Jessica R. Barson

Opioids in the hypothalamic paraventricular nucleus stimulate ethanol intake.

BACKGROUND Specialized hypothalamic systems that increase food intake might also increase ethanol intake. To test this possibility, morphine and receptor-specific opioid agonists were microinjected in the paraventricular nucleus (PVN) of rats that had learned to drink ethanol. To cross-validate the results, naloxone methiodide (m-naloxone), an opioid antagonist, was microinjected with the expectation that it would have the opposite effect of morphine and the specific opioid agonists. METHODS Sprague-Dawley rats were trained, without sugar, to drink 4 or 7% ethanol and were then implanted with chronic brain cannulas aimed at the PVN. After recovery, those drinking 7% ethanol, with food and water available, were injected with 2 doses each of morphine or m-naloxone. To test for receptor specificity, 2 doses each of the mu-receptor agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-Enkephalin (DAMGO), delta-receptor agonist D-Ala-Gly-Phe-Met-NH2 (DALA), or kappa-receptor agonist U-50,488H were injected. DAMGO was also tested in rats drinking 4% ethanol without food or water available. As an anatomical control for drug reflux, injections were made 2 mm dorsal to the PVN. RESULTS A main result was a significant increase in ethanol intake induced by PVN injection of morphine. The opposite effect was produced by m-naloxone. The effects of morphine and m-naloxone were exclusively on intake of ethanol, even though food and water were freely available. In the analysis with specific receptor agonists, PVN injection of the delta-agonist DALA significantly increased 7% ethanol intake without affecting food or water intake. This is in contrast to the kappa-agonist U-50,488H, which decreased ethanol intake, and the mu-agonist DAMGO, which had no effect on ethanol intake in the presence or absence of food and water. In the anatomical control location 2 mm dorsal to the PVN, no drug caused any significant changes in ethanol, food, or water intake, providing evidence that the active site was close to the cannula tip. CONCLUSIONS The delta-opioid receptor agonist in the PVN increased ethanol intake in strong preference over food and water, while the kappa-opioid agonist suppressed ethanol intake. Prior studies show that learning to drink ethanol stimulates PVN expression and production of the peptides enkephalin and dynorphin, which are endogenous agonists for the delta- and kappa-receptors, respectively. These results suggest that enkephalin via the delta-opioid system can function locally within a positive feedback circuit to cause ethanol intake to escalate and ultimately contribute to the abuse of ethanol. This is in contrast to dynorphin via the kappa-opioid system, which may act to counter this escalation. Naltrexone therapy for alcoholism may act, in part, by blocking the enkephalin-triggered positive feedback cycle.

Positive relationship between dietary fat, ethanol intake, triglycerides and hypothalamic peptides: Counteraction by lipid-lowering drugs

Studies in both humans and animals suggest a positive relationship between the intake of ethanol and intake of fat, which may contribute to alcohol abuse. This relationship may be mediated, in part, by hypothalamic orexigenic peptides such as orexin (OX), which stimulate both consumption of ethanol and fat, and circulating triglycerides (TGs), which stimulate these peptides and promote consummatory behavior. The present study investigated this vicious cycle between ethanol and fat, to further characterize its relation to TGs and to test the effects of lowering TG levels. In Experiment 1, the behavioral relationship between fat intake and ethanol was confirmed. Adult male Sprague-Dawley rats, chronically injected intraperitoneally with ethanol (1g/kg) and tested in terms of their preference for a high-fat diet (HFD) compared with low-fat diet (LFD), showed a significant increase in their fat preference, compared with rats injected with saline, in measures of 2h and 24h intake. Experiment 2 tested the relationship of circulating TGs in this positive association between ethanol and fat, in rats chronically consuming 9% ethanol versus water and given acute meal tests (25kcal) of a HFD versus LFD. Levels of TGs were elevated in response to both chronic drinking of ethanol versus water and acute eating of a high-fat versus low-fat meal. Most importantly, ethanol and a HFD showed an interaction effect, whereby their combination produced a considerably larger increase in TG levels (+172%) compared to ethanol with a LFD (+111%). In Experiment 3, a direct manipulation of TG levels was found to affect ethanol intake. After intragastric administration of gemfibrozil (50mg/kg) compared with vehicle, TG levels were lowered by 37%, and ethanol intake was significantly reduced. In Experiment 4, the TG-lowering drug gemfibrozil also caused a significant reduction in the expression of the orexigenic peptide, OX, in the perifornical lateral hypothalamus. These results support the existence of a vicious cycle between ethanol and fat, whereby each nutrient stimulates intake of the other. Within this vicious cycle, ethanol and fat act synergistically to increase TG levels, which in turn stimulate peptides that promote further consumption, and these phenomena are reversed by gemfibrozil, which lowers TG levels.

Anterior thalamic paraventricular nucleus is involved in intermittent access ethanol drinking: role of orexin receptor 2

The paraventricular nucleus of the thalamus (PVT) has been shown to participate in hedonic feeding and is thought to influence drug seeking. This understudied nucleus contains anterior (aPVT) and posterior (pPVT) subregions, which receive dense projections from hypothalamic orexin/hypocretin (OX) but exhibit anatomical and functional differences. This study sought to characterize in Long‐Evans rats the involvement of these PVT subregions and their OX receptor activity in consumption of the drug, ethanol. Compared with those maintained on water and chow only (water group), rats trained to drink pharmacologically relevant levels of ethanol (ethanol group) showed increased neuronal activation in the PVT, specifically the aPVT but not pPVT, as indicated by c‐Fos immunoreactivity. Similar results were obtained in rats administered ethanol via oral gavage, indicating that this site‐specific effect was due to ethanol exposure. In support of the involvement of OX, the ethanol group also showed increased mRNA levels of this neuropeptide in the hypothalamus and of OX 2 receptor (OX2R) but not OX 1 receptor (OX1R), again in the aPVT but not pPVT. Similarly, ethanol gavage increased double labeling of c‐Fos with OX2R but not OX1R, specifically in the aPVT. Evidence directly supporting a role for aPVT OX2R in ethanol consumption was provided by results with local injections, showing ethanol intake to be enhanced by OX‐A or OX‐B in the aPVT but not pPVT and reduced by a local antagonist of OX2R but not OX1R. These results focus attention on the aPVT and specifically its OX2R in mediating a positive feedback relationship with ethanol intake.

Differential Effects of Acute and Chronic Ethanol Exposure on Orexin Expression in the Perifornical Lateral Hypothalamus

BACKGROUND Recent reports support the involvement of hypothalamic orexigenic peptides in stimulating ethanol intake. Our previous studies have examined the effects of ethanol on hypothalamic peptide systems of the paraventricular nucleus (PVN) and identified a positive feedback loop in which PVN peptides, such as enkephalin and galanin, stimulate ethanol intake and ethanol, in turn, stimulates the expression of these peptides. Recently, orexin (OX), a peptide produced mainly by cells in the perifornical lateral hypothalamus (PFLH), has been shown to play an important role in mediating the rewarding aspects of ethanol intake. However, there is little evidence showing the effects that ethanol itself may have on the OX peptide system. In order to understand the feedback relationship between ethanol and the OX system, the current investigation was designed to measure OX gene expression in the PFLH following acute as well as chronic ethanol intake. METHODS In the first experiment, Sprague-Dawley rats were trained to voluntarily consume a 2 or 9% concentration of ethanol, and the expression of OX mRNA in the PFLH was measured using quantitative real-time polymerase chain reaction (qRT-PCR). The second set of experiments tested the impact of acute oral gavage of 0.75 and 2.5 g/kg ethanol solution on OX expression in the PFLH using qRT-PCR, as well as radiolabeled in situ hybridization. Further tests using digoxigenin-labeled in situ hybridization and immunofluorescence histochemistry allowed us to more clearly distinguish the effects of acute ethanol on OX cells in the lateral hypothalamic (LH) versus perifornical (PF) regions. RESULTS The results showed chronic consumption of ethanol versus water to dose-dependently reduce OX mRNA in the PFLH, with a larger effect observed in rats consuming 2.5 g/kg/d (-70%) or 1.0 g/kg/d (-50%) compared to animals consuming 0.75 g/kg/d (-40%). In contrast to chronic intake, acute oral ethanol compared to water significantly enhanced OX expression in the PFLH, and this effect occurred at the lower (0.75 g/kg) but not higher (2.5 g/kg) dose of ethanol. Additional analyses of the OX cells in the LH versus PF regions identified the former as the primary site of ethanols stimulatory effect on the OX system. In the LH but not the PF, acute ethanol increased the density of OX-expressing and OX-immunoreactive neurons. The increase in gene expression was detected only at the lower dose of ethanol (0.75 g/kg), whereas the increase in OX peptide was seen only at the higher dose of ethanol (2.5 g/kg). CONCLUSION These results lead us to propose that OX neurons, while responsive to negative feedback signals from chronic ethanol consumption, are stimulated by acute ethanol administration, most potently in the LH where OX may trigger central reward mechanisms that promote further ethanol consumption.

Complementary Roles of Orexin and Melanin-Concentrating Hormone in Feeding Behavior

Transcribed within the lateral hypothalamus, the neuropeptides orexin/hypocretin (OX) and melanin-concentrating hormone (MCH) both promote palatable food intake and are stimulated by palatable food. While these two neuropeptides share this similar positive relationship with food, recent evidence suggests that this occurs through different albeit complementary effects on behavior, with OX promoting food seeking and motivation for palatable food and MCH functioning during ongoing food intake, reinforcing the consumption of calorically dense foods. Further differences are evident in their effects on physiological processes, which are largely opposite in nature. For example, activation of OX receptors, which is neuronally excitatory, promotes waking, increases energy expenditure, and enhances limbic dopamine levels and reward. In contrast, activation of MCH receptors, which is neuronally inhibitory, promotes paradoxical sleep, enhances energy conservation, reduces limbic dopamine, and increases depressive behavior. This review describes these different effects of the neuropeptides, developing the hypothesis that they stimulate the consumption of palatable food through excessive seeking in the case of OX and through excessive energy conservation in the case of MCH. It proposes that OX initiates food intake and subsequently stimulates MCH which then acts to prolong the consumption of palatable, energy-dense food.

Similarities in hypothalamic and mesocorticolimbic circuits regulating the overconsumption of food and alcohol

Historically, studies of food intake regulation started with the hypothalamus and gradually expanded to mesocorticolimbic regions, while studies of drug use began with mesocorticolimbic regions and now include the hypothalamus. As research on ingestive behavior has progressed, it has uncovered more and more similarities between the regulation of palatable food and drug intake. It has also identified specific neurochemicals involved in palatable food and drug intake. Hypothalamic orexigenic neurochemicals specifically involved in controlling fat ingestion, including galanin, enkephalin, orexin and melanin-concentrating hormone, show positive feedback with this macronutrient, with these peptides both increasing fat intake and being further stimulated by its intake. This positive relationship offers some explanation for why foods high in fat are so often overconsumed. Research in Bart Hoebels laboratory in conjunction with our own has shown that consumption of ethanol, a drug of abuse that also contains calories, is similarly driven by these neurochemical systems involved in fat intake, consistent with evidence closely relating fat and ethanol consumption. Both fat and ethanol intake are also regulated by dopamine and acetylcholine acting in mesocorticolimbic nuclei. This close relationship of fat and ethanol is likely driven in part by circulating lipids, which are increased by fat and ethanol intake, known to increase expression and levels of the neurochemicals, and found to promote further intake of fat and ethanol. Compellingly, recent studies suggest that these systems may already be dysregulated in animals prone to consuming excess fat or ethanol, even before they have ever been exposed to these substances. Further understanding of these systems involved in consummatory behavior will allow researchers to develop effective therapies for the treatment of overeating as well as drug abuse.

Opioids in the hypothalamus control dopamine and acetylcholine levels in the nucleus accumbens

The experimental question is whether hypothalamic opioids, known to stimulate consummatory behavior, control a link to the nucleus accumbens (NAc). It was hypothesized that opioids injected in the hypothalamic paraventricular nucleus (PVN) alter the balance of dopamine (DA) and acetylcholine (ACh) in the NAc in a manner that fosters appetite for food or ethanol. Rats were implanted with two guide shafts, one in the NAc to measure extracellular DA and ACh by microdialysis and the other in the PVN for microinjection of opioid mu- and delta-agonists, an antagonist, or saline vehicle. The compounds tested were morphine, the mu-receptor agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-Enkephalin (DAMGO), the delta-receptor agonist D-Ala-Gly-Phe-Met-NH2 (DALA), and the opioid antagonist naloxone methiodide (m-naloxone). Morphine in the PVN increased the release of accumbens DA (+41%) and decreased ACh (-35%). Consistent with this, the opioid antagonist m-naloxone decreased DA (-24%) and increased ACh (+19%). In terms of receptor involvement, DAMGO dose-dependently increased DA to up to 209% of baseline. Simultaneously, ACh levels were markedly decreased to 55% of baseline. The agonist DALA produced a smaller but significant, 34% increase in DA, without affecting ACh. In contrast, control injections of saline had no significant effect. These results demonstrate that mu- and delta-opioids in the PVN contribute to the control of accumbens DA and ACh release and suggest that this circuit from the PVN to the NAc may be one of the mechanisms underlying opiate-induced ingestive behavior as well as naltrexone therapy for overeating and alcoholism.

Opioids in the nucleus accumbens stimulate ethanol intake

The nucleus accumbens (NAc) participates in the control of both motivation and addiction. To test the possibility that opioids in the NAc can cause rats to select ethanol in preference to food, Sprague-Dawley rats with ethanol, food, and water available, were injected with two doses each of morphine, the mu-receptor agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-Enkephalin (DAMGO), the delta-receptor agonist D-Ala-Gly-Phe-Met-NH2 (DALA), the k-receptor agonist (+/-)-trans-U-50488 methanesulfonate (U-50,488H), or the opioid antagonist naloxone methiodide (m-naloxone). As an anatomical control for drug reflux, injections were also made 2mm above the NAc. The main result was that morphine in the NAc significantly increased ethanol and food intake, whereas m-naloxone reduced ethanol intake without affecting food or water intake. Of the selective receptor agonists, DALA in the NAc increased ethanol intake in preference to food. This is in contrast to DAMGO, which stimulated food but not ethanol intake, and the k-agonist U-50,488H, which had no effect on intake. When injected in the anatomical control site 2mm dorsal to the NAc, the opioids had no effects on ethanol intake. These results demonstrate that ethanol intake produced by morphine in the NAc is driven in large part by the delta-receptor. In light of other studies showing ethanol intake to increase enkephalin expression in the NAc, the present finding of enkephalin-induced ethanol intake suggests the existence of a positive feedback loop that fosters alcohol abuse. Naltrexone therapy for alcohol abuse may then act, in part, in the NAc by blocking this opioid-triggered cycle of alcohol intake.

Effect of Chronic Ethanol on Enkephalin in the Hypothalamus and Extra‐Hypothalamic Areas

BACKGROUND Ethanol may be consumed for reasons such as reward, anxiety reduction, or caloric content, and the opioid enkephalin (ENK) appears to be involved in many of these functions. Previous studies in Sprague-Dawley rats have demonstrated that ENK in the hypothalamic paraventricular nucleus (PVN) is stimulated by voluntary consumption of ethanol. This suggests that this opioid peptide may be involved in promoting the drinking of ethanol, consistent with our recent findings that PVN injections of ENK analogs stimulate ethanol intake. To broaden our understanding of how this peptide functions throughout the brain to promote ethanol intake, we measured, in rats trained to drink 9% ethanol, the expression of the ENK gene in additional brain areas outside the hypothalamus, namely, the ventral tegmental area (VTA), nucleus accumbens shell (NAcSh) and core (NAcC), medial prefrontal cortex (mPFC), and central nucleus of the amygdala (CeA). METHODS In the first experiment, the brains of rats chronically drinking 1 g/kg/d ethanol, 3 g/kg/d ethanol, or water were examined using real-time quantitative polymerase chain reaction (qRT-PCR). In the second experiment, a more detailed, anatomic analysis of changes in gene expression, in rats chronically drinking 3 g/kg/d ethanol compared to water, was performed using radiolabeled in situ hybridization (ISH). The third experiment employed digoxigenin-labeled ISH (DIG) to examine changes in the density of cells expressing ENK and, for comparison, dynorphin (DYN) in rats chronically drinking 3 g/kg/d ethanol versus water. RESULTS With qRT-PCR, the rats chronically drinking ethanol plus water compared to water alone showed significantly higher levels of ENK mRNA, not only in the PVN but also in the VTA, NAcSh, NAcC, and mPFC, although not in the CeA. Using radiolabeled ISH, levels of ENK mRNA in rats drinking ethanol were found to be elevated in all areas examined, including the CeA. The experiment using DIG confirmed this effect of ethanol, showing an increase in density of ENK-expressing cells in all areas studied. It additionally revealed a similar change in DYN mRNA in the PVN, mPFC, and CeA, although not in the NAcSh or NAcC. CONCLUSIONS While distinguishing the NAc as a site where ENK and DYN respond differentially, these findings lead us to propose that these opioids, in response to voluntary ethanol consumption, are generally elevated in extra-hypothalamic as well as hypothalamic areas, possibly to carry out specific area-related functions that, in turn, drive animals to further consume ethanol. These functions include calorie ingestion in the PVN, reward and motivation in the VTA and NAcSh, response-reinforcement learning in the NAcC, stress reduction in the CeA, and behavioral control in the mPFC.

Effect of dietary fatty acid composition on food intake, triglycerides, and hypothalamic peptides

While a high-fat diet when compared to low-fat diet is known to produce overeating and health complications, less is known about the effects produced by fat-rich diets differing in their specific composition of fat. This study examined the effects of a high-fat diet containing relatively high levels of saturated compared to unsaturated fatty acids (HiSat) to a high-fat diet with higher levels of unsaturated fatty acids (USat). A HiSat compared to USat meal caused rats to consume more calories in a subsequent chow test meal. The HiSat meal also increased circulating levels of triglycerides (TG) and expression of the orexigenic peptides, galanin (GAL) in the hypothalamic paraventricular nucleus (PVN) and orexin (OX) in the perifornical lateral hypothalamus (PFLH). A similar increase in TG levels and PVN GAL and PFLH OX was also seen in rats given chronic access to the HiSat compared to USat diet, while neuropeptide Y (NPY) and agouti-related protein (AgRP) in the arcuate nucleus showed decreased expression. The importance of TG in producing these changes was supported by the finding that the TG-lowering medication gemfibrozil as compared to vehicle, when peripherally administered before consumption of a HiSat meal, significantly decreased the expression of OX, while increasing the expression of NPY and AgRP. These findings substantiate the importance of the fat composition in a diet, indicating that those rich in saturated compared to unsaturated fatty acids may promote overeating by increasing circulating lipids and specific hypothalamic peptides, GAL and OX, known to preferentially stimulate the consumption of a fat-rich diet.