Food restriction markedly increases dopamine D2 receptor (D2R) in a rat model of obesity as assessed with in-vivo muPET imaging ([11C] raclopride) and in-vitro ([3H] spiperone) autoradiography

Introduction: Dopamine (DA) regulates food intake by modulating food reward and motivation but its involvement in obesity is much less understood. Recent evidence points to the involvement of leptin in the DA‐related modulation of food intake. Here we assess DA D2 receptors (D2R) in a genetic rodent obesity model characterized by leptin‐receptor deficiency and assess the influence of food restriction on these receptors. Methods: We compared D2R levels between Zucker Obese (fa/fa) and Lean (Fa/Fa) rats at 1 and 4 months of age and in two different feeding conditions (restricted and unrestricted food access) using in‐vivo μPET imaging ([¹¹C] raclopride, which is a method sensitive to competition with endogenous DA) and in‐vitro ([³H] spiperone washed to ensure no competition with endogenous DA) autoradiography (ARG). Results: Both ARG and μPET showed that D2R were higher at 1 month than at 4 months of age and that food restricted animals had higher D2R than unrestricted animals. However there were significant differences in the results obtained at 4 months between ARG and μPET. ARG showed that at 1 month and at 4 months unrestricted lean rats (Le U) had significantly higher D2R binding than obese unrestricted rats (Ob U) but showed no differences between restricted obese (Ob R) and restricted lean rats (Le R). It also showed that D2R decline between 1 and 4 months of age was significantly attenuated in food restricted rats [both obese and lean]. In contrast, μPET showed that at 4 months of age, Ob U showed greater D2R availability than Le U rats but like ARG showed no differences between Ob R and Le R rats. Conclusion: The lower D2R binding in Ob U than Le U rats observed with ARG most likely reflects decreases in striatal D2 receptors levels whereas the increased availability observed with μPET is likely to reflect reduced DA release (resulting in decreased competition with endogenous DA). Lack of a significant difference between Ob R and Le R suggests that the differences in dopamine activity and D2R levels between Ob and Le Zucker rats are modulated by access to food. The ARG finding of an attenuation of the age‐related loss of D2R binding corroborates previous studies of the salutary effects of food restriction in the aging process. Because [¹¹C] raclopride is sensitive to competition with endogenous DA, the higher D2R binding in obese rats with raclopride despite the lower D2R levels shown with spiperone could reflect lower extracellular DA in the Ob rats and merits further investigation. Synapse 62:50–61, 2008. Published 2007 Wiley‐Liss, Inc.     

Obese rats with deficient leptin signaling exhibit heightened sensitivity to olfactory food cues

The Zucker rat is used as a model of genetic obesity, and while Zucker rats have been well studied for their reduced sensitivity to leptin signaling and subsequent weight gain, little work has examined their responses to environmental signals that are associated with “hedonic” feeding. This study evaluated the effects of a high‐fat food olfactory cue (bacon) in stimulating nose‐poke food‐seeking behavior on first exposure (novel) and after a period of access for consumption (familiar) in lean and obese Zucker rats at either 4 or 12 months of age, and under ad‐lib fed (unrestricted; U) or chronically food‐restricted (70% of ad‐lib; R) conditions. Baseline nose‐poke levels were comparable amongst all groups. At 4 months of age, only ObU rats displayed increased behavioral activation to familiar food cues. Twelve‐month‐old Ob rats, regardless of diet, exhibited substantially greater food‐seeking behavior when exposed to both the novel and familiar olfactory cues. A strong positive correlation between body weight and nose‐poke entries for the familiar food cue was observed at both ages, while this correlation for the novel food cue was significant in 12–month‐old rats only. Similarly, there were strong positive correlations between food intake and poke entries for the familiar food cue was observed at both ages, while this correlation for the novel food cue was significant in 12–month‐old rats only. Although it is possible that differences in olfactory sensitivity contribute to these behavioral effects, our findings support the interactions between food intake, obesity, and food‐seeking behavior and are consistent with leptin inhibiting the brain's reactivity to food cues and suggest that the enhanced sensitivity to the food cues with leptin deficiency is likely to contribute to overeating and weight gain. Synapse, 2013. © 2012 Wiley Periodicals, Inc.     

Leptin increases striatal dopamine D2 receptor binding in leptin‐deficient obese (ob/ob) mice

Peripheral and central leptin administration have been shown to mediate central dopamine (DA) signaling. Leptin‐receptor deficient rodents show decreased DA D2 receptor (D2R) binding in striatum and unique DA profiles compared to controls. Leptin‐deficient mice show increased DA activity in reward‐related brain regions. The objective of this study was to examine whether basal D2R‐binding differences contribute to the phenotypic behaviors of leptin‐deficient ob/ob mice, and whether D2R binding is altered in response to peripheral leptin treatment in these mice. Leptin decreased body weight, food intake, and plasma insulin concentration in ob/ob mice but not in wild‐type mice. Basal striatal D2R binding (measured with autoradiography [³H] spiperone) did not differ between ob/ob and wild‐type mice but the response to leptin did. In wild‐type mice, leptin decreased striatal D2R binding, whereas, in ob/ob mice, leptin increased D2R binding. Our findings provide further evidence that leptin modulates D2R expression in striatum and that these effects are genotype/phenotype dependent. Synapse 64:503–510, 2010. © 2010 Wiley‐Liss, Inc.     

Activation of PAR1 in the lateral hypothalamus of rats enhances food intake and REMS through CB1R

The activation of protease-activated receptor 1 (PAR1) in cultured rat hippocampal neurons triggers synaptic retrograde signaling through the endocannabinoid 2-arachidonoylglycerol, thereby activating the cannabinoid receptor 1 (CB1R). CB1R is a metabotropic receptor activated by marihuana and endocannabinoids that suppresses neurotransmitter release. Also, activation of the CB1R increases rapid eye movement sleep (REMS) and food intake. The lateral hypothalamus is a crucial structure to modulate both feeding and waking. To evaluate the effect of PAR1 stimulation in the lateral hypothalamus on food intake and on the sleep-waking cycle, we implanted rats with electrodes, for recording sleep, and cannulae, to administer S1820, a selective PAR1 agonist peptide, bilaterally into the lateral hypothalamus. To determine whether the effects induced by PAR1 stimulation were mediated by CB1R activation, we administered AM251, a CB1R inverse agonist, to block S1820 effects. Our results show that the stimulation of PAR1 into the lateral hypothalamus increases both food intake and REMS and such effects were prevented by AM251, indicating that PAR1 modulates both food intake and the sleep-waking cycle, in the lateral hypothalamus, through CB1R activation. This study shows novel behavioral changes induced by PAR1 activation and further supports the notion that endocannabinoids are food intake and REMS promoters.     

CB1 cannabinoid receptor expression in brain regions associated with zebra finch song control

Cannabinoids have been used for millennia through various preparations of Cannabis sativa. Despite this long history of use, the physiological significance of cannabinoid signaling in the vertebrate CNS is not well understood. High CB1 cannabinoid receptor densities in mammalian telencephalon and the results of behavioral studies suggest that cannabinoids play a role in cognitive function, learning, and memory. Since a network of discrete brain regions in zebra finch telencephalon controls song learning, we hypothesized that cannabinoid signaling may be relevant to songbird vocal development and behavior. Radioligand binding experiments using the cannabinoid agonist [3H]CP-55940 allowed identification of a dense population of high-affinity cannabinoid binding sites in zebra finch neuronal membranes. Northern blotting and RT-PCR experiments demonstrated expression of a predominant zebra finch CB1 mRNA of approximately 5.5 kb. Expression of this CB1 mRNA appears to change over the course of vocal development within the caudal telencephalon. As zebra finch caudal telencephalon contains the higher vocal center (HVC) and the robust nucleus of the archistriatum (RA), regions involved in song learning and production, we further investigated CB1 expression in these areas using in situ hybridization. In situ hybridization revealed that CB1 mRNA is expressed at high levels within both HVC and RA. Overall, these data demonstrate the presence of CB1 signaling systems within songbird telencephalon, notably within regions known to be involved in song learning and production. High-level CB1 expression in song regions suggests a potential role for cannabinoid signaling in zebra finch vocal development.     

Overexpression of cannabinoid CB2 receptor in the brain induces hyperglycaemia and a lean phenotype in adult mice

It is well known that the endocannabinoid system, through cannabinoid CB1 receptor activation, has an important role in the main aspects of energy balance (i.e. food intake, energy expenditure and glucose and fat metabolism), orchestrating all the machinery involved in body weight control and energy homeostasis. A number of studies have revealed a crucial role of brain CB1 receptors in these processes. However, functional cannabinoid CB2 receptors have also been described in the brain, with no studies addressing their putative role in body weight control and glucose homeostasis. We have tested this hypothesis by analysing fasting-induced feeding, body weight, some hypothalamic neuropeptides, glucose tolerance and plasma hormones in an animal model specifically overexpressing CB2 receptors in the central nervous system. We found that specific overexpression of CB2 receptors in the brain promoted higher basal glucose levels, decreased fasting-induced feeding and, eventually, led to a lean phenotype and glucose intolerance. These findings could not be attributed to decreased locomotor activity, increased anxiety or depressive-like behaviours. The expression of relevant neuropeptides such as pro-opiomelanocortin and galanin in the arcuate nucleus of the hypothalamus was altered but not those of the CB1 receptor. Indeed, no changes in CB1 expression were found in the liver, skeletal muscle and adipose tissue. However, cannabinoid CB1 and CB2 receptor expression in the endocrine pancreas and glucagon plasma levels were decreased. No changes in plasma adiponectin, leptin, insulin and somatostatin were found. Taken together, these results suggest a role for central cannabinoid CB2 receptors in body weight control and glucose homeostasis.     

Effect of food deprivation and leptin repletion on the plasma levels of estrogen (E2) and NADPH-d reactivity in the ventromedial and arcuate nuclei of the hypothalamus in the female rats

The exact role of leptin in fasting has not been completely elucidated. To determine whether leptin can act in fasting to influence plasma estrogen levels and nitric oxide synthase reactivity in food regulating centers of the brain, we fasted female rats for 4 days and treated them i.p. with vehicle or 100 microg of recombinant mouse leptin as 1 ml on the 3rd and 4th day twice daily (10.00 and 17.00 h). Proestrus blood was collected at 10.00, 14.00, 18.00 and at 22.00 h, plasma obtained and assayed for estrogen (E2) and leptin levels. Verification of ovulation occurrence was by examining the oviduct for extruded ovum. The rat brains were removed and processed for nitric oxide synthase reactivity in the ventromedial hypothalamus (VMH) and arcuate nucleus (ARC) using NADPH-diaphorase histochemistry, a marker for neurons expressing NOS enzyme. Leptin effect on dependable variables such as food intake, water intake and body weight gain was also investigated. Four days fasting significantly decreased body weight, estrogen and postfast leptin levels, nitric oxide reactivity in the VMH and ARC nucleus and stopped ovulation in many (4 out of 5) rats fasted and given vehicle. Leptin treatment significantly increased plasma estrogen and postfast leptin levels, restored ovulation in many (4 out of 5) rats and increased nitric oxide reactivity in the VMH and ARC. Leptin significantly inhibited food intake, water intake and gain in body weight during recommenced feeding. These observations suggest that leptin could act in the pituitary-ovarian axis during fasting to improve reproductive function by partly stimulating estrogen secretion.     

Bidirectional regulation of mu-opioid and CB1-cannabinoid receptor in rats self-administering heroin or WIN 55,212-2

This study examines the effect of intravenous self-administration (SA) of either heroin or the cannabinoid receptor agonist WIN 55,212-2 on levels and functionality of µ-opioid (MOR) and CB1-cannabinoid receptors (CB1R) in reward-related brain areas, such as the prefrontal cortex (PFC), nucleus accumbens (NAc), caudate putamen (CP), hippocampus (Hippo), amygdala (Amy), hypothalamus (Hypo) and ventral tegmental area (VTA). [³H]DAMGO and [³H]CP-55,940 autoradiography and agonist-stimulated [³⁵S]GTPγS binding were performed on brain sections of rats firmly self-administering heroin or WIN 55,212-2. Animals failing to acquire heroin or cannabinoid SA behaviour as well as drug-naïve animals never exposed to experimental apparatus or procedure (home-control group) were used as controls. With respect to control groups, which displayed very similar values, rats SA heroin showed increased MOR binding in the NAc (+174%), CP (+165%), Hippo (+121%), VTA (+175%), an enhanced CB1R density localized in the Amy (+147%) and VTA (+37%), and a widespread increased CB1 receptor functionality in the PFC (+95%), NAc (+313%), CP (+265%), Hippo (+38%), Amy (+221%). In turn, cannabinoid SA differently modulates CB1R binding in the Amy (+47%), Hypo (+94%), Hippo (−23%), VTA (−15%), and increases MOR levels (PFC: +124%; NAc: +68%; CP: +80%; Hippo: +73%; Amy: +99%) and efficiency (Hippo: +518%; Amy: +173%; Hypo: +188%). These findings suggest that voluntary chronic intake of opioids or cannabinoids induces reciprocal but differential regulation of MORs and CB1Rs density and activity in brain structures underlying drug-taking and drug-seeking behaviour, which could represent long-term neuroadaptations contributing to the development of drug addiction and dependence.