The Rat Mammary Gland as a Novel Site of Expression of Melanin-Concentrating Hormone Receptor 1 mRNA and Its Protein Immunoreactivity.

Lactation is a complex physiological process, depending on orchestrated central and peripheral events, including substantial brain plasticity. Among these events is a novel expression of pro-melanin-concentrating hormone (Pmch) mRNA in the rodent hypothalamus, such as the ventral part of the medial preoptic area (vmMPOA). This expression reaches its highest levels around postpartum day 19 (PPD19), when dams transition from lactation to the weaning period. The appearance of this lactation-related Pmch expression occurs simultaneously with the presence of one of the Pmch products, melanin-concentrating hormone (MCH), in the serum. Given the relevance of the MPOA to maternal physiology and the contemporaneity between Pmch expression in this structure and the weaning period, we hypothesized that MCH has a role in the termination of lactation, acting as a mediator between central and peripheral changes. To test this, we investigated the presence of the MCH receptor 1 (MCHR1) and its gene expression in the mammary gland of female rats in different stages of the reproductive cycle. To that end, in situ hybridization, RT-PCR, RT-qPCR, nucleotide sequencing, immunohistochemistry, and Western blotting were employed. Although Mchr1 expression was detected in the epidermis and dermis of both diestrus and lactating rats, parenchymal expression was exclusively found in the functional mammary gland of lactating rats. The expression of Mchr1 mRNA oscillated through the lactation period and reached its maximum in PPD19 dams. Presence of MCHR1 was confirmed with immunohistochemistry with preferential location of MCHR1 immunoreactive cells in the alveolar secretory cells. As was the case for gene expression, the MCHR1 protein levels were significantly higher in PPD19 than in other groups. Our data demonstrate the presence of an anatomical basis for the participation of MCH peptidergic system on the control of lactation through the mammary gland, suggesting that MCH could modulate a prolactation action in early postpartum days and the opposite role at the end of the lactation.

Ciliary melanin-concentrating hormone receptor 1 (MCHR1) is widely distributed in the murine CNS in a sex-independent manner.

Melanin-concentrating hormone (MCH) is a ubiquitous vertebrate neuropeptide predominantly synthesized by neurons of the diencephalon that can act through two G protein-coupled receptors, called MCHR1 and MCHR2. The expression of Mchr1 has been investigated in both rats and mice, but its synthesis remains poorly described. After identifying an antibody that detects MCHR1 with high specificity, we employed immunohistochemistry to map the distribution of MCHR1 in the CNS of rats and mice. Multiple neurochemical markers were also employed to characterize some of the neuronal populations that synthesize MCHR1. Our results show that MCHR1 is abundantly found in a subcellular structure called the primary cilium, which has been associated, among other functions, with the detection of free neurochemical messengers present in the extracellular space. Ciliary MCHR1 was found in a wide range of areas, including the olfactory bulb, cortical mantle, striatum, hippocampal formation, amygdala, midline thalamic nuclei, periventricular hypothalamic nuclei, midbrain areas, and in the spinal cord. No differences were observed between male and female mice, and interspecies differences were found in the caudate-putamen nucleus and the subgranular zone. Ciliary MCHR1 was found in close association with several neurochemical markers, including tyrosine hydroxylase, calretinin, kisspeptin, estrogen receptor, oxytocin, vasopressin, and corticotropin-releasing factor. Given the role of neuronal primary cilia in sensing free neurochemical messengers in the extracellular fluid, the widespread distribution of ciliary MCHR1, and the diverse neurochemical populations who synthesize MCHR1, our data indicate that nonsynaptic communication plays a prominent role in the normal function of the MCH system.

Sensing of triacylglycerol in the gut: different mechanisms for fatty acids and 2-monoacylglycerol.

Sensing of dietary triacylglycerol in the proximal small intestine results in physiological, hormonal and behavioural responses. However, the exact physiological pathways linking intestinal fat sensing to food intake and the activation of brain circuits remain to be identified. In this study we examined the role of triacylglycerol digestion for intestinal fat sensing, and compared the effects of the triacylglycerol digestion products, fatty acids and 2-monoacylglycerol, on behavioural, hormonal and dopaminergic responses in behaving mice. Using an operant task in which mice are trained to self-administer lipid emulsions directly into the stomach, we show that inhibiting triacylglycerol digestion disrupts normal behaviour of self-administration in mice, indicating that fat sensing is conditional to digestion. When administered separately, both digestion products, 2-monoacylglycerol and fatty acids, were sensed by the mice, and self-administration patterns of fatty acids were affected by the fatty acid chain length. Peripheral plasma concentrations of the gut hormones GLP-1, GIP, PYY, CCK and insulin did not offer an explanation of the differing behavioural effects produced by 2-monoacylglycerol and fatty acids. However, combined with behavioural responses, striatal dopamine effluxes induced by gut infusions of oleic acid were significantly greater than those produced by equivalent infusions of 2-oleoylglycerol. Our data demonstrate recruitment of different signalling pathways by fatty acids and 2-monoacylglycerol, and suggest that the structural properties of fat rather than total caloric value determine intestinal sensing and the assignment of reward value to lipids.

Glucose utilization rates regulate intake levels of artificial sweeteners.

It is well established that animals including humans attribute greater reinforcing value to glucose-containing sugars compared to their non-caloric counterparts, generally termed 'artificial sweeteners'. However, much remains to be determined regarding the physiological signals and brain systems mediating the attribution of greater reinforcing value to sweet solutions that contain glucose. Here we show that disruption of glucose utilization in mice produces an enduring inhibitory effect on artificial sweetener intake, an effect that did not depend on sweetness perception or aversion. Indeed, such an effect was not observed in mice presented with a less palatable, yet caloric, glucose solution. Consistently, hungry mice shifted their preferences away from artificial sweeteners and in favour of glucose after experiencing glucose in a hungry state. Glucose intake was found to produce significantly greater levels of dopamine efflux compared to artificial sweetener in dorsal striatum, whereas disrupting glucose oxidation suppressed dorsal striatum dopamine efflux. Conversely, inhibiting striatal dopamine receptor signalling during glucose intake in sweet-naïve animals resulted in reduced, artificial sweetener-like intake of glucose during subsequent gluco-deprivation. Our results demonstrate that glucose oxidation controls intake levels of sweet tastants by modulating extracellular dopamine levels in dorsal striatum, and suggest that glucose utilization is one critical physiological signal involved in the control of goal-directed sweetener intake.

A gut lipid messenger links excess dietary fat to dopamine deficiency.

Excessive intake of dietary fats leads to diminished brain dopaminergic function. It has been proposed that dopamine deficiency exacerbates obesity by provoking compensatory overfeeding as one way to restore reward sensitivity. However, the physiological mechanisms linking prolonged high-fat intake to dopamine deficiency remain elusive. We show that administering oleoylethanolamine, a gastrointestinal lipid messenger whose synthesis is suppressed after prolonged high-fat exposure, is sufficient to restore gut-stimulated dopamine release in high-fat-fed mice. Administering oleoylethanolamine to high-fat-fed mice also eliminated motivation deficits during flavorless intragastric feeding and increased oral intake of low-fat emulsions. Our findings suggest that high-fat-induced gastrointestinal dysfunctions play a key role in dopamine deficiency and that restoring gut-generated lipid signaling may increase the reward value of less palatable, yet healthier, foods.

Flavor-independent maintenance, extinction, and reinstatement of fat self-administration in mice.

BACKGROUND: Mounting evidence suggests that overeating may be conceptualized within the same behavioral and neurobiological framework as drug addiction. One potentially important difference between overeating versus drug abuse refers to the sensory stimulation of oral receptors by palatable foods, a feature that may be required for reinforcement during intake. Likewise, postingestive effects and caloric content of food also contribute to reinforcing behavior and might influence the development of compulsive eating behavior. The purpose of the current study was to establish whether intragastric self-administration of fat emulsions, that is, bypassing the oral cavity, recapitulates some of the behavioral and neurobiological hallmarks of psychostimulant self-administration. METHODS: We used behavioral assays in mice to assess acquisition, maintenance, extinction, and reinstatement of intragastric self-administration of lipid emulsions to determine the extent to which postoral fat self-administration recapitulates psychostimulant self-administration. Striatal dopamine efflux during behavioral tasks was determined by brain microdialysis coupled to chromatographic-electrochemical analyses. RESULTS: We show that in direct analogy to drug self-administration, 1) decreases in fat dose concentration were met with compensatory increases in response rates aimed at maintaining constant hourly caloric intake; 2) rates of responding markedly increased during both extinction and progressive ratio schedules of reinforcement; and 3) elevations in striatal dopamine levels observed during maintenance were markedly attenuated during extinction sessions, only to be restored on reinstatement. CONCLUSIONS: Our data thus support the contention that stimulation of oral receptors by caloric foods may not be required for the expression of certain addiction-related neurobehavioral markers.

Hypothalamic melanin concentrating hormone neurons communicate the nutrient value of sugar.

Sugars that contain glucose, such as sucrose, are generally preferred to artificial sweeteners owing to their post-ingestive rewarding effect, which elevates striatal dopamine (DA) release. While the post-ingestive rewarding effect, which artificial sweeteners do not have, signals the nutrient value of sugar and influences food preference, the neural circuitry that mediates the rewarding effect of glucose is unknown. In this study, we show that optogenetic activation of melanin-concentrating hormone (MCH) neurons during intake of the artificial sweetener sucralose increases striatal dopamine levels and inverts the normal preference for sucrose vs sucralose. Conversely, animals with ablation of MCH neurons no longer prefer sucrose to sucralose and show reduced striatal DA release upon sucrose ingestion. We further show that MCH neurons project to reward areas and are required for the post-ingestive rewarding effect of sucrose in sweet-blind Trpm5(-/-) mice. These studies identify an essential component of the neural pathways linking nutrient sensing and food reward. DOI: http://dx.doi.org/10.7554/eLife.01462.001.

AgRP neurons regulate development of dopamine neuronal plasticity and nonfood-associated behaviors.

It is not known whether behaviors unrelated to feeding are affected by hypothalamic regulators of hunger. We found that impairment of Agouti-related protein (AgRP) circuitry by either Sirt1 knockdown in AgRP-expressing neurons or early postnatal ablation of these neurons increased exploratory behavior and enhanced responses to cocaine. In AgRP circuit-impaired mice, ventral tegmental dopamine neurons exhibited enhanced spike timing-dependent long-term potentiation, altered amplitude of miniature postsynaptic currents and elevated dopamine in basal forebrain. Thus, AgRP neurons determine the set point of the reward circuitry and associated behaviors.

The gut-brain dopamine axis: a regulatory system for caloric intake.

Post-ingestive factors are known to strongly modulate feeding behavior by providing feedback signals to the central nervous system on the current physiological state of the organism. Of particular interest is the identification of the physiological pathways that permit the brain to sense post-ingestive signals. We will review recent evidence supporting the concept that direct stimulation of the gastrointestinal tract with nutrients induces release of the catecholamine neurotransmitter dopamine. In addition, changes in dopamine efflux produced by direct stimulation of the gastrointestinal tract were found to reflect the caloric load of the infusates, suggesting that dopamine signaling may function as a central caloric sensor that mediates adjustments in intake according to the caloric density of a meal. Consistent with the above, blockade of dopamine signaling disrupts flavor-nutrient associations and impairs the regulatory capacity to maintain constant caloric intake during intra-gastric feeding. Future research must determine the exact pathways linking gut nutrient administration to dopamine efflux. Current evidence points to parallel contributions by pre- and post-absorptive pathways, indicating that dopamine systems constitute a site of convergence through which distinct physiological signals can exert control over ingestive behaviors.

Regulation of fat intake in the absence of flavour signalling.

Animals, including humans, can achieve precise regulation of caloric intake by adjusting consumption in response to covert changes in energy density. It remains unknown, however, whether the presence of flavour cues are required for the ability to maintain constant caloric intake. Also unknown are the brain circuits that may function as the central calorie monitors that control adaptive adjustments in energy intake. Here we show that mice trained to lick a dry spout in order to receive intra-gastric infusions of a fat emulsion maintained constant hourly caloric intake by adjusting the number of dry licks in response to changes in caloric density. Animals also increased dry licking according to hunger levels, and developed conditioned preferences for dry sippers associated with high calorie infusions. Importantly, striatal dopamine levels were closely associated with the amount of calories ingested, rather than with the number of dry licks produced. Dopamine levels in dorsal and ventral striatum also reflected caloric density in mice passively receiving intra-gastric infusions of fat emulsions. Consistent with the above, systemic administration of the dopamine receptor blocker haloperidol markedly increased the production of dry licks needed to obtain high-calorie infusions, as if the caloric density of the infusions had been diluted. Conversely, haloperidol markedly decreased the production of dry licks needed to obtain low-calorie infusions. Taken together, our results support the proposition that brain dopamine circuits function as one central sensor of calorie ingestion, since (1) extracellular striatal dopamine levels fluctuate in proportion to the caloric density of nutrients infused in the gut; and (2) inhibiting dopamine receptor signalling disrupts the animals' ability to maintain constant caloric intake across experimental sessions.

Effects of ad libitum ingestion of monosodium glutamate on weight gain in C57BL6/J mice.

Although the umami compound monosodium glutamate (MSG) is a widely used flavor enhancer, controversy still persists regarding the effects of MSG intake on body weight. It has been claimed, in particular, that chronic MSG intake may result in excessive body weight gain and obesity. In this study we assessed the effects of chronic (16 weeks) ad libitum MSG on body weight and metabolism of C57BL6/J mice. Adult male mice were divided in four experimental groups and fed with either a low-fat (LF) or high-fat (HF) diet and with either two bottles of plain water or one bottle containing 1% MSG and another one containing water according to a factorial design. Mice were monitored weekly for body weight and food/fluid intake for 15 weeks. At the end of the experiments, the circulating levels of leptin, insulin, total protein, total cholesterol, triglyceride, blood urea nitrogen, and non-esterified fatty acids were also analyzed. Our results show that MSG intake did not influence body weight in either LF or HF groups. Interestingly, although animals overall displayed strong preferences for MSG against water, preferences were relatively higher in LF compared to HF group. Consistent with the body weight data, while significant differences in leptin, insulin, total cholesterol, and non-esterified fatty acids were found between HF and LF groups, such an effect was not influenced by MSG intake. Finally, indirect calorimetry measurements revealed similar energy expenditure levels between animals being presented water only and MSG only. In summary, our data does not support the notion that ad libitum MSG intake should trigger the development of obesity or other metabolic abnormalities.

Metabolic sensing in brain dopamine systems.

The gustatory system allows the brain to monitor the presence of chemicals in the oral cavity and initiate appropriate responses of acceptance or rejection. Among such chemicals are the nutrients that must be rapidly recognized and ingested for immediate oxidation or storage. In the periphery, the gustatory system consists of a highly efficient sensing mechanism, where distinct cell types express receptors that bind specifically to chemicals associated with one particular taste quality. These specialized receptors connect to the brain via dedicated pathways, the stimulation of which triggers stereotypic behavioral responses as well as neurotransmitter release in brain reward dopamine systems. However, evidence also exists in favor of the concept that the critical regulators of long-term nutrient choice are physiological processes taking place after ingestion and independently of gustation. We will appraise the hypothesis that organisms can develop preferences for nutrients independently of oral taste stimulation. Of particular interest are recent findings indicating that disrupting nutrient utilization interferes with activity in brain dopamine pathways. These findings establish the metabolic fate of nutrients as previously unanticipated reward signals that regulate the reinforcing value of foods. In particular, it suggests a role for brain dopamine reward systems as metabolic sensors, allowing for signals generated by the metabolic utilization of nutrients to regulate neurotransmitter release and food reinforcement.

Nutrient selection in the absence of taste receptor signaling.

When allowed to choose between different macronutrients, most animals display a strong attraction toward carbohydrates compared with proteins. It remains uncertain, however, whether this food selection pattern depends primarily on the sensory properties intrinsic to each nutrient or, alternatively, metabolic signals can act independently of the hedonic value of sweetness to stimulate elevated sugar intake. Here we show that Trpm5(-/-) mice, which lack the cellular mechanisms required for sweet and several forms of l-amino acid taste transduction, develop a robust preference for d-glucose compared with isocaloric l-serine independently of the perception of sweetness. Moreover, a close relationship was found between glucose oxidation and taste-independent nutrient intake levels, with animals increasing intake as a function of glucose oxidation rates. Furthermore, microdialysis measurements revealed nutrient-specific dopaminergic responses in accumbens and dorsal striatum during intragastric infusions of glucose or serine. Specifically, intragastric infusions of glucose induced significantly higher levels of dopamine release compared with isocaloric serine in both ventral and dorsal striatum. Intragastric stimulation of dopamine release seemed to depend on glucose utilization, because administration of an anti-metabolic glucose analog resulted in lower dopamine levels in striatum, an effect that was reversed by intravenous glucose infusions. Together, our findings suggest that carbohydrate-specific preferences can develop independently of taste quality or caloric load, an effect associated with the ability of a given nutrient to regulate glucose metabolism and stimulate brain dopamine centers.