Abstract
While hypothalamic kisspeptin (KP) neurons play well-established roles in the estrogen-dependent regulation of reproduction, little is known about extrahypothalamic KP-producing (KPLS) neurons of the lateral septum. As established previously, Kiss1 expression in this region is low and regulated by estrogen receptor- and GABAB receptor-dependent mechanisms. Our present experiments on Kiss1-Cre/ZsGreen knock-in mice revealed that transgene expression in the LS begins at Postnatal Day (P)33–36 in females and P40–45 in males and is stimulated by estrogen receptor signaling. Fluorescent cell numbers continue to increase in adulthood and are higher in females. Viral tracing uncovered that the bulk of KPLS fibers joins the medial forebrain bundle and terminates in the hypothalamic supramammillary nucleus. Smaller subsets innervate the medial amygdala or project to other limbic structures. One-quarter of gonadotropin-releasing hormone (GnRH)-immunoreactive perikarya in the preoptic area and their dendrites receive appositions from KPLS axons. OVX adult Kiss1-Cre/ZsGreen mice treated for 4 d with 17β-estradiol or vehicle were used for RNA sequencing studies of laser-microdissected KPLS neurons. The transcriptome included markers of GABAergic and neuropeptidergic (Penk, Cartpt, Vgf) cotransmission and 571 estrogen-regulated transcripts. Estrogen treatment upregulated the acetylcholine receptor transcript Chrm2 and, in slice electrophysiology experiments, caused enhanced muscarinic inhibition of KPLS neurons. Finally, we provided immunohistochemical evidence for homologous neurons in the postmortem human brain, suggesting that KPLS neurons may contribute to evolutionarily conserved regulatory mechanisms. Future studies will need to investigate the putative roles of KPLS neurons in the estrogen-dependent control of GnRH neurons and/or various hypothalamic/limbic functions.
- gonadotropin-releasing hormone
- kisspeptin
- laser capture microdissection
- lateral septum
- reproduction
- RNA sequencing
Significance Statement
Gonadotropin-releasing hormone (GnRH) neurons of the hypothalamus stimulate pituitary gonadotropin secretion to promote gonadal functions. Sex steroids of the gonads serve as feedback signals to control the hypothalamic output of GnRH. According to current consensus, steroid feedback is mainly sensed and communicated to the GnRH neuronal system by sex steroid-sensitive hypothalamic cell groups through the neuropeptide kisspeptin (KP). Anatomical, developmental, spatial transcriptomic, and electrophysiological characterization of an extrahypothalamic KP system in this study unveils the previously unexplored contribution of lateral septal KP cells to the sex steroid-dependent afferent control of GnRH neurons. Identification of analogous KP neurons in the postmortem human brain suggests that this estrogen-dependent system may play an evolutionarily conserved role in the central regulation of reproduction.
Introduction
The hypothalamic neuropeptide kisspeptin (KP) plays critical roles in the central regulation of puberty and fertility. Inactivating mutations of the genes encoding KP (KISS1) or its receptor (KISS1R) cause hypogonadotropic hypogonadism in humans (de Roux et al., 2003; Seminara et al., 2003) and mice (Funes et al., 2003; Seminara et al., 2003; d'Anglemont de Tassigny et al., 2007; Lapatto et al., 2007), and conversely, activating mutations account for clinical cases of precocious puberty (Teles et al., 2008; Silveira et al., 2010). Hypothalamic KP neurons promote fertility via stimulating luteinizing hormone (LH) secretion (Gottsch et al., 2004; Dhillo et al., 2005; Messager et al., 2005; Plant et al., 2006), and this effect is mediated by gonadotropin-releasing hormone (GnRH) neurons expressing Kiss1r (Han et al., 2005; Irwig et al., 2005).
Immunohistochemical and in situ hybridization (ISH) studies localized the majority of KP-synthesizing neurons to the rostral periventricular area of the third ventricle (KPRP3V neurons) and the hypothalamic arcuate nucleus (KPARC neurons; Lehman et al., 2010). Both hypothalamic populations integrate and communicate a variety of external and internal cues to GnRH neurons, including the positive and negative feedback effects of estrogens on the reproductive axis (Herbison, 2020; Moenter et al., 2020). KPRP3V neurons have been implicated in the positive feedback effect of 17β-estradiol (E2) triggering the preovulatory surge of LH in female rodents (Herbison, 2008, 2020; Moenter et al., 2020). They directly innervate GnRH perikarya and dendrites (Kinoshita et al., 2005; Clarkson and Herbison, 2006; Kallo et al., 2012), express the activity marker c-Fos during the LH surge (Adachi et al., 2007; Clarkson et al., 2008), and respond to E2 with robust transcriptional changes, including increased Kiss1 expression (Stephens and Kauffman, 2021; Gocz et al., 2022a). KPARC neurons represent the largest KP population in all mammals studied before (Lehman et al., 2010). Estrogen deficiency in postmenopausal women causes profound morphofunctional changes of KPARC neurons, characterized by neuronal hypertrophy (Rance et al., 1990; Hrabovszky et al., 2011) and increased neurokinin B (NKB; Rance and Young, 1991; Hrabovszky et al., 2011), KP (Rometo et al., 2007; Hrabovszky et al., 2011), and substance P (Rance and Young, 1991; Hrabovszky and Liposits, 2013) biosynthesis. KPARC neurons (a.k.a. “KNDy” neurons) synthesize NKB and dynorphin in various mammals, including rodent species (Lehman et al., 2010) and act primarily upon GnRH nerve terminals (d'Anglemont de Tassigny et al., 2008) to mediate sex steroid feedback control of the pulsatile secretion of GnRH and LH (Mittelman-Smith et al., 2012; Herbison, 2020; Liu et al., 2021).
While hypothalamic KP signaling has been studied extensively during the past two decades, extrahypothalamic KP cell groups in the medial amygdala (MeA), the bed nucleus of the stria terminalis, and the lateral septum (KPLS neurons) have attracted only moderate attention (Lehman et al., 2010; Kim et al., 2011; Di Giorgio et al., 2014; Yeo et al., 2016; Stephens and Kauffman, 2017; Stephens et al., 2018; Hatcher et al., 2023).
In this study, we investigate the KPLS neuron population using a KP-Cre/ZsGreen mouse model. Previous ISH experiments on knock-out mice have established that Kiss1 expression in the LS is regulated positively by E2 via estrogen receptor-α (ERα) signaling and Kiss1 levels can be enhanced further by the removal of GABAB receptor (GABABR) signaling (Di Giorgio et al., 2014; Stephens et al., 2018). While Kiss1 mRNA is virtually undetectable in the LS of wild-type gonadectomized (GDX) mice, GABABR-deficient mice maintain some Kiss1 expression following gonadectomy (Stephens et al., 2018). In our present study, we provide evidence that KP expression of the LS is linked to late pubertal development and ER signaling. KPLS cell numbers are significantly higher in females than in males and increase further in adulthood. We use viral tracing and 3D reconstruction of optically cleared whole brains to establish that KPLS neurons form appositions to ∼27% of GnRH neurons and abundantly innervate the hypothalamic supramammillary nucleus (SUM) and the MeA. We characterize the unique transcriptome profile of laser capture-microdissected KPLS neurons with ∼15,000 transcripts of which 571 respond with altered expression to a 4 d E2 treatment of OVX mice. KPLS neurons exhibit low Kiss1 and very high Penk, Cartpt, and Vgf expression. We confirm with slice electrophysiology that the upregulation of the Chrm2 transcript by E2 causes enhanced muscarinic inhibition of KPLS neurons in OVX mice treated with E2 for 4 d (OVX + E2), in comparison with vehicle-treated controls (OVX + Veh). Finally, we provide immunohistochemical evidence for the existence of KPLS neurons in the human brain, indicating that this system may subserve evolutionarily conserved functions.
Materials and Methods
Animals
Animal experiments were carried out in accordance with the Institutional Ethical Codex, Hungarian Act of Animal Care and Experimentation (1998, XXVIII, section 243/1998) and the European Union guidelines (directive 2010/63/EU) and approved by the Animal Care and Use Committees of the Institute of Experimental Medicine and the University of Lille. All efforts were made to minimize potential pain or suffering and to reduce the number of animals used.
KP-Cre/ZsGreen mice (N = 209) were housed under standard conditions (lights on between 0700 and 1900 h, temperature 22 ± 1°C, chow and water ad libitum) in the animal facility of the Institute of Experimental Medicine. Reverse light/dark cycles were used for studies involving sexual behavior. KP-Cre/ZsGreen mice were generated by crossing heterozygous Kiss-Cre (Yeo et al., 2016) males with females of the Ai6 RCL-ZsGreen [B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze/J] indicator line (Jackson Laboratory; RRID, IMSR_JAX:007906). Cre-dependent recombination in this knock-in model causes permanent expression of the fluorescent marker protein which does not require further presence of Cre recombinase.
Adult [Postnatal Day (P)80] cycling female Kiss1::Cre mice (N = 3; RRID, IMSR_JAX:017701; Gottsch et al., 2011) used for viral tracing studies were housed in the animal facility of the PLBS UAR 2014-US41, under specific pathogen-free conditions and controlled temperatures (21–22°C), with a 12 h light/dark cycle and ad libitum access to food and water. This second knock-in mouse model had to be introduced based on the results of pilot experiments in which we were unable to label KPLS neurons of the Kiss-Cre strain (Yeo et al., 2016) likely due to low regional levels of Cre expression.
Developmental studies of KPLS cell numbers with fluorescent microscopy
Male (N = 40) and female (N = 42) KP-Cre/ZsGreen mice from eight different age groups (N = 4–7/group) between P20–24 and P180–200 were anesthetized with a cocktail of ketamine (50 mg/kg)/xylazine (10 mg/kg)/promethazine (5.0 mg/kg) in saline and perfused transcardially with 50 ml of ice-cold 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS; 0.1 M), pH 7.4. The brains were postfixed for 2 h, infiltrated with a 20% sucrose solution in PBS, snap-frozen in powdered dry ice, and stored at 80°C until sectioned at 40 µm in the coronal plane with a Leica SM 2000R freezing microtome (Leica Biosystems). The sections were stored at −20°C in 24-well tissue culture plates filled with antifreeze solution (30% ethylene glycol, 25% glycerol, 0.05 M phosphate buffer), pH 7.4. To determine cell counts, we analyzed all sections of the LS between Plates 18 and 34 (from the bregma 1.53 to −0.35 mm) of the mouse brain atlas of Paxinos and Franklin (2013) with a Zeiss AxioImager M1 microscope (Carl Zeiss) using a 10× objective lens and a filter set for fluorescein isothiocyanate. Representative fluorescent images of the LS were captured with an AxioCam MRc5 digital camera (Carl Zeiss) using the AxioVision Se64 Rel.4.9.1 software. The final composite images were generated with Adobe Illustrator (Adobe Systems).
Surgical ovariectomy and estrogen treatment of adult female KP-Cre/ZsGreen mice
The OVX + Veh and the OVX + E2 adult female KP-Cre/ZsGreen models with low and high E2 levels, respectively, were generated as described previously (Gocz et al., 2022a,b). These mice were used in RNA sequencing (RNA-seq), slice electrophysiology, immunohistochemistry, and fluorescent ISH studies. In brief, P70–120 mice were surgically OVX in ketamine (25 mg/kg)/xylazine (5 mg/kg)/promethazine (2.5 mg/kg) anesthesia. To prevent infections, we injected Augmentin (GlaxoSmithKline; 30 µg/10 g bw in saline) into the peritoneal cavity during the surgery, and we applied Baneocin ointment (Sandoz) to the wound before the skin was clipped. On postovariectomy Day 9, the animals were reanesthetized and implanted subcutaneously with a single silicone capsule (Sanitech; l = 10 mm; id = 1.57 mm; od = 3.18 mm) containing either 100 μg/ml E2 (Sigma Chemical) in sunflower oil (OVX + E2 group) or oil vehicle (OVX + Veh group). The mice were anesthetized and killed 4 d later, between 0900 and 1100 h. Recent ELISA studies in our laboratory using the ES180S-100 Calbiotech Kit with a 1 pg/ml sensitivity established that this E2 treatment produces 7.59 ± 0.7 pg/ml (mean ± SEM) serum E2 concentration in adult OVX female mice (Gocz et al., 2022b) which falls within the high physiological range of intact cycling females (∼6 pg/ml in diestrus and ∼8 pg/ml in proestrus; Nilsson et al., 2015). This 4 d E2 treatment was shown to cause a robust seven- to eightfold increase in uterine weight of OVX + E2 mice versus the OVX + Veh controls (Gocz et al., 2022b).
Immunohistochemical detection of ERα in KPLS neurons of adult OVX + Veh mice
Detection and quantitative analysis of ERα immunoreactivity in KPLS neurons of KP-Cre/ZsGreen mice relied on the OVX + Veh model to maximize sensitivity. The mice (N = 4) were perfused with 4% paraformaldehyde, postfixed for 2 h, and cryoprotected with 20% sucrose as described for developmental studies. The brains were sectioned serially at 30 µm. For pretreatment, the sections were thoroughly rinsed in PBS and incubated with a mixture of 1% H2O2 and 0.5% Triton X-100 for 20 min. To maximize the immunofluorescent labeling for ERα, we carried out tyramide signal amplification (TSA) using the following incubation steps: rabbit polyclonal ERα antibodies (C1355; Merck Millipore; catalog #06-935; 1:10,000; 12 h; RT), biotin-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories; 1:500; 3 h; RT), ABC Elite reagent (VectorLabs; 1:1,000 in 0.05 M Tris–HCl buffer; 60 min; RT), and, finally, Cy3-tyramide (diluted 1:3,000 with 0.05 M Tris–HCl buffer, containing 0.003% H2O2; 15 min; RT), pH 7.6. The sections were mounted with a paint brush on glass microscope slides and coverslipped with Mowiol. Scanned digital images were prepared with fluorescent microscopy for quantitative analysis. KPLS neurons were recognized by their green fluorescence. Negative control sections that were processed with the omission of the primary antibodies remained entirely devoid of the Cy3 labeling for ERα.
Gonadectomy of female and male juvenile mice
To address the need of sex steroid exposure for the developmental increase in KPLS cell numbers, we anesthetized P21–31 female (N = 8) and male (N = 4) KP-Cre/ZsGreen mice with ketamine (25 mg/kg)/xylazine (5 mg/kg)/promethazine (2.5 mg/kg) and GDX using sterile surgical techniques. All animals were killed at P70–80 by transcardiac perfusion with 4% paraformaldehyde. The brains were postfixed for 2 h, infiltrated with 20% sucrose, and then processed for section preparation and the fluorescent microscopic analysis of KPLS cell numbers as described above.
Sex steroid treatments to increase KPLS cell numbers in juvenile mice
To study the effect of a 4 d E2 treatment of juvenile (P20) female (N = 4) and male (N = 3) KP-Cre/ZsGreen mice on the number of KPLS neurons, the same type of E2-filled silicone capsules was used as for adult mice. Other age-matched males were implanted for 4 d with another type of silicone capsule (Sanitech; l = 10 mm; id = 1.47 mm; od = 1.96 mm) that was filled with T (N = 4) or DHT (N = 4) powder in an attempt to advance the occurrence of KPLS neurons. After 4 d of steroid treatment, the mice were killed by transcardiac perfusion with 4% paraformaldehyde. Processing of the brains for cell counting with fluorescent microscopy was carried out as described above.
Studies of c-Fos expression in KPLS neurons of mice exposed to opposite-sex odor or after mating
Animal models
Experiments to address the effects of opposite-sex odor and/or mating on the expression of the neuronal activity marker c-Fos in KPLS neurons included sexually experienced male and estrous female KP-Cre/ZsGreen mice trained via multiple mating sessions (Fig. 1f). Females were housed in groups of five per cage and males individually, both in a reversed 12 h light/dark cycle (lights on at 1900 h) with water and food ad libitum.
Female mouse models
Adult P100–120 females (N = 15) used to study the behavioral induction of c-Fos expression were surgically OVX (Day 0) and subjected weekly to a hormone regimen to induce estrus (Marco-Manclus et al., 2020). In brief, the OVX female mice were first injected subcutaneously with 1 µg estradiol benzoate (Sigma-Aldrich; in 100 µl sunflower oil) on Day 10 (1530 h) and with progesterone (Sigma-Aldrich; 100 µg in 100 µl sunflower oil) on Day 12 (between 1130 and 1200 h). Four hours after progesterone treatment (before lights on), the females were placed in a behavioral arena with an experienced stimulus male which was allowed to mate and reach ejaculation within the upcoming 30 min period. The same hormone treatments and mating session were repeated 1 week later for training and another week later for the actual behavioral test and c-Fos experiment. The females were allowed to habituate for 30 min to the mating arena in the 2 d preceding each session. The females with induced estrus were divided into one of the following three experimental groups: control estrous females (FCtrl) were transferred into an empty mating arena for 30 min. Females exposed to male odor (FOdor) were transferred into the mating arena where they were exposed for 30 min to soiled bedding collected from male cages. The third group of females (FMated) were allowed to mate with their partner from the previous training sessions.
Male mice
P100–120 males (N = 12) were preselected based on their sexual performance in mating tests. To be included in behavioral studies, they had to produce vaginal plugs in two sequential mating sessions, 1 week apart, with sexually receptive females. For habituation, the males were transferred into a test arena for 30 min in 2 sequential days before the final behavioral experiment. For each male, the behavioral test started with a 10 min habituation to the mating arena. Control males (MCtrl; N = 4) were left in the cage for an additional 30 min. MOdor males (N = 4) were exposed for 30 min to soiled bedding collected from the home cage of estrous females. MMated males (N = 4) mated and ejaculated during a 30 min test session with a receptive stimulus female. Stimulus females were prepared as described above via hormone-priming OVX mice. Hormone treatments and mating sessions were repeated at least twice at weekly intervals before these females were used for the behavioral tests (Marco-Manclus et al., 2020).
Tissue collection
Ninety minutes after bedding exposure or mating, male and female mice were anesthetized with a cocktail of ketamine (50 mg/kg)/xylazine (10 mg/kg)/promethazine (5.0 mg/kg) and perfused transcardially with 4% paraformaldehyde. Video recordings and the presence of vaginal plugs confirmed that males ejaculated.
Analysis of activated neurons
All male and female brains perfused with 4% paraformaldehyde were postfixed for 2 h and then infiltrated with 20% sucrose. Free-floating 30-µm-thick sections were collected from the LS with a freezing microtome (Leica Biosystems). Activated neurons were identified using the immunofluorescent detection of c-Fos with sheep polyclonal antibodies (S-033-500; Biosensis). The primary antibodies (1:20,000; 48 h; 4°C) were reacted with Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories; 1:1,000; 12 h; RT). The sections were mounted with a paint brush on microscope slides and coverslipped with Mowiol. The fluorescent microscopic analysis of c-Fos–positive KPLS neurons was performed by an investigator blind to group assignment. The percentage of c-Fos–positive KPLS neurons was determined in each animal, and the results presented as mean ± SEM of female (N = 5/group) and male (N = 4/group) mice. The effects of sex and behavior were addressed with two-way ANOVA.
Studying the projections of KPLS neurons in gonadally intact female Kiss::Cre mice
Animals
To visualize the major efferent connections of KPLS neurons, we carried out multiple microinjections of a Cre-inducible AAV1/2-FLEXon-shScrambled-green fluorescent protein (GFP) virus specifically to the LS of gonadally intact adult (∼P70) female Kiss::Cre mice (RRID, IMSR_JAX:017701; N = 3; Gottsch et al., 2011). As opposed to the Kiss-Cre strain (Yeo et al., 2016), KPLS neurons of these mice could be transfected with high efficiency in pilot studies, a difference likely due to lower Cre expression in the former transgenic strain.
Stereotaxic surgeries
Virus-injected mice were deeply anesthetized with isoflurane (3% in 1 L/min air flow) in an induction chamber and placed in a stereotaxic apparatus (Kopf Instruments) equipped with a mask to maintain anesthesia during surgery (isoflurane 1% in 0.7 L/min airflow). The mice were given subcutaneous injections of carprofen (5 mg/kg) after anesthesia was confirmed. Thereafter, a Neuro Hamilton syringe (Hamilton; Ref, 65459-01) hooked to an infusion pump was slowly inserted into the LS. Three consecutive injections were performed using the following coordinates: AP, +1 mm; ML, +0.38 mm; DV, −2.7 mm//AP, +0.5 mm; ML, −0.38 mm; DV, −2.7 mm//AP, +0 mm; ML, +0 mm; DV, −2.5 mm. The dorsoventral coordinate was taken from the skull surface. All stereotaxic coordinates were determined according to the Mouse Brain in Stereotaxic Coordinates (Paxinos and Franklin, 2013). Each injection consisted of 200 nl of AAV1/2-[FLEXon]-CMV-eGFP:Scramble[miR30-shRNA] (5.2 × 109 gc/µl; #P220803-1014zpb; VectorBuilder) over a 10 min period. Three weeks after this surgery, the mice were anesthetized and killed in the morning of estrus using transcardiac perfusion with 4% paraformaldehyde in 0.01 M PBS, pH 7.4. The brains were harvested and postfixed in the same fixative solution overnight.
Whole-mount immunolabeling and tissue clearing
Dual-immunofluorescent detection of GFP and GnRH used a modified iDISCO+ protocol (Renier et al., 2014; Belle et al., 2017). Labeled KPLS neurons and their projections were studied using light sheet microscopy (LSM) followed by 3D reconstruction and analysis. First, the brains were gradually dehydrated with increasing concentrations (20, 40, 60, and 80%) of methanol in PBS, followed by 2 × 100% methanol, 1 h each, delipidated overnight in 66% dichloromethane (DCM)/33% methanol at 4°C with agitation, rinsed twice in methanol, and bleached overnight at 4°C in methanol with 5% H2O2. After gradual rehydration (100, 80, 60, 40, and 20% methanol in PBS, followed by two rinses in PBS, 1 h each), the samples were incubated for 4 d at 37°C with rotation in permeabilization/blocking solution containing 1% Triton X-100, 0.2% gelatin, and 0.05% sodium azide in PBS (PBS-GT; Belle et al., 2017). For immunolabeling, the samples were incubated with primary antibodies (rabbit anti-GFP, 1:5,000, A6455, Invitrogen; rat anti-GnRH, 1:5,000, EH#1044; Skrapits et al., 2015) in PBS-GT for 10 d at 37°C with rotation, rinsed 6 × 1 h in PBS + 1% Triton X-100, and then transferred for 7 d to secondary antibodies from Invitrogen (goat anti-rabbit Alexa Fluor 647, 1:1,000, A-21245; goat anti-rat Alexa Fluor 568, 1:1,000, A-11077) in PBS-GT at 37°C with rotation. Brains were next washed 6 × 1 h in PBS + 1% Triton X-100 to remove free antibodies. For tissue clearing, the samples were gradually dehydrated with increasing concentrations of methanol (20, 40, 60, and 80% methanol in PBS, followed by 2 × 100% methanol, 1 h each). Methanol was washed out with an incubation in 66% DCM/33% methanol overnight at 4°C with agitation, followed by 2 × 1 h in 100% DCM at 20°C with agitation. The samples were finally transferred to dibenzylether (DBE) for refractive index matching during 2 h at 20°C with rotation. A fresh solution of DBE was used for storage, and samples were kept at 20°C in the dark until imaging.
Light sheet imaging and analysis
3D imaging was performed on the Ultramicroscope I (LaVision BioTec) equipped with a 1.1×/0.1 NA and a 4×/0.3 NA objectives and an Andor Neo 5.5 sCMOS camera. The light sheet was generated by a laser (wavelength 488, 568, or 647 nm, Coherent Sapphire Laser, LaVision BioTec) and two cylindrical lenses. Samples were placed in an imaging reservoir made of 100% quartz (LaVision BioTec) filled with DBE and illuminated from the side by the laser light. The ImspectorPro software (LaVision BioTec) was used for image acquisition: the z-step was set to 5 µm (1.1× objective) or 2 µm (4× objective), and the laser numerical aperture was set to 0.30. The resulting sequences of TIFF files were processed with Imaris Converter and Imaris Stitcher (Oxford Instruments). Imaris 10 (Oxford Instruments) was used for visualization and analysis of the datasets and for the preparation of the MP4 video file (Movie 1).
Tissue clearing reversal for confocal imaging
To allow confocal analysis after light sheet imaging, the optical clearing was reversed in one of the virally labeled samples. This brain was first rinsed 2 × 2 h in 100% methanol to wash out the DBE, before gradual rehydration in methanol/PBS (100, 80, 60, 40, and 20% methanol in PBS, followed by two rinses in PBS, 1 h each). Reagents for tissue clearing and reversal were purchased from Thermo Fisher Scientific (DBE, DCM, 37% formaldehyde, sodium azide), Sigma-Aldrich (10× DPBS, Triton X-100), VWR (methanol), and Acros Organics (gelatin). The sample then underwent cryoprotection with 20% sucrose and sectioned coronally at 30 µm with a Leica CM1860 UV cryostat. Scanned digital images of the slide-mounted sections were analyzed to obtain an estimate of the level of connectivity between AAV-labeled KPLS neurons and preoptic GnRH neurons. Axosomatic and axodendritic contacts were illustrated in confocal images, and the percentage of the innervated GnRH somata was determined via the analysis of 411 GnRH-immunoreactive cell bodies.
Transcriptomic studies of KPLS neurons in adult OVX + Veh and OVX + E2 mice
Section preparation for laser capture microdissection
For all experiments, reagents were of molecular biology grade. Buffers were pretreated overnight with diethylpyrocarbonate (DEPC; Merck; 1 ml/L) and autoclaved or prepared using DEPC-treated and autoclaved water as diluent. The working area was cleaned with RNaseZAP (Merck). To minimize possible adverse effects of fixation on RNA integrity and recovery, ice-cold 0.5% paraformaldehyde in 0.1 M PBS (80 ml) was used for transcardiac perfusion of (P80–100) adult OVX + Veh (N = 9) and OVX + E2 (N = 12) KP-Cre/ZsGreen mice, followed by ice-cold 20% sucrose in PBS (50 ml). The brains were snap-frozen on pulverized dry ice and stored at −80°C until sectioned with a cryostat. Twelve-micrometer-thick sections containing the LS were thaw-mounted onto PEN membrane glass slides (Membrane Slide 1.0 PEN, Carl Zeiss), air-dried in the cryostat chamber, and preprocessed for laser capture microdissection (LCM) as reported previously (Gocz et al., 2022a,b). In brief, the slides were immersed sequentially in 50% EtOH (20 s), n-buthanol:EtOH (25:1; 90 s), and xylene substitute:n-butanol (25:1; 60 s). Then, they were stored at −80°C in slide mailers with silica gel desiccants or processed immediately for LCM.
LCM
Fluorescently tagged KP neurons (n = 360/library) were microdissected individually and pressure-catapulted into 0.5 ml tube caps (Adhesive Cap 500, Carl Zeiss) with a single laser pulse using a 40× objective lens and the PALM MicroBeam system and RoboSoftware (Carl Zeiss). Each neuronal pool was collected proportionally from three brains and stored in LCM tube caps at −80°C until RNA extraction.
RNA extraction
RNA samples were prepared with the Arcturus Paradise PLUS FFPE RNA Isolation Kit (Applied Biosystems).
RNA-seq library preparation
Sequencing libraries were prepared with the TruSeq Stranded Total RNA Library Preparation Gold kit (Illumina), using 16 PCR cycles for cDNA fragment enrichment. cDNA samples were subjected to Bioanalyzer analysis, and a 1 nM library mix (20 µl) containing proportionally the indexed libraries was sequenced with an Illumina NextSeq500 instrument using the NextSeq500/550 High Output v2.5 kit (75 cycles).
Bioinformatics
Trimmomatic 0.39 (settings, LEADING:3, TRAILING:3, SLIDINGWINDOW:4:15, MINLEN:36; Bolger et al., 2014) and Cutadapt 4.4 (Martin, 2011) were used to remove low-quality and adapter sequences, respectively. Remaining reads were mapped to the Ensembl mm109 mouse reference genome using STAR (v 2.7.10b; Dobin et al., 2013). Read assignment to genes, read summarization, and gene level quantification were performed by featureCounts (Subread v 2.0.6; Liao et al., 2014). The counts per million (CPM) read values were calculated with the edgeR R package (Robinson et al., 2010).
Identification of the cholinergic fiber network around KPLS neurons in adult female mice
Daily vaginal smears were obtained from adult (P70–100) cycling female KP-Cre/ZsGreen mice (N = 3). The mice were anesthetized, and their brains were perfusion-fixed with 4% paraformaldehyde in diestrous phase and, following a 2 h postfixation, infiltrated with 20% sucrose. Serial sections (30 µm) of the LS were prepared with a cryostat for the immunofluorescent detection of the cholinergic marker enzyme vesicular acetylcholine transporter (VAChT) using the following incubation steps: guinea pig polyclonal VAChT antibody (#139 103; Synaptic Systems; 1:1,000; 12 h; RT), biotin-conjugated anti-guinea pig IgG (Jackson ImmunoResearch Laboratories; 1:500; 2 h; RT), ABC Elite reagent (VectorLabs; 1:1,000 in 0.05 M Tris–HCl buffer; 60 min; RT), and Cy3-tyramide (diluted 1:1,000 with 0.05 M Tris–HCl buffer, containing 0.003% H2O2; 30 min; RT), pH 7.6. Control sections processed with the omission of the primary antibodies remained devoid of Cy3 labeling.
Slice electrophysiology to study muscarinic regulation of KPLS neurons in OVX + Veh and OVX + E2 mice
Brain slice preparation
Brain slices were prepared as described earlier (Farkas et al., 2010) with slight modifications. Briefly, P70–120 OVX + Veh (N = 5) and OVX + E2 (N = 11) KP/ZsGreen female mice (Yeo et al., 2016) were decapitated in deep isoflurane anesthesia. The brains were immersed in ice-cold low–Na cutting solution bubbled with carbogen (mixture of 95% O2 and 5% CO2). The cutting solution contained the following (in mM): 205 saccharose, 2.5 KCl, 26 NaHCO3, 5 MgCl2, 1.25 NaH2PO4, 1 CaCl2, and 10 glucose. Brain blocks including the LS were dissected. The 220-μm-thick coronal slices were prepared with a VT-1000S vibratome (Leica Biosystems) and transferred into oxygenated artificial cerebrospinal fluid (aCSF; 33°C) containing the following (in mM): 130 NaCl, 3.5 KCl, 26 NaHCO3, 1.2 MgSO4, 1.25 NaH2PO4, 2.5 CaCl2, and 10 glucose. The solution was then allowed to equilibrate to room temperature for 1 h.
Whole-cell patch–clamp experiments
Recordings were carried out in oxygenated aCSF at 33°C using Axopatch-200B patch–clamp amplifier, Digidata-1550B data acquisition system, and pClamp 10.7 software (Molecular Devices). Neurons were visualized with a BX51WI IR-DIC microscope (Olympus). KP-ZsGreen neurons showing green fluorescence in the LS were identified by a brief illumination (CoolLED, pE-100) at 470 nm using an epifluorescent filter set. The patch electrodes (OD = 1.5 mm, thin wall; Worcester Polytechnic Institute) were pulled with a Flaming-Brown P-97 puller (Sutter Instrument). Electrode resistance was 2–3 MΩ. The intracellular pipette solution contained the following (in mM): 130 K-gluconate, 10 KCl, 10 NaCl, 10 HEPES, 0.1 MgCl2, 1 EGTA, 4 Mg-ATP, and 0.3 Na-GTP, pH 7.2–7.3, with KOH. Osmolarity was adjusted to 300 mOsm with d-sorbitol. Pipette offset potential, series resistance (Rs), and capacitance were compensated before recording. Only cells with low holding current (<50 pA) and stable baseline were used. Input resistance (Rin), Rs, and membrane capacitance (Cm) were also measured before each recording by using 5 mV hyperpolarizing pulses. To ensure consistent recording qualities, we accepted only cells with Rs < 20 MΩ, Rin > 300 MΩ, and Cm > 10 pF.
Spontaneous firing activity and resting membrane potential (Vrest) of KPLS neurons were recorded in whole-cell current–clamp mode at 0 pA holding current. Measurements started with a control recording (1 min). Then a single bolus of muscarinic ACh-R agonist muscarine (100 μM; Chen et al., 2022) was pipetted into the aCSF-filled measurement chamber, respectively, and the recording continued for a further 7 min.
Pretreatment with the extracellularly applied selective antagonist of the M2 subtype of mACh-R methoctramine (Methoct; 2 μM, Sigma-Aldrich; Seeger and Alzheimer, 2001) or the voltage-gated Na-channel blocker tetrodotoxin (TTX; 660 nM, Tocris Bioscience) started 10 min before adding muscarine. These inhibitors were continuously present in the aCSF during the electrophysiological recording. Each neuron served as its own control when drug effects were evaluated.
Data analyses
Recordings were stored and analyzed off-line. Event detection was performed using the Clampfit module of the pClamp 10.7 software (Molecular Devices). Mean firing rates were calculated as the number of action potentials (APs) divided by the control and treatment time periods, respectively. All the recordings were self-controlled in each neuron, and the effects were expressed as percentage changes relative to the control periods. Duration of the effect of muscarine was defined as the period from the point all the APs disappeared until appearance of the first AP after the silent phase. Treatment group data were expressed as mean ± SEM.
Fluorescent ISH studies in OVX + Veh mice
ISH studies were used to confirm the presence of highly expressed transcripts encoding three neuropeptides (Cartpt, Penk, and Vgf) and the G-protein–coupled receptor Glp1r.
Preparation of cRNA hybridization probes
Fluorescent ISH procedures to detect Cartpt, Penk, Vgf, and Glp1r mRNAs with digoxigenin-labeled cRNA hybridization probes were adapted from previous studies (Hrabovszky et al., 2004; Wittmann et al., 2013). The probes, added at 1:100 to the hybridization buffer, were generated by in vitro transcription of cDNA templates in the presence of digoxigenin-11-UTP (Merck; Hrabovszky et al., 2004), and they targeted bases 61–466 of murine Cartpt (NM_013732), bases 387–1,411 of murine Penk (NM_001002927.3), and bases 11–1,402 of murine Glp1r mRNAs (Ruska et al., 2022). The Vgf probe was transcribed from a ∼320 bp rat cDNA fragment (Hahm et al., 2002).
Histological procedures
In this study, we used OVX + Veh mice (N = 6) which showed higher levels of expression for these specific transcripts than the OVX + E2 model. Two different types of the tissue were used. Four percent paraformaldehyde-perfused and 20% sucrose-infiltrated brains (N = 3) were used to detect Cartpt and Penk mRNAs. This fixative preserved the ZsGreen fluorescence during the hybridization procedure but compromised somewhat the sensitivity of ISH detection. Twenty-micrometer-thick coronal sections were prepared with a freezing microtome (Leica Biosystems) and mounted with a paint brush on Superfrost Plus Microscope Slides (Thermo Fisher Scientific). A different strategy was used to maximize the signals for Vgf and Glp1r mRNAs. In these cases, unfixed brains (N = 3) were snap-frozen and 16-µm-thick fresh–frozen sections were prepared with a Leica CM1860 UV cryostat (Leica Biosystems), mounted directly on microscope slides, fixed briefly with 4% paraformaldehyde, subjected to nuclear staining with 1 µg/ml 4,6-diamidino-2-phenylindole (DAPI), coverslipped with 0.1 M PBS, and photographed to register the relative position of ZsGreen neurons and DAPI-labeled nuclei. The coverslips were floated off, and the sections were processed through prehybridization and hybridization steps. The ISH method was adapted from procedures detailed previously (Hrabovszky et al., 2004; Wittmann et al., 2013). After posthybridization, the sections were incubated with anti-digoxigenin antibodies conjugated to horseradish peroxidase (anti-digoxigenin-POD; Fab fragment; 1/100; Merck; 16 h), followed by the deposition of biotin tyramide (1:500; 30 min; TSA Plus Biotin Kit; catalog #NEL749A001KT, Akoya Biosciences) to be reacted with Alexa Fluor 555 streptavidin (Thermo Fisher Scientific; 1:500; 1 h). To study and illustrate Vgf and Glp1r mRNA expression in KPLS neurons, we photographed the Alexa Fluor 555 and DAPI signals with a fluorescent microscope and precisely superimposed with Adobe Photoshop CS5 (Adobe Systems) to the archived ZsGreen/DAPI images obtained before hybridization. The final composite images were created with Adobe Illustrator (Adobe Systems). ZsGreen neurons containing the fluorescent Penk and Cartpt mRNA signals were studied, and images were prepared with confocal microscopy.
Immunohistochemical detection of KP neurons in the septum of male human subjects
Human tissues
Adult human brain tissues (N = 6) from 38-, 52-, 53-, 54-, 56-, and 69-year-old male individuals without known neurological disorders were collected from autopsies at the Department of Pathology and Experimental Cancer Research, Semmelweis University. Sample collection was carried out between 12 and 24 h after death. Ethic permissions were obtained from the Regional and Institutional Committee of Science and Research Ethics of Semmelweis University (SE-TUKEB 251/2016), in accordance with the World Medical Association Declaration of Helsinki and the Hungarian Law (1997 CLIV and 18/1998/XII.27. EÜM Decree/) which latter does not require prior written consent from the deceased person. The septum was dissected, cut into 4-mm-thick slices in the coronal plane, immersion-fixed with buffered 4% paraformaldehyde for 48 h, infiltrated with 20% sucrose for 72 h, embedded in Jung tissue freezing medium (Leica Biosystems) and snap-frozen on powdered dry ice. Then, 30-μm-thick coronal sections were collected with a freezing microtome (Leica Biosystems) into tissue culture plates filled with antifreeze solution (30% ethylene glycol; 25% glycerol; 0.05 M phosphate buffer), pH 7.4, for long-term storage at −20°C.
Detection of human KPLS neurons with peroxidase immunohistochemistry
The sections were rinsed in PBS, pretreated with a mixture of 1% H2O2 and 0.5% Triton X-100 for 20 min and subjected to antigen retrieval with 0.1 M citrate buffer, pH 6.0, at 80°C for 30 min. Every 24th section was immunostained with a well-characterized sheep polyclonal antiserum (GQ2; 1:75,000; 48 h; 4°C) against human kisspeptin-54 (aa 68–121 of NP_002247.3; Dhillo et al., 2005; Hrabovszky et al., 2010) using biotinylated secondary antibodies (Jackson ImmunoResearch Laboratories; 1:500; 2 h; RT), the ABC Elite reagent (VectorLabs; 1:1,000; 60 min; RT), and the nickel-intensified diaminobenzidine (NiDAB) chromogen [10 mg diaminobenzidine, 30 mg nickel–ammonium–sulfate and 0.003% H2O2 in 20 ml Tris–HCl buffer solution (0.05 M)], pH 8.0. Immunostained sections were mounted on 75 × 50 mm microscope slides from 0.3% polyvinyl alcohol; air-dried; dehydrated with 70, 95, and 100% ethanol (5 min each); cleared with xylenes (2 × 5 min); and coverslipped with DPX mounting medium (Merck) for light microscopic analysis and photography with a Zeiss AxioImager M1 microscope (Carl Zeiss), using an AxioCam MRc5 digital camera (Carl Zeiss) and the AxioVision Se64 Rel.4.9.1 software.
Generation and use of human preproKP antisera for positive control experiments
As a positive control for staining human brains with the GQ2 antibodies, we have generated control antibodies against aa 70–93 of the human preproKP. Excluding the C-terminal RFamide motif from the antigen eliminated potential cross reactions with other members of the RFamide peptide family. The synthetic peptide, including an additional N-terminal cysteine, was conjugated to keyhole limpet hemocyanin using the Sulfo-SMCC cross-linker. Antibody production has been carried out in accordance with the Council Directive of 24 November 1986 of the European Communities (86/609/EEC) and approved by the Animal Welfare Committee of the Institute of Experimental Medicine (No. PE/EA/1510-7/2018). Five rats were immunized intraperitoneally with 100 μg antigen/injection in 300 μl volumes. The ratio of the aqueous phase and the adjuvant was always 1:9. Initial immunization was carried out with Freund's complete adjuvant, and subsequent boosts were applied at 2 week intervals with Freund's incomplete adjuvant. Serum samples were withdrawn 1 week after each boost and tested with immunohistochemistry on sections of the infundibular nucleus which contains the majority of KP-immunoreactive neurons in the human hypothalamus (Hrabovszky et al., 2010, 2021). Two rats provided excellent polyclonal antibodies (SST#01 and SST#02) after terminal bleeding. Detection of KP neurons with SST#01 (1:5,000; 48 h; 4°C) using biotinylated secondary antibodies (Jackson ImmunoResearch Laboratories; 1:500; 2 h; RT), the ABC reagent (VectorLabs; 1:1,000; 60 min; RT), and NiDAB chromogen were studied with light microscopy to serve as a control approach to confirm the presence of authentic KP synthesis in the human LS.
Dual-immunofluorescent experiments with the combined use of the GQ2 and SST#01 antisera
Dual-immunofluorescent experiments on 20-μm-thick coronal sections from the infundibular nucleus of a 69-year-old male subject were used to confirm that the two polyclonal antibodies recognize the same KP neuron population. Following suppression of tissue autofluorescence with Sudan Black (Sigma-Aldrich; Mihaly et al., 2000), the sections were incubated in GQ2 antibodies (1:4,000; 16 h; RT), followed by Cy3-conjugated anti-sheep antibodies (Jackson ImmunoResearch Laboratories; 1:1,000; 2 h; RT). Subsequently, the SST#01 antibodies (1:1,000; 16 h; RT) were applied and detected with Alexa Fluor 488-conjugated anti-rat secondary antibodies (Jackson ImmunoResearch; 1:1000; 2 h; RT). The dual-labeled sections were mounted on microscope slides and analyzed using fluorescent and confocal microscopy.
Fluorescent and confocal microscopy
For fluorescent microscopy, sections were mounted from 0.1 M Tris–HCl buffer, pH 7.6, air-dried and coverslipped with the aqueous mounting medium Mowiol. Images were captured using a Zeiss AxioImager M1 microscope (Carl Zeiss), an AxioCam MRc5 digital camera (Carl Zeiss), and the AxioVision Se64 Rel.4.9.1 software, using filter sets for fluorescein isothiocyanate (for ZsGreen) and rhodamine (for Cy3 and Alexa Fluor 555).
Confocal images were prepared with a Zeiss LSM 780 confocal microscope (Carl Zeiss). We used line-by-line sequential scanning with laser excitation lines 488 nm for Alexa Fluor 488 or ZsGreen; 561 nm for Cy3, Alexa Fluor 555, or Alexa Fluor 568; and 650 nm for Alexa Fluor 647. Detection wavelengths were set to 493–550 nm for Alexa Fluor 488 or ZsGreen; to 566–681 nm for Cy3, Alexa Fluor 555, or Alexa Fluor 568; and to 665–705 nm for Alexa Fluor 647. Plan-Apochromat 20× and 63× lenses were used, with pinhole sizes set to obtain 1.5–1.9 and ∼0.8-µm-thin optical slices, respectively. Confocal z-stack images were generated with the Zen 2012 software (Carl Zeiss). Single optical slices and orthographic side views of neuronal contacts were used to demonstrate the absence of gap between virally labeled KPLS fibers and the innervated GnRH-immunoreactive neurons. The Adobe Illustrator program (Adobe System) was used to prepare composite images for illustrations.
Experimental design and statistics
Transcriptomic studies
Transcripts with the highest mean CPMs were identified in KPLS, KPARC, and KPRP3V transcriptomes using functional categories of the KEGG BRITE database of the Kyoto Encyclopedia of Genes and Genomes (KEGG; Kanehisa and Goto, 2000) and a Neuropeptide database (http://www.neuropeptides.nl/). DE analysis was performed with the DESeq2 R package (Love et al., 2014), following correction for unwanted variations with RUVSeq (Risso et al., 2014). Differences in mRNA expression levels were quantified by log2 fold changes (log2FC). To take multiple testing into account, we corrected p values by the method of Benjamini and Hochberg (Benjamini et al., 2001). Volcano plot and heat map, made with the EnhancedVolcano and Pheatmap program packages, respectively, were used to illustrate all transcripts that were expressed differentially.
Studies of c-Fos expression in response to mating-related stimuli
Group data were expressed as mean ± SEM of 4–5 mice. Statistical comparison of the groups was carried out using two-way ANOVA, with animal sex and behavior, as main effects.
Statistics in studies using slice electrophysiology
Statistical significance was determined with two-tailed paired and unpaired Student's t tests and one-way ANOVA followed by Dunnett's post hoc test using the Prism 3.0 software (GraphPad). Differences were considered significant at p < 0.05. Detailed statistics are provided in Extended Data Figure 6-1.
Results
KP-Cre/ZsGreen neurons start to emerge during pubertal development and exhibit higher numbers in females than in males
We first studied the postnatal ontogeny of KPLS neurons in the KP-Cre/ZsGreen knock-in mouse model in which Kiss1 promoter-driven expression of Cre recombinase switches on transcription of the fluorescent marker transgene in the ROSA 26 locus (Yeo et al., 2016). In recent studies, we have used these mice for electrophysiological, anatomical, and transcriptomic studies of KPARC and KRRP3V neurons (Gocz et al., 2022a,b).
Female and male mice (N = 82) from eight different age groups (N = 4–7/group) were perfused transcardially with 4% paraformaldehyde, and their brains were sectioned serially with a freezing microtome between Plates 18 and 34 (from the bregma 1.53 to −0.35 mm) of the mouse brain atlas (Paxinos and Franklin, 2013). The number of KP-Cre/ZsGreen neurons detectable in the intermediate and dorsal subnuclei of the LS (referred to as KPLS neurons) was analyzed using fluorescent microscopy. KP neurons were virtually absent in the LS before P30. We observed the first obvious increase in the number of KPLS neurons at P33–36 in female and P40–45 in male mice. Fluorescent cell numbers increased gradually during late pubertal development and after to reach a plateau in P140–160 females and P180–200 males (Fig. 1a,b). KPLS neurons were more numerous in females than in age-matched males. Sex difference was statistically significant (p < 0.05; Mann–Whitney's U test with one-sided alternative hypothesis) at P33–36 (p = 0.022) and all subsequent timepoints (P40–45, p = 0.037; P53–60, p = 0.004; P90–100, p = 0.009; P140–160, p = 0.011; P180–200, p = 0.028; Fig. 1a,b).
Developmental profile, sexual dimorphism, and sex steroid-dependent emergence of the KP-Cre/ZsGreen neuronal phenotype in the lateral septum. a, Representative fluorescent photomicrographs illustrate different stages of KPLS neuron (arrowheads) development in female and male KP-Cre/ZsGreen mice. b, Bar graphs reveal that the fluorescent neurons start to occur at P33–36 in female and P40–45 in male mice and exhibit higher numbers in females (*p < 0.05 by Mann–Whitney's U test). c, The immunohistochemical (IHC) localization of nuclear ERα signal (magenta) in most KPLS neurons (green) of adult female mice that were OVX and treated with oil supports the idea that the classical estrogen receptor plays a crucial role in the molecular regulation of KPLS neurons. The arrowhead indicate dual-labeled neurons. d, Females OVX at P21–31 develop KPLS neurons, although P70–80 cell numbers tend to be lower than those of the age-matched intact controls (p = 0.1838; Mann–Whitney's U test, one-sided). In contrast, male mice ORCHX at P21–31 do not develop KPLS neurons by P70–80. e, KPLS neuron development can be advanced by a 4 d E2 treatment of P20 juvenile female and male mice. Treatment of male mice with testosterone (T) also produces KPLS neurons, albeit less efficiently than E2. In this effect, T is likely converted to E2 to act on estrogen receptors because KPLS neurons cannot be observed in male mice treated with the nonaromatizable androgen DHT. f, g, Schematic of the experimental design (f) and results of c-Fos colocalization experiments (g) illustrate that the percentage of c-Fos–expressing KPLS neurons is higher in males than in estrous females and remains unaffected 90 min after exposure to opposite-sex odor or mating (*p = 0.014; F = 7.23; two-way ANOVA). Scale bars: 250 µm in a; 400 µm in left and 50 µm in right panels in c. GDX, gonadectomy; LSD, lateral septum, dorsal subnucleus; LSI, lateral septum, intermediate subnucleus; LV, lateral ventricle; MS, medial septum.
The majority of KP-Cre/ZsGreen neurons express ERα immunoreactivity in OVX + Veh mice
Earlier ISH experiments on E2-treated knock-out mice established that Kiss1 mRNA expression in the LS is regulated positively by ERα signaling (Di Giorgio et al., 2014; Stephens et al., 2018). The late pubertal emergence of the KP-Cre/ZsGreen phenotype raised the possibility that E2 plays a critical role in the development and transcriptional regulation of the KPLS neuronal system. Indeed, fluorescent microscopic analysis of serial LS sections from OVX + Veh mice (N = 4) provided evidence that 72.5 ± 7.6% (mean ± SEM) of ZsGreen-tagged KPLS neurons exhibit immunohistochemical signal for the classical ERα (Fig. 1c).
P21–31 gonadectomy prevents the development of the KP-Cre/ZsGreen phenotype in males but not in females
To address the hypothesis that KPLS neuron development requires the pubertal rise of gonadal sex steroids, we studied the effect of P21–31 gonadectomy on adult KP-Cre/ZsGreen cell numbers. Supporting this hypothesis in males, we found that P70–80 adult mice orchidectomized (ORCHX) at P21–31 to prevent the pubertal rise of testicular steroids did not contain KPLS neurons (Fig. 1d). As opposed to males, females OVX at P21–31 developed nearly normal KPLS cell numbers by P70–80 (Fig. 1d).
Estrogen receptor-acting sex steroids advance the emergence of KP-Cre/ZsGreen neurons in juvenile females and males
To provide direct evidence for the involvement of sex steroids in the development of the KP-Cre/ZsGreen neuronal phenotype, we implanted P20 female mice (N = 4) with a subcutaneous silicone capsule containing 100 μg/ml E2 in sunflower oil for a 4 d treatment, whereas control females (N = 4) received implants filled with vehicle. Treatment with E2, but not its vehicle, resulted in fluorescent ZsGreen signal in 87.75 ± 39.44 LS neurons at P24 (Fig. 1e), resembling the cell counts of intact P90–100 adult females (108.14 ± 22.25; Fig. 1b). KPLS neurons also occurred in E2-treated P20 male mice (N = 3) by P24 (Fig. 1e), whereas intact (N = 3) or oil-treated (N = 3) P24 males were devoid of KPLS neurons (Fig. 1e). Implants releasing the male sex hormone testosterone (T) were also able to induce an increase in detectable KPLS neurons in juvenile male mice (N = 4), although cell numbers were significantly lower (11.5 ± 1.32; mean ± SEM) than after E2 treatment (44.67 ± 4.91; p = 0.029, Mann–Whitney's U test with one-sided alternative hypothesis). Given that tissue aromatases can convert T partly to E2 which can act on estrogen receptors, the putative involvement of androgen receptors in the effect of T was clarified using a group of male mice (N = 4) implanted at P20 with a silicone capsule releasing the nonaromatizable androgen dihydrotestosterone (DHT). We did not observe fluorescent KPLS neurons in mice subjected to a 4 day DHT treatment, indicating that sex steroids increase the number of KP-Cre/ZsGreen neurons solely by ER signaling also in juvenile males (Fig. 1e). These experiments provided evidence that estrogen receptors play a critical role in KPLS neuron development in both sexes.
c-Fos expression in KPLS neurons is higher in males than in females and remains unaffected by opposite-sex odor or mating
As the LS is known to serve as a source for a sexually dimorphic lordosis-inhibiting pathway (Tsukahara and Yamanouchi, 2001; Tsukahara et al., 2014), we raised the possibility that the KPLS system is part of the circuitry regulating reproductive behavior. Experiments to address the effects of opposite-sex odor and mating on the expression of the neuronal activity marker c-Fos in KPLS neurons included sexually experienced male and estrous female mice trained via multiple mating sessions (Fig. 1f). The animals were killed 90 min after completion of the behavioral test by transcardiac perfusion with 4% paraformaldehyde, and the brains were processed for the immunofluorescent detection of c-Fos. Fluorescent microscopic analysis of c-Fos–labeled KP-Cre/ZsGreen neurons followed by two-way ANOVA established that sex (p = 0.014; F = 7.23), but not behavior (p = 0.708; F = 0.351), exerted a significant effect on the percentages of c-Fos–positive KPLS neurons, which was higher in males than in females. Opposite-sex odor or mating had no effect on the colocalization of the neuronal activity marker (Fig. 1g). This observation failed to prove, without excluding the possibility that KPLS neurons play a role in sexual behavior.
Viral tracing reveals that KPLS neurons innervate the MeA and the hypothalamic SUM
To visualize the major efferent connections of KPLS neurons, we carried out multiple microinjections of a Cre-inducible AAV1/2-FLEXon-shScrambled-GFP virus specifically to the LS of three gonadally intact adult (P80) female Kiss::Cre mice (Gottsch et al., 2011). Three weeks after viral injections, the brains were fixed with 4% paraformaldehyde and processed for the dual-immunofluorescent detection of GFP and GnRH using a modified iDISCO+ protocol (Renier et al., 2014; Belle et al., 2017). Labeled KPLS neurons and their projections were studied using LSM. The immunohistochemical detection of GFP labeled selectively the KPLS neuron population without visualizing KPRP3V and KPARC neurons, allowing us to follow the major fiber projections in optically cleared whole brains (Fig. 2; Movie 1). A subset of the KPLS fibers turned rostrally to innervate the LS (Fig. 2d), whereas the bulk turned ventrally (Fig. 2a) and joined the medial forebrain bundle (mfb) to form caudal projections (Fig. 2a,b,f,g). A group of KPLS axons detached from the mfb and innervated the MeA (Fig. 2f), whereas the largest fiber population terminated in the SUM (Fig. 2a,g; Movie 1).
Viral tracing reveals that KPLS neurons innervate limbic and hypothalamic regions and GnRH neurons. a–c, Sagittal (a), transversal (b), and coronal (c) projection views of KPLS neurons and their fibers in a whole female mouse brain processed for optical clearing with the iDISCO+ protocol and 3D imaging with LSM. Three intracerebral injections of the Cre-inducible AAV1/2-FLEXon-shScrambled-GFP virus to the lateral septum (LS) selectively label the KPLS neuron population without infecting KP neurons of the RP3V and the ARC. Neurons and their fibers are visualized using the immunofluorescent detection of GFP (in red). The major fiber tract arising from the LS projects caudally via the medial forebrain bundle (mfb) to innervate the medial amygdala (MeA) and the hypothalamic supramammillary nucleus (SUM), among other regions. d–g, Representative images illustrate labeled fibers from four different coronal planes. h, Horizontal projection view of a 3D reconstructed brain labeled for both GFP (magenta) and GnRH (green) to study the relationship between the two neuronal systems. i, A coronal optical section through the preoptic area shows virally tagged KP neurons of the LS and the overlap between KPLS fiber projections and GnRH neurons. j, Representative 19-µm-thick z-stack confocal image illustrates the close anatomical relationship between KPLS fibers and GnRH neurons in the preoptica area. k’–m’, Individual 0.8-µm-thick optical slices of framed regions from j reveal cases of direct apposition between KPLS axons (magenta) and the dendritic (k) and somatic (l, m) compartments of GnRH neurons (green). The absence of gap between the juxtaposed profiles also illustrated in orthographic projection views (k’’–m’’ and k”’–m”’) indicates that KPLS neurons contribute to the afferent control of the GnRH neuronal system. AA, anterior amygdaloid area; AH, anterior hypothalamic area; DBB, diagonal band of Broca; LH, lateral hypothalamic area; LPoA, lateral preoptic area; MB, mammillary bodies; MPoA, medial preoptic area; opt, optic tract; VMH, ventromedial hypothalamic nucleus. Scale bars: 1,000 µm in a, b, c, and h; 400 µm in d, e, and i; 200 µm in f and g; 5 µm in j, k, and l.
MP4 file generated from LSM images of an optically cleared whole mouse brain illustrates the anatomical location and the major fiber projections of lateral septal kisspeptin (KPLS) neurons labeled virally with GFP. MeA, medial amygdala; mfb, medial forebrain bundle; SUM, supramammillary nucleus; VDB, ventral diagonal band of Broca. [View online]
KPLS fibers establish axodendritic and axosomatic contacts with GnRH neurons in the medial preoptic area
Light sheet microscopic analysis revealed that the major KPLS fiber track traversed the medial preoptic area vertically and intermingled with GnRH-immunoreactive cell bodies and processes (Fig. 2h,i). To study whether the two neuronal systems interact, the cleared brain shown in Figure 2a–i was rehydrated, infiltrated with sucrose, and sectioned at 30 µm with a cryostat for confocal microscopic analysis. We found that KPLS fibers often establish en passant-type direct appositions to GnRH-immunoreactive dendritic processes and somata (Fig. 2j–m). A quantitative analysis of serial sections from this brain identified 411 GnRH-immunoreactive cell bodies out of which 113 (27.5%) were contacted by GFP-immunoreactive fibers arising from KPLS neurons. These anatomical observations raised the intriguing possibility that the KPLS system contributes to the afferent control of GnRH neurons, as shown previously for hypothalamic fibers mostly arising from KPRP3V (Kallo et al., 2012) and for extrahypothalamic fibers arising from KPMeA (Pineda et al., 2017) neurons.
KPLS neurons exhibit a unique transcriptome profile with low levels of Kiss1 mRNA
A LCM/RNA-seq (“LCM-Seq”) method developed recently in our laboratory (Rumpler et al., 2023) has enabled us to study and identify the estrogen-regulated transcripts of KPARC (Gocz et al., 2022b) and KPRP3V (Gocz et al., 2022a) neurons. Here we used the same OVX + Veh and OVX + E2 animal models and LCM-Seq protocols to characterize the transcriptome landscape of KPLS neurons, allowing us to compare the gene expression profiles of the three different KP cell types. The animals were perfused transcardially with 0.5% paraformaldehyde followed by 20% sucrose. The LS was sectioned serially with a cryostat, and ZsGreen-tagged KPLS neurons were isolated with LCM as reported previously (Gocz et al., 2022a,b). As the low number of KPLS cells in individual mice of this animal model may result in suboptimal detection sensitivity and quantification precision/accuracy of the LCM-Seq protocol (Rumpler et al., 2023), ∼360 fluorescently tagged neurons were pooled proportionally from three brains to prepare each RNA sample and cDNA library for Illumina sequencing (Gocz et al., 2022a,b). For comparison of different KP cell types, meta-analysis of the hypothalamic KP neuron transcriptomes was carried out from open-access public repositories associated with the original publications (Gocz et al., 2022a,b).
Bioinformatic analysis revealed a marked difference between KPLS neurons and the two hypothalamic KP cell types in the level of Kiss1 expression which was virtually undetectable in KPLS neurons of OVX + Veh mice and reached low levels only in OVX + E2 samples with a mean of 7.0 in CPM units. In contrast, Kiss1 levels were high in both KPARC (CPMOVX + Veh, 570.5; CPMOVX + E2, 36.3) and KPRP3V neurons (CPMOVX + Veh, 95.5; CPMOVX + E2, 777.5) (Fig. 3b; Extended Data Fig. 3-1). Differential expression (DE) analysis with DeSeq2 identified thousands of transcripts expressed differentially between KP neurons of the LS and the hypothalamus in either the OVX + Veh or the OVX + E2 models (Fig. 3c; Extended Data Fig. 3-1). For each KP cell population, the top 20 (with the highest mean CPMs, considering all OVX + Veh and OVX + E2 samples) transcription factors, transporters, ion channels, and receptors (defined in the KEGG BRITE database) were selected and analyzed (Extended Data Fig. 3-2). The top 20 lists combined from the three KP cell types included 31 ion channels, 28 transporters, 32 transcription factors, 29 neuropeptides, and 31 receptors with little similarity in expression levels and estrogen-dependent regulation between KPLS, KPARC, and KPRP3V neurons (Fig. 3d; Extended Data Fig. 3-2).
Transcriptome analysis with LCM-Seq reveals the unique gene expression profile of KPLS neurons. a, The use of the OVX + Veh and OVX + E2 animal models and the LCM-Seq protocol to investigate the transcriptome profile of KPLS neurons in the present and KPARC and KPRP3V neurons in earlier gene expression studies allows comparison of the three distinct KP cell types. b, The spider chart illustrates that Kiss1 expression is upregulated in KPLS and KPRP3V neurons and downregulated in KPARC neurons by E2 treatment. Notably, KPLS neurons express considerably lower Kiss1 levels than either KPRP3V or KPARC neurons. c, The quantification table summarizes the number of genes expressed at significantly different levels (p.adj. < 0.05 by DESeq2 analysis) between KPLS neurons and the two hypothalamic KP cell types (Extended Data Fig. 3-1). d, Heat maps compiled from the lists of the top 20 ion channels, transporters, transcription factors, neuropeptides, and receptors from each spatially defined KP cell type reveal large differences in expression levels (in CPM) and in patterns of estrogenic regulation (Extended Data Fig. 3-2).
Figure 3-1
Transcripts expressed differentially between KPLS and hypothalamic (KPARC or KPRP3V) KP neurons in OVX + Veh and in OVX + E2 mice, arranged from highest to lowest log2 fold change (ratio of CPMs: KPLS to KPARC or KPLS to KPRP3V). Download Figure 3-1, XLSX file.
Figure 3-2
Top 20 ion channels, transporters, transcription factors, neuropeptides/granins and receptors in KPLS, KPARC and KPRP3V neurons. Data represent the highest mean CPMs from all OVX + Veh and OVX + E2 mice. Download Figure 3-2, XLSX file.
LCM-Seq unveils 571 estrogen-regulated transcripts in KPLS neurons
DE analysis of the OVX + Veh and the OVX + E2 transcriptomes with the DESeq2 R package and RUVSeq normalization revealed 571 transcripts that changed significantly (p.adj. < 0.05) within KPLS neurons in response to E2 treatment (Fig. 4; Extended Data Fig. 4-1). E2 upregulated ∼80% of the changing transcripts (Fig. 4a,b). Overrepresentation analysis (ORA) identified significant changes in the neuroactive ligand–receptor interaction, glutamatergic synapse, and retrograde endocannabinoid signaling functional (KEGG) pathways (Fig. 4c). Individual E2-regulated transcripts fell into diverse functional categories defined in KEGG BRITE and Neuropeptide (http://www.neuropeptides.nl/) databases, including ion channels (N = 22), transporters (N = 19), transcription factors (N = 23), neuropeptides/granins (N = 8), and receptors (N = 15; Fig. 4d). For a full listing of E2-regulated KPLS neuron transcripts, see Extended Data Figure 4-1. Meta-analysis of the KPARC and KPRP3V transcriptome databases with DESeq2 (using RuvSeq) identified 2,458 and 547 transcripts, respectively, that were regulated significantly by the same E2 treatment (Fig. 5; Extended Data Fig. 4-1). Comparison of regulated transcripts in the three cell types confirmed the unique molecular properties and regulatory patterns of KPLS, KPARC, and KPRP3V neurons. Only 133 (∼23%) of the 571 E2-dependent KPLS transcripts were also regulated by E2 in KPARC and 58 (∼10%) in KPRP3V neurons (Fig. 5). In 46 instances, opposite regulation was found between KPLS neurons and either the KPARC or the KPRP3V cell type. This phenomenon was reminiscent of several inverse regulatory changes we observed earlier while comparing E2-induced transcriptomic changes in the KPARC and KPRP3V cell types (Gocz et al., 2022a). E2-dependent transcripts of each KP cell type and overlaps between their E2-dependent transcripts are listed in Extended Data Figure 4-1.
E2 treatment of OVX mice induces profound transcriptomic changes within KPLS neurons. a, Heat map illustrates that ∼80% of the 571 transcripts altered by the 4 d E2 treatment are upregulated (dendrogram generated using UPGMA hierarchical clustering). b, Differentially expressed genes are shown in a volcano plot. Neuropeptides/granins and receptors are tagged with gene symbols. c, ORA identifies three significantly altered functional (KEGG) pathways. E2-regulated transcripts of the neuroactive ligand–receptor interaction, glutamatergic synapse, and retrograde endocannabinoid signaling categories are illustrated graphically. d, Individual E2-regulated transcripts fall into diverse functional categories defined in KEGG BRITE and Neuropeptide databases. Many of them encode ion channels (N = 22), transporters (N = 19), transcription factors (N = 23), neuropeptides/granins (N = 8), and receptors (N = 15). Numbers in dots refer to relative transcript abundances in CPM units. Dot areas change in proportion to transcriptional responses, with the larger dot representing 100%. For full listing of E2-regulated KPLS neuron transcripts, see Extended Data Figure 4-1.
Figure 4-1
Full listing and functional categorization (ion channels, transporters, transcrip- tion factors, neuropeptides/granins and receptors) of transcripts responding to E2 treatment in KPLS neu- rons, estrogen regulated transcripts of KPARC and KPRP3V neurons and estrogen-dependent transcripts shared between two different KP cell types. Download Figure 4-1, XLSX file.
Distinct E2-dependent gene sets of KPLS, KPARC, and KPRP3V neurons predict unique functional properties of each KP cell type. Venn diagram illustrates 166 out of the total 571 KPLS neuron transcripts that are also regulated by E2 in KPARC and/or KPRP3V neurons. For full listing of the regulated transcripts in each cell type, see Extended Data Figure 4-1. White labels highlight genes that exhibit inverse regulatory patterns in different KP cell types.
Estrogenic upregulation of the Chrm2 transcript enhances muscarinic inhibition of KPLS neurons
The presence of muscarinic and nicotinic receptor transcripts in the KPLS neuron transcriptome indicated that acetylcholine plays an important role in the afferent control in KPLS neurons. Indeed, we found that KPLS neurons are embedded in a dense fiber network immunoreactive to the cholinergic marker VAChT (Fig. 6a). Among the muscarinic and nicotinic receptor transcripts, Chrm2 showed the highest expression level and a strong positive regulation by E2 (Fig. 6b), allowing us to predict enhanced muscarinic inhibition of KPLS neurons in the presence of estrogens. To provide functional support for this hypothesis, the effects of muscarine on the spontaneous firing and resting membrane potential (Vrest) of KPLS neurons were studied with whole-cell patch–clamp electrophysiology (Fig. 6c). KPLS neurons were spontaneously active, with phasic-irregular (∼ 90%) or tonic (∼10%) firing, at a rate of 3.1 ± 0.55 Hz in OVX + Veh (n = 15) and 2.9 ± 0.58 Hz (n = 12) in OVX + E2 mice (not different statistically; p = 0.8035 by Student's t test). A single bolus of muscarine (100 μM) abolished the firing of KPLS neurons. This inhibition was transient and lasted longer in OVX + E2 mice (157 ± 28.4 s in OVX + Veh and 290 ± 30.3 s in OVX + E2 mice; p = 0.0016; Dunnett's post hoc test; Fig. 6d,e,h; Extended Data Fig. 6-1). The firing rate calculated for the 7 min period after the application of muscarine dropped to 26.7 ± 6.58% of the control rate in OVX + E2 and to 54.8 ± 7.33% in OVX + Veh mice (p = 0.0408; Dunnett's post hoc test; Fig. 6d,e,i; Extended Data Fig. 6-1). Pretreatment of slices with the M2 muscarinic receptor antagonist Methoct (2 μM) entirely prevented the silencing of KPLS neurons by muscarine, providing evidence that this receptor subtype has major contribution to inhibition (Fig. 6f,h,i; Extended Data Fig. 6-1).
Estrogen-induced upregulation of the M2 acetylcholine receptor enhances muscarinic inhibition of KPLS neurons. a, Confocal microscopic analysis reveals that KPLS neurons (green ZsGreen signal) are embedded in a dense network of cholinergic fibers immunoreactive to the VAChT (magenta). b, KPLS neurons express several muscarinic acetylcholine receptors. High levels and estrogenic upregulation of Chrm2 suggest that muscarine exerts heavier inhibition on KPLS neurons via M2 cholinergic receptor in the presence of E2. c, The use of whole-cell patch–clamp electrophysiology allows the comparative analysis of muscarinic responses in OVX + Veh versus OVX + E2 mice. d, e, Representative traces obtained from OVX + Veh and OVX + E2 mice illustrate that a single bolus of muscarine (100 μM) hyperpolarizes KPLS neurons and transiently abolishes their firing. Note that the silent period lasts longer in the OVX + E2 model (e), expressed also as a decrease in firing rates for the 7 min period after muscarine administration. f, The presence of the M2 muscarinic acetylcholine receptor antagonist Methoct (2 μM) in the aCSF medium prevents the muscarine-induced hyperpolarization and silencing of KPLS neurons. g, Muscarine-induced hyperpolarization of KPLS neurons persists in the presence of TTX (660 nM), suggesting that muscarine acts directly on these cells. h, i, Bar graphs reveal that the length of the silent period (h) and the percentage decrease in the firing rates (i) differ significantly between OVX + E2 mice and OVX + Veh mice as well as between OVX + E2 mice with and without Methoct pretreatment. j, The bar graph reveals that the amplitude of muscarine-induced hyperpolarization is significantly larger in OVX + E2 versus OVX + Veh mice, nearly disappears in the presence of Methoct and persists if APs are inhibited by TTX. Arrows indicate the time when muscarine is added to the aCSF. *p < 0.05. For source data, see Extended Data Figure 6-1. Scale bars, 10 µm in a and 200 µm in c.
Figure 6-1
Source data and statistical analyses of slice electrophysiology results. Download Figure 6-1, XLSX file.
The resting membrane potentials were similar in the two endocrine models (−48 ± 1.3 mV in OVX + Veh and −48 ± 1.5 mV in OVX + E2 mice; p = 0.9008 by Student's t test). A bolus of muscarine (100 μM) transiently hyperpolarized KPLS neurons (Fig. 6d,e,j), and this effect disappeared in the presence of 2 μM Methoct (Fig. 6f,j). Notably, the amplitude of hyperpolarization was significantly greater in the OVX + E2 model (ΔVrest; −8.0 ± 0.98 mV in OVX + Veh vs −12.2 ± 1.14 mV in OVX + E2 mice; p = 0.0094; Dunnett's post hoc test; Fig. 6j; Extended Data Fig. 6-1). Control experiments showing that muscarine hyperpolarizes KPLS neurons of OVX + E2 mice in the presence of the AP inhibitor TTX confirmed that the muscarinic inhibition of KPLS neurons is direct (660 nM; ΔVrest = −10.6 ± 2.00 mV; Fig. 6g,j). Together, these observations provided support for the hypothesis that the upregulation of Chrm2 by E2 results in a stronger muscarinic inhibition of KPLS neurons in the OVX + E2 model (for all statistics, see Extended Data Fig. 6-1).
Fluorescent ISH results reveal that large percentages of KPLS neurons synthesize other neuropeptide cotransmitters and are heavily regulated by glucagon-like peptide-1
High expression of several neuropeptide transcripts suggested that KPLS neurons use additional cotransmitters to communicate with their target cells. From the most heavily expressed transcripts, we selected Cartpt (CPMOVX + Veh:135.9; CPMOVX + E2:69.7), Penk (CPMOVX + Veh:826.3; CPMOVX + E2:615.7), and Vgf (CPMOVX + Veh:121.5; CPMOVX + E2:83.2) for colocalization experiments with ISH. Among the G-protein–coupled receptors, the robust Glp1r expression (CPMOVX + Veh:188.3; CPMOVX + E2:187.9) was identified as a unique feature of the KPLS neuron population, suggesting that these neurons are heavily regulated by glucagon-like peptide-1 signaling. Results of fluorescent ISH studies with digoxigenin-labeled cRNA probes revealed that 43.1 ± 3.5% (mean ± SEM) of KPLS neurons synthesize cocaine- and amphetamine-regulated transcript (CART; encoded by Cartpt), 77.0 ± 6.6% synthesize neuropeptides derived from the proenkephalin precursor (PENK; encoded by Penk), 19.3 ± 9.7% synthesize neuropeptides derived from the VGF precursor, and 49.1 ± 9.9% synthesize glucagon-like peptide-receptor-1 (Fig. 7a–f).
Neuroanatomical studies confirm that KPLS neurons express transcripts for neuropeptide cotransmitters (Cartpt, Penk, Vgf) and the glucagone-like peptide 1 receptor (Glpr1) and reveal the presence of KPLS neurons in the human brain. a–f, Confirming RNA-seq results, fluorescent ISH (FISH) detection of Cartpt (a), Penk (b), Vgf (d), and Glp1r (e) mRNAs with digoxigenin-labeled cRNA probes provides morphological evidence for abundant expression of these transcripts in large subsets (c, f) of KPLS neurons. Arrowheads in a–e point to KPLS neurons (ZsGreen) expressing the FISH signal. g, Immunohistochemical detection of KP (black nickel–diaminobenzidine chromogen) in postmortem brains of 53-year-old (left) and 54-year-old (right) male subjects using sheep (GQ2) and rat (SST#01) polyclonal antibodies illustrates the presence of KP neurons in the human LS. h, Dual-immunofluorescent labeling of KP neurons in the hypothalamic infundibular nucleus (Inf.; 69-year-old male) with the GQ2 and SST#01 antibodies serves as a positive immunohistochemical control for detection specificity. Scale bars: 50 µm in a, b, d, e, h; 20 µm in high-power; and 500 µm in low-power panels in g. fx, fornix; LSI, lateral septum, intermediate subnucleus; LSV, lateral septum, ventral subnucleus; LV, lateral ventricle; 3V, third ventricle.
Detection of KPLS neurons in the human brain suggests evolutionarily conserved roles
In previous studies from our laboratory, we provided evidence that the human hypothalamus contains the same two hypothalamic KP neuron populations that were reported in a variety of other species (Hrabovszky et al., 2010, 2021). To address if KPLS neurons are also present in the human brain, we carried out immunohistochemical studies of KP neurons in immersion-fixed postmortem samples collected from four male individuals. Detection of the human KP54 peptide with the widely used GQ2 antiserum (Dhillo et al., 2005; Hrabovszky et al., 2010, 2021) and a newly generated rat polyclonal antibody (SST#01) directed to a different segment of human prepoKP revealed a few scattered KP-immunoreactive neurons and a moderately dense KP fiber plexus in the human LS (Fig. 7g). A positive control experiment to confirm antibody specificity verified that the GQ2 and SST#01 antisera recognize the same KP neuronal elements in the human hypothalamus (Fig. 7h). These immunohistochemical results strongly suggest that KPLS neurons play evolutionarily conserved roles in the regulation of currently unknown limbic functions.
Discussion
While much attention has been paid over the past two decades to the reproductive significance of the two hypothalamic KP neuron populations (KPARC and KPRP3V neurons), earlier studies on transgenic mice have also identified KP neurons outside the hypothalamus, including the LS, the MeA, the periaquaductal gray, and the mammillary nuclei (Yeo et al., 2016). The localization of Kiss1 mRNA expressing neurons to the same anatomical sites with sensitive radioisotopic ISH approaches validated the earlier observations made on genetically modified models (Lehman et al., 2010; Kim et al., 2011; Di Giorgio et al., 2014; Yeo et al., 2016; Stephens and Kauffman, 2017; Stephens et al., 2018; Hatcher et al., 2023).
Our findings that 72.5 ± 7.6% of KPLS neurons in OVX + E2 mice express ERα immunoreactivity and these neurons exhibit E2-dependent development and transcriptome profile are in accordance with the results of earlier ISH studies which showed that E2 positively regulates Kiss1 mRNA expression in this brain area (Di Giorgio et al., 2014; Stephens et al., 2018) via acting on ERα.
Microscopic analysis of ZsGreen-labeled KPLS neurons in our study revealed a conspicuous sexual dimorphism, with significantly higher cell numbers in females than in males during both adolescence and adulthood. Our observations on P20 mice treated for 4 d with different sex steroids provided evidence that E2 signaling through ERα plays an equally important role in the development of KPLS neurons in males and females. It was somewhat unexpected that gonadectomy at P21–31 only prevented the pubertal activation of the Kiss1 promoter in males. The seemingly E2-independent development in females might be related to an earlier ovarian stimulation during minipuberty (Chachlaki et al., 2022). Alternatively, sex steroid-independent mechanisms may also activate the Kiss1 promoter in the developing female mouse brain. Accordingly, it was proposed that ERs are not involved in the mechanism, whereby GABABR suppresses the Kiss1 promoter in the LS (Di Giorgio et al., 2014; Stephens et al., 2018).
In our developmental studies, the first large increases in KPLS cell numbers were observed in P33–36 female and P40–45 male mice, corresponding to late stages of pubertal development. It is interesting to note that immunohistochemically detectable KPRP3V neurons which also show E2-dependent development start to emerge at P15 and their number at P30 already resembles those of P60 adult female mice (Clarkson et al., 2009). Considering that the two studies used different detection techniques, the dissimilar developmental profiles of KPRP3V and KPLS neurons need to be confirmed using identical methods. Although not pursued, we found that P21–31 gonadectomy was unable to stop the later emergence of the KP-Cre/ZsGreen phenotype in the LS of female mice. We note that an even earlier ovariectomy at P15 reduced by 70–90% but did not prevent entirely the estrogen-dependent development of KPRP3V neurons (Clarkson et al., 2009). The developmental escape of KP neurons in both cases could be related to a transient adenohypophyseal follicle-stimulating hormone secretion and follicle stimulation during the earlier minipuberty peaking at P12 (Chachlaki et al., 2022).
The higher number of KPLS neurons in female versus male mice adds a new aspect to the sexual dimorphism characteristic to various KP cell types. A large female dominance of the KPRP3V neuron population (Kauffman et al., 2007; Herbison, 2008, 2020; Clarkson et al., 2009) is in accordance with the presence of positive estrogen feedback in female rodents only. Sexual dimorphism has also been observed in the human hypothalamus which contains more KPARC neurons in females than in males (Hrabovszky et al., 2010, 2021). Interestingly, ISH detection of Kiss1 mRNA revealed an opposite pattern of dimorphism in the rodent MeA, with higher cell numbers in intact males versus diestrous females (Kim et al., 2011). The sex difference of the KPMeA system likely reflects the dissimilar sex steroid milieu rather than a developmental imprinting because it disappears if GDX females and males are treated with the same E2 dose (Kim et al., 2011).
We found that KPLS neurons innervated most heavily the hypothalamic SUM. This target nucleus plays critical roles in hippocampal processes like theta rhythm and learning/memory and regulates a variety of behaviors, such as arousal, reward-seeking, exploration, social memory, and anxiety (Lopez-Ferreras et al., 2020; Kesner et al., 2021). High levels of Glp1r expression was a unique feature of KPLS neurons not observed previously in the KPARC and KPRP3V transcriptomes (Gocz et al., 2022a,b). Glutamatergic neurons of the SUM which synthesize GLP1R (Kesner et al., 2021) now also appear to receive a strong afferent input from Glp1r-expressing KPLS neurons, suggesting that the anxiogenic effect of circulating GLP-1 in the SUM (Kesner et al., 2021) may use at least two independent mechanisms.
Based on earlier suggestions that the LS gives rise to a sexually dimorphic lordosis-inhibiting pathway (Tsukahara and Yamanouchi, 2001; Tsukahara et al., 2014), we tested the possibility that the activity of KPLS neurons changes in response to sexual behavior. This study detected significantly higher percentages of c-Fos–positive KPLS neurons in male mice than in estrous females but failed to provide evidence for the altered activity of these neurons in response to either opposite-sex smell or mating. These findings neither support nor exclude the contribution of KPLS neurons to the regulation of sexual behavior.
In our study, we characterized thoroughly the transcriptome profile of KPLS neurons using a versatile “LCM-Seq” technique developed in our laboratory (Rumpler et al., 2023). High sensitivity achieved via the bulk sequencing strategy (360 neurons/pool) allowed us to distinguish over 15,000 different transcripts. Somewhat unexpectedly, KPLS neurons exhibited only weak Kiss1 phenotype (0 CPM in OVX + Veh and 7 CPM in OVX + E2 mice) which was an important difference from both the KPARC and the KPRP3V cell types. In contrast, the abundance of Gad1, Gad2, Slc32a1, Penk, Cartpt, and Vgf among the expressed transcripts indicates that GABA, enkephalin, CART, and VGF-derived neuropeptides play paramount roles in the efferent communication of KPLS neurons with limbic and hypothalamic target cells, including GnRH neurons.
The KPLS neuron transcriptome can now be compared with the gene expression profiles of KPARC (Gocz et al., 2022b) and KPRP3V (Gocz et al., 2022a) neurons we studied earlier using the same methodologies and animal models (Gocz et al., 2022a,b) as well as to the elegant data on the E2-dependent active KPRP3V (Stephens and Kauffman, 2021) and KPMeA (Hatcher et al., 2023) neuron translatomes from the Kauffman and Stephens laboratories, respectively. Comparisons revealed that KP cell types have distinct transcriptome landscapes with often different and occasionally even opposite transcriptional regulation by E2. Meta-analysis of the deposited sequencing files using DESeq2 with RUVseq normalization identified 2,891, 905, 571, and 101 E2-regulated transcripts in KPARC (Gocz et al., 2022b), KPRP3V (Gocz et al., 2022a), KPLS (present study), and KPMeA (Hatcher et al., 2023) neurons, respectively, indicating that transcriptional responses to E2 are strongest in KPARC neurons and relatively weak in KP cells of extrahypothalamic regions. An obvious regulatory difference between KP cell types included the upregulation of Pgr (encoding progesterone receptor) in hypothalamic but not in extrahypothalamic KP neurons. KPLS neurons exhibit a unique transcription factor profile, with several homeobox (Meis2, Otx2, Lhx6, Lhx8) and zinc finger (Zic1-Zic5) proteins. Some of its major transcription factors (e.g., Myt1l, Esr1), however, are detectable in multiple KP cell types (Gocz et al., 2022a,b). Using DE analysis of RNA-seq libraries from OVX + Veh and OVX + E2 mice, we have identified hundreds of E2-regulated transcripts in KPLS neurons, including Kiss1. The low level of Kiss1 and its upregulation by E2 in KPLS neurons were in agreement with the results of previous ISH studies (Di Giorgio et al., 2014; Stephens et al., 2018). E2 also increased the expression of transcripts related to dopaminergic, glutamatergic, and cholinergic neurotransmission. We propose that these changes may also contribute to the fine-tuning of limbic functions subserved by the KPLS system. In accord with our predictions, studies using whole-cell current–clamp electrophysiology provided functional evidence that the increased Chrm2 expression of the OVX + E2 versus the OVX + Veh model coincides with an enhanced muscarinic inhibition of KPLS neurons.
Each KP neuron population studied so far with transcriptomics now appear to exhibit a very unique amino acid neurotransmitter and neuropeptide cotransmitter profile (Stephens and Kauffman, 2021; Gocz et al., 2022a,b; Hatcher et al., 2023). While KPARC neurons are dominantly glutamatergic (Gocz et al., 2022a,b), KPMeA neurons express glutamatergic markers, and somewhat lower amounts of GABAergic markers (Hatcher et al., 2023), KPRP3V (Stephens and Kauffman, 2021; Gocz et al., 2022a), and KPLS neurons (present study) appear to use GABA as a major amino acid neurotransmitter. Highly expressed peptide cotransmitters include Tac2, Pdyn, Vgf, Pomc, and Gal in KPARC neurons (Gocz et al., 2022a,b); Penk, Kiss1, Vgf, Gal, Nts, Cartpt, and Pdyn in KPRP3V neurons (Stephens and Kauffman, 2021; Gocz et al., 2022a); Penk, Vgf, Cartpt, and Sst in KPLS neurons (present study); and Vgf, Cck, Penk, Prl, Cartpt, and Sst in KPMeA neurons (Hatcher et al., 2023).
3D reconstruction of KPLS fiber projections and immunohistochemically labeled GnRH neurons in whole brains cleared optically with the iDISCO protocol revealed a considerable overlap between the two neuronal systems. Confocal microscopic analysis of preoptic sections identified direct contacts without intervening gaps between virally labeled KPLS axons and GnRH neurons. While synaptic interaction cannot be studied at this spatial resolution, the presence of such appositions on 27.5% of GnRH-immunoreactive cell bodies suggests that the KPLS system contributes considerably to the afferent control of GnRH neurons, as shown previously for the KPMeA system (Pineda et al., 2017). The contribution of the LS to the sex steroid-dependent stimulation of LH secretion has been known for four decades (Carreras et al., 1984). Studies of male rats have established that septal lesions decrease (Carreras et al., 1984), whereas implantation of crystalline T and DHT to the LS increases plasma LH levels (Roca et al., 2001). The direct connectivity we observed between KPLS neurons and the GnRH system may represent an important anatomical pathway, whereby the sex steroid-sensitive and sexually dimorphic neurocircuitry of the LS regulates adenohypophyseal LH secretion.
Earlier studies of postmortem human brains in our laboratory localized the majority of KP neurons to the hypothalamic infundibular nucleus (Hrabovszky et al., 2010, 2021). This system appears to correspond to the KPARC (KNDy) neurons of mice and rats, although also differs considerably in coexpressed neuropeptides; while human KP neurons rarely contain dynorphin, they synthesize substance P, CART, and enkephalins (Hrabovszky et al., 2021). Humans also contain an additional KP system in the rostral hypothalamus (paraventricular nucleus) which may correspond to KPRP3V neurons of rodents (Rumpler et al., 2021). Our present study used two different polyclonal antibodies as a positive control approach and provided evidence that, similarly to mice, humans also contain KP neurons in the LS. This observation suggests that the functions subserved by KPLS neurons are evolutionarily conserved.
In conclusion, in this study we report the detailed ontogeny, sexual dimorphism, neuroanatomy, neurochemistry, E2-dependent transcriptomic regulation, and contribution to the afferent regulation of GnRH neurons of the KPLS neuronal system in mice. We also provide anatomical evidence that homologous KP neurons exist in the postmortem human brain. Together, these data raise the intriguing possibility that KPLS neurons regulate the GnRH neuronal system and exert estrogen-dependent and sex-specific control on currently unknown hypothalamic and limbic functions, with likely human relevances.
Data Availability
RNA sequencing files for KPLS neurons are available in BioProject with the accession number PRJNA1017786. Reanalyzed source files for sequenced KPARC and KPRP3V neurons are referenced in original publications (Gocz et al., 2022a,b). Scripts will be available upon request.
Footnotes
This work was supported by grants to Project Number RRF-2.3.1-21-2022-00011, titled National Laboratory of Translational Neuroscience implemented with the support provided by the Recovery and Resilience Facility of the European Union within the framework of Programme Széchenyi Plan Plus, the National Research, Development and Innovation Office (K138137 to E.H. and PD134837 to K.S.), and the Hungarian Research Network (SA-104/2021). S.S.-T. and B.Gö. are awardees of the New National Excellence Programme of Hungary. The work of S.S.-T. received support by the award of the National Academy of Scientist Education program.
↵*S.S.-T. and B. Göcz contributed equally to his work.
The authors declare no competing financial interests.
- Correspondence should be addressed to Gergely Rácz at racz.gergely{at}semmelweis.hu or Erik Hrabovszky at hrabovszky.erik{at}koki.hun-ren.hu.
