Changes in neuropeptide Y receptors and pro-opiomelanocortin in the anorexia (anx/anx) mouse hypothalamus

Abstract

The pro-opiomelanocortinergic (POMCergic) system originating in the hypothalamic arcuate nucleus extends projections widely over the brain and has been shown to be intricately linked and parallel to the arcuate neuropeptide Y (NPY) system. Both NPY and POMC-derived peptides (melanocortins) have been strongly implicated in the control of feeding behavior, with the former exerting orexigenic effects and the latter having anorexigenic properties. Mice homozygous for the lethal anorexia (anx) mutation are hypophagic, emaciated, and exhibit anomalous processing of NPY exclusively in the arcuate nucleus, providing an interesting model to study NPY-POMC interactions. In the present study, several morphological markers were used to investigate the histochemistry and morphology of the POMC system in anx/anx mice. In situ hybridization demonstrated decreased numbers of POMC mRNA-expressing neurons in the anx/anx arcuate nucleus. In parallel, mRNA levels for both the NPY Y1 and Y5 receptors, which are expressed in POMC neurons, were decreased. Also, expression of the NPY Y2 autoreceptor was attenuated. Immunohistochemistry using antibodies against adrenocorticotropic hormone to demonstrate POMC cell bodies, against alpha-melanocyte-stimulating hormone to demonstrate axonal projections and against the NPY Y1 receptor to demonstrate dendritic arborizations, showed strikingly decreased immunoreactivities for all these markers. The present data suggest that degeneration of the arcuate POMC system is a feature characteristic of the anx/anx mouse. The possible relationship to the NPYergic phenotype of this animal is discussed.

Fig. 1.

Dark-field micrograph of the arcuate nucleus from a wild-type (a) and ananx/anx (b) mouse after hybridization for POMC mRNA. Note fewer silver grains over individual cell profiles (arrows) in the mutant animal. Scale bar, 50 μm.

Fig. 2.

Dark-field micrographs of arcuate nuclei from wild-type (a, c, e) andanx/anx (b,d, f) mice after hybridization for Y1R (a, b), Y2R (c,d), and Y5R (e, f) mRNAs. There is an attenuation of signal for all three mRNAs in mutant animals. Scale bar, 50 μm.

Fig. 3.

Relative POMC, Y1R, Y2R, and Y5R mRNA levels in wild-type (filled bars) andanx/anx (stippled bars) mice, as measured by quantitative autoradiography. Levels of all four mRNA transcripts are significantly lower in mutant mice. Statistical analysis was performed by using ANOVA and Mann–Whitney U test. ***p < 0.001.

Fig. 4.

Fluorescence micrographs of sections from the arcuate nucleus of a wild-type (a, c) and an anx/anx (b,d) mouse stained with antiserum against ACTH. The number of ACTH-IR cell bodies (a, arrows) is reduced in mutant mice. c and d represent a higher magnification of a and b, respectively. Asterisk indicates third ventricle.ME, Median eminence. Scale bars: (in a),a, b, 50 μm; (in c)c, d, 50 μm.

Fig. 5.

Fluorescence micrographs of sections from the periventricular area (a, b) and arcuate nucleus (c–f) of wild-type (a,c, e) andanx/anx (b,d, f) mice stained with antiserum against α-MSH. The number of α-MSH-IR terminals (arrows) are reduced in mutant mice. eand f represent a higher magnification ofc and d, respectively.Asterisk indicates third ventricle. Arc, Arcuate nucleus; DMH, dorsomedial hypothalamic nucleus;SCh, suprachiasmatic nucleus; VMH, ventromedial hypothalamic nucleus. Scale bars: (in a)a, b, e, f, 50 μm; (in c) c, d, 100 μm.

Fig. 6.

a, Quantification of the number of ACTH-IR neuron profiles in the arcuate nucleus. There is a significant reduction in the number of stained profiles inanx/anx mice compared with wild-type controls. b, Density of α-MSH-IR terminals in the periventricular area of anx/anx compared with wild-type mice. Density was quantified as the number of crossings between the lines of a grid inserted in the microscope eyepiece and stained terminals. The number of line crossings is significantly reduced in mutant mice. Statistical analysis was performed by using ANOVA and Mann–Whitney U test. ***p< 0.001.

Fig. 7.

Fluorescence (a, b) and confocal (c, d) micrographs of sections from the arcuate nucleus of a wild-type (a,c) and an anx/anx(b, d) mouse stained with antiserum against the Y1R. Note dramatic reduction in dendritic arborizations (arrowheads), as well as attenuation in fluorescence and number of cell bodies (arrows) inanx/anx mice. Asteriskindicates third ventricle. ME, Median eminence. Scale bars: (in a) a, b, 50 μm; (in c) c, d, 10 μm.

Fig. 8.

Fluorescence micrographs of sections from the paraventricular hypothalamic nucleus of a wild-type (a, c) and ananx/anx (b,d) mouse stained with antiserum against the Y1R. (Micrographs in a and b are magnified inc and d, respectively). There is a reduction in both cell bodies (arrows) and dendritic arborizations. Asterisk indicates third ventricle.mp, Medial parvocellular nucleus; lm, lateral magnocellular nucleus. Scale bar, 50 μm.