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Hypothalamic POMC neurons are required for glucose and energy homeostasis. POMC neurons have a wide synaptic connection with neurons both within and outside the hypothalamus, and their activity is controlled by a balance between excitatory and inhibitory synaptic inputs. Brain glucose-sensing plays an essential role in the maintenance of normal body weight and metabolism; however, the effect of glucose on synaptic transmission in POMC neurons is largely unknown. Here we identified three types of POMC neurons (EPSC(+), EPSC(-), and EPSC(+/-)) based on their glucose-regulated spontaneous excitatory postsynaptic currents (sEPSCs), using whole-cell patch-clamp recordings. Lowering extracellular glucose decreased the frequency of sEPSCs in EPSC(+) neurons, but increased it in EPSC(-) neurons. Unlike EPSC(+) and EPSC(-) neurons, EPSC(+/-) neurons displayed a bi-phasic sEPSC response to glucoprivation. In the first phase of glucoprivation, both the frequency and the amplitude of sEPSCs decreased, whereas in the second phase, they increased progressively to the levels above the baseline values. Accordingly, lowering glucose exerted a bi-phasic effect on spontaneous action potentials in EPSC(+/-) neurons. Glucoprivation decreased firing rates in the first phase, but increased them in the second phase. These data indicate that glucose induces distinct excitatory synaptic plasticity in different subpopulations of POMC neurons. This synaptic remodeling is likely to regulate the sensitivity of the melanocortin system to neuronal and hormonal signals.

Hypothalamic proopiomelanocortin (POMC) neurons constitute a critical anorexigenic node in the central nervous system (CNS) for maintaining energy balance. These neurons directly affect energy expenditure and feeding behavior by releasing bioactive neuropeptides but are also subject to signals directly related to nutritional state such as the adipokine leptin. To further investigate the interaction of diet and leptin on hypothalamic POMC peptide levels, we exposed 8- to 10-wk-old male POMC-Discosoma red fluorescent protein (DsRed) transgenic reporter mice to either 24-48 h (acute) or 2 wk (chronic) food restriction, high-fat diet (HFD), or leptin treatment. Using semiquantitative immunofluorescence and radioimmunoassays, we discovered that acute fasting and chronic food restriction decreased the levels of adrenocorticotropic hormone (ACTH), α-melanocyte-stimulating hormone (α-MSH), and β-endorphin in the hypothalamus, together with decreased DsRed fluorescence, compared with control ad libitum-fed mice. Furthermore, acute but not chronic HFD or leptin administration selectively increased α-MSH levels in POMC fibers and increased DsRed fluorescence in POMC cell bodies. HFD and leptin treatments comparably increased circulating leptin levels at both time points, suggesting that transcription of Pomc and synthesis of POMC peptide products are not modified in direct relation to the concentration of plasma leptin. Our findings indicate that negative energy balance persistently downregulated POMC peptide levels, and this phenomenon may be partially explained by decreased leptin levels, since these changes were blocked in fasted mice treated with leptin. In contrast, sustained elevation of plasma leptin by HFD or hormone supplementation did not significantly alter POMC peptide levels, indicating that enhanced leptin signaling does not chronically increase Pomc transcription and peptide synthesis.

The microbial pattern-recognizing Toll-like receptors (TLRs) are major signal transducers known to shape and influence the postnatal maturation of host intestinal epithelium. Perturbations in this intricate host-microbe cross-talk have been reported to be associated with uncontrolled epithelial cell growth and thus potential cancer development by mechanisms which are largely unknown. We therefore generated transgenic mice carrying a constitutively active TLR4 (CD4-TLR4) linked to an intestinal epithelial cell-specific promoter. Ex vivo analysis of transgenic crypt-villus organoid cultures revealed an increased proliferative capacity and a lowered cyclooxygenase 2 (Cox-2) expression in these organoids compared with wild-type control cultures. Introducing the CD4-TLR4 transgene into APC(Min/+) mice (CD4-TLR4-APC(Min/+)), a model of colorectal carcinoma, resulted in a dramatic drop in tumor load as compared with control APC(Min/+) mice. Intestinal tumors from CD4-TLR4-APC(Min/+) mice displayed reduced Cox-2 protein, elevated interferon β expression and increased caspase-3 activity, which correlated with increased apoptosis in vivo. Thus, our data reveal that host microbiota-mediated signal transduction via TLR4 in intestinal epithelial cells is far more complex than what is previously reported.

Mutations in regulatory regions including enhancers are an important source of variation and innovation during evolution. Enhancers can evolve by changes in the sequence, arrangement and repertoire of transcription factor binding sites, but whole enhancers can also be lost or gained in certain lineages in a process of turnover. The proopiomelanocortin gene (Pomc), which encodes a prohormone, is expressed in the pituitary and hypothalamus of all jawed vertebrates. We have previously described that hypothalamic Pomc expression in mammals is controlled by two enhancers-nPE1 and nPE2-that are derived from transposable elements and that presumably replaced the ancestral neuronal Pomc regulatory regions. Here, we show that nPE1 and nPE2, even though they are mammalian novelties with no homologous counterpart in other vertebrates, nevertheless can drive gene expression specifically to POMC neurons in the hypothalamus of larval and adult transgenic zebrafish. This indicates that when neuronal Pomc enhancers originated de novo during early mammalian evolution, the newly created cis- and trans-codes were similar to the ancestral ones. We also identify the neuronal regulatory region of zebrafish pomca and confirm that it is not homologous to the mammalian enhancers. Our work sheds light on the process of gene regulatory evolution by showing how a locus can undergo enhancer turnover and nevertheless maintain the ancestral transcriptional output.

Competition between adult males for limited resources such as food and receptive females is shaped by the male pattern of pituitary growth hormone (GH) secretion that determines body size and the production of urinary pheromones involved in male-to-male aggression. In the brain, dopamine (DA) provides incentive salience to stimuli that predict the availability of food and sexual partners. Although the importance of the GH axis and central DA neurotransmission in social dominance and fitness is clearly appreciated, the two systems have always been studied unconnectedly. Here we conducted a cell-specific genetic dissection study in conditional mutant mice that selectively lack DA D2 receptors (D2R) from pituitary lactotropes (lacDrd2KO) or neurons (neuroDrd2KO). Whereas lacDrd2KO mice developed a normal GH axis, neuroDrd2KO mice displayed fewer somatotropes; reduced hypothalamic Ghrh expression, pituitary GH content, and serum IGF-I levels; and exhibited reduced body size and weight. As a consequence of a GH axis deficit, neuroDrd2KO adult males excreted low levels of major urinary proteins and their urine failed to promote aggression and territorial behavior in control male challengers, in contrast to the urine taken from control adult males. These findings reveal that central D2Rs mediate a neuroendocrine-exocrine cascade that controls the maturation of the GH axis and downstream signals that are critical for fitness, social dominance, and competition between adult males.

Evidence that lipocalin 2 (LCN2) is oncogenic has grown in recent years and comes from both animal models and expression analysis from a variety of human cancers. In the intestine, LCN2 is overexpressed in colitis patients and its overexpression is a negative prognostic indicator in colorectal cancer. Functionally, LCN2 has a number of different activities that may contribute to its oncogenic potential, including increasing matrix metalloproteinase activity, control of iron availability and stimulating inflammation. In this report, we examined APCmin intestinal tumorigenesis in an LCN2-deficient background. We found that the loss of LCN2 increased tumor multiplicity specifically in the duodenum, suggesting a potential tumor-suppressive activity. Concurrently, however, LCN2 increased the average small intestinal tumor size particularly in the distal small intestine. We found that this increase was correlated to tumor iron(II) content, suggesting that an iron-scavenging role is important for LCN2 oncogenic activity in the intestine.

Central proopiomelanocortin (POMC) neurons form a potent anorexigenic network, but our understanding of the integration of this hypothalamic circuit throughout the central nervous system (CNS) remains incomplete. POMC neurons extend projections along the rostrocaudal axis of the brain, and can signal with both POMC-derived peptides and fast amino acid neurotransmitters. Although recent experimental advances in circuit-level manipulation have been applied to POMC neurons, many pivotal questions still remain: how and where do POMC neurons integrate metabolic information? Under what conditions do POMC neurons release bioactive molecules throughout the CNS? Are GABA and glutamate or neuropeptides released from POMC neurons more crucial for modulating feeding and metabolism? Resolving the exact stoichiometry of signals evoked from POMC neurons under different metabolic conditions therefore remains an ongoing endeavor. In this review, we analyze the anatomical atlas of this network juxtaposed to the physiological signaling of POMC neurons both in vitro and in vivo. We also consider novel genetic tools to further characterize the function of the POMC circuit in vivo. Our goal is to synthesize a global view of the POMC network, and to highlight gaps that require further research to expand our knowledge on how these neurons modulate energy balance.

Obesity is a chronic metabolic disorder affecting half a billion people worldwide. Major difficulties in managing obesity are the cessation of continued weight loss in patients after an initial period of responsiveness and rebound to pretreatment weight. It is conceivable that chronic weight gain unrelated to physiological needs induces an allostatic regulatory state that defends a supranormal adipose mass despite its maladaptive consequences. To challenge this hypothesis, we generated a reversible genetic mouse model of early-onset hyperphagia and severe obesity by selectively blocking the expression of the proopiomelanocortin gene (Pomc) in hypothalamic neurons. Eutopic reactivation of central POMC transmission at different stages of overweight progression normalized or greatly reduced food intake in these obesity-programmed mice. Hypothalamic Pomc rescue also attenuated comorbidities such as hyperglycemia, hyperinsulinemia, and hepatic steatosis and normalized locomotor activity. However, effectiveness of treatment to normalize body weight and adiposity declined progressively as the level of obesity at the time of Pomc induction increased. Thus, our study using a novel reversible monogenic obesity model reveals the critical importance of early intervention for the prevention of subsequent allostatic overload that auto-perpetuates obesity.

The proopiomelanocortin (Pomc) gene encodes a prepropeptide with essential functions in the response to stress and energy balance, which is expressed in the pituitary and hypothalamus of vertebrate animals. Neuronal expression of Pomc is controlled by two distal enhancers named nPE1 and nPE2. Using transgenic mice, we observed that both enhancers drive identical expression patterns in the mammalian hypothalamus, starting at embryonic day 10.5, when endogenous Pomc expression commences. This overlapping enhancer activity is maintained throughout hypothalamic development and into adulthood. We also found that nPE1 and nPE2 were exapted as neuronal enhancers into the POMC locus after the sequential insertion of two unrelated retroposons. Thus, nPE1 and nPE2 are functional analogs and represent an authentic first example of convergent molecular evolution of cell-specific transcriptional enhancers. In this Commentary we discuss the following questions that remain unanswered: (1) how does transcriptional control of POMC operate in hypothalamic neurons of non-mammalian vertebrates? (2) What evolutionary forces are maintaining two discrete neuronal POMC enhancers under purifying selection for the last ~100 million years in all placental mammals? (3) What is the contribution of MaLRs to genome evolution?

The proopiomelanocortin gene (POMC) is expressed in a group of neurons present in the arcuate nucleus of the hypothalamus. Neuron-specific POMC expression in mammals is conveyed by two distal enhancers, named nPE1 and nPE2. Previous transgenic mouse studies showed that nPE1 and nPE2 independently drive reporter gene expression to POMC neurons. Here, we investigated the evolutionary mechanisms that shaped not one but two neuron-specific POMC enhancers and tested whether nPE1 and nPE2 drive identical or complementary spatiotemporal expression patterns. Sequence comparison among representative genomes of most vertebrate classes and mammalian orders showed that nPE1 is a placental novelty. Using in silico paleogenomics we found that nPE1 originated from the exaptation of a mammalian-apparent LTR retrotransposon sometime between the metatherian/eutherian split (147 Mya) and the placental mammal radiation (≈ 90 Mya). Thus, the evolutionary origin of nPE1 differs, in kind and time, from that previously demonstrated for nPE2, which was exapted from a CORE-short interspersed nucleotide element (SINE) retroposon before the origin of prototherians, 166 Mya. Transgenic mice expressing the fluorescent markers tomato and EGFP driven by nPE1 or nPE2, respectively, demonstrated coexpression of both reporter genes along the entire arcuate nucleus. The onset of reporter gene expression guided by nPE1 and nPE2 was also identical and coincidental with the onset of Pomc expression in the presumptive mouse diencephalon. Thus, the independent exaptation of two unrelated retroposons into functional analogs regulating neuronal POMC expression constitutes an authentic example of convergent molecular evolution of cell-specific enhancers.

Hypothalamic pro-opiomelanocortin (POMC) neurons are the major source of anorectic melanocortin peptides in the brain. A recent study (Mineur et al., 2011) demonstrates that nicotine directly stimulates arcuate POMC neurons through nicotinic acetylcholinergic α3β4 receptors, suggesting a new mechanism to understand the inverse relationship between tobacco smoking and body weight., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Dopamine (DA) D2 receptors expressed in DA neurons (D2 autoreceptors) exert a negative feedback regulation that reduces DA neuron firing, DA synthesis and DA release. As D2 receptors are mostly expressed in postsynaptic neurons, pharmacological and genetic approaches have been unable to definitively address the in vivo contribution of D2 autoreceptors to DA-mediated behaviors. We found that midbrain DA neurons from mice deficient in D2 autoreceptors (Drd2(loxP/loxP); Dat(+/IRES-cre), referred to as autoDrd2KO mice) lacked DA-mediated somatodendritic synaptic responses and inhibition of DA release. AutoDrd2KO mice displayed elevated DA synthesis and release, hyperlocomotion and supersensitivity to the psychomotor effects of cocaine. The mice also exhibited increased place preference for cocaine and enhanced motivation for food reward. Our results highlight the importance of D2 autoreceptors in the regulation of DA neurotransmission and demonstrate that D2 autoreceptors are important for normal motor function, food-seeking behavior, and sensitivity to the locomotor and rewarding properties of cocaine.

The central melanocortin system, consisting of melanocortin peptides, agouti gene related peptide and their receptors plays a critical role in the homeostatic control of energy balance. Loss of function mutations in the genes encoding proopiomelanocortin or melanocortin MC(4) receptors cause profound obesity and hyperphagia. However, little is known about the functional relationship of melanocortin neurocircuits to the temporal organization of meal-taking behavior. We used an operant paradigm that combined lever pressing for food pellet deliveries with free water intake monitored by lickometers to quantify meal patterns in mutant mice that selectively lack proopiomelanocortin expression in hypothalamic neurons (nPOMCKO). Compared to wildtype siblings, nPOMCKO mice consumed 50% more food and water daily and exhibited a more stereotyped feeding pattern characterized by reduced inter-meal and inter-mouse variations. Average meals were larger in size but shorter in duration, with no change in meal number. Consequently, intermeal intervals were prolonged in nPOMCKO mice. Similar patterns were observed in pre-obese juvenile and frankly obese adult mice suggesting that neither age nor degree of obesity was responsible for the altered phenotypes. Spontaneous locomotion and wheel running were decreased in nPOMCKO mice, but circadian variations in locomotor and feeding activity were conserved. These data show that hyperphagia in male nPOMCKO mice is due to increased meal size but not meal number, and this pattern is established by age of 5weeks. The combination of larger, more rapidly consumed meals and prolonged intermeal intervals suggests that proopiomelanocortin peptides are necessary for normal meal termination, but not the maintenance of satiety., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The gene encoding the prohormone proopiomelanocortin (POMC) is mainly expressed in two regions in vertebrates, namely corticotrophs and melanotrophs in the pituitary and a small population of neurons in the arcuate nucleus of the hypothalamus. In this latter region, POMC-derived peptides participate in the control of energy balance and sensitivity to pain. Neuronal expression of POMC is conferred by two enhancers, nPE1 and nPE2, which are conserved in most mammals, but no transcription factors are yet known to bind to these enhancers. In this work, by means of a one-hybrid screening, we identify that nPE2 possesses an element recognized by transcription factors of the nuclear receptor superfamily. This element, named NRBE, is conserved in all known nPE2 enhancers and is necessary to confer full enhancer strength to nPE2-driven reporter gene expression in transgenic mice assays, indicating that the phylogenetic conservation of the element is indicative of its functional importance. In a search for candidate nuclear receptors that might control POMC we observed that estrogen receptor alpha (ESR1) - a known regulator of energy balance at the hypothalamic level - can bind to the NRBE element in vitro. In addition we observed by immunofluorescence that ESR1 is coexpressed with POMC in around 25-30% of hypothalamic neurons of males and females during late embryonic stages and adulthood. Thus, our results indicate that hypothalamic expression of POMC is controlled by nuclear receptors and establish ESR1 as a candidate regulator of POMC., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Arachidonic acid (AA) is generated in the anterior pituitary gland upon stimulation by the ACTH secretagogue, CRH. Using the patch clamp technique, we examined the action of AA on the excitability of single pituitary corticotropes obtained from a transgenic mouse strain that expresses the enhanced green fluorescent protein driven by the proopiomelanocortin promoter. CRH evoked depolarization, but AA caused hyperpolarization. Under voltage clamp condition, AA caused a rapid inhibition of the delayed rectifier K(+) current and then increased a background K(+) current. Inhibition of AA metabolism did not prevent the activation of the K(+) current by AA, suggesting a direct action of AA. The sensitivity of the AA-activated K(+) current to fluoxetine, chlorpromazine, extracellular acidification, diphenylbutylpiperidine antipsychotics, and the membrane permeable cAMP analog [8-(4-chlorophenylthio)-cAMP] suggest that the current is mediated via TWIK-related K(+) channel (TREK)-1 channels. Activation of the CRH receptors that are coupled to the adenylate cyclase pathway suppressed the activation of TREK-1 current by AA and reversed the AA-mediated hyperpolarization. Intracellular acidification (pH 7.0) increased the basal amplitude of TREK-1 current and resulted in hyperpolarizaton. CRH suppressed the basal TREK-1 current in cells with intracellular acidification and caused depolarization. Our finding indicates that TREK-1 channels are important in setting the resting potential in corticotropes. The opposing actions of CRH and AA on the excitability of corticotropes raise the possibility that AA may act as a negative feedback regulator to reduce the stimulatory action of CRH and thus prevent excessive ACTH release during chronic stress.

Evidence showing expression of endogenous opioids in the mammalian retina is sparse. In the present study we examined a transgenic mouse line expressing an obligate dimerized form of Discosoma red fluorescent protein (DsRed) under the control of the pro-opiomelanocortin promoter and distal upstream regulatory elements to assess whether pro-opiomelanocortin peptide (POMC), and its opioid cleavage product, beta-endorphin, are expressed in the mouse retina. Using double label immunohistochemistry we found that DsRed fluorescence was restricted to a subset of GAD-67-positive cholinergic amacrine cells of both orthotopic and displaced subtypes. About 50% of cholinergic amacrine cells colocalized DsRed and a large fraction of DsRed-expressing amacrine cells was positive for beta-endorphin immunostaining, whereas beta-endorphin-immunoreactive neurons were absent in retinas of POMC null mice. Our findings contribute to a growing body of evidence demonstrating that opioid peptides are an integral component of vertebrate retinas, including those of mammals., ((c) 2010 Wiley-Liss, Inc.)

The role of dopaminergic receptors in the control of GH release remains controversial. The dopamine receptor 2 (D2R) knockout mouse represents a useful model to study the participation of the D2R on growth and GHRH-GH regulation. These knockout mice have hyperprolactinemia and lactotrope hyperplasia, but unexpectedly, they are also growth retarded. In D2R knockout mice there is a significant decrease in somatotrope population, which is paralleled by decreased GH content and output from pituitary cells. The sensitivity of GHRH-induced GH and cAMP release is similar between genotypes, even though the response amplitude is lower in knockouts. We point to an involvement of D2R signaling at the hypothalamic level as dopamine did not release GH acting at the pituitary level, and both somatostatin and GHRH mRNA expression are altered in knockout mice. The similarity of the pituitary defect in the D2R knockout mouse to that of GHRH deficient models suggests a probable mechanism. Loss of dopamine signaling via hypothalamic D2Rs at a critical age may cause inadequate GHRH secretion subsequently leading to inappropriate somatotrope lineage development. Furthermore, GH pulsatility, which depends on a regulated temporal balance between GHRH and somatostatin output might be compromised in D2R knockout mice, leading to lower IGF-I, and growth retardation., (Copyright (c) 2010 S. Karger AG, Basel.)

Proopiomelanocortin (Pomc) neurons play a central role in energy homeostasis. Despite the complexity of Pomc posttranslational processing, regulation of Pomc gene expression often takes center stage. Complementary papers that zero in on distinct carboxypeptidases (Plum et al., 2009; Wallingford et al., 2009) now refocus the spotlight on regulated peptide cleavage.

Proopiomelanocortin (POMC) neurons have been intensively studied because of their essential role in regulating energy balance and body weight. Many effects of POMC neurons can be attributed to their release of cognate neuropeptides from secretory granules in axon terminals. However, these neurons also synaptically release non-peptide neurotransmitters. The aim of this study was to settle the controversy whether there are separate populations of POMC neurons that release GABA or glutamate. Transgenic mice expressing a red fluorescent protein [Discosoma red (DsRed)] driven by Pomc neuronal regulatory elements (POMC-DsRed) were crossed to mice that expressed green fluorescent protein (gfp) in GABAergic neurons (GAD67-gfp). Approximately 40% of POMC neurons in the arcuate nucleus of the double-transgenic mice expressed the GAD67-gfp transgene. In vitro neurotransmitter release was detected using whole-cell electrophysiologic recordings in cultured GAD67-gfp-positive and GAD67-gfp-negative POMC neurons that had formed recurrent synapses (autapses). Autapses from GAD67-gfp-positive neurons were uniformly GABAergic. In contrast, autapses from the GAD67-gfp-negative POMC neurons exclusively exhibited postsynaptic currents mediated by glutamate. Together, these results indicate that there are two subpopulations of POMC neurons in the arcuate nucleus differentiated by their amino acid neurotransmitter phenotype. Whole-cell voltage-clamp recordings from POMC neurons in live brain slices indicated that GABAergic and glutamatergic POMC neurons are under similar presynaptic and postsynaptic regulation, although the GABAergic POMC neurons are smaller and have higher input resistance. GABAergic and glutamatergic POMC neurons may mediate distinct aspects of POMC neuron function, including the regulation of energy homeostasis.

In 5-month-old male and female dopamine receptor 2 (D2R) knockout mice food intake per animal was unaltered while food per g BW was increased. We wished to evaluate the effect of D2R disruption on different components of energy balance and food intake regulation. We determined hypothalamic orexin precursor (PPO) expression, its receptor OX1, serum leptin levels, hypothalamic leptin receptor (OBR), circulating and pituitary alpha MSH levels, as well as central MC3 and MC4 receptors and NPY mRNA in wildtype and D2R knockout mice (KO). Loss of D2R caused a marked increase in serum prolactin levels, to higher levels in females compared to male KO mice. On the other hand, it produced a female-specific increase in circulating alphaMSH, and hypothalamic alphaMSH content, while neurointermediate alphaMSH content was decreased in both sexes. No differences were found in hypothalamic NPY, MC3R or MC4R concentration. Hypothalamic PPO mRNA expression was significantly decreased only in female KOs, while OX1 mRNA was not different between genotypes. Serum leptin levels were also similar in both genotypes. Our results show that in female and not in male mice disruption of the D2R produces two potentially anorexigenic events: an increase in serum and hypothalamic alphaMSH, and a decrease in hypothalamic orexin expression. Very high prolactin levels, which are orexigenic, probably counterbalance these effects, so that food intake is slightly altered. In males, on the other hand, hypothalamic PPO, and serum or hypothalamic alphaMSH are not modified, and increased prolactin levels may account for increased food intake per g BW. These results suggest a sexually dimorphic participation of the D2R in food intake regulation.

Background: Behavioral sensitization is a result of neuroadaptation to repeated drug administration and is hypothesized to reflect an increased susceptibility to drug abuse. Proopiomelanocortin (POMC) derived peptides including beta-endorphin and alpha-melanocyte stimulating hormone have been implicated in development of behavioral sensitization and the reinforcing effects of alcohol and other drugs of abuse. This study used a genetically engineered mouse strain that is deficient for neural POMC to directly determine if any POMC peptides are necessary for the development of ethanol-induced locomotor sensitization., Methods: Adult female mice deficient for POMC in neurons only (Pomc(-/-)Tg/Tg, KO) and wildtype (Pomc(+/+)Tg/Tg, WT) littermates were injected once daily with either saline or ethanol (i.p.) for 12 to 13 days. On ethanol test day (day 13 or 14) all mice from both treatment groups received an i.p. injection of ethanol immediately before a 15-minute analysis of locomotor activity. Blood ethanol concentration (BEC) was measured on ethanol test day immediately following the test session. Baseline locomotor activity was measured for 15 minutes after a saline injection 2 days later in both groups., Results: There was no significant difference in BEC between genotypes (WT = 2.11 +/- 0.06; KO = 2.03 +/- 0.08 mg/ml). Both WT and nPOMC-deficient mice treated repeatedly with ethanol demonstrated a significant increase in locomotor activity on test day when compared to repeated saline-treated counterparts. In addition, mice of both genotypes in the repeated saline groups showed a significant locomotor stimulant response to acute ethanol injection., Conclusions: Central POMC peptides are not required for either the acute locomotor stimulatory effect of ethanol or the development of ethanol-induced locomotor sensitization. While these peptides may modulate other ethanol-associated behaviors, they are not essential for development of behavioral sensitization.

The activation of G protein-coupled receptors results in a cascade of events that include acute signaling, desensitization, and internalization, and it is thought that not all agonists affect each process to the same extent. The early steps in opioid receptor signaling, including desensitization, have been characterized electrophysiologically using brain slice preparations, whereas most previous studies of opioid receptor trafficking have been conducted in heterologous cell models. This study used transgenic mice that express an epitope-tagged (FLAG) micro-opioid receptor (FLAGMOR) targeted to catecholamine neurons by regulatory elements from the tyrosine hydroxylase gene. Brain slices from these mice were used to study tagged MOR receptors in neurons of the locus ceruleus. Activation of the FLAGMOR with [Met5]enkephalin (ME) produced a hyperpolarization that desensitized acutely to the same extent as native MOR in slices from wild-type mice. A series of opioid agonists were then used to study desensitization and receptor trafficking in brain slices, which was monitored with a monoclonal antibody against the FLAG epitope (M1) conjugated to Alexa 594. Three patterns of receptor trafficking and desensitization were observed: 1) ME, etorphine, and methadone resulted in both receptor desensitization and internalization; 2) morphine and oxymorphone caused significant desensitization without evidence for internalization; and 3) oxycodone was ineffective in both processes. These results show that two distinct forms of signaling were differentially engaged depending on the agonist used to activate the receptor, and they support the hypothesis that ligand-specific regulation of opioid receptors occurs in neurons maintained in brain slices from adult animals.

Deficits in prepulse inhibition (PPI) of startle, an operational measure of sensorimotor gating, are characteristics of schizophrenia and related neuropsychiatric disorders. Previous studies in mice demonstrate a contribution of dopamine (DA) D(1)-family receptors in modulating PPI and DA D(2) receptors (D2R) in mediating the PPI-disruptive effects of amphetamine. To examine further the contributions of DA receptor subtypes in PPI, we used a combined pharmacological and genetic approach. In congenic C57BL/6 J wild-type mice, we tested whether the D1R antagonist SCH23390 or the D2/3R antagonist raclopride would attenuate the effects of the indirect DA agonist cocaine (40 mg/kg). Both the D1R and D2/3R antagonists attenuated the cocaine-induced PPI deficit. We also tested the effect of cocaine on PPI in wild-type and DA D1R, D2R, or D3R knockout mice. The cocaine-induced PPI deficit was influenced differently by the three DA receptor subtypes, being absent in D1R knockout mice, partially attenuated in D2R knockout mice, and exaggerated in D3R knockout mice. Thus, the D1R is necessary for the PPI-disruptive effects of cocaine, while the D2R partially contributes to these effects. Conversely, the D3R appears to inhibit the PPI-disruptive effects of cocaine. Uncovering neural mechanisms involved in PPI will further our understanding of substrates of sensorimotor gating and could lead to better therapeutics to treat complex cognitive disorders such as schizophrenia.

Signal transducer and activator of transcription (Stat)-3 signals mediate many of the metabolic effects of the fat cell-derived hormone, leptin. In mice, brain-specific depletion of either the long form of the leptin receptor (Lepr) or Stat3 results in comparable obese phenotypes as does replacement of Lepr with an altered leptin receptor locus that codes for a Lepr unable to interact with Stat3. Among the multiple brain regions containing leptin-sensitive Stat3 sites, cells expressing feeding-related neuropeptides in the arcuate nucleus of the hypothalamus have received much of the focus. To determine the contribution to energy homeostasis of Stat3 expressed in agouti-related protein (Agrp)/neuropeptide Y (Npy) arcuate neurons, Stat3 was deleted specifically from these cells, and several metabolic indices were measured. It was found that deletion of Stat3 from Agrp/Npy neurons resulted in modest weight gain that was accounted for by increased adiposity. Agrp/Stat3-deficient mice also showed hyperleptinemia, and high-fat diet-induced hyperinsulinemia. Stat3 deletion in Agrp/Npy neurons also resulted in altered hypothalamic gene expression indicated by increased Npy mRNA and decreased induction of suppressor of cytokine signaling-3 in response to leptin. Agrp mRNA levels in the fed or fasted state were unaffected. Behaviorally, mice without Stat3 in Agrp/Npy neurons were mildly hyperphagic and hyporesponsive to leptin. We conclude that Stat3 in Agrp/Npy neurons is required for normal energy homeostasis, but Stat3 signaling in other brain areas also contributes to the regulation of energy homeostasis.

Behavioral sensitization to psychostimulants manifests as an increased locomotor response with repeated administration. Dopamine systems are accepted to play a fundamental role in sensitization, but the role of specific dopamine receptor subtypes has not been completely defined. This study used the combination of dopamine D2 receptor-deficient mice and a D1-like antagonist to examine dopamine D1 and D2 receptor involvement in acute and sensitized locomotor responses to methamphetamine. Absence of the dopamine D2 receptor resulted in attenuation of the acute stimulant effects of methamphetamine. Mutant and wild-type mice exhibited sensitization that lasted longer within the time period of the challenge test in the mutant animals. Pretreatment with the D1-like receptor antagonist SCH 23390 produced more potent reductions in the acute and sensitized locomotor responses to methamphetamine in D2 receptor-deficient mice than in wild-type mice; however, the expression of locomotor sensitization when challenged with methamphetamine alone was equivalently attenuated by previous treatment with SCH 23390. These data suggest that dopamine D2 receptors play a key role in the acute stimulant and sensitizing effects of methamphetamine and act in concert with D1-like receptors to influence the acquisition of methamphetamine-induced behavioral sensitization, traits that may influence continued methamphetamine use.

Brainstem A2/C2 neurons are catecholamine (CA) neurons within the solitary tract nucleus (NTS) that influence many homeostatic functions, including cardiovascular reflexes, food intake, and stress. Because NTS is a major interface between sensory visceral afferents and the CNS, NTS CA neurons are ideally suited to coordinate complex responses by their projections to multiple brain regions. To test how NTS CA neurons process visceral afferent information carried by solitary tract (ST) afferents, we identified CA neurons using transgenic mice expressing TH-EGFP (enhanced green fluorescent protein under the control of the tyrosine hydroxylase promoter) and recorded synaptic responses to ST activation in horizontal slices. ST shocks evoked large-amplitude, short-latency, glutamatergic EPSCs (ST-EPSCs) in 90% of NTS CA neurons. Within neurons, ST-EPSCs had constant latency, rarely failed, and depressed substantially at high ST frequencies, indicating that NTS CA neurons receive direct monosynaptic connections from afferent terminals. NTS CA neurons received direct ST inputs from only one or two afferent fibers, with one-half also receiving smaller amplitude indirect inputs. Up to 90% of ST shocks evoked action potentials in NTS CA neurons. However, transmission of sensory afferent information through NTS CA neurons critically depended on the expression of an A-type potassium current (I(KA)), which when active attenuated ST-activated action potentials to a 37% success rate. The satiety peptide, cholecystokinin, presynaptically facilitated glutamate transmission in one-half of NTS CA neurons. Thus, NTS CA neurons are directly driven by visceral afferents with output being modulated by presynaptic peptide receptors and postsynaptic potassium channels.

The stress response involves complex physiological mechanisms that maximize behavioral efficacy during attack or defense and is highly conserved in all vertebrates. Key mediators of the stress response are pituitary hormones encoded by the proopiomelanocortin gene (POMC). Despite conservation of physiological function and expression pattern of POMC in all vertebrates, phylogenetic footprinting analyses at the POMC locus across vertebrates failed to detect conserved noncoding sequences with potential regulatory function. To investigate whether ortholog POMC promoters from extremely distant vertebrates are functionally conserved, we used 5'-flanking sequences of the teleost fish Tetraodon nigroviridis POMCalpha gene to produce transgenic mice. Tetraodon POMCalpha promoter targeted reporter gene expression exclusively to mouse pituitary cells that normally express Pomc. Importantly, transgenic expression in mouse corticotrophs was increased after adrenalectomy. To understand how conservation of precise gene expression mechanisms coexists with great sequence divergence, we investigated whether very short elements are still conserved in all vertebrate POMC promoters. Multiple local sequence alignments that consider phylogenetic relationships of ortholog regions identified a unique 10-bp motif GTGCTAA(T/G)CC that is usually present in two copies in POMC 5'-flanking sequences of all vertebrates. Underlined nucleotides represent totally conserved sequences. Deletion of these paired motifs from Tetraodon POMCalpha promoter markedly reduced its transcriptional activity in a mouse corticotropic cell line and in pituitary POMC cells of transgenic mice. In mammals, the conserved motifs correspond to reported binding sites for pituitary-specific nuclear proteins that participate in POMC transcriptional regulation. Together, these results demonstrate that mechanisms that participate in pituitary-specific and hormonally regulated expression of POMC have been preserved since mammals and teleosts diverged from a common ancestor 450 million years ago despite great promoter sequence divergence.

The proopiomelanocortin gene (POMC) is expressed in the pituitary gland and the ventral hypothalamus of all jawed vertebrates, producing several bioactive peptides that function as peripheral hormones or central neuropeptides, respectively. We have recently determined that mouse and human POMC expression in the hypothalamus is conferred by the action of two 5' distal and unrelated enhancers, nPE1 and nPE2. To investigate the evolutionary origin of the neuronal enhancer nPE2, we searched available vertebrate genome databases and determined that nPE2 is a highly conserved element in placentals, marsupials, and monotremes, whereas it is absent in nonmammalian vertebrates. Following an in silico paleogenomic strategy based on genome-wide searches for paralog sequences, we discovered that opossum and wallaby nPE2 sequences are highly similar to members of the superfamily of CORE-short interspersed nucleotide element (SINE) retroposons, in particular to MAR1 retroposons that are widely present in marsupial genomes. Thus, the neuronal enhancer nPE2 originated from the exaptation of a CORE-SINE retroposon in the lineage leading to mammals and remained under purifying selection in all mammalian orders for the last 170 million years. Expression studies performed in transgenic mice showed that two nonadjacent nPE2 subregions are essential to drive reporter gene expression into POMC hypothalamic neurons, providing the first functional example of an exapted enhancer derived from an ancient CORE-SINE retroposon. In addition, we found that this CORE-SINE family of retroposons is likely to still be active in American and Australian marsupial genomes and that several highly conserved exonic, intronic and intergenic sequences in the human genome originated from the exaptation of CORE-SINE retroposons. Together, our results provide clear evidence of the functional novelties that transposed elements contributed to their host genomes throughout evolution., Competing Interests: Competing interests. FSJdS, MJL, and MR have intellectual property and patent interests in the POMC neuronal-specific enhancers and have received income from the licensing of this intellectual property and related research material to financially interested companies.

Phosphorylation of specific sites in the second intracellular loop and in the C-terminal domain have previously been suggested to cause desensitization and internalization of the mu-opioid receptor (MOP-R). To assess sites of MOP-R phosphorylation in vivo, affinity-purified, phosphoselective antibodies were raised against either phosphothreonine-180 in the second intracellular loop (MOR-P1) or the C-terminal domain of MOP-R containing phosphothreonine-370 and phosphoserine-375 (MOR-P2). We found that MOR-P2-immunoreactivity (IR) was significantly increased within the striatum of wild-type C57BL/6 mice after injection of the agonist fentanyl. Pretreatment with the antagonist naloxone blocked the fentanyl-induced increase. Furthermore, mutant mice lacking MOP-R showed only non-specific nuclear MOR-P2-IR before or after fentanyl treatment, confirming the specificity of the MOR-P2 antibodies. To assess whether MOP-R phosphorylation occurs following endogenous opioid release, we induced chronic neuropathic pain by partial sciatic nerve ligation (pSNL), which caused a significant increase in MOR-P2-IR in the striatum. pSNL also induced signs of mu opioid receptor tolerance demonstrated by a rightward shift in the morphine dose response in the tail withdrawal assay and by a reduction in morphine conditioned place preference (CPP). Mutant mice selectively lacking all forms of the beta-endorphin peptides derived from the proopiomelanocortin (Pomc) gene did not show increased MOR-P2-IR, decreased morphine antinociception, or reduced morphine CPP following pSNL. In contrast gene deletion of either proenkephalin or prodynorphin opioids did not block the effects of pSNL. These results suggest that neuropathic pain caused by pSNL in wild-type mice activates the release of the endogenous opioid beta-endorphin, which subsequently induces MOP-R phosphorylation and opiate tolerance.

Corticotropin-releasing factor (CRF), a neuropeptide released during stress, has been reported to modulate startle behavior, including reducing the threshold for acoustic startle responding and reducing prepulse inhibition (PPI). The central mechanisms mediating CRF system regulation of startle and PPI are still unclear. Some antipsychotic drugs attenuate CRF-induced deficits in PPI in rats and mice. Here we tested the hypothesis that indirect activation of DA(1)-receptors (D(1)) and DA(2)-receptors (D(2)) contributes to the effects of CRF on PPI. We compared the effect of central administration of h/r-CRF (0.2-0.6 nmol) on PPI in mice with either a D(1) or D(2) receptor null mutation (knockout, KO) or in mice pretreated with D(1) or D(2) receptor antagonists SCH23390 (1 mg/kg) or haloperidol (1 mg/kg). D(1) and D(2) KO mice exhibited no significant differences in their sensitivity to CRF-induced disruptions of PPI. Similarly, neither SCH23390 nor haloperidol pretreatment altered the CRF-induced disruption in PPI, although both increased PPI at baseline. CRF-induced increases in startle also remained unchanged by any of the DA receptor manipulations. These results indicate that neither D(1)- nor D(2)-receptor activation is necessary for CRF to exert its effects on acoustic startle and PPI in mice.

Proopiomelanocortin (POMC) is synthesized predominantly in pituitary corticotrophs, melanotrophs, and arcuate hypothalamic neurons. Corticotroph-derived ACTH mediates basal and stress-induced glucocorticoid secretion, but it is uncertain whether POMC peptides produced in the brain also regulate the hypothalamic-pituitary-adrenal axis. To address this question, we generated neuron-specific POMC-deficient mice by transgenic (Tg) replacement of pituitary POMC in a global Pomc(-/-) background. Selective restoration of pituitary POMC prevented the adrenal insufficiency and neonatal mortality characteristic of Pomc(-/-) mice. However, adult Pomc(-/-)Tg/+ mice expressing the pituitary-specific transgene exhibited adrenal cortical hypertrophy, elevated basal plasma corticosterone, elevated basal but attenuated stress-induced ACTH secretion, and inappropriately elevated CRH expression in the hypothalamic paraventricular nucleus. In addition, Pomc(-/-)Tg/+, Pomc(+/-)Tg/+, and Pomc(+/-) mice, which all displayed varying degrees of elevated CRH, frequently developed melanotroph adenomas after 1 yr of age, whereas Pomc(-/-) mice, with maximal CRH expression and glucocorticoid disinhibition, developed corticotroph and melanotroph adenomas. These results indicate that neuronal POMC peptides are necessary to regulate CRH within physiological limits and that a chronic reduction or absence of hypothalamic POMC leads to trophic stimulation of pituitary cells directly or indirectly through elevated CRH levels.

Complex behaviors such as those associated with reward to unconditioned positive reinforcers are polygenic processes. In studies using genetically modified mice specific for the endogenous opioid systems an observed phenotype in a complex behavior is likely to be dependent on interacting genes which, in inbred mouse lines, influence that phenotype. To address this issue we examined operant responding for palatable food reinforcers in mice lacking the expression of beta-endorphin, enkephalin or both peptides congenic to two different genetic backgrounds; C57BL/6J and DBA/2J. These two inbred strains were chosen because their endogenous opioid states differ and they respond differently to exogenous opioids in many behavioral assays. We found that wildtype and mutant C57BL/6J mice acquired operant responding for food reinforcers faster than DBA/2J mice, regardless of their opioid genotype. Although wildtype DBA/2J mice had a significant deficit in acquisition of bar-pressing behavior to reach a pre-established performance criterion, no subsequent deficit was observed under two different schedules of reinforcement. Additionally, we found that mice lacking enkephalin had decreased motivation to bar press for palatable food reinforcers under a progressive ratio regardless of sex or background strain. In contrast, the only subset of beta-endorphin-deficient mice that had decreased motivation to bar press under a progressive ratio was males on the C57BL/6J background. Of the two classical endogenous opioid peptides with preferential activation of the mu opioid receptor, the knockout models would suggest that enkephalins play a more consistent role than beta-endorphin in mediating the motivation for food reward when tested under a progressive ratio.

Endogenous opioids within the central nervous system are postulated to mediate hedonic aspects of feeding behavior. To identify the relevant endogenous opioid receptor ligands, mice lacking one or two of the opioid peptide families beta-endorphin, enkephalins or dynorphins were tested for sucrose preference in a two-bottle free-choice drinking paradigm under drug-naïve conditions and following treatment with an opioid antagonist (1 mg/kg naloxone i.p.) or saline. Basal sucrose consumption was unaltered in all of the knockout genotypes compared to their congenic wild-type C57BL/6 littermates during 0.5 and 6 h access to a bottle containing 2, 4, 8, or 16% sucrose and a second bottle containing water. Moreover, all mutant genotypes and wildtype mice exhibited a similar compensatory decrease in overnight food intake following the extra caloric load from 6 h sucrose access. Although these basal responses to sucrose were unaffected by the knockout genotypes, naloxone reduced sucrose consumption by 50% compared to saline treatment during the first 0.5 h in wild-type and beta-endorphin knockout mice, but had no effect in enkephalin knockouts, beta-endorphin and enkephalin double knockouts, or dynorphin knockouts. These data suggest that naloxone reduces sucrose consumption in wild-type mice by blocking endogenous enkephalin and/or dynorphin signaling, but not beta-endorphin. Dynorphin knockouts in the current study had bar-pressing responses for a palatable food reinforcer in an operant procedure under free-feeding conditions similar to wild-type mice while we found in a previous study that beta-endorphin and enkephalin knockout mice had reduced motivation to respond [Hayward MD, Pintar JE, Low MJ. Selective reward deficit in mice lacking beta-endorphin and enkephalin. J Neurosci 2002;22:8251-8258.]. We conclude from these studies directly comparing three strains of opioid peptide knockout mice that enkephalin and dynorphin can modulate sucrose preference but are not necessary to support sucrose consumption. However, dynorphin was not necessary to support wildtype levels of operant responding suggesting that only enkephalin and beta-endorphin modulate conditioned food reinforcement.

MDMA or 'ecstasy' (3,4-methylenedioxymethamphetamine) is a commonly used psychoactive drug that has unusual and distinctive behavioral effects in both humans and animals. In rodents, MDMA administration produces a unique locomotor activity pattern, with high activity characterized by smooth locomotor paths and perseverative thigmotaxis. Although considerable evidence supports a major role for serotonin release in MDMA-induced locomotor activity, dopamine (DA) receptor antagonists have recently been shown to attenuate these effects. Here, we tested the hypothesis that DA D1, D2, and D3 receptors contribute to MDMA-induced alterations in locomotor activity and motor patterns. DA D1, D2, or D3 receptor knockout (KO) and wild-type (WT) mice received vehicle or (+/-)-MDMA and were tested for 60 min in the behavioral pattern monitor (BPM). D1 KO mice exhibited significant increases in MDMA-induced hyperactivity in the late testing phase as well as an overall increase in straight path movements. In contrast, D2 KO mice exhibited reductions in MDMA-induced hyperactivity in the late testing phase, and exhibited significantly less sensitivity to MDMA-induced perseverative thigmotaxis. At baseline, D2 KO mice also exhibited reduced activity and more circumscribed movements compared to WT mice. Female D3 KO mice showed a slight reduction in MDMA-induced hyperactivity. These results confirm differential modulatory roles for D1 and D2 and perhaps D3 receptors in MDMA-induced hyperactivity. More specifically, D1 receptor activation appears to modify the type of activity (linear vs circumscribed), whereas D2 receptor activation appears to contribute to the repetitive circling behavior produced by MDMA.

The present study investigated the localization and density of somatostatin (SRIF) receptor subtypes (sst(1-5)) and SRIF-nitric oxide (NO()) interactions in the retina of wildtype [WT, (+/+)] and somatostatin deficient mice [SRIF (-/-)]. Immunohistochemistry and radioligand binding studies with subsequent autoradiography were performed. Monoclonal antibodies [SRIF, protein kinase C (rod bipolar cells marker), microtubule associated protein 1A (ganglion cell marker)] and polyclonal antibodies (anti-sst(1), sst(2A), sst(4) receptor) were applied to 10-14 microm sections of retinas fixed in paraformaldehyde. NADPH-diaphorase reactivity was assessed histochemically. [(125)I]LTT SRIF-28 alone or in the presence of MK678 (sst(2) agonist) and [(125)I]Tyr(3)-octreotide were employed to quantify sst(1-5), sst(1/4)and sst(2/5) receptor densities, respectively. sst(1), sst(2A), and sst(4) receptor immunoreactivities were observed in processes of the inner plexiform layer (IPL), rod bipolar, and in ganglion cells and processes, respectively, in WT and SRIF (-/-) mice. Specific [(125)I]LTT SRIF-28 and [(125)I]Tyr(3)-octreotide binding was increased significantly in SRIF (-/-) mice. NADPH-diaphorase staining was localized in photoreceptors and amacrine cells, but not rod bipolar and ganglion cells. Also, NADPH-diaphorase staining was not colocalized with sst(1), sst(2A) or sst(4) receptor immunoreactivity. These results demonstrate an upregulation of SRIF receptors in mice lacking SRIF, but no evident SRIF-NO(*) interaction was observed in the mouse retina.

Recently, the importance of the dopaminergic D2 receptor (D2R) subtype in normal body growth and neonatal GH secretion has been highlighted. Disruption of D2R alters the GHRH-GH-IGF-I axis and impairs body growth in adult male mice. The D2R knockout (KO) dwarf mouse has not been well characterized; we therefore sought to determine somatotrope function in the adult pituitary. Using immunohistochemistry and confocal microscopy, we found a significant decrease in the somatotrope population in pituitaries from KO mice (P=0.043), which was paralleled by a decreased GH output from pituitary cells cultured in vitro. In cells from adult mice the response amplitude to GHRH differed between genotypes (lower in KO), but this difference was less dramatic after taking into account the lower basal release and hormone content in the KO cells. Furthermore, there were no significant differences in cAMP generation in response to GHRH between genotypes. By Western blot, GHRH-receptor in pituitary membranes from KO mice was reduced to 46% of the level found in wildtype (WT) mice (P=0.016). Somatostatin induced a concentration-dependent decrease in GH and prolactin (PRL) secretion in both genotypes, and 1x10(-7) M ghrelin released GH in cells from both genotypes (P=0.017) in a proportionate manner to basal levels. These results suggest that KO somatotropes maintain a regulated secretory function. Finally, we tested the direct effect of dopamine on GH and PRL secretion in cells from both genotypes at 20 days and 6 months of life. As expected, we found that dopamine could reduce PRL levels at both ages in WT mice but not in KO mice, but there was no consistent effect of the neurotransmitter on GH release in either genotype at the ages studied. The present study demonstrates that in the adult male D2R KO mouse, there is a reduction in pituitary GH content and secretory activity. Our results point to an involvement of D2R signaling at the hypothalamic level as dopamine did not release GH acting at the pituitary level either in 1-month-old or adult mice. The similarity of the pituitary defect in the D2R KO mouse to that of GHRH-deficient models suggests a probable mechanism. A loss of dopamine signaling via hypothalamic D2Rs at a critical age causes the reduced release of GHRH from hypophyseotropic neurons leading to inadequate clonal expansion of the somatotrope population. Our data also reveal that somatotrope cell number is much more sensitive to changes in neonatal GHRH input than their capacity to develop properly regulated GH-secretory function.

The present study was undertaken to examine the role of the dopamine (DA) D2 receptor in the ethanol-evoked DA response in the ventral striatum. We performed microdialysis experiments using the D2 null mutant and wild-type controls and measured the effect of an intraperitoneal (i.p.) injection of either saline or ethanol (2 g/kg) on dialysate DA concentrations in the ventral striatum. Dialysate ethanol concentrations were also determined in the samples from the ventral striatum. In addition, the effects of quinpirole, a D2/D3 agonist, were examined in both the ventral and dorsal striatum. Basal dialysate concentrations of DA were significantly reduced in both the ventral and dorsal striatum of the D2 knockouts compared with wild-type controls. Ethanol administration significantly enhanced ventral striatal DA in both groups, but the increase in dialysate DA concentration was 3.5-fold higher in the wild-type controls. The time course of dialysate ethanol concentrations was similar in the two groups. Saline injection did not alter DA concentrations in either the ventral or dorsal striatum. However, quinpirole (0.3 mg/kg) administration significantly depressed striatal dialysate DA concentrations in the wild-type mice, but not in the D2 knockouts. The results suggest that the D2 receptor is necessary for normal development and regulation of striatal extracellular DA concentrations, but the mechanism for this alteration is unclear. In addition, the blunted ethanol-evoked DA response in the D2 knockouts may contribute, in part, to some of the behavioral deficits previously observed in response to ethanol.

A paucity of in vitro models has hampered studies of molecular mechanisms of FSH subunit gene expression. Consequently, we used an in vivo transgenic strategy to map the location of regulatory elements in the cloned 10 kb human FSHbeta gene. Analyses of transgenic mouse lines revealed that successive 5' truncations of the hFSHbeta promoter region to -350 bp relative to the transcriptional initiation site retained gonadotroph-specific expression and the sexually dimorphic pattern of male greater than female FSHbeta mRNA levels found normally in rodent pituitary. Truncation of the 3' flanking sequences from positions +3142 to +2138 bp relative to the translational stop codon in exon 3 resulted in a complete loss of transgene expression, suggesting the presence of critical regulatory elements mapping to the 1 kb genomic segment downstream of position +2138, in addition to the proximal 5' promoter elements. In silico phylogenetic comparisons of mammalian FSHbeta genes revealed five islands of highly conserved sequence homology corresponding precisely to the proximal 5' promoter region, exon 2, the 5' translated region of exon 3, and two regions at the 3' untranslated end of exon 3 that include putative polyadenylation and transcriptional termination signals. Sequence analyses of the 5' proximal promoter revealed the presence of several putative homeodomain binding sites as well as GATA, SMAD, AP-1, NF-1, NF-Y and steroid hormone transcription factor binding sites within the highly conserved -350 bp promoter region. Notably absent from these 5' sequences, however, are consensus binding sites for either Egr-1 or Lim-2 transcription factors known to be critical for the gonadotroph-specific expression of the LHbeta gene. These findings support the hypothesis that one of the mechanisms underlying the differential regulation of the LHbeta, FSHbeta, and common alpha-gonadotropin subunits within pituitary gonadotrophs may be differences in sequence-specific binding requirements for distinct combinations of transcription factors.

Null mutations of the proopiomelanocortin gene (Pomc) cause obesity in humans and rodents, but the contributions of central versus pituitary POMC deficiency are not fully established. To elucidate these roles, we introduced a POMC transgene (Tg) that selectively restored peripheral melanocortin and corticosterone secretion in Pomc mice. Rather than improving energy balance, the genetic replacement of pituitary POMC in PomcTg mice aggravated their metabolic syndrome with increased caloric intake and feed efficiency, reduced oxygen consumption, increased subcutaneous, visceral, and hepatic fat, and severe insulin resistance. Pair-feeding of PomcTg mice to the daily intake of lean controls normalized their rate of weight gain but did not abolish obesity, indicating that hyperphagia is a major but not sole determinant of the phenotype. Replacement of corticosterone in the drinking water of Pomc mice recapitulated the hyperphagia, excess weight gain and fat accumulation, and hyperleptinemia characteristic of genetically rescued PomcTg mice. These data demonstrate that CNS POMC peptides play a critical role in energy homeostasis that is not substituted by peripheral POMC. Restoration of pituitary POMC expression to create a de facto neuronal POMC deficiency exacerbated the development of obesity, largely via glucocorticoid modulation of appetite, metabolism, and energy partitioning.

Dopamine receptor type 2 (D2R) knockout mice (KO) have chronic hyperprolactinemia, pituitary hyperplasia, and a moderate decrease in MSH content. They are also growth retarded evidencing an alteration in the GH-IGF-I axis. In D2R KO, lactotropes do not show dense secretory granules but degranulated cells and fewer somatotropes, gonadotropes and thyrotropes. Prolactin levels are always higher in female than in male knockouts, and in accordance, pituitary hyperplasia is observed at 8 months only in females. After 16 months of age, highly vascularized adenomas develop, especially in females. Prominent vascular channels in the hyperplastic and adenomatous pituitaries, as well as extravasated red blood cells not contained in capillaries is also a common finding. Prolactin is not the factor that enhances the hyperplastic phenotype in females while estrogen is a permissive factor. VEGF-A expression is increased in pituitaries from D2R KO. VEGF-A is expressed in follicle stellate cells. Because D2R receptors are found in lactotropes and not in follicle stellate cells, it may be inferred that a paracrine-derived factor from lactotropes is acting on follicle stellate cells to increase VEGF-A expression. VEGF-A does not induce pituitary cell proliferation, even though it enhances prolactin secretion. But it may act on adjacent endothelial cells and participate in the angiogenic process that increases the availability of different growth factors and mitogens. The D2R knockout mouse represents a unique animal model to study dopamine-resistant prolactinomas, and VEGF-A may be an alternative therapeutic target in this pathology.

The proopiomelanocortin gene (POMC) encodes several bioactive peptides, including adrenocorticotropin hormone, alpha-, beta-, and gamma-melanocyte-stimulating hormone, and the opioid peptide beta-endorphin, which play key roles in vertebrate physiology. In the human, mouse, and chicken genomes, there is only one POMC gene. By searching public genome projects, we have found that Tetraodon (Tetraodon nigroviridis), Fugu (Takifugu rubripes), and zebrafish (Danio rerio) possess two POMC genes, which we called POMCalpha and POMCbeta, and we present phylogenetic and mapping evidence that these paralogue genes originated in the whole-genome duplication specific to the teleost lineage over 300 MYA. In addition, we present evidence for two types of subfunction partitioning between the paralogues. First, in situ hybridization experiments indicate that the expression domains of the ancestral POMC gene have been subfunctionalized in Tetraodon, with POMCalpha expressed in the nucleus lateralis tuberis of the hypothalamus, as well as in the rostral pars distalis and pars intermedia (PI) of the pituitary, whereas POMCbeta is expressed in the preoptic area of the brain and weakly in the pituitary PI. Second, POMCbeta genes have a beta-endorphin segment that lacks the consensus opioid signal and seems to be under neutral evolution in tetraodontids, whereas POMCalpha genes possess well-conserved peptide regions. Thus, POMC paralogues have experienced subfunctionalization of both expression and peptide domains during teleost evolution. The study of regulatory regions of fish POMC genes might shed light on the mechanisms of enhancer partitioning between duplicate genes, as well as the roles of POMC-derived peptides in fish physiology.

Mice lacking the D2 dopamine receptor (D2(-/-)) and congenic to the C57BL/6J background were tested for opioid-mediated locomotor activity to examine the involvement of the D2 dopamine receptor in opioid pharmacology. Morphine-stimulated locomotor activity did not significantly differ between the two genotypes. The opioid antagonist naloxone dose-dependently decreased spontaneous motor activity in wild-type mice but was without significant effect in D2(-/-) mice. The magnitude of food-conditioned increases in locomotor activity in wild-type mice and D2(-/-) mice was similar but naloxone did not decrease conditioned motor activity in D2(-/-) mice. Spontaneous locomotor activity of mice lacking the endogenous opioids beta-endorphin and/or enkephalin was also tested and we found that naloxone did not reduce activity in mice specifically lacking enkephalin. We suggest that the D2 dopamine receptor is necessary for modulation of spontaneous locomotor activity stimulated by the endogenous opioid enkephalin.

Endocannabinoid release from a single neuron has been shown to cause presynaptic inhibition of transmitter release at many different sites. Here, we demonstrate that hypothalamic proopiomelanocortin (POMC) neurons release endocannabinoids continuously under basal conditions, unlike other release sites at which endocannabinoid production must be stimulated. The basal endocannabinoid release selectively inhibited GABA release onto POMC neurons, although exogenous administration of cannabinoid agonists also inhibited glutamate release. The CB1 cannabinoid receptor antagonist AM 251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] blocked endocannabinoid-mediated inhibition of GABA release without affecting excitatory synaptic currents, whereas the CB1 receptor agonist WIN 55,212-2 [R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol [1,2,3-de]-1,4-benzoxazin-6-yl)(1-naphthalenyl) methanone monomethanesulfonate] inhibited both inhibitory and excitatory synaptic currents in POMC neurons. These data demonstrate that endogenously released cannabinoids and exogenously applied CB1 receptor agonists can have markedly different effects on synaptic inputs. Furthermore, the data suggest a novel form of endocannabinoid-mediated retrograde inhibition, whereby the regulation of a subset of inputs requires either the removal of tonic presynaptic inhibition caused by endocannabinoids or the engagement of a mechanism that actively inhibits endocannabinoid production.

Rationale: The rewarding effects of lateral hypothalamic brain stimulation, various natural rewards, and several drugs of abuse are attenuated by D1 or D2 dopamine receptor (D1R or D2R) antagonists. Much of the evidence for dopaminergic involvement in rewards is based on pharmacological agents with limited or "relative" selectivity for dopamine receptor subtypes. Genetically engineered animal models provide a complementary approach to pharmacological investigations., Objectives: In the present study, we explored the contribution of dopamine D2Rs to (1) brain stimulation reward (BSR) and (2) the potentiation of this behavior by morphine and amphetamine using D2R-deficient mice., Methods: Wild-type (D2Rwt), heterozygous (D2Rhet), and D2R knockout (D2Rko) mice were trained to turn a wheel for rewarding brain stimulation. Once equivalent rate-frequency curves were established, morphine-induced (0, 1.0, 3.0, and 5.6 mg/kg s.c.) and amphetamine-induced (0, 1.0, 2.0, and 4.0 mg/kg i.p.) potentiations of BSR were determined., Results: The D2Rko mice required approximately 50% more stimulation than the D2Rwt mice did. With the equi-rewarding levels of stimulation current, amphetamine potentiated BSR equally across the three genotypes. In contrast, morphine potentiated rewarding stimulation in the D2Rwt, had no effect in the D2Rhet, and antagonized rewarding stimulation in the D2Rko mice., Conclusions: D2R elimination decreases, but does not eliminate, the rewarding effects of lateral hypothalamic stimulation. After compensation for this deficit, amphetamine continues to potentiate BSR, while morphine does not.

Vascular endothelial growth factor (VEGF)-A is an important angiogenic cytokine in cancer and pathological angiogenesis and has been related to the antiangiogenic activity of dopamine in endothelial cells. We investigated VEGF expression, localization, and function in pituitary hyperplasia of dopamine D2 receptor (D2R)-knockout female mice. Pituitaries from knockout mice showed increased protein and mRNA VEGF-A expression when compared with wild-type mice. In wild-type mice, prolonged treatment with the D2R antagonist, haloperidol, enhanced pituitary VEGF expression and prolactin release, suggesting that dopamine inhibits pituitary VEGF expression. VEGF expression was also increased in pituitary cells from knockout mice, even though these cells proliferated less in vitro when compared with wild-type cells, as determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium proliferation assay, proliferating cell nuclear antigen expression, and [(3)H]thymidine incorporation. In contrast to other animal models, estrogen did not increase pituitary VEGF protein and mRNA expression and lowered serum prolactin secretion in vivo and in vitro in both genotypes. VEGF (10 and 30 ng/ml) did not modify pituitary cell proliferation in either genotype and increased prolactin secretion in vitro in estrogen-pretreated cells of both genotypes. But conditioned media from D2R(-/-) cells enhanced human umbilical vein cell proliferation, and this effect could be partially inhibited by an anti-VEGF antiserum. Finally, using dual-labeling immunofluorescence and confocal laser microscopy, we found that in the hyperplastic pituitaries, VEGF-A was mostly present in follicle-stellate cells. In conclusion, pituitary VEGF expression is under dopaminergic control, and even though VEGF does not promote pituitary cellular proliferation in vitro, it may be critical for pituitary angiogenesis through paracrine actions in the D2R knockout female mice.

The nucleus tractus solitarius (NTS) receives dense terminations from cranial visceral afferents, including those from the gastrointestinal (GI) system. Although the NTS integrates peripheral satiety signals and relays this signal to central feeding centers, little is known about which NTS neurons are involved or what mechanisms are responsible. Proopiomelanocortin (POMC) neurons are good candidates for GI integration, because disruption of the POMC gene leads to severe obesity and hyperphagia. Here, we used POMC-enhanced green fluorescent protein (EGFP) transgenic mice to identify NTS POMC neurons. Intraperitoneal administration of cholecystokinin (CCK) induced c-fos gene expression in NTS POMC-EGFP neurons, suggesting that they are activated by afferents stimulated by the satiety hormone. We tested the synaptic relationship of these neurons to visceral afferents and their modulation by CCK and opioids using patch recordings in horizontal brain slices. Electrical activation of the solitary tract (ST) evoked EPSCs in NTS POMC-EGFP neurons. The invariant latencies, low failure rates, and substantial paired-pulse depression of the ST-evoked EPSCs indicate that NTS POMC-EGFP neurons are second-order neurons directly contacted by afferent terminals. The EPSCs were blocked by the glutamate antagonist 2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[f]quinoxaline. CCK increased the amplitude of the ST-stimulated EPSCs and the frequency of miniature EPSCs, effects attenuated by the CCK1 receptor antagonist lorglumide. In contrast, the orexigenic opioid agonists [D-Ala(2), N-Me-Phe(4), Gly-ol(5)]-enkephalin and met-enkephalin inhibited both ST-stimulated EPSCs and the frequency of miniature EPSCs. These findings identify a potential satiety pathway in which visceral afferents directly activate NTS POMC-EGFP neurons with excitatory inputs that are appropriately modulated by appetite regulators.

The proopiomelanocortin (POMC) gene is expressed in the pituitary and arcuate neurons of the hypothalamus. POMC arcuate neurons play a central role in the control of energy homeostasis, and rare loss-of-function mutations in POMC cause obesity. Moreover, POMC is the prime candidate gene within a highly significant quantitative trait locus on chromosome 2 associated with obesity traits in several human populations. Here, we identify two phylogenetically conserved neuronal POMC enhancers designated nPE1 (600 bp) and nPE2 (150 bp) located approximately 10 to 12 kb upstream of mammalian POMC transcriptional units. We show that mouse or human genomic regions containing these enhancers are able to direct reporter gene expression to POMC hypothalamic neurons, but not the pituitary of transgenic mice. Conversely, deletion of nPE1 and nPE2 in the context of the entire transcriptional unit of POMC abolishes transgene expression in the hypothalamus without affecting pituitary expression. Our results indicate that the nPEs are necessary and sufficient for hypothalamic POMC expression and that POMC expression in the brain and pituitary is controlled by independent sets of enhancers. Our study advances the understanding of the molecular nature of hypothalamic POMC neurons and will be useful to determine whether polymorphisms in POMC regulatory regions play a role in the predisposition to obesity.