A serotonin-dependent mechanism explains the leptin regulation of bone mass, appetite, and energy expenditure.

Leptin inhibition of bone mass accrual requires the integrity of specific hypothalamic neurons but not expression of its receptor on these neurons. The same is true for its regulation of appetite and energy expenditure. This suggests that leptin acts elsewhere in the brain to achieve these three functions. We show here that brainstem-derived serotonin (BDS) favors bone mass accrual following its binding to Htr2c receptors on ventromedial hypothalamic neurons and appetite via Htr1a and 2b receptors on arcuate neurons. Leptin inhibits these functions and increases energy expenditure because it reduces serotonin synthesis and firing of serotonergic neurons. Accordingly, while abrogating BDS synthesis corrects the bone, appetite and energy expenditure phenotypes caused by leptin deficiency, inactivation of the leptin receptor in serotonergic neurons recapitulates them fully. This study modifies the map of leptin signaling in the brain and identifies a molecular basis for the common regulation of bone and energy metabolisms. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online.

Gαi/o-coupled receptor signaling restricts pancreatic ß-cell expansion.

Gi-GPCRs, G protein-coupled receptors that signal via Gα proteins of the i/o class (Gαi/o), acutely regulate cellular behaviors widely in mammalian tissues, but their impact on the development and growth of these tissues is less clear. For example, Gi-GPCRs acutely regulate insulin release from pancreatic ß cells, and variants in genes encoding several Gi-GPCRs--including the α-2a adrenergic receptor, ADRA2A--increase the risk of type 2 diabetes mellitus. However, type 2 diabetes also is associated with reduced total ß-cell mass, and the role of Gi-GPCRs in establishing ß-cell mass is unknown. Therefore, we asked whether Gi-GPCR signaling regulates ß-cell mass. Here we show that Gi-GPCRs limit the proliferation of the insulin-producing pancreatic ß cells and especially their expansion during the critical perinatal period. Increased Gi-GPCR activity in perinatal ß cells decreased ß-cell proliferation, reduced adult ß-cell mass, and impaired glucose homeostasis. In contrast, Gi-GPCR inhibition enhanced perinatal ß-cell proliferation, increased adult ß-cell mass, and improved glucose homeostasis. Transcriptome analysis detected the expression of multiple Gi-GPCRs in developing and adult ß cells, and gene-deletion experiments identified ADRA2A as a key Gi-GPCR regulator of ß-cell replication. These studies link Gi-GPCR signaling to ß-cell mass and diabetes risk and identify it as a potential target for therapies to protect and increase ß-cell mass in patients with diabetes.

CREB mediates brain serotonin regulation of bone mass through its expression in ventromedial hypothalamic neurons.

Serotonin is a bioamine regulating bone mass accrual differently depending on its site of synthesis. It decreases accrual when synthesized in the gut, and increases it when synthesized in the brain. The signal transduction events elicited by gut-derived serotonin once it binds to the Htr1b receptor present on osteoblasts have been identified and culminate in cAMP response element-binding protein (CREB) regulation of osteoblast proliferation. In contrast, we do not know how brain-derived serotonin favors bone mass accrual following its binding to the Htr2c receptor on neurons of the hypothalamic ventromedial nucleus (VMH). We show here--through gene expression analysis, serotonin treatment of wild-type and Htr2c(-/-) hypothalamic explants, and cell-specific gene deletion in the mouse--that, following its binding to the Htr2c receptor on VMH neurons, serotonin uses a calmodulin kinase (CaMK)-dependent signaling cascade involving CaMKKß and CaMKIV to decrease the sympathetic tone and increase bone mass accrual. We further show that the transcriptional mediator of these events is CREB, whose phosphorylation on Ser 133 is increased by CaMKIV following serotonin treatment of hypothalamic explants. A microarray experiment identified two genes necessary for optimum sympathetic activity whose expression is regulated by CREB. These results provide a molecular understanding of how serotonin signals in hypothalamic neurons to regulate bone mass accrual and identify CREB as a critical determinant of this function, although through different mechanisms depending on the cell type, neuron, or osteoblast in which it is expressed.

Serotonin and the orchestration of energy balance.

The phylogenetically ancient signaling molecule serotonin is found in all species that possess nervous systems and orchestrates diverse behavioral and physiological processes in the service of energy balance. In some instances, the manner in which serotonin signaling influences these processes appears comparable among invertebrate and vertebrate species. Within mammalian species, central nervous system serotonergic signaling influences both behavioral and physiological determinants of energy balance. Within the gastrointestinal tract, serotonin mediates diverse sensory, motor, and secretory functions. Further examinations of serotonergic influences on peripheral organ systems are likely to uncover novel functions consistent with an apparently pervasive association between serotonergic signaling and physiological substrates of energy balance.

A robust automated system elucidates mouse home cage behavioral structure.

Patterns of behavior exhibited by mice in their home cages reflect the function and interaction of numerous behavioral and physiological systems. Detailed assessment of these patterns thus has the potential to provide a powerful tool for understanding basic aspects of behavioral regulation and their perturbation by disease processes. However, the capacity to identify and examine these patterns in terms of their discrete levels of organization across diverse behaviors has been difficult to achieve and automate. Here, we describe an automated approach for the quantitative characterization of fundamental behavioral elements and their patterns in the freely behaving mouse. We demonstrate the utility of this approach by identifying unique features of home cage behavioral structure and changes in distinct levels of behavioral organization in mice with single gene mutations altering energy balance. The robust, automated, reproducible quantification of mouse home cage behavioral structure detailed here should have wide applicability for the study of mammalian physiology, behavior, and disease.

Essential function of HIPK2 in TGFbeta-dependent survival of midbrain dopamine neurons.

Transforming growth factor beta (TGFbeta) is a potent trophic factor for midbrain dopamine (DA) neurons, but its in vivo function and signaling mechanisms are not entirely understood. We show that the transcriptional cofactor homeodomain interacting protein kinase 2 (HIPK2) is required for the TGFbeta-mediated survival of mouse DA neurons. The targeted deletion of Hipk2 has no deleterious effect on the neurogenesis of DA neurons, but leads to a selective loss of these neurons that is due to increased apoptosis during programmed cell death. As a consequence, Hipk2(-/-) mutants show an array of psychomotor abnormalities. The function of HIPK2 depends on its interaction with receptor-regulated Smads to activate TGFbeta target genes. In support of this notion, DA neurons from Hipk2(-/-) mutants fail to survive in the presence of TGFbeta3 and Tgfbeta3(-/-) mutants show DA neuron abnormalities similar to those seen in Hipk2(-/-) mutants. These data underscore the importance of the TGFbeta-Smad-HIPK2 pathway in the survival of DA neurons and its potential as a therapeutic target for promoting DA neuron survival during neurodegeneration.

Impact of serotonin 2C receptor null mutation on physiology and behavior associated with nigrostriatal dopamine pathway function.

The impact of serotonergic neurotransmission on brain dopaminergic pathways has substantial relevance to many neuropsychiatric disorders. A particularly prominent role has been ascribed to the inhibitory effects of serotonin 2C receptor (5-HT(2C)R) activation on physiology and behavior mediated by the mesolimbic dopaminergic pathway, particularly in the terminal region of the nucleus accumbens. The influence of this receptor subtype on functions mediated by the nigrostriatal dopaminergic pathway is less clear. Here we report that a null mutation eliminating expression of 5-HT(2C)Rs produces marked alterations in the activity and functional output of this pathway. 5-HT(2C)R mutant mice displayed increased activity of substantia nigra pars compacta (SNc) dopaminergic neurons, elevated baseline extracellular dopamine concentrations in the dorsal striatum (DSt), alterations in grooming behavior, and enhanced sensitivity to the stereotypic behavioral effects of d-amphetamine and GBR 12909. These psychostimulant responses occurred in the absence of phenotypic differences in drug-induced extracellular dopamine concentration, suggesting a phenotypic alteration in behavioral responses to released dopamine. This was further suggested by enhanced behavioral responses of mutant mice to the D(1) receptor agonist SKF 81297. Differences in DSt D(1) or D(2) receptor expression were not found, nor were differences in medium spiny neuron firing patterns or intrinsic membrane properties following dopamine stimulation. We conclude that 5-HT(2C)Rs regulate nigrostriatal dopaminergic activity and function both at SNc dopaminergic neurons and at a locus downstream of the DSt.

Serotonin activates murine alveolar macrophages through 5-HT2C receptors.

Serotonin (5-HT), known as neuromodulator, regulates immune responses and inflammatory cascades. The expression and function of 5-HT receptors on alveolar macrophages (AM), which are the major fraction of pulmonary immune cells, remain elusive. Therefore, we determined the expression of 5-HT type 2 receptors and investigated the effects evoked by stimulation with 5-HT in AM compared with alveolar epithelial cells (AEC). Quantitative PCR (qPCR) analysis revealed expression of the receptors 5-HT(2A) and 5-HT(2B) in AEC and of 5-HT(2C) in AM. In AM, 5-HT (10(-5) M) induced a rise in intracellular calcium concentration ([Ca(2+)](i)) that was initiated by release of Ca(2+) from intracellular stores and depended on extracellular Ca(2+) in a sustained phase. This 5-HT-induced increase in [Ca(2+)](i) was not observed in AM treated with the 5-HT(2C)-selective inhibitor RS-102221 and in AM derived from 5-HT(2C)-deficient mice. AM stimulated with 5-HT (10(-5) M) showed increased expression of CCL2 (MCP-1) mRNA as assayed by qPCR at 4 h and augmented production of CCL2 protein as determined by dot-blot assay and ELISA at 24 h. Notably, in 5-HT(2C)-deficient AM, CCL2 production was not induced by 5-HT treatment. Moreover, transcriptional responses to 5-HT exposure assayed by microarray experiments were only observed in AM from wild-type animals and not in AM derived from 5-HT(2C)-deficient mice. Taken together, these data demonstrate the presence of functional 5-HT(2C) receptors on AM and suggest a role of 5-HT as novel modulator of AM function. These effects are exclusively driven by the 5-HT(2C) receptor, thereby providing the potential for selective intervention.

Precise pattern of recombination in serotonergic and hypothalamic neurons in a Pdx1-cre transgenic mouse line.

BACKGROUND: Multicellular organisms are characterized by a remarkable diversity of morphologically distinct and functionally specialized cell types. Transgenic techniques for the manipulation of gene expression in specific cellular populations are highly useful for elucidating the development and function of these cellular populations. Given notable similarities in developmental gene expression between pancreatic ß-cells and serotonergic neurons, we examined the pattern of Cre-mediated recombination in the nervous system of a widely used mouse line, Pdx1-cre (formal designation, Tg(Ipf1-cre)89.1Dam), in which the expression of Cre recombinase is driven by regulatory elements upstream of the pdx1 (pancreatic-duodenal homeobox 1) gene. METHODS: Single (hemizygous) transgenic mice of the pdx1-creCre/0 genotype were bred to single (hemizygous) transgenic reporter mice (Z/EG and rosa26R lines). Recombination pattern was examined in offspring using whole-mount and sectioned histological preparations at e9.5, e10.5, e11.5, e16.5 and adult developmental stages. RESULTS: In addition to the previously reported pancreatic recombination, recombination in the developing nervous system and inner ear formation was observed. In the central nervous system, we observed a highly specific pattern of recombination in neuronal progenitors in the ventral brainstem and diencephalon. In the rostral brainstem (r1-r2), recombination occurred in newborn serotonergic neurons. In the caudal brainstem, recombination occurred in non-serotonergic cells. In the adult, this resulted in reporter expression in the vast majority of forebrain-projecting serotonergic neurons (located in the dorsal and median raphe nuclei) but in none of the spinal cord-projecting serotonergic neurons of the caudal raphe nuclei. In the adult caudal brainstem, reporter expression was widespread in the inferior olive nucleus. In the adult hypothalamus, recombination was observed in the arcuate nucleus and dorsomedial hypothalamus. Recombination was not observed in any other region of the central nervous system. Neuronal expression of endogenous pdx1 was not observed. CONCLUSIONS: The Pdx1-cre mouse line, and the regulatory elements contained in the corresponding transgene, could be a valuable tool for targeted genetic manipulation of developing forebrain-projecting serotonergic neurons and several other unique neuronal sub-populations. These results suggest that investigators employing this mouse line for studies of pancreatic function should consider the possible contributions of central nervous system effects towards resulting phenotypes.

Social circuits: peptidergic regulation of mammalian social behavior.

Mammals have developed patterns of social relationships that enhance the survival of individuals and maximize the reproductive success of species. Although social stimuli and social responses are highly complex, recent studies are providing substantial insights into their neural substrates. Neural pathways employing the nonapeptides vasopressin and oxytocin play a particularly prominent role both in social recognition and the expression of appropriate social responses. New insights into social neuroscience are discussed, along with the relevance of this rapidly developing field to human relationships and disease processes.

Alpha1-adrenergic receptors prevent a maladaptive cardiac response to pressure overload.

An alpha1-adrenergic receptor (alpha1-AR) antagonist increased heart failure in the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT), but it is unknown whether this adverse result was due to alpha1-AR inhibition or a nonspecific drug effect. We studied cardiac pressure overload in mice with double KO of the 2 main alpha1-AR subtypes in the heart, alpha 1A (Adra1a) and alpha 1B (Adra1b). At 2 weeks after transverse aortic constriction (TAC), KO mouse survival was only 60% of WT, and surviving KO mice had lower ejection fractions and larger end-diastolic volumes than WT mice. Mechanistically, final heart weight and myocyte cross-sectional area were the same after TAC in KO and WT mice. However, KO hearts after TAC had increased interstitial fibrosis, increased apoptosis, and failed induction of the fetal hypertrophic genes. Before TAC, isolated KO myocytes were more susceptible to apoptosis after oxidative and beta-AR stimulation, and beta-ARs were desensitized. Thus, alpha1-AR deletion worsens dilated cardiomyopathy after pressure overload, by multiple mechanisms, indicating that alpha1-signaling is required for cardiac adaptation. These results suggest that the adverse cardiac effects of alpha1-antagonists in clinical trials are due to loss of alpha1-signaling in myocytes, emphasizing concern about clinical use of alpha1-antagonists, and point to a revised perspective on sympathetic activation in heart failure.

Dysregulation of striatal dopamine signaling by amphetamine inhibits feeding by hungry mice.

Amphetamine (AMPH) releases monoamines, transiently stimulates locomotion, and inhibits feeding. Using a genetic approach, we show that mice lacking dopamine (DA-deficient, or DD, mice) are resistant to the hypophagic effects of a moderate dose of AMPH (2 microg/g) but manifest normal AMPH-induced hypophagia after restoration of DA signaling in the caudate putamen by viral gene therapy. By contrast, AMPH-induced hypophagia in response to the same dose of AMPH is not blunted in mice lacking the ability to make norepinephrine and epinephrine (Dbh(-/-)), dopamine D(2) receptors (D2r(-/-)), dopamine D(1) receptors (D1r(-/-)), serotonin 2C receptors (Htr2c(-/Y)), neuropeptide Y (Npy(-/-)), and in mice with compromised melanocortin signaling (A(y)). We suggest that, at this moderate dose of AMPH, dysregulation of striatal DA is the primary cause of AMPH-induced hypophagia and that regulated striatal dopaminergic signaling may be necessary for normal feeding behaviors.

Serotonin activates the hypothalamic-pituitary-adrenal axis via serotonin 2C receptor stimulation.

The dynamic interplay between serotonin [5-hydroxytryptamine (5-HT)] neurotransmission and the hypothalamic-pituitary-adrenal (HPA) axis has been extensively studied over the past 30 years, but the underlying mechanism of this interaction has not been defined. A possibility receiving little attention is that 5-HT regulates upstream corticotropin-releasing hormone (CRH) signaling systems via activation of serotonin 2C receptors (5-HT(2C)Rs) in the paraventricular nucleus of the hypothalamus (PVH). Through complementary approaches in wild-type rodents and 5-HT(2C)R-deficient mice, we determined that 5-HT(2C)Rs are necessary for 5-HT-induced HPA axis activation. We used laser-capture PVH microdissection followed by microarray analysis to compare the expression of 13 5-HTRs. Only 5-HT(2C)R and 5-HT(1D)R transcripts were consistently identified as present in the PVH, and of these, the 5-HT(2C)R was expressed at a substantially higher level. The abundant expression of 5-HT(2C)Rs in the PVH was confirmed with in situ hybridization histochemistry. Dual-neurohistochemical labeling revealed that approximately one-half of PVH CRH-containing neurons coexpressed 5-HT(2C)R mRNA. We observed that PVH CRH neurons consistently depolarized in the presence of a high-affinity 5-HT(2C)R agonist, an effect blocked by a 5-HT(2C)R antagonist. Supporting the importance of 5-HT(2C)Rs in CRH neuronal activity, genetic inactivation of 5-HT(2C)Rs produced a downregulation of CRH mRNA and blunted CRH and corticosterone release after 5-HT compound administration. These findings thus provide a mechanistic explanation for the longstanding observation of HPA axis stimulation in response to 5-HT and thereby give insight into the neural circuitry mediating the complex neuroendocrine responses to stress.

Synergistic impairment of glucose homeostasis in ob/ob mice lacking functional serotonin 2C receptors.

To investigate how serotonin and leptin interact in the regulation of energy balance and glucose homeostasis, we generated a genetic mouse model, the OB2C mouse, which lacks functional serotonin 2C receptors and the adipocyte hormone leptin. The OB2C mice exhibited a dramatic diabetes phenotype, evidenced by a synergistic increase in serum glucose levels and water intake. The severity of the animals' diabetes phenotype would not have been predicted from the phenotypic characterization of mice bearing mutations of either the leptin (OB mutant mice) or the serotonin 2C receptor gene (2C mutant mice). The synergistic impairment in glucose homeostasis developed at an age when OB2C mice did not differ in body weight from OB mice, suggesting that this impairment was not an indirect consequence of increased adiposity. We also demonstrated that the improvement in glucose tolerance in wild-type mice treated with the serotonin releaser and reuptake inhibitor fenfluramine was blunted in 2C mutant mice. These pharmacological and genetic findings provide evidence that the serotonin 2C receptor has direct effects on glucose homeostasis.

A novel method for automatic quantification of psychostimulant-evoked route-tracing stereotypy: application to Mus musculus.

RATIONALE: Route-tracing stereotypy is a powerful behavioral correlate of striatal function that is difficult to quantify. Measurements of route-tracing stereotypy in an automated, high throughput, easily quantified, and replicable manner would facilitate functional studies of this central nervous system region. OBJECTIVE: We examined how t-pattern sequential analysis (Magnusson Behav Res Meth Instrum Comput 32:93-110, 2000) can be used to quantify mouse route-tracing stereotypies. This method reveals patterns by testing whether particular sequences of defined states occur within a specific time interval at a probability greater than chance. RESULTS: Mouse home-cage locomotor patterns were recorded after psychostimulant administration (GBR 12909, 0, 3, 10, and 30 mg/kg; d-amphetamine, 0, 2.5, 5, and 10 mg/kg). After treatment with GBR 12909, dose-dependent increases in the number of found patterns and overall pattern length and depth were observed. Similar findings were seen after treatment with d-amphetamine up to the dosage where focused stereotypies dominated behavioral response. For both psychostimulants, detected patterns displayed similar morphological features. Pattern sets containing a few frequently repeated patterns of greater length/depth accounted for a greater percentage of overall trial duration in a dose-dependant manner. This finding led to the development of a t-pattern-derived route-tracing stereotypy score. Compared to scores derived by manual observation, these t-pattern-derived route-tracing stereotypy scores yielded similar results with less within-group variability. These findings remained similar after reanalysis with removal of patterns unmatched after human scoring and after normalization of locomotor speeds at low and high ranges. CONCLUSIONS: T-pattern analysis is a versatile and robust pattern detection and quantification algorithm that complements currently available observational phenotyping methods.

Neurobehavioral assessment in the information age.

The elucidation of the human and mouse genomes provides new opportunities for exploring the genetic underpinnings of complex mammalian behaviors. This information also provides new windows into the pathophysiology and treatment of neuropsychiatric diseases. Optimal use of the rapidly escalating numbers of mouse lines engineered for these purposes is hindered, however, by practical and theoretical limitations of common behavioral analyses. New strategies combining automated behavioral monitoring and information technologies are currently under development in both academic and industrial settings. These hold promise, both for improving the throughput of mouse behavioral assessment and for providing new insights into the neurobiology of mammalian behavioral regulation.

Brain-derived neurotrophic factor regulates energy balance downstream of melanocortin-4 receptor.

The melanocortin-4 receptor (MC4R) is critically involved in regulating energy balance, and obesity has been observed in mice with mutations in the gene for brain-derived neurotrophic factor (BDNF). Here we report that BDNF is expressed at high levels in the ventromedial hypothalamus (VMH) where its expression is regulated by nutritional state and by MC4R signaling. In addition, similar to MC4R mutants, mouse mutants that expresses the BDNF receptor TrkB at a quarter of the normal amount showed hyperphagia and excessive weight gain on higher-fat diets. Furthermore, BDNF infusion into the brain suppressed the hyperphagia and excessive weight gain observed on higher-fat diets in mice with deficient MC4R signaling. These results show that MC4R signaling controls BDNF expression in the VMH and support the hypothesis that BDNF is an important effector through which MC4R signaling controls energy balance.

A null mutation of the serotonin 6 receptor alters acute responses to ethanol.

The anatomical distribution and pharmacology of serotonin 6 receptors (5-HT6Rs) implicate them as contributors to the serotonergic regulation of complex behavior. To complement the limited range of pharmacological tools available to examine 5-HT6R function, we have generated a mouse line bearing a constitutive null mutation of the 5-HT6R gene. No perturbations of baseline behavior were noted in a wide array of assays pertinent to multiple neurobehavioral processes. However, 5-HT6R mutant mice demonstrated reduced responses to the ataxic and sedative effects of ethanol. No differences in ethanol metabolism were evident between wild-type and 5-HT6R mutant mice. These findings implicate 5-HT6Rs in the serotonergic modulation of responses to ethanol.

Age-related changes in cerebellar and hypothalamic function accompany non-microglial immune gene expression, altered synapse organization, and excitatory amino acid neurotransmission deficits.

We describe age-related molecular and neuronal changes that disrupt mobility or energy balance based on brain region and genetic background. Compared to young mice, aged C57BL/6 mice exhibit marked locomotor (but not energy balance) impairments. In contrast, aged BALB mice exhibit marked energy balance (but not locomotor) impairments. Age-related changes in cerebellar or hypothalamic gene expression accompany these phenotypes. Aging evokes upregulation of immune pattern recognition receptors and cell adhesion molecules. However, these changes do not localize to microglia, the major CNS immunocyte. Consistent with a neuronal role, there is a marked age-related increase in excitatory synapses over the cerebellum and hypothalamus. Functional imaging of these regions is consistent with age-related synaptic impairments. These studies suggest that aging reactivates a developmental program employed during embryogenesis where immune molecules guide synapse formation and pruning. Renewed activity in this program may disrupt excitatory neurotransmission, causing significant behavioral deficits.

Elucidation of The Behavioral Program and Neuronal Network Encoded by Dorsal Raphe Serotonergic Neurons.

Elucidating how the brain's serotonergic network mediates diverse behavioral actions over both relatively short (minutes-hours) and long period of time (days-weeks) remains a major challenge for neuroscience. Our relative ignorance is largely due to the lack of technologies with robustness, reversibility, and spatio-temporal control. Recently, we have demonstrated that our chemogenetic approach (eg, Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)) provides a reliable and robust tool for controlling genetically defined neural populations. Here we show how short- and long-term activation of dorsal raphe nucleus (DRN) serotonergic neurons induces robust behavioral responses. We found that both short- and long-term activation of DRN serotonergic neurons induce antidepressant-like behavioral responses. However, only short-term activation induces anxiogenic-like behaviors. In parallel, these behavioral phenotypes were associated with a metabolic map of whole brain network activity via a recently developed non-invasive imaging technology DREAMM (DREADD Associated Metabolic Mapping). Our findings reveal a previously unappreciated brain network elicited by selective activation of DRN serotonin neurons and illuminate potential therapeutic and adverse effects of drugs targeting DRN neurons.