Here, we took advantage of the house mouse, a species whose ejaculation is dependent on copulatory patterns that resemble human sexual dynamics, in particular repeated vaginal thrusting preceding ejaculation. This feature of mouse copulation stands in contrast to rat sexual behavior, where ejaculation is dependent on the execution of multiple individual penile insertions, spaced in time, each one with the potential of triggering ejaculation. To investigate spinal circuits involved in sexual behavior, we used the pelvic muscle controlling the last step of this complex behavior, the BSM, as an entry point into the system. We successfully labeled the motoneurons that innervate the BSM and their presumably presynaptic partners with the pseudorabies strategy that was indicated in the proposal. Amongst the BSM motoneurons and the presumably presynaptic partners that were positive for Gal+ we labeled multiple other parasympathetic and sympathetic centers that have been shown to be involved in ejaculation, such as the intermediolateral nucleus and the central autonomic nucleus. Since the pseudorabies strategy however cannot make any claim regarding a monosynaptic connection between the BSM motoneurons and the Gal+ neurons we decided to take a viral approach that has been described by Silvia Arbers group (Stepien et al. 2010) which uses the co-injection of a retrograde traveling adenoassociated virus (AAV) fused to a G-protein and a G-deleted rabies such as that by a single injection, the motoneurons and presynaptic partners are labeled thereby proving a monosynaptic connection. Even though we tried over a year to have this working, we failed to obtain positive results and hence changed plans and went on injecting an AAV that labeled the synapses of our Gal+ cells. By injecting fluorogold into the BSM and checking for overlap of synaptic boutons and fluorogold labeled BSM motoneurons, we were able to get a hint about the monosynaptic connection between these two cell groups. To prove the latter functionally, we teamed up with the group of Sandrine Bertrand and performed in vitro whole cell recordings of BSM motoneurons in line with optogenetic stimulation of Gal+ neurons. The latter led to specific activity in BSM motoneurons but not motoneurons that innervate the leg thereby proving a monosynaptic connection between the BSM motoneurons and the Gal+ cells.
We went on to show that the Gal+ neurons are recipients of genital sensory input, and that their electrical stimulation evokes dominant BSM activity (measured through an EMG) but only after spinalization. Interestingly, the evoked activity was dependent on the sexual arousal of the male prior to spinalization, indicating that the dynamics of the spinal cord circuitry controlling BSM activity also represent the internal state of the animal. However, contrary to what is observed in the rat, we failed to elicit emission with stimulation of the Gal+ population, but only observed expulsion-like BSM activity. Through cfos labeling we were furthermore able to show that the Gal+ neurons seem to be active as soon as the male becomes sexually aroused. Finally, the genetic ablation of this population led to profound impact on the copulatory length and structure. All these results point towards an unexpected and more intricate role of the spinal cord in the control of sexual behavior, beyond the relay of genital information and the production of the ejaculatory reflex.
