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.

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.

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.

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.

Deletion of Mecp2 in Sim1-expressing neurons reveals a critical role for MeCP2 in feeding behavior, aggression, and the response to stress.

Rett Syndrome (RTT) is an autism spectrum disorder caused by mutations in the X-linked gene encoding methyl-CpG binding protein 2 (MeCP2). In order to map the neuroanatomic origins of the complex neuropsychiatric behaviors observed in patients with RTT and to uncover endogenous functions of MeCP2 in the hypothalamus, we removed Mecp2 from Sim1-expressing neurons in the hypothalamus using Cre-loxP technology. Loss of MeCP2 in Sim1-expressing neurons resulted in mice that recapitulated the abnormal physiological stress response that is seen upon MeCP2 dysfunction in the entire brain. Surprisingly, we also uncovered a role for MeCP2 in the regulation of social and feeding behaviors since the Mecp2 conditional knockout (CKO) mice were aggressive, hyperphagic, and obese. This study demonstrates that deleting Mecp2 in a defined brain region is an excellent approach to map the neuronal origins of complex behaviors and provides new insight about the function of MeCP2 in specific neurons.

Central serotonin and melanocortin pathways regulating energy homeostasis.

It is now established that the hypothalamus is essential in coordinating endocrine, autonomic, and behavioral responses to changes in energy availability. However, the interaction of key peptides, neuropeptides, and neurotransmitters systems within the hypothalamus has yet to be delineated. Recently, we investigated the mechanisms through which central serotonergic (5-hydroxytryptamine, 5-HT) systems recruit leptin-responsive hypothalamic pathways, such as the melanocortin systems, to affect energy balance. Through a combination of functional neuroanatomy, feeding, and electrophysiology studies in rodents, we found that 5-HT drugs require functional melanocortin pathways to exert their effects on food intake. Specifically, we observed that anorectic 5-HT drugs activate pro-opiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (Arc). We provide evidence that the serotonin 2C receptor (5-HT(2C)R) is expressed on POMC neurons and contributes to this effect. Finally, we found that 5-HT drug-induced hypophagia is attenuated by pharmacological or genetic blockade of downstream melanocortin 3 and 4 receptors. We review candidate brain regions expressing melanocortin 3 and 4 receptors that play a role in energy balance. A model is presented in which activation of the melanocortin system is downstream of 5-HT and is necessary to produce the complete anorectic effect of 5-HT drugs. The data reviewed in this paper incorporate the central 5-HT system to the growing list of metabolic signals that converge on melanocortin neurons in the hypothalamus.