Periodic Reporting for period 1 - SEG (Anatomical and functional characterization of the neural circuits controlling ejaculation)
Reporting period: 2019-02-25 to 2021-02-24
Despite the importance of sex for the existence and quality of human life, its underlying neurobiological mechanisms are poorly understood. Sex fits the basic structure of pleasure cycles and can be divided into appetitive, consummatory and inhibitory phases. This proposal focuses on the consummatory (ejaculation) phase in mice.
During sexual behaviour, copulation-related sensory information and modulatory signals from the brain must be integrated and converted into the motor and secretory outputs that characterize ejaculation. Studies in humans and rats suggest the existence of a group of interneurons in the lumbar spinal cord that mediates this step: the spinal ejaculation generator (SEG). The SEG is thought to control motor neuron activity innervating the bulbospongiosus muscle (BSM): the BSM surrounds the base of the penis, and its rhythmic contraction is necessary for ejaculation to occur. The SEG has access to peripheral information via the sensory branch of the pudendal nerve and descending input from the brain. Experiments in the rat suggest that the SEG can trigger ejaculation and might also be involved in the establishment of the post-ejaculatory refractory period (PERP), during which male mice won’t perform any sexual behaviour.
However, these ideas remain controversial, in part because the methods used have poor anatomical and cellular specificity and low temporal resolution. Hence, we employed cutting-edge molecular based strategies with high specificity (viruses combined with transgenic mice) and high temporal resolution (optogenetics and electrophysiology) to gain mechanistic insights about the neural circuits controlling ejaculation. Thereby, we found that BSM-motor neurons receive direct synaptic input from a group of galanin-expressing (Gal+) interneurons located in the upper lumbar spinal cord and that this population is progressively activated during sexual behavior and the recipient of genital sensory input. Electrical and optogenetic activation of the Gal+ neurons evoked BSM-motor neuron and BSM-muscle activity after spinalization, but the effects were dependent on the behavioral state of the male and drastically decreased with repeated stimulation. Moreover, genetic ablation of the Gal+ neurons severely impacted the latency to ejaculate and the structure of the copulatory sequence. Taken together, our results imply an unexpected involvement of the spinal cord in the integration of signals during copulation and in the post-ejaculatory refractory period, suggesting a more central and intricate involvement of the periphery in the control of copulatory behavior than previously suspected.
During sexual behaviour, copulation-related sensory information and modulatory signals from the brain must be integrated and converted into the motor and secretory outputs that characterize ejaculation. Studies in humans and rats suggest the existence of a group of interneurons in the lumbar spinal cord that mediates this step: the spinal ejaculation generator (SEG). The SEG is thought to control motor neuron activity innervating the bulbospongiosus muscle (BSM): the BSM surrounds the base of the penis, and its rhythmic contraction is necessary for ejaculation to occur. The SEG has access to peripheral information via the sensory branch of the pudendal nerve and descending input from the brain. Experiments in the rat suggest that the SEG can trigger ejaculation and might also be involved in the establishment of the post-ejaculatory refractory period (PERP), during which male mice won’t perform any sexual behaviour.
However, these ideas remain controversial, in part because the methods used have poor anatomical and cellular specificity and low temporal resolution. Hence, we employed cutting-edge molecular based strategies with high specificity (viruses combined with transgenic mice) and high temporal resolution (optogenetics and electrophysiology) to gain mechanistic insights about the neural circuits controlling ejaculation. Thereby, we found that BSM-motor neurons receive direct synaptic input from a group of galanin-expressing (Gal+) interneurons located in the upper lumbar spinal cord and that this population is progressively activated during sexual behavior and the recipient of genital sensory input. Electrical and optogenetic activation of the Gal+ neurons evoked BSM-motor neuron and BSM-muscle activity after spinalization, but the effects were dependent on the behavioral state of the male and drastically decreased with repeated stimulation. Moreover, genetic ablation of the Gal+ neurons severely impacted the latency to ejaculate and the structure of the copulatory sequence. Taken together, our results imply an unexpected involvement of the spinal cord in the integration of signals during copulation and in the post-ejaculatory refractory period, suggesting a more central and intricate involvement of the periphery in the control of copulatory behavior than previously suspected.
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.
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.
Our project and the arising results have not only potential implications for the treatment of sexual dysfunction, but also opens further avenues in the research field of male orgasm. Our established juxtacellular recordings of the Gal+ cells (SEG), for example, have the potential to further characterize the afferent stimuli which activate the SEG, a question which is completely unsolved till now, but which will lay an important foundation for the development of new therapies geared towards alleviating sexual malaise. Furthermore, knowing the neurological site that seems to reflect the sexual state of a male mouse could help to further strive the development of new pharmacological agents aimed at ameliorating both hypo and hyperactive sexual disorders. In addition, the functional knowledge and techniques pioneered here can not only be used to test functional efficiency of so far known pharmacological agents used for sexual disorders but may also be applied to improve existing assisted reproductive techniques in both humans and other mammalian species. Finally, our project has significant implications for the whole field of spinal cord research as our knowledge about the microcircuits processing somatosensory, sensory or motor signals are still quite sparse.