections collected at 0.2 mm intervals from bregma -1.0 to -2.6, i.e. the full AP extent of the BA, the absolute number of CTB+ BA-NAc neuronal cell bodies was maximal at -1.8 to -2.2 (consistent across groups), and they were distributed across the medial-lateral extent of the BA (Fig. 1C, F, I; Fig. S1B). A similar profile was observed using CTB+ BA-NAc neuron density (Fig. S3A). It should be noted that when CTB-555 was injected more anteriorly in the NAc, namely at +1.6, there was a corresponding anterior shift in the distribution profile of CTB+ BA-NAc cell bodies (Fig. S4); this indicates that BA-NAc neurons are organized topographically along the AP axis, demonstrating both the importance of the projection region targeted and the specificity of the current findings to the NAc region that we targeted.
In these same sections, the AP distribution profiles of total BA cells immunostained for c-Fos were determined: in both SR mice and SA mice, this number was substantially greater than in No S mice and maximal at -1.4 to -1.8. Whilst the AP profiles of BA c-Fos+cells were overall rather similar in SR and SA mice, there were more such cells at -1.6 and -2.6 in SA than SR mice (Fig. 1D, G, I). Broadly similar profiles were obtained using BA c-Fos+ cell density, except that the increase in SA versus SR mice was now specific to -2.6 (Fig. S3B). Anecdotally, in SR mice, most of the c-Fos+ neurons were in the medial BA with a minority in the lateral BA; for SA mice, the distribution was even more medially focused (Fig. 1I); however, the medial-lateral distribution was not quantified. Focusing specifically on c-Fos+ neurons in the BA-NAc neuron pathway (Fig. 1E, H, I): in SR mice the number was highest at -1.6 to -2.0 (intermediate (int-)BA), and SA mice had a similarly high number of c-Fos+ BA-NAc neurons at these same bregma levels. In SA mice specifically, this relatively high number of c-Fos+ neurons was also present at -2.4 to -2.6, such that SA mice had more c-Fos+ BA-NAc neurons than SR mice at these posterior bregma levels. Similar profiles were obtained using c-Fos+ BA-NAc neuron density (Fig. S3C). In terms of percentage of total CTB+ int-BA-NAc neurons, compared with 3.1% ± 0.5% in No S mice, 14.8% ± 2.1% were activated by SR and 17.2% ± 1.2% by SA; in posterior BA-NAc neurons, the values were 2.4% ± 0.63%, 8.4% ± 0.8% and 13.1% ± 0.9%, respectively. To facilitate comparison with previous studies that have focused on physical stimuli, another group of mice was exposed for 90 min to the fox odor-excrement constituent trimethylthiazoline (TMT) (see e.g. ): compared with both SA and SR, fewer BA cells and fewer BA-NAc neurons were c-Fos+ in mice exposed to TMT (Fig. S5).
Therefore, in terms of projectors to the NAc core/shell at bregma +1.0-1.2, it was not the case that the AP extent of the BA was divided into separate subregions that contained BA-NAc neurons responsive to either SR or to SA. The intermediate BA (int-BA) had the highest number of NAc-projecting neurons activated by a (pro-)estrous female (SR), and this same region had a similarly high number of neurons activated by an aggressive-dominant male (SA). In the posterior BA (post-BA), more BA-NAc neurons were activated by SA than SR.