Transient expression of the neuropeptide galanin modulates peripheral­to­central connectivity in the somatosensory thalamus during whisker development in mice.

The significance of transient neuropeptide expression during postnatal brain development is unknown. Here, we show that galanin expression in the ventrobasal thalamus of infant mice coincides with whisker map development and modulates subcortical circuit wiring. Time-resolved neuroanatomy and single-nucleus RNA-seq identified complementary galanin (Gal) and galanin receptor 1 (Galr1) expression in the ventrobasal thalamus and the principal sensory nucleus of the trigeminal nerve (Pr5), respectively. Somatodendritic galanin release from the ventrobasal thalamus was time-locked to the first postnatal week, when Gal1R+ Pr5 afferents form glutamatergic (Slc17a6+) synapses for the topographical whisker map to emerge. RNAi-mediated silencing of galanin expression disrupted glutamatergic synaptogenesis, which manifested as impaired whisker-dependent exploratory behaviors in infant mice, with behavioral abnormalities enduring into adulthood. Pharmacological probing of receptor selectivity in vivo corroborated that target recognition and synaptogenesis in the thalamus, at least in part, are reliant on agonist-induced Gal1R activation in inbound excitatory axons. Overall, we suggest a neuropeptide-dependent developmental mechanism to contribute to the topographical specification of a fundamental sensory neurocircuit in mice.

Brain-wide mapping of efferent projections of glutamatergic (Onecut3+ ) neurons in the lateral mouse hypothalamus.

AIM: This study mapped the spatiotemporal positions and connectivity of Onecut3+ neuronal populations in the developing and adult mouse brain. METHODS: We generated fluorescent reporter mice to chart Onecut3+ neurons for brain-wide analysis. Moreover, we crossed Onecut3-iCre and Mapt-mGFP (Tau-mGFP) mice to visualize axonal projections. A dual Cre/Flp-dependent AAV construct in Onecut3-iCre cross-bred with Slc17a6-FLPo mice was used in an intersectional strategy to map the connectivity of glutamatergic lateral hypothalamic neurons in the adult mouse. RESULTS: We first found that Onecut3 marks a hitherto undescribed Slc17a6+ /Vglut2+ neuronal cohort in the lateral hypothalamus, with the majority expressing thyrotropin-releasing hormone. In the adult, Onecut3+ /Vglut2+ neurons of the lateral hypothalamus had both intra- and extrahypothalamic efferents, particularly to the septal complex and habenula, where they targeted other cohorts of Onecut3+ neurons and additionally to the neocortex and hippocampus. This arrangement suggests that intrinsic reinforcement loops could exist for Onecut3+ neurons to coordinate their activity along the brain's midline axis. CONCLUSION: We present both a toolbox to manipulate novel subtypes of hypothalamic neurons and an anatomical arrangement by which extrahypothalamic targets can be simultaneously entrained.

A hypothalamic dopamine locus for psychostimulant-induced hyperlocomotion in mice.

The lateral septum (LS) has been implicated in the regulation of locomotion. Nevertheless, the neurons synchronizing LS activity with the brain's clock in the suprachiasmatic nucleus (SCN) remain unknown. By interrogating the molecular, anatomical and physiological heterogeneity of dopamine neurons of the periventricular nucleus (PeVN; A14 catecholaminergic group), we find that Th+/Dat1+ cells from its anterior subdivision innervate the LS in mice. These dopamine neurons receive dense neuropeptidergic innervation from the SCN. Reciprocal viral tracing in combination with optogenetic stimulation ex vivo identified somatostatin-containing neurons in the LS as preferred synaptic targets of extrahypothalamic A14 efferents. In vivo chemogenetic manipulation of anterior A14 neurons impacted locomotion. Moreover, chemogenetic inhibition of dopamine output from the anterior PeVN normalized amphetamine-induced hyperlocomotion, particularly during sedentary periods. Cumulatively, our findings identify a hypothalamic locus for the diurnal control of locomotion and pinpoint a midbrain-independent cellular target of psychostimulants.

Ontogenetic rules for the molecular diversification of hypothalamic neurons.

The hypothalamus is an evolutionarily conserved endocrine interface that, among other roles, links central homeostatic control to adaptive bodily responses by releasing hormones and neuropeptides from its many neuronal subtypes. In its preoptic, anterior, tuberal and mammillary subdivisions, a kaleidoscope of magnocellular and parvocellular neuroendocrine command neurons, local-circuit neurons, and neurons that project to extrahypothalamic areas are intermingled in partially overlapping patches of nuclei. Molecular fingerprinting has produced data of unprecedented mass and depth to distinguish and even to predict the synaptic and endocrine competences, connectivity and stimulus selectivity of many neuronal modalities. These new insights support eminent studies from the past century but challenge others on the molecular rules that shape the developmental segregation of hypothalamic neuronal subtypes and their use of morphogenic cues for terminal differentiation. Here, we integrate single-cell RNA sequencing studies with those of mouse genetics and endocrinology to describe key stages of hypothalamus development, including local neurogenesis, the direct terminal differentiation of glutamatergic neurons, transition cascades for GABAergic and GABAergic cell-derived dopamine cells, waves of local neuronal migration, and sequential enrichment in neuropeptides and hormones. We particularly emphasize how transcription factors determine neuronal identity and, consequently, circuit architecture, and whether their deviations triggered by environmental factors and hormones provoke neuroendocrine illnesses.

Molecular design of hypothalamus development.

A wealth of specialized neuroendocrine command systems intercalated within the hypothalamus control the most fundamental physiological needs in vertebrates1,2. Nevertheless, we lack a developmental blueprint that integrates the molecular determinants of neuronal and glial diversity along temporal and spatial scales of hypothalamus development3. Here we combine single-cell RNA sequencing of 51,199 mouse cells of ectodermal origin, gene regulatory network (GRN) screens in conjunction with genome-wide association study-based disease phenotyping, and genetic lineage reconstruction to show that nine glial and thirty-three neuronal subtypes are generated by mid-gestation under the control of distinct GRNs. Combinatorial molecular codes that arise from neurotransmitters, neuropeptides and transcription factors are minimally required to decode the taxonomical hierarchy of hypothalamic neurons. The differentiation of γ-aminobutyric acid (GABA) and dopamine neurons, but not glutamate neurons, relies on quasi-stable intermediate states, with a pool of GABA progenitors giving rise to dopamine cells4. We found an unexpected abundance of chemotropic proliferation and guidance cues that are commonly implicated in dorsal (cortical) patterning5 in the hypothalamus. In particular, loss of SLIT-ROBO signalling impaired both the production and positioning of periventricular dopamine neurons. Overall, we identify molecular principles that shape the developmental architecture of the hypothalamus and show how neuronal heterogeneity is transformed into a multimodal neural unit to provide virtually infinite adaptive potential throughout life.

Unified Classification of Molecular, Network, and Endocrine Features of Hypothalamic Neurons.

Peripheral endocrine output relies on either direct or feed-forward multi-order command from the hypothalamus. Efficient coding of endocrine responses is made possible by the many neuronal cell types that coexist in intercalated hypothalamic nuclei and communicate through extensive synaptic connectivity. Although general anatomical and neurochemical features of hypothalamic neurons were described during the past decades, they have yet to be reconciled with recently discovered molecular classifiers and neurogenetic function determination. By interrogating magnocellular as well as parvocellular dopamine, GABA, glutamate, and phenotypically mixed neurons, we integrate available information at the molecular, cellular, network, and endocrine output levels to propose a framework for the comprehensive classification of hypothalamic neurons. Simultaneously, we single out putative neuronal subclasses for which future research can fill in existing gaps of knowledge to rationalize cellular diversity through function-determinant molecular marks in the hypothalamus.

Hypothalamic cell diversity: non-neuronal codes for long-distance volume transmission by neuropeptides.

Volume transmission is a mode of intercellular communication using cerebral liquor to deliver signal molecules over long distances and allow their action for extended periods. For hypothalamic neuropeptides, nerve endings amongst ependymal cells are seen as a site of release into the cerebrospinal fluid. Recent single-cell RNA-seq data identify tanycytes and ventricular ependyma as alternative sources by being unexpectedly rich in neuroactive substances. This notion, coupled with circuit analysis showing regionalized innervation of periventricular ependyma by intrahypothalamic neurons, could allow for the integration of hypothalamic neuronal activity patterns with brain-wide activity changes upon metabolic challenges through phasic volume transmission primed by neuron-ependyma coupling. Here, we discuss emerging data for an ependymal interface and its breaches in neuropsychiatric disease.

Hypothalamic CNTF volume transmission shapes cortical noradrenergic excitability upon acute stress.

Stress-induced cortical alertness is maintained by a heightened excitability of noradrenergic neurons innervating, notably, the prefrontal cortex. However, neither the signaling axis linking hypothalamic activation to delayed and lasting noradrenergic excitability nor the molecular cascade gating noradrenaline synthesis is defined. Here, we show that hypothalamic corticotropin-releasing hormone-releasing neurons innervate ependymal cells of the 3rd ventricle to induce ciliary neurotrophic factor (CNTF) release for transport through the brain's aqueductal system. CNTF binding to its cognate receptors on norepinephrinergic neurons in the locus coeruleus then initiates sequential phosphorylation of extracellular signal-regulated kinase 1 and tyrosine hydroxylase with the Ca2+-sensor secretagogin ensuring activity dependence in both rodent and human brains. Both CNTF and secretagogin ablation occlude stress-induced cortical norepinephrine synthesis, ensuing neuronal excitation and behavioral stereotypes. Cumulatively, we identify a multimodal pathway that is rate-limited by CNTF volume transmission and poised to directly convert hypothalamic activation into long-lasting cortical excitability following acute stress.

Molecular diversity of corticotropin-releasing hormone mRNA-containing neurons in the hypothalamus.

Hormonal responses to acute stress rely on the rapid induction of corticotropin-releasing hormone (CRH) production in the mammalian hypothalamus, with subsequent instructive steps culminating in corticosterone release at the periphery. Hypothalamic CRH neurons in the paraventricular nucleus of the hypothalamus are therefore considered as 'stress neurons'. However, significant morphological and functional diversity among neurons that can transiently produce CRH in other hypothalamic nuclei has been proposed, particularly as histochemical and molecular biology evidence associates CRH to both GABA and glutamate neurotransmission. Here, we review recent advances through single-cell RNA sequencing and circuit mapping to suggest that CRH production reflects a state switch in hypothalamic neurons and thus confers functional competence rather than being an identity mark of phenotypically segregated neurons. We show that CRH mRNA transcripts can therefore be seen in GABAergic, glutamatergic and dopaminergic neuronal contingents in the hypothalamus. We then distinguish 'stress neurons' of the paraventricular nucleus that constitutively express secretagogin, a Ca2+ sensor critical for the stimulus-driven assembly of the molecular machinery underpinning the fast regulated exocytosis of CRH at the median eminence. Cumulatively, we infer that CRH neurons are functionally and molecularly more diverse than previously thought.

Molecular interrogation of hypothalamic organization reveals distinct dopamine neuronal subtypes.

The hypothalamus contains the highest diversity of neurons in the brain. Many of these neurons can co-release neurotransmitters and neuropeptides in a use-dependent manner. Investigators have hitherto relied on candidate protein-based tools to correlate behavioral, endocrine and gender traits with hypothalamic neuron identity. Here we map neuronal identities in the hypothalamus by single-cell RNA sequencing. We distinguished 62 neuronal subtypes producing glutamatergic, dopaminergic or GABAergic markers for synaptic neurotransmission and harboring the ability to engage in task-dependent neurotransmitter switching. We identified dopamine neurons that uniquely coexpress the Onecut3 and Nmur2 genes, and placed these in the periventricular nucleus with many synaptic afferents arising from neuromedin S+ neurons of the suprachiasmatic nucleus. These neuroendocrine dopamine cells may contribute to the dopaminergic inhibition of prolactin secretion diurnally, as their neuromedin S+ inputs originate from neurons expressing Per2 and Per3 and their tyrosine hydroxylase phosphorylation is regulated in a circadian fashion. Overall, our catalog of neuronal subclasses provides new understanding of hypothalamic organization and function.

Comparative anatomical distribution of neuronal calcium-binding protein (NECAB) 1 and -2 in rodent and human spinal cord.

Neuronal calcium-binding protein 1 and -2 (NECAB1/2) localize to multiple excitatory neuron populations in the mouse spinal cord. Here, we analyzed rat and human spinal cord, combining in situ hybridization and immunohistochemistry, complementing newly collated data on mouse spinal cord for direct comparisons. Necab1/2 mRNA transcripts showed complementary distribution in rodent's spinal cord. Multiple-labeling fluorescence histochemistry with neuronal phenotypic markers localized NECAB1 to a dense fiber plexus in the dorsal horn, to neurons mainly in superficial layers and to commissural interneurons in both rodent species. NECAB1-positive (+) motor neurons were only found in mice. NECAB1 distribution in the human spinal cord was similar with the addition of NECAB1-like immunoreactivity surrounding myelinated axons. NECAB2 was mainly present in excitatory synaptic boutons in the dorsal horn of all three species, and often in calbindin-D28k(+) neuronal somata. Rodent ependymal cells expressed calbindin-D28k. In humans, they instead were NECAB2(+) and/or calretinin(+). Our results reveal that the association of NECAB2 to excitatory neuronal circuits in the spinal cord is evolutionarily conserved across the mammalian species investigated so far. In contrast, NECAB1 expression is more heterogeneous. Thus, our study suggests that the phenotypic segregation of NECAB1 and -2 to respective excitatory and inhibitory spinal systems can underpin functional modalities in determining the fidelity of synaptic neurotransmission and neuronal responsiveness, and might bear translational relevance to humans.

H1N1 influenza virus induces narcolepsy-like sleep disruption and targets sleep-wake regulatory neurons in mice.

An increased incidence in the sleep-disorder narcolepsy has been associated with the 2009-2010 pandemic of H1N1 influenza virus in China and with mass vaccination campaigns against influenza during the pandemic in Finland and Sweden. Pathogenetic mechanisms of narcolepsy have so far mainly focused on autoimmunity. We here tested an alternative working hypothesis involving a direct role of influenza virus infection in the pathogenesis of narcolepsy in susceptible subjects. We show that infection with H1N1 influenza virus in mice that lack B and T cells (Recombinant activating gene 1-deficient mice) can lead to narcoleptic-like sleep-wake fragmentation and sleep structure alterations. Interestingly, the infection targeted brainstem and hypothalamic neurons, including orexin/hypocretin-producing neurons that regulate sleep-wake stability and are affected in narcolepsy. Because changes occurred in the absence of adaptive autoimmune responses, the findings show that brain infections with H1N1 virus have the potential to cause per se narcoleptic-like sleep disruption.

Gut Commensal E. coli Proteins Activate Host Satiety Pathways following Nutrient-Induced Bacterial Growth.

The composition of gut microbiota has been associated with host metabolic phenotypes, but it is not known if gut bacteria may influence host appetite. Here we show that regular nutrient provision stabilizes exponential growth of E. coli, with the stationary phase occurring 20 min after nutrient supply accompanied by bacterial proteome changes, suggesting involvement of bacterial proteins in host satiety. Indeed, intestinal infusions of E. coli stationary phase proteins increased plasma PYY and their intraperitoneal injections suppressed acutely food intake and activated c-Fos in hypothalamic POMC neurons, while their repeated administrations reduced meal size. ClpB, a bacterial protein mimetic of α-MSH, was upregulated in the E. coli stationary phase, was detected in plasma proportional to ClpB DNA in feces, and stimulated firing rate of hypothalamic POMC neurons. Thus, these data show that bacterial proteins produced after nutrient-induced E. coli growth may signal meal termination. Furthermore, continuous exposure to E. coli proteins may influence long-term meal pattern.

Replacing SNAP-25b with SNAP-25a expression results in metabolic disease.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a key molecule in the soluble N-ethylmaleimide-sensitive factor attachment protein (SNARE) complex mediating fast Ca(2+)-triggered release of hormones and neurotransmitters, and both splice variants, SNAP-25a and SNAP-25b, can participate in this process. Here we explore the hypothesis that minor alterations in the machinery mediating regulated membrane fusion can increase the susceptibility for metabolic disease and precede obesity and type 2 diabetes. Thus, we used a mouse mutant engineered to express normal levels of SNAP-25 but only SNAP-25a. These SNAP-25b-deficient mice were exposed to either a control or a high-fat/high-sucrose diet. Monitoring of food intake, body weight, hypothalamic function, and lipid and glucose homeostases showed that SNAP-25b-deficient mice fed with control diet developed hyperglycemia, liver steatosis, and adipocyte hypertrophy, conditions dramatically exacerbated when combined with the high-fat/high-sucrose diet. Thus, modified SNARE function regulating stimulus-dependent exocytosis can increase the vulnerability to and even provoke metabolic disease. When combined with a high-fat/high-sucrose diet, this vulnerability resulted in diabesity. Our SNAP-25b-deficient mouse may represent a diabesity model.

The number of preproghrelin mRNA expressing cells is increased in mice with activity-based anorexia.

Plasma levels of ghrelin, an orexigenic peptide, are increased during conditions of chronic starvation, such as in patients with anorexia nervosa. However, it is not known whether such increase can be related to the number of preproghrelin mRNA-expressing cells in the stomach, and if chronic starvation may activate a tentative central ghrelin production. In this work, in situ hybridization technique was used to analyze the presence and number of preproghrelin mRNA-expressing cells in the stomach and the hypothalamus of mice with activity-based anorexia (ABA) induced by the combination of running wheel activity with progressive, during 10 days, feeding-time restriction (FTR) and compared with sedentary FTR, ABA pair-fed (PF) and ad libitum-fed control mice. All food-restricted mice lost more than 20% of body weight. Body weight loss was similar in ABA and PF mice, but it was more pronounced than in FTR mice. Food intake was also lower in ABA than in FTR mice. Preproghrelin mRNA-expressing cells in the stomach were increased proportionally to the body weight loss in all food-restricted groups with the highest number in ABA mice. No preproghrelin mRNA-producing cells were detectable in the hypothalamus of either control or food-restricted mice. Thus, the increased number of gastric preproghrelin mRNA-producing cells during chronic starvation proportionally to the body weight loss and reduced food intake may underlie increased plasma ghrelin. Hyperactivity-induced anorexia appears to further increase the number of preproghrelin mRNA-producing cells in the stomach. No evidence was found for ghrelin expression in the hypothalamus, not even in any of the present experimental models.

Neuronal calcium-binding proteins 1/2 localize to dorsal root ganglia and excitatory spinal neurons and are regulated by nerve injury.

Neuronal calcium (Ca(2+))-binding proteins 1 and 2 (NECAB1/2) are members of the phylogenetically conserved EF-hand Ca(2+)-binding protein superfamily. To date, NECABs have been explored only to a limited extent and, so far, not at all at the spinal level. Here, we describe the distribution, phenotype, and nerve injury-induced regulation of NECAB1/NECAB2 in mouse dorsal root ganglia (DRGs) and spinal cord. In DRGs, NECAB1/2 are expressed in around 70% of mainly small- and medium-sized neurons. Many colocalize with calcitonin gene-related peptide and isolectin B4, and thus represent nociceptors. NECAB1/2 neurons are much more abundant in DRGs than the Ca(2+)-binding proteins (parvalbumin, calbindin, calretinin, and secretagogin) studied to date. In the spinal cord, the NECAB1/2 distribution is mainly complementary. NECAB1 labels interneurons and a plexus of processes in superficial layers of the dorsal horn, commissural neurons in the intermediate area, and motor neurons in the ventral horn. Using CLARITY, a novel, bilaterally connected neuronal system with dendrites that embrace the dorsal columns like palisades is observed. NECAB2 is present in cell bodies and presynaptic boutons across the spinal cord. In the dorsal horn, most NECAB1/2 neurons are glutamatergic. Both NECAB1/2 are transported into dorsal roots and peripheral nerves. Peripheral nerve injury reduces NECAB2, but not NECAB1, expression in DRG neurons. Our study identifies NECAB1/2 as abundant Ca(2+)-binding proteins in pain-related DRG neurons and a variety of spinal systems, providing molecular markers for known and unknown neuron populations of mechanosensory and pain circuits in the spinal cord.

Anorexia and hypothalamic degeneration.

Anorexia, meaning poor appetite, occurs in many human conditions, for example, anorexia nervosa, cachexia, and failure to thrive in infants. A key player in the regulation of appetite/food intake in general, as well as conditions of anorexia, is the hypothalamus, in particular, the AGRP/NPY and POMC/CART neurons in the arcuate nucleus. In this chapter, we review the hypothalamic aberrances seen in the anorectic anx/anx mouse. This mouse displays deviations in neuropeptidergic/-transmitter systems, including selective hypothalamic degeneration and inflammation that have been associated with mitochondrial dysfunction. In addition, we discuss data from other animal models, as well as clinical data relating hypothalamic inflammation/degeneration, neurogenesis, and mitochondrial dysfunction to conditions of disturbed regulation of food intake.

Secretagogin is expressed in sensory CGRP neurons and in spinal cord of mouse and complements other calcium-binding proteins, with a note on rat and human.

BACKGROUND: Secretagogin (Scgn), a member of the EF-hand calcium-binding protein (CaBP) superfamily, has recently been found in subsets of developing and adult neurons. Here, we have analyzed the expression of Scgn in dorsal root ganglia (DRGs) and trigeminal ganglia (TGs), and in spinal cord of mouse at the mRNA and protein levels, and in comparison to the well-known CaBPs, calbindin D-28k, parvalbumin and calretinin. Rat DRGs, TGs and spinal cord, as well as human DRGs and spinal cord were used to reveal phylogenetic variations. RESULTS: We found Scgn mRNA expressed in mouse and human DRGs and in mouse ventral spinal cord. Our immunohistochemical data showed a complementary distribution of Scgn and the three CaBPs in mouse DRG neurons and spinal cord. Scgn was expressed in ~7% of all mouse DRG neuron profiles, mainly small ones and almost exclusively co-localized with calcitonin gene-related peptide (CGRP). This co-localization was also seen in human, but not in rat DRGs. Scgn could be detected in the mouse sciatic nerve and accumulated proximal to its constriction. In mouse spinal cord, Scgn-positive neuronal cell bodies and fibers were found in gray matter, especially in the dorsal horn, with particularly high concentrations of fibers in the superficial laminae, as well as in cell bodies in inner lamina II and in some other laminae. A dense Scgn-positive fiber network and some small cell bodies were also found in the superficial dorsal horn of humans. In the ventral horn, a small number of neurons were Scgn-positive in mouse but not rat, confirming mRNA distribution. Both in mouse and rat, a subset of TG neurons contained Scgn. Dorsal rhizotomy strongly reduced Scgn fiber staining in the dorsal horn. Peripheral axotomy did not clearly affect Scgn expression in DRGs, dorsal horn or ventral horn neurons in mouse. CONCLUSIONS: Scgn is a CaBP expressed in a subpopulation of nociceptive DRG neurons and their processes in the dorsal horn of mouse, human and rat, the former two co-expressing CGRP, as well as in dorsal horn neurons in all three species. Functional implications of these findings include the cellular refinement of sensory information, in particular during the processing of pain.

Hypothalamic mitochondrial dysfunction associated with anorexia in the anx/anx mouse.

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.

Evidence of hypothalamic degeneration in the anorectic anx/anx mouse.

Mice homozygous for the anorexia (anx) mutation are characterized by poor food intake and death by three to five weeks after birth. By P21 these mice display lower density of hypothalamic neuropeptides, including Agouti gene-related protein (AGRP). The AGRP/neuropeptide Y (NPY) system of the anx/anx mice develops normally until postnatal day (P) 12, then the normal increase in fiber density ceases, in some areas even distinctly decreases. This overlaps with activation of microglia, indicating an inflammatory and/or degenerative process. Here we studied, by in situ hybridization and immunohistochemistry (IHC), the expression of major histocompatibility complex (MHC) class I-related molecules and markers for cellular reactivity in hypothalamus of anx/anx mice. MHC class I transcript and -related proteins were found in arcuate nucleus (Arc), presumably both in neurons and glia, the latter also in areas innervated by AGRP (NPY) neurons. In the anx/anx hypothalamus, using TUNEL labeling, significantly higher number of apoptotic cells were found compared with +/+ mice, and active caspase 6 immunoreactivity was detected in degenerating NPY-fibers as well as signs of "microglia-associated cell death". In addition, Y1 receptor-labeled processes and soma of pro-opiomelanocortin (POMC) neurons, were markedly decreased at P21. These results support the hypothesis of degeneration of hypothalamic arcuate neuron populations in the anx/anx mice, whereby the AGRP system may be first affected, the changes in the POMC system being secondary in this process.