Fig 1.
Close anatomical association between POMC and AgRP fibers and oxytocin neurons in the paraventricular nucleus.
(A, C). Representative images of OT (PS38) immunoreactive neurons and (A) POMC- and (C) AgRP-immunoreactive fibers in the paraventricular nucleus of the hypothalamus (PVH) of adult wild-type mice. (B, D). Quantification of the shortest distance between (B) POMC- or (D) AgRP-immunoreactive fibers and OT-immunoreactive cell bodies processes within the PVH (n = 5 animals per group). V3, third ventricle. Scale bar, 50 μm. The data underlying this Figure can be found in S1 Data.
Fig 2.
Neonatal chemogenetic inhibition of OT neurons disrupts melanocortin circuits.
(A) Description of the animal model. Created in BioRender. Bouret, S. (2025) https://BioRender.com/v6d7iia. (B) Representative images of OT neurons containing mCitrine in the paraventricular nucleus (PVH). (C–F) Representative images and quantification of (C, E) POMC and (D, F) AgRP fibers innervating the (C, D) PVH and (E, F) dorsomedial nucleus (DMH) of adult R26-LSL-hM4Di-DREADD (control) and Ot-Cre::R26-LSL-hM4Di-DREADD injected with Compound-21 neonatally, or during juvenile life, or during adulthood (n = 6–7 animals per group). (G, H) Representative images and quantification of OT (PS38+) neurons and fibers innervating the (G) PVH and (H) lateral hypothalamic area (LHA), respectively, in adult R26-LSL-hM4Di-DREADD (control) and Ot-Cre::R26-LSL-hM4Di-DREADD injected with Compound-21 neonatally, or during juvenile life, or during adulthood (n = 4–5 animals per group). Data are presented as mean + SEM. ***P < 0.001. Scale bar, 200 μm. The data underlying this Figure can be found in S1 Data.
Fig 3.
Normal development of melanocortin circuits in nitric oxide-deficient mice.
(A) Representative images of single optical sections of the hypothalamus of P10 mice showing OT and nitric oxide co-labeling in the paraventricular nucleus (PVH). (B–E) Representative images and quantification of (B, D) POMC and (C, E) AgRP fibers innervating the (B, C) PVH and (D, E) dorsomedial nucleus (DMH) of adult wild-type (control) and nNOS KO mice (n = 4-5 animals per group). (F, G) Representative images and quantification of OT (PS38+) neurons and fibers innervating the (F) PVH and (G) lateral hypothalamic area (LHA), respectively, in adult wild-type (control) and nNOS KO mice (n = 5 animals per group). Data are presented as mean + SEM. Scale bars, 40 μm (A), 200 μm (B–G). The data underlying this Figure can be found in S1 Data.
Fig 4.
Blocking exocytosis of OT neurons perturbs the development of melanocortin circuits.
(A) Description of the animal model. Created in BioRender. Bouret, S. (2025) https://BioRender.com/v6d7iia. (B–E) Representative images and quantification of (B, D) POMC and (C, E) AgRP fibers innervating the (B, C) paraventricular nucleus (PVH) and (D, E) dorsomedial nucleus (DMH) of adult BoNT/B (control) and Ot-Cre::BoNT/B mice (n = 4−8 animals per group). (F, G) Representative images and quantification of OT (PS38+) neurons and fibers innervating the (F) PVH and (G) lateral hypothalamic area (LHA), respectively, in adult BoNT/B (control) and Ot-Cre::BoNT/B mice (n = 8−4 animals per group). Data are presented as mean + SEM. *P < 0.05, **P < 0.01. Scale bar, 200 μm. The data underlying this Figure can be found in S1 Data.
Fig 5.
Distribution of oxytocin fibers and expression pattern of oxytocin receptor in the developing hypothalamus.
(A) Description of the animal model and (B) representative images showing immuno- (PS 38 antibody) and genetic (Ot mGFP) labeling of OT neurons and fibers at the levels of the paraventricular nucleus (PVH) and arcuate nucleus (ARH) at P0, P25, and P60. (C, D) Representative images showing oxytocin receptor (Otr) mRNA at the level of (C) the arcuate/ventromedial/dorsomedial nuclei and (D) the paraventricular nucleus of a P10 WT mouse. (E) Relative expression of Otr mRNA in the ARH, VMH, DMH, and PVH of WT mice at P0, P5, P10, P25, and P60 (n = 3-8 animals per group). (F) Representative images and (G) quantification of Otr co-expression in Pomc and Agrp neurons at P0, P5, P10, P25, and P60 (n = 6–8 animals per group). Data are presented as means ± SEM. LHA, lateral hypothalamic area; me, median eminence; SON, supraoptic nucleus. Scale bar 50 μm. The data underlying this Figure can be found in S1 Data. Fig 5A is created in BioRender. Bouret, S. (2025) https://BioRender.com/d5iyc5j.
Fig 6.
Oxytocin promotes the growth of arcuate nucleus axons.
Representative images and quantification of βIII-tubulin–immunopositive fibers from isolated cultures of neonatal ARH incubated for 48 hours with vehicle or oxytocin (100 ng/ml) (n = 4–8 per group). Data are presented as means + SEM. The data underlying this Figure can be found in S1 Data.
Fig 7.
Neonatal blockade of oxytocin signaling disrupts melanocortin circuits.
(A) Description of the animal model. Created in BioRender. Bouret, S. (2025) https://BioRender.com/d5iyc5j. (B–E) Representative images and quantification of (B, D) POMC and (C, E) AgRP fibers innervating the (B, C) paraventricular nucleus (PVH) and (D, E) dorsomedial nucleus (DMH) of adult wild-type mice injected with saline (control) or an oxytocin receptor antagonist (L-368,899, OTRA) neonatally (n = 8−5 animals per group). (F, G) Representative images and quantification of OT (PS38+) neurons and fibers innervating the (F) PVH and (G) lateral hypothalamic area (LHA), respectively, in adult mice injected with saline (control) or an oxytocin receptor antagonist (L-368,899, OTRA) neonatally (n = 7−6 animals per group). Data are presented as means + SEM. **P < 0.01. Scale bar 200 μm. The data underlying this Figure can be found in S1 Data.
Fig 8.
Neonatal blockade of oxytocin signaling causes lifelong metabolic dysregulations.
(A) Body weight, (B) body composition, (C) cumulative and (D) average food intake, (E) 24 h and (F) average respiratory exchange ratio, (G) average ambulatory activity, (H) 24 h and (I) average energy expenditure, (J) average Z rearing, (K) fasting glycemia, (L) glucose tolerance test (GTT) and (M) area under the GTT curve in adult male mice injected with saline (control) or an oxytocin receptor antagonist (L-368,899, OTRA) neonatally (n = 6 animals per group). Data are presented as means ± SEM. *P < 0.05, **P < 0.01. The data underlying this Figure can be found in S1 Data.
Fig 9.
Neonatal chemogenetic activation of OT neurons rescues POMC circuit deficits in Magel2 KO mice.
(A) Description of the animal model and its validation through specific activation of OT neurons (cFos immunoreactivity) following Compound-21 injection. Created in BioRender. Bouret, S. (2025) https://BioRender.com/bc3g0nq. (B–E) Representative images and quantification of (B, D) POMC and (C, E) AgRP fibers innervating the (B, C) paraventricular nucleus (PVH) and (D, E) dorsomedial nucleus (DMH) of adult Magel2 KO, R26-hM3Dq-DREADD (control) and Magel2 KO, Ot-Cre::R26-hM3Dq-DREADD mice injected with Compound-21 neonatally (n = 5−6 animals per group). (F, G) Representative images and quantification of OT (PS38+) neurons and fibers innervating the (F) PVH and (G) lateral hypothalamic area (LHA), respectively, in adult Magel2 KO, R26-hM3Dq-DREADD (control) and Magel2 KO, Ot-Cre::R26-hM3Dq-DREADD mice injected with Compound-21 neonatally (n = 5−3 animals per group). Data are presented as mean + SEM. *P < 0.05. Scale bar, 200 μm. The data underlying this Figure can be found in S1 Data.