Sexually naïve estrous female mice seek out male urinary pheromones; however, they initially display little receptive (lordosis) behavior in response to male mounts. Vomeronasal - accessory olfactory bulb inputs to the medial amygdala (Me) regulate courtship in female rodents. We used a reversible inhibitory chemogenetic technique (Designer Receptors Exclusively Activated by Designer Drugs; DREADDs) to assess the contribution of Me signaling to females’ preference for male pheromones and improvement in receptivity normally seen with repeated testing. Sexually naïve females received bilateral Me injections of an adeno-associated virus carrying an inhibitory DREADD. Females were later ovariectomized, treated with ovarian hormones, and given behavioral tests following intraperitoneal injections of saline or clozapine-N-oxide (CNO; which hyperpolarizes infected Me neurons). CNO attenuated females’ preference to investigate male vs female urinary odors. Repeated CNO treatment also slowed the increase in lordosis otherwise seen in females given saline. However, when saline was given to females previously treated with CNO, their lordosis quotients were as high as other females repeatedly given saline. No disruptive behavioral effects of CNO were seen in estrous females lacking DREADD infections of the Me. Finally, CNO attenuated the ability of male pheromones to stimulate Fos expression in the Me of DREADD-infected mice but not in non-infected females. Our results affirm the importance of Me signaling in females’ chemosensory preferences and in the acute expression of lordosis. However, they provide no indication that Me signaling is required for the increase in receptivity normally seen after repeated hormone priming and testing with a male.

Previous research has shown that repeated testing with a stimulus male is required for ovariectomized, hormone-primed female mice to become sexually receptive (show maximal lordosis quotients; LQs) and that drug-induced, epigenetic enhancement of estradiol receptor function accelerated the improvement in LQs otherwise shown by estrous females with repeated testing. We asked whether pre-exposure to male pheromones (‘pheromone priming’) would also accelerate the improvement in LQs with repeated tests and whether optogenetic inhibition of accessory olfactory bulb (AOB) projection neurons could inhibit lordosis in sexually experienced estrous female mice. In Experiment 1, repeated priming with soiled male bedding failed to accelerate the progressive improvement in LQs shown by estrous female mice across 5 tests, although the duration of each lordosis response and females’ investigation of male body parts during the first test was augmented by such priming. In Experiment 2, acute optogenetic inhibition of AOB inputs to the forebrain during freely moving behavioral tests significantly reduced LQs, suggesting that continued AOB signaling to the forebrain during mating is required for maximal lordotic responsiveness even in sexually experienced females. Our results also suggest that pheromonal stimulation, by itself, cannot substitute for the full complement of sensory stimulation received by estrous females from mounting males that normally leads to the progressive improvement in their LQs with repeated testing.