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.