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.

1. 1. Ahima RS
   2. Prabakaran D
   3. Mantzoros C
   4. Qu D
   5. Lowell B
   6. Maratos-Flier E
   7. Flier JS

   (1996) Role of leptin in the neuroendocrine response to fasting

   Nature 382:250–252.

   https://doi.org/10.1038/382250a0

   • PubMed
   • Google Scholar
2. 1. Bellefontaine N
   2. Chachlaki K
   3. Parkash J
   4. Vanacker C
   5. Colledge W
   6. d’Anglemont de Tassigny X
   7. Garthwaite J
   8. Bouret SG
   9. Prevot V

   (2014) Leptin-dependent neuronal NO signaling in the preoptic hypothalamus facilitates reproduction

   The Journal of Clinical Investigation 124:2550–2559.

   https://doi.org/10.1172/JCI65928

   • PubMed
   • Google Scholar
3. 1. Berglund ED
   2. Vianna CR
   3. Donato J
   4. Kim MH
   5. Chuang JC
   6. Lee CE
   7. Lauzon DA
   8. Lin P
   9. Brule LJ
   10. Scott MM
   11. Coppari R
   12. Elmquist JK

   (2012) Direct leptin action on POMC neurons regulates glucose homeostasis and hepatic insulin sensitivity in mice

   The Journal of Clinical Investigation 122:1000–1009.

   https://doi.org/10.1172/JCI59816

   • PubMed
   • Google Scholar
4. 1. Biro FM
   2. Khoury P
   3. Morrison JA

   (2006) Influence of obesity on timing of puberty

   International Journal of Andrology 29:272–277.

   https://doi.org/10.1111/j.1365-2605.2005.00602.x

   • PubMed
   • Google Scholar
5. 1. Boehm U
   2. Zou Z
   3. Buck LB

   (2005) Feedback loops link odor and pheromone signaling with reproduction

   Cell 123:683–695.

   https://doi.org/10.1016/j.cell.2005.09.027

   • PubMed
   • Google Scholar
6. 1. Bray GA
   2. York DA

   (1979) Hypothalamic and genetic obesity in experimental animals: an autonomic and endocrine hypothesis

   Physiological Reviews 59:719–809.

   https://doi.org/10.1152/physrev.1979.59.3.719

   • PubMed
   • Google Scholar
7. 1. Bronson FH
   2. Marsteller FA

   (1985) Effect of short-term food deprivation on reproduction in female mice

   Biology of Reproduction 33:660–667.

   https://doi.org/10.1095/biolreprod33.3.660

   • PubMed
   • Google Scholar
8. 1. Burt Solorzano CM
   2. McCartney CR

   (2010) Obesity and the pubertal transition in girls and boys

   Reproduction 140:399–410.

   https://doi.org/10.1530/REP-10-0119

   • PubMed
   • Google Scholar
9. 1. Cagampang FR
   2. Maeda K
   3. Yokoyama A
   4. Ota K

   (1990) Effect of food deprivation on the pulsatile LH release in the cycling and ovariectomized female rat

   Hormone and Metabolic Research = Hormon- Und Stoffwechselforschung = Hormones et Metabolisme 22:269–272.

   https://doi.org/10.1055/s-2007-1004900

   • PubMed
   • Google Scholar
10. 1. Cameron JL
    2. Weltzin TE
    3. McConaha C
    4. Helmreich DL
    5. Kaye WH

    (1991) Slowing of pulsatile luteinizing hormone secretion in men after forty-eight hours of fasting

    The Journal of Clinical Endocrinology and Metabolism 73:35–41.

    https://doi.org/10.1210/jcem-73-1-35

    • PubMed
    • Google Scholar
11. 1. Canteras NS
    2. Simerly RB
    3. Swanson LW

    (1992) Projections of the ventral premammillary nucleus

    The Journal of Comparative Neurology 324:195–212.

    https://doi.org/10.1002/cne.903240205

    • PubMed
    • Google Scholar
12. 1. Chachlaki K
    2. Garthwaite J
    3. Prevot V

    (2017) The gentle art of saying NO: how nitric oxide gets things done in the hypothalamus

    Nature Reviews. Endocrinology 13:521–535.

    https://doi.org/10.1038/nrendo.2017.69

    • PubMed
    • Google Scholar
13. 1. Chachlaki K
    2. Messina A
    3. Delli V
    4. Leysen V
    5. Maurnyi C
    6. Huber C
    7. Ternier G
    8. Skrapits K
    9. Papadakis G
    10. Shruti S
    11. Kapanidou M
    12. Cheng X
    13. Acierno J
    14. Rademaker J
    15. Rasika S
    16. Quinton R
    17. Niedziela M
    18. L’Allemand D
    19. Pignatelli D
    20. Dirlewander M
    21. Lang-Muritano M
    22. Kempf P
    23. Catteau-Jonard S
    24. Niederländer NJ
    25. Ciofi P
    26. Tena-Sempere M
    27. Garthwaite J
    28. Storme L
    29. Avan P
    30. Hrabovszky E
    31. Carleton A
    32. Santoni F
    33. Giacobini P
    34. Pitteloud N
    35. Prevot V

    (2022) NOS1 mutations cause hypogonadotropic hypogonadism with sensory and cognitive deficits that can be reversed in infantile mice

    Science Translational Medicine 14:1–15.

    https://doi.org/10.1126/scitranslmed.abh2369

    • PubMed
    • Google Scholar
14. 1. Chan JL
    2. Heist K
    3. DePaoli AM
    4. Veldhuis JD
    5. Mantzoros CS

    (2003) The role of falling leptin levels in the neuroendocrine and metabolic adaptation to short-term starvation in healthy men

    The Journal of Clinical Investigation 111:1409–1421.

    https://doi.org/10.1172/JCI17490

    • PubMed
    • Google Scholar
15. 1. Cravo RM
    2. Margatho LO
    3. Osborne-Lawrence S
    4. Donato J
    5. Atkin S
    6. Bookout AL
    7. Rovinsky S
    8. Frazão R
    9. Lee CE
    10. Gautron L
    11. Zigman JM
    12. Elias CF

    (2011) Characterization of Kiss1 neurons using transgenic mouse models

    Neuroscience 173:37–56.

    https://doi.org/10.1016/j.neuroscience.2010.11.022

    • PubMed
    • Google Scholar
16. 1. Cravo RM
    2. Frazao R
    3. Perello M
    4. Osborne-Lawrence S
    5. Williams KW
    6. Zigman JM
    7. Vianna C
    8. Elias CF

    (2013) Leptin signaling in Kiss1 neurons arises after pubertal development

    PLOS ONE 8:e58698.

    https://doi.org/10.1371/journal.pone.0058698

    • PubMed
    • Google Scholar
17. 1. Czieselsky K
    2. Prescott M
    3. Porteous R
    4. Campos P
    5. Clarkson J
    6. Steyn FJ
    7. Campbell RE
    8. Herbison AE

    (2016) Pulse and surge profiles of luteinizing hormone secretion in the mouse

    Endocrinology 157:4794–4802.

    https://doi.org/10.1210/en.2016-1351

    • PubMed
    • Google Scholar
18. 1. d’Anglemont de Tassigny X
    2. Campagne C
    3. Dehouck B
    4. Leroy D
    5. Holstein GR
    6. Beauvillain J-C
    7. Buee-Scherrer V
    8. Prevot V

    (2007) Coupling of neuronal nitric oxide synthase to NMDA receptors via postsynaptic density-95 depends on estrogen and contributes to the central control of adult female reproduction

    Journal of Neuroscience 27:6103–6114.

    https://doi.org/10.1523/JNEUROSCI.5595-06.2007

    • Google Scholar
19. 1. de Luca C
    2. Kowalski TJ
    3. Zhang Y
    4. Elmquist JK
    5. Lee C
    6. Kilimann MW
    7. Ludwig T
    8. Liu S-M
    9. Chua SC Jr

    (2005) Complete rescue of obesity, diabetes, and infertility in db/db mice by neuron-specific LEPR-B transgenes

    The Journal of Clinical Investigation 115:3484–3493.

    https://doi.org/10.1172/JCI24059

    • PubMed
    • Google Scholar
20. 1. Donato Jose Jr
    2. Silva RJ
    3. Sita LV
    4. Lee S
    5. Lee C
    6. Lacchini S
    7. Bittencourt JC
    8. Franci CR
    9. Canteras NS
    10. Elias CF

    (2009) The ventral premammillary nucleus links fasting-induced changes in leptin levels and coordinated luteinizing hormone secretion

    The Journal of Neuroscience 29:5240–5250.

    https://doi.org/10.1523/JNEUROSCI.0405-09.2009

    • PubMed
    • Google Scholar
21. 1. Donato J
    2. Frazão R
    3. Fukuda M
    4. Vianna CR
    5. Elias CF

    (2010) Leptin induces phosphorylation of neuronal nitric oxide synthase in defined hypothalamic neurons

    Endocrinology 151:5415–5427.

    https://doi.org/10.1210/en.2010-0651

    • PubMed
    • Google Scholar
22. 1. Donato J
    2. Cravo RM
    3. Frazão R
    4. Gautron L
    5. Scott MM
    6. Lachey J
    7. Castro IA
    8. Margatho LO
    9. Lee S
    10. Lee C
    11. Richardson JA
    12. Friedman J
    13. Chua S
    14. Coppari R
    15. Zigman JM
    16. Elmquist JK
    17. Elias CF

    (2011) Leptin’s effect on puberty in mice is relayed by the ventral premammillary nucleus and does not require signaling in Kiss1 neurons

    The Journal of Clinical Investigation 121:355–368.

    https://doi.org/10.1172/JCI45106

    • PubMed
    • Google Scholar
23. 1. Donato J
    2. Lee C
    3. Ratra DV
    4. Franci CR
    5. Canteras NS
    6. Elias CF

    (2013) Lesions of the ventral premammillary nucleus disrupt the dynamic changes in Kiss1 and GnRH expression characteristic of the proestrus-estrus transition

    Neuroscience 241:67–79.

    https://doi.org/10.1016/j.neuroscience.2013.03.013

    • PubMed
    • Google Scholar
24. 1. Evans MC
    2. Wallace EG
    3. Ancel CM
    4. Anderson GM

    (2023) Leptin, but not Estradiol, Signaling in PACAP Neurons Modulates Puberty Onset

    Endocrinology 164:bqad097.

    https://doi.org/10.1210/endocr/bqad097

    • PubMed
    • Google Scholar
25. 1. Farooqi IS
    2. Matarese G
    3. Lord GM
    4. Keogh JM
    5. Lawrence E
    6. Agwu C
    7. Sanna V
    8. Jebb SA
    9. Perna F
    10. Fontana S
    11. Lechler RI
    12. DePaoli AM
    13. O’Rahilly S

    (2002) Beneficial effects of leptin on obesity, T cell hyporesponsiveness, and neuroendocrine/metabolic dysfunction of human congenital leptin deficiency

    The Journal of Clinical Investigation 110:1093–1103.

    https://doi.org/10.1172/JCI15693

    • PubMed
    • Google Scholar
26. 1. Farooqi IS
    2. Wangensteen T
    3. Collins S
    4. Kimber W
    5. Matarese G
    6. Keogh JM
    7. Lank E
    8. Bottomley B
    9. Lopez-Fernandez J
    10. Ferraz-Amaro I
    11. Dattani MT
    12. Ercan O
    13. Myhre AG
    14. Retterstol L
    15. Stanhope R
    16. Edge JA
    17. McKenzie S
    18. Lessan N
    19. Ghodsi M
    20. De Rosa V
    21. Perna F
    22. Fontana S
    23. Barroso I
    24. Undlien DE
    25. O’Rahilly S

    (2007) Clinical and molecular genetic spectrum of congenital deficiency of the leptin receptor

    The New England Journal of Medicine 356:237–247.

    https://doi.org/10.1056/NEJMoa063988

    • PubMed
    • Google Scholar
27. 1. García-Galiano D
    2. Pineda R
    3. Roa J
    4. Ruiz-Pino F
    5. Sánchez-Garrido MA
    6. Castellano JM
    7. Aguilar E
    8. Navarro VM
    9. Pinilla L
    10. Tena-Sempere M

    (2012) Differential modulation of gonadotropin responses to kisspeptin by aminoacidergic, peptidergic, and nitric oxide neurotransmission

    American Journal of Physiology. Endocrinology and Metabolism 303:E1252–E1263.

    https://doi.org/10.1152/ajpendo.00250.2012

    • PubMed
    • Google Scholar
28. 1. Garcia-Galiano D
    2. Borges BC
    3. Donato J
    4. Allen SJ
    5. Bellefontaine N
    6. Wang M
    7. Zhao JJ
    8. Kozloff KM
    9. Hill JW
    10. Elias CF

    (2017) PI3Kα inactivation in leptin receptor cells increases leptin sensitivity but disrupts growth and reproduction

    JCI Insight 2:e96728.

    https://doi.org/10.1172/jci.insight.96728

    • PubMed
    • Google Scholar
29. 1. Garthwaite J
    2. Charles SL
    3. Chess-Williams R

    (1988) Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain

    Nature 336:385–388.

    https://doi.org/10.1038/336385a0

    • PubMed
    • Google Scholar
30. 1. Gaskins GT
    2. Glanowska KM
    3. Moenter SM

    (2013) Activation of neurokinin 3 receptors stimulates GnRH release in a location-dependent but kisspeptin-independent manner in adult mice

    Endocrinology 154:3984–3989.

    https://doi.org/10.1210/en.2013-1479

    • PubMed
    • Google Scholar
31. 1. Gomez JL
    2. Bonaventura J
    3. Lesniak W
    4. Mathews WB
    5. Sysa-Shah P
    6. Rodriguez LA
    7. Ellis RJ
    8. Richie CT
    9. Harvey BK
    10. Dannals RF
    11. Pomper MG
    12. Bonci A
    13. Michaelides M

    (2017) Chemogenetics revealed: DREADD occupancy and activation via converted clozapine

    Science 357:503–507.

    https://doi.org/10.1126/science.aan2475

    • PubMed
    • Google Scholar
32. 1. Han X
    2. Burger LL
    3. Garcia-Galiano D
    4. Sim S
    5. Allen SJ
    6. Olson DP
    7. Myers MG
    8. Elias CF

    (2020) Hypothalamic and cell-specific transcriptomes unravel a dynamic neuropil remodeling in leptin-induced and typical pubertal transition in female mice

    iScience 23:101563.

    https://doi.org/10.1016/j.isci.2020.101563

    • PubMed
    • Google Scholar
33. 1. Han SY
    2. Morris PG
    3. Kim JC
    4. Guru S
    5. Pardo-Navarro M
    6. Yeo SH
    7. McQuillan HJ
    8. Herbison AE

    (2023) Mechanism of kisspeptin neuron synchronization for pulsatile hormone secretion in male mice

    Cell Reports 42:111914.

    https://doi.org/10.1016/j.celrep.2022.111914

    • PubMed
    • Google Scholar
34. 1. Hill JW
    2. Elias CF
    3. Fukuda M
    4. Williams KW
    5. Berglund ED
    6. Holland WL
    7. Cho YR
    8. Chuang JC
    9. Xu Y
    10. Choi M
    11. Lauzon D
    12. Lee CE
    13. Coppari R
    14. Richardson JA
    15. Zigman JM
    16. Chua S
    17. Scherer PE
    18. Lowell BB
    19. Brüning JC
    20. Elmquist JK

    (2010) Direct insulin and leptin action on pro-opiomelanocortin neurons is required for normal glucose homeostasis and fertility

    Cell Metabolism 11:286–297.

    https://doi.org/10.1016/j.cmet.2010.03.002

    • PubMed
    • Google Scholar
35. 1. Hill JW
    2. Elias CF

    (2018) Neuroanatomical framework of the metabolic control of reproduction

    Physiological Reviews 98:2349–2380.

    https://doi.org/10.1152/physrev.00033.2017

    • PubMed
    • Google Scholar
36. 1. Krashes MJ
    2. Koda S
    3. Ye C
    4. Rogan SC
    5. Adams AC
    6. Cusher DS
    7. Maratos-Flier E
    8. Roth BL
    9. Lowell BB

    (2011) Rapid, reversible activation of AgRP neurons drives feeding behavior in mice

    The Journal of Clinical Investigation 121:1424–1428.

    https://doi.org/10.1172/JCI46229

    • PubMed
    • Google Scholar
37. 1. Leranth C
    2. MacLusky NJ
    3. Shanabrough M
    4. Naftolin F

    (1988) Immunohistochemical evidence for synaptic connections between pro-opiomelanocortin-immunoreactive axons and LH-RH neurons in the preoptic area of the rat

    Brain Research 449:167–176.

    https://doi.org/10.1016/0006-8993(88)91035-9

    • PubMed
    • Google Scholar
38. 1. Leshan RL
    2. Björnholm M
    3. Münzberg H
    4. Myers MGJ

    (2006) Leptin receptor signaling and action in the central nervous system

    Obesity 14 Suppl 5:208S–212S.

    https://doi.org/10.1038/oby.2006.310

    • PubMed
    • Google Scholar
39. 1. Leshan RL
    2. Louis GW
    3. Jo YH
    4. Rhodes CJ
    5. Münzberg H
    6. Myers MG

    (2009) Direct innervation of GnRH neurons by metabolic- and sexual odorant-sensing leptin receptor neurons in the hypothalamic ventral premammillary nucleus

    The Journal of Neuroscience 29:3138–3147.

    https://doi.org/10.1523/JNEUROSCI.0155-09.2009

    • PubMed
    • Google Scholar
40. 1. Louis GW
    2. Greenwald-Yarnell M
    3. Phillips R
    4. Coolen LM
    5. Lehman MN
    6. Myers MG

    (2011) Molecular mapping of the neural pathways linking leptin to the neuroendocrine reproductive axis

    Endocrinology 152:2302–2310.

    https://doi.org/10.1210/en.2011-0096

    • PubMed
    • Google Scholar
41. 1. Mahany EB
    2. Han X
    3. Borges BC
    4. da Silveira Cruz-Machado S
    5. Allen SJ
    6. Garcia-Galiano D
    7. Hoenerhoff MJ
    8. Bellefontaine NH
    9. Elias CF

    (2018) Obesity and high-fat diet induce distinct changes in placental gene expression and pregnancy outcome

    Endocrinology 159:1718–1733.

    https://doi.org/10.1210/en.2017-03053

    • PubMed
    • Google Scholar
42. 1. Manfredi-Lozano M
    2. Roa J
    3. Ruiz-Pino F
    4. Piet R
    5. Garcia-Galiano D
    6. Pineda R
    7. Zamora A
    8. Leon S
    9. Sanchez-Garrido MA
    10. Romero-Ruiz A
    11. Dieguez C
    12. Vazquez MJ
    13. Herbison AE
    14. Pinilla L
    15. Tena-Sempere M

    (2016) Defining a novel leptin-melanocortin-kisspeptin pathway involved in the metabolic control of puberty

    Molecular Metabolism 5:844–857.

    https://doi.org/10.1016/j.molmet.2016.08.003

    • PubMed
    • Google Scholar
43. 1. Martin C
    2. Navarro VM
    3. Simavli S
    4. Vong L
    5. Carroll RS
    6. Lowell BB
    7. Kaiser UB

    (2014) Leptin-responsive GABAergic neurons regulate fertility through pathways that result in reduced kisspeptinergic tone

    The Journal of Neuroscience 34:6047–6056.

    https://doi.org/10.1523/JNEUROSCI.3003-13.2014

    • PubMed
    • Google Scholar
44. 1. Moore AM
    2. Abbott G
    3. Mair J
    4. Prescott M
    5. Campbell RE

    (2018) Mapping GABA and glutamate inputs to gonadotrophin-releasing hormone neurones in male and female mice

    Journal of Neuroendocrinology 30:1–13.

    https://doi.org/10.1111/jne.12657

    • PubMed
    • Google Scholar
45. 1. Münzberg H
    2. Huo L
    3. Nillni EA
    4. Hollenberg AN
    5. Bjørbaek C

    (2003) Role of signal transducer and activator of transcription 3 in regulation of hypothalamic proopiomelanocortin gene expression by leptin

    Endocrinology 144:2121–2131.

    https://doi.org/10.1210/en.2002-221037

    • PubMed
    • Google Scholar
46. 1. Parfitt DB
    2. Church KR
    3. Cameron JL

    (1991) Restoration of pulsatile luteinizing hormone secretion after fasting in rhesus monkeys (Macaca mulatta): dependence on size of the refeed meal

    Endocrinology 129:749–756.

    https://doi.org/10.1210/endo-129-2-749

    • PubMed
    • Google Scholar
47. 1. Porter DT
    2. Goodman RL
    3. Hileman SM
    4. Lehman MN

    (2021) Evidence that synaptic plasticity of glutamatergic inputs onto KNDy neurones during the ovine follicular phase is dependent on increasing levels of oestradiol

    Journal of Neuroendocrinology 33:e12945.

    https://doi.org/10.1111/jne.12945

    • PubMed
    • Google Scholar
48. 1. Quaresma PGF
    2. Wasinski F
    3. Mansano NS
    4. Furigo IC
    5. Teixeira PDS
    6. Gusmao DO
    7. Frazao R
    8. Donato J

    (2021) Leptin receptor expression in GABAergic cells is not sufficient to normalize metabolism and reproduction in mice

    Endocrinology 162:1–16.

    https://doi.org/10.1210/endocr/bqab168

    • PubMed
    • Google Scholar
49. 1. Quennell JH
    2. Mulligan AC
    3. Tups A
    4. Liu X
    5. Phipps SJ
    6. Kemp CJ
    7. Herbison AE
    8. Grattan DR
    9. Anderson GM

    (2009) Leptin indirectly regulates gonadotropin-releasing hormone neuronal function

    Endocrinology 150:2805–2812.

    https://doi.org/10.1210/en.2008-1693

    • PubMed
    • Google Scholar
50. 1. Roa J
    2. Herbison AE

    (2012) Direct regulation of GnRH neuron excitability by arcuate nucleus POMC and NPY neuron neuropeptides in female mice

    Endocrinology 153:5587–5599.

    https://doi.org/10.1210/en.2012-1470

    • PubMed
    • Google Scholar
51. 1. Ross RA
    2. Leon S
    3. Madara JC
    4. Schafer D
    5. Fergani C
    6. Maguire CA
    7. Verstegen AMJ
    8. Brengle E
    9. Kong D
    10. Herbison AE
    11. Kaiser UB
    12. Lowell BB
    13. Navarro VM

    (2018) PACAP neurons in the ventral premammillary nucleus regulate reproductive function in the female mouse

    eLife 7:e35960.

    https://doi.org/10.7554/eLife.35960

    • PubMed
    • Google Scholar
52. 1. Ruka KA
    2. Burger LL
    3. Moenter SM

    (2013) Regulation of arcuate neurons coexpressing kisspeptin, neurokinin B, and dynorphin by modulators of neurokinin 3 and κ-opioid receptors in adult male mice

    Endocrinology 154:2761–2771.

    https://doi.org/10.1210/en.2013-1268

    • PubMed
    • Google Scholar
53. 1. Sáenz de Miera C
    2. Feng J
    3. Elias CF
    4. Qi N

    (2023) Remote neuronal activation coupled with automated blood sampling to induce and measure circulating luteinizing hormone in mice

    Journal of Visualized Experiments 198:e65875.

    https://doi.org/10.3791/65875

    • PubMed
    • Google Scholar
54. 1. Scimonelli T
    2. Celis ME

    (1990) A central action of alpha-melanocyte-stimulating hormone on serum levels of LH and prolactin in rats

    The Journal of Endocrinology 124:127–132.

    https://doi.org/10.1677/joe.0.1240127

    • PubMed
    • Google Scholar
55. 1. Scott MM
    2. Lachey JL
    3. Sternson SM
    4. Lee CE
    5. Elias CF
    6. Friedman JM
    7. Elmquist JK

    (2009) Leptin targets in the mouse brain

    The Journal of Comparative Neurology 514:518–532.

    https://doi.org/10.1002/cne.22025

    • PubMed
    • Google Scholar
56. 1. Shamas S
    2. Khan SUH
    3. Khan MY
    4. Shabbir N
    5. Zubair H
    6. Shafqat S
    7. Wahab F
    8. Shahab M

    (2015) Fasting induced kisspeptin signaling suppression is regulated by glutamate mediated cues in adult male rhesus macaque (Macaca mulatta)

    Neuropeptides 52:39–45.

    https://doi.org/10.1016/j.npep.2015.06.005

    • PubMed
    • Google Scholar
57. 1. Steyn FJ
    2. Wan Y
    3. Clarkson J
    4. Veldhuis JD
    5. Herbison AE
    6. Chen C

    (2013) Development of a methodology for and assessment of pulsatile luteinizing hormone secretion in juvenile and adult male mice

    Endocrinology 154:4939–4945.

    https://doi.org/10.1210/en.2013-1502

    • PubMed
    • Google Scholar
58. 1. Tong Q
    2. Ye C
    3. McCrimmon RJ
    4. Dhillon H
    5. Choi B
    6. Kramer MD
    7. Yu J
    8. Yang Z
    9. Christiansen LM
    10. Lee CE
    11. Choi CS
    12. Zigman JM
    13. Shulman GI
    14. Sherwin RS
    15. Elmquist JK
    16. Lowell BB

    (2007) Synaptic glutamate release by ventromedial hypothalamic neurons is part of the neurocircuitry that prevents hypoglycemia

    Cell Metabolism 5:383–393.

    https://doi.org/10.1016/j.cmet.2007.04.001

    • PubMed
    • Google Scholar
59. 1. True C
    2. Kirigiti MA
    3. Kievit P
    4. Grove KL
    5. Smith MS

    (2011) Leptin is not the critical signal for kisspeptin or luteinising hormone restoration during exit from negative energy balance

    Journal of Neuroendocrinology 23:1099–1112.

    https://doi.org/10.1111/j.1365-2826.2011.02144.x

    • PubMed
    • Google Scholar
60. 1. Uenoyama Y
    2. Tsukamura H

    (2023) KNDy neurones and GnRH/LH pulse generation: Current understanding and future aspects

    Journal of Neuroendocrinology 35:e13285.

    https://doi.org/10.1111/jne.13285

    • PubMed
    • Google Scholar
61. 1. Vanacker C
    2. Defazio RA
    3. Sykes CM
    4. Moenter SM

    (2021) A role for glial fibrillary acidic protein (GFAP)-expressing cells in the regulation of gonadotropin-releasing hormone (GnRH) but not arcuate kisspeptin neuron output in male mice

    eLife 10:e68205.

    https://doi.org/10.7554/eLife.68205

    • PubMed
    • Google Scholar
62. 1. Vong L
    2. Ye C
    3. Yang Z
    4. Choi B
    5. Chua S
    6. Lowell BB

    (2011) Leptin action on GABAergic neurons prevents obesity and reduces inhibitory tone to POMC neurons

    Neuron 71:142–154.

    https://doi.org/10.1016/j.neuron.2011.05.028

    • PubMed
    • Google Scholar
63. 1. Wang L
    2. Vanacker C
    3. Burger LL
    4. Barnes T
    5. Shah YM
    6. Myers MG
    7. Moenter SM

    (2019) Genetic dissection of the different roles of hypothalamic kisspeptin neurons in regulating female reproduction

    eLife 8:e43999.

    https://doi.org/10.7554/eLife.43999

    • Google Scholar
64. 1. Wang L
    2. Guo W
    3. Shen X
    4. Yeo S
    5. Long H
    6. Wang Z
    7. Lyu Q
    8. Herbison AE
    9. Kuang Y

    (2020) Different dendritic domains of the GnRH neuron underlie the pulse and surge modes of GnRH secretion in female mice

    eLife 9:e53945.

    https://doi.org/10.7554/eLife.53945

    • PubMed
    • Google Scholar
65. 1. Welt CK
    2. Chan JL
    3. Bullen J
    4. Murphy R
    5. Smith P
    6. DePaoli AM
    7. Karalis A
    8. Mantzoros CS

    (2004) Recombinant human leptin in women with hypothalamic amenorrhea

    The New England Journal of Medicine 351:987–997.

    https://doi.org/10.1056/NEJMoa040388

    • PubMed
    • Google Scholar
66. 1. Williams KW
    2. Sohn JW
    3. Donato J
    4. Lee CE
    5. Zhao JJ
    6. Elmquist JK
    7. Elias CF

    (2011) The acute effects of leptin require PI3K signaling in the hypothalamic ventral premammillary nucleus

    The Journal of Neuroscience 31:13147–13156.

    https://doi.org/10.1523/JNEUROSCI.2602-11.2011

    • PubMed
    • Google Scholar
67. 1. Xu Y
    2. Kim ER
    3. Zhao R
    4. Myers MG Jr
    5. Munzberg H
    6. Tong Q

    (2013) Glutamate release mediates leptin action on energy expenditure

    Molecular Metabolism 2:109–115.

    https://doi.org/10.1016/j.molmet.2013.01.004

    • PubMed
    • Google Scholar
68. 1. Yeo SH
    2. Herde MK
    3. Herbison AE

    (2021) Morphological assessment of GABA and glutamate inputs to GnRH neurons in intact female mice using expansion microscopy

    Journal of Neuroendocrinology 33:1–14.

    https://doi.org/10.1111/jne.13021

    • PubMed
    • Google Scholar
69. 1. Yu WH
    2. Walczewska A
    3. Karanth S
    4. McCann SM

    (1997) Nitric oxide mediates leptin-induced luteinizing hormone-releasing hormone (LHRH) and LHRH and leptin-induced LH release from the pituitary gland

    Endocrinology 138:5055–5058.

    https://doi.org/10.1210/endo.138.11.5649

    • PubMed
    • Google Scholar
70. 1. Zuure WA
    2. Roberts AL
    3. Quennell JH
    4. Anderson GM

    (2013) Leptin signaling in GABA neurons, but not glutamate neurons, is required for reproductive function

    The Journal of Neuroscience 33:17874–17883.

    https://doi.org/10.1523/JNEUROSCI.2278-13.2013

    • PubMed
    • Google Scholar

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

• 207

  views

• 8

  downloads

• 0

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.