Stimulus exposure protocols
All protocols were conducted between 09:00-13:00 h, i.e. in the first
half of the dark phase. Table S1 summarizes the conditions used for
stimulus pre-exposure and test in this and the other experiments.
Social reward (SR). At the onset of the dark phase on days 15 and
16 post-surgery, adult female BL/6 mice were screened for reproductive
stage: vaginal lavage was conducted by gently pipetting and triturating
50 µL sterile ddH2O at the opening of the vagina. The
derived cell suspension was transferred onto a glass slide and placed at
37°C until dry. The cells were then stained with 50 µL 0.1% cresyl
violet, cover-slipped and assessed at the microscope . Females that were
at proestrus or oestrus were included as social reward stimuli. An
unfamilar (pro-)estrous female was introduced into the home cage of the
male for test onset (t0). Behavioural events were
observed during 90 min using an ethogram (Table S2); it was noted
whether each of mount, copulate and penis lick occurred. If males showed
at least one mount within min 1-20 the protocol was continued; if not,
which was attributable to the female repeatedly leaving the male, the
protocol was discontinued and the male was presented with another female
on the following day. After 90 min (t90), the female was
returned to its home cage and the male was processed for brain
perfusion-fixation.
Social aversion (SA) . The protocol comprised 3 stages conducted
within a period of 6 days in the colony room. In the habituation stage
on days 15-17 post-surgery, the home cage of the male was divided by
placing a transparent, perforated plastic divider along its length for
20 min per day. In the pre-exposure stage on day 18, the same divider
was placed in the cage of an aggressive, ex-breeder CD-1 male aged 8-10
months and weighing 38-55 g (Janvier Labs, France). The BL/6 mouse was
introduced into the same compartment as the CD-1 mouse and received a
cumulative total of 60 s physical attack maximum or at least 30 s
physical attack during 10 min maximum (see ethogram, Table S2). The BL/6
mouse was then returned to the home cage and left undisturbed for 24-48
h. This primed the BL/6 male so that distal and proximal exposure to the
CD-1 mouse was aversive in the final test stage. In the social aversion
test on day 20, the same divider was placed in the cage of the same CD-1
mouse: the BL/6 mouse was placed in the opposite compartment to the CD-1
mouse for test onset (t0) and exposed to distal aversion
(visual, olfactory, auditory) for 20 min; it was then placed in the same
compartment as the CD-1 mouse (proximal) for 30-60 s cumulative physical
attack during 10 min maximum, and then remained in this compartment
whilst the CD-1 mouse was transferred to the other compartment (distal
aversion) until t90 (Table S2). The BL/6 mouse was then
returned to its home cage and processed for brain perfusion-fixation.
Aversive odour (AO). As a physical aversive stimulus,
2,5-dihydro-2,4,5-trimethylthiazoline (TMT, 1 g/mL; Angene
International, Hong Kong), a constituent of fox urine/feces with an
odour innately aversive to rodents , was used. The BL/6 mice were
transferred to a remote and isolated room on days 15-16 post-surgery and
allowed to acclimatise. On day 17, a paper tissue was inserted into a 50
mL Falcon tube and 500 µL TMT were pipetted onto the tissue, and the
tube without cap was placed on top of the grid of the home cage and
covered with the cage lid (t0). The mouse could not
physically contact the tissue but the TMT odor permeated the cage. The
following behaviours occurred and were maintained throughout the
majority of the protocol: piloerection, inactivity, huddling in a corner
or in the sleeping house. At t90 the mouse was processed
for brain perfusion-fixation.
No stimulus (No S). To provide a comparison group of basal c-Fos
activity in BA-NAc neurons, No S mice remained undisturbed in the colony
room in their home cage until being processed for brain
perfusion-fixation. On each day that stimulus exposure was run, 1-3 No S
mice were also included in the expeiment.