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Hypothalamic circuits for the selection of defensive and mating behavior in females

Periodic Reporting for period 2 - YinYang (Hypothalamic circuits for the selection of defensive and mating behavior in females)

Reporting period: 2019-09-01 to 2021-02-28

In the mood for sex

The way we interact with the world is tightly coupled to our internal state. For example, a restaurant sign will make us salivate when we are hungry. In most animal species, the willingness to have sex is also tightly regulated by sex hormones, such that sex mostly happens when fertilization is likely to occur. This is, nature has evolved ways of matching sexual behavior to the reproductive capacity of an individual (mostly of females). In our laboratory, we use the female mouse as our model system. Similarly to humans, the reproductive cycle of mice, called the estrous cycle, is regulated by the shifting levels of the hormones estrogen and progesterone. It was already known that these hormones are strong modulators of behaviour in mice. Depending on the female’s mouse internal state, responses to brief social interactions with males can result in radically different outcomes, ranging from receptivity to downright aggression. This implies that sex hormones work not only on the level of the reproductive organs, but also on the brain. What is not yet fully known is precisely how these hormones influence the activity of neural circuits in the brain. We are interested in understanding the neuronal mechanisms that allow this sex hormone dependent change in behavior.

We focus our efforts in the hypothalamus, which is known to regulate many instinctive behaviours, including feeding, sleep and sexual behaviour. Therefore, our first step was to record the activity of neurons in an hypothalamic area that is dedicated to socio-sexual behaviour while females were interacting either with males or with other females: the ventromedial hypothalamus. We performed extracellular recordings of neurons in this region and discovered that the activity of some neurons changed depending on the reproductive state of the female. But these recordings are very limited, specifically because we do not know the identity of the recorded neurons, impeding us from obtaining more detailed information about the underlying mechanisms.

Therefore, in this present grant we proposed to study a particular genetically delineated population of neurons: neurons expressing the receptor for the female hormone progesterone (PR+ neurons). We chose this population because 1) they have receptors for estrogen and progesterone, meaning that their activity is sensitive to the fluctuating levels of sex hormones that occur during the estrous cycle; 2) specific ablation of these neurons cause deficits in female sexual behavior; 3) preliminary experiments in our laboratory have shown that these neurons are active during mating, but also during rejection, when females are not sexually receptive.

These results lead us to our current hypothesis abut the role of the ventromedial hypothalamus in the control of female sexual behavior: we propose that the ventromedial hypothalamus is involved in the switch between rejection/defensive behavior to receptivity. For that we propose that there might be at least two PR+ subpopulations, one involved in receptivity and the other in rejection/defensive behaviour and their activity might be bidirectionally modulated by sex hormones. Our project is divided in three main parts:

Aim1: Characterization of the individual functional specificity of individual PR+ neurons: we observed that PR+ neurons are active during mating, but also during rejections. We are interested in understanding if individual neurons are involved in the two behavioral outputs or if different cells are involved in different actions.
Aim2: We aim to identify the connectivity of different PR+ populations.
Aim3: Causally prove that distinct PR+ populations are involved in different behavioral outputs. For that we will use optogenetics to artificially stimulate different PR+ populations.
Aim4: Uncover the molecular mechanisms by which different PR+ neurons lead to a different output. For this we will use in vitro studies to understand what molecular pathways are being modulated by sex hormones.

We believe we are studying a fundamental process that goes beyond the specifics of mouse sexual behavior. Even though humans believe their sexual behavior is not tied to the reproductive cycle, there is enough data that shows that female behavior is affected by sex hormones. Also, the hypothalamic region that we are studying exists in all vertebrates and the mouse ventromedial hypothalamus is very similar in structure and molecular identity to the primate. Second, we believe that sex competes with our strong natural defensive behavior. To be safe, our brain needs to balance out two processes that are happening simultaneously: attraction and defense. So even though you would normally lean towards a ‘fight or flight’ response when coming across a stranger that could be potentially dangerous, your hormonal state gets you ‘in the mood’ while lowering your baseline defenses. The ventromedial hypothalamus and its PR+ connect to downstream areas that are involved in the "fight or flight" responses. Therefore, we are probably studying a more general circuit that allows individuals to maintain their personal space, but that can be modulated when the animal is in the right phase of the reproductive cycle to mate.
Initially we had monitored the activity of PR+ neurons of the VMH using fiber photometry, a tool that uses calcium sensors to infer neuronal activity. Fiber photometry gives us access to the bulk activity of a neuronal population, this is, we do not have single cell resolution. While doing these recordings, we observed that PR+ neurons were active when the female accepted mounting from the male, but also when she rejected. This made us hypothesize that there are at least two populations of PR+ neurons, one that is involved in mating and the other in rejection. Sex hormones bidirectionally modulate the activity of these two populations, such that when the female is receptive the animal mates and rejects when not in the right phase of the cycle. Since the beginning of the project, we have advanced in all 4 different aims proposed which intende to test this hypothesis.

Aim1. Characterize the functional specificity of individual PR+ VMHvl neurons across the reproductive cycle while females interact with male mice, using calcium based methods and microendoscopy.
Contrary to fiber photometry, microendoscopy allows to monitor the activity of individual neurons. We are performing the same type of experiments as before, allowing females to interact with females and males while they are in the receptive or non receptive phase of the cycle. We also allow females to mate with a male when sexually receptive. We have now collected the data for 5 individual females and we are in the process of data analysis.

Aim2. Map the connectivity of the different PR+ VMHvl neuronal populations to the distinct compartments of the PAG using viral tracing and optogenetic-assisted mapping.
Using immediate early genes as read out for neuronal activity we have uncovered that the most anterior portion of the VMHvl is more active in response to male stimuli when the female is non receptive. This observation, together with other published data that shows that the most anterior part of the VMHvl in males is involved in defensive behavior, lead us to hypothesize that this subregion is involved in the rejection response to males when females are not receptive. We are currently mapping the outputs of this subpopulation.

Aim3. Test the impact of different VMHvl-to-PAG pathways in vivo by activating and silencing them using optogenetic methods.
We have recently started to perform optogenetic manipulations of the anterior VMHvl and testing its involvement in rejection behavior.

Aim4. Determine how sex hormones modulate the functional properties of different PR+ neurons with respect to dendritic structure, synaptic input and intrinsic excitability.
We have immediately started this aim, first probing the electrophysiological properties of individual PR+ neurons in vitro. With these experiments we have uncovered that different subpopulations of PR+ neurons receive different excitatory drive across the reproductive cycle. We also have strong hints about the molecular pathway that is being modulated across the cycle and which causes PR+ neurons to operate on a different regime if the female is receptive or not.
Within the first half of the project we have already uncovered several important aspects about the VMHvl function which substantially add to the current state of the art.

- With the experiments performed in the first half of this project, we have uncovered that the VMHvl is also involved in rejection behavior, while the classical view of this structure was that it was only involved in mating behavior.
- We have uncovered that the VMHvl is not an homogeneous structure, this is, there is an anterior posterior axis within the VMHvl. Within that axis the properties of PR+ neurons are different and vary differently across the cycle.
- We have uncovered that the PR+ population is different from the PR negative population.
- We have compelling preliminary data that suggests the involvement of a particular molecular pathway in the change on the properties of PR+ neurons across the cycle.

Until the end of the project we intende to test the function of the observations we made and test the hypothesis that the PR+ of the VMHvl are involved in rejection and not only in mating as it was previously accepted.
Behavioral switch across the reproductive cycle