Hypocretin/orexin neurons encode social discrimination and exhibit a sex-dependent necessity for social interaction.

The hypothalamus plays a crucial role in the modulation of social behavior by encoding internal states. The hypothalamic hypocretin/orexin neurons, initially identified as regulators of sleep and appetite, are important for emotional and motivated behaviors. However, their role in social behavior remains unclear. Using fiber photometry and behavioral analysis, we show here that hypocretin neurons differentially encode social discrimination based on the nature of social encounters. The optogenetic inhibition of hypocretin neuron activity or blocking of hcrt-1 receptors reduces the amount of time mice are engaged in social interaction in males but not in females. Reduced hcrt-1 receptor signaling during social interaction is associated with altered activity in the insular cortex and ventral tegmental area in males. Our data implicating hypocretin neurons as sexually dimorphic regulators within social networks have significant implications for the treatment of neuropsychiatric diseases with social dysfunction, particularly considering varying prevalence among sexes.

Developmental or adult-onset deletion of neurotensin receptor-1 from dopamine neurons differentially reduces body weight.

Central neurotensin signaling via neurotensin receptor-1 (NtsR1) modulates various aspects of physiology, including suppressing feeding and promoting locomotor activity that can support weight loss. However, it remains unclear when and where NtsR1 expression contributes to control of body weight vs. other effects. We previously showed that activating ventral tegmental area (VTA) dopamine (DA) neurons that express NtsR1 promotes weight loss. We therefore hypothesized that deleting NtsR1 from DA neurons would promote weight gain by increasing food intake and decreasing physical activity. In contrast, developmental deletion of NtsR1 from DA neurons (by crossing DATCre mice with NtsR1flox/flox mice) had no impact on the feeding or body weight of mice fed a chow diet, though it augmented locomotor activity. Developmental deletion of NtsR1 from DA neurons protected mice from diet-induced obesity, but not via altering feeding, physical activity, or energy expenditure. Given that NtsR1 may exert distinct roles within development vs. adulthood, we then examined the impact of adult-onset deletion of NtsR1 from VTA DA neurons. We injected adult NtsR1flox/flox mice in the VTA with adeno associated virus to Cre-dependently delete NtsR1 in the VTA (VTAR1Null mice) and compared them to mice with intact NtsR1 (Controls). Again, in contrast to our hypothesis, VTAR1Null mice gained less weight than Controls while on normal chow or high fat diets. Moreover, VTAR1Null mice exhibited blunted feeding after fasting, suggesting a role for NtsR1 in adult VTA DA neurons in coordinating energy need and intake. Altogether, these data suggest that intact expression of NtsR1 in DA neurons is necessary for appropriate regulation of body weight, but a lack of NtsR1 in the developing vs. adult DA system protects from weight gain via different mechanisms. These findings emphasize the need for temporal and site-specific resolution to fully understand the role of NtsR1 within the brain.

Depleting hypothalamic somatostatinergic neurons recapitulates diabetic phenotypes in mouse brain, bone marrow, adipose and retina.

AIMS/HYPOTHESIS: Hypothalamic inflammation and sympathetic nervous system hyperactivity are hallmark features of the metabolic syndrome and type 2 diabetes. Hypothalamic inflammation may aggravate metabolic and immunological pathologies due to extensive sympathetic activation of peripheral tissues. Loss of somatostatinergic (SST) neurons may contribute to enhanced hypothalamic inflammation. METHODS: The present data show that leptin receptor-deficient (db/db) mice exhibit reduced hypothalamic SST neurons, particularly in the periventricular nucleus. We model this finding, using adeno-associated virus delivery of diphtheria toxin subunit A (DTA) driven by an SST-cre system to deplete these neurons in Sstcre/gfp mice (SST-DTA). RESULTS: SST-DTA mice exhibit enhanced hypothalamic c-Fos expression and brain inflammation as demonstrated by microglial and astrocytic activation. Bone marrow from SST-DTA mice undergoes skewed haematopoiesis, generating excess granulocyte-monocyte progenitors and increased proinflammatory (C-C chemokine receptor type 2; CCR2hi) monocytes. SST-DTA mice exhibited a 'diabetic retinopathy-like' phenotype: reduced visual function by optokinetic response (0.4 vs 0.25 cycles/degree; SST-DTA vs control mice); delayed electroretinogram oscillatory potentials; and increased percentages of retinal monocytes. Finally, mesenteric visceral adipose tissue from SST-DTA mice was resistant to catecholamine-induced lipolysis, displaying 50% reduction in isoprenaline (isoproterenol)-induced lipolysis compared with control littermates. Importantly, hyperglycaemia was not observed in SST-DTA mice. CONCLUSIONS/INTERPRETATION: The isolated reduction in hypothalamic SST neurons was able to recapitulate several hallmark features of type 2 diabetes in disease-relevant tissues.

Activation of ventral tegmental area neurotensin Receptor-1 neurons promotes weight loss.

Dopamine (DA) neurons in the ventral tegmental area (VTA) modulate physical activity and feeding behaviors that are disrupted in obesity. Yet, the heterogeneity of VTA DA neurons has hindered determination of which ones might be leveraged to support weight loss. We hypothesized that increased activity in the subset of VTA DA neurons expressing neurotensin receptor-1 (NtsR1) might promote weight loss behaviors. To test this, we used Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to activate VTA NtsR1 neurons in normal weight and diet-induced obese mice. Acute activation of VTA NtsR1 neurons (24hr) significantly decreased body weight in normal weight and obese mice by reducing food intake and increasing physical activity. Moreover, daily activation of VTA NtsR1 neurons in obese mice sustained weight loss over 7 days. Activating VTA NtsR1 neurons also suppressed how much mice worked to obtain sucrose rewards, even when there was high motivation to consume. However, VTA NtsR1 neural activation was not reinforcing, nor did it invoke liabilities associated with whole-body NtsR1 agonism such as anxiety, vasodepressor response or hypothermia. Activating VTA NtsR1 neurons therefore promotes dual behaviors that support weight loss without causing adverse effects, and is worth further exploration for managing obesity.

The Role of Central Neurotensin in Regulating Feeding and Body Weight.

The small peptide neurotensin (Nts) is implicated in myriad processes including analgesia, thermoregulation, reward, arousal, blood pressure, and modulation of feeding and body weight. Alterations in Nts have recently been described in individuals with obesity or eating disorders, suggesting that disrupted Nts signaling may contribute to body weight disturbance. Curiously, Nts mediates seemingly opposing regulation of body weight via different tissues. Peripherally acting Nts promotes fat absorption and weight gain, whereas central Nts signaling suppresses feeding and weight gain. Thus, because Nts is pleiotropic, a location-based approach must be used to understand its contributions to disordered body weight and whether the Nts system might be leveraged to improve metabolic health. Here we review the role of Nts signaling in the brain to understand the sites, receptors, and mechanisms by which Nts can promote behaviors that modify body weight. New techniques permitting site-specific modulation of Nts and Nts receptor-expressing cells suggest that, even in the brain, not all Nts circuitry exerts the same function. Intriguingly, there may be dedicated brain regions and circuits via which Nts specifically suppresses feeding behavior and weight gain vs other Nts-attributed physiology. Defining the central mechanisms by which Nts signaling modifies body weight may suggest strategies to correct disrupted energy balance, as needed to address overweight, obesity, and eating disorders.

Lateral hypothalamic area neuropeptides modulate ventral tegmental area dopamine neurons and feeding.

Understanding how the brain coordinates energy status with the motivation to eat is crucial to identify strategies to improve disordered body weight. The ventral tegmental area (VTA), known as the core of the mesolimbic system, is of particular interest in this regard because it controls the motivation to consume palatable, calorie-dense foods and to engage in volitional activity. The VTA is largely composed of dopamine (DA) neurons, but modulating these DA neurons has been alternately linked with promoting and suppressing feeding, suggesting heterogeneity in their function. Subsets of VTA DA neurons have recently been described based on their anatomical distribution, electrophysiological features, connectivity and molecular expression, but to date there are no signatures to categorize how DA neurons control feeding. Assessing the neuropeptide receptors expressed by VTA DA neurons may be useful in this regard, as many neuropeptides mediate anorexic or orexigenic responses. In particular, the lateral hypothalamic area (LHA) releases a wide variety of feeding-modulating neuropeptides to the VTA. Since VTA neurons intercept LHA neuropeptides known to either evoke or suppress feeding, expression of the cognate neuropeptide receptors within the VTA may point to VTA DA neuronal mechanisms to promote or suppress feeding, respectively. Here we review the role of the VTA in energy balance and the LHA neuropeptide signaling systems that act in the VTA, whose receptors might be used to classify how VTA DA neurons contribute to energy balance.

Mapping the populations of neurotensin neurons in the male mouse brain.

Neurotensin (Nts) is a neuropeptide implicated in the regulation of many facets of physiology, including cardiovascular tone, pain processing, ingestive behaviors, locomotor drive, sleep, addiction and social behaviors. Yet, there is incomplete understanding about how the various populations of Nts neurons distributed throughout the brain mediate such physiology. This knowledge gap largely stemmed from the inability to simultaneously identify Nts cell bodies and manipulate them in vivo. One means of overcoming this obstacle is to study NtsCre mice crossed onto a Cre-inducible green fluorescent reporter line (NtsCre;GFP mice), as these mice permit both visualization and in vivo modulation of specific populations of Nts neurons (using Cre-inducible viral and genetic tools) to reveal their function. Here we provide a comprehensive characterization of the distribution and relative densities of the Nts-GFP populations observed throughout the male NtsCre;GFP mouse brain, which will pave the way for future work to define their physiologic roles. We also compared the distribution of Nts-GFP neurons with Nts-In situ Hybridization (Nts-ISH) data from the adult mouse brain. By comparing these data sets we can distinguish Nts-GFP populations that may only transiently express Nts during development but not in the mature brain, and hence which populations may not be amenable to Cre-mediated manipulation in adult NtsCre;GFP mice. This atlas of Nts-GFP neurons will facilitate future studies using the NtsCre;GFP line to describe the physiological functions of individual Nts populations and how modulating them may be useful to treat disease.

Long-Acting Neurotensin Synergizes With Liraglutide to Reverse Obesity Through a Melanocortin-Dependent Pathway.

Neurotensin (NT), a gut hormone and neuropeptide, increases in circulation after bariatric surgery in rodents and humans and inhibits food intake in mice. However, its potential to treat obesity and the subsequent metabolic dysfunctions have been difficult to assess owing to its short half-life in vivo. Here, we demonstrate that a long-acting, pegylated analog of the NT peptide (P-NT) reduces food intake, body weight, and adiposity in diet-induced obese mice when administered once daily for 6 days. Strikingly, when P-NT was combined with the glucagon-like peptide 1 mimetic liraglutide, the two peptides synergized to reduce food intake and body weight relative to each monotherapy, without inducing a taste aversion. Further, P-NT and liraglutide coadministration improved glycemia and reduced steatohepatitis. Finally, we show that the melanocortin pathway is central for P-NT-induced anorexia and necessary for the full synergistic effect of P-NT and liraglutide combination therapy. Overall, our data suggest that P-NT and liraglutide combination therapy could be an enhanced treatment for obesity with improved tolerability compared with liraglutide monotherapy.

Hippocampal Subgranular Zone FosB Expression Is Critical for Neurogenesis and Learning.

Neural proliferation in the dentate gyrus (DG) is closely linked with learning and memory, but the transcriptional programming that drives adult proliferation remains incompletely understood. Our lab previously elucidated the critical role of the transcription factor ΔFosB in the dorsal hippocampus (dHPC) in learning and memory, and the FosB gene has been suggested to play a role in neuronal proliferation. However, the subregion-specific and potentially cell-autonomous role of dHPC ΔFosB in neurogenesis-dependent learning has not been studied. Here, we crossed neurotensin receptor-2 (NtsR2) Cre mice, which express Cre within the subgranular zone (SGZ) of dHPC DG, with floxed FosB mice to show that knockout of ΔFosB in hippocampal SGZ neurons reduces antidepressant-induced neurogenesis and impedes hippocampus-dependent learning in the novel object recognition task. Taken together, these data indicate that FosB gene expression in SGZ is necessary for both hippocampal neurogenesis and memory formation.

Distinct Subsets of Lateral Hypothalamic Neurotensin Neurons are Activated by Leptin or Dehydration.

The lateral hypothalamic area (LHA) is essential for ingestive behavior but it remains unclear how LHA neurons coordinate feeding vs. drinking. Most LHA populations promote food and water consumption but LHA neurotensin (Nts) neurons preferentially induce water intake while suppressing feeding. We identified two molecularly and projection-specified subpopulations of LHA Nts neurons that are positioned to coordinate either feeding or drinking. One subpopulation co-expresses the long form of the leptin receptor (LepRb) and is activated by the anorectic hormone leptin (NtsLepRb neurons). A separate subpopulation lacks LepRb and is activated by dehydration (NtsDehy neurons). These molecularly distinct LHA Nts subpopulations also differ in connectivity: NtsLepRb neurons project to the ventral tegmental area and substantia nigra compacta but NtsDehy neurons do not. Intriguingly, the LHA Nts subpopulations cannot be discriminated via their classical neurotransmitter content, as we found that all LHA Nts neurons are GABAergic. Collectively, our data identify two molecularly- and projection-specified subpopulations of LHA Nts neurons that intercept either leptin or dehydration cues, and which conceivably could regulate feeding vs. drinking behavior. Selective regulation of these LHA Nts subpopulations might be useful to specialize treatment for ingestive disorders such as polydipsia or obesity.

Activation of lateral hypothalamic area neurotensin-expressing neurons promotes drinking.

Animals must ingest water via drinking to maintain fluid homeostasis, yet the neurons that specifically promote drinking behavior are incompletely characterized. The lateral hypothalamic area (LHA) as a whole is essential for drinking behavior but most LHA neurons indiscriminately promote drinking and feeding. By contrast, activating neurotensin (Nts)-expressing LHA neurons (termed LHA Nts neurons) causes mice to immediately drink water with a delayed suppression of feeding. We therefore hypothesized that LHA Nts neurons are sufficient to induce drinking behavior and that these neurons specifically bias for fluid intake over food intake. To test this hypothesis we used designer receptors exclusively activated by designer drugs (DREADDs) to selectively activate LHA Nts neurons and studied the impact on fluid intake, fluid preference and feeding. Activation of LHA Nts neurons stimulated drinking in water-replete and dehydrated mice, indicating that these neurons are sufficient to promote water intake regardless of homeostatic need. Interestingly, mice with activated LHA Nts neurons drank any fluid that was provided regardless of its palatability, but if given a choice they preferred water or palatable solutions over unpalatable (quinine) or dehydrating (hypertonic saline) solutions. Notably, acute activation of LHA Nts neurons robustly promoted fluid but not food intake. Overall, our study confirms that activation of LHA Nts neurons is sufficient to induce drinking behavior and biases for fluid intake. Hence, LHA Nts neurons may be important targets for orchestrating the appropriate ingestive behavior necessary to maintain fluid homeostasis. This article is part of the Special Issue entitled 'Hypothalamic Control of Homeostasis'.

Determination of neurotensin projections to the ventral tegmental area in mice.

Pharmacologic treatment with the neuropeptide neurotensin (Nts) modifies motivated behaviors such as feeding, locomotor activity, and reproduction. Dopamine (DA) neurons of the ventral tegmental area (VTA) control these behaviors, and Nts directly modulates the activity of DA neurons via Nts receptor-1. While Nts sources to the VTA have been described in starlings and rats, the endogenous sources of Nts to the VTA of mice remain incompletely understood, impeding determination of which Nts circuits orchestrate specific behaviors in this model. To overcome this obstacle we injected the retrograde tracer Fluoro-Gold into the VTA of mice that express GFP in Nts neurons. Identification of GFP-Nts cells that accumulate Fluoro-Gold revealed the Nts afferents to the VTA in mice. Similar to rats, most Nts afferents to the VTA of mice arise from the medial and lateral preoptic areas (POA) and the lateral hypothalamic area (LHA), brain regions that are critical for coordination of feeding and reproduction. Additionally, the VTA receives dense input from Nts neurons in the nucleus accumbens shell (NAsh) of mice, and minor Nts projections from the amygdala and periaqueductal gray area. Collectively, our data reveal multiple populations of Nts neurons that provide direct afferents to the VTA and which may regulate specific aspects of motivated behavior. This work lays the foundation for understanding endogenous Nts actions in the VTA, and how circuit-specific Nts modulation may be useful to correct motivational and affective deficits in neuropsychiatric disease.

Identification of Neurotensin Receptor Expressing Cells in the Ventral Tegmental Area across the Lifespan.

Neurotensin (Nts) promotes activation of dopamine (DA) neurons in the ventral tegmental area (VTA) via incompletely understood mechanisms. Nts can signal via the G protein-coupled Nts receptors 1 and 2 (NtsR1 and NtsR2), but the lack of methods to detect NtsR1- and NtsR2-expressing cells has limited mechanistic understanding of Nts action. To overcome this challenge, we generated dual recombinase mice that express FlpO-dependent Cre recombinase in NtsR1 or NtsR2 cells. This strategy permitted temporal control over recombination, such that we could identify NtsR1- or NtsR2-expressing cells and determine whether their distributions differed between the developing and adult brain. Using this system, we found that NtsR1 is transiently expressed in nearly all DA neurons and in many non-DA neurons in the VTA during development. However, NtsR1 expression is more restricted within the adult brain, where only two thirds of VTA DA neurons expressed NtsR1. By contrast, NtsR2 expression remains constant throughout lifespan, but it is predominantly expressed within glia. Anterograde tract tracing revealed that NtsR1 is expressed by mesolimbic, not mesocortical DA neurons, suggesting that VTA NtsR1 neurons may represent a functionally unique subset of VTA DA neurons. Collectively, this work reveals a cellular mechanism by which Nts can directly engage NtsR1-expressing DA neurons to modify DA signaling. Going forward, the dual recombinase strategy developed here will be useful to selectively modulate NtsR1- and NtsR2-expressing cells and to parse their contributions to Nts-mediated behaviors.

Role of central neurotensin in regulating feeding: Implications for the development and treatment of body weight disorders.

The peptide neurotensin (Nts) was discovered within the brain over 40years ago and is implicated in regulating analgesia, body temperature, blood pressure, locomotor activity and feeding. Recent evidence suggests, however, that these disparate processes may be controlled via specific populations of Nts neurons and receptors. The neuronal mediators of Nts anorectic action are now beginning to be understood, and, as such, modulating specific Nts pathways might be useful in treating feeding and body weight disorders. This review considers mechanisms through which Nts normally regulates feeding and how disruptions in Nts signaling might contribute to the disordered feeding and body weight of schizophrenia, Parkinson's disease, anorexia nervosa, and obesity. Defining how Nts specifically mediates feeding vs. other aspects of physiology will inform the design of therapeutics that modify body weight without disrupting other important Nts-mediated physiology.

Lateral Hypothalamic Neurotensin Neurons Orchestrate Dual Weight Loss Behaviors via Distinct Mechanisms.

The central mechanism by which neurotensin (Nts) potentiates weight loss has remained elusive. We leveraged chemogenetics to reveal that Nts-expressing neurons of the lateral hypothalamic area (LHA) promote weight loss in mice by increasing volitional activity and restraining food intake. Intriguingly, these dual weight loss behaviors are mediated by distinct signaling pathways: Nts action via NtsR1 is essential for the anorectic effect of the LHA Nts circuit, but not for regulation of locomotor or drinking behavior. Furthermore, although LHA Nts neurons cannot reduce intake of freely available obesogenic foods, they effectively restrain motivated feeding in hungry, weight-restricted animals. LHA Nts neurons are thus vital mediators of central Nts action, particularly in the face of negative energy balance. Enhanced action via LHA Nts neurons may, therefore, be useful to suppress the increased appetitive drive that occurs after lifestyle-mediated weight loss and, hence, to prevent weight regain.

Neurotensin Receptor-1 Identifies a Subset of Ventral Tegmental Dopamine Neurons that Coordinates Energy Balance.

Dopamine (DA) neurons in the ventral tegmental area (VTA) are heterogeneous and differentially regulate ingestive and locomotor behaviors that affect energy balance. Identification of which VTA DA neurons mediate behaviors that limit weight gain has been hindered, however, by the lack of molecular markers to distinguish VTA DA populations. Here, we identified a specific subset of VTA DA neurons that express neurotensin receptor-1 (NtsR1) and preferentially comprise mesolimbic, but not mesocortical, DA neurons. Genetically targeted ablation of VTA NtsR1 neurons uncouples motivated feeding and physical activity, biasing behavior toward energy expenditure and protecting mice from age-related and diet-induced weight gain. VTA NtsR1 neurons thus represent a molecularly defined subset of DA neurons that are essential for the coordination of energy balance. Modulation of VTA NtsR1 neurons may therefore be useful to promote behaviors that prevent the development of obesity.

Loss of Action via Neurotensin-Leptin Receptor Neurons Disrupts Leptin and Ghrelin-Mediated Control of Energy Balance.

The hormones ghrelin and leptin act via the lateral hypothalamic area (LHA) to modify energy balance, but the underlying neural mechanisms remain unclear. We investigated how leptin and ghrelin engage LHA neurons to modify energy balance behaviors and whether there is any crosstalk between leptin and ghrelin-responsive circuits. We demonstrate that ghrelin activates LHA neurons expressing hypocretin/orexin (OX) to increase food intake. Leptin mediates anorectic actions via separate neurons expressing the long form of the leptin receptor (LepRb), many of which coexpress the neuropeptide neurotensin (Nts); we refer to these as NtsLepRb neurons. Because NtsLepRb neurons inhibit OX neurons, we hypothesized that disruption of the NtsLepRb neuronal circuit would impair both NtsLepRb and OX neurons from responding to their respective hormonal cues, thus compromising adaptive energy balance. Indeed, mice with developmental deletion of LepRb specifically from NtsLepRb neurons exhibit blunted adaptive responses to leptin and ghrelin that discoordinate the mesolimbic dopamine system and ingestive and locomotor behaviors, leading to weight gain. Collectively, these data reveal a crucial role for LepRb in the proper formation of LHA circuits, and that NtsLepRb neurons are important neuronal hubs within the LHA for hormone-mediated control of ingestive and locomotor behaviors.

To ingest or rest? Specialized roles of lateral hypothalamic area neurons in coordinating energy balance.

Survival depends on an organism's ability to sense nutrient status and accordingly regulate intake and energy expenditure behaviors. Uncoupling of energy sensing and behavior, however, underlies energy balance disorders such as anorexia or obesity. The hypothalamus regulates energy balance, and in particular the lateral hypothalamic area (LHA) is poised to coordinate peripheral cues of energy status and behaviors that impact weight, such as drinking, locomotor behavior, arousal/sleep and autonomic output. There are several populations of LHA neurons that are defined by their neuropeptide content and contribute to energy balance. LHA neurons that express the neuropeptides melanin-concentrating hormone (MCH) or orexins/hypocretins (OX) are best characterized and these neurons play important roles in regulating ingestion, arousal, locomotor behavior and autonomic function via distinct neuronal circuits. Recently, another population of LHA neurons containing the neuropeptide Neurotensin (Nts) has been implicated in coordinating anorectic stimuli and behavior to regulate hydration and energy balance. Understanding the specific roles of MCH, OX and Nts neurons in harmonizing energy sensing and behavior thus has the potential to inform pharmacological strategies to modify behaviors and treat energy balance disorders.

Ventral tegmental area neurotensin signaling links the lateral hypothalamus to locomotor activity and striatal dopamine efflux in male mice.

Projections from the lateral hypothalamic area (LHA) innervate components of the mesolimbic dopamine (MLDA) system, including the ventral tegmental area (VTA) and nucleus accumbens (NAc), to modulate motivation appropriately for physiologic state. Neurotensin (NT)-containing LHA neurons respond to multiple homeostatic challenges and project to the VTA, suggesting that these neurons could link such signals to MLDA function. Indeed, we found that pharmacogenetic activation of LHA NT neurons promoted prolonged DA-dependent locomotor activity and NAc DA efflux, suggesting the importance of VTA neurotransmitter release by LHA NT neurons for the control of MLDA function. Using a microdialysis-mass spectrometry technique that we developed to detect endogenous NT in extracellular fluid in the mouse brain, we found that activation of LHA NT cells acutely increased the extracellular concentration of NT (a known activator of VTA DA cells) in the VTA. In contrast to the prolonged elevation of extracellular NAc DA, however, VTA NT concentrations rapidly returned to baseline. Intra-VTA infusion of NT receptor antagonist abrogated the ability of LHA NT cells to increase extracellular DA in the NAc, demonstrating that VTA NT promotes NAc DA release. Thus, transient LHA-derived NT release in the VTA couples LHA signaling to prolonged changes in DA efflux and MLDA function.