Figure 6: Mrgprb4-lineage neurons are required for dopamine release in the nucleus accumbens during sexual mounts.

A) Graphic depicting experimental setup. Female Mrgprb4^Cre; Rosa^DTA mice or littermate Rosa^DTA controls, which had been injected with GRAB_DA to the NAc over two weeks prior to testing were paired with males for a mating assay. Females were ovariectomized and hormone primed to be in a state of behavioral estrus for testing. GRAB_DA signals were recorded for the entire pairing and analyzed surrounding male mounts. B) Average z-score traces surrounding mount onset (T=0) for littermate Rosa^DTA controls (gray) (N=12) and C) Mrgprb4^Cre; Rosa^DTA females (purple) (N=7) D) Area under the curve for Z-scored signals at pre-mount baseline (−5 to −2s), 0–3s post-mount, and 3–5s post-mount for each animal. Repeated measures two-way ANOVA revealed a significant interaction between genotype and time (p=0.037), and Sidak’s multiple comparison test revealed significant difference in GRABDA signal between baseline and 0–3s post mount for control (**p=0.0024) but not Mrgprb4^Cre; Rosa^DTA females (p=0.9945). E) Schematic of a miniaturized one-photon fluorescence microscope and calcium imaging. A DAT-Cre animal was unilaterally injected with jGCaMP8m virus into the left hemisphere of the ventral tegmental area (VTA; AP:−3.20 mm, ML: −0.40 mm, DV: −4.40 mm). DAPI-stained (blue) coronal section of DAT-Cre mouse showing GCaMP8m fluorescence (green) and the location of a 0.6mm-diameter gradient index (GRIN) lens implant (dashed lines). The inset image indicates the imaging field from the lens. F) A cell contour map (left) and CNMFe-processed image of the neurons located in the VTA (middle) with corresponding raw calcium traces (right). G) Sample video frames of anogenital sniffing and mounting. The animal poses were manually annotated on a frame-by-frame by two human annotators. H) Raw calcium traces from 7 dopamine cells in VTA (black lines) with two manual behavior annotations: anogenital sniffing (blue; 14 events) and mounting (red; 16 events). Median z-scored calcium signals of each dopamine cell at the time of anogenital sniffing (blue; left) or mounting (red; right). I) Representative calcium activity of VTA dopamine neurons during anogenital sniffing or mounting (n=23 cells, 3 mice).