Mouse Forebrain Research Articles

Breast carcinoma-amplified sequence 2 regulates adult neurogenesis via \u03b2-catenin

BackgroundBreast carcinoma-amplified sequence 2 (BCAS2) regulates β-catenin gene splicing. The conditional knockout of BCAS2 expression in the forebrain (BCAS2 cKO) of mice confers impaired learning and memory along with decreased β-catenin expression. Because β-catenin reportedly regulates adult neurogenesis, we wondered whether BCAS2 could regulate adult neurogenesis via β-catenin.MethodsBCAS2-regulating neurogenesis was investigated by characterizing BCAS2 cKO mice. Also, lentivirus-shBCAS2 was intracranially injected into the hippocampus of wild-type mice to knock down BCAS2 expression. We evaluated the rescue effects of BCAS2 cKO by intracranial injection of adeno-associated virus encoding BCAS2 (AAV-DJ8-BCAS2) and AAV-β-catenin gene therapy.ResultsTo show that BCAS2-regulating adult neurogenesis via β-catenin, first, BCAS2 cKO mice showed low SRY-box 2-positive (Sox2+) neural stem cell proliferation and doublecortin-positive (DCX+) immature neurons. Second, stereotaxic intracranial injection of lentivirus-shBCAS2 knocked down BCAS2 in the hippocampus of wild-type mice, and we confirmed the BCAS2 regulation of adult neurogenesis via β-catenin. Third, AAV-DJ8-BCAS2 gene therapy in BCAS2 cKO mice reversed the low proliferation of Sox2+ neural stem cells and the decreased number of DCX+ immature neurons with increased β-catenin expression. Moreover, AAV-β-catenin gene therapy restored neuron stem cell proliferation and immature neuron differentiation, which further supports BCAS2-regulating adult neurogenesis via β-catenin. In addition, cells targeted by AAV-DJ8 injection into the hippocampus included Sox2 and DCX immature neurons, interneurons, and astrocytes. BCAS2 may regulate adult neurogenesis by targeting Sox2+ and DCX+ immature neurons for autocrine effects and interneurons or astrocytes for paracrine effects.ConclusionsBCAS2 can regulate adult neurogenesis in mice via β-catenin.

Polymorphisms in the hypoxia inducible factor binding site of the macrophage migration inhibitory factor gene promoter in schizophrenia.

Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine that promotes neurogenesis and neuroprotection. MIF is predominantly expressed in astrocytes in the brain. The serum MIF level and microsatellites/single nucleotide polymorphisms (SNPs) in the MIF gene promoter region are known to be associated with schizophrenia (SCZ). Interestingly, previous studies reported that hypoxia, an environmental risk factor for SCZ, induced MIF expression through binding of the hypoxia inducible factor (HIF)-1 to the hypoxia response element (HRE) in the MIF promoter. We investigated the involvement of MIF in SCZ while focusing on the HIF pathway. First, we conducted an association study of the SNP rs17004038 (C>A) in the HRE of the MIF promoter between 1758 patients with SCZ and 1507 controls. Next, we investigated the effect of hypoxia on MIF expression in primary cultured astrocytes derived from neonatal mice forebrain. SNP rs17004038 was significantly associated with SCZ (p = 0.0424, odds ratio = 1.445), indicating that this SNP in the HRE of the MIF promoter was a genetic risk factor for SCZ. Hypoxia induced MIF mRNA expression and MIF protein production and increased HIF-1 binding to the MIF promoter, while the activity of the MIF promoter was suppressed by mutations in the HRE and by deletion of the HRE in astrocytes. These results suggest that SNP rs17004038 in the HRE of the MIF promoter was significantly associated with SCZ and may be involved in the pathophysiology of SCZ via suppression of hypoxia and HIF pathway-induced MIF expression.

Assessment of the In Vivo Relationship Between Cerebral Hypometabolism, Tau Deposition, TSPO Expression, and Synaptic Density in a Tauopathy Mouse Model: a Multi-tracer PET Study

Cerebral glucose hypometabolism is a typical hallmark of Alzheimer’s disease (AD), usually associated with ongoing neurodegeneration and neuronal dysfunction. However, underlying pathological processes are not fully understood and reproducibility in animal models is not well established. The aim of the present study was to investigate the regional interrelation of glucose hypometabolism measured by [18F]FDG positron emission tomography (PET) with various molecular targets of AD pathophysiology using the PET tracers [18F]PI-2620 for tau deposition, [18F]DPA-714 for TSPO expression associated with neuroinflammation, and [18F]UCB-H for synaptic density in a transgenic tauopathy mouse model. Seven-month-old rTg4510 mice (n = 8) and non-transgenic littermates (n = 8) were examined in a small animal PET scanner with the tracers listed above. Hypometabolism was observed throughout the forebrain of rTg4510 mice. Tau pathology, increased TSPO expression, and synaptic loss were co-localized in the cortex and hippocampus and correlated with hypometabolism. In the thalamus, however, hypometabolism occurred in the absence of tau-related pathology. Thus, cerebral hypometabolism was associated with two regionally distinct forms of molecular pathology: (1) characteristic neuropathology of the Alzheimer-type including synaptic degeneration and neuroinflammation co-localized with tau deposition in the cerebral cortex, and (2) pathological changes in the thalamus in the absence of other markers of AD pathophysiology, possibly reflecting downstream or remote adaptive processes which may affect functional connectivity. Our study demonstrates the feasibility of a multitracer approach to explore complex interactions of distinct AD-pathomechanisms in vivo in a small animal model. The observations demonstrate that multiple, spatially heterogeneous pathomechanisms can contribute to hypometabolism observed in AD mouse models and they motivate future longitudinal studies as well as the investigation of possibly comparable pathomechanisms in human patients.

Pax6 loss alters the morphological and electrophysiological development of mouse prethalamic neurons.

ABSTRACTPax6 is a well-known regulator of early neuroepithelial progenitor development. Its constitutive loss has a particularly strong effect on the developing prethalamus, causing it to become extremely hypoplastic. To overcome this difficulty in studying the long-term consequences of Pax6 loss for prethalamic development, we used conditional mutagenesis to delete Pax6 at the onset of neurogenesis and studied the developmental potential of the mutant prethalamic neurons in vitro. We found that Pax6 loss affected their rates of neurite elongation, the location and length of their axon initial segments, and their electrophysiological properties. Our results broaden our understanding of the long-term consequences of Pax6 deletion in the developing mouse forebrain, suggesting that it can have cell-autonomous effects on the structural and functional development of some neurons.

Molecular divergence of mammalian astrocyte progenitor cells at early gliogenesis.

During mammalian brain development, how different astrocytes are specified from progenitor cells is not well understood. In particular, whether astrocyte progenitor cells (APCs) start as a relatively homogenous population or whether there is early heterogeneity remains unclear. Here, we have dissected subpopulations of embryonic mouse forebrain progenitors using single-cell transcriptome analyses. Our sequencing data revealed two molecularly distinct APC subgroups at the start of gliogenesis from both dorsal and ventral forebrains. The two APC subgroups were marked, respectively, by specific expression of Sparc and Sparcl1, which are known to function in mature astrocytes with opposing activities for regulating synapse formation. Expression analyses showed that SPARC and SPARCL1 mark APC subgroups that display distinct temporal and spatial patterns, correlating with major waves of astrogliogenesis during development. Our results uncover an early molecular divergence of APCs in the mammalian brain and provide a useful transcriptome resource for the study of glial cell specification.

Increased expression of SLC25A1/CIC causes an autistic-like phenotype with altered neuron morphology.

N ε-lysine acetylation within the lumen of the endoplasmic reticulum is a recently characterized protein quality control system that positively selects properly folded glycoproteins in the early secretory pathway. Overexpression of the endoplasmic reticulum acetyl-CoA transporter AT-1 in mouse forebrain neurons results in increased dendritic branching, spine formation and an autistic-like phenotype that is attributed to altered glycoprotein flux through the secretory pathway. AT-1 overexpressing neurons maintain the cytosolic pool of acetyl-CoA by upregulation of SLC25A1, the mitochondrial citrate/malate antiporter and ATP citrate lyase, which converts cytosolic citrate into acetyl-CoA. All three genes have been associated with autism spectrum disorder, suggesting that aberrant cytosolic-to-endoplasmic reticulum flux of acetyl-CoA can be a mechanistic driver for the development of autism spectrum disorder. We therefore generated a SLC25A1 neuron transgenic mouse with overexpression specifically in the forebrain neurons. The mice displayed autistic-like behaviours with a jumping stereotypy. They exhibited increased steady-state levels of citrate and acetyl-CoA, disrupted white matter integrity with activated microglia and altered synaptic plasticity and morphology. Finally, quantitative proteomic and acetyl-proteomic analyses revealed differential adaptations in the hippocampus and cortex. Overall, our study reinforces the connection between aberrant cytosolic-to-endoplasmic reticulum acetyl-CoA flux and the development of an autistic-like phenotype.

Transcriptional heterogeneity of ventricular zone cells in the ganglionic eminences of the mouse forebrain.

The ventricular zone (VZ) of the nervous system contains radial glia cells that were originally considered relatively homogenous in their gene expression, but a detailed characterization of transcriptional diversity in these VZ cells has not been reported. Here, we performed single-cell RNA sequencing to characterize transcriptional heterogeneity of neural progenitors within the VZ and subventricular zone (SVZ) of the ganglionic eminences (GEs), the source of all forebrain GABAergic neurons. By using a transgenic mouse line to enrich for VZ cells, we characterize significant transcriptional heterogeneity, both between GEs and within spatial subdomains of specific GEs. Additionally, we observe differential gene expression between E12.5 and E14.5 VZ cells, which could provide insights into temporal changes in cell fate. Together, our results reveal a previously unknown spatial and temporal genetic diversity of VZ cells in the ventral forebrain that will aid our understanding of initial fate decisions in the forebrain.

P140Cap Controls Female Fertility in Mice Acting via Glutamatergic Afference on Hypothalamic Gonadotropin-Releasing Hormone Neurons.

p140Cap, encoded by the gene SRCIN1 (SRC kinase signaling inhibitor 1), is an adaptor/scaffold protein highly expressed in the mouse brain, participating in several pre- and post-synaptic mechanisms. p140Cap knock-out (KO) female mice show severe hypofertility, delayed puberty onset, altered estrus cycle, reduced ovulation, and defective production of luteinizing hormone and estradiol during proestrus. We investigated the role of p140Cap in the development and maturation of the hypothalamic gonadotropic system. During embryonic development, migration of Gonadotropin-Releasing Hormone (GnRH) neurons from the nasal placode to the forebrain in p140Cap KO mice appeared normal, and young p140Cap KO animals showed a normal number of GnRH-immunoreactive (-ir) neurons. In contrast, adult p140Cap KO mice showed a significant loss of GnRH-ir neurons and a decreased density of GnRH-ir projections in the median eminence, accompanied by reduced levels of GnRH and LH mRNAs in the hypothalamus and pituitary gland, respectively. We examined the number of kisspeptin (KP) neurons in the rostral periventricular region of the third ventricle, the number of KP-ir fibers in the arcuate nucleus, and the number of KP-ir punctae on GnRH neurons but we found no significant changes. Consistently, the responsiveness to exogenous KP in vivo was unchanged, excluding a cell-autonomous defect on the GnRH neurons at the level of KP receptor or its signal transduction. Since glutamatergic signaling in the hypothalamus is critical for both puberty onset and modulation of GnRH secretion, we examined the density of glutamatergic synapses in p140Cap KO mice and observed a significant reduction in the density of VGLUT-ir punctae both in the preoptic area and on GnRH neurons. Our data suggest that the glutamatergic circuitry in the hypothalamus is altered in the absence of p140Cap and is required for female fertility.

Nests of dividing neuroblasts sustain interneuron production for the developing human brain.

The human cortex contains inhibitory interneurons derived from the medial ganglionic eminence (MGE), a germinal zone in the embryonic ventral forebrain. How this germinal zone generates sufficient interneurons for the human brain remains unclear. We found that the human MGE (hMGE) contains nests of proliferative neuroblasts with ultrastructural and transcriptomic features that distinguish them from other progenitors in the hMGE. When dissociated hMGE cells are transplanted into the neonatal mouse brain, they reform into nests containing proliferating neuroblasts that generate young neurons that migrate extensively into the mouse forebrain and mature into different subtypes of functional interneurons. Together, these results indicate that the nest organization and sustained proliferation of neuroblasts in the hMGE provide a mechanism for the extended production of interneurons for the human forebrain.

Phosphorylation-dependent proteome of Marcks in ependyma during aging and behavioral homeostasis in the mouse forebrain.

Ependymal cells (ECs) line the ventricular surfaces of the mammalian central nervous system (CNS) and their development is indispensable to structural integrity and functions of the CNS. We previously reported that EC-specific genetic deletion of the myristoylated alanine-rich protein kinase C substrate (Marcks) disrupts barrier functions and elevates oxidative stress and lipid droplet accumulation in ECs causing precocious cellular aging. However, little is known regarding the mechanisms that mediate these changes in ECs. To gain insight into Marcks-mediated mechanisms, we performed mass spectrometric analyses on Marcks-associated proteins in young and aged ECs in the mouse forebrain using an integrated approach. Network analysis on annotated proteins revealed that the identified Marcks-associated complexes are in part involved in protein transport mechanisms in young ECs. In fact, we found perturbed intracellular vesicular trafficking in cultured ECs with selective deletion of Marcks (Marcks-cKO mice), or upon pharmacological alteration to phosphorylation status of Marcks. In comparison, Marcks-associated protein complexes in aged ECs appear to be involved in regulation of lipid metabolism and responses to oxidative stress. Confirming this, we found elevated signatures of inflammation in the cerebral cortices and the hippocampi of young Marcks-cKO mice. Interestingly, behavioral testing using a water maze task indicated that spatial learning and memory is diminished in young Marcks-cKO mice similar to aged wildtype mice. Taken together, our study provides first line of evidence for potential mechanisms that may mediate differential Marcks functions in young and old ECs, and their effect on forebrain homeostasis during aging.

High Level Forebrain Expression of Active Tau Kinase p38γ Exacerbates Cognitive Dysfunction in Aged APP-transgenic Alzheimer’s Mice

Persistent improvement of cognitive deficits in Alzheimer’s disease (AD), a common form of dementia, is an unattained therapeutic objective. Gene therapy holds promise for treatment of familial and sporadic forms of AD. p38γ, a member of the p38 mitogen-activated protein (MAP) kinase family, inhibits amyloid-β toxicity through regulation of tau phosphorylation. We recently showed that a gene delivery approach increasing p38γ resulted in markedly better learning and memory performance in mouse models of AD at advanced stages of amyloid-β- and tau-mediated cognitive impairment. Notably, low-to-moderate expression of p38γ had beneficial outcomes on cognition. The impact of high levels of p38γ on neuronal function remain unclear. Therefore, we addressed the outcomes of high levels of active p38γ on brain function, by direct injection of p38γ-encoding adeno-associated virus (AAV) into the forebrain of aged mice of an APP transgenic AD mouse model. While motor function in p38γ-expressing APP transgenic mice 2 months post-injection was comparable to control treated APP mice, their activity was markedly reduced in the open field test and included frequent bouts of immobility. Moreover, their learning and memory function was markedly impaired compared to control-treated aged APP mice. These results suggest that high neuronal levels of active p38γ emphasize a stress kinase role of p38γ, perturbing circuit function in motivation, navigation, and spatial learning. Overall, this work shows excessive neuronal p38γ levels can aggravate circuit dysfunction and advises adjustable expression systems will be required for sustainable AD gene therapy based on p38γ activity.

Bulk and Mosaic Deletions of <i>Egfr</i> Reveal Regionally Defined Gliogenesis in the Developing Mouse Forebrain

Bulk and Mosaic Deletions of <i>Egfr</i> Reveal Regionally Defined Gliogenesis in the Developing Mouse Forebrain

Succinyl-CoA Synthetase Deficiency in Mouse Forebrain Results in Hyper-Succinylation With Perturbed Neuronal Transcriptional Regulation and Metabolism

Succinyl-CoA Synthetase Deficiency in Mouse Forebrain Results in Hyper-Succinylation With Perturbed Neuronal Transcriptional Regulation and Metabolism

Contribution of astrocytes to familial risk and clinical manifestation of schizophrenia.

Previous studies have implicated several brain cell types in schizophrenia (SCZ), but the genetic impact of astrocytes is unknown. Considering their high complexity in humans, astrocytes are likely key determinants of neurodevelopmental diseases, such as SCZ. Human induced pluripotent stem cell (hiPSC)‐derived astrocytes differentiated from five monozygotic twin pairs discordant for SCZ and five healthy subjects were studied for alterations related to high genetic risk and clinical manifestation of SCZ in astrocyte transcriptomics, neuron‐astrocyte co‐cultures, and in humanized mice. We found gene expression and signaling pathway alterations related to synaptic dysfunction, inflammation, and extracellular matrix components in SCZ astrocytes, and demyelination in SCZ astrocyte transplanted mice. While Ingenuity Pathway Analysis identified SCZ disease and synaptic transmission pathway changes in SCZ astrocytes, the most consistent findings were related to collagen and cell adhesion associated pathways. Neuronal responses to glutamate and GABA differed between astrocytes from control persons, affected twins, and their unaffected co‐twins and were normalized by clozapine treatment. SCZ astrocyte cell transplantation to the mouse forebrain caused gene expression changes in synaptic dysfunction and inflammation pathways of mouse brain cells and resulted in behavioral changes in cognitive and olfactory functions. Differentially expressed transcriptomes and signaling pathways related to synaptic functions, inflammation, and especially collagen and glycoprotein 6 pathways indicate abnormal extracellular matrix composition in the brain as one of the key characteristics in the etiology of SCZ.

Single-cell delineation of lineage and genetic identity in the mouse brain

During neurogenesis, mitotic progenitor cells lining the ventricles of the embryonic mouse brain undergo their final rounds of cell division, giving rise to a wide spectrum of postmitotic neurons and glia1,2. The link between developmental lineage and cell-type diversity remains an open question. Here we used massively parallel tagging of progenitors to track clonal relationships and transcriptomic signatures during mouse forebrain development. We quantified clonal divergence and convergence across all major cell classes postnatally, and found diverse types of GABAergic neuron that share a common lineage. Divergence of GABAergic clones occurred during embryogenesis upon cell-cycle exit, suggesting that differentiation into subtypes is initiated as a lineage-dependent process at the progenitor cell level.

Parallel functional testing identifies enhancers active in early postnatal mouse brain.

Enhancers are cis-regulatory elements that play critical regulatory roles in modulating developmental transcription programs and driving cell-type-specific and context-dependent gene expression in the brain. The development of massively parallel reporter assays (MPRAs) has enabled high-throughput functional screening of candidate DNA sequences for enhancer activity. Tissue-specific screening of in vivo enhancer function at scale has the potential to greatly expand our understanding of the role of non-coding sequences in development, evolution, and disease. Here, we adapted a self-transcribing regulatory element MPRA strategy for delivery to early postnatal mouse brain via recombinant adeno-associated virus (rAAV). We identified and validated putative enhancers capable of driving reporter gene expression in mouse forebrain, including regulatory elements within an intronic CACNA1C linkage disequilibrium block associated with risk in neuropsychiatric disorder genetic studies. Paired screening and single enhancer in vivo functional testing, as we show here, represents a powerful approach towards characterizing regulatory activity of enhancers and understanding how enhancer sequences organize gene expression in the brain.

EFhd2 brain interactome reveals its association with different cellular and molecular processes.

EFhd2 is a conserved calcium-binding protein that is highly expressed in the central nervous system. We have shown that EFhd2 interacts with tau protein, a key pathological hallmark in Alzheimer's disease and related dementias. However, EFhd2's physiological and pathological functions in the brain are still poorly understood. To gain insights into its physiological function, we identified proteins that co-immunoprecipitated with EFhd2 from mouse forebrain and hindbrain, using tandem mass spectrometry (MS). In addition, quantitative mass spectrometry was used to detect protein abundance changes due to the deletion of the Efhd2gene in mouse forebrain and hindbrain regions. Our data show that mouse EFhd2 is associated with cytoskeleton components, vesicle trafficking modulators, cellular stress response-regulating proteins, and metabolic proteins. Moreover, proteins associated with the cytoskeleton, vesicular transport, calcium signaling, stress response, and metabolic pathways showed differential abundance in Efhd2(-/-) mice. This study presents, for the first time, an EFhd2 brain interactome that it is associated with different cellular and molecular processes. These findings will help prioritize further studies to investigate the mechanisms by which EFhd2modulates these processes in physiological and pathological conditions of the nervous system.

Morphological differences in the commissural connections of the forebrain in white outbred mice and BALB/C mice

BACKGROUND: The study of the mechanisms of interaction of paired structures of the mammalian brain is a fundamental problem of modern neuroscience, which is of great applied importance. Even mild underdevelopment of the corpus callosum in humans can lead to autism. It is known that the intensity of intraspecific interactions in BALB/c mice is lower than in white outbred ones, while some BALB/c substrains are characterized by underdevelopment of the corpus callosum.
 AIM: To compare the morphological parameters of the large brain commissures in white outbred mice and BALB/c mice grown in the Rappolovo nursery (Leningrad region).
 MATERIALS AND METHODS: The morphology of the corpus callosum was studied in 13 male white outbred mice and 7 male BALB/c mice at the age of 8 months.
 RESULTS: In mice of both subpopulations, the area of the anterior commissure of the left hemisphere was smaller than that of the right hemisphere (p 0.05). There were no differences between subpopulations in this parameter. The area of the left section of the corpus callosum trunkus in outbred mice was larger than the right one (p 0.001), while in BALB/c mice the areas of the left and right slices did not differ. Despite the absence of significant differences in the area of the anterior part (rostrum et genu) of the corpus callosum the density of the location of oligodendrocytes in this brain structure in the mice of the two subpopulations was different. The number of oligodendrocytes in 0.01 mm2 on the left section of the anterior part of the corpus callosum in BALB/c mice was greater than in white outbred mice (p 0.05). A similar trend was revealed when comparing slices of the right hemisphere (p = 0.065).
 CONCLUSIONS: The large area of the right parasagittal slice of the anterior commissure suggests that some of its constituent fibers do not cross the midline, but end within the same hemisphere, which may be the morphological basis for the functional dominance of the temporal cortex of the left hemisphere in mice of both subpopulations. The corpus callosum in BALB/c mice is developed symmetrically, and in white outbred ones asymmetrically. This feature may be the morphological basis for the functional dominance of the parietal cortex of the right hemisphere in outbred animals.

Reduced Insulin-Like Growth Factor-I Effects in the Basal Forebrain of Aging Mouse.

It is known that aging is frequently accompanied by a decline in cognition. Furthermore, aging is associated with lower serum IGF-I levels that may contribute to this deterioration. We studied the effect of IGF-I in neurons of the horizontal diagonal band of Broca (HDB) of young (≤6 months old) and old (≥20-month-old) mice to determine if changes in the response of these neurons to IGF-I occur along with aging. Local injection of IGF-I in the HDB nucleus increased their neuronal activity and induced fast oscillatory activity in the electrocorticogram (ECoG). Furthermore, IGF-I facilitated tactile responses in the primary somatosensory cortex elicited by air-puffs delivered in the whiskers. These excitatory effects decreased in old mice. Immunohistochemistry showed that cholinergic HDB neurons express IGF-I receptors and that IGF-I injection increased the expression of c-fos in young, but not in old animals. IGF-I increased the activity of optogenetically-identified cholinergic neurons in young animals, suggesting that most of the IGF-I-induced excitatory effects were mediated by activation of these neurons. Effects of aging were partially ameliorated by chronic IGF-I treatment in old mice. The present findings suggest that reduced IGF-I activity in old animals participates in age-associated changes in cortical activity.

Conditional Inactivation of Limbic Neuropeptide Y-1 Receptors Increases Vulnerability to Diet-Induced Obesity in Male Mice.

NPY and its Y1 cognate receptor (Y1R) have been shown to be involved in the regulation of stress, anxiety, depression and energy homeostasis. We previously demonstrated that conditional knockout of Npy1r gene in the excitatory neurons of the forebrain of adolescent male mice (Npy1rrfb mice) decreased body weight growth and adipose tissue and increased anxiety. In the present study, we used the same conditional system to examine whether the targeted disruption of the Npy1r gene in limbic areas might affect susceptibility to obesity and associated disorders during adulthood in response to a 3-week high-fat diet (HFD) regimen. We demonstrated that following HFD exposure, Npy1rrfb male mice showed increased body weight, visceral adipose tissue, and blood glucose levels, hyperphagia and a dysregulation of calory intake as compared to control Npy1r2lox mice. These results suggest that low expression of Npy1r in limbic areas impairs habituation to high caloric food and causes high susceptibility to diet-induced obesity and glucose intolerance in male mice, uncovering a specific contribution of the limbic Npy1r gene in the dysregulation of the eating/satiety balance.