Brief social experiences (e.g., sexual experiences) can lead to long-term changes in internal states and influence social behaviors such as mating and aggression. For example, after successful mating and ejaculation, many species rapidly exhibit a suppression of mating tendencies for hours, days, or longer, an effect known as sexual gratification. This inhibition is not only essential for avoiding excessive risk-taking or finding new sexual partners, but is also essential for increasing fertilization rates. However, little is known about how such experience-dependent changes manifest in neural circuits and have long-term effects on behavioral decisions.
On February 10, 2023, Beijing Brain Science and Brain-inspired Research CenterLi Ying’s research team published in the top journal "Science" Research paper titled "Hyperexcited limbic neurons represent sexual satiety and reduce mating motivation". The study found that ejaculation experience selectively activates estrogen receptor 2 (Esr2)-expressing neurons - BNSTEsr2 - in the bed nucleus of the stria terminalis (BNST) and causes a sustained decrease in firing threshold for several days, during which time mice display sexual satisfaction. . Suppression of hyperexcitable BNSTEsr2 causes rapid mating recovery in satiated mice of both sexes. In males, this hyperexcitability reduces mating motivation and is mediated in part by larger HCN currents. Therefore, BNSTEsr2 not only encodes specific mating actions, but also represents a sustained state of satisfaction, and changes in neuronal ion channels contribute to long-term changes in sexual experience-dependent mating drives.
The research work was completed by Li Ying’s research group at the Beijing Brain Science and Brain-inspired Research Center. Self-recruited postdoctoral fellow Zhou Xiaojuan, Ph.D. Li Ang, and technician Mi Xue from Beijing Brain Science and Brain-inspired Research Center are the co-first authors of the research paper, and researcher Li Ying is the corresponding author.
1. Ejaculation is the key event that drives sexual satiety in both sexes
So, what are the key factors that lead to sexual satiety in mice? Researchers designed a control group of male and female mice that experienced multiple penetrations but separated from their partners before ejaculation (did not experience ejaculation) and found that the control group did not show sexual satiation, suggesting that ejaculation is the driver Key events in the sexual satiation state of both sexes (Figure 1).
Figure 1. Ejaculation is the key event that leads animals of both sexes to a state of sexual satiation.
In order to study the neural mechanism of sexual satiety in mice of both sexes, the researchers reviewed the literature and found that in rats and long-tailed mice, ejaculation-induced Fos protein expression was present in multiple brain areas of the vomeronasal pathway, including the stria terminalis. posterior bed nucleus (BNST), central amygdala (MeA), and medial parafascicular thalamic nucleus (mSPF), but not the medial preoptic area (mPOA). Using fluorescence in situ hybridization (FISH), the researchers found that male mice that completed ejaculation had the largest change in the number of Fos+ neurons in the BNST compared with male mice that only experienced 3-5 penetrations but no ejaculation. Therefore, researchers focused on the BNST brain area for in-depth research.
Previous mononuclear cell sequencing (RNA-seq) experiments have shown that BNST contains two types of neuron subpopulations with sexual dimorphism: BNSTEsr2 and BNSST18. Calcium imaging in freely moving mice revealed that a large number of BNSTEsr2 neurons (>50%) were strongly activated when males ejaculated and when females felt ejaculation, but showed no obvious response during other stages of mating. In contrast, another population of St18-expressing neurons in this region (BNSTSt18) was primarily activated during social sniffing and other mating processes, with no apparent response during ejaculation (Fig. 2).
Figure 2. Ejaculation selectively activates Esr2-expressing neurons in BNSTpr in both sexes.
2. Hyperexcitable BNSTEsr2 neurons continuously encode sexual satiety in both sexes
After male and female separation, the response of BNSTEsr2 neurons activated by ejaculation was significantly reduced, but some neurons still maintained strong spontaneous activity 30 minutes after the end of ejaculation, suggesting that these neurons may encode a sustained satiety state. To further examine whether BNSTEsr2 neuron activity encodes sexual satiation, the researchers compared spontaneous firing when alone in male rats that had only engaged in social contact, mated, but were not sexually satiated, with male rats that had become sexually satiated after mating. , found that only male rats that reached sexual satiety showed greater amplitude and frequency of spontaneous Ca2+ activity, and this phenomenon could last for several days throughout the sexual satiety period. When male mice regain the ability to mate, spontaneous Ca2+ activity returns to baseline levels and rises again after mating again and sexual satiation.
Consistent with male mice, the spontaneous Ca2+ activity of BNSTEsr2 neurons in female mice also increased significantly 24 hours after mating, independent of whether pregnancy was successful. This increase typically persists throughout pseudopregnancy, pregnancy, and lactation, decreasing to baseline levels after mating behavior resumes.
3. Activating BNSTEsr2 neurons in male mice inhibits mating motivation
The researchers expressed the inhibitory chemical genetic virus hM4Di in the bilateral BNST of Esr2-Cre mice, and injected clozapine-N-oxide (CNO) intraperitoneally into sexually satiated male mice to inhibit BNSTEsr2 neurons. Activity. The experiment found that six out of seven male rats could resume their mating behavior and successfully ejaculate within 30 minutes. The same operation can also significantly restore the sexual receptivity of female rats that have just completed mating and are in the sexual satiety period.
In order to further distinguish whether BNSTEsr2 neurons can inhibit mating behavior or mating motivation, in male mice, the researchers expressed the opsin with ultra-high light sensitivity (SOUL) and conducted experiments at different mating stages. Cranial optical stimulation non-invasively activates BNSTEsr2 neurons. Experiments have found that activating BNSTEsr2 neurons during the sniffing phase rather than after the onset of mating can inhibit the onset of mating in normal male mice. These findings further indicate that BNSTEsr2 neurons inhibit mating motivation rather than mating actions. played an important role.
4. The increased expression of HCN ion channels in BNSTEsr2 neurons is involved in regulating sexual satiety in male mice.
To understand the mechanism of sustained changes in BNSTEsr2 neural activity, the researchers used whole-cell patch clamp methods to record the electrophysiological properties of BNSTEsr2 neurons in sexually inexperienced, sexually satiated, and mating-recovered mice of both sexes. In both male and female mice, sexually satiated (36–48 hours after mating) mice had more BNSTEsr2 neurons exhibiting higher excitability compared with sexually inexperienced and mating-recovered mice. Despite comparable input resistance and membrane capacitance, resting membrane potentials (RMPs) are highest and rheobases (the most negative step current required to induce all or no discharge) are lowest in sexually satiated BNSTEsr2 neurons, suggesting smaller Depolarization can trigger an action potential. This change was largely restored in behaviorally reinstated mice, further supporting the role of hyperexcitable BNSTEsr2 neurons in encoding the satiated state.
Analyzing the existing BNST single-cell sequencing results, the researchers found that injection of negative current into BNSTEsr2 neurons showed a depolarization "sag" voltage, mainly mediated by the hyperpolarization-activated cyclic nucleotide-gated (Hcn) cation channel. guide. Interestingly, in male mice, the magnitude of this “droop” voltage was significantly greater in sexually satiated mice compared with BNSTEsr2 neurons in sexually inexperienced or sexually reinstated mice, whereas this phenomenon was observed in female mice. Not significant. Systematically reanalyzing published snRNA-seq data sets in BNST, the researchers found that Hcn1, but not other subunits, was highly enriched in Esr2+ neurons in BNSTp relative to other cells. In addition, through the FISH method, the researchers observed a large number of HCN1+Esr2+ neurons in BNSTEsr2 neurons of male mice.
Using CRISPR/Cas9 technology to selectively knock out the Hcn1 gene in BNSTEsr2 neurons, the researchers found that male mice showed a significant loss of sexual satiety. In order to further test the role of HCN channels in regulating sexual satiety, the researchers administered ZD-7288 (HCN inhibitor) or artificial cerebrospinal fluid (vehicle control) to the intracranial BNST of sexually satiated mice about 1 hour later. 5/6 male-typical sexual behaviors were restored. These results indicate that activation of HCN channels is necessary to maintain the sexually satiated state after ejaculation in male mice (Fig. 3).
Figure 3. After CRISPR/Cas9-mediated knockout of HCN1 in BNSTprEsr2 neurons, male mice can continue to mate.
5. Summary
To sum up, this work has made an important breakthrough in revealing the important scientific issue of how sexual experience changes the internal state of the brain. This research helps us understand the neural mechanisms by which brief experiences are persistently represented in the brain, and how this representation affects animal behavior in the long term. In addition, this work also found that brief sexual experience can cause changes in the expression of ion channels in neurons, providing a new perspective for us to understand how sexual experience causes lasting changes in the brain (Figure 4).
Figure 4. Explanation of the mechanism of BNSTEsr2 neurons involved in regulating mating motivation.