The Journal of Neuroscience, August 15, 1999, 19(16):7130–7139 Changes in Neuropeptide Y Receptors and Pro-Opiomelanocortin in the Anorexia (anx/anx) Mouse Hypothalamus Christian Broberger,1 Jeanette Johansen,2 Hjalmar Brismar,3 Carolina Johansson,2 Martin Schalling,2 and Tomas Ho¨ kfelt1 Departments of 1Neuroscience, 2Molecular Medicine, and 3Woman and Child Health, Karolinska Institutet, 171 77 Stockholm, Sweden The pro-opiomelanocortinergic (POMCergic) system originating NPY Y1 and Y5 receptors, which are expressed in POMC in the hypothalamic arcuate nucleus extends projections widely neurons, were decreased. Also, expression of the NPY Y2 over the brain and has been shown to be intricately linked and autoreceptor was attenuated. Immunohistochemistry using an- parallel to the arcuate neuropeptide Y (NPY) system. Both NPY tibodies against adrenocorticotropic hormone to demonstrate and POMC-derived peptides (melanocortins) have been POMC cell bodies, against a-melanocyte-stimulating hormone strongly implicated in the control of feeding behavior, with the to demonstrate axonal projections and against the NPY Y1 former exerting orexigenic effects and the latter having anorex- receptor to demonstrate dendritic arborizations, showed strik- igenic properties. Mice homozygous for the lethal anorexia (anx) ingly decreased immunoreactivities for all these markers. The mutation are hypophagic, emaciated, and exhibit anomalous present data suggest that degeneration of the arcuate POMC processing of NPY exclusively in the arcuate nucleus, providing system is a feature characteristic of the anx/anx mouse. The an interesting model to study NPY–POMC interactions. In the possible relationship to the NPYergic phenotype of this animal present study, several morphological markers were used to is discussed. investigate the histochemistry and morphology of the POMC Key words: adrenocorticotropic hormone; a-melanocyte- system in anx/anx mice. In situ hybridization demonstrated stimulating hormone; arcuate nucleus; feeding; immunohisto- decreased numbers of POMC mRNA-expressing neurons in the chemistry; in situ hybridization; paraventricular nucleus of the anx/anx arcuate nucleus. In parallel, mRNA levels for both the hypothalamus; trophism The neurochemical basis for the central control of food intake has The projections of the arcuate NPY and POMC neurons follow been illuminated by recent investigations (Leibowitz, 1995; seemingly parallel routes (Broberger et al., 1998b) and may ter- Sawchenko, 1998; Woods et al., 1998; Elmquist et al., 1999; Kalra minate on common postsynaptic targets. NPY neurons also in- et al., 1999). Two messenger molecules expressed in discrete nervate the POMC cell bodies, which express the inhibitory NPY populations of the hypothalamic arcuate nucleus appear to play Y1 receptor (Y1R) (Csiffa´ry et al., 1990; Zhang et al., 1994; antagonistic roles in energy balance control, namely neuropeptide Broberger et al., 1997b; Fuxe et al., 1997). A similar expression Y (NPY) (Tatemoto, 1982; Tatemoto et al., 1982) and pro- pattern has been suggested for the Y5 receptor (Y5R) (Gerald et opiomelanocortin (POMC) (Mains et al., 1977; Roberts and Her- al., 1996; Naveilhan et al., 1998a). Activation of the Y1R and the bert, 1977; Nakanishi et al., 1979), the precursor protein for the Y5R (Flood and Morley, 1989; Stanley et al., 1992; Gerald et al., melanocortin peptides adrenocorticotropic hormone (ACTH) 1996) stimulates food intake, possibly through inhibition of a a and -melanocyte-stimulating hormone ( -MSH). Intracerebral POMC signaling. In addition, NPY neurons also express agouti injections of NPY stimulate food intake (Clark et al., 1984; gene-related protein (AGRP) (Shutter et al., 1997; Broberger et Stanley and Leibowitz, 1985), whereas melanocortin treatment al., 1998a,b; Hahn et al., 1998), an antagonist of the melanocortins induces anorexia (Poggioli et al., 1986; Fan et al., 1997; Grill et (Ollmann et al., 1997). Thus, both anatomical and pharmacolog- al., 1998), and mice deficient for the melanocortin-4 receptor ical evidence suggest that the balance between NPY and POMC develop obesity (Huszar et al., 1997). Furthermore, fasting in- transmission may be correlated to the level of food intake. creases arcuate NPY mRNA levels and decreases POMC mRNA The anorexia (anx) mutation is a recessive autosomal mutation (Brady et al., 1990). in mice that phenotypically results in decreased food intake, emaciation, and premature death during the fourth postnatal week (Maltais et al., 1984). A possible explanation for this phe- Received Feb. 11, 1999; revised April 28, 1999; accepted June 3, 1999. notype was provided by the recent demonstration of NPY abnor- This study was supported by the Swedish Medical Research Council (Grants malities in anx/anx mice that appear to be restricted to the arcuate 14X-2887 and 13X-10909 for T.H. and M.S., respectively), Marianne and Marcus Wallenberg’s Foundation, a Bristol-Myers Squibb Unrestricted Neuroscience grant system, characterized by a decrease in the number of its NPY- to T.H., National Alliance for Research on Schizophrenia and Depression, and immunoreactive (-IR) nerve terminals and a strongly increased funds from Karolinska Institutet and the Karolinska Hospital. We thank Drs. John Walsh and Helen Wong, (CURE, University of California at Los Angeles, Los perikaryal staining in the arcuate nucleus, a pattern suggestive Angeles, CA) for kindly providing Y1 receptor antiserum (antibody/Radioimmu- of cell body accumulation and impaired transport and/or release noassay Core Grant DK41301). (Broberger et al., 1997a). The same pattern was also noted with Correspondence should be addressed to Dr. Christian Broberger, Department of Neuroscience, Karolinska Institutet, 171 77 Stockholm, Sweden. AGRP histochemistry (Broberger et al., 1998b). In accordance Copyright © 1999 Society for Neuroscience 0270-6474/99/197130-10$05.00/0 with the orexigenic function of arcuate NPY neurons described Broberger et al. • Arcuate POMC in anx/anx Mice J. Neurosci., August 15, 1999, 19(16):7130–7139 7131 above, diminished NPY–AGRP signaling could be causally re- in distilled water, exposed for 1 (POMC probe) or 8 (Y1R, Y2R, and 2 lated to the anorectic phenotype of anx/anx mice. Y5R probes) weeks in light-free metal containers at 20°C, developed in Kodak D19 for 3 min, and fixed in Kodak 3000 for 6–7 min. Sections The present study investigated the histochemistry of POMC were rinsed in distilled water, counterstained with cresyl violet, and neurons in anx/anx mice, using several markers expressed by this coverslipped with Entellan (Merck). All sections were examined under population, to gain understanding of how the melanocortinergic bright- and dark-field illumination using a Nikon (Tokyo, Japan) system functions in a potentially “hypoNPYergic” state. These Microphot-FX microscope. Photomicrographs were taken with Kodak markers include mRNAs for POMC, the Y1R and the Y5R T-max 100 film. a Quantification of autoradiograms. Measurements were performed on a studied by in situ hybridization, as well as ACTH, -MSH, and MacIntosh IIx (Apple Computer Inc., Cupertino, CA) equipped with a the Y1R studied by immunohistochemistry. We have also inves- Quick Capture frame grabber card (Data Translation, Marlboro, MA), tigated the expression of the autoinhibitory Y2 receptor coex- a Northern Light precision illuminator (Imaging Research, St. Ca- pressed with NPY in arcuate neurons (Broberger et al., 1997b) in tharines, Ontario, Canada), and a Dage-MTI CCD-72 series camera anx/anx mice. (Dage-MTI, Michigan City, IN) equipped with a Nikon 55 mm lens. To process images, NIH Image software (courtesy of Wayne Rasband, Part of these results have been presented previously in abstract National Institute of Mental Health, Bethesda, MD) was used. Each form (Broberger et al., 1998c). image was an average of eight video frames digitized to a 512 3 512 matrix with 256 gray levels for each picture element. To adjust for MATERIALS AND METHODS possible defect in the illumination or optical pathway, an image of the empty illumination screen was taken through a neutral filter and used for Animals. Animals were housed together in ventilated cages in an animal background shading correction. The gray levels of eight 14C-plastic room maintained on a 12 hr light/dark schedule with lights on at 7.00 standards (Amersham) (Miller, 1991) exposed to the autoradiography A.M., at a temperature of 125°C. The anx/anx mice were characterized film together with the tissue sections were determined and used in a genotypically using nearby located simple sequence length polymor- third-degree polynominal approximation to construct a gray level to phisms and phenotypically on the basis of body weight and neurological abnormalities determined before dissection–perfusion. Both male and activity transfer function. The borders of the measuring fields were female mice were analyzed. For in situ hybridization, 10 anx/anx mice interactively defined, and the average gray level was calculated using the and eight wild-type littermates were used. For immunohistochemistry, Macintosh computer and Microsoft Excel (Apple). The average gray six anx/anx mice and seven wild-type littermates were used. Dissection level of control tissue was set to 100%, and changes in gray levels in the was performed on postnatal days 20 or 21. For in situ hybridization arcuate nucleus from sections of anx/anx mice were expressed as per- experiments, brains were dissected from decapitated animals and rapidly