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.