{"paper_id":"1d8944fa-38b4-40f8-adc4-caf8a7063c02","body_text":"Neural substrates of female sexual rejection: hypothalamic pathways to the \nperiaqueductal gray \nInês C. Dias 1*, Nicolas Gutierrez-Castellanos 2, Liliana Ferreira 1, Ana Rasteiro 1, \nMargarida A. Duarte1, Susana Q. Lima1* \n1. Champalimaud Research, Champalimaud Foundation, Lisboa, Portugal. \n2. Department of Cell Biology, Functional Biology, and Physical Anthropology, University of \nValencia, 46100, Burjassot, Spain. \n* correspondance: susana.lima@neuro.fchampalimaud.org & \nines.dias@research.fchampalimaud.org \nAbstract \nSelecting an appropriate behavioral response according to one’s internal state is \nessential for well-being. Across the reproductive cycle, fluctuating levels of sex \nhormones align female behavior with reproductive capacity by modulating neuronal \ncircuits that express hormone receptors. Sex hormone receptor-expressing neurons \npresent along the anterior-posterior axis of the ventrolateral region of the ventromedial \nhypothalamus (VMHvl) are key regulators of female sexual behavior. While posterior \nprogesterone receptor-expressing neurons of the VMHvl (pVMHvl PR+) are fundamental \nfor female sexual receptivity during the receptive phase of the reproductive cycle, we \nhave recently shown that anterior VMHvl PR+ (aVMHvl PR+) neurons are involved in \nrejection behavior when non-receptive. Here, we mapped the connectional architecture \nof aVMHvl PR+ neurons using viral tracing approaches. As expected, these neurons \nstrongly project to several hypothalamic areas. Furthermore, consistent with previous \nreports, we show that aVMHvl PR+ neurons robustly project to several columns of the \nperiaqueductal gray (PAG) along its anterior-posterior axis. Artificial activation of \naVMHvlPR+ somas selectively recruits the dorsomedial PAG (dmPAG). Optogenetic \nactivation of aVMHvl PR+ axons in the dmPAG partially recapitulates the rejection \nphenotype observed upon activation of aVMHvl PR+ somas, increasing the rate of \nrejections in receptive females. These findings reveal a putative pathway regulating \nfemale rejection behavior within a more complex circuit, ensuring that mating does not \noccur during non fertile periods.  \n1 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\nIntroduction \nMating is costly for females, who may be harmed by seminal toxins or copulatory plugs, \nas well as directly through behaviors such as sexual coercion and harassment 1,2. The \nphysical act of mating can also cause injury 3,4 , reducing the likelihood that females will \nre-mate with other males. These costs can stem from sexual conflict, a phenomenon \nthat occurs when the evolutionary interests of males and females do not fully align and \nthe reproductive strategies that maximize fitness in one sex impose costs on the other 5. \nSuch conflicts can emerge before copulation as well, for example through disputes over \nmating rates or partner choice, or after copulation, in the form of competition over sperm \nutilization. In addition, mating incurs energetic and physiological costs 6 and increases \nexposure to predation and disease 7,8. Together, these factors illustrate how mating can \nimpose significant burdens on females, thereby shaping the dynamics of sexual \nencounters. \nFor the house mouse, as in many other species, mating is restricted to occasions when \nall favorable conditions for successful fertilization are met, and prevented otherwise. To \nmaximize the likelihood that copulation coincides with peak fertility, females often \nemploy a peri-ovulation mating strategy, in which sexual behavior and receptivity are \nconcentrated around ovulation. However, the progression to copulatory behavior \nultimately depends on the integration of external cues with the female's reproductive \nstate, effectively forming a “permissive mating checklist” 9,10, which in addition to female \nfertility, also includes sufficient food and water and reduced environmental stress11–13.  \nOne of the most extensively studied regions regarding the expression of sexual \nreceptivity and lordosis, the female receptive posture, is the ventrolateral region of the \nventromedial hypothalamus (VMHvl), an area densely populated with neurons \nexpressing sex hormone receptors 14–23. While female receptive behavior has received \ngreat attention, the rejection behavior of non-receptive females has been largely \noverlooked and until recently, failure to copulate was mostly attributed to inactive VMHvl \n“lordosis” circuitry, which involves the posterior region of the VMHvl (pVMHvl) 20–23. \nHowever, using naturally cycling females, our lab has recently shown the importance of \nactive sexual rejection when females are non-receptive, and uncovered the role of \n2 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\nProgesterone receptor-expressing (PR+) neurons in the anterior VMHvl (aVMHvl PR+) in \nthe control of this behavior 24. When non-receptive, the activity of aVMHvl PR+ neurons is \nheightened and correlated with rejection behavior in response to male’s attempts of \ncopulation, and these neurons can bidirectionally regulate rejection behavior. The \nactivity of this population seems to be dynamically regulated across the reproductive \ncycle by the amount of synaptic input it receives 24. Still, the downstream projections of \naVMHvlPR+ neurons and their specific contribution to the expression of sexual rejection \nremain unknown. \nStudies using classic non-specific anterograde and retrograde tracing approaches have \nprovided a complex map of inputs and outputs to and from hypothalamic nuclei, \nincluding the VMHvl 25,26, as well as strong interconnectivity between them 27. However, \nhow the different genetically defined neural subpopulations present in the VMHvl, or the \nanatomical diversity across the anterior-posterior (AP) axis, fit in these complex \nconnectivity profiles has only recently started to be unravelled 28. Investigating the \nconnectivity of VMHvl neurons expressing Estrogen receptor (VMHvl Esr1+), Lo and \ncolleagues found that the pVMHvl Esr1+ subpopulation preferentially sends afferents to \namygdalar/hypothalamic nuclei, while aVMHvl Esr1+ neurons preferentially projects to the \nmidbrain periaqueductal gray (PAG), a premotor structure28.  \nIn contrast, the characterization of the outputs of VMHvl PR+ neurons remains incomplete  \n(but see Inoue et al., 2019; Yang et al., 2013)20,28. Although it is commonly assumed that \nEsr1+ and PR+ neurons are a fully overlapping ensemble, suggesting that the \nprojections could be similar across the cells expressing these receptors, this degree of \noverlap is controversial. Some studies report that over 92% of PR+ neurons co-label for \nEsr1 in both sexes 20,29, while others show that only ~50% of PR+ neurons co-express \nEsr1, and 40-60% of Esr1+ neurons co-label for PR 30. Whereas these differences could \npotentially be explained by different colocalization ratios in different species (e.g. guinea \npig vs rat vs mouse), they highlight the need to better characterize the connectivity \npattern of VMHvlPR+ neurons across the AP axis in the female mouse. \nHere, we first use viral tracing methods to map the efferent connectivity of aVMHvl PR+ \nand pVMHvlPR+ neurons. These neurons share a high degree of output regions, however \n3 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\nwe identified anatomically and functionally distinct projection patterns, in particular to \nthe PAG. Using optogenetics, we show that the specific pathway from aVMHvl PR+ \nneurons to the dmPAG partially recapitulates the rejection phenotype previously \nobserved upon activation of the aVMHvl PR+ somas, increasing the rate of rejections in \nreceptive females, suggesting that the aVMHvl→dmPAG pathway is involved in sexual \nrejection in non-receptive females. \nResults \nProjections of anterior VMHvlPR+ neurons  \nTo investigate the output connectivity of the aVMHvl PR+ subpopulation of neurons, we \nunilaterally injected Cre-dependent adeno-associated vectors (AAV) carrying \nsynaptophysin tagged with GFP (SynGFP, a synaptic vesicle protein present in axonal \nterminals)31 in the anterior VMHvl of PR-Cre female mice (Fig. 1A). A quantitative \nwhole-brain analysis of the density of SynGFP+ puncta revealed that the terminals of \naVMHvlPR+ neurons are present in more than 150 brain regions (Table S1), strongly \nprojecting to hypothalamic, midbrain and thalamic regions (Fig. 1B). The output \nconnectivity of aVMHvl PR+ neurons differs from that of pVMHvl PR+ neurons, whose \nterminals are mainly present in hypothalamic regions (Fig. S1A,B). Importantly, 17 out of \nthe 161 identified outputs of the aVMHvl PR+ neurons are specific outputs for this \nsubpopulation (for detail see Table S1 and Table S2). These aVMHvl PR+ specific targets \ninclude areas within the thalamus, such as some nuclei of the geniculate complex (IntG, \nIGL, SGN and LGd) and ventral posterolateral nucleus of the thalamus (VPLpc), or \nwithin the cerebral cortex, such as perirhinal area (PERI), postrhinal area (VISpor), key \nstructures for multisensory integration32–35.  \nDespite the differences in connectivity between the anterior and posterior neurons, \nthese two subpopulations innervate many shared regions. However, within these shared \nregions some are anterior-preferred outputs, such as the PAG, the anterior \nhypothalamic nucleus (AHN), the accessory supraoptic group (ASO), the \nsubparaventricular zone (SBPV) and the precommissural nucleus (PRC) (Fig. 1C-E, \nTable S1, Fig. S1E-F), while others are posterior-preferred, such as the posterodorsal \n4 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\npreoptic nucleus (PD), the medial preoptic nucleus (MPN), the medial preoptic area \n(MPOA), and the ventral premammillary nucleus (PMv) (Fig. S1C-F, Table S1 and S2).  \n \nFigure 1 - Outputs of anterior VMHvl PR+ neurons. (A) Schematic of virus injection aVMHvl (left) and \nrepresentative histological section showing PR-Cre neurons expressing Synaptophysin-GFP (SynGFP) at \nthe injection site (right). Scale bar: 500 μm. (B) Broad quantification of the projections of aVMHvl PR+ \nneurons to main brain divisions: thalamus (Tha), striatum/pallidus (Stri/Pall), olfactory (Olf), \nmidbrain/hindbrain/medulla (Mid/Hind/Med), hypothalamus (Hyp), hippocampus (Hippo), cortex (Cort), \ncerebellum (Cereb).  (C) Quantification of the projections of aVMHvl PR+ neurons (N = 5). The analysis of \nthe signal across 30 principal targets revealed strong projections from the aVMHvl PR+ neurons to \nhypothalamic regions, including the anterior hypothalamic nucleus (AHN), retrochiasmatic area (RCH), \naccessory supraoptic group (ASO), subparaventricular zone (SBPV), medial preoptic nucleus (MPN), \nmedial preoptic area (MPOA), ventral premammillary nucleus (PMv), dorsal premammillary nucleus \n(PMd); to midbrain regions, such as the periaqueductal gray (PAG), precommissural nucleus (PRC), \n5 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\nnucleus of the posterior commissure (NPC); to thalamic regions, such as the peripeduncular nucleus \n(PP); and bed nucleus of the stria terminalis (BNST). (D) Schematic summary with the main output \nregions of the aVMHvl PR+. Arrow thickness represents relative projection strength. (E) Representative \nimages of Synaptophysin-GFP signal, Scale bar: 500 μm (top) with inset on regions of interest (below). \nScale bar: 200 μm.  \nAnterior VMHvlPR+ neurons innervate specific subregions of the PAG \nAs previously shown 28,36, the PAG is one of the main output regions of the VMHvl, and \nof its PR+ subpopulation in particular 20,21. Here, we sought to investigate in more detail \nthe projection patterns of aVMHvl PR+ within the PAG (Fig. 2A,B).   To do so, we quantified \nthe synaptophysin-GFP density  along  the  AP axis of the PAG after specifically \ntransfecting aVMHvlPR+ neurons (Fig. 2C,D). \nWe observed that aVMHvl PR+ neurons project to the entire AP extent of the PAG, with \nstronger density in its medial and posterior portions (Fig. 2C,D). When investigating \neach AP level in detail, we observe a high density of aVMHvl PR+ projections to the \ndmPAG, dlPAG and lPAG columns in the dorso-ventral and medio-lateral axis (Fig. 2C). \nInterestingly, we observed that the projection pattern of aVMHvl PR+ neurons differs from \nthat of pVMHvl PR+ neurons, since the posterior population projects mainly to the most \nposterior region of the PAG (Fig. S2A-D), preferentially targeting the lPAG and the \nvlPAG (Fig. S2C). \nActivation of anterior VMHvlPR+ somas lead to localized activation of the PAG \nTo determine if and how the anatomical connectivity between the aVMHvl PR+ neurons \ntranslates into the activation of the PAG, we optogenetically activated the somas of \naVMHvlPR+ neurons in vivo and used immunohistochemistry to detect the presence of \nthe cFos protein, a proxy for neuronal activation, in brain sections spanning the whole \nAP axis of the PAG (Fig. 2E). \nWe observed that the artificial activation of aVMHvl PR+ neurons induces a visible \nincrease in cFos positive somas across the AP axis of most PAG columns. However, the \ncFos increase on the medial part of the dmPAG was particularly high, reaching \nstatistical significance when compared to control animals (Fig. 2F-J, Fig. S2E-L) and \nsuggesting that this region of the PAG might be involved in sexual rejection behavior. \n6 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\n \n \n7 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\nFigure 2 - Optogenetic activation of aVMHvl PR+ neurons elicited an increase in cFos expression in \nthe medial region in the AP axis of the dorsomedial column of the PAG (dmPAG). (A) Schematic of \nthe quantified projections from the aVMHvl PR+ neurons to the PAG. (B) Representative image showing \nSynGFP expression in the PAG. Scale bar: 1000 μm (inset: scale bar 250 μm). (C) Heatmaps of binned \nSynGFP signal across different AP levels, normalized to injection size and averaged across mice (N=5). \nScale bar: 1000 μm. (D) Quantification of aVMHvl PR+ to PAG projections shown in C. (E) Schematic \nrepresentation of the optogenetic activation of aVMHvl PR+ neurons (top left), an example histological \nimage of the injection site and fiber placement (bottom left), and the light stimulation protocol (right). Scale \nbar: 400 μm. (F) Representative histological images showing a cFos increase in the dmPAG of ChR2 \nfemales compared to the control group. Scale bar: 500 μm. (G-J) cFos density in the different columns of \nthe medial PAG. (G) Mann-Whitney test, p = 0.04 and (J-L) posterior. *p < 0.05, Mann-Whitney test. Gray: \nCtrl, N = 6, blue: ChR2, N = 8. \n \nIn vivo optogenetic manipulation of anterior VMHvlPR+ axon terminals in the \ndmPAG elicits rejection behavior in sexually receptive females \nTo investigate the role of the aVMHvl PR+ - dmPAG pathway in female sexual rejection \nbehavior, we optogenetically activated the axon terminals of aVMHvl PR+ neurons \nprojecting to the dmPAG of naturally cycling, sexually receptive females \n(proestrous-estrous, PE) during a sexual encounter. To do so, adult PR-Cre females \nwere bilaterally injected in the aVMHvl with a viral construct carrying either ChR2 \n(ChR2) or YFP (Ctrl), and implanted with a single fiber-optic cannula above the dmPAG \n(Fig. 3A, Fig. S3A). After at least 3 weeks of recovery, PE females were allowed to \ninteract with a stud male. The stimulation pattern consisted of cycles of 1 s light ON (20 \nHz, 5 ms, 3-4 mW) and 4 s light OFF (stimulation protocol based on Ahmadlou et al., \n2024; Cao et al., 2025, see Methods for details, Fig. 3B) 37,38. To assess the efficiency of \nour stimulation protocol, we unilaterally injected a viral construct expressing ChR2 in the \naVMHvl of PR-Cre females and performed ex vivo whole-cell patch clamp recordings in \nbrain slices containing the PAG (Fig. 3C). The stimulation reliably evoked EPSCs in \nPAG neurons with high fidelity ex vivo (Fig. 3D).  \nContrary to the observed when optogenetically activating aVMHvl PR+ somas24, activation \nof dmPAG terminals did not alter the behavior of test females during the Appetitive 1 \nphase of the social interaction (from male in until the first male mount attempt), when \ncompared to Ctrl animals (Fig. 3E-G). Males in both groups also investigated the female \nat similar rates during the Appetitive phase 1 (Fig. 3H). \n8 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\nThe duration of the Appetitive phase 2 (from the first male mount attempt until the first \nsuccessful penile intromission) for ChR2 females showed a slight, not significant (Fig. \n3I) increase compared to controls, and was not correlated with the number of rejections \n(Fig. S3). The distance between the couple in the ChR2 group during this phase was \nsignificantly increased (Fig. 3J), with no difference in the rate of male-directed \nanogenital investigations by the females (Fig. 3K). The rate of male mount attempts was \nsignificantly decreased in the ChR2 couples (Fig. 3L). These differences may arise from \nthe fact that a significantly higher proportion of ChR2 females (ChR2: 6 out of 9) \ndisplayed sexual rejection (boxing and kicking) compared to Ctrl females (Ctrl 0 out of \n7) (Fig. 3M), with a significantly higher rejection rate as well (Fig. 3N). No difference was \nobserved in the rate of escapes performed by the females of the two groups (Fig. 3O). \nDuring the Appetitive phase 2, males in both groups also investigated the female at \nsimilar rates (Fig. 3P), suggesting that the disruption in the social interaction is not \ncaused by a decrease in motivation from the males interacting with the ChR2 females. \nAlthough the artificial activation of the aVMHvl PR+ - dmPAG pathway is sufficient to shift \nthe behavior of PE females towards sexual rejection, the manipulation was not sufficient \nto prevent copulation (Fig. 3Q). All females, independently of displaying rejections \nduring the Appetitive phase 2 or not, accepted mounting by the male and allowed him to \nejaculate. However, when we examined the behavior during the Consummatory phase \n(from the first successful penile intromission until the male ejaculates), we observed \ndifferences between the behavior of the two groups. A larger percentage of ChR2 \nfemales performed rejection behavior during the Consummatory phase, compared to \nthe Ctrl females - 8 out of 9 ChR2 females displayed rejections, while only 2 out 7 Ctrl \nrejected (Fig. 3R,S). Even though more ChR2 females rejected, the rejection rate \nbetween the two groups was not different (Fig. 3S). The duration of the phase also did \nnot differ between the two groups (Fig. 3T), nor the distance (Fig. 3U). The rate of \nmale-directed anogenital investigations performed by the female also did not differ (Fig. \n3V). However, in the ChR2 couples, the males performed a significantly higher rate of \nmount attempts (Fig. 3W), even though a large percentage of those mounts attempts \ndid not end in penetration (Fig. 3X). The rate of successful mounts was not altered (Fig. \n3Y), nor did the rate of anogenital investigations performed by the male (Fig. 3Z). \n9 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\nInterestingly, we observed a positive correlation between the number of rejections \nduring the Consummatory phase and the duration of the phase (Fig. S3C), but no \nrelationship between the number of rejections during the Appetitive phase 2 and the \nduration of the Consummatory phase (Fig. S3D). During the Consummatory phase, the \nnumber of mount attempts performed by the male is positively correlated with the \nnumber of rejections performed by the ChR2 females (Fig. S3E), suggesting that \nrejection behavior leads to an increase in motivation of the male to mate (Fig. 3W). \nFinally, a closer look at the pattern of mount attempts and rejections showed that the \nCtrl females rejected towards the end of the session, while the ChR2 females rejected \nearlier, after the first mount attempt performed by the male (Fig. S3F). This observation \nis supported by survival analysis of the latency to the first rejection - ChR2 females \nexhibited a significantly higher probability of rejecting than Ctrl (~15-fold higher hazard), \nshifted towards earlier time points (Fig. 3ZA). \nThe aVMHvl PR+ - medial dmPAG terminal stimulation did not have an effect in other \nbehavioral metrics, such as total distance traveled, speed and center occupancy when \nthe female was alone in the cage (Fig. S3G,H), suggesting it does not affect locomotion \nand it is not anxiogenic; importantly, the stimulation of ChR2 females did not affect \nsocial encounters with female conspecifics (Fig. S3I), supporting the specificity of the \nmanipulation to the mating context. In addition, activation of aVMHvl PR+ terminals in the \ndmPAG did not increase cFos in aVMHvl PR+ neurons, suggesting that the change in \nbehavior is unlikely to be due to the recruitment of other output regions of the aVMHvlPR+ \nneurons via back propagation of action potentials (Fig. S3J-L). \n10 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\n \n \n11 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\nFigure 3 - Optogenetic activation of the aVMHvl PR+-dmPAG pathway leads to an increase in \nrejection behavior during App2. (A) Schematic representations and representative histological sections \nof the ChR2 virus injection in the aVMHvl (left) and the mono fiber-optic cannula implantation in the \ndmPAG (dashed lines represent optical fiber placement). Scale bars: 250 µm. (B) Behavioral design (see \nMethods). (C) Schematic representation of whole-cell recording of PAG neurons while stimulating \naVMHvlPR+ axons expressing ChR2 (top) with an example of evoked EPSCs with a 20 Hz, 5 ms, 4 mW \nlight stimulation protocol (bottom). (D) EPSC fidelity (EPSC per light pulse) during the light stimulation \nprotocol (n = 7 neurons from N = 4 mice). (E) Duration of Appetitive phase 1 (App1), equivalent to the \nlatency to the first mount attempt by the male from the moment he enters the experimental arena. (F) \nAverage distance between the couple. (G) Rate of male-directed anogenital investigations by the female \nduring App1. (H) Rate of female-directed anogenital investigations by the male. (I) Duration of Appetitive \nphase 2 (App2), from the first mount attempt to the first successful mount by the male. (J) Average \ndistance between the couple during App2. Mann–Whitney U test, p = 0.023. (K) Rate of male-directed \nanogenital investigations displayed by females during the App2 phase. (L) Rate of mount attempts \nperformed by the male. Unpaired t-test, p = 0.025. (M) Percentage of females that displayed at least one \nrejection event during App2. N-1 Two-proportion test, two-tailed p = 0.008. (N) Rate of rejection events \nduring App2. Mann–Whitney U test, p = 0.015. (O) Rate of escapes performed by the female during \nApp2. (P) Rate of male-directed anogenital investigations by the female during App2. (Q) Percentage of \nfemales that had sex. (R) Percentage of females that displayed at least one rejection event during \nConsummatory phase (Cons). N-1 Two-proportion test, two-tailed, p = 0.017. (S) Rate of rejection events \nduring Cons. Mann–Whitney U test, p = 0.052. (T) Duration of the Cons phase, from the first successful \nmount by the male to ejaculation or session termination. (U) Average distance between the couple during \nCons. (V) Rate of male-directed anogenital investigations displayed by females during the Cons phase. \n(W) Rate of mount attempts performed by the male. Unpaired t-test, p = 0.018. (X) Percentage of \nsuccessful mounts during Cons. (Y) Rate of mount with thrusts (successful mounts) performed by the \nmale. (Z) Rate of male-directed anogenital investigations by the female during Cons. (ZA) Survival \nanalysis of the latency to the first rejection. ChR2 females showed a significantly higher probability of \nrejecting earlier than controls (Cox proportional hazards model, HR = 15.0, 95% CI = 1.8–123.7, p = \n0.01). *p < 0.05, **p < 0.01. Boxplots represent the median and interquartile range (IQR); whiskers extend \nto 1.53 IQR. Gray: Ctrl N = 7, blue: ChR2 N = 9. \n \nDiscussion \nIn this study, using anatomical tracing techniques, we characterized in detail the \nwhole-brain output pattern of aVMHvl PR+ neurons, showing projections to more than 150 \nbrain regions, strongly projecting to hypothalamic, midbrain and thalamic regions. \nMoreover, our analysis revealed that the efferent connectivity of aVMHvl PR+ neurons \npresents shared and distinct output targets when compared to the efferent connectivity \nof pVMHvl PR+ neurons. One prominent difference was found in the PAG: whereas \naVMHvlPR+ neurons sends strong projections across the whole AP axis of the dmPAG, \ndlPAG and lPAG, the axon terminals of pVMHvl PR+ are mostly present in the most \nposterior lPAG and the vlPAG regions. Using optogenetics, we found that the activation \n12 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\nof aVMHvlPR+ neurons leads to the specific activation of dmPAG neurons and modulates \nthe behavior of sexually receptive females during a sexual encounter by eliciting an \nincrease in rejection behavior, partially recapitulating the behavioral phenotype \nobserved when directly stimulation the somas of aVMHvlPR+ neurons. \nThe differences in connectivity of PR+ neurons across the AP axis of the VMHvl \nreported in the present study are largely consistent with those previously described for \nthe Esr1-expressing population 28. For instance, while posterior Esr1+ neurons engage \nmore prominently in intra-hypothalamic connectivity, anterior Esr1+ neurons produce \nmajor additional outputs to the midbrain. Moreover, we have identified several areas \nthat receive strong input from PR+ neurons, and that had been previously shown to \nreceive no or few projections from the VMHvl Esr+ subpopulation, which may reflect \nspecific output regions of the PR+ population. These input recipients include the ASO, \nthe supraoptic nucleus (SO) and the peripeduncular nucleus (PP) for aVMHvl PR+ \nneurons; and the posterodorsal preoptic nucleus (PD) and Barrington’s nucleus (B) for \npVMHvlPR+ neurons.These areas are involved in a wide array of different functions, \nranging from the control of vasopressinergic systems (Riva 1999), to male and female \nsexual behavior 39–42 and bladder control 43. Notably, the PD has been shown to be \nselectively activated by ejaculation and lesions in this region reduce mounting behavior \nand delay ejaculation 44,45, while the PP in female rats has been shown to play a \nfunctional role in the control of lordosis 41. This highlights functionally meaningful output \nregions in the context of sexual behavior that are specific projections of the PR+ \nneurons.  \nThese AP connectivity differences may underlie aspects of the functional differences \nthat have been reported for the PR-expressing population throughout the VMHvl AP \naxis in the context of female sexual behavior 21,24. Other spatially organized functional \nheterogeneities have been observed for other molecularly defined subpopulations of \nVMHvl neurons. For instance, while regulation of female sexual receptivity seems to be \nlocalized to the most posterior-lateral part of the VMHvl19, which neurons mainly express \nCckar23, its posterior-medial division is involved in aggressive behavior toward intruders \n13 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\nin mothers 19. And while the pVMHvl ER+,Npy2r– population was shown to be involved in \nsexual receptivity, the pVMHvlER+,Npy2r+ population is involved in maternal aggression22. \nIn this study we focused on the projections from the aVMHvl PR+ neurons to the PAG. \nThe PAG is a major midbrain hub for translating a vast array of anatomical inputs, \nincluding hypothalamic signals, into coordinated motor outputs underlying social \nbehaviours, including defense and mating, through its projections to spinal premotor \ncircuits46,47. The PAG is a very large and complex structure with widespread \ntranscriptional and functional heterogeneity 48,49. The dorsal, lateral and ventrolateral \nsubregions of the PAG are known to control different behaviors ranging from escape \nand active avoidance from a threat 50–53 (dorsomedial and dorsolateral PAG or \ndm/dlPAG), to mating, hunting and attack 54–59 (lateral PAG or lPAG), and immobility, \nfreezing, and lordosis 49,57,60–63 (ventrolateral PAG or vlPAG). We observed differences in \nthe output connectivity of PR+ neurons across the VMH AP axis, namely while the \nterminals of anterior neurons could be detected spanning the whole PAG (with \nprominent projections to the dmPAG, dlPAG and lPAG), posterior neurons terminate \nmore posterior in lPAG and vlPAG. These differences in connectivity agree with the \nfunctional heterogeneity that has been reported for the PR+ population, as different \nsubregions of the PAG are involved in the control of distinct behaviors.  \nThe analysis of cFos density in the PAG after the optogenetic experiment revealed that \nartificial activation of aVMHvl PR+ neurons specifically activates the medial dmPAG. We \nobserved a slight increase in cFos density in all PAG subdivisions, but it was more \npronounced in the medial dmPAG, which is consistent with the previously proposed \nhypothesis that the PAG columns are co-activated during diverse behaviors48. In Vaughn \net al. 64, the authors investigate the transcriptional and spatial logic of PAG function \nduring instinctive behaviors, not only across the different columns, but also along the \nentire AP axis. They observed that after mating, in the female brain, the more posterior \npart of PAG is active, while aggression in males (not studied in females) recruits the \nmore anterior columns. This is consistent with our results that show that pVMHvl \n(involved in sexual receptivity) neurons project mainly to the posterior PAG, while the \naVMHvlPR+ population projects strongly throughout the AP axis. These results suggest \n14 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\nthat the aVMHvl PR+-medial dmPAG pathway is a putative pathway for the control of \nsexual rejection behavior. We tested this hypothesis by optogenetically stimulating the \naxons of the aVMHvl PR+ neurons terminating in the medial dmPAG, in receptive females \n(in PE phase). Overall, the optogenetic manipulation resulted in disruptions of the \nsexual interaction, in particular, we observed an increase in the number of females that \nexhibited sexual rejections, similar to what we observed when we stimulated the somas \nof aVMHvlPR+ neurons24. However, contrary to the soma stimulation, we were not able to \nfully disrupt copulation, as all couples had sex and all the males ejaculated. While the \nsoma stimulation leads to the activation of all projections from aVMHvl PR+, terminal \nactivation was localized to the dmPAG. The fact that we did not observe any cFos \nactivation in aVMHvl PR+ after the stimulation of its dmPAG terminals suggests that there \nwas no backpropagation 65, thus the manipulation was specific to the location where the \noptical fiber was present. However, definite confirmation would require \npharmacologically silencing the silencing of the cell bodies while stimulating the axon \nterminals. As such, the most parsimonious explanation for the lesser behavioral \ndisruption lies in the fact that in the present study we manipulated a single output of the \naVMHvlPR+ population, the dmPAG. The more extreme phenotype after soma stimulation \nmight have resulted from the fact that we did not manipulate other aVMHvl PR+ terminals \nin the PAG, or projections to other brain regions, such as other hypothalamic areas. \nRecent studies have shown that distinct projections of a neural population play separate \nroles in behavior. For instance, Liu et al., have shown that downstream pathways of the \nMPN mediate different aspects of social isolation 66, and Lee et al., have shown that \ndifferent DRN projections mediate distinct behaviors in social homeostasis, despite \nsubstantial collateralization 67. Therefore, these results raise the question of whether \nthere are other pathways involved in the regulation of rejection behavior or if it is \nspecific to the aVMHvl PR+-dmPAG. We believe that complementary mechanisms and \nadditional neural pathways are necessary for the full expression of the non-receptive \nstate. In males, oxytocin neurons in the retrochiasmatic supraoptic nucleus (SO) were \nshown to be essential for social avoidance 68. In addition, GABAergic neurons of the \nAHN have been identified to mediate anxiety-associated investigatory behaviors, during \nrisk assessment, in the context of predatory threat 69. In this study, we show that the \n15 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\naVMHvlPR+ neurons project to these nuclei, raising the hypothesis that the \naVMHvlPR+-SO or aVMHvl PR+-AHN pathways could also be mediating social avoidance \nin females.  \nAltogether, our findings advance our understanding of the circuit logic through which \naVMHvlPR⁺ neurons influence female sexual behavior. By linking the spatially organized \noutputs of these neurons to functionally distinct PAG subregions, this study identifies the \naVMHvlPR+–dmPAG pathway as a candidate circuit component contributing to sexual \nrejection during non-receptive states within a larger, multi-node circuit. \n \nMethod details \nAnimals and and reproductive/estrous cycle monitoring \nData were collected from adult (2–9 months old) \nB6129S(Cg)-Pgrtm1.1(Cre)Shah/AndJ75 (PR-Cre 20; JAX stock #017915), expressing \nCre recombinase under the control of the PR promoter.  \nAnimals were kept under controlled temperature of 23±1 °C, reversed photoperiod of 12 \nh light/dark cycle (light available from 8 pm to 8 am) and group-housed conditions \n(unless specified otherwise) in standard cages with environmental enrichment elements \n(cardboard igloo, shredded paper and soft nesting material). Food and water were \nprovided ad libitum . Females were weaned at 20–21 days of age and group-housed \nwith two to five animals. For optogenetic experiments, after reaching 6 weeks of age, \nfemales were exposed to adult C57BL/6 (JAX stock #000664) male soiled bedding once \nper week to stimulate the natural reproductive cycle. Cages were not changed on \nexperimental days. \nC57BL/6J male mice that had at least 3 prior ejaculation experiences within 3–4 weeks \nwere used as studs for sexual behavior. For the training sessions, we used hormonally \nprimed OVX females. The hormones were dissolved in sesame oil (Sigma-Aldrich, \nS3547-1L): estrogen (0,1 mg/ml, β-Estradiol 3-benzoate, Sigma-Aldrich, E8515-200MG) \nand progesterone (5 mg/ml, Sigma-Aldrich, P0130-25G). Females received 0,1 ml of \n16 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\nestrogen 2 days prior to the sexual behavior and 0,1 ml of estrogen 4 h before the \nencounter, subcutaneously in the back while under anesthesia (with 3 % isoflurane in \noxygen). All mice were kept in Specific Pathogen Free level 1 conditions. \nProcedures were executed in accordance with the standards approved by the \nCommission for Experimentation and Animal Welfare of the Champalimaud Centre for \nthe Unknown (Órgão para o Bem Estar Animal; ORBEA) and by the Portuguese \nNational Authority for Animal Health (Direcção Geral de Alimentação e Veterinária; \nDGAV) (references 0421/000/000/2018 and 122571/24-S). \nFor the optogenetic experiments, females were habituated to Papanicolaou (Pap) \nsmear collection daily for at least 4–5 days (approx. length of one complete reproductive \ncycle) before the experimental day. Pap smear collection consisted of manual restraint \nof the female followed by lavage of the vaginal region, using a pipette (Axygen, \nTE-204-Y, with a soft tip so that the vaginal region is not stimulated): 10 μL 0.1 mM \nPBS, which was then collected onto a slide 70. These smears were then stained using \nthe Papanicolaou staining protocol 70, observed under a Zeiss AxioScope A1 brightfield \nmicroscope with a 10 x objective, and the reproductive state was determined by manual \nassessment of the proportion of different cell types in the stained smear 13,70–72. Females \nwere classified as D/diestrus or non-receptive when the predominant cell type was \nleukocytes, and as PE/between proestrus and estrus or receptive when there was a \nhigher number of anucleated cornified cells along with an equal or lower proportion of \nnucleated epithelial cells in the smear. Experiments were performed immediately after \npap smear collection and staining if the female was assessed as being in the PE or D \nstate (specified in each experiment). Females that did not exhibit proper reproductive \ncycles during the habituation phase were not included in the study. All female mice used \nin this study were drug and test naive and had not been used for any previous \nprocedures. Females had no previous sexual experience (sexually naive). \nStereotaxic surgeries \nFor anatomical tracing, females were unilaterally injected on a stereotaxic (Kopf, Model \n962) with 13.8 nL of AA1-CAG-Floxed-SynGFPrev-WPre (prepared in-house) in the \n17 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\naVMHvl (AP: −1.05, ML: –0.50, DV: −5.70; N = 5) or pVMHvl (AP: −1.3, ML: –0.70, DV: \n−6.00; N = 6) at 4.6 nL/pulse using a nanoliter injector (Nanoject II, Drummond \nScientific, 3-000-205A) at 0.1 Hz. Females were perfused 3 weeks after viral injection. \nFor optogenetic activation of aVMHvl PR+ axons during male interactions, females were \nbilaterally injected with 100 nL of AAV9-EF1a-double \nfloxed-hChR2(H134R)-EYFP-WPRE-HGHpA (Addgene, 20298) for the ChR2 group (N \n= 9) or 75 nL of AAV9-CAG-GFP (prepared in-house) for the Ctrl group (N = 7) in the \naVMHvl (AP: −0.85, ML: ±0.50, DV: −5.60) at 4.6 nL/pulse using a nanoliter injector at \n0.1 Hz. Subsequently, 400 μm diameter mono fiber-optic cannulas (Doric, \nMFC_400/430-0.48_3mm_SM3_FLT) were implanted above the medial dmPAG on a \n26º angle (AP: −4.30, ML: +0.90, DV: −1.95) and were fixed using a light-cured dental \ncement (Optibond Universal, Kerr Dental, 36519) and a nano optimized fluid composite \n(Tetric Evoflow Universal, Ivoclar Vivadent, 595953).  \nA 300 μL intraperitoneal injection of saline and a 100 μL subcutaneous injection of \nbuprenorphine was administered 0.5 h before the end of each surgery. Cages were left \non a heating pad (Vet-Tech, HE008A) for 24 h. Females were thereafter singly housed \nand allowed to recover for at least 2 weeks before pap smear habituation was initiated. \nElectrophysiological recordings \nFor ex vivo electrophysiological recordings of PAG cells during optogenetic activation of \naVMHvlPR+ axons, adult PR-Cre female mice (N = 4) were injected unilaterally with 150 \nnL of AAV9-EF1a-double floxed-hChR2(H134R)-EYFP-WPRE-HGHpA (Addgene, \n20298) into the aVMHvl. Three weeks after viral injection, mice were deeply \nanesthetized using isofluorane and decapitated. Following decapitation, brains were \nquickly removed and placed in an “ice-cold” solution containing (in mM): 0.66 kynurenic \nacid, 3.63 pyruvate, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 10 D-Glucose, 230 Sucrose, \n0.5 CaCl2, 10 MgSO4, and bubbled with carbogen (5% CO2 and 95% O2). Coronal \nbrain slices of 300 µm thickness containing the PAG were obtained using a vibratome \n(Leica, VT1200) and continuously perfused with oxygenated artificial cerebrospinal fluid \n(ACSF) containing (in mM): 127 NaCl, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 25 \n18 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\nD-Glucose, 2 CaCl2, and 1 MgCl2, at 34º C for 30 min and stored in the same solution \nat room temperature. Slices were then transferred to a recording chamber containing \ncirculating oxygenated ACSF and visualized using a SliceScope Pro microscope \n(Scientifica). Whole-cell recordings were performed using pipettes (resistance 3-5 mV) \nfilled with an internal solution containing (in mM): 135 K-gluconate, 10 HEPES, 10 \nNa-phosphocreatine, 3 Na-L-ascorbate, 4 MgCl2,4 Na-ATP, 0.4 Na-GTP (pH 7.2 \nadjusted with NaOH and osmolarity; 292 mOsm). Recordings were obtained with a \nMulticlamp 700B amplifier (Molecular Devices) and digitized at 10 KHz with a Digidata \n1440a digitizer (Molecular Devices). Optogenetic activation was delivered using a 450 \nnm LED mounted on the back of the microscope (CoolLED, pR-300 Lite), with light \nbeing delivered to the slice through a 40 x objective lens at a focal distance of \napproximately 2 mm (Olympus,  LUMPLFLN 40XW). Light pulses of 5 ms were \ndelivered at 4 mW and 20 Hz. \nIn vivo optogenetic manipulations \nLight was delivered to the cannula using a 450 nm LED (Doric Lenses, \nLDFLS_450/075) via a 400 μm mono fiber-optic patch cords (Doric Lenses, \nD207-1405). Light stimulation (5 ms, 20 Hz, 3-4 mW) was delivered for 1 s every 5 s (1 \ns on – 4 s off) throughout the session, as used in other studies of optogenetic terminal \nstimulation of hypothalamic neurons 37,38. Before each session, the LED power was \nmeasured at each tip of the patch cord using an optical power meter (Thorlabs, Inc., \nPM130D) and set to 3–4 mW.  \nAll experiments were performed on PE females. The experimental session began with \nan habituation of 5 min in the experimental arena alone, followed by an open field test \n(OFT). Afterwards, a female was added into the arena and they were allowed to freely \ninteract for 20 min. The female was removed and a male stud was added into the arena. \nThe session was terminated either right after the male ejaculated, 30 min after the first \nmount attempt by the male if the female did not allow the male to successfully mount \nher until then, or 1 h after first mount with intromission if the male did not ejaculate until \nthen. All our males were screened for sexual performance and we based the interval on \nprevious studies of the lab 73,74 where we observed that after the first mount attempt, \n19 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\nmost males ejaculated in less than 10 min. If the female did not allow the male to \nsuccessfully mount her, a follow up pap smear was collected immediately after the \nconclusion of the experiment to assess if the female had transitioned to the next phase \nof the reproductive cycle, which would explain her non-receptive behavior. Only females \nthat had follow-up pap smears still in the PE state were included. If the male did not \nexhibit a mount attempt within 30 min of introduction into the cage, he was removed and \nanother male was added to the cage. \nVideo recording was performed using 2 cameras (Point Grey, Flea3, Monochrome), one \ntop view and one front view, at 30 frames/second (fps). A custom data acquisition and \nsynchronization board (constructed in-house) was used for triggering the LED and video \ncameras and was controlled using a custom program written in Bonsai 2.4.0.  \nOn the day of euthanasia, females underwent 10 min of the experimental light \nstimulation protocol in the experimental cage before being returned to their home cage. \nPerfusion was performed after 90 min for cFos detection.  \nBehavioral annotations \nBehaviors during the sexual behavior assays were manually annotated frame-by-frame \nusing Python Video Annotator 3.9.21 (Software Platform, Champalimaud Research). \nThe following behavioral events were scored: \n● Male entry - point event - when the male was added to the cage and all four of \nhis paws hit the cage floor. \n● Anogenital investigation - point event - when the nose of the animal is in the \nvicinity of the anogenital region of the other animal. This could be a male to female \nanogenital investigation event or vice-versa. \n● Mount attempt - point event - when the male puts his paws on the back of the \nfemale in order to mount her, but fails to perform shallow thrusts (probing) or thrusting. \n● Mount with intromission and intravaginal thrust - window of time - starts when the \nmale puts his paws on the back of the female, same as a mount attempt, and is able to \n20 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\nperform intromission and intravaginal thrusts. Ends when the male removes his paws \nfrom the back of the female. \n● Ejaculation - point event - when the male enters into the last thrust before \nshivering and falling to the side. \n● Rejection - point event - when the female lifts her hind- or fore-paw to hit the \nmale. \n● Escape - point event - when the female begins to run away from a mount attempt \nof a male and is successful in doing so within 15 frames of the mount attempt. \nThe distance between the two animals was calculated using the frame-by-frame 2-body \ncentroid tracking node of Bonsai.  \nHistology \nFemales were deeply anesthetized with sodium pentobarbital (120 mg/kg of body \nweight) and perfused transcardially using 0.1 M PBS followed by fixation using ice-cold \n0.4% paraformaldehyde in 0.1 M PBS. The brains were removed and stored in 30% \nsucrose-PBS + 0.1% sodium azide solution until being sliced. The heads of the females \nused in the optogenetic experiments were severed and stored whole at 4℃ in 0.4% PFA \nfor 24–48 h before the brains were removed. Brains were coronally sliced on a sliding \nmicrotome (Leica, SM2000 R) or a cryostat (Leica, CM1800) at 45–50 μm thickness.  \nAnatomical tracing: The brain sections required no immunostaining, as the SynGFP \nexhibits strong native fluorescence. After sliced, the brain sections were mounted on \nglass slides (Thermo Fisher Scientific) using Mowiol (Sigma-Aldrich) as a mounting \nmedium, cover-slipped (Thermo Scientific Menzel) and kept at 4℃ until imaging.  \nOptogenetics experiment: floating brain slices underwent a double immunostaining for \nEYFP and cFos using a primary antibody cocktail of rabbit anti-cFos (1:2000, Synaptic \nSystems, Cat. No. 226 008) and goat anti-GFP (1:1000, Abcam, ab6673) followed by a \nsecondary antibody cocktail of Alexa Fluor 594 donkey anti-rabbit (1:1000, Invitrogen, \nab150076) and Alexa Fluor Plus 488 donkey anti-goat (1:1000, Invitrogen, A32814). \n21 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\nSlices were mounted on glass slides (Thermo Fisher Scientific) using Mowiol \n(Sigma-Aldrich) as a mounting medium, cover-slipped (Thermo Scientific Menzel) and \nkept at 4℃ until imaging.  \nImaging \nAnatomical tracing: The whole-brain sections were imaged using a slide scanner (Zeiss \nMicroscopy, ZeissAxioScan.Z1).  \nOptogenetics experiment. For brain sections in which colocalization analysis was \nconducted, a confocal laser scanning microscope with Airyscan (Zeiss, LSM 710) \nequipped with a 10 x/0.45 apochromat lens (Zeiss) was used for acquisition. Brain \nslices containing the aVMHvl were identified by overlaying the Allen brain atlas 75 (© \n2004 Allen Institute for Brain Science. Allen Mouse Brain Atlas) on brain slice images in \nAdobe Illustrator 2020 (Adobe).  \nQuantification and statistical analysis  \nBrain section image analysis of Synaptophysin-GFP \nThe obtained images were processed using the Zen software (Zen 2.6, Zeiss \nMicroscopy). Shading correction and stitching were applied and finally images were \nresized to 50% and exported as portable network graphics (.png).  \nThe images were then analyzed following the QUINT workflow 76 with some adaptations. \nThe process involved the following steps: \n (1) Pre-processing: Images were processed in Nutil using the Resize function to meet \nthe QuickNII input requirement of a maximum image size of 16 MP. The same resize \nfactor was applied to all images within a batch, ensuring all images met the \nrequirements.  \n(2) Atlas registration: For each animal, an extensible markup language (XML) descriptor \nfile containing all its brain sections was generated using the “FileBuider.bat” tool \nprovided with QuickNII (RRID:SCR_016865). Image registration to the Allen brain atlas \n22 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\nas reference for the brain regions (© 2004 Allen Institute for Brain Science. Allen Mouse \nBrain Atlas)75 was performed using QuickNII.  \n(3) Segmentation: Segmentation of labelled features was performed using ilastik. The \nsegmentation was performed in two steps. First, using Pixel Classification, as described \nin Yates et al. (2019) 76, obtaining a probability map. The second step would be Object \nClassification, however it was not appropriate for our type of signal (puncta). To \novercome this, we used a customized MATLAB script to save the segmentation images \nobtained in the previous step. A probability threshold of 0.55 was applied to first binarize \nthe image, and then the image was saved as RGB color mode, with the signal in the red \nchannel only.  \n(4) Quantification: Quantification of features per atlas region was performed by using \nthe Quantifier feature of Nutil, as described 76. This step generated a set of report files \nand customized atlas images superimposed with the object pixels (signal). \nCustomized MATLAB scripts were used to analyze the data of the reports. Several \nexclusion criteria were applied in sequential order: (1) areas that had 0 object pixels for \nall animals of the batch were excluded; (2) brain regions that 4 out of 5 animals (for \naVMHvl) or 5 out of 6 animals (for pVMHvl) had 0 object pixels were excluded; (3) brain \nregions that had less than 9 object pixels in more than 4 animals (for both batches), it \nwas considered below signal and those areas were excluded. After this, brain areas \nwith subregions were clustered together, summing the object pixels and the area (e.g. \n'Primary motor area, Layer 2/3', 'Primary motor area, Layer 5', 'Primary motor area, \nLayer 6a', 'Primary motor area, Layer 6b' were clustered to ‘Primary motor area’). (4) \nSignal on fiber tracts was not included in the analysis.  \nAs a result, we obtained the area (in pixels) and the signal (in pixels) for each brain \nregion. By determining the pixel size in μm2, per batch, we calculated the area in μm2 \nand the density of the signal. Finally, the density was normalized to the number of \nsomas in the injection site ((# of fluorescent pixels/area (mm2))/# of somas). The \nnumber of somas was manually counted, using the images with their original size.   \n23 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\nFor the detailed analysis of Synaptophysin-GFP in the periaqueductal gray (PAG), brain \nsections spanning the full AP extent of the PAG were selected. The number of AP levels \nwas defined by the number of slices of the animal that contained less sections (per \nbatch). Slices were selected so they would match between all animals (per AP level). If \nthere were missing slices, the synGFP density values were interpolated and plotted as a \nfunction of AP levels, in MATLAB. Bregma value per section was obtained from \nQuickNII.  \nTo generate the PAG heatmaps, we performed image registration in MATLAB to align \nthe PAG slices. We used the segmentation images obtained in step 3 of the adapted \nQUINT workflow, as previously described. Per anteroposterior level referenced in \nBregma, the segmentation map of one animal was fixed and used as reference. To the \nsegmentation maps from the other animals a geometric transformation to the reference \nimage was applied. With that, we obtained heatmaps with the  SynGFP signal (in pixels) \nfor each AP level that had the same geometric position. The signal was binned, \nnormalized to the number of somas in the injection site, averaged between animals and \nplotted as an image. \nBehavioral analysis for optogenetic experiments \nWe tracked the position of the animals’ center of mass using Bonsai. The distance \nbetween the couple, duration of the phases and rate of events were calculated using \nPython. Box plots were generated using the boxplot function, scatter plots were \ngenerated using the plot function and the survival analysis was calculated using the \nKaplanMeierFitter in Python. \n cFos quantification \nThe location of brain regions was manually determined by overlaying vectorized Allen \nBrain Atlas (© 2004 Allen Institute for Brain Science. Allen Mouse Brain Atlas) 75 \nreference sections in Adobe Illustrator (Adobe).  \ncFos positive cells were manually counted in aVMHvl and PAG and normalized to the \narea, calculated in ImageJ 1.52n. Two brain slices containing aVMHvl from each animal \n24 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\nwere used for the quantification of cFos expression. Three brain slices containing PAG \n(one anterior (~ -3.40 to -3.52 mm), one medial (~ -4.36 to -4.60 mm) and one posterior \n(~ -4.96 to -5.02 mm)) from each animal were used for the quantification cFos \nexpression, using Paxino’s Mouse Brain Atlas as reference 77. Cell counting was \nperformed manually using ImageJ. \nStatistical analysis \nStatistical analyses were performed using GraphPad Prism 9 Software. Normality of the \nresiduals was tested using the Shapiro-Wilk test. In data that passed the normality test, \nindependent-samples Student’s t-test or paired t-test were used to evaluate differences \nbetween groups. If data did not follow a normal distribution, analysis was performed \nusing a non-parametric Mann–Whitney U test for unpaired samples. \nBox plots indicate median and the interquartile range (IQR, ± 25th – 75th percentile) \nand the whisker edges represent the minimum and maximum data limits excluding \noutliers using the Tukey criterion (outliers are depicted outside the box plot). Error bars \nand shaded error bars represent mean and standard error of the mean. For comparing \nfractions (i.e. percentage of animals that had sex or rejected), we used the N-1 \nTwo-proportion test. For the survival function analysis, we used the Cox \nproportional-hazards model. Statistical significance was set at p < 0.05. Statistic details \n(test and p values) can be found in the results section. \nAll mice were randomly assigned into experimental groups. Each experimental batch \nconsisted of age and weight matched groups of females. Each experimental batch was \nbalanced between control and test groups. Blinding of the data was conducted before \nany manual scoring (both for behavioral annotations and for cFos quantification). No \nsample size estimation was conducted prior to this study.  \nFor optogenetics experiments, biological replicates consisted of independent mice (N =  \nnumber of mice) tested only once. These results were analyzed with unpaired statistics. \nFor neural recording and manipulation experiments, mice were excluded only when \nvirus or fiber location was visibly off-target. \n25 \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted January 21, 2026. ; https://doi.org/10.64898/2026.01.20.700523doi: bioRxiv preprint \n\nReferences \n1. 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