Preoptic kisspeptin-nNOS-GnRH (KiNG) neuronal network regulates LH rhythmicity through activation-inhibition in mice

Domain: neuroscience
Published: 2026-02-10
Editorial outcome: major_revision

Abstract
Gonadotropin-releasing hormone (GnRH) neurons are the final target of a complex network regulating reproduction. The balance between excitatory and inhibitory inputs is essential for rhythmic GnRH secretion, including pulses and surges, yet the underlying mechanisms remain unresolved. Here, using adult animals of both sexes, we test the hypothesis that excitatory kisspeptin and inhibitory neuronal nitric oxide (NO) synthase (nNOS)-derived inputs orchestrate GnRH release within a microcircuit of kisspeptin, nNOS and GnRH neurons (the “KiNG” network). We focus on nNOS neurons of the organum vasculosum of the lamina terminalis (OV) and the median preoptic nucleus (MePO), which interact with kisspeptin and exhibit cycle-dependent kisspeptin receptor (Kiss1r) expression. Using a highly-sensitive NO/cGMP biosensor together with electrophysiological, genetic, chemogenetic and pharmacological approaches we demonstrate that kisspeptin induces NO-dependent cGMP production in the OV/MePO, including in GnRH neurons, which in turn fine-tunes the GnRH/LH response, providing mechanistic insights into how the KiNG network shapes pulse and surge generation.

Introduction
Gonadotropin-releasing hormone (GnRH) neurons of the hypothalamus orchestrate reproduction. While their cell bodies are mainly distributed in the preoptic region, their nerve terminals, which lie in the median eminence of the hypothalamus, release the GnRH decapeptide in a rhythmic manner into the pituitary portal capillary system1,2. Blood-borne GnRH acts on gonadotrophs in the anterior pituitary, stimulating the synthesis and secretion of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The release pattern of GnRH includes both pulse and surge phases in females but only pulses in males. This GnRH rhythmicity arises from the integration of a complex array of excitatory and inhibitory inputs from both the central nervous system (e.g., kisspeptin and nitric oxide [NO]) and peripheral organs (e.g., estrogen and testosterone from the gonads). The correct balance of these signals is indispensable for the timely and appropriate release of LH and FSH, which are key to the establishment and proper function of the hypothalamic-pituitary-gonadal (HPG) axis.

The stimulatory effect of kisspeptin-kisspeptin receptor (Kiss1-Kiss1r) signaling on GnRH activity and secretion is fundamental to inducing LH release3, crucial for the establishment and maintenance of reproduction4–6. However, this function requires GnRH/LH pulses rather than prolonged and sustained kisspeptin-induced GnRH activation, which shuts down the axis, suggesting the existence of an “OFF” switch. It is known that kisspeptin neurons of the anteroventral periventricular nucleus (AVPV) are important components of the mechanism mediating the positive feedback effects of estrogen on the preovulatory GnRH/LH surge, while the subpopulation of kisspeptin neurons in the arcuate nucleus (ARH), frequently referred to as KNDy neurons due to their coexpression of kisspeptin, neurokinin B and dynorphin, has been associated with the control of pulsatile LH secretion during the negative feedback phase of estrogen7,8. While the activatory role of kisspeptin is well established, the negative regulators of the GnRH system that terminate the kisspeptin response remain largely understudied. For example, the KNDy neuronal hypothesis can explain the control of kisspeptin neurons themselves but not how GnRH neurons are controlled9, since dynorphin is not necessary for the termination of the GnRH/LH pulses in rodents10 and in addition, is lacking in human KNDy neurons11. There is, therefore, a knowledge gap regarding the intricate regulatory network governing GnRH neuronal activity and hence the mechanisms that shape the GnRH surge and pulse.

In a recent review, we postulated the existence of a Kisspeptin-nNOS-GnRH or “KiNG” neuronal network that would function by alternating between neuronal activation and tonic inhibition in order to modulate LH pulse and surge generation12. Similar to kisspeptin, neurons expressing the neuronal NO synthase (nNOS, coded by Nos1), which produce the highly diffusible gaseous messenger NO, are estradiol-sensitive and express the estrogen receptor alpha (ERα)13. nNOS neurons are morphologically and functionally associated with GnRH neuronal cell bodies and dendrites in the median preoptic nucleus (MePO) and the organum vasculosum of the lamina terminalis (OV); NO acts as an inhibitory signal that rapidly integrates and transmits peripheral signals to the brain, particularly to GnRH neurons14. Once produced, NO diffuses to neighboring cells where it binds and activates soluble guanylate cyclase (sGC), leading to the generation of the second messenger cyclic guanosine monophosphate (cGMP). The amplitude and duration of this NO/cGMP signal are further shaped by phosphodiesterases (PDEs) such as PDE5, which degrade cGMP and thereby terminate the response15. Because GnRH neurons are widely dispersed across the preoptic area a diffusible messenger like NO is well positioned to coordinate their activity. The importance of NO signaling in the regulation of GnRH functionality is increasingly apparent (for review see ref. 14) and loss-of-function mutations in Nos1 have recently been identified in a cohort of patients with congenital hypogonadotropic hypogonadism16, demonstrating the key role of nNOS in reproduction. This hypogonadotropic hypogonadal phenotype also occurs in mice genetically deficient for Nos1 (Nos1−/−)16–18, which exhibit both abnormal estrous cyclicity and blunted LH levels in proestrus16 while the intracerebroventricular injection of NOS blockers inhibits GnRH/LH secretion19, suggesting that nNOS activity and NO release are involved in both the positive and negative feedback effects of estrogen. In fact, estradiol positively regulates nNOS activity in the OV/MePO by promoting the physical association of the nNOS enzyme with the NMDA receptor20,21 and subsequently, the phosphorylation of nNOS at Ser-141222, an action that requires Kiss1-Kiss1r signaling17. Kisspeptin-NO interactions have also been identified as a mechanism shaping GnRH pulsatility by modulating the refractory period of GnRH neuronal activity in response to kisspeptinergic stimuli23, supporting the so-called KiNG neuronal network hypothesis, i.e., the possibility that a microcircuit of excitatory and inhibitory inputs from kisspeptin and nNOS neurons could orchestrate GnRH neuronal activity and release12.

In this report, we test this hypothesis by focusing on the preoptic OV/MePO nNOS population (nNOSOV/MePO), surrounding the GnRH cell bodies. Using in situ hybridization and various in vivo genetic and chemogenetic approaches, we examine the role of the nNOSOV/MePO neurons in the control of pulsatile and kisspeptin-induced LH secretion, and uncover mechanistic details of the KiNG neuronal network, thanks to a viral tool engineered to measure in vivo NO/cGMP release.

Results
Cycle-dependent Kiss1r expression in preoptic Nos1-expressing neurons

nNOS enzymatic activity in the OV/MePO, and hence NO levels, have been suggested to be controlled by the classic Kiss1/Kiss1r/AKT pathway17, being low during diestrus I (Di) and reaching their maximum during proestrus (Pro)22. In order to more accurately analyze the anatomical relationship between neurons expressing Kiss1, Kiss1r, and Nos1 mRNA in the hypothalamic preoptic area and evaluate plausible differential dynamics across the estrous cycle, we took advantage of RNAscope technology. Fluorescence in situ hybridization in the OV and MePO of cycling females revealed the expression of Kiss1r mRNA in a fair proportion of Nos1-positive cells (Fig. 1A, B, Suppl. Fig. 2A). Interestingly, even though the percentage of Nos1- or Kiss1r-expressing cells remained unaltered across the estrous cycle (Fig. 1C, D), the percentage of Nos1-expressing cells also positive for Kiss1r mRNA was significantly increased in proestrus, while no marked difference was seen between the metestrus (mE) and diestrus stages (OV: 44.0 ± 2.5% in mE, 45.1 ± 1.3 % in Di vs. 55.2 ± 1.1 % in Pro, p=0.02 and p=0.01, respectively; MePO: 45.4 ± 1.6% in mE, 44.9 ± 4.5% in Di vs. 58.8 ± 3.4% in Pro, p=0.02 and p=0.06, respectively) (Fig. 1B). Similar to metestrous and diestrous females, male mice demonstrated lower levels of co-expression between the Nos1 and Kiss1r mRNAs (OV: 36.5 ± 2.5 % and MePO: 30.0 ± 3.1%) (Fig. 1B).

These changes were specific for the nNOS population of the OV/MePO (nNOSOV/MePO). In the hypothalamic AVPV, the percentage of Nos1-expressing cells co-expressing Kiss1r mRNA was found to be unaltered between diestrus and proestrus (41.9 ± 1.8 % in Di vs. 44.5 ± 2.2% in Pro) (Suppl. Figs. 1A, C–E and 2B). Even though Kiss1 and Nos1 mRNAs were found to be separately expressed in the vast majority of cells in the AVPV (Suppl. Fig. 1B, E–G), <10% of Kiss1-positive cells were seen to express low amounts of Nos1 mRNA at both stages of the estrous cycle (7.8 ± 2.1 % in Di, vs. 6.1 ± 1.6 % in Pro, p=0.5), with Kiss1 mRNA showing an almost 50% increase in proestrus (13.2 ± 1.8 % in Di, vs. 21.3 ± 1.8 % in Pro, p=0.03) (Suppl. Fig. 1F).

Kisspeptin drives NO-dependent cGMP production in OV/MePO neurons

Previous reports have revealed that the intraperitoneal administration of the decapeptide form of kisspeptin (kisspeptin-10, kp-10) promotes an increase in nNOS phosphorylation at Ser1412 in the OV/MePO, accompanied by an increase in LH levels17, an effect that we also observe in our model (Fig. 1E–H) in keeping with the frequent coexpression of Kiss1r in these neurons (Fig. 1). Phosphorylation at Ser1412 is believed to be associated with enhanced nNOS enzymatic activity24. However, the association of Ser1412-phosphorylation of nNOS with hypothalamic NO production has not been demonstrated in vivo. We therefore engineered an AAV9 viral tool expressing the cGMP-dependent biosensor, FlincG3 (Fig. 2A), which we have previously used to accurately measure levels of NO released by transfected cells in vitro16,25, under the control of the CMV promoter. Stereotaxic injection of the virus expressing the FlincG3 construct into the OV/MePO of adult male mice allowed us to perform ex-vivo cGMP imaging on living brain slices (Fig. 2B, C). Live imaging revealed the presence of basal levels of FlincG3 fluorescence in the OV (Fig. 2D). Bath application of a high concentration of the NO donor PAPA/NO (5μM) resulted in an increase in fluorescence as a consequence of local NO-stimulated cGMP production (Fig. 2D, E). This increase was prevented when PAPA/NO was applied together with the sGC inhibitor ODQ (10μM), confirming that the FlincG3 fluorescence was a result of NO/cGMP production (Fig. 2D, E). We then took advantage of this technology to verify whether kisspeptin-induced nNOS activation actually results in NO/cGMP release. Bath application of kp-10 (10nM) resulted in a >200% increase in the maximum FlincG3 ΔF/F0 (change in fluorescence relative to baseline) values in the OV (mean baseline ΔF/F0 = 0.006 ± 0.002 vs. mean kp-10 ΔF/F0 = 0.03 ± 0.002) (Fig. 2F). Bath application of ODQ before kp-10 administration completely blocked the kp-10-induced increase in FlincG3 fluorescence (Fig. 2G).

In order to determine whether cGMP is synthesized in cells different from those expressing the nNOS protein, and thus whether NO acts in a paracrine manner to stimulate the synthesis of cGMP, we analyzed the anatomical distribution of cGMP-producing cells in the OV/MePO of cycling females after local stereotaxic injections of the FlincG3 construct (Fig. 2H). Females were perfused during diestrus, when nNOS enzymatic activity and NO release are at their nadir, after a bolus intraperitoneal injection of saline or kp-10 (1 nmol; Fig. 2H). Kp-10 administration led to an increase in Ser1412-phosphorylation of the nNOS protein in the OV/MePO, as well as circulating LH levels, in agreement with previous reports (Fig. 1E–H)17. In accordance with our live imaging data, kisspeptin treatment was also accompanied by massive cGMP production in the OV/MePO (Fig. 2I–L) as attested to by the 250% increase in FlincG3 integrated fluorescence intensity (Int Dens saline = 47.6 ± 13.8 vs. kp-10 = 168.8 ± 28.7) (Fig. 2J). Interestingly, FlincG3 fluorescence was rarely seen in nNOS-immunoreactive cells (OV: 1.6 ± 0.5 % and MePO: 4.0 ± 0.7 % of nNOS neurons showing FlincG3 signal) but was instead observed in cells surrounding nNOS-expressing neurons, including, strikingly, GnRH neurons (14.4 ± 3.2% of GnRH neurons showing FlincG3 signal) (Fig. 2L). Notably, the FlincG3 intensity was positively correlated with the levels of phosphorylation of the nNOS enzyme following kp-10 administration (Fig. 2I, K). Our observations demonstrate that cGMP production is coupled with the phosphorylation of nNOS protein at Ser1412 in vivo, and further support the hypothesis that kisspeptin, by modulating nNOS enzymatic activity, can directly increase local NO/cGMP levels. Altogether, these results provide further support for the existence of a kisspeptin-NO-GnRH communication network in the OV/MePO, as postulated in the KiNG hypothesis.

nNOSOV/MePO activation triggers surge-like LH release

According to the mathematical model we have previously proposed26, the potential cumulative activation of nNOSOV/MePO neurons, such as we have shown to occur in the presence of kisspeptin, could result in the build-up of NO to levels capable of influencing neighboring GnRH neurons and synchronizing their activity. In order to assess whether the activation of nNOSOV/MePO neurons is sufficient to induce LH release, we stereotaxically delivered a viral vector carrying a designer receptor exclusively activated by designer drugs (DREADD), AAV-hSyn-DIO-hM3D(Gq)-mCherry, into the MePO of Nos1cre (Nos1cre, B6.129-Nos1tm1(cre)Mgmj/J) female and male mice, allowing the selective expression of the activating DREADD hM3D(Gq) in nNOSOV/MePO neurons (Suppl. Fig. 3A, F). This approach provided an average efficiency of 77.8 ± 0.1% (n=4) of nNOSOV/MePO neurons targeted with the activating DREADD, as indicated by RFP fluorescence (Fig. 3B). Tail blood was collected every 10 min for 180 min during diestrus in females, when nNOSOV/MePO activity is minimum. During the test, mice received an intraperitoneal injection of saline 5 min before the assessment of pulsatile LH release (min -5) and a CNO injection (1 mg/kg) to activate the DREADD at minute 65 or 105 (Suppl. Fig. 3B, G). As previously reported for intact female mice during diestrus, basal LH (0.25 ± 0.08 ng/ml) and the area under the curve (AUC) (25.0 ± 3.3 ng/ml), reflecting total LH output during the blood sampling protocol, were low, with mice displaying one pulse of LH over the first 60 min of blood sampling (Suppl. Fig. 3B). Interestingly, the selective activation of nNOSOV/MePO neurons upon CNO administration led to a significant increase in both basal LH (0.55 ± 0.1 ng/ml), and AUC (50.5 ± 6.4 ng/ml) levels (Suppl. Fig. 3C–E). Male mice displayed a comparable shift in LH secretory dynamics following nNOSOV/MePO neuron activation, with a clear transition from low-amplitude pulsatility to a prolonged elevation in circulating LH (Suppl. Fig. 3H–J), indicating that stimulation of nNOSOV/MePO neurons is sufficient to promote surge-like LH release in both sexes.

To determine whether this rise in LH secretion was mediated by NO, we repeated the same experimental paradigm in a separate group of female diestrous mice pre-treated with saline or the NOS inhibitor L-NAME (50 mg/kg), administered 30 min prior to tail bleeding (Fig. 3A). As expected, CNO administration elicited a marked increase in mean LH and AUC (Fig. 3C–G), with LH concentration continuing to rise up to 100 min post CNO injection (minute 180: 5.1 ± 1.5 ng/ml) (Fig. 3C). Strikingly, inhibition of endogenous NO synthesis significantly blunted this response (minute 180: 1.9 ± 0.2 ng/ml) (Fig. 3D–G), indicating that NO production by nNOSOV/MePO neurons is both necessary and sufficient to trigger an LH surge-like release.

Kisspeptin regulation of GnRH activity involves neuronal NO

NO/cGMP is a known regulator of the GnRH neuron excitability27, and our results identify nNOSOV/MePO neurons as a source of endogenous NO-induced cGMP production in the vicinity of GnRH neurons. To evaluate the functional impact of manipulating the endogenous NO/cGMP pathway we performed electrophysiological recordings of GnRH neurons in the hypothalamus of female mice at diestrus and male mice following the pharmacological or genetic manipulation of the nNOS/NO signaling cascade, using a Nos1-deficient transgenic mouse line (Nos1−/−). In agreement with our previous report showing elevated GnRH neuron firing in Nos1−/− mice during minipuberty (the early postnatal activation of the HPG axis)16, spontaneous firing in GnRH neurons was also markedly increased in adult female Gnrh::Gfp; Nos1−/− mice at diestrus compared to their Gnrh::Gfp; Nos1+/+ littermates (Suppl. Fig. 4A–C). This increased and sustained GnRH neuron firing was associated with a significantly reduced AUC and mean LH levels in both female and male Nos1−/− mice (Suppl. Fig. 5), in agreement with the hypogonadotropic hypogonadal phenotype associated with nNOS deficiency16,28.

To further verify the hypothesis that local kisspeptin-nNOS interactions contribute to the regulation of GnRH neuron activity we examined kp-10 evoked GnRH neuron activity under our recording conditions. Bath or local application of kp-10 (10 nM) was able to stimulate GnRH neuron firing in hypothalamic slices from both Gnrh::Gfp; Nos1+/+ and Gnrh::Gfp; Nos1−/− female diestrous and male mice (Fig. 4A, B, D, E and Suppl. Fig. 4D–G). Intriguingly, the ex vivo application of the NOS inhibitor L-NAME (1 mM), either before or after kp-10, resulted in an enhancement of the kp-10-induced increase in GnRH neuron firing (Fig. 4A, C and Suppl. Fig. 4D, E). Similarly, inhibition of sGC, the canonical NO receptor, with ODQ (10μM) also increased the GnRH neuron firing rate, reinforcing the idea that NO acts through the NO–sGC–cGMP cascade to control the responsiveness of GnRH neurons to kisspeptin (Fig. 4A, C and Suppl. Fig. 4F, G). Conversely, the ex vivo bath application of an NO donor (DEA/NO; 100μM) abolished this increase in the kp-10-induced response of GnRH neurons in Gnrh::Gfp; Nos1−/− mice, reducing the firing rate to that seen in Gnrh::Gfp; Nos1+/+ mice upon kp-10 application (Fig. 4B, E). Similar results were obtained when impeding the degradation of endogenous cGMP through the administration of sildenafil, a selective PDE5 inhibitor, in Gnrh::Gfp; Nos1−/− mice (Fig. 4B, E), suggesting that cGMP accumulation can also regulate GnRH neuronal activity. Together, our results support a role for NO/cGMP signaling as a negative regulator of GnRH neuronal activity, fine-tuning the excitatory effects of kisspeptin via local NO production by nNOSOV/MePO neurons.

nNOSOV/MePO neurons modulate kisspeptin-driven LH release

Since, according to our results, kisspeptin promotes NO/cGMP production that in turn is required for the precise regulation of kisspeptin-induced GnRH neuronal activity, we focused our investigations on whether nNOSOV/MePO neuronal activity could itself be involved in the fine-tuning of kisspeptin-mediated LH release. To this end, we first injected the viral tool AAV-hSyn-DIO-hM4D(Gi)-mCherry carrying an inhibitory DREADD hM4(Gi)29 into the MePO of Nos1cre male mice to selectively target nNOSOV/MePO neurons. DREADD activation by CNO administration had no effect on the LH secretion pattern in either female or male mice, consistent with the very low basal activity of nNOSOV/MePO neurons under these conditions (Suppl. Fig. 6). Kp-10 was then intraperitoneally administered before and after CNO administration (Fig. 5A). As expected, the application of kp-10 (1 nmol) during basal nNOSOV/MePO neuronal activity (i.e., before CNO) elicited a rapid increase in LH release (peak LH = 2.8 ± 0.1 ng/ml) (Fig. 5B–G). Intriguingly, the inhibition of the nNOSOV/MePO population by CNO resulted in an enhanced kp-10-induced LH response (peak LH = 4.6 ± 0.5 ng/ml) (Fig. 3B–G), indicating that nNOSOV/MePO neurons provide inhibitory feedback to GnRH neurons, triggered specifically by kisspeptin activation. In agreement with this, the AUC (Fig. 5C), mean (Fig. 5D) and basal LH levels (Fig. 5E) were all increased in the presence of kisspeptin following CNO administration.

To specifically assess the role of kisspeptin signaling in nNOSOV/MePO neurons, we generated and injected an AAV expressing a Cre-dependent shRNA targeting Kiss1r into the OV/MePO of Nos1cre mice (Fig. 5H, I). This strategy resulted in a ~60% reduction in Kiss1r expression specifically in nNOS neurons (Fig. 5J; Suppl. Fig. 7). The overall signal for Kiss1r in the OV/MePO trended towards a decrease, consistent with its selective loss from about half of the nNOS neurons, since ~40% of them normally express the receptor in Di/mE females and males, and with the relatively small percentage of Kiss1r-expressing cells that are also Nos1 positive in this nucleus (Fig. 5K; Suppl. Fig. 7). Remarkably, the selective knockdown of Kiss1r reproduced the effect of nNOSOV/MePO neuron inhibition, as peripheral kisspeptin administration elicited an exaggerated LH surge (Fig. 5L, M), indicating that Kiss1r-dependent signaling in these neurons is required to restrain kisspeptin-induced LH release. These results, together with our electrophysiological data, strengthen the importance of nNOSOV/MePO neuron activity and their responsiveness to kisspeptin in the fine-tuning of GnRH neuronal firing and subsequent LH release.

Kisspeptin drives LH release via dual activation-inhibition by NO

In order to further explore the physiological significance of kisspeptin-NO interactions for the GnRH secretion profile, we assessed whether in vivo pharmacological manipulation of NO/cGMP signaling could modulate the kisspeptin-induced LH response. To this end, intact wildtype (WT) male mice were intraperitoneally administered saline, the NOS inhibitor L-NAME (50 mg/kg) or sildenafil (15 mg/kg), 30 min before blood sampling, and were then subjected to two kp-10 (1 nmol) administrations at an interval of one hour (Fig. 6A). Treatments did not modify basal LH levels prior to kp-10 administration (Fig. 6B). As expected, kp-10 administration induced a rapid, sharp increase in LH secretion in saline-treated animals. The administration of L-NAME did not impact the ability of kp-10 to efficiently stimulate GnRH/LH secretion, since, within 5 min of kp-10 administration, both groups had identical LH levels (Fig. 6F). However, in the absence of NO, the LH profile during the resolution phase was significantly altered. In agreement with what was seen upon pharmacogenetic inhibition of nNOSOV/MePO neurons, the administration of L-NAME induced an overshooting of the kisspeptin-induced LH response, which continued to rise even when LH values in saline-treated animals were rapidly decreasing (Fig. 6E), supporting the view that kisspeptin-induced NO release is necessary to end kisspeptin-induced GnRH/LH secretion. Interestingly, the administration of sildenafil, which prolongs the activity of the NO/cGMP cascade, led to a faster clearing of the kisspeptin effect, with LH levels eventually dropping below those in saline-treated animals (Fig. 6G). A second administration of kp-10, at the same dose as the initial one, 60 min later, resulted in a potentiated LH response to kisspeptin in animals treated with saline (Fig. 6F). Animals treated with L-NAME also displayed an enhanced LH response to the second kp-10 administration, similar to what was seen during the first kp-10 administration (Fig. 6E, F). Consistently, L-NAME treatment increased mean LH levels and AUC over the 180-min period (Fig. 6C, D). Intriguingly, sildenafil treatment, by amplifying the effects of endogenous NO through the inhibition of cGMP degradation, prevented the potentiation of LH release during the second administration of kp-10 compared to the first one, leading to an attenuated peak value of LH levels in comparison to the saline-treated animals (Fig. 6E, F). Of note, sildenafil treatment resulted in a faster decline in LH levels, with lower nadir levels at the end of the experiment (Fig. 6G). Similar experiments performed in female mice revealed the same response pattern, with L-NAME enhancing the kisspeptin-induced LH response, and sildenafil producing a mild constraining trend, with no measurable change, likely reflecting cycle-dependent saturation of cGMP signaling (Suppl. Fig. 8). Overall, our data support a role for NO/cGMP signaling in both amplification and resolution phases of the kisspeptin-induced LH response.

Methods
Animals
Experiments were performed on adult (3-4 months old) male and female C57BL/6J mice (Charles River Laboratories), Nos1cre mice (B6.129-Nos1tm1(cre)Mgmj/J), Nos1-deficient (B6.129S4-Nos1tm1Plh/J) mice and Gnrh::Gfp mice (a generous gift of Dr. Daniel J. Spergel, Section of Endocrinology, Department of Medicine, University of Chicago, IL). Nos1−/−; Gnrh::Gfp mice were generated in our animal facility by crossing Nos1−/+ mice with Gnrh::Gfp mice. All mice were housed under specific pathogen-free conditions in a temperature-controlled room (21-22°C) with a 12 h light/dark cycle and an average humidity of 42%. Mice had ad libitum access to food and water. Animal studies were approved by the Institutional Ethics Committees for the Care and Use of Experimental Animals of the Universities of Lille and the University of Geneva; all experiments were performed in accordance with the guidelines for animal use specified by the European Union Council Directive of September 22, 2010 (2010/63/EU) and were approved by the French Department of Research (APAFIS#2617-2015110517317420v5; APAFIS#29172-2020121811279767v5) and the State of Geneva (License number 369).

Pharmacological agents
Synthetic kisspeptin-10 [rodent metastin (45–54) amide; YNWNSFGLRY-NH2] was purchased from GeneCust and administered at 1 nmol per animal in vivo, or at 10 nM ex vivo. The NOS inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, Sigma #N5751; 50 mg/kg in vivo, 1 mM ex vivo), the cGMP-specific PDE5 inhibitor Sildenafil citrate (Sildenafil, Sigma-Aldrich #SML3033; 15 mg/kg in vivo, 30 μM ex vivo), and the Dimethyl Sulfoxide (DMSO, Millipore #317275) were purchased from Merck Life Science (Milano, IT). The selective inhibitor of NO-sensitive guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, #0880; 10 μM) and Clozapine N-oxide dihydrochloride (CNO, # 6329; 1 mg/kg) were purchased from Tocris Bioscience (Bristol, UK). The NO donors (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate] PAPA/NONOate, # ALX-430-016; 5 μM) and the DEA/NO, 2-(N,N-diethylamino)-diazenolate-2-oxide.diethyl-ammonium salt (DEA NONOate, # ALX-430-034; 100 mM) was from Enzo Life Sciences (Exeter, UK).

Stereotaxic injections
The AAV9 hM4D DREADD (AAV-hSyn-DIO-hM4D(Gi)-mCherry) (Addgene; 44362-AAV9), the AAV9 hM3D DREADD (AAV-hSyn-DIO-hM3D(Gq)-mCherry) (Addgene; 44361-AAV9) and the AAV1/2 Kiss1r shRNA or Scrambled shRNA (AAV-CMV-DIO-shKiss1r(3x)-EGFP, # P210901-1002gjb; AAV-FLEX-shScramble-EGFP, # VB220803-1150vgq, VectorBuilder) were stereotaxically injected into the OV/MEPO of Nos1cre female and male mice after dilution in PBS to reach a titer of 4.5 × 1012 vg/mL. The AAV9 FlincG3 (AAV-CMV-FlincG3) (titer = 10 × 1012 vg/mL; Genecust) was stereotaxically injected into the OV/MEPO of wild-type female mice. Animals were kept under general anesthesia (induction 4% in air 2 L/min, then 1.5% in air 0.3 L/min), after local injection of lidocaine (30 microliters of a 0.5% solution, s.c.) and pre-emptive Meloxicam treatment (5 mg/kg). The needle of a 2 μL Hamilton Neuros syringe (Hamilton; catalog #65459-01) was placed into the target region. After 5-min, 300 nL of the solution containing viral particles was delivered at a rate of 80 nL per minute. 10 minutes after the completion of the delivery, the needle was slowly removed, and the skin was sutured. To target the OV/MEPO, the following coordinates were used: anterior-posterior (A-P) = + 1.3 mm; medial-lateral (M-L) = 0 mm; dorsal-ventral (D-V) = -4.8 mm.

Mice were allowed to recover for three weeks after brain surgery to ensure sufficient viral transduction. Following the in vivo experiments, all animals were sacrificed and the brains were processed for immunohistochemistry to verify the efficiency and specificity of viral expression. Animals in which the infection covered less than one-third of the OV/MePO were excluded from the analysis. The average transduction efficiency and off-target infection rate were determined by counting nNOS-labeled neurons, RFP (mCherry) /GFP-positive neurons, and double-labeled cells within a manually defined subregion of the OV and MePO for each animal. To validate the downregulation of the kisspeptin receptor, an independent cohort of mice was injected with AAV Kiss1r shRNA or scrambled shRNA, and brains were processed for fluorescence in situ hybridization as described below.

Examination of physiology
Adult female mice (3–6 months of age) were examined daily for two consecutive estrous cycles to confirm regular cycling before use in experiments. Vaginal smears were collected and analyzed under a light microscope to determine the stage of the estrous cycle, and all experimental procedures were performed on the indicated day of the cycle.

Assessment of LH secretion dynamics and LH assay
In this study, LH release was used as a physiological readout of hypothalamic GnRH output, consistent with previous work demonstrating that pituitary LH secretion faithfully reflects changes in GnRH neuronal activity and release dynamics. For the pulsatility test, mice were habituated to handling and tail-tip blood sampling for at least 10 days prior to the experiment. When a viral vector was administered stereotaxically, animals were allowed to recover for two weeks before the start of habituation. Blood samples for measuring circulating LH concentrations were collected in the morning and, for females, only during the diestrus stage. Mice were gently restrained, and tail-tip blood was sampled at 5- or 10-minute intervals, as indicated in the figure legend. For each time point, 4 μL of whole blood were collected into Eppendorf tubes preloaded with 56 μL of 0.01 M PBS containing 0.05% Tween-20 and 0.2% BSA, thoroughly mixed, and kept on ice until storage at –80°C. LH concentration was measured using a highly sensitive Enzyme-linked Immunosorbent Assay (ELISA) recently described in Kreisman et al.45. Briefly, we used a 96-well high-affinity binding microplate (Corning) coated with 50 μL of primary capture antibody (bovine LHβ subunit, 518B7; L. Sibley; University of California, UC Davis) diluted at 1 µg/ml in 0.01 M PBS. The mouse LH-RP was provided by Dr. Albert F. Parlow (National Hormone and Pituitary Program, Torrance, California, USA) and used to generate a standard curve with a two fold serial dilution. The detection primary antibody (Mouse Monoclonal LH antibody, cat: Medix, 5303 SPRN SPRN-5) was biotinylated using the EZ-Link NHS-PEG4 Biotinylation Kit (Thermo Scientific, catalog No. PI21455) and used at a concentration of 1 µg/ml, diluted in 0.01 M PBS-0.05% Tween, 5% skim milk powder. For the final step, the poly-HRP Streptavidin (Thermo Fisher, Cat# N200) was diluted 1:8000 in 0.01 M PBS-0.05% Tween, then revealed with 100 μL of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific, cat. # 34028). The colorimetric reaction was stopped with stop solution of 50 μL of 3 M HCl. Optical density (OD) was read at 490 nm and 650 nm for background subtraction.

Fluorescence in situ hybridization
FISH was performed on frozen brain sections of 3-month-old female mice with the RNAscope Multiplex Fluorescent Kit v2 (323100, Advanced Cell Diagnostics). Estral cyclicity was assessed during 2 weeks by daily vaginal smears. Animals were anesthetized with isoflurane and then sacrificed by decapitation in the late afternoon of the first day of diestrus or proestrus. Brains were dissected and included in OCT medium (Tissue-Tek), frozen on dry ice, and stored at –80°C until sectioning, while the edematous swelling of the uterus was visually checked to confirm the estrous stage. Tissues were cryosectioned (Leica cryostat) coronally at 16 μm on Superfrost Plus slides and stored at -80°C. Sections were processed according to the manufacturer’s protocol, using specific probes to detect Nos1 (437651-C2, NM_008712.2, target region 2-1097), Kiss1r (408001, NM_053244.5, target region 21–1599), Kiss1 (476291, XM_006529679.2, target region 121-1376) and Gnrh1 (476281-C3, XM_006518564.3, target region 81-914) mRNAs. Hybridization with a probe against the Bacillus subtilis dihydrodipicolinate reductase (DapB) gene (320878) was used as a negative control.

RNAscope preparations were analyzed on the LSM 710 Zeiss confocal microscope. Excitation wavelengths of 405/443 nm, 488/518 nm, 561/699, and 633/699 were selected to image the different channels. All images were taken with the objective EC Plan-Neofluar M27 (thread type) using the 40X oil objective with a numerical aperture of 0.50, and a zoom of 1.4. Z-stack images were taken every 1 μm, for a total of 10 μm per section. All the images are presented as maximal intensity projections (MIP) of three-dimensional volumes along the optical axis. Images to be used for figures were pseudocolored and adjusted for brightness and contrast using Photoshop (Adobe Systems, San Jose, CA).

Cell counting. The nomenclature of brain regions used in this article and the definition of the region of interest was based on the ones described in the Allen brain atlas46. The cell counts were undertaken unilaterally on maximal intensity projections (MIP) images. For each mouse, the number of single-labeled Nos1 positive, Kiss1 positive and Kiss1r-positive neurons, and double-labeled Nos1/Kiss1r and Nos1/Kiss1 neurons in the region of the OV/MePO (represented by plate 50 of the Allen Mouse Brain Atlas) and in the region of the AVPV (plate 53) were performed manually. To count DAPI nuclear staining, accounting for the total number of cells in the area, the particles were automatically counted on the images after binarization and segmentation using ImageJ. The representation of the percentage of Nos1, Kiss1 and Kiss1r mRNA positive cells in the region was obtained by normalizing over the DAPI nuclear staining in the areas of interest.

Immunohistochemistry
Tissue preparation. Mice were anesthetized with a lethal dose of pentobarbital (DOLÉTHAL®, 5 mg/kg) and locally treated with Lidocaine (30 μl of a 0.5% solution). Animals were perfused thoroughly with cold saline, until exsanguination, then perfused with cold fixative solution [4% paraformaldehyde (PFA), in 0.1 M PB, pH 7.4]. The brain was extracted and post-fixed with the same fixative solution for 2 h at 4°C, then cryoprotected in PB 0.1 M 20% sucrose for 24 h at 4°C. Afterwards, the brain was embedded in OCT medium (Tissue-Tek), frozen on dry ice, and stored at –80°C until sectioning. Tissues were cryosectioned (Leica cryostat) coronally at 35 μm (free-floating sections).

Antibodies. The goat polyclonal anti-nNOS antibody (1:1000; Cat# OSN00004G, Lot# VF3001511) and the rabbit polyclonal anti-Ser1412 phospho-nNOS antibody (1:500; Cat# PA1-032, Lot# XE350390) were purchased from Thermo Scientific. The rabbit polyclonal anti-RFP was purchased from Rockland (1:1000; Cat# 600-401-379). The rat polyclonal anti-GnRH (1:2500; Cat# EH1044) was a generous gift from Professor Hrabovszky (Laboratory of Reproductive Neurobiology, Institute of Experimental Medicine, Budapest, Hungary). The chicken polyclonal anti-GFP (1:1000; Cat# GFP-1020) was purchased from Aves Lab. The secondary antibodies were all purchased from Invitrogen and used at a final concentration of 1:500. Alexa Fluor 647-conjugated donkey anti-goat (1:500; Cat# A21447) was used for nNOS, and 568-conjugated donkey anti-rat (1:500; Cat# A78946) for GnRH immunolabeling. Alexa Fluor 568-conjugated donkey anti-rabbit (1:500; Cat# A10042) was used to detect phospho-nNOS and RFP (mCherry) expression, and the Alexa Fluor 488-conjugated donkey anti-chicken (1:500) was used to amplify the GFP expression of the Kiss1r shRNA/Scrambled vector.

Immunolabeling. Sections were washed 3 times for 5 min each in PB 0.1 M and then incubated in blocking solution (5% NDS, 0.3% Triton X-100 in PB 0.1 M) for 1 hour at room temperature. Sections were incubated for 48–72 h at 4°C with the primary antibodies diluted in blocking solution, then rinsed 3 times for 5 min each in PB 0.1 M. The sections were incubated with secondary antibodies diluted in PB 0.1 M for 1 h at room temperature, then rinsed in PB 0.1 M 3 times for 5 min each, and finally incubated 7 min with DAPI (1:5000 in PB 0.1 M) for nuclear staining. After washing, sections were mounted on glass slides and coverslipped under Mowiol medium.

Digital image acquisition. Images were acquired using a Leica Stellaris 5 confocal microscope. Excitation wavelengths of 405, 488, 561, and 633 nm were selected to image the different channels. The images were acquired with a 20x/0.8 objective (numerical aperture NA = 0.80) with a zoom of 1.0. Z-stack images were taken every 1 μm, for a total of 20 μm per section. All images are presented as maximal intensity projections (MIP) of three-dimensional volumes along the optical axis. Images to be used for figures were adjusted for brightness and contrast using Photoshop (Adobe Systems, San Jose, CA).

Cell counting. The quantification of nNOS neurons co-expressing P-nNOS was undertaken by counting the number of single-labeled nNOS-positive neurons and double-labeled nNOS/P-nNOS in the OV, represented by plate 50 of the Allen Mouse Brain Atlas. Cell counts were carried out unilaterally, represented as the percentage of nNOS neurons positive for P-nNOS immunoreactivity, and the values from each mouse were averaged to determine mean counts and SEM for each group. FlincG3 fluorescence was quantified by analyzing the integrated density. Using ImageJ analysis software, a region of interest of identical size was defined on every image in the region of the OV/MEPO, represented by plate 50 of the Allen Mouse Brain Atlas. The defined region was binarized, and the integrated intensity was then calculated for each image, which reflects the total number of pixels in the skeletonized image. In the same region of interest, the single-labeled nNOS-positive neurons and the double-labeled nNOS/FlincG3 neurons were also manually quantified. All values are presented as the averaged values from each mouse to determine mean counts and SEM for each age group.

Live FlincG3 imaging
Brain slice preparation. FlincG3 recordings were performed on acute brain slices from 8–12-week-old male mice. Animals were anesthetized with isoflurane and decapitated, and the brains were rapidly removed and placed in ice-cold artificial cerebrospinal fluid (aCSF) containing (in mM): NaCl 120, KCl 2, NaHCO₃ 26, KH₂PO₄ 1.18, MgSO₄ 1.19, glucose 11, and CaCl₂ 2, continuously bubbled with 95% O₂/5% CO₂. Coronal slices (200 μm) encompassing the preoptic area were cut using a VT1200 vibratome (Leica). Slices were transferred to a recovery chamber containing oxygenated aCSF and maintained at 34°C for 1 h, then allowed to cool to room temperature (≈22°C) before imaging.

Live recording. Slices were placed in a 500 μL recording chamber mounted on an inverted microscope (Zeiss Axiovert 135 TV, Carl Zeiss, UK) and superfused continuously (1.5 mL min⁻¹) with pre-warmed (37°C) recording solution containing (in mM): NaCl 140, KCl 2, KH₂PO₄ 1.18, glucose 5.5, HEPES 10, and CaCl₂ 1.5 (pH 7.4; 285–290 mOsm kg⁻¹). The solution was equilibrated with pure O₂ and supplemented with superoxide dismutase (100 U mL⁻¹). Imaging was performed using a Zeiss AxioObserver Z1 equipped with an Orca LT camera (Hamamatsu) and a 20× air objective (NA 0.8, Zeiss), under control of Zen Imaging Software (Zeiss). FlincG3 fluorescence was excited at 495 nm and collected at 519 nm. Exposure time was 300 ms, and illumination intensity was set to 30%. All pharmacological compounds were administered by superfusion.

Fluorescence data analysis. Fluorescence measurements were background-corrected and displayed as the change in intensity relative to the baseline divided by the baseline intensity (ΔF/F0) in Image-Pro Premier (version 9.1, Media Cybernetics). As appropriate, traces were corrected for NO-independent, post-NMDA undershoots and baseline drift47 in Origin (version 10.1, OriginLab Corp., Northampton, MA). Results are given as means ± S.E. Each experiment was performed on a separate brain slice and repeated at least twice to ensure reproducibility; n values refer to the number of responders.

Electrophysiology
Brain slice preparation. Electrophysiological recordings were performed on acute brain slices obtained from 8–12-week-old female and male mice. Animals were anesthetized with isoflurane and decapitated, and the brains were rapidly removed and placed in ice-cold artificial cerebrospinal fluid (aCSF). Coronal slices (200–250 μm) containing the preoptic area were cut using a VT1200 vibratome (Leica). For Protocol 1, the aCSF composition was (in mM): NaCl 120, KCl 3.2, NaH₂PO₄ 1, NaHCO₃ 26, MgCl₂ 1, CaCl₂ 2, and D-glucose 10 (300 mOsm, pH 7.4). For Protocol 2, the aCSF contained (in mM): NaCl 124, KCl 3, CaCl₂ 2, MgSO₄ 1.3, NaHCO₃ 26, NaH₂PO₄ 1.25, and D-glucose 10 (300 mOsm, pH 7.4). All solutions were continuously bubbled with 95% O₂/5% CO₂ for at least 1 h before use. Slices were incubated in oxygenated aCSF at 34°C (Protocol 1) or 37°C (Protocol 2) for 1 h to recover, then maintained at room temperature until patch-clamp recording.

Patch-clamp recording. Individual coronal brain slices containing the preoptic area were transferred to a submerged recording chamber (Warner Instruments) and continuously perfused with oxygenated artificial cerebrospinal fluid (aCSF; 95% O₂/5% CO₂) at a rate of 2–3 mL/min. The bath temperature was maintained at 32.8°C using a TC-344C temperature controller (Warner Instruments). GnRH neurons were visualized under 10× and 40× objectives using an upright fluorescence microscope equipped with infrared differential interference contrast optics (DM-LFSA, Leica) and an ORCA-Flash 4.0 digital CMOS camera (Hamamatsu). Additional recordings were obtained with an LN scope (Luigs & Neumann) equipped with a BX51 fluorescence tower (Olympus) and an ORCA-Fusion camera (Hamamatsu). Recording pipettes (resistance 6–8 MΩ) were pulled from borosilicate glass capillaries (1.5 mm outer diameter, 1.12 mm inner diameter; World Precision Instruments) using a P-1000 puller (Sutter Instruments). Only cells showing <20% change in access resistance throughout the recording were included; junction potentials were corrected via pipette offset. Neurons were considered responsive if their firing frequency changed by >20% during drug application.

[Protocol 1] The following compounds were tested: kisspeptin-10 (10 nM), L-NAME (1 mM), ODQ (10 μM), and DEA/NO (100 μM). Recordings were performed at 32.8°C. The pipette solution contained (in mM): K-gluconate 140, KCl 10, EGTA 1, Na₂-ATP 2, and HEPES 10 (pH 7.3 with KOH; 290 mOsm). Following stable baseline activity, drugs were bath-applied or locally delivered using a Valve Control System (Warner Instruments). Whole-cell recordings were performed in current-clamp mode using a Multiclamp 700B amplifier (Molecular Devices). Signals were digitized with a Digidata 1550B interface and acquired using pClamp 11.4 software (Molecular Devices). Data were analyzed offline with Clampfit 11.4 (Molecular Devices) to determine mean firing rate and membrane potential before, during, and after drug application.

[Protocol 2] The following compounds were tested: kisspeptin-10 (10 nM), ODQ (10 μM), and sildenafil (30 μM). Recordings were performed at 37°C using a temperature-controlled Luigs & Neumann chamber. The pipette solution contained (in mM): K-gluconate 120, KCl 10, HEPES 10, ATP 4, GTP 0.3, phosphocreatine 10, and EGTA 1 (pH 7.2–7.3; 280–290 mOsm). Following stable baseline activity, drugs were bath-applied. Whole-cell recordings were performed in current-clamp mode using a Multiclamp 700B amplifier (Molecular Devices). Signals were digitized with the PulseQ acquisition system (Luigs & Neumann) running in Igor Pro (Wavemetrics) and sampled at 50 kHz, low-pass filtered at 4 kHz. Data were analyzed in MATLAB to extract firing frequency and membrane potential under each condition. Both male and female mice were used. Because the kisspeptin-10–induced change in firing rate did not differ between sexes (two-way repeated-measures ANOVA; Treatment × Sex interaction WT: F(1,15) = 1.35, p=0.26; KO: F(1,16) = 1.98, p=0.18), data from males and females are shown together in the summary graphs. However, statistical analyses were performed within each experimental subgroup, as some pharmacological conditions were applied in only one sex.

Statistical analyses
All analyses were performed using Prism 10 (Graphpad Software, San Diego, CA) and assessed for normality (Shapiro–Wilk test) and variance, when appropriate. Sample sizes were chosen according to standard practice in the field. Experimenters were blinded to group allocation during data collection and analysis. Details on biological replicates and sample sizes are provided in the figure legends. For normally distributed data, comparisons between two groups were made using a two-tailed unpaired t-test, or a paired t-test when appropriate. Comparisons among more than two groups were made using a one-way or two-way ANOVA, followed by Tukey’s or Holm-Sidak’s multiple-comparison post hoc test. Repeated-measures including missing values were analysed with a Mixed-effects model followed by Dunnett’s test. For data not following a normal distribution, comparisons between two independent groups were made using the Mann–Whitney test. Comparisons among more than two groups were made using the Kruskal–Wallis test, followed by Dunn’s post hoc test for multiple comparisons. All data are presented as mean ± s.e.m. Statistical significance was defined as P < 0.05. The significance levels shown in the figures are: ns, P > 0.05; P < 0.05 (*); P < 0.01 (**); P < 0.001 (***). Details on animal age, sex, and number of independent experiments are reported in the main text or figure legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript by Delli et al, they employ electrophysiological, genetic, chemogenetic and pharmacological approaches to investigate a microcircuit involving kisspeptin, nitric oxide synthase and GnRH in the regulation of the GnRH and LH pulse and surge.\nUsing a series of elaborate experiments, they demonstrate that nitric oxide production from nitric oxide synthase neurons in the hypothalamic organum vasculosum laminae terminalis and the median preoptic nucleus is needed to fine-tune the GnRH/LH response to kisspeptin, to turn off GnRH release, thereby generating pulses.\nThe experiments are extremely robust and very well-described. The manuscript is clearly written and the data representation clear. The data are highly novel.\nMy comments are minor:\n1. All the experiments involving kisspeptin administration use kisspeptin-10. Could the authors explain this choice over kisspeptin-54? Based on what is known about kisspeptin biology, would they expect different results with kisspeptin-54?\n2. Figure 4: the half-life of kisspeptin-10 is very short (~4 minutes). Could the authors explain why they selected an interval of 60 minutes between the two doses?\n3. Their data are very convincing from a physiological perspective. However, could the authors elaborate more about the therapeutic implications. Whilst there is no doubt that kisspeptin-based medicines have significant potential to treat reproductive disorders, for what conditions might manipulating NO be useful?\nVery minor comments:\n1. Figures: for consistency, could the vertical bars indicating the intervention administered be in the same colour, e.g., grey for kisspeptin throughout all the figures?\n2. For consistency, please use ‘kisspeptin-10’ throughout as in some places, the hyphen is missing.\n3. Two reference styles are used in the manuscript, e.g., in the second paragraph of the introduction. For simplicity, please stick to one.\n4. In the results, when referring to a drug administration, please include the dose/concentration.", "reviewer_2": "The study by Delli et al illustrates that NO production within the OV/MePO is contributing to fine tuning the GnRH/LH response to kisspeptin and is able to turn off GnRH release but acting as an inhibitory signal. The authors apply a range of sophisticated tools to substantiate their findings and data interpretation. Their data provides news insights into the tripartite ‘KiNG’ neuronal network illustrating new mechanisms for regulation of the GnRH/LH pulse and surge release.\nAddressing the following point would reinforce the arguments that cGMP signalling is involved in modulating neuronal and thus GnRH function. The paper postulates that the NO-cGMP axis is responsible for regulating the hormones. However, assessing direct effects on neuronal activity shows clearly that NO is altering firing. Would the authors be able to show that the block of guanylyl cyclase by ODQ or inducing cGMP signalling by 8-bromo cGMP application or indeed activation of the cyclase by BAY compounds is able to mimic the effects of direct NO modulation (L-NAME, DEA). In other words, is the effect a direct cGMP-mediate change on neuronal firing or related to other NO signalling routes?\nFigure 2 F shows increases in FlincG fluorescence in response to PAPA. It seems that the time course of onset of PAPA or kp10 induced changes is a bout 2.5mins. The trace in F is cut off at about 30min, at which time one would otherwise expect to see an increase. Could this time course be extended past 40min?\nSome minor issues:\nLine 24: ….orchestrates GnRH…delete ‘s’\nLines 52, 79: formate references\nLine 275: …sildenafil treatment, by promoting additional NO production…’ rephrase, as sildenafil does not change NO production.\nLine 812: correct ‘FlicG3’\nLine 861: correct ‘1nMol’ into 1nM", "reviewer_3": "The manuscript by Delli et al. examined the role of neuronal nitric oxide (NO) synthase (nNOS) neurons in regulation of the reproductive system. The authors show that Nos1-expressing neurons express kisspeptin receptor (Kiss1r) and kisspeptin treatment induces nNOS activation and production of cGMP in the organum vasculosum of the lamina terminalis (OV) and medial preoptic area (MePO). Using a chemogenetic approach targeted to Nos-1 expressing cells in OV/MePO, the authors show that activation of Nos1-expressing cells increase LH release, while inhibition of Nos-1 expressing cells potentiates the effects of kisspeptin treatment on LH release. Modulation of NO production leads to a change in kisspeptin-induced GnRH neuron firing activity in brain slices and LH release in vivo. The findings suggest that NO could play a role in modulation of GnRH neuron activity and LH release.\nThe findings are interesting and explore a new aspect of regulation of GnRH neurons and reproductive functions. However, some results from this study do not support the proposed model. In addition, the experiments do not fully prove the direct action of the kisspeptin on nNOSOV/MePO neurons and the regulation of GnRH neurons and LH release.\nMajor comments:\n1. This study proposes a model by which kisspeptin neurons activate a brake by promoting NO release and thus suppressing GnRH neurons and LH release. However, the result from Nos1Cre + hM3DGq experiment (Fig 1I-N) contradicts the proposed model. Activation of Nos1 cells lead to a significant increase in LH secretion, suggesting a stimulatory role of nNOSOV/MePO neurons. Results from an inhibitory DREADD experiment, electrophysiological recordings, and a pharmacological experiment suggest an inhibitory role of NO on GnRH neurons and LH release. The authors do not address the discrepancy in the effects of NO in the discussion and therefore weaken the proposed model.\n2. In line with the above, the model proposed by the authors suggests that kisspeptin acts on Kiss1r on nNOSOV/MePO neurons. However, without an experiment showing that kisspeptin acts directly at nNOSOV/MePO neurons, the effects observed in this study could be indirect via intermediate neurons. Moreover, kp-10 was delivered via ip injection in the majority of experiments. Therefore, it is possible that the effects of kp-10 observed in this study is not directly at nNOSOV/MePO neurons. To confirm that kisspeptin acts directly at nNOSOV/MePO neurons via Kiss1r to modulate GnRH neurons, an experiment in which Kiss1r is knocked out specifically in nNOSOV/MePO neurons is needed.\n3. The authors state in Line 167-168 that this work “demonstrate for the first time that the phosphorylation of nNOS protein at Ser1412 is coupled with cGMP production in vivo”. This is a proposition based on the results from two separate experiments showing that kisspeptin treatment increase phosphorylation at Ser1412 (Fig 1F-G) and kisspeptin increases cGMP production (Fig 2). A single experiment showing increased P-nNOS and FlincG3 (for example, staining in the brains from the same experiment) would better support the statement.\nIn addition, FlincG3 fluorescence was found in GnRH neurons, but the authors did not quantify the percentage of GnRH neurons that show colocalization with FlincG3.\n4. The authors state that activation of nNOSOV/MePO neurons is sufficient to trigger LH surge-like release. Based on previous reports, the level of LH surge during proestrous (14.50 ng/ml) or ovariectomized animals with estradiol supplementation (7.70 ng/ml; Czieselsky et al., 2016; DOI: 10.1210/en.2016-1351) are much higher than the LH values observed in this study (peak at around 1.5 ng/ml). Therefore, it is misleading to state that the LH increase is at the level of LH surge.\n5. The authors state that “genetic nNOS deficiency results in exacerbated kp-10 induced GnRH neuron firing” (line 220-221). However, the increase in GnRH neuron firing activity caused by kp-10 is about 100% from basal firing activity in both Nos1+/+ (~0.3Hz to 2Hz) and Nos1-/- (~2Hz to ~3.8Hz). Since the basal firing of GnRH neurons is different between the mouse models, the authors should take this information into account regarding the response to kp-10 treatment.\n6. The use of pharmacological treatment (L-NAME and DEA/NO) in electrophysiological experiments is not sufficient to show that NO is involved in kisspeptin-induced activation of GnRH neurons. It could simply be the additive effects from kisspeptin and NO that are coincidentally observed within the same GnRH neurons. It is possible that the modification of NO production itself could have effects on GnRH neuron activity. An experiment where GnRH neurons are treated with just L-NAME or DEA/NO should be included to address this possibility.\n7. In Nos1Cre+hM3Dq (Fig 1I) and Nos1Cre+hM4Gi (Fig 3G) experiments, there is no control for the potential impact of CNO on LH release. It is possible that CNO (or clozapine) is responsible for the change in LH release independent of impact of NO. A control group where Nos1Cre animals are injected with a control virus (an AAV reporter virus without DREADD receptor) and treated with CNO should be include in the study.\n8. The experiment in Fig 4 was performed in male mice. However, other main experiments in the manuscript were performed in female mice. The authors did not explain the rationale behind this. It is possible that the response is different between males and females. Therefore, the authors should also perform the experiment in Fig 4 in female mice to be able to fairly compare the results between experiments.\n9. In the discussion, the authors make several statements without the direct evidence. For example, in line 299-300 the authors state “kisspeptin was seen to use NO to contain the firing activity of the GnRH neurons and potentially terminate its own effects”. From the electrophysiological experiment in this study, it is not sufficient to prove that kisspeptin use NO to contain firing activity of the GnRH neurons (comment #5).\n10. The authors use the terminology “GnRH/LH release” throughout the manuscript. For example, “activation of nNOS neurons of the OV/MePO is sufficient to trigger GnRH/LH surge-like release” (line 172-173). It is true that LH release reflects GnRH release in most case. However, in this study, GnRH release is not directly measured at any point and, therefore, could be misleading to readers.\nMinor comments:\n1. The authors show results on the effect of kisspeptin on Ser1412-phosphorylation of the nNOS protein in Fig 1F-H but do not mention this finding in the results section of the manuscript.\n2. The experiment in Fig 1I-N should be moved to later section of the results section to improve the flow of the manuscript.\n3. Line 125: the numbers do not match the data in the supplemental figure 1G\n4. Line 126: the numbers do not match the data in the supplemental figure 1F\n5. Line 183: “77,8” should be “77.8”\n6. The Y axis in Fig 2F-H should use “.” instead of “,”\n7. Line 290-291: The authors note that the highest expression of Kiss1r-expressing nNOS neurons is during proestrus. In this study, only mice in diestrus and proestrus were studied, but not metestrus. Therefore, in order to make such a statement the authors should include mice in metestrus."}, "author_response": "We thank the reviewer for this comment which allows to raise indeed the important point of pharmacological differences between kisspeptin-10 (kp-10) and kisspeptin-54 (kp-54). The choice to use kp-10 in our experiments was initially guided by its wide usage in animal experiments and lower cost due to its shorter amino acid sequence. However, it is important to acknowledge that kp-10 and kp-54 differ notably in their pharmacokinetics and bioactivity. As it has been demonstrated by Jayasena et al., Human Reprod 2015 (PMID: 26089302) and d’Anglemont de Tassigny et al., PLOS ONE 2017 (PMID: 28464043), Kp-54 has a substantially longer in vivo plasma half-life (~27 minutes) compared to kp-10 (~4 minutes), leading to much higher steady-state circulating levels during infusion at the same molar dose. Additionally, while both peptides bind the Kiss1 receptor with similar affinities, kp-54 is generally considered more potent in stimulating gonadotrophin release across species. This longer persistence means that in paradigms involving repeated dosing, residual kp-54 could overlap with the response to a subsequent injection, whereas kp-10 clears more rapidly and allows temporally separated stimulation.\nTo directly address this point in our model, we conducted a comparative experiment using both kp-10 and kp-54 (Fig. R1). Interestingly, we found that nNOS phosphorylation levels (P-nNOS) in the OV/MePO area increased with both peptides at similar timings, within 2 minutes post-kisspeptin administration, consistent with a strong, rapid calcium-dependent activation of nNOS. LH concentrations showed a comparable rise at 30 minutes post-injection. These findings indicate that kp-54 and kp-10 activate the same neuronal targets with similar efficacy. Considering the known pharmacokinetic differences between kp-10 and kp-54 it would be interesting to investigate the temporal dynamics of this process in more detail in a follow-up study.\nFigure R1. (A) Immunolabeling for P-nNOS (red) in the OV/MePO of female mice in diestrus upon intraperitoneal administration of kp-10 or kp-54. (B) Quantification of the ratio of P-nNOS immunoreactive cells on the total number of nNOS cells and (C) measurement of the circulating LH in mice before or 30 minutes after receiving a peripheral injection of kp-10 or kp-54.\n2. Figure 4: the half-life of kisspeptin-10 is very short (~4 minutes). Could the authors explain why they selected an interval of 60 minutes between the two doses?\nWhile kp-10 has a short plasma half-life, its downstream biological effects — including GnRH release, LH elevation, and nNOS phosphorylation — persist well beyond its circulation time. In our pulsatility experiments, we observed that LH levels remain elevated for over 60 minutes following a single kp-10 injection. This suggests that the downstream signaling cascade remains active even after kp clearance.\nTo allow sufficient time for resolution of the first response ensure that the second kp-10 injection reflected a de novo activation of the system rather than a cumulative response, we selected a 60-minute interval between injections. At the same time, this delay allows the hormonal response to return toward baseline, avoiding overlap between the two LH peaks.\n3. Their data are very convincing from a physiological perspective. However, could the authors elaborate more about the therapeutic implications. Whilst there is no doubt that kisspeptin-based medicines have significant potential to treat reproductive disorders, for what conditions might manipulating NO be useful?\nOur study highlights NO as a key downstream effector of kisspeptin signaling, and we believe that modulating NO signaling could offer novel therapeutic avenues, especially in contexts where kisspeptin responsiveness is impaired or bypassed, or where current treatments may fall short.\nFor example, in a preclinical model of polycystic ovary syndrome (PCOS), it was demonstrated that hypothalamic NO signaling, acting downstream of kisspeptin, was impaired, and that restoring NO levels pharmacologically was sufficient to rescue LH secretion, reproductive cycles, and sexual behavior (Silva et al., PNAS 2023 PMID: 35867816). This provides direct preclinical evidence that NO-targeted therapies could benefit women with PCOS, especially those who do not respond to upstream interventions like kisspeptin analogs or metabolic sensitizers.\nIn addition, recent findings from Bentefour and Bakker (Nat Commun, 2024; PMID: 38688927) show that NO can activate GnRH neurons independently of kisspeptin and that its impairment contributes to female sexual dysfunction, further supporting the potential of NO-based interventions across the female lifespan.\nOur own work (Chachlaki et al., Sci Transl Med, 2022; PMID: 36197968) shows that NO signaling is also critical during minipuberty, a key developmental window for reproductive programming. This is especially relevant for preterm infants, where disrupted early-life NO homeostasis may impact NO-dependent regulation of GnRH neurons, with long-term consequences for reproductive and mental health.\nImportantly, the feasibility of targeting NO, supported by the clinical use of sildenafil (i.e., Viagra), in both reproductive and sexual health contexts, as well as in neonatal care against pulmonary dysfunction, further highlights that modulating NO signaling offers a promising avenue to complement or extend kisspeptin-based therapies. We have inlcuded this clinical potential in our discussion, lines 469-480\nVery minor comments:\n1. Figures: for consistency, could the vertical bars indicating the intervention administered be in the same colour, e.g., grey for kisspeptin throughout all the figures?\nThe vertical bars and overall indicative colors have been updated in all figures.\n2. For consistency, please use ‘kisspeptin-10’ throughout as in some places, the hyphen is missing.\nThe hyphen has been added.\n3. Two reference styles are used in the manuscript, e.g., in the second paragraph of the introduction. For simplicity, please stick to one.\nThe reference style has been updated.\n4. In the results, when referring to a drug administration, please include the dose/concentration.\nThe doses have been updated in the text and also in the material and method section.\nReviewer #2 (Remarks to the Author):\nThe study by Delli et al illustrates that NO production within the OV/MePO is contributing to fine tuning the GnRH/LH response to kisspeptin and is able to turn off GnRH release but acting as an inhibitory signal. The authors apply a range of sophisticated tools to substantiate their findings and data interpretation. Their data provides news insights into the tripartite ‘KiNG’ neuronal network illustrating new mechanisms for regulation of the GnRH/LH pulse and surge release.\nAddressing the following point would reinforce the arguments that cGMP signalling is involved in modulating neuronal and thus GnRH function. The paper postulates that the NO-cGMP axis is responsible for regulating the hormones. However, assessing direct effects on neuronal activity shows clearly that NO is altering firing.\nWould the authors be able to show that the block of guanylyl cyclase by ODQ or inducing cGMP signalling by 8-bromo cGMP application or indeed activation of the cyclase by BAY compounds is able to mimic the effects of direct NO modulation (L-NAME, DEA). In other words, is the effect a direct cGMP-mediate change on neuronal firing or related to other NO signalling routes?\nWe thank the reviewer for this excellent mechanistic question. In fact, previous work from our group has already addressed this question in detail. In particular, Clasadonte et al. Endocrinology 2008 (PMID: 18006627) demonstrated that exogenous application of 8-pCPTcGMP, a permeable analog of the by-product of sGC, mimicked the L-Arg-mediated inhibitory effects on GnRH neuron firing, while blocking sGC with ODQ enhances GnRH neuron firing, supporting a tonic inhibitory role of the NO–cGMP pathway in adult GnRH neurons.\nBuilding on this framework and on the reviewer’s question, we have now specifically examined the effect of kp-10 in the presence or absence of ODQ, and the new data are now included in Figure 4 and Extended Figure 4. We observed that inhibition of the NO–cGMP pathway with ODQ significantly amplified the response to kp-10, mimicking the effects of NO moduation with L-NAME, indicating that cGMP-dependent NO signaling imposes a negative feedback on kisspeptin-induced neuronal activation. In contrast, inhibition of phosphodiesterase 5 through the application of sildenafil, and thus prolongation of the NO/cGMP response, results in the blunting of the kp-10 mediated increase in GnRH neuron firing, further supporting the key role of NO/cGMP signaling for the fine-tuning of the kisspeptin effect on GnRH neuron activity. These results are presented in lines 234-250 and further discussed in lines 396-414.\nFigure 2 F shows increases in FlincG fluorescence in response to PAPA. It seems that the time course of onset of PAPA or kp-10 induced changes is a bout 2.5mins. The trace in F is cut off at about 30min, at which time one would otherwise expect to see an increase. Could this time course be extended past 40min?\nIn the specific example shown in Figure 2F, the recording was unfortunately stopped around 30 minutes, and we are unable to provide additional data beyond that point for this particular trace. The reviewer is right, ideally, a longer time course would be valuable. The trace shown in Figure 2F was selected because, in the majority of our experiments, PAPA/NO was applied between two Kp-10 stimulations rather than in isolation. As a result, very few traces captured a clean PAPA/NO response alone, without overlapping Kissspetin-10 effects. To avoid confusion and ensure clear interpretation, we chose to represent each condition separately in the main figure panels.\nTo further reassure the reviewer, we include below an additional representative trace (not shown in the manuscript) from another recording (0 to 20 min; Panel A) in which kisspeptin-10 was applied followed by PAPA/NO + ODQ that extended beyond the original window (35 to 55 min; Panel B). This example confirms that no delayed increase in cGMP occurs, supporting an efficient inhibition of NO/cGMP release by ODQ under our experimental conditions and no late-phase signal observed in the presence of ODQ. Indeed this is also the case in Figure 2 H, showing that the window of the response is well contained within the first 15 minutes.\nFigure R2. Data plots show ΔF/F0 in responder cells (N = 3 mice, n = 5- 7 cells) upon application of (A) 10 nM kp-10 (black bars) or (B) 5 μM PAPA/NO (black bars) in the absence or presence of 10 μm ODQ (red bars).\nSome minor issues:\nLine 24: ….orchestrates GnRH…delete ‘s’\nLines 52, 79: formate references\nLine 275: …sildenafil treatment, by promoting additional NO production…’ rephrase, as sildenafil does not change NO production.\nLine 812: correct ‘FlicG3’\nLine 861: correct ‘1nMol’ into 1nM\nWe appreciate the reviewer’s attention to detail. In line 861 we corrected to 1 nmole to clearly distinguish from 1 nM (nanomolar). The rest of the suggested corrections have been implemented.\nReviewer #3 (Remarks to the Author):\nThe manuscript by Delli et al. examined the role of neuronal nitric oxide (NO) synthase (nNOS) neurons in regulation of the reproductive system. The authors show that Nos1-expressing neurons express kisspeptin receptor (Kiss1r) and kisspeptin treatment induces nNOS activation and production of cGMP in the organum vasculosum of the lamina terminalis (OV) and medial preoptic area (MePO). Using a chemogenetic approach targeted to Nos-1 expressing cells in OV/MePO, the authors show that activation of Nos1-expressing cells increase LH release, while inhibition of Nos-1 expressing cells potentiates the effects of kisspeptin treatment on LH release. Modulation of NO production leads to a change in kisspeptin-induced GnRH neuron firing activity in brain slices and LH release in vivo. The findings suggest that NO could play a role in modulation of GnRH neuron activity and LH release.\nThe findings are interesting and explore a new aspect of regulation of GnRH neurons and reproductive functions. However, some results from this study do not support the proposed model. In addition, the experiments do not fully prove the direct action of the kisspeptin on nNOSOV/MePO neurons and the regulation of GnRH neurons and LH release.\nMajor comments:\n1. This study proposes a model by which kisspeptin neurons activate a brake by promoting NO release and thus suppressing GnRH neurons and LH release. However, the result from Nos1Cre + hM3DGq experiment (Fig 1I-N) contradicts the proposed model. Activation of Nos1 cells lead to a significant increase in LH secretion, suggesting a stimulatory role of nNOSOV/MePO neurons. Results from an inhibitory DREADD experiment, electrophysiological recordings, and a pharmacological experiment suggest an inhibitory role of NO on GnRH neurons and LH release. The authors do not address the discrepancy in the effects of NO in the discussion and therefore weaken the proposed model.\nWe thank the reviewer for this important observation. We agree that the increase in LH levels following chemogenetic activation of Nos1 neurons (original Figure 1I–N, updated Figure 3 & Suppl. Fig. 3) may, at first glance, appear to contradict the model we propose, where NO release exerts an inhibitory effect on GnRH neuron activity.\nHowever, our interpretation is grounded in a dynamic model of NO signaling, supported both by our previous mathematical modeling work (Bellefontaine et al., 2014; PMID: 24812663) and by electrophysiological data from the current study and prior work (Clasadonte et al. Endocrinology 2008; PMID: 18006627). As stated in lines 185–188 of the manuscript, our model proposes that the cumulative activation of nNOS OV/MePO neurons, such as that triggered by CNO in the case of the + hM3DGq experiment or by the exogenous administration of kisspeptin, leads to a transient rise in NO levels. While this NO surge temporarily silences GnRH neurons via a direct inhibitory effect — clearly seen in our whole-cell recordings — this inhibition serves a coordinating or synchronizing function, especially given the ability of highly diffusible NO to reach distant GnRH neurons. Given the rapid kinetics of NO clearance (on the order of milliseconds), we propose that once NO levels drop, these silenced GnRH neurons resume firing simultaneously, a critical prerequisite for generating the preovulatory GnRH/LH surge. Thus, while the acute activation of nNOS neurons (and NO release) serves as a \"brake\" at the single-neuron level, the ultimate consequence at the circuit level may be stimulatory, by allowing the coordination of GnRH output. In fact, this is also in agreement with experiments showing that the administration of L-NAME during proestrous blunts the LH surge (Hanchate et al., 2012; PMID: 22262891). This biphasic response — local inhibition enabling system-level synchronization — is central to the dynamic logic of the model, and not inherently contradictory.\nWe have now discussed this in lines 405–420, in addition to summarizing it in the concluding paragraph as well as the new Figure 7 to make this mechanism more explicit.\n2. In line with the above, the model proposed by the authors suggests that kisspeptin acts on Kiss1r on nNOSOV/MePO neurons. However, without an experiment showing that kisspeptin acts directly at nNOSOV/MePO neurons, the effects observed in this study could be indirect via intermediate neurons. Moreover, kp-10 was delivered via ip injection in the majority of experiments. Therefore, it is possible that the effects of kp-10 observed in this study is not directly at nNOSOV/MePO neurons. To confirm that kisspeptin acts directly at nNOSOV/MePO neurons via Kiss1r to modulate GnRH neurons, an experiment in which Kiss1r is knocked out specifically in nNOSOV/MePO neurons is needed.\nWe agree with the reviewer that, in the absence of a cell-specific functional experiment, we cannot fully exclude the possibility that the effects of kisspeptin observed in our study are partially mediated through intermediate neuronal populations. However, several lines of evidence strongly suggest that kisspeptin acts—at least in part—directly on nNOS OV/MePO neurons. First, peripheral administration of kp-10 leads to increased phosphorylation of nNOS as well as elevated cGMP levels (ex vivo), both indicating enhanced activity of the NO pathway in the OV/MePO region. Second, we have shown that nNOS neurons in this region express Kiss1r in a cycle-dependent manner, supporting their capacity to respond directly to kisspeptin in vivo. In addition, to specifically address whether kisspeptin acts directly on nNOS OV/MePO neurons via Kiss1r, we have now employed a Cre-dependent AAV vector (pAAV[FLEXon]-CMV>LL:rev(EGFP:mKiss1r):LL:WPRE) designed to silence Kiss1r expression selectively in Nos1 expressing neurons of Nos1cre mice. This vector expresses three distinct shRNAs targeting mouse Kiss1r (mKiss1r) under a FLEX (Cre-On) configuration, ensuring specificity to Cre-positive neurons. Upon stereotaxic injection into the OV/MePO, this viral tool led to a partial decrease in total Kiss1r mRNA levels corresponding to an approximately 60% decrease specifically in nNOS neurons, as confirmed by RNAscope. Notably, this level of knockdown was sufficient to exacerbate the LH response to peripheral kisspeptin administration, demonstrating that Kiss1r expression in nNOS OV/MePO neurons is functionally required to constrain kisspeptin-driven activation of the reproductive axis. These results are shown in the new Figure 5 and discussed in lines 271-284 and 421-429.\n3. The authors state in Line 167-168 that this work “demonstrate for the first time that the phosphorylation of nNOS protein at Ser1412 is coupled with cGMP production in vivo”. This is a proposition based on the results from two separate experiments showing that kisspeptin treatment increase phosphorylation at Ser1412 (Fig 1F-G) and kisspeptin increases cGMP production (Fig 2). A single experiment showing increased P-nNOS and FlincG3 (for example, staining in the brains from the same experiment) would better support the statement.\nIn addition, FlincG3 fluorescence was found in GnRH neurons, but the authors did not quantify the percentage of GnRH neurons that show colocalization with FlincG3.\nWe thank the reviewer for this valuable suggestion. In response, we have now included a new Figure 2 (I, K), showing the co-localization of the FlincG3 signal and P-nNOS (Ser1412) in the same brain sections. This additional experiment allows us to visually appreciate the local production of cGMP surrounding activated nNOS neurons, while also allowing for a correlation analysis between FlincG3 and the levels of phosphorylation of nNOS, thus strengthening our conclusion that nNOS phosphorylation is functionally coupled to cGMP generation in vivo. These results have been now presented in lines 175-177.\nRegarding the presence of FlincG3 signal in GnRH neurons, , suggesting a direct action of NO, as we have noted, only a small proportion of these neurons was found to be positive (14.4% ± 3.2). FlincG3 fluorescence reflects a very dynamic signal (as seen by our live imaging experiments). The time point of sacrifice represents only a snapshot, which complicates accurate quantification of cGMP in a population of cells with asynchronous activity, such as the GnRH neurons. To address this, future experiments using GnRHcre animals injected with a Cre-dependent FlincG3 construct would be required, allowing specific and real-time monitoring of NO/cGMP signaling in GnRH neurons. We have included those findings in line 175.\n4. The authors state that activation of nNOSOV/MePO neurons is sufficient to trigger LH surge-like release. Based on previous reports, the level of LH surge during proestrous (14.50 ng/ml) or ovariectomized animals with estradiol supplementation (7.70 ng/ml; Czieselsky et al., 2016; DOI: 10.1210/en.2016-1351) are much higher than the LH values observed in this study (peak at around 1.5 ng/ml). Therefore, it is misleading to state that the LH increase is at the level of LH surge.\nWe agree that the LH levels we obtain following chemogenetic activation of nNOS OV/MePO neurons (~1.5–2.0 ng/mL) are lower than the physiological preovulatory surge reported in proestrous mice or in ovariectomized animals treated with estradiol (~7.7 ng/mL), as described in Czieselsky et al. (2016).\nIn our study, we use the term “surge-like” in the functional rather than quantitative sense: to describe the pattern of the response—its strong fold-increase over baseline, and, critically, its sustained elevation. As shown in Supplementary Figure 3, LH levels remain elevated for at least 100 minutes after nNOS OV/MePO activation, which mirrors the dynamic profile characteristic of a “surge-like” response even if the absolute amplitude is lower than that of the endogenous preovulatory surge. We believe that the revised manuscript conveys this interpretation without implying equivalence with the physiological surge.\n5. The authors state that “genetic nNOS deficiency results in exacerbated kp-10 induced GnRH neuron firing” (line 220-221). However, the increase in GnRH neuron firing activity caused by kp-10 is about 100% from basal firing activity in both Nos1+/+ (~0.3Hz to 2Hz) and Nos1-/- (~2Hz to ~3.8Hz). Since the basal firing of GnRH neurons is different between the mouse models, the authors should take this information into account regarding the response to kp-10 treatment.\nWe appreciate the reviewer’s comment regarding the difference in basal firing rates between Nos1 +/+ and Nos1 -/- mice, and we agree that this difference must be considered when interpreting the response to kisspeptin. As shown in Suppl. Fig. 5B GnRH neurons from Nos1 -/- female mice in Di display a higher basal firing rate (~1.75 Hz vs. ~0.36 Hz in Nos1 +/+), and in both sexes they reach a higher absolute firing rate following kisspeptin exposure as seen in Fig. 4 (~2.1 Hz and. ~2.8 Hz in Nos1 +/+). This pattern indicates that in the native environment, GnRH neurons operate at low tonic activity, whereas in the absence of NO, the GnRH neurons are closer to their maximal firing capacity. As such, the proportional increase (~400% in the Nos1 +/+ vs ~ 150% in the Nos1 -/-) following kp-10 administration is necessarily reduced due to ceiling effects, rather than a reduced responsiveness. This ceiling effect also explains why the response cannot be interpreted as a simple “additive” mechanism (in response to the reviewer’s comment #6): in conditions where kisspeptin robustly drives GnRH neuron firing close to saturation, NO’s role is to dampen or contain the kisspeptin-induced activation. This interpretation is further supportef by the extended recordings (Extended Fig. 4A–B) showing that inhibition of NOS with L-NAME increased basal GnRH neuron firing and potentiated the response to kisspeptin, whereas following washout of L-NAME, the second kisspeptin application returned toward pre-L-NAME levels, indicating partial recovery of endogenous NO tone. In contrast, in the presence of the irreversible sGC inhibitor ODQ (Extended Fig. 4C–D), a second kisspeptin application further enhanced firing, consistent with sustained blockade of the NO/cGMP pathway. These complementary observations confirm that NO signaling dynamically restrains kisspeptin-driven activation and that loss of this inhibitory pathway leads to cumulative excitatory responses.\nIn addition, in an independent series of recordings performed in male Nos1 -/- mice (new Fig. 4) the genetic Nos1 deficiency or pharmacological blockade of the NO/cGMP cascade still resulted in an increased kispspetin-induced GnRH firing, confirming that this regulatory mechanism is conserved across sexes.\n6. The use of pharmacological treatment (L-NAME and DEA/NO) in electrophysiological experiments is not sufficient to show that NO is involved in kisspeptin-induced activation of GnRH neurons. It could simply be the additive effects from kisspeptin and NO that are coincidentally observed within the same GnRH neurons. It is possible that the modification of NO production itself could have effects on GnRH neuron activity. An experiment where GnRH neurons are treated with just L-NAME or DEA/NO should be included to address this possibility.\nWe thank the reviewer for raising this point. We respectfully clarify that the aim of our electrophysiological experiments was not to characterize the independent effects of NO on GnRH neuron activity, but rather to determine whether modulation of the NO pathway alters the response of these neurons to kisspeptin. Our study is therefore focused on the interaction between kisspeptin and NO signaling, rather than on the additive or basal actions of each molecule alone.\nTo formally assess additive versus synergistic effects, one would need complete concentration–response curves for both kisspeptin and NO donors across a wide range of subthreshold doses. However, this is not the question we sought to address. In our experiments, saturating doses of kisspeptin were applied to reliably evoke the canonical excitatory response, allowing us to test how pharmacological blockade or stimulation of the NO pathway modulates this response.\nAs shown in Figure 4 and reported in lines 236-250, the inhibition of NOS (by L-NAME) or sGC (by ODQ) significantly amplified the kisspeptin-induced increase in GnRH neuron firing, while the prolongation of cGMP signaling (sildenafil) attenuated it. These results are not compatible with a simple additive mechanism, since an additive action of two independent excitatory inputs would not be expected to enhance excitability when one pathway is blocked. Instead, they demonstrate that NO signaling dynamically constrains kisspeptin-induced activation, consistent with its role as a negative feedback component of the kisspeptin–GnRH signaling cascade, as also clearly shown in Constantin et al. Proc Natl Acad Sci U S A 2021(PMID: 33443156). In that study, it was demonstrated that NO is an integral component of the kisspeptin response, actively terminating and resetting the calcium response in GnRH neurons via the canonical cGMP–PKG–PIP2 replenishment pathway. This establishes that NO is functionally required for shaping kisspeptin’s temporal effects on GnRH activity, being a regulatory feedback signal embedded in the kisspeptin-response machinery.\nFinally, previous studies have already established that acute application of NO donors (DEA/NO or L-arginine) suppresses GnRH firing, whereas NO/ cGMP inhibition alone (L-NAME or ODQ) has a minimal effect on basal activity (Clasadonte et al. Endocrinology 2008 PMID: 18006627), in agreement with our Extended Fig. 4, indicating that NO is not tonically active but triggered specifically by the kisspeptinergic excitatory drive. We have clarified this concept in the revised Discussion, lines 396-405.\n7. In Nos1Cre+hM3Dq (Fig 1I) and Nos1Cre+hM4Gi (Fig 3G) experiments, there is no control for the potential impact of CNO on LH release. It is possible that CNO (or clozapine) is responsible for the change in LH release independent of impact of NO. A control group where Nos1Cre animals are injected with a control virus (an AAV reporter virus without DREADD receptor) and treated with CNO should be include in the study.\nWhile it is well established in the literature that CNO at commonly used doses does not significantly impact LH release in the absence of DREADD expression (e.g., Coutinho et al., 2020, PMID: 31630145; Wang et al., 2020, PMID: 32644040), we nonetheless performed our own control experiments to directly address this concern. In our study, n= 5 female ROSA26CAGSloxSTOPloxhMD3 (hM3D loxP/loxP mice) mice in the estrous stage (10 weeks old Gq Gq ) were subjected to a 1 h tail-tip blood sampling for LH measurement, with blood collected at 6-min intervals. Thereafter all mice received an intraperitoneal injection of 1 mg/kg CNO and subjected to iterative bleeding for 1 more hour. As demonstrated in the figure below, CNO in the absence of hMD3Gq expression did not elicit any significant change in circulating LH levels, indicating that the observed hormonal effects in our experimental groups are specific to the DREADD-mediated manipulation of nNOS OV/MePO neurons, rather than to off-target effects of CNO or its metabolites.\nFigure R3. Representative LH pulse profiles of 5 female mice and subsequent analysis of the (B) Area Under the Curve (AUC) (Wilcoxon paired t-test p>0.9; N=5), and (B) baseline LH (Wilcoxon paired t-test p= 0.01; N= 5), before (white) or after (black) CNO administration (60th minute). Values indicate means ± SD ns: non-significant\n8. The experiment in Fig 4 was performed in male mice. However, other main experiments in the manuscript were performed in female mice. The authors did not explain the rationale behind this. It is possible that the response is different between males and females. Therefore, the authors should also perform the experiment in Fig 4 in female mice to be able to fairly compare the results between experiments.\nThe rationale for performing the experiment in Figure 4 (new Fig. 6) in male mice was based on both technical feasibility and experimental consistency. More specifically, we have consistently observed across our dataset that robust manipulations such as chemogenetics (e.g., DREADDs) yield clear and reproducible effects in both sexes. In contrast, acute pharmacological interventions in intact cycling females are more variable, due to cycle-dependent hormonal fluctuations that can modulate neural responsiveness to kisspeptin, NO, or other modulators. This inherent variability can obscure pharmacological effects unless tightly controlled. These constraints initially led us to select male mice, whose hormonal background is more stable and allows for more straightforward interpretation of pharmacological responses.\nNonetheless, in response to the reviewer’s concerns, we performed the same experiment shown in Extended Figure 6 in a cohort of Di female mice. While variability was indeed higher the data revealed the same trend as in males, supporting the mechanisms proposed by our hypothesis. In fact, in female mice, the impact of NO inhibition on the kp-10-induced LH surge was already apparent 5 minutes after kp-10 administration (Extended Fig 6 A, E-F), whereas in males the divergence between saline and L-NAME appears around 10 minutes after injection (Fig. 6 A, E-F). Although differences didn’t reach statistical significance, in females both kp1 and kp2 showed a clear trend toward higher LH levels under L-NAME compared with saline (kp1 minute 60: unpaired t-test saline vs L-NAME, t = 2.02 p= 0.07; and kp2, minute 120: unpaired t-test saline (10) vs L-NAME, t = 1.90 p= 0.08; n= 6). Notably, this divergence became more pronounced (10) approximately 50 minutes after kp-10 administration (minute 100), which corresponds to the time point at which LH normally returns to baseline under control conditions.\nInterestingly, in contrast to what we observed in male mice, sildenafil had no significant effect compared to saline, suggesting that, under basal conditions, the NO/cGMP pathway is probably already maximally engaged in females (one-way- ANOVA, kp1 saline vs L-NAME, p= 0.03; saline vs Sildenafil, p= 0.9; N=6). We have now included these data in the revised manuscript (Extended Figure 6) and lines 316-320 and 452-458 in the main text. Importantly, all other key experiments—including the RNAscope, electrophysiology and the excitatory DREADD manipulations—are now performed in both sexes, consistently showing comparable results.\n9. In the discussion, the authors make several statements without the direct evidence. For example, in line 299-300 the authors state “kisspeptin was seen to use NO to contain the firing activity of the GnRH neurons and potentially terminate its own effects”. From the electrophysiological experiment in this study, it is not sufficient to prove that kisspeptin use NO to contain firing activity of the GnRH neurons (comment #5).\nWe agree that our electrophysiological data do not directly demonstrate that kisspeptin directly terminates its own effects via NO in a causal, temporally resolved manner. However, our findings do strongly support a functional interplay between kisspeptin signaling and NO production in shaping the firing activity of GnRH neurons.\nMore specifically, our study shows that:\n• Kisspeptin induces phosphorylation of nNOS at Ser1412 and enhances local cGMP production in the OV/MePO, including in GnRH neurons.\n• Blocking NO synthesis or signaling (via L-NAME or ODQ) exacerbates the kisspeptin-induced firing of GnRH neurons, without affecting basal firing.\n• Conversely, application of an NO donor or a phosphodiesterase 5 inhibitor suppresses the heightened kisspeptin-induced activation in Nos1 -/- mice.\n• Pharmacogenetic inhibition of nNOS neurons or L-NAME treatment results in a prolonged LH response to kisspeptin, suggesting impaired resolution of the kisspeptin effect.\nThese data collectively support a model in which NO signaling, induced downstream of kisspeptin, contains or limits the extent of kisspeptin-induced excitation of GnRH neurons and LH release. While this may not constitute direct \"proof\" of a causal negative feedback loop in real time, it is consistent with the proposed model and with previous studies showing that NO contributes to the termination of kisspeptin-induced responses via intracellular mechanisms such as PIP2 replenishment (Constantin et al. Proc Natl Acad Sci U S A 2021(PMID: 33443156).\nWe have modified the above sentence (lines 338-340) and we believe that our updated discussion covers these points.\n10. The authors use the terminology “GnRH/LH release” throughout the manuscript. For example, “activation of nNOS neurons of the OV/MePO is sufficient to trigger GnRH/LH surge-like release” (line 172-173). It is true that LH release reflects GnRH release in most case. However, in this study, GnRH release is not directly measured at any point and, therefore, could be misleading to readers.\nThe reviewer is right to point out that our study does not include direct measurements of GnRH release and that referring to \"GnRH/LH release\" could potentially be misleading. Our intention was to reflect the established physiological relationship between GnRH secretion and pituitary LH output, as the reviewer also recognizes, where LH pulsatility and surges are widely accepted as reliable readouts of upstream GnRH activity.\nHowever, to ensure precision and avoid overstating the data, we have revised the manuscript to remove the terminology “GnRH/LH release” and instead refer specifically to “LH release” or “LH responses”, except where GnRH neuron activity was directly assessed through electrophysiological recordings or when we hypothesize possible network dynamics. We have also added a brief note to clarify that LH release is used here as a functional proxy for GnRH output, consistent with established neuroendocrine practice (lines 563-565).\nMinor comments:\n1. The authors show results on the effect of kisspeptin on Ser1412-phosphorylation of the nNOS protein in Fig 1F-H but do not mention this finding in the results section of the manuscript.\nThis result was indeed mentioned in the original Results section and can be found in the revised manuscript as follows: “(…) an effect that we also observe in our model (Fig. 1 E-H) (…)” (lines 140-141) and “Kp-10 administration led to an increase in Ser1412-phosphorylation of the nNOS protein in the OV/MePO, as well as circulating LH levels, in agreement with previous reports (Fig. 1E–H)” (lines 166–168).\n2. The experiment in Fig 1I-N should be moved to later section of the results section to improve the flow of the manuscript.\nWe thank the reviewer for their suggestion. The experiment has now been moved to new Figure 3 and Suppl. Fig. 3.\n3. Line 125: the numbers do not match the data in the supplemental figure 1G\n4. Line 126: the numbers do not match the data in the supplemental figure 1F\nWe thank the reviewer for their careful observations. Upon review, we noted that the y-axis labeling in Supplemental Figure 1G was indeed incorrect and has now been corrected. In addition, we realized that in the text referencing Supplemental Figure 1F, the values were originally expressed as fractions rather than percentages, which has now been updated to ensure consistency with the figure.\n5. Line 183: “77,8” should be “77.8”\n“77,8” has now been corrected to “77.8.”\n6. The Y axis in Fig 2F-H should use “.” instead of “,”\nThe Y-axis notation in Fig. 2F–H has now been corrected\n7. Line 290-291: The authors note that the highest expression of Kiss1r-expressing nNOS neurons is during proestrus. In this study, only mice in diestrus and proestrus were studied, but not metestrus. Therefore, in order to make such a statement the authors should include mice in metestrus.\nWe thank the reviewer for this helpful comment. In response, we have now included metestrus-stage females in our analysis, as requested. This addition allowed us to more fully capture the cycle-dependent dynamics of Kiss1r expression in nNOS neurons. Our updated data shown in Figure 1 B-D confirm that Kiss1r expression peaks during proestrus, with significantly lower levels observed in both diestrus and metestrus, thereby strengthening the conclusion regarding estrogen-sensitive regulation. In addition, to broaden the physiological relevance and include both sexes, we also analyzed male mice, which provide a stable, low-estrogen baseline. Kiss1r expression in nNOS neurons was significantly lower in males compared to proestrus females, consistent with the hormone-dependent pattern observed across the female cycle. The manuscript text and figure legends have been updated to reflect these new data, lines 121-126 and 327-330."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I thank the authors for their thorough responses to my original comments, which I am pleased have been adequately addressed. I have no further comments prior to acceptance", "reviewer_2": "The authors have addressed my comments in full and to satisfaction.", "reviewer_3": "The authors have addressed all the comments. There is no additional comment for the manuscript."}, "author_response": "We would like to thank all the reviewers for allowing us to improve our manuscript by adressing their comments."}]

Decision letter