Formation of projection pathways from the arcuate nucleus of the hypothalamus to hypothalamic regions implicated in the neural control of feeding behavior in mice

Abstract

The arcuate nucleus of the hypothalamus (ARH) is a critical component of forebrain pathways that regulate a variety of neuroendocrine functions, including an important role in relaying leptin signals to other parts of the hypothalamus. However, neonatal rodents do not lose weight in response to leptin treatment in the same way as do adults, suggesting that certain aspects of leptin signaling pathways in the hypothalamus may not be mature. We tested this possibility by using DiI axonal labeling to examine the development of projections from the ARH to other parts of the hypothalamus in neonatal mice, paying particular attention to the innervation of the paraventricular nucleus (PVH), the dorsomedial nucleus (DMH), and the lateral hypothalamic area (LHA), each of which have been implicated in the regulation of feeding. The results indicate that ARH projections are quite immature at birth and appear to innervate the DMH, PVH, and LHA in succession, within distinct temporal domains. The projections from the ARH to the DMH develop rapidly and are established by the sixth postnatal day (P6), whereas those to the PVH develop significantly later, with the mature pattern of innervation first apparent between postnatal day 8 (P8)-P10. Furthermore, the ability of leptin to activate Fos in the PVH, DMH, and LHA appears to be age-dependent and correlates with the arrival of ARH projections to each nucleus. Taken together, these findings provide new insight into development of hypothalamic circuits and suggest an anatomical basis for the delayed postnatal regulation of food intake and body weight by leptin.

Figure 1.

A, Low magnification images of a Nissl-stained section showing a representative DiI implant in the ARH of a P10 mouse. The arrow indicates the location of the lateral margin of the ARH. B, Low magnification confocal image showing the appearance and distribution of DiI in a P10 mouse.

Figure 2.

Confocal images of arcuate DiI-labeled fibers in the DMH (A–C), PVH (D–F), and LHA (G–I) of mice on P4 (A), P6 (B, D), P8 (E, G), P12 (C, H), P14 (F), and P16 (I). A maximal projection image was derived from eight confocal images collected (10× objective) through a total distance of 16 μm in an 80-μm-thick section. fx, Fornix; V3, third ventricle.

Figure 3.

Quantitative comparison of DiI-labeled fibers within the DMH (A), LHA (B), parvicellular (C), and magnocellular (D) components of the PVH in mice at different stages of postnatal development. Bars depicts the mean of the total numbers if pixels in user-defined regions of thresholded, binarized images of DiI-labeled fibers, derived from a maximum projection images of 10 confocal image planes and collected with a 10×objective from three animals with comparable DiI implants centered in the ARH. ND, Not detectable.

Figure 4.

Confocal images of arcuate DiI-labeled fibers in the MPN (A,B), AVPV (C,D), BST (E,F), and LSv (G,H) of P10 (A,C), P12 (E,G), P14 (B), and P16 (D,F,H) mice. A maximal projection image was derived from eight confocal images collected (10× objective) through a total distance of 16 μm in an 80-μm-thick section. aco, Anterior commissures; BST, bed nuclei of the stria terminalis; LSv, ventral part of the lateral septal nucleus; MPN, median preoptic nucleus; V3, third ventricle; VL, lateral ventricle.

Figure 5.

A, Low magnification images of a Nissl-stained section showing a representative DiI implant in the DMH of a P6 mouse. B, Low magnification confocal image showing the appearance and distribution of DiI in a P6 mouse. C, D, Confocal images of DiI-labeled fibers in the PVH (C) and LHA (D) after DiI placement in the DMH of P6 mice (A, B). A maximal projection image was derived from eight confocal images collected (10× objective) through a total distance of 16 μm in an 80-μm-thick section.

Figure 7.

Number of Fos-IR cells in the ARH (A), PVH (B), and LHA (C) of mice that were administrated intraperitoneally with 10 mg/kg of leptin (white bars) or vehicle (black bars) on P6, P10, or P16. Values are the mean ± SEM (n = 3). Differences between groups were determined by ANOVA. *p < 0.05 compared with vehicle-treated mice.

Figure 6.

Confocal images illustrating the expression of Fos-immunoreactivity (Fos-IR) in the ARH (A–C), PVH (D–F), and LHA (G–I) 90 min (A–C, G–I) or 120 min (D–F) after intraperitoneal administration of leptin (10 mg/kg) on P6 (B, E, H) or P16 (C, F, I) mice. A, D, and G show typical Fos-IR in mice injected with vehicle (here on P10). A maximal projection image was derived from 10 confocal images collected (25× objective) through a total distance of 20μm.

Figure 8.

Leptin activates POMC neurons during the postnatal life. A, Representative confocal image showing induction of Fos-IR (green fluorescence) in arcuate neurons containing α-melanocyte stimulating hormone (α-MSH, red fluorescence; arrows) of a P10 mouse 90 min after leptin administration. B, Line drawing of a representative section through the ARH to illustrate the distribution of leptin-induced Fos-IR in POMC neurons (red squares). Cells containing only POMC (empty squares) or Fos (circles) are also represented.