Hindbrain REV-ERB nuclear receptors regulate sensitivity to diet-induced obesity and brown adipose tissue pathophysiology.

OBJECTIVE: The dorsal vagal complex (DVC) of the hindbrain is a major point of integration for central and peripheral signals that regulate a wide variety of metabolic functions to maintain energy balance. The REV-ERB nuclear receptors are important modulators of molecular metabolism, but their role in the DVC has yet to be established. METHODS: Male REV-ERBα/ß floxed mice received stereotaxic injections of a Cre expressing virus to the DVC to create the DVC REV-ERBα/ß double knockout (DVC RDKO). Control littermates received stereotaxic injections to the DVC of a green fluorescent protein expressing virus. Animals were maintained on a normal chow diet or a 60% high-fat diet to observe the metabolic phenotype arising from DVC RDKO under healthy and metabolically stressed conditions. RESULTS: DVC RDKO animals on high-fat diet exhibited increased weight gain compared to control animals maintained on the same diet. Increased weight gain in DVC RDKO animals was associated with decreased basal metabolic rate and dampened signature of brown adipose tissue activity. RDKO decreased gene expression of calcitonin receptor in the DVC and tyrosine hydroxylase in the brown adipose tissue. CONCLUSIONS: These results suggest a previously unappreciated role of REV-ERB nuclear receptors in the DVC for maintaining energy balance and metabolic rate potentially through indirect sympathetic outflow to the brown adipose tissue.

The locus coeruleus contributes to the anorectic, nausea, and autonomic physiological effects of glucagon-like peptide-1.

Increasing the therapeutic potential and reducing the side effects of U.S. Food and Drug Administration-approved glucagon-like peptide-1 receptor (GLP-1R) agonists used to treat obesity require complete characterization of the central mechanisms that mediate both the food intake-suppressive and illness-like effects of GLP-1R signaling. Our studies, in the rat, demonstrate that GLP-1Rs in the locus coeruleus (LC) are pharmacologically and physiologically relevant for food intake control. Furthermore, agonism of LC GLP-1Rs induces illness-like behaviors, and antagonism of LC GLP-1Rs can attenuate GLP-1R-mediated nausea. Electrophysiological and behavioral pharmacology data support a role for LC GLP-1Rs expressed on presynaptic glutamatergic terminals in the control of feeding and malaise. Collectively, our work establishes the LC as a site of action for GLP-1 signaling and extends our understanding of the GLP-1 signaling mechanism necessary for the development of improved obesity pharmacotherapies.

Partitioning of the denitrification pathway and other nitrite metabolisms within global oxygen deficient zones.

Oxygen deficient zones (ODZs) account for about 30% of total oceanic fixed nitrogen loss via processes including denitrification, a microbially mediated pathway proceeding stepwise from NO3- to N2. This process may be performed entirely by complete denitrifiers capable of all four enzymatic steps, but many organisms possess only partial denitrification pathways, either producing or consuming key intermediates such as the greenhouse gas N2O. Metagenomics and marker gene surveys have revealed a diversity of denitrification genes within ODZs, but whether these genes co-occur within complete or partial denitrifiers and the identities of denitrifying taxa remain open questions. We assemble genomes from metagenomes spanning the ETNP and Arabian Sea, and map these metagenome-assembled genomes (MAGs) to 56 metagenomes from all three major ODZs to reveal the predominance of partial denitrifiers, particularly single-step denitrifiers. We find niche differentiation among nitrogen-cycling organisms, with communities performing each nitrogen transformation distinct in taxonomic identity and motility traits. Our collection of 962 MAGs presents the largest collection of pelagic ODZ microorganisms and reveals a clearer picture of the nitrogen cycling community within this environment.

Blooms of the harmful algae Margalefidinium polykrikoides and Alexandrium monilatum alter the York River Estuary microbiome.

Harmful algal blooms (HABs) cause damage to fisheries, aquaculture, and human health around the globe. However, the impact of HABs on water column microbiomes and biogeochemistry is poorly understood. This study examined the impacts of consecutive blooms of the ichthyotoxic dinoflagellates Margalefidinium polykrikoides and Alexandrium monilatum on the water microbiome in the York River Estuary, Chesapeake Bay, USA. The samples dominated by single dinoflagellate species and by a mix of the two dinoflagellates had different microbiome compositions than the ones with low levels of both species. The M. polykrikoides bloom was co-dominated by Winogradskyella and had increased concentrations of dissolved organic carbon. The A. monilatum bloom had little impact on the prokaryotic portion of the whole community but was associated with a specific group of prokaryotes in the particle-attached (>3 µm) fraction including Candidatus Nitrosopumilus, Candidatus Actinomarina, SAR11 Clade Ia, Candidatus Bealeia, and Rhodobacteraceae HIMB11. Thus, blooms of these two algal species impacted the estuarine microbiome in different ways, likely leading to shifts in estuarine carbon and nutrient cycling, with M. polykrikoides potentially having a greater impact on carbon cycling in the estuarine ecosystem than A. monilatum.

The role of glia in the physiology and pharmacology of glucagon-like peptide-1: implications for obesity, diabetes, neurodegeneration and glaucoma.

The medical applications of glucagon-like peptide-1 receptor (GLP-1R) agonists is evergrowing in scope, highlighting the urgent need for a comprehensive understanding of the mechanisms through which GLP-1R activation impacts physiology and behaviour. A new area of research aims to elucidate the role GLP-1R signalling in glia, which play a role in regulating energy balance, glycemic control, neuroinflammation and oxidative stress. Once controversial, existing evidence now suggests that subsets of glia (e.g. microglia, tanycytes and astrocytes) and infiltrating macrophages express GLP-1Rs. In this review, we discuss the implications of these findings, with particular focus on the effectiveness of both clinically available and novel GLP-1R agonists for treating metabolic and neurodegenerative diseases, enhancing cognition and combating substance abuse. LINKED ARTICLES: This article is part of a themed issue on GLP1 receptor ligands (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.4/issuetoc.

Microbially mediated nitrogen removal and retention in the York River Estuary.

Denitrification, anaerobic ammonium oxidation and dissimilatory nitrate reduction to ammonium (DNRA) are important microbial processes determining the fate of nitrogen (N) in estuaries. This study examined these processes in sediments of the York River Estuary, a tributary of Chesapeake Bay, and investigated environmental and microbial drivers of the rates of denitrification and DNRA. Nitrate reduction followed a consistent pattern throughout the year and across the estuary with nitrogen removal, primarily through denitrification, decreasing from the head of the estuary to the mouth and nitrogen retention, through DNRA, following the opposite pattern. At the mouth of the estuary, nitrogen retention was consistently higher than nitrogen removal. Denitrification rates showed strong linear relationships with concentrations of organic matter, nitrate and chlorophyll a, and the abundance of the nirS gene. DNRA rates were best correlated with the relative abundance of three bacterial families, Anaerolineaceae,Ectothiorhodospiraceae and Prolixibacteraceae, which carry the nrfA gene. The controls responsible for retention or removal of N from an estuary are complex, involving both geochemical and microbial factors. The N retained within estuaries may support primary production and seasonal algae blooms and result in estuarine eutrophication.

GIP Receptor Agonism Attenuates GLP-1 Receptor Agonist-Induced Nausea and Emesis in Preclinical Models.

Glucagon-like peptide 1 receptor (GLP-1R) agonists decrease body weight and improve glycemic control in obesity and diabetes. Patient compliance and maximal efficacy of GLP-1 therapeutics are limited by adverse side effects, including nausea and emesis. In three different species (i.e., mice, rats, and musk shrews), we show that glucose-dependent insulinotropic polypeptide receptor (GIPR) signaling blocks emesis and attenuates illness behaviors elicited by GLP-1R activation, while maintaining reduced food intake, body weight loss, and improved glucose tolerance. The area postrema and nucleus tractus solitarius (AP/NTS) of the hindbrain are required for food intake and body weight suppression by GLP-1R ligands and processing of emetic stimuli. Using single-nuclei RNA sequencing, we identified the cellular phenotypes of AP/NTS cells expressing GIPR and GLP-1R on distinct populations of inhibitory and excitatory neurons, with the greatest expression of GIPR in γ-aminobutyric acid-ergic neurons. This work suggests that combinatorial pharmaceutical targeting of GLP-1R and GIPR will increase efficacy in treating obesity and diabetes by reducing nausea and vomiting.

The long-acting amylin/calcitonin receptor agonist ZP5461 suppresses food intake and body weight in male rats.

The peptide hormone amylin reduces food intake and body weight and is an attractive candidate target for novel pharmacotherapies to treat obesity. However, the short half-life of native amylin and amylin analogs like pramlintide limits these compounds' potential utility in promoting sustained negative energy balance. Here, we evaluate the ability of the novel long-acting amylin/calcitonin receptor agonist ZP5461 to reduce feeding and body weight in rats, and also test the role of calcitonin receptors (CTRs) in the dorsal vagal complex (DVC) of the hindbrain in the energy balance effects of chronic ZP5461 administration. Acute dose-response studies indicate that systemic ZP5461 (0.5-3 nmol/kg) robustly suppresses energy intake and body weight gain in chow- and high-fat diet (HFD)-fed rats. When HFD-fed rats received chronic systemic administration of ZP5461 (1-2 nmol/kg), the compound initially produced reductions in energy intake and weight gain but failed to produce sustained suppression of intake and body weight. Using virally mediated knockdown of DVC CTRs, the ability of chronic systemic ZP5461 to promote early reductions in intake and body weight gain was determined to be mediated in part by activation of DVC CTRs, implicating the DVC as a central site of action for ZP5461. Future studies should address other dosing regimens of ZP5461 to determine whether an alternative dose/frequency of administration would produce more sustained body weight suppression.

The Mesencephalic Trigeminal Nucleus Controls Food Intake and Body Weight via Hindbrain POMC Projections.

The mesencephalic trigeminal nucleus (Mes5) processes oral sensory-motor information, but its role in the control of energy balance remains unexplored. Here, using fluorescent in situ hybridization, we show that the Mes5 expresses the melanocortin-4 receptor. Consistent with MC4R activation in other areas of the brain, we found that Mes5 microinjection of the MC4R agonist melanotan-II (MTII) suppresses food intake and body weight in the mouse. Furthermore, NTS POMC-projecting neurons to the Mes5 can be chemogenetically activated to drive a suppression in food intake. Taken together, these findings highlight the Mes5 as a novel target of melanocortinergic control of food intake and body weight regulation, although elucidating the endogenous role of this circuit requires future study. While we observed the sufficiency of Mes5 MC4Rs for food intake and body weight suppression, these receptors do not appear to be necessary for food intake or body weight control. Collectively, the data presented here support the functional relevance of the NTS POMC to Mes5 projection pathway as a novel circuit that can be targeted to modulate food intake and body weight.

Corrination of a GLP-1 Receptor Agonist for Glycemic Control without Emesis.

Glucagon-like peptide-1 receptor (GLP-1R) agonists used to treat type 2 diabetes mellitus often produce nausea, vomiting, and in some patients, undesired anorexia. Notably, these behavioral effects are caused by direct central GLP-1R activation. Herein, we describe the creation of a GLP-1R agonist conjugate with modified brain penetrance that enhances GLP-1R-mediated glycemic control without inducing vomiting. Covalent attachment of the GLP-1R agonist exendin-4 (Ex4) to dicyanocobinamide (Cbi), a corrin ring containing precursor of vitamin B12, produces a "corrinated" Ex4 construct (Cbi-Ex4). Data collected in the musk shrew (Suncus murinus), an emetic mammal, reveal beneficial effects of Cbi-Ex4 relative to Ex4, as evidenced by improvements in glycemic responses in glucose tolerance tests and a profound reduction of emetic events. Our findings highlight the potential for clinical use of Cbi-Ex4 for millions of patients seeking improved glycemic control without common side effects (e.g., emesis) characteristic of current GLP-1 therapeutics.

GABA neurons in the nucleus tractus solitarius express GLP-1 receptors and mediate anorectic effects of liraglutide in rats.

The glucagon-like peptide-1 receptor (GLP-1R) agonist liraglutide is approved for the treatment of obesity; however, there is still much to be learned regarding the neuronal sites of action that underlie its suppressive effects on food intake and body weight. Peripherally administered liraglutide in rats acts in part through central GLP-1Rs in both the hypothalamus and the hindbrain. Here, we extend findings supporting a role for hindbrain GLP-1Rs in mediating the anorectic effects of liraglutide in male rats. To dissociate the contribution of GLP-1Rs in the area postrema (AP) and the nucleus tractus solitarius (NTS), we examined the effects of liraglutide in both NTS AAV-shRNA-driven Glp1r knockdown and AP-lesioned animals. Knockdown of NTS GLP-1Rs, but not surgical lesioning of the AP, attenuated the anorectic and body weight-reducing effects of acutely delivered liraglutide. In addition, NTS c-Fos responses were maintained in AP-lesioned animals. Moreover, NTS Glp1r knockdown was sufficient to attenuate the intake- and body weight-reducing effects of chronic daily administered liraglutide over 3 weeks. Development of improved obesity pharmacotherapies requires an understanding of the cellular phenotypes targeted by GLP-1R agonists. Fluorescence in situ hybridization identified Glp1r transcripts in NTS GABAergic neurons, which when inhibited using chemogenetics, attenuated the food intake- and body weight-reducing effects of liraglutide. This work demonstrates the contribution of NTS GLP-1Rs to the anorectic potential of liraglutide and highlights a phenotypically distinct (GABAergic) population of neurons within the NTS that express the GLP-1R and are involved in the mediation of liraglutide signaling.

Hindbrain melanocortin 3/4 receptors modulate the food intake and body weight suppressive effects of the GLP-1 receptor agonist, liraglutide.

Simultaneously targeting multiple energy balance control systems is a promising direction for the development of obesity pharmacotherapies. Here, we explore the interaction between the GLP-1 and melanocortin system within the dorsal vagal complex (DVC) of the caudal brainstem. Using a pharmacological approach, we demonstrate that the full anorectic potential of liraglutide, an FDA-approved GLP-1 analog for the treatment of obesity, requires DVC melanocortin 3/4 receptor (MC3/4R) signaling. Specifically, the food intake and body weight suppressive effects of liraglutide were attenuated by DVC administration of the MC3/4R antagonist SHU9119. In contrast, the anorectic effects of liraglutide were enhanced by combined activation of DVC MC3/4Rs using the agonist MTII. Our findings highlight the modulation of liraglutide-induced anorexia by DVC MC3/4R signaling, thereby suggesting a site of action at which two important energy balance control systems interact.

Quantifying and identifying microplastics in the effluent of advanced wastewater treatment systems using Raman microspectroscopy.

Microplastics in wastewater treatment plant (WWTP) effluent have been identified and quantified, but few studies have examined the microplastics in advanced treatment systems. A new method for isolating, quantifying, and determining the polymer type of microplastics was developed that included chemical digestion coupled with Raman microspectroscopy to investigate microplastics in the effluent of reverse osmosis nanofiltration and activated carbon filtration systems. This method allows for the removal of organics and the quantification and identification of all microplastics present in the sample. A large number of microplastics, the majority of which were smaller than 10 µm, were identified in the effluent of the advanced filtration systems with polyethylene the most common polymer identified. This study not only reports a new method for microplastic identification and quantification but also shows the importance of measuring the smallest fraction of microplastics, those smaller than 20 µm, which have previously been understudied.

Challenges to Body Fluid Homeostasis Differentially Recruit Phasic Dopamine Signaling in a Taste-Selective Manner.

The internal environment of an organism must remain stable to ensure optimal performance and ultimately survival. The generation of motivated behaviors is an adaptive mechanism for defending homeostasis. Although physiological state modulates motivated behaviors, the influence of physiological state on phasic dopamine signaling, an underlying neurobiological substrate of reward-driven behavior, is underexplored. Here, we use sodium depletion and water restriction, manipulations of body fluid homeostasis, to determine the flexibility and specificity of dopamine responses. Changes in dopamine concentration were measured using fast-scan cyclic voltammetry in the nucleus accumbens shell of male rats in response to intraoral infusions of fluids that either satisfied or did not satisfy homeostatic need. Increases in dopamine concentration during intraoral infusions were observed only under conditions of physiological deficit. Furthermore, dopamine increases were selective and limited to those that satisfied the need state of the animal. Thus, dopamine neurons track fluid balance and respond to salt and water stimuli in a state- and taste-dependent manner. Using Fluoro-Gold tracing and immunohistochemistry for c-Fos and Foxp2, a marker of sodium-deprivation responsive neurons, we revealed brainstem populations of neurons that are activated by sodium depletion and project directly to the ventral tegmental area. The identified projections may modulate dopamine neuron excitability and consequently the state-specific dopamine release observed in our experiments. This work illustrates the impact of physiological state on mesolimbic dopamine signaling and a potential circuit by which homeostatic disruptions are communicated to mesolimbic circuitry to drive the selective reinforcement of biologically-required stimuli under conditions of physiological need.SIGNIFICANCE STATEMENT Motivated behaviors arise during physiological need and are highly selective for homeostasis-restoring stimuli. Although phasic dopamine signaling has been shown to contribute to the generation of motivated behaviors, the state and stimulus specificity of phasic dopamine signaling is less clear. These studies use thirst and sodium appetite to show that dopamine neurons dynamically track body fluid homeostasis and respond to water and salt stimuli in a state- and taste-dependent manner. We also identify hindbrain sodium deprivation-responsive neurons that project directly to the ventral tegmental area, where dopamine neuron cell bodies reside. This work demonstrates command of homeostasis over dopamine signaling and proposes a circuit by which physiological need drives motivated behavior by state- and taste-selective recruitment of phasic dopamine signaling.

Central GLP-1 receptor activation modulates cocaine-evoked phasic dopamine signaling in the nucleus accumbens core.

Drugs of abuse increase the frequency and magnitude of brief (1-3s), high concentration (phasic) dopamine release events in terminal regions. These are thought to be a critical part of drug reinforcement and ultimately the development of addiction. Recently, metabolic regulatory peptides, including the satiety signal glucagon-like peptide-1 (GLP-1), have been shown to modulate cocaine reward-driven behavior and sustained dopamine levels after cocaine administration. Here, we use fast-scan cyclic voltammetry (FSCV) to explore GLP-1 receptor (GLP-1R) modulation of dynamic dopamine release in the nucleus accumbens (NAc) during cocaine administration. We analyzed dopamine release events in both the NAc shell and core, as these two subregions are differentially affected by cocaine and uniquely contribute to motivated behavior. We found that central delivery of the GLP-1R agonist Exendin-4 suppressed the induction of phasic dopamine release events by intravenous cocaine. This effect was selective for dopamine signaling in the NAc core. Suppression of phasic signaling in the core by Exendin-4 could not be attributed to interference with cocaine binding to one of its major substrates, the dopamine transporter, as cocaine-induced increases in reuptake were unaffected. The results suggest that GLP-1R activation, instead, exerts its suppressive effects by altering dopamine release - possibly by suppressing the excitability of dopamine neurons. Given the role of NAc core dopamine in the generation of conditioned responses based on associative learning, suppression of cocaine-induced dopamine signaling in this subregion by GLP-1R agonism may decrease the reinforcing properties of cocaine. Thus, GLP-1Rs remain viable targets for the treatment and prevention of cocaine seeking, taking and relapse.

Physiological state tunes mesolimbic signaling: Lessons from sodium appetite and inspiration from Randall R. Sakai.

Sodium deficit poses a life-threatening challenge to body fluid homeostasis and generates a sodium appetite - the behavioral drive to ingest sodium. Dr. Randall R. Sakai greatly contributed to our understanding of the hormonal responses to negative sodium balance and to the central processing of these signals. Reactivity to the taste of sodium solutions and the motivation to seek and consume sodium changes dramatically with body fluid balance. Here, we review studies that collectively suggest that sodium deficit recruits the mesolimbic system to play a role in the behavioral expression of sodium appetite. The recruitment of the mesolimbic system likely contributes to intense sodium seeking and reinforces sodium consumption observed in deficient animals. Some of the hormones that are released in response to sodium deficit act directly on both dopamine and nucleus accumbens elements. Moreover, the taste of sodium in sodium deficient rats evokes a pattern of dopamine and nucleus accumbens activity that is similar to responses to rewarding stimuli. A very different pattern of activity is observed in non-deficient rats. Given the well-characterized endocrine response to sodium deficit and its central action, sodium appetite becomes an ideal model for understanding the role of mesolimbic signaling in reward, reinforcement and the generation of motivated behavior.

Physiological state gates acquisition and expression of mesolimbic reward prediction signals.

Phasic dopamine signaling participates in associative learning by reinforcing associations between outcomes (unconditioned stimulus; US) and their predictors (conditioned stimulus; CS). However, prior work has always engendered these associations with innately rewarding stimuli. Thus, whether dopamine neurons can acquire prediction signals in the absence of appetitive experience and update them when the value of the outcome changes remains unknown. Here, we used sodium depletion to reversibly manipulate the appetitive value of a hypertonic sodium solution while measuring phasic dopamine signaling in rat nucleus accumbens. Dopamine responses to the NaCl US following sodium depletion updated independent of prior experience. In contrast, prediction signals were only acquired through extensive experience with a US that had positive affective value. Once learned, dopamine prediction signals were flexibly expressed in a state-dependent manner. Our results reveal striking differences with respect to how physiological state shapes dopamine signals evoked by outcomes and their predictors.

The Aversive Agent Lithium Chloride Suppresses Phasic Dopamine Release Through Central GLP-1 Receptors.

Unconditioned rewarding stimuli evoke phasic increases in dopamine concentration in the nucleus accumbens (NAc) while discrete aversive stimuli elicit pauses in dopamine neuron firing and reductions in NAc dopamine concentration. The unconditioned effects of more prolonged aversive states on dopamine release dynamics are not well understood and are investigated here using the malaise-inducing agent lithium chloride (LiCl). We used fast-scan cyclic voltammetry to measure phasic increases in NAc dopamine resulting from electrical stimulation of dopamine cell bodies in the ventral tegmental area (VTA). Systemic LiCl injection reduced electrically evoked dopamine release in the NAc of both anesthetized and awake rats. As some behavioral effects of LiCl appear to be mediated through glucagon-like peptide-1 receptor (GLP-1R) activation, we hypothesized that the suppression of phasic dopamine by LiCl is GLP-1R dependent. Indeed, peripheral pretreatment with the GLP-1R antagonist exendin-9 (Ex-9) potently attenuated the LiCl-induced suppression of dopamine. Pretreatment with Ex-9 did not, however, affect the suppression of phasic dopamine release by the kappa-opioid receptor agonist, salvinorin A, supporting a selective effect of GLP-1R stimulation in LiCl-induced dopamine suppression. By delivering Ex-9 to either the lateral or fourth ventricle, we highlight a population of central GLP-1 receptors rostral to the hindbrain that are involved in the LiCl-mediated suppression of NAc dopamine release.

Optical suppression of drug-evoked phasic dopamine release.

Brief fluctuations in dopamine concentration (dopamine transients) play a key role in behavior towards rewards, including drugs of abuse. Drug-evoked dopamine transients may result from actions at both dopamine cell bodies and dopamine terminals. Inhibitory opsins can be targeted to dopamine neurons permitting their firing activity to be suppressed. However, as dopamine transients can become uncoupled from firing, it is unknown whether optogenetic hyperpolarization at the level of the soma is able to suppress dopamine transients. Here, we used in vivo fast-scan cyclic voltammetry to record transients evoked by cocaine and raclopride in nucleus accumbens (NAc) of urethane-anesthetized rats. We targeted halorhodopsin (NpHR) specifically to dopamine cells by injecting Cre-inducible virus into ventral tegmental area (VTA) of transgenic rats that expressed Cre recombinase under control of the tyrosine hydroxylase promoter (TH-Cre(+) rats). Consistent with previous work, co-administration of cocaine and raclopride led to the generation of dopamine transients in NAc shell. Illumination of VTA with laser strongly suppressed the frequency of transients in NpHR-expressing rats, but not in control rats. Laser did not have any effect on amplitude of transients. Thus, optogenetics can effectively reduce the occurrence of drug-evoked transients and is therefore a suitable approach for studying the functional role of such transients in drug-associated behavior.

Leptin signaling in the medial nucleus tractus solitarius reduces food seeking and willingness to work for food.

The adipose-derived hormone leptin signals in the medial nucleus tractus solitarius (mNTS) to suppress food intake, in part, by amplifying within-meal gastrointestinal (GI) satiation signals. Here we show that mNTS leptin receptor (LepRb) signaling also reduces appetitive and motivational aspects of feeding, and that these effects can depend on energy status. Using the lowest dose that significantly suppressed 3-h cumulative food intake, unilateral leptin (0.3 µg) administration to the mNTS (3 h before testing) reduced operant lever pressing for sucrose under increasing work demands (progressive ratio reinforcement schedule) regardless of whether animals were energy deplete (food restricted) or replete (ad libitum fed). However, in a separate test of food-motivated responding in which there was no opportunity to consume food (conditioned place preference (CPP) for an environment previously associated with a palatable food reward), mNTS leptin administration suppressed food-seeking behavior only in chronically food-restricted rats. On the other hand, mNTS LepRb signaling did not reduce CPP expression for morphine reinforcement regardless of energy status, suggesting that mNTS leptin signaling differentially influences motivated responding for food vs opioid reward. Overall results show that mNTS LepRb signaling reduces food intake and appetitive food-motivated responding independent of energy status in situations involving orosensory and postingestive contact with food, whereas food-seeking behavior independent of food consumption is only reduced by mNTS LepRb activation in a state of energy deficit. These findings reveal a novel appetitive role for LepRb signaling in the mNTS, a brain region traditionally linked with processing of meal-related GI satiation signals.