Reproduction is strongly influenced by nutritional status due to the high energy demand required for this function. Food scarcity leads to suppression of fertility (Ahima et al., 1996; Bronson and Marsteller, 1985; Cagampang et al., 1990; Cameron et al., 1991; Parfitt et al., 1991). Similarly, excess adiposity may induce ovulatory dysfunction, higher risks of pregnancy complications and earlier age at puberty onset (Biro et al., 2006; Burt Solorzano and McCartney, 2010; Mahany et al., 2018). Metabolic cues signal energy status to the hypothalamo–pituitary–gonadal (HPG) axis, but the sites of action and mechanisms are not fully elucidated.

The adipocyte-derived hormone leptin is an essential metabolic signal acting in the brain to regulate the reproductive axis. Mice and humans with mutations in the leptin or leptin receptor genes are infertile and remain in a prepubertal state (Bray and York, 1979; Farooqi et al., 2007; Farooqi et al., 2002). Specific restoration of the leptin receptor (Lepr) gene only in neurons of Lepr-deficient mice rescues infertility (de Luca et al., 2005; Quennell et al., 2009). It is well accepted that leptin action in the modulation of the HPG axis is indirect. Gonadotropin-releasing hormone (GnRH) neurons do not express Lepr (Quennell et al., 2009) but they do receive direct innervation from Lepr-expressing neurons (Louis et al., 2011). The neural circuitry involved, however, is incompletely known.

An often-overlooked hypothalamic site, rich in neurons expressing the long form of LepR (LepRb), is the ventral premammillary nucleus (PMv) (Donato et al., 2011; Han et al., 2020; Leshan et al., 2009; Louis et al., 2011; Ross et al., 2018; Scott et al., 2009). In rats, bilateral lesions of the PMv disrupt estrous cycles and the ability of leptin to increase luteinizing hormone (LH) secretion following a fast (Donato et al., 2009). Endogenous restoration of LepRb exclusively in PMv neurons rescues pubertal maturation and fertility in Lepr-null female mice (Donato et al., 2011).

Leptin responsive neurons in the PMv project directly to and make contacts with GnRH cell bodies and their terminals in the adjacencies of the median eminence (Boehm et al., 2005; Canteras et al., 1992; Donato et al., 2011; Leshan et al., 2009). These cells also project to the anteroventral periventricular (AVPV) and arcuate (Arc) nuclei, home of kisspeptin (Kiss1 gene) neurons, the main GnRH regulatory population (Canteras et al., 1992; Donato et al., 2011). Projections from the PMv make apparent contacts with Kiss1 expressing cells in the AVPV (Donato et al., 2011), and stimulation of PMv neurons directly activates kisspeptin cells in both the AVPV and the Arc (Ross et al., 2018).

The PMv is essentially glutamatergic (Vong et al., 2011). LepRb neurons coexpress the vesicular glutamate transporter Slc17a6 (Vglut2) (Donato et al., 2011), and acute administration of leptin depolarizes most of these neurons (Williams et al., 2011). Glutamatergic terminals are abundant in GnRH (Moore et al., 2018; Yeo et al., 2021) and in kisspeptin (Porter et al., 2021) neurons, while states of low leptin levels, as in fasting, reduce the number of glutamatergic synaptic inputs to kisspeptin neurons of male macaques (Shamas et al., 2015). Prior data, however, have shown that congenital deletion of Lepr in glutamatergic neurons has virtually no effect on pubertal development or reproductive function (Martin et al., 2014; Zuure et al., 2013).

In the attempt to untangle these apparent inconsistencies, we used chemogenetics to determine if remote stimulation of PMv neurons induces LH release in diestrous mice. We next used several transgenic mouse models to determine whether disruption of glutamate neurotransmission from Lepr neurons impairs leptin-induced puberty and normal reproductive function. Overall, our results indicate that glutamatergic signaling from leptin responsive PMv neurons is required for normal reproductive development and function.

Understanding the role of leptin in the control of reproduction has largely advanced in the last few years, but many open questions remain regarding the neural pathways and the molecular mechanisms involved. While overlapping neural pathways exist, the PMv has arisen as an essential hub in this circuitry. The PMv is a glutamatergic nucleus (Donato et al., 2011; Vong et al., 2011) rich in leptin-responsive cells (Leshan et al., 2009; Louis et al., 2011; Scott et al., 2009; Williams et al., 2011). Nevertheless, recent studies have shown that deletion of LepRb in glutamatergic cells does not disrupt puberty or fertility, as opposed to large reproductive deficits when LepRb is deleted from GABAergic cells (Martin et al., 2014; Zuure et al., 2013). We have therefore investigated in more detail the role of glutamatergic neurotransmission in LepRb PMv neurons.

Leptin treatment prevents a fall in LH levels in states of negative energy balance in rodents and humans (Ahima et al., 1996; Chan et al., 2003; Donato et al., 2009; Welt et al., 2004). In ob/ob (Lep^ob) mice, which are infertile and hypogonadotropic, leptin administration increases LH levels, while this treatment is ineffective in PMv lesioned Lep^ob mice (Donato et al., 2011), indicating that intact PMv is required for the leptin stimulatory effect on LH in leptin-deficient mice. In the current study, we expanded this observation showing that in fed mice, acute unilateral activation of leptin responsive PMv neurons using stimulatory DREADDs transiently elevated LH levels in a normal nutritional state (i.e., not in negative energy balance). This increase was correlated with the number of activated PMv LepRb neurons, and it was only observed following a substantial neuronal stimulation (Fos activation). The animals that did not show an LH increase despite having the virus targeted to the PMv had low Fos-ir in the PMv, possibly due to virus inactivity or low CNO entry into the brain (Gomez et al., 2017).

It is well accepted that clozapine has high affinity to DREADDs (Gomez et al., 2017). Thus, the hM3Dq injected animals showing clozapine-induced LH release strongly support our observations using CNO, that is targeted activation of PMv neurons leads to increase in circulating LH. Of note, optogenetic activation of a pituitary adenylate-cyclase-activating polypeptide (PACAP) expressing subpopulation of PMv neurons leads to increased activity of kisspeptin neurons. Whether this increases LH release is not known. Instead, the authors showed that deletion of PACAP in the PMv leads to an exacerbation of LH response to acute leptin administration (Ross et al., 2018), what reinforces the role of LepRb PMv neurons in LH secretion. Further studies are required to define the interaction between glutamate and PACAP neurotransmission in downstream targets of PMv neurons.

The use of stereotaxic injections inevitably leads to heterogeneity in the injection sites. The main caveat to our study was the activation in some animals of neighboring hypothalamic areas containing leptin receptor, abundant in the mediobasal hypothalamus (Louis et al., 2011; Scott et al., 2009). Larger injections in a few animals, and perhaps the presence of dendrites from LepRb neurons adjacent to PMv, led to contamination of other sites, mainly the Arc. For this reason, we investigated the distribution of Fos-ir in Arc neuronal populations involved in stimulation of LH secretion (i.e., POMC and kisspeptin).

POMC neurons have been involved in modulation of the reproductive axis (Leranth et al., 1988; Manfredi-Lozano et al., 2016; Scimonelli and Celis, 1990). The POMC-derived peptide α-melanocyte-stimulating hormone has a stimulatory role on reproduction via actions on kisspeptin and GnRH neurons (Manfredi-Lozano et al., 2016; Roa and Herbison, 2012). Therefore, direct activation of POMC neurons, although small in number, may have contributed to the higher LH peak observed in these animals. On the other hand, kisspeptin is a direct stimulator of GnRH but only about 10% of KNDy neurons in female mice express LepRb (Cravo et al., 2013; Cravo et al., 2011; Donato et al., 2011; Louis et al., 2011; True et al., 2011). Restoration of LepRb in kisspeptin neurons does not restore fertility in Lepr-deficient mice (Cravo et al., 2013; Cravo et al., 2011). The very low number of Fos-ir KNDy neurons we observed makes it unlikely that contamination or downstream activation of KNDy neurons contributed to the LH secretion. Of note, in kisspeptin signaling deficient mice, glutamate receptor agonists induce LH secretion, indicating that alternative glutamatergic pathways bypass KNDy neurons to regulate the reproductive axis (García-Galiano et al., 2012). Alternatively, chemogenetic activation of leptin sensitive PMv neurons might primarily act via direct activation of GnRH cell bodies in the preoptic region or their terminals in the median eminence, as discussed below for the Lepr^ΔVglut2 females. Uncharacterized mCherry-expressing Fos neurons were observed in the posterior Arc, but lack of prior knowledge of a role in reproduction curtails the evaluation of a plausible role in LH release.

Due to lack of a specific model to target the PMv neurons, we investigated the reproductive phenotype in animals with Vglut2 deletion in LepRb expressing neurons, rendering them incapable of transmitting fast-acting downstream signals (Xu et al., 2013). These animals develop obesity (Xu et al., 2013). To avoid the effects of high adiposity in reproductive outcome, body weight was monitored during all reproductive phenotype assessment. While the reproductive effect in males was not obvious, Lepr^ΔVglut2 females showed delayed pubertal completion and blunted estrous cycles. The reproductive deficits were observed prior to the manifestation of any changes in body weight, indicating that these alterations are independent of metabolic dysregulation (Donato et al., 2011). It is important to stress that Vglut2 is expressed in several other LepRb neurons including the dorsomedial and the ventromedial nuclei of the hypothalamus (Xu et al., 2013). Based on extensive literature and circuit tracing, the PMv remains the prime candidate to relay leptin’s effect in reproductive function.

To test if glutamatergic neurotransmission in LepRb PMv neurons is required for sexual maturation we used LepR-null;Vglut2-floxed females. This strategy aimed to ‘bypass’ the potential redundancy played by alternative neural pathways. Our prior studies showed that selective reexpression of LepRb in the PMv-induced puberty and fertility in LepR-null female mice (Donato et al., 2011; Mahany et al., 2018). Here, we observed that deletion of Vglut2 in PMv neurons blocked this effect. Female gonads remained in a prepubertal state, with undeveloped uterine horns and no ovarian corpora lutea.

Despite prior reports showing that lack of leptin signaling in glutamatergic cells has no effects on reproduction (Martin et al., 2014; Zuure et al., 2013), we show that glutamatergic neurotransmission in leptin responsive cells of the PMv is necessary for typical pubertal development and reproduction. Due to the fundamental role of reproduction for species survival, redundancy of neural pathways is expected, and developmental adaptations may have occurred in those studies. Of note, a recent study showed that reexpression of LepRb only in GABAergic cells is not sufficient for restoration of reproduction in otherwise LepR-null mice, emphasizing the need for other populations (Quaresma et al., 2021). Similarly, alternative metabolic signals may take over control in the absence of leptin signaling in this specific pathway (Hill et al., 2010; Hill and Elias, 2018).

Leptin depolarizes about 75% of PMv LepRb neurons (Williams et al., 2011). Depolarizing glutamatergic neurons may directly activate downstream neurons. The LepRb PMv neurons project to the preoptic area, where they directly contact GnRH neurons (Leshan et al., 2009; Louis et al., 2011). Alternatively, LepRb neurons may activate astrocytes, which in turn stimulate GnRH neurons and LH pulsatility (Vanacker et al., 2021) or GnRH fibers at the median eminence (Donato et al., 2011). In fact, Lepr^ΔVglut2 females showed increased GnRH-ir fibers located in the adjacencies of the median eminence. In these females, LH release in response to kisspeptin was reduced suggesting peptide retention and deficits in GnRH release. Similar results were obtained after reactivation of leptin signaling in the PMv of LepRb-null mice (Donato et al., 2011). In Lep^ob mice, GnRH fiber density is higher, but it decreases following leptin injection (Bellefontaine et al., 2014). A large density of synaptic contacts exists in the GnRH terminals in the vicinity of the ME, and specific inhibition of these terminals blocks LH pulses (Wang et al., 2020). We did not observe alterations in gonadotropin expression in the pituitary gland, but we found a reduction in Tac2 levels, coding for Neurokinin B (NKB), in the hypothalamus of Lepr^ΔVglut2 females. These findings indicate that blockade of glutamatergic neurotransmission in LepRb neurons disrupts key components of the GnRH pulse generator (Han et al., 2023; Ruka et al., 2013; Uenoyama and Tsukamura, 2023), leading ultimately to reproductive deficits. Indeed, previous research showed that activation of the Neurokinin 3 receptor (NK3R) by an NKB agonist-induced GnRH release consistently from the ME even in kisspeptin knockout animals, but not the preoptic region where GnRH cell bodies are located (Gaskins et al., 2013), suggesting that Tac2 alone could be a direct mediator of the glutamatergic effect on GnRH release.

LepRb PMv neurons also project to the Arc and AVPV, where they contact Kiss1 neurons (Donato et al., 2011; Ross et al., 2018). However, the interaction and dynamics between the PMv and the kisspeptin populations remain unclear. In rats, the proestrus increase in Kiss1 mRNA and Fos-ir in AVPV is blunted following bilateral lesions of the PMv. In the Arc, Kiss1 mRNA is not altered during a leptin-induced LH release or in PMv lesioned animals (Donato et al., 2009; Donato et al., 2013). In mice, Arc Kiss1 neurons are downstream of PACAP PMv neurons in reproductive function (Ross et al., 2018). Thus, complementary to fast-acting glutamatergic neurotransmission, neuropeptides released from PMv cells contribute to the long-term regulation of reproduction by leptin and its integration with other modulatory signals, for example, the neuropeptide PACAP (Evans et al., 2023; Ross et al., 2018).

LepRb PMv neurons also express nitric oxide synthase (nNOS, Nos1 gene) required for nitric oxide production (Donato et al., 2011; Donato et al., 2010; Leshan et al., 2009). Loss-of-function mutation in NOS1/Nos1 gene in humans and mice cause infertility and blockade of nitric oxide neurotransmission disrupts leptin actin in reproductive axis (Bellefontaine et al., 2014; Chachlaki et al., 2022; Yu et al., 1997). Of note, glutamatergic neurotransmission is associated with activation of nitric oxide production via actions on glutamate N-methyl-D-aspartate (NMDA) receptor (Chachlaki et al., 2017; d’Anglemont de Tassigny et al., 2007; Garthwaite et al., 1988). Whether the reproductive deficits caused by disruption of glutamatergic neurotransmission in LepRb neurons are upstream of nitric oxide needs further investigation.

All data generated or analyzed during this study are included in the manuscript. Source data files have been provided for Figures 1–7.

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Author details

1. Cristina Sáenz de Miera

   Department of Molecular and Integrative Physiology, University of Michigan–Ann Arbor, Ann Arbor, United States

   Contribution

   Methodology, Writing – review and editing, Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Writing - original draft, Project administration

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8047-035X

2. Nicole Bellefontaine

   Department of Molecular and Integrative Physiology, University of Michigan–Ann Arbor, Ann Arbor, United States

   Contribution

   Conceptualization, Formal analysis

   Competing interests

   No competing interests declared

3. Susan J Allen

   Department of Molecular and Integrative Physiology, University of Michigan–Ann Arbor, Ann Arbor, United States

   Contribution

   Writing - original draft, Project administration

   Competing interests

   No competing interests declared

4. Martin G Myers

   1. Department of Molecular and Integrative Physiology, University of Michigan–Ann Arbor, Ann Arbor, United States
   2. Elizabeth W. Caswell Diabetes Institute, University of Michigan–Ann Arbor, Ann Arbor, United States
   3. Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes, University of Michigan–Ann Arbor, Ann Arbor, United States

   Contribution

   Investigation, Project administration

   Competing interests

   No competing interests declared

5. Carol F Elias

   1. Department of Molecular and Integrative Physiology, University of Michigan–Ann Arbor, Ann Arbor, United States
   2. Elizabeth W. Caswell Diabetes Institute, University of Michigan–Ann Arbor, Ann Arbor, United States
   3. Department of Obstetrics and Gynecology, University of Michigan–Ann Arbor, Ann Arbor, United States

   Contribution

   Methodology, Writing – review and editing, Conceptualization, Supervision, Funding acquisition, Formal analysis, Investigation, Visualization, Writing - original draft, Project administration

   For correspondence

   cfelias@umich.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9878-9203

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