Analysis of SR-FP data
LABView files of video recording and of optical signal data were used to
score social events using an ethogram (Table S2). Per test phase, the
number and total duration of social episodes, and from this the mean
duration of each social episode, were calculated. The data for the first
SR test were used for statistical analysis unless they included
substantial artefacts in which case the second test was used (in the
case of one mouse). Social events were manually time stamped onto the
optical signal data. The time of each social event that resulted in the
onset of a social episode, e.g. approach, approach+mount,
approach+copulation, was designated as t=0 s. The fibre photometry data
were analyzed with custom-written MATLAB programs . Optical signal data
were demodulated at 970 Hz, down-sampled to a sampling frequency of 20
Hz, and smoothed using an exponential smoothing function with damping
factor 0.5, thereby excluding some of the broad-spectrum signal noise.
The social event-specific mean Ca2+ (or EGFP) activity
during the 5 s prior to event onset at t=0 s provided the measure of
baseline activity. For 10 s after social-event onset, regardless of the
duration of the social episode that it initiated, for each 0.05 s time
bin (t), the z -scored (normalized) signal intensity (F )
was calculated using the formula (F (t ) -F 0)/SD 0, whereF0 and SD0 denote mean and
standard deviation of 5-s baseline activity (mean z-scored
Ca2+ baseline signal = 0). After the onset and offset
of a social episode, if the onset of the next social episode occurred
within 10 s, this latter episode was not analyzed; this was done in
order to achieve some separation between baseline signal and social
episode-related signal. For each mouse and each phase (distal SR,
proximal SR), the mean peri-event/episode histogram was calculated, and
these mouse-specific histograms were used to calculate the overall mean
histograms per phase.