Neonatal leptin exposure specifies innervation of presympathetic hypothalamic neurons and improves the metabolic status of leptin-deficient mice

Abstract

The paraventricular nucleus of the hypothalamus (PVH) consists of distinct functional compartments regulating neuroendocrine, behavioral, and autonomic activities that are involved in the homeostatic control of energy balance. These compartments receive synaptic inputs from neurons of the arcuate nucleus of the hypothalamus (ARH) that contains orexigenic agouti-related peptide (AgRP) and anorexigenic pro-opiomelanocortin (POMC) neuropeptides. The axon outgrowth from the ARH to PVH occurs during a critical postnatal period and is influenced by the adipocyte-derived hormone leptin, which promotes its development. However, little is known about leptin's role in specifying patterns of cellular connectivity in the different compartments of the PVH. To address this question, we used retrograde and immunohistochemical labeling to evaluate neuronal inputs onto sympathetic preautonomic and neuroendocrine neurons in PVH of leptin-deficient mice (Lep(ob)/Lep(ob)) exposed to a postnatal leptin treatment. In adult Lep(ob)/Lep(ob) mice, densities of AgRP- and α-melanocortin stimulating hormone (αMSH)-immunoreactive fibers were significantly reduced in neuroendocrine compartments of the PVH, but only AgRP were reduced in all regions containing preautonomic neurons. Moreover, postnatal leptin treatment significantly increased the density of AgRP-containing fibers and peptidergic inputs onto identified preautonomic, but not onto neuroendocrine cells. Neonatal leptin treatment neither rescued αMSH inputs onto neuroendocrine neurons, nor altered cellular ratios of inhibitory and excitatory inputs. These effects were associated with attenuated body weight gain, food intake and improved physiological response to sympathetic stimuli. Together, these results provide evidence that leptin directs cell type-specific patterns of ARH peptidergic inputs onto preautonomic neurons in the PVH, which contribute to normal energy balance regulation.

Figure 1.

Fluorogold injection site in the thoracic spinal cord. A, Schematic overview of the thoracic level of the spinal cord. B, Illustration of a representative injection site of Fluorogold (green channel) in the intermediolateral column of the thoracic spinal cord visualized using DAPI nuclear staining (red channel).

Figure 2.

Leptin impacts the peptidergic innervation of preautonomic divisions in adult mouse PVH. A, Representative confocal images through the level of the PVH (delineated using antisera against HuC/D, red channel), illustrating the pattern of retrograde labeling following injection of Fluorogold (green channel) in the spinal cord of WT mice at the upper thoracic level (T1–T2). Preautonomic neurons were observed in the rostral and caudal aspects of the ventral medial parvicellular (rmpv and cmpv), dorsal parvicellular (dp) and lateral parvicellular (lp) parts. Outlined regions identify volumes of quantification. V3, Third ventricle. Scale bar, 100 μm. B, Quantitative comparison of the density of AgRP- and αMSH-immunoreactive fibers innervating the mpv, dp, and lp autonomic parts of the PVH in WT (n = 6), Lep^ob/Lep^ob vehicle-treated (n = 5), and Lep^ob/Lep^ob leptin-treated mice (n = 6). The values represent mean ± SEM of fiber length per set of volume sampled. Significance between groups was determined using one-way ANOVA followed by pairwise post hoc tests; ***p < 0.001, **p < 0.01, and *p < 0.05 between vehicle- or leptin-treated Lep^ob/Lep^ob mice and their WT littermates. mpv, Ventral zone of the medial parvicellular part; dp, dorsal parvicellular; lp, lateral parvicellular parts of the PVH.

Figure 3.

Leptin impacts the axonal terminal densities of preautonomic divisions in adult mouse PVH. A, B, Single confocal image: plane of HuC/D, AgRP, synaptophysin (A) and HuC/D, αMSH, synapsin 1/2 (B) triple-labeled immunofluorescence at the level of the PVH of WT mouse. Arrows indicate examples of dual-labeled AgRP and synaptophysin (A), or αMSH- and synapsin 1/2-immunopositive (B) terminals in close contact with PVH soma (identified using antisera against HuC/D). Scale bar, 10 μm. C, D, Quantitative comparison of the number of AgRP (C) and αMSH-IR (D) varicosities in close apposition to spinal cord projecting neurons of the PVH in WT (n = 6), Lep^ob/Lep^ob vehicle-treated (n = 5), and Lep^ob/Lep^ob leptin-treated mice (n = 6). The values represent mean ± SEM of density of inputs. Significance between groups was determined using one-way ANOVA followed by pairwise post hoc tests; ***p < 0.001, **p < 0.01, and *p < 0.05 between vehicle- or leptin-treated Lep^ob/Lep^ob mice and their WT littermates.

Figure 4.

Postnatal leptin exposure does not rescue peptidergic innervation of neurosecretory divisions in adult mouse PVH. A, Representative confocal images through the level of the PVH (delineated using antisera against HuC/D, red channel) illustrate the pattern of retrograde labeling following injection of Fluorogold in the blood compartment (green channel). Fluorogold labeling is observed in the medial parvicellular part, dorsal zone (mpd) and the posterior magnocellular part, lateral zone (pml) of the PVH. Outlined regions identify volumes of quantification. V3, Third ventricle. Scale bar, 100 μm. B, Quantitative comparison of the density of AgRP- and αMSH-immunoreactive fibers innervating the mpd and pml neuroendocrine parts of the PVH in WT (n = 6), Lep^ob/Lep^ob vehicle-treated (n = 5), and Lep^ob/Lep^ob leptin-treated mice (n = 6). The values represent mean ± SEM of fiber length per set of volume sampled. Significance between groups was determined using one-way ANOVA followed by pairwise post hoc tests; ***p < 0.001, **p < 0.01 and *p < 0.05 between vehicle- or leptin-treated Lep^ob/Lep^ob mice and their WT littermates.

Figure 5.

Leptin modulates neuropeptidergic terminal densities to neurosecretory neurons in the adult PVH. A, High magnification confocal images of AgRP, αMSH, FG and HuC/D immunofluorescence in the neurosecretory division of the PVH. Three-dimensional rendering view of the image volume depicting the quantification of terminal densities (AgRP, blue objects and αMSH, pink objects) in close contact with neuronal cells (HuC/D) filled with FG retrograde tracer (orange objects, right). Post-processing, reconstruction, and quantitative analyses of confocal images were performed using Improvision Volocity software. Scale bar, 20 μm. B, Quantitative comparison of AgRP- and αMSH-IR varicosities in close apposition to neurosecretory neurons of the PVH in WT (n = 6), Lep^ob/Lep^ob vehicle-treated (n = 5), and Lep^ob/Lep^ob leptin-treated mice (n = 6). The values represent mean ± SEM of number of inputs per volume analyzed. Significance between groups was determined using one-way ANOVA followed by pairwise post hoc tests; ***p < 0.001 and *p < 0.05 between vehicle- or leptin-treated Lep^ob/Lep^ob mice and their WT littermates. mpd, Medial parvicellular part, dorsal zone; pml, posterior magnocellular part, lateral zone of the PVH.

Figure 6.

GABAergic and glutamatergic innervation of functionally identified adult PVH neuronal compartments. A, Confocal images illustrating GABAergic (VGAT) and Glutamatergic (vGlut2) immunoreactive appositions on preautonomic neurons visualized by the fluorescent dye Fluorogold following spinal cord injection. Scale bar, 5 μm. B, C, Quantitative comparison of the density or ratio of vGlut2-IR over VGAT-IR terminals onto preautonomic (B) or neurosecretory (C) neurons in the PVH in WT (n = 6), Lep^ob/Lep^ob vehicle-treated (n = 5), and Lep^ob/Lep^ob leptin-treated mice (n = 6). The values represent mean ± SEM of number of inputs per volume sampled. Significance between groups was determined using one-way ANOVA followed by pairwise post hoc tests **p < 0.01, *p < 0.05 between vehicle- or leptin-treated Lep^ob/Lep^ob mice and their WT littermates. am, Anterior magnocellular part; dp, dorsal parvicellular part; f, forniceal part; mm, medial magnocellular part; mpv, medial parvicellular part, ventral zone, rostral (rmpv) and caudal aspects (cmpv); mpd, medial parvicellular part, dorsal zone; lp, lateral parvicellular part; pmm, posterior magnocellular part, medial zone; pml, posterior magnocellular part, lateral zone; pv, periventricular part of the PVH; IML, intermediolateral column; PP, posterior pituitary; ME, median eminence. Schematics are adapted from Biag et al. (2012).

Figure 7.

Postnatal leptin exposure reduces body weight gain and food intake of adult leptin-deficient mice. A, B, Preweaning (A) and postweaning (B) growth curves of WT vehicle-treated (WT Veh), Lep^ob/Lep^ob vehicle-treated (Lep^ob/Lep^ob Veh), and Lep^ob/Lep^ob leptin-treated (Lep^ob/Lep^ob Lep), male mice (n > 25 per group). C–E, Food intake measurement normalized to body weight (BW) per 24 h (C) and during the light period (D) and dark period (E) of 11-week-old WT Vehicle (n = 15), Lep^ob/Lep^ob Vehicle (n = 8), and Lep^ob/Lep^ob Leptin male mice (n = 12). F, Graphical representation of the relative distribution of total food intake during the light and dark periods. ^#p < 0.05 between vehicle- and leptin-treated Lep^ob/Lep^ob mice. Basal glucose levels in ad libitum fed (G) or 14 h fasting (H) male WT (n = 15), Lep^ob/Lep^ob vehicle-treated (n = 9), and Lep^ob/Lep^ob leptin-treated male mice (n = 12) at 6–8 weeks of age. I, Following a bolus of glucose (intraperitoneal injection), glucose tolerance test (GTT) was analyzed in 6- to 7-week-old mice. J, Insulin tolerance test (ITT) was assessed following an insulin bolus in 7- to 8-week-old WT (n = 15), Lep^ob/Lep^ob Vehicle (n = 9), and Lep^ob/Lep^ob Leptin male mice (n = 12). All data are mean ± SEM. One-way or two-way ANOVA and pairwise post hoc tests were performed for each dependent variable. *p < 0.05, **p < 0.01, and ***p < 0.001. In A and B, only the significance between vehicle- or leptin-treated Lep^ob/Lep^ob mice is represented.

Figure 8.

Postnatal leptin treatment improves the thermogenic response of leptin-deficient mice during a cold or a nutritional challenge. A, Core temperature during a 210 min cold challenge at +4°C of 9- to 10-week-old WT Vehicle (n = 15), Lep^ob/Lep^ob Vehicle (n = 8), and Lep^ob/Lep^ob Leptin male mice (n = 12). B, C, Basal core body temperatures of ad libitum fed (B) or 14 h fasting (C) WT Veh (n = 15), Lep^ob/Lep^ob Vehicle (n = 8), and Lep^ob/Lep^ob Leptin (n = 12) male mice at 8–10 weeks. D, Basal core temperature difference (delta temperature) between fed and fasted states. E, Representative confocal images of epididymal WAT labeled using an antisera against PerilipinA/B (top) and bright-field images of BAT stained with hematoxylin and eosin on paraffin-embedded sections (bottom) from 20-week-old WT Vehicle (n = 4), Lep^ob/Lep^ob Vehicle (n = 4), and Lep^ob/Lep^ob Leptin (n = 4) male mice. Scale bars: 50 μm for WAT and 200 μm for BAT. F, Quantification of WAT cell surface area. G, Plasma levels of NEFA at 20 weeks. All data are mean ± SEM. One-way or two-way ANOVA and pairwise post hoc tests were performed for each dependent variable. *p < 0.05, **p < 0.01, and ***p < 0.001. In A, only the significance between vehicle- or leptin-treated Lep^ob/Lep^ob mice is represented.

Figure 9.

Postnatal leptin treatment increases in vivo leptin anorectic response in Lep^ob/Lep^ob mice. In vivo leptin sensitivity in adult mice exposed to different neonatal leptin environments. Twelve-week-old mice (n = 7–9 mice per group) were injected intraperitoneally twice daily with Sodium Citrate (Vehicle) for 3 d, then with leptin (2 mg/kg) for 3 d. Food intake was measured twice a day during the two injection periods. A, Average 24 h food intake during injection periods. B, Food intake following leptin treatment is represented as a percentage of inhibition versus control treatment. All data are mean ± SEM. Two-way ANOVA and pairwise post hoc test were performed for each dependent variable. *p < 0.05, **p < 0.01, ***p < 0.001.