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.