Developmental changes in hypothalamic leptin receptor: relationship with the postnatal leptin surge and energy balance neuropeptides in the postnatal rat

Abstract

In the adult brain, leptin regulates energy homeostasis primarily via hypothalamic circuitry that affects food intake and energy expenditure. Evidence from rodent models has demonstrated that during early postnatal life, leptin is relatively ineffective in modulating these pathways, despite the high circulating levels and the presence of leptin receptors within the central nervous system. Furthermore, in recent years, a neurotrophic role for leptin in the establishment of energy balance circuits has emerged. The precise way in which leptin exerts these effects, and the site of leptin action, is unclear. To provide a detailed description of the development of energy balance systems in the postnatal rat in relation to leptin concentrations during this time, endogenous leptin levels were measured, along with gene expression of leptin receptors and energy balance neuropeptides in the medial basal hypothalamus, using in situ hybridization. Expression of leptin receptors and both orexigenic and anorexigenic neuropeptides increased in the arcuate nucleus during the early postnatal period. At postnatal day 4 (P4), we detected dense leptin receptor expression in ependymal cells of the third ventricle (3V), which showed a dramatic reduction over the first postnatal weeks, coinciding with marked morphological changes in this region. An acute leptin challenge robustly induced suppressor of cytokine signaling-3 expression in the 3V of P4 but not P14 animals, revealing a clear change in the location of leptin action over this period. These findings suggest that the neurotrophic actions of leptin may involve signaling at the 3V during a restricted period of postnatal development.

Fig. 1.

Postnatal profiles of leptin (A), glucose (B) and insulin (C) in the male rat over the early postnatal period.

Fig. 2.

Semiquantitative analysis of leptin receptors ObR (A, B) and ObRb (C, D) mRNA expression in the arcuate nucleus (ARC; A, C) and ventromedial hypothalamic nucleus (VMH; B, D) in the postnatal rat brain. Results are expressed as a percentage of P4 values. Significant differences in receptor mRNA expression across postnatal ages are indicated as *P < 0.05, **P < 0.01, ***P < 0.001, compared with P4 values.

Fig. 3.

Semiquantitative analysis of ObR mRNA in the third ventricle (3V), showing the dramatic reduction in this region over the early postnatal weeks, and inset of representative autoradiograph sections illustrating changes in ObR distribution in the medial basal hypothalamus. Significant differences in 3V mRNA expression in across postnatal ages are indicated as ***P < 0.001, compared with P4 values. ND indicates no detectable signal.

Fig. 4.

Changes in hypothalamic leptin receptor (ObR) mRNA distribution over the early postnatal period. A: representative brightfield photomicrograph indicating hypothalamic regions analyzed. B: representative darkfield photomicrographs of emulsion-coated sections illustrating the changing distribution in the 3V, ARC, and VMH at P4 (i), P10 (ii), and P19 (iii). C: higher magnification of the 3V boxed region indicated in A showing morphological changes over the early postnatal period at P4 (i), P10 (ii), and P19 (iii). Scale bars: 100 μm (A and B), and 50 μm (C).

Fig. 5.

Semiquantitative analysis of neuropeptide mRNA expression in the rat hypothalamus during the early postnatal period. Proopiomelanocortin (POMC) (A), cocaine- and amphetamine-regulated transcript (CART) (B), neuropeptide Y (NPY) (C) and Agouti-related peptide (AgRP) (D) mRNA expression in the arcuate nucleus (ARC). Results are expressed as a percentage of P4 values. Significant differences in mRNA expression across postnatal ages are indicated as *P < 0.05, **P < 0.01, ***P < 0.001, compared with P4 values.

Fig. 6.

Suppressor of cytokine signaling-3 (SOCS-3) induction in the postnatal hypothalamus in response to an acute leptin injection (3 μg/g ip). Representative images of saline- or leptin-injected animals at P4 or P14 are shown in (A), and semiquantification of SOCS-3 mRNA induction in the 3V (B), ARC (C), and VMH (D). Solid bars denote saline-injected animals; open bars denote leptin-injected animals. Significant differences in SOCS3 mRNA expression are indicated as **P < 0.01, ***P < 0.001, compared with P4 saline-injected values, or P14 saline-injected animals in the case of VMH data.

Fig. 7.

Representative brightfield photomicrographs illustrating the hypothalamic regions of interest (A) and the corresponding darkfield photomicrographs of emulsion-coated sections (B) demonstrating the change in distribution of SOCS3 induction by leptin at P4 and P14. At P4, a high density of silver grains is apparent over the 3V in leptin-injected animals, and this effect is absent at P14. Induction of SOCS3 is apparent in the ARC at both ages. Scale bars = 100 μm.

Fig. 8.

Representative brightfield photomicrographs of nestin (A, B) and SOCS3 (C, D) gene expression in 3V ependymal cells. Nestin expression is seen in essentially all ependymal 3V cells in both saline- and leptin-injected animals, as indicated by the density of silver grains over this entire region. In contrast, SOCS3 expression was minimal in saline-injected animals but clearly increases in response to leptin injection (D) both in ependyaml cells and those adjacent to the 3V. Scale bars = 20 μm.