In vivo evidence for BA-NAc neuron population activity
during interaction with social reward
To complement the c-Fos-based ex vivo evidence that social
stimuli engage intermediate BA-NAc neurons, in a third experiment we
investigated the in vivo activity of the int-BA-NAc neuron
population during distal or proximal interactions with social reward,
using calcium-sensor fibre photometry (Fig. 3A-C). For SA, a pilot study
demonstrated that Ca2+ activity could not be recorded
reliably during proximal exposure to an aggressive-dominant male due to
risk of injury by (and damage to) the patch cord, and the experiment was
therefore conducted with SR specifically. BL/6 male mice were injected
in int-BA (-1.6/-2.0) with the AAV vector rAAV-FLEX-GCaMP6, or with
rAAV-FLEX-EGFP in the case of signal-control mice, and in NAc core/shell
(+1.0/1.3) with rAAV-retro-Cre, thereby achieving pathway-specific
BA-NAc expression of intracellular calcium (Ca2+)
indicator GcaMP6 (n=10) or of EGFP (n=4). An optic fibre was positioned
with its tip in the int-BA to enable GcaMP6- or EGFP-fluorescence
detection (Fig. 3C, S9). Whilst the SR stimulus was a (pro-)estrous
female as in the c-Fos experiments, SR exposure was modified such that
there were separate distal and proximal test phases (Table S1), allowing
comparison of these two forms of sensory stimulation. Calcium (or EGFP)
activity was recorded during a distal-SR phase (male separated from
female via a perforated divider) and a proximal-SR phase (male and
female with full contact). The SR tests were video recorded and the
onset and offset of social events/episodes (Table S2) were time stamped
manually onto the fibre photometry signal trace.
For each measure, number of social events that initiated social episodes
(Fig. S10A), total duration of social episodes (Fig. S10B) and the
average duration of each social episode (Fig. S10C), values were higher
in the proximal-SR phase than in the distal-SR phase. The peri-event
histograms for Ca2+ activity relative to onset of an
episode of social behaviour are given in Fig. 3D, E. In the distal-SR
phase (Fig. 3D), although social episodes were limited to the male
facing the female on the opposite side of the divider i.e. distal
contact, and had an average duration of 1-2 s, there was an increase in
Ca2+ activity at sec 3 and 4 after distal contact
onset. In the proximal-SR phase (Fig. 3E), social episodes comprised
contact and sexual behaviors (Table S2): all 10 males mounted the female
and 9 of 10 copulated and penis licked. There was an increase in
Ca2+ activity at sec 2 after the onset of proximal
contact, and episodes had an average duration of 6-7 s. There was no
time point at which there was a significant difference in post-event
Ca2+ activity between the proximal-SR and distal-SR
phases. Control mice expressed BA-NAc neuron EGFP, and this neuron
activity-independent signal was applied to check for signal artefacts
related to specific motor behaviours: EGFP mice had social interactions
similar to those of GcaMP6 mice (Fig. S10A-C); the peri-event mean EGFP
signal remained relatively stable during the distal-SR (Fig. S10D) and
proximal-SR (Fig. S10E) phases, indicating that the GcaMP6 signal
changes were unlikely to have been motor-activity artefacts related to
socio-sexual behaviours.
These in vivo social behaviour-related Ca2+activity data complement the ex vivo data in demonstrating that
int-BA-NAc neurons are indeed engaged by social stimuli, in this case
both distal and proximal interactions with a (pro-)estrous female.