Christa M. Patterson

Hypothalamic Neural Projections Are Permanently Disrupted in Diet-Induced Obese Rats

The arcuate nucleus of the hypothalamus (ARH) is a key component of hypothalamic pathways regulating energy balance, and leptin is required for normal development of ARH projections. Diet-induced obesity (DIO) has a polygenic mode of inheritance, and DIO individuals develop the metabolic syndrome when a moderate amount of fat is added to the diet. Here we demonstrate that rats selectively bred to develop DIO, which are known to be leptin resistant before they become obese, have defective ARH projections that persist into adulthood. Furthermore, the ability of leptin to activate intracellular signaling in ARH neurons in vivo and to promote ARH neurite outgrowth in vitro is significantly reduced in DIO neonates. Thus, animals that are genetically predisposed toward obesity display an abnormal organization of hypothalamic pathways involved in energy homeostasis that may be the result of diminished responsiveness of ARH neurons to the trophic actions of leptin during postnatal development.

Ventral Tegmental Area Leptin Receptor Neurons Specifically Project to and Regulate Cocaine- and Amphetamine-Regulated Transcript Neurons of the Extended Central Amygdala

Leptin acts via its receptor (LepRb) to regulate neural circuits in concert with body energy stores. In addition to acting on a number of hypothalamic structures, leptin modulates the mesolimbic dopamine (DA) system. To determine the sites at which LepRb neurons might directly influence the mesolimbic DA system, we examined the distribution of LepRb neurons and their projections within mesolimbic brain regions. Although the ventral tegmental area (VTA) contains DA LepRb neurons, LepRb neurons are absent from the amygdala and striatum. Also, LepRb-EGFPf mice (which label projections from LepRb neurons throughout the brain) reveal that few LepRb neurons project to the nucleus accumbens (NAc). In contrast, the central amygdala (CeA) and its rostral extension receive copious projections from LepRb neurons. Indeed, LepRb-specific anterograde tracing demonstrates (and retrograde tracing confirms) that VTA LepRb neurons project to the extended CeA (extCeA) but not the NAc. Consistently, leptin promotes cAMP response element-binding protein phosphorylation in the extCeA, but not NAc, of leptin-deficient animals. Furthermore, transgenic mice expressing the trans-synaptic tracer wheat germ agglutinin in LepRb neurons reveal the innervation of CeA cocaine- and amphetamine-regulated transcript (CART) neurons by LepRb neurons, and leptin suppresses the increased CeA CART expression of leptin-deficient animals. Thus, LepRb VTA neurons represent a subclass of VTA DA neurons that specifically innervates and controls the extCeA; we hypothesize that these neurons primarily modulate CeA-directed behaviors.

Molecular mapping of mouse brain regions innervated by leptin receptor-expressing cells

Leptin acts via the long form of the leptin receptor (LepRb) on specialized sets of neurons in the brain to modulate diverse functions in concert with energy stores. Previous studies have revealed the distribution of LepRb-expressing neurons in the brain but not the regions to which LepRb neurons project to mediate downstream leptin actions. We utilized LepRb-cre in combination with cre-inducible enhanced green fluorescent protein (EGFP) and farnesylated EGFP (EGFPf) mouse reporter strains to visualize LepRb neurons and their projections, respectively, throughout the brain. The areas containing LepRb soma and projections were relatively circumscribed, as many brain regions contained no detectable EGFP or EGFPf. The highest concentrations of LepRb neurons and LepRb projections were found in the hypothalamus, where the ventral premamillary (PMv), dorsomedial (DMH), and arcuate (ARC) nuclei contained the greatest number of cell bodies, in addition to substantial EGFPf-reactivity. Furthermore, both LepRb soma and projections were present in a few midbrain and brainstem nuclei. Several brain regions including the hypothalamic paraventricular nucleus (PVH), the anteroventral periventricular nucleus (AVPe), and the central nucleus of the amygdala (CeA) contained few LepRb neurons but substantial EGFPf, suggesting that these regions represent targets of LepRb neurons that lie elsewhere in the brain. In some nuclei that contained both soma and projections, the distribution of soma and projections differed, suggesting that these areas transmit leptin-encoded information in a neuroanatomically directional manner.

Leptin action through hypothalamic nitric oxide synthase-1–expressing neurons controls energy balance

Few effective measures exist to combat the worldwide obesity epidemic, and the identification of potential therapeutic targets requires a deeper understanding of the mechanisms that control energy balance. Leptin, an adipocyte-derived hormone that signals the long-term status of bodily energy stores, acts through multiple types of leptin receptor long isoform (LepRb)-expressing neurons (called here LepRb neurons) in the brain to control feeding, energy expenditure and endocrine function. The modest contributions to energy balance that are attributable to leptin action in many LepRb populations suggest that other previously unidentified hypothalamic LepRb neurons have key roles in energy balance. Here we examine the role of LepRb in neuronal nitric oxide synthase (NOS1)-expressing LebRb (LepRbNOS1) neurons that comprise approximately 20% of the total hypothalamic LepRb neurons. Nos1cre-mediated genetic ablation of LepRb (LeprNos1KO) in mice produces hyperphagic obesity, decreased energy expenditure and hyperglycemia approaching that seen in whole-body LepRb-null mice. In contrast, the endocrine functions in LeprNos1KO mice are only modestly affected by the genetic ablation of LepRb in these neurons. Thus, hypothalamic LepRbNOS1 neurons are a key site of action of the leptin-mediated control of systemic energy balance.

Three weeks of postweaning exercise in DIO rats produces prolonged increases in central leptin sensitivity and signaling

In rats selectively bred to develop diet-induced obesity (DIO) 3 wk of postweaning exercise reduces weight and adipose regain for 10 wk after exercise cessation, despite intake of 31% fat high-energy (HE) diet. To test the hypothesis that this effect is due to increased central leptin sensitivity, 4-wk-old DIO rats were fed the HE diet and left sedentary (Sed), exercised for 3 wk, and then remained sedentary for 10 additional weeks (Ex/Sed) or continued exercise for a full 13 wk (Ex). After 3 wk, leptin (5 mg/kg ip) induced a 36% decrease in 24-h food intake in Ex rats, while Sed rats had no change in 24-h intake. Ex rats also had 23% more leptin-induced phospho-STAT3 (pSTAT3)-expressing neurons in the arcuate nucleus (ARC) and 95% and 68% higher (125)I-labeled leptin receptor binding in the ventromedial and dorsomedial nuclei than did Sed rats, respectively. At 7 wk after onset, leptin decreased 24-h intake by 20% in Ex and 24% in Ex/Sed rats without altering Sed intake. After a total of 13 wk, compared with Sed rats, Ex and Ex/Sed rats had 58% and 38% less fat, respectively, but leptin failed to decrease food intake in any group. Nevertheless, Ex, but not Ex/Sed rats, still had 32% more ARC leptin-induced pSTAT3-expressing neurons than Sed rats. These data suggest that brief postweaning exercise in DIO rats that are inherently leptin resistant causes a sustained resistance to obesity on HE diet, which is, in part, due to increased central leptin sensitivity.

Leptin Acts via Lateral Hypothalamic Area Neurotensin Neurons to Inhibit Orexin Neurons by Multiple GABA-Independent Mechanisms

The adipocyte-derived hormone leptin modulates neural systems appropriately for the status of body energy stores. Leptin inhibits lateral hypothalamic area (LHA) orexin (OX; also known as hypocretin)-producing neurons, which control feeding, activity, and energy expenditure, among other parameters. Our previous results suggest that GABAergic LHA leptin receptor (LepRb)-containing and neurotensin (Nts)-containing (LepRbNts) neurons lie in close apposition with OX neurons and control Ox mRNA expression. Here, we show that, similar to leptin, activation of LHA Nts neurons by the excitatory hM3Dq DREADD (designer receptor exclusively activated by designer drugs) hyperpolarizes membrane potential and suppresses action potential firing in OX neurons in mouse hypothalamic slices. Furthermore, ablation of LepRb from Nts neurons abrogated the leptin-mediated inhibition, demonstrating that LepRbNts neurons mediate the inhibition of OX neurons by leptin. Leptin did not significantly enhance GABAA-mediated inhibitory synaptic transmission, and GABA receptor antagonists did not block leptin-mediated inhibition of OX neuron activity. Rather, leptin diminished the frequency of spontaneous EPSCs onto OX neurons. Furthermore, leptin indirectly activated an ATP-sensitive potassium (KATP) channel in OX neurons, which was required for the hyperpolarization of OX neurons by leptin. Although Nts did not alter OX activity, galanin, which is coexpressed in LepRbNts neurons, inhibited OX neurons, whereas the galanin receptor antagonist M40 (galanin-(1–12)-Pro3-(Ala-Leu)2-Ala amide) prevented the leptin-induced hyperpolarization of OX cells. These findings demonstrate that leptin indirectly inhibits OX neurons by acting on LHA LepRbNts neurons to mediate two distinct GABA-independent mechanisms of inhibition: the presynaptic inhibition of excitatory neurotransmission and the opening of KATP channels.

Individual Differences in Cue-Induced Motivation and Striatal Systems in Rats Susceptible to Diet-Induced Obesity

Pavlovian cues associated with junk-foods (caloric, highly sweet, and/or fatty foods), like the smell of brownies, can elicit craving to eat and increase the amount of food consumed. People who are more susceptible to these motivational effects of food cues may have a higher risk for becoming obese. Further, overconsumption of junk-foods leading to the development of obesity may itself heighten attraction to food cues. Here, we used a model of individual susceptibility to junk-foods diet-induced obesity to determine whether there are pre-existing and/or diet-induced increases in attraction to and motivation for sucrose-paired cues (ie, incentive salience or ‘wanting’). We also assessed diet- vs obesity-associated alterations in mesolimbic function and receptor expression. We found that rats susceptible to diet-induced obesity displayed heightened conditioned approach prior to the development of obesity. In addition, after junk-food diet exposure, those rats that developed obesity also showed increased willingness to gain access to a sucrose cue. Heightened ‘wanting’ was not due to individual differences in the hedonic impact (‘liking’) of sucrose. Neurobiologically, Mu opioid receptor mRNA expression was lower in striatal ‘hot-spots’ that generate eating or hedonic impact only in those rats that became obese. In contrast, prolonged exposure to junk-food resulted in cross-sensitization to amphetamine-induced locomotion and downregulation of striatal D2R mRNA regardless of the development of obesity. Together these data shed light on individual differences in behavioral and neurobiological consequences of exposure to junk-food diets and the potential contribution of incentive sensitization in susceptible individuals to greater food cue-triggered motivation.

A Parabrachial-Hypothalamic Cholecystokinin Neurocircuit Controls Counterregulatory Responses to Hypoglycemia

Summary Hypoglycemia engenders an autonomically mediated counterregulatory (CR)-response that stimulates endogenous glucose production to maintain concentrations within an appropriate physiological range. Although the involvement of the brain in preserving normoglycemia has been established, the neurocircuitry underlying centrally mediated CR-responses remains unclear. Here we demonstrate that lateral parabrachial nucleus cholecystokinin (CCKLPBN) neurons are a population of glucose-sensing cells (glucose inhibited) with counterregulatory capacity. Furthermore, we reveal that steroidogenic-factor 1 (SF1)-expressing neurons of the ventromedial nucleus of the hypothalamus (SF1VMH) are the specific target of CCKLPBN glucoregulatory neurons. This discrete CCKLPBN→SF1VMH neurocircuit is both necessary and sufficient for the induction of CR-responses. Together, these data identify CCKLPBN neurons, and specifically CCK neuropeptide, as glucoregulatory and provide significant insight into the homeostatic mechanisms controlling CR-responses to hypoglycemia.

Leptin-inhibited PBN neurons enhance responses to hypoglycemia in negative energy balance

Hypoglycemia initiates the counter-regulatory response (CRR), in which the sympathetic nervous system, glucagon and glucocorticoids restore glucose to appropriate concentrations. During starvation, low leptin levels restrain energy utilization, enhancing long-term survival. To ensure short-term survival during hypoglycemia in fasted animals, the CRR must overcome this energy-sparing program and nutrient depletion. Here we identify in mice a previously unrecognized role for leptin and a population of leptin-regulated neurons that modulate the CRR to meet these challenges. Hypoglycemia activates neurons of the parabrachial nucleus (PBN) that coexpress leptin receptor (LepRb) and cholecystokinin (CCK) (PBN LepRbCCK neurons), which project to the ventromedial hypothalamic nucleus. Leptin inhibits these cells, and Cckcre-mediated ablation of LepRb enhances the CRR. Inhibition of PBN LepRb cells blunts the CRR, whereas their activation mimics the CRR in a CCK-dependent manner. PBN LepRbCCK neurons are a crucial component of the CRR system and may be a therapeutic target in hypoglycemia.

Large Litter Rearing Enhances Leptin Sensitivity and Protects Selectively Bred Diet-Induced Obese Rats from Becoming Obese

Because rearing rats in large litters (LLs) protects them from becoming obese, we postulated that LL rearing would protect rats selectively bred to develop diet-induced obesity (DIO) from becoming obese by overcoming their inborn central leptin resistance. Male and female DIO rats were raised in normal litters (NLs; 10 pups/dam) or LLs (16 pups/dam) and assessed for anatomical, biochemical, and functional aspects of leptin sensitivity at various ages when fed low-fat chow or a 31% fat high-energy (HE) diet. LL rearing reduced plasma leptin levels by postnatal day 2 (P2) and body weight gain by P8. At P16, LL DIO neonates had increased arcuate nucleus (ARC) binding of leptin to its extracellular receptors and at P28 an associated increase of their agouti-related peptide and alpha-MSH axonal projections to the paraventricular nucleus. Reduced body weight persisted and was associated with increased ARC leptin receptor binding and sensitivity to the anorectic effects of leptin, reduced adiposity, and enhanced insulin sensitivity in LL DIO rats fed chow until 10 wk of age. The enhanced ARC leptin receptor binding and reduced adiposity of LL DIO rats persisted after an additional 5 wk on the HE diet. Female LL DIO rats had similar reductions in weight gain on both chow and HE diet vs. normal litter DIO rats. We postulate that LL rearing enhances DIO leptin sensitivity by lowering plasma leptin levels and thereby increasing leptin receptor availability and that this both enhances the ARC-paraventricular nucleus pathway development and protects them from becoming obese.