Neuropeptide Y (NPY) and the endogenous melanocortin receptor antagonist, agouti gene-related protein (AGRP), coexist in the arcuate nucleus, and both exert orexigenic effects. The present study aimed primarily at determining the brain distribution of AGRP. AGRP mRNAexpressing cells were limited to the arcuate nucleus, representing a major subpopulation (95%) of the NPY neurons, which also was confirmed with immunohistochemistry. AGRP-immunoreactive (-ir) terminals all contained NPY and were observed in many brain regions extending from the rostral telencephalon to the pons, including the parabrachial nucleus. NPY-positive, AGRP-negative terminals were observed in many areas. AGRP-ir terminals were reduced dramatically in all brain regions of mice treated neonatally with monosodium glutamate as well as of mice homozygous for the anorexia mutation. Terminals immunoreactive for the melanocortin peptide ␣-melanocyte-stimulating hormone formed a population separate from, but parallel to, the AGRP-ir terminals. Our results show that arcuate NPY neurons, identified by the presence of AGRP, project more extensively in the brain than previously known and indicate that the feeding regulatory actions of NPY may extend beyond the hypothalamus.Neuropeptide Y (NPY) (1)-producing neurons in the hypothalamic arcuate nucleus (2, 3) appear to be involved in the regulation of feeding behavior. Intracerebral injections of NPY induce food intake (4, 5), and starved animals increase NPY expression (6, 7). In genetic models of feeding disorders, arcuate NPY overexpression has been linked to hyperphagia and obesity (8), and NPY deficiency has been linked to hypophagia and anorexia (9). However, surprisingly little information is available on the arcuate NPY projections, and targets for these neurons so far have been demonstrated only in a limited number of hypothalamic nuclei (10-12). Recently, Hahn et al. (13) and we (14) have reported that many NPY neurons in the rodent arcuate coexpress agouti gene-related protein (AGRP) (15), an endogenous antagonist of the anorexigenic melanocortin peptides, e.g., ␣-melanocytestimulating hormone (␣MSH) (16,17). The present study aimed at investigating whether AGRP expression is limited to arcuate NPY neurons, using double-labeling immunohistochemistry and in situ hybridization. If so, AGRP staining could be used to map the terminal projections of arcuate NPY neurons. To further ascertain the arcuate origin of AGRPimmunoreactive (ir) terminals, we also analyzed mice treated neonatally with monosodium glutamate (MSG), which induces necrosis of arcuate neurons (18), including the NPY cells (11,19). In addition, we studied AGRP in the mutant anorexia (anx͞anx) mouse, where abnormalities in arcuate NPY histochemistry have been demonstrated (9). MATERIALS AND METHODSAnimals. C57Bl6 mice (B&K Universal, Sollentuna, Sweden) were injected s.c. with MSG (Sigma) at doses of 2.2, 2.5, 2.8, 3.2, 3.4, 3.6, 3.8, 4.0, and 4.2 mg͞g or 0.9% NaCl (vehicle) on postnatal days 2-10, respectively (20). Four MSG...

The megencephaly mouse, mceph/mceph, displays dramatically increased brain volume and hypertrophic brain cells. Despite overall enlargement, the mceph/mceph brain appears structurally normal, without oedema, hydrocephaly or leukodystrophy, and with only minor astrocytosis. Furthermore, it presents striking disturbances in expression of trophic and neuromodulating factors within the hippocampus and cortex. Using a positional cloning approach we have identified the mceph mutation. We show that mceph/mceph mice carry an 11-base-pair deletion in the gene encoding the Shaker-like voltage-gated potassium channel subtype 1, Kcna1. The mutation leads to a frame shift and the predicted MCEPH protein is truncated at amino acid 230 (out of 495), terminating with six aberrant amino acids. The expression of Kcna1 mRNA is increased in the mceph/mceph brain. However, the C-terminal domains of the corresponding Kv1.1 protein are absent. The putative MCEPH protein retains only the N-terminal domains for channel assembly and may congregate nonfunctional complexes of multiple Shaker-like subunits. Indeed, whereas Kcna2 and Kcna3 mRNA expression is normal, the mceph/mceph hippocampus displays decreased amounts of Kv1.2 and Kv1.3 proteins, suggesting interactions at the protein level. We show that mceph/mceph mice have disturbed brain electrophysiology and experience recurrent behavioural seizures, in agreement with the abnormal electrical brain activity found in Shaker mutants. However, in contrast to the commonly demonstrated epilepsy-induced neurodegeneration, we find that the mceph mutation leads to seizures with a concomitant increase in brain size, without overt neural atrophy.

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The anorectic anx/anx mouse exhibits disturbed feeding behavior and aberrances, including neurodegeneration, in peptidergic neurons in the appetite regulating hypothalamic arcuate nucleus. Poor feeding in infants, as well as neurodegeneration, are common phenotypes in human disorders caused by dysfunction of the mitochondrial oxidative phosphorylation system (OXPHOS). We therefore hypothesized that the anorexia and degenerative phenotypes in the anx/anx mouse could be related to defects in the OXPHOS. In this study, we found reduced efficiency of hypothalamic OXPHOS complex I assembly and activity in the anx/anx mouse. We also recorded signs of increased oxidative stress in anx/anx hypothalamus, possibly as an effect of the decreased hypothalamic levels of fully assembled complex I, that were demonstrated by native Western blots. Furthermore, the Ndufaf1 gene, encoding a complex I assembly factor, was genetically mapped to the anx interval and found to be down-regulated in anx/ anx mice. These results suggest that the anorexia and hypothalamic neurodegeneration of the anx/anx mouse are associated with dysfunction of mitochondrial complex I.neuropeptides | reactive oxygen species | agouti-gene related protein | food intake | neuroinflammation T he anorectic anx/anx mouse (1) is an attractive model for disturbed feeding behavior, as it exhibits phenotypes associated with failure to thrive in infants and young children (2), anorexia nervosa (3), and cachexia (4). This mouse arose by a spontaneous mutation (anorexia, allele symbol anx) and is characterized by poor appetite and reduced stomach content, and dies (likely because of the severe starvation) around 3 wk after birth (1). The anx/ anx mice eat significantly less than their wild-type littermates, and by postnatal day (P) 21 they weigh half as much, rendering them an emaciated appearance (1). These mice also exhibit a number of neurological abnormalities, such as body tremors, head weaving, hyperactivity, and uncoordinated gait (1).Several neurotransmitter (5, 6) and neuropeptidergic (7-11) systems involved in the regulation of food intake and energy metabolism are disturbed in the anx/anx mouse. The majority of these findings are centered around the hypothalamus, the origin of a neuronal network important for the control of initiation and termination of food intake, as well as diet-induced thermogenesis and energy expenditure. The arcuate nucleus of hypothalamus (Arc), situated at the interface between the periphery and brain, receives signals about energy status from the periphery, such as circulating leptin and insulin levels, resulting in anorexigenic or orexigenic behavior. Two main populations of food intake-regulating neurons reside in the Arc, both expressing leptin and insulin receptors (12). One group coexpresses the two orexigenic neuropeptides neuropeptide Y (NPY) and agoutigene related protein (AGRP) (11, 13), whereas the other population expresses anorexigenic proopiomelanocortin (POMC) and cocaine-and amphetamine-regulated transcript (CART) (14)...

Agouti-related protein (AgRP) is a key orexigenic neuropeptide expressed in the hypothalamic arcuate nucleus and a marker for neurons conveying hormonal signals of hunger to the brain. Mice homozygous for the anorexia (anx) mutation are characterized by decreased food intake, starvation, and death by 3-5 weeks of age. At this stage immunoreactivity for AgRP is increased in cell bodies but decreased in the nerve terminals. We studied when during early postnatal development the aberrant phenotype of the AgRP system becomes apparent in anx/anx mice and possible underlying mechanisms. AgRP and ionized calcium binding adapter molecule (Iba1), a marker for activated microglia, as well as Toll-like receptor 2 (TLR-2), were studied by immunohistochemistry at postnatal days P1, P5, P10, P12, P15 and P21 in anx/anx and wild-type mice. We found that the AgRP system in the anx/anx mouse develops similarly to the wild type until P12, when AgRP fibers in anx/anx mice cease to increase in density in the main projection areas. At P21, AgRP fiber density in anx/anx mice was significantly reduced vs. P15, in certain regions. At P21, many strongly AgRP-positive cell bodies were observed in the anx/anx arcuate nucleus vs. only few and weakly fluorescent ones in the wild type. The decrease in AgRP fiber density in anx/anx mice overlapped with an increase in Iba1 and TLR-2 immunoreactivities. Thus, the aberrant appearance of the AgRP system in the anx/anx mouse in the early postnatal development could involve a microglia-associated process and the innate immune system.

The hypothalamus contains integrative systems that support life, including physiological processes such as food intake, energy expenditure, and reproduction. Here, we show that anorexia nervosa (AN) patients, contrary to normal weight and constitutionally lean individuals, respond with a paradoxical reduction in hypothalamic levels of glutamate/ glutamine (Glx) upon feeding. This reversal of the Glx response is associated with decreased wiring in the arcuate nucleus and increased connectivity in the lateral hypothalamic area, which are involved in the regulation on a variety of physiological and behavioral functions including the con-trol of food intake and energy balance. The identification of distinct hypothalamic neurochemical dysfunctions and associated structural variations in AN paves the way for the development of new diagnostic and treatment strategies in conditions associated with abnormal body mass index and a maladaptive response to negative energy balance.

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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.