A parabrachial hub for the prioritization of survival behavior.

Long-term sustained pain in the absence of acute physical injury is a prominent feature of chronic pain conditions. While neurons responding to noxious stimuli have been identified, understanding the signals that persist without ongoing painful stimuli remains a challenge. Using an ethological approach based on the prioritization of adaptive survival behaviors, we determined that neuropeptide Y (NPY) signaling from multiple sources converges on parabrachial neurons expressing the NPY Y1 receptor to reduce sustained pain responses. Neural activity recordings and computational modeling demonstrate that activity in Y1R parabrachial neurons is elevated following injury, predicts functional coping behavior, and is inhibited by competing survival needs. Taken together, our findings suggest that parabrachial Y1 receptor-expressing neurons are a critical hub for endogenous analgesic pathways that suppress sustained pain states.

Reverse engineering placebo analgesia.

Placebo analgesia is a widely observed clinical phenomenon. Establishing a robust mouse model of placebo analgesia is needed for careful dissection of the underpinning circuit mechanisms. However, previous studies failed to observe consistent placebo effects in rodent models of chronic pain. We wondered whether strong placebo analgesia can be reverse engineered using general anesthesia-activated neurons in the central amygdala (CeA GA ) that can potently suppress pain. Indeed, in both acute and chronic pain models, pairing a context with CeA GA -mediated pain relief produced robust context-dependent analgesia, exceeding that induced by morphine in the same paradigm. We reasoned that if the analgesic effect was dependent on reactivation of CeA GA neurons by conditioned contextual cues, the analgesia would still be an active treatment, rather than a placebo effect. CeA GA neurons indeed receive monosynaptic inputs from temporal lobe areas that could potentially relay contextual cues directly to CeA GA . However, in vivo imaging showed that CeA GA neurons were not re-activated in the conditioned context, despite mice displaying a strong analgesic phenotype, supporting the notion that the cue-induced pain relief is true placebo analgesia. Our results show that conditioning with activation of a central pain-suppressing circuit is sufficient to engineer placebo analgesia, and that purposefully linking a context with an active treatment could be a means to harness the power of placebo for pain relief.

Anti-inflammatory effects of hunger are transmitted to the periphery via projection-specific AgRP circuits.

Caloric restriction has anti-inflammatory effects. However, the coordinated physiological actions that lead to reduced inflammation in a state of caloric deficit (hunger) are largely unknown. Using a mouse model of injury-induced peripheral inflammation, we find that food deprivation reduces edema, temperature, and cytokine responses that occur after injury. The magnitude of the anti-inflammatory effect that occurs during hunger is more robust than that of non-steroidal anti-inflammatory drugs. The effects of hunger are recapitulated centrally by activity in nutrient-sensing hypothalamic agouti-related protein (AgRP)-expressing neurons. We find that AgRP neurons projecting to the paraventricular nucleus of the hypothalamus rapidly and robustly reduce inflammation and mediate the majority of hunger's anti-inflammatory effects. Intact vagal efferent signaling is required for the anti-inflammatory action of hunger, revealing a brain-to-periphery pathway for this reduction in inflammation. Taken together, these data begin to unravel a potent anti-inflammatory pathway engaged by hypothalamic AgRP neurons to reduce inflammation.

General Anesthesia Activates a Central Anxiolytic Center in the BNST.

Low doses of general anesthetics like ketamine and dexmedetomidine have anxiolytic properties independent of their sedative effects. How these different drugs exert these anxiolytic effects is not well understood. We discovered a population of GABAergic neurons in the oval division of the bed nucleus of the stria terminalis that is activated by multiple anesthetics and the anxiolytic drug diazepam (ovBNST GA ). A majority of ovBNST GA neurons express neurotensin receptor 1 (Ntsr1) and innervate brain regions known to regulate anxiety and stress responses. Optogenetic activation ovBNST GA or ovBNST Ntsr1 neurons significantly attenuated anxiety-like behaviors in both naïve animals and mice with inflammatory pain, while inhibition of these cells increased anxiety. Notably, activation of these neurons decreased heart rate and increased heart rate variability, suggesting that they reduce anxiety through modulation of the autonomic nervous system. Our study identifies ovBNST GA /ovBNST Ntsr1 neurons as one of the brain's endogenous anxiolytic centers and a potential therapeutic target for treating anxiety-related disorders. HIGHLIGHTS: General anesthetics and anxiolytics activate a population of neurons in the ovBNSTAnesthesia-activated ovBNST neurons bidirectionally modulate anxiety-like behaviorMost anesthesia-activated ovBNST neurons express neurotensin receptor 1 ovBNST Ntsr1 neuron activation shifts autonomic responses to an anxiolytic state.

A microbiome-dependent gut-brain pathway regulates motivation for exercise.

Exercise exerts a wide range of beneficial effects for healthy physiology1. However, the mechanisms regulating an individual's motivation to engage in physical activity remain incompletely understood. An important factor stimulating the engagement in both competitive and recreational exercise is the motivating pleasure derived from prolonged physical activity, which is triggered by exercise-induced neurochemical changes in the brain. Here, we report on the discovery of a gut-brain connection in mice that enhances exercise performance by augmenting dopamine signalling during physical activity. We find that microbiome-dependent production of endocannabinoid metabolites in the gut stimulates the activity of TRPV1-expressing sensory neurons and thereby elevates dopamine levels in the ventral striatum during exercise. Stimulation of this pathway improves running performance, whereas microbiome depletion, peripheral endocannabinoid receptor inhibition, ablation of spinal afferent neurons or dopamine blockade abrogate exercise capacity. These findings indicate that the rewarding properties of exercise are influenced by gut-derived interoceptive circuits and provide a microbiome-dependent explanation for interindividual variability in exercise performance. Our study also suggests that interoceptomimetic molecules that stimulate the transmission of gut-derived signals to the brain may enhance the motivation for exercise.

Specificity of Varenicline in Blocking Mesolimbic Circuit Activation to Natural and Drug Rewards.

The mesolimbic dopamine (DA) system reinforces behaviors that are critical for survival. However, drug dependence can occur when drugs of abuse, such as nicotine, highjack this reinforcement system. Pharmacologically targeting the DA system to selectively block drug reinforcement requires a detailed understanding of the neural circuits and molecular pathways that lead to the reward-based activation of mesolimbic circuits. Varenicline is an approved smoking cessation drug that has been shown to block nicotine-evoked DA increases in the nucleus accumbens (NAc) through action on nicotinic acetylcholine receptors. Because these receptors have been implicated in the reinforcement of other addictive substances, we explored the possibility that varenicline could broadly affect reward processing. We used in vivo fiber photometry to monitor midbrain DA neuron activity and striatal DA levels following either natural or drug rewards in mice treated with varenicline. We demonstrate that varenicline pretreatment enhances the suppression of nicotine-evoked DA release by attenuating DA neuron activity in the VTA. Varenicline's ability to attenuate DA release is highly specific to nicotine, and varenicline slightly elevates DA release when co-administered with morphine or ethanol. Furthermore, varenicline has no effect on DA release in response to naturally rewarding behavior such as food intake or exercise. These results demonstrate the exquisite specificity with which varenicline blocks nicotine reward and highlight the complexity with which different rewards activate the mesolimbic DA system.

Reverse-translational identification of a cerebellar satiation network.

The brain is the seat of body weight homeostasis. However, our inability to control the increasing prevalence of obesity highlights a need to look beyond canonical feeding pathways to broaden our understanding of body weight control1-3. Here we used a reverse-translational approach to identify and anatomically, molecularly and functionally characterize a neural ensemble that promotes satiation. Unbiased, task-based functional magnetic resonance imaging revealed marked differences in cerebellar responses to food in people with a genetic disorder characterized by insatiable appetite. Transcriptomic analyses in mice revealed molecularly and topographically -distinct neurons in the anterior deep cerebellar nuclei (aDCN) that are activated by feeding or nutrient infusion in the gut. Selective activation of aDCN neurons substantially decreased food intake by reducing meal size without compensatory changes to metabolic rate. We found that aDCN activity terminates food intake by increasing striatal dopamine levels and attenuating the phasic dopamine response to subsequent food consumption. Our study defines a conserved satiation centre that may represent a novel therapeutic target for the management of excessive eating, and underscores the utility of a 'bedside-to-bench' approach for the identification of neural circuits that influence behaviour.

Ventral striatal islands of Calleja neurons control grooming in mice.

The striatum comprises multiple subdivisions and neural circuits that differentially control motor output. The islands of Calleja (IC) contain clusters of densely packed granule cells situated in the ventral striatum, predominantly in the olfactory tubercle (OT). Characterized by expression of the D3 dopamine receptor, the IC are evolutionally conserved, but have undefined functions. Here, we show that optogenetic activation of OT D3 neurons robustly initiates self-grooming in mice while suppressing other ongoing behaviors. Conversely, optogenetic inhibition of these neurons halts ongoing grooming, and genetic ablation reduces spontaneous grooming. Furthermore, OT D3 neurons show increased activity before and during grooming and influence local striatal output via synaptic connections with neighboring OT neurons (primarily spiny projection neurons), whose firing rates display grooming-related modulation. Our study uncovers a new role of the ventral striatum's IC in regulating motor output and has important implications for the neural control of grooming.

Time and metabolic state-dependent effects of GLP-1R agonists on NPY/AgRP and POMC neuronal activity in vivo.

OBJECTIVE: Long-acting glucagon-like peptide-1 receptor agonists (GLP-1RAs), like liraglutide and semaglutide, are viable treatments for diabetes and obesity. Liraglutide directly activates hypothalamic proopiomelanocortin (POMC) neurons while indirectly inhibiting Neuropeptide Y/Agouti-related peptide (NPY/AgRP) neurons ex vivo. While temporal control of GLP-1R agonist concentration as well as accessibility to tissues/cells can be achieved with relative ease ex vivo, in vivo this is dependent upon the pharmacokinetics of these agonists and relative penetration into structures of interest. Thus, whether liraglutide or semaglutide modifies the activity of POMC and NPY/AgRP neurons in vivo as well as mechanisms required for any changes in cellular activity remains undefined. METHODS: In order to resolve this issue, we utilized neuron-specific transgenic mouse models to examine changes in the activity of POMC and NPY/AgRP neurons after injection of either liraglutide or semaglutide (intraperitoneal - I.P. and subcutaneous - S·C.). POMC and NPY/AgRP neurons were targeted for patch-clamp electrophysiology as well as in vivo fiber photometry. RESULTS: We found that liraglutide and semaglutide directly activate and increase excitatory tone to POMC neurons in a time-dependent manner. This increased activity of POMC neurons required GLP-1Rs in POMC neurons as well as a downstream mixed cation channel comprised of TRPC5 subunits. We also observed an indirect upregulation of excitatory input to POMC neurons originating from glutamatergic cells that also required TRPC5 subunits. Conversely, GLP-1Ra's decreased excitatory input to and indirectly inhibited NPY/AgRP neurons through activation of K-ATP and TRPC5 channels in GABAergic neurons. Notably, the temporal activation of POMC and inhibition of NPY/AgRP neuronal activity after liraglutide or semaglutide was injected [either intraperitoneal (I.P.) or subcutaneous (S·C.)] was dependent upon the nutritional state of the animals (fed vs food-deprived). CONCLUSIONS: Our results support a mechanism of liraglutide and semaglutide in vivo to activate POMC while inhibiting NPY/AgRP neurons, which depends upon metabolic state and mirrors the pharmacokinetic profile of these compounds in vivo.

Hypothalamic detection of macronutrients via multiple gut-brain pathways.

Food intake is tightly regulated by complex and coordinated gut-brain interactions. Nutrients rapidly modulate activity in key populations of hypothalamic neurons that regulate food intake, including hunger-sensitive agouti-related protein (AgRP)-expressing neurons. Because individual macronutrients engage specific receptors in the gut to communicate with the brain, we reasoned that macronutrients may utilize different pathways to reduce activity in AgRP neurons. Here, we revealed that AgRP neuron activity in hungry mice is inhibited by site-specific intestinal detection of different macronutrients. We showed that vagal gut-brain signaling is required for AgRP neuron inhibition by fat. In contrast, spinal gut-brain signaling relays the presence of intestinal glucose. Further, we identified glucose sensors in the intestine and hepatic portal vein that mediate glucose-dependent AgRP neuron inhibition. Therefore, distinct pathways are activated by individual macronutrients to inhibit AgRP neuron activity.

Interneuron Desynchronization Precedes Seizures in a Mouse Model of Dravet Syndrome.

Recurrent seizures, which define epilepsy, are transient abnormalities in the electrical activity of the brain. The mechanistic basis of seizure initiation, and the contribution of defined neuronal subtypes to seizure pathophysiology, remains poorly understood. We performed in vivo two-photon calcium imaging in neocortex during temperature-induced seizures in male and female Dravet syndrome (Scn1a+/-) mice, a neurodevelopmental disorder with prominent temperature-sensitive epilepsy. Mean activity of both putative principal cells and parvalbumin-positive interneurons (PV-INs) was higher in Scn1a+/- relative to wild-type controls during quiet wakefulness at baseline and at elevated core body temperature. However, wild-type PV-INs showed a progressive synchronization in response to temperature elevation that was absent in PV-INs from Scn1a+/- mice. Hence, PV-IN activity remains intact interictally in Scn1a+/- mice, yet exhibits decreased synchrony immediately before seizure onset. We suggest that impaired PV-IN synchronization may contribute to the transition to the ictal state during temperature-induced seizures in Dravet syndrome.SIGNIFICANCE STATEMENT Epilepsy is a common neurological disorder defined by recurrent, unprovoked seizures. However, basic mechanisms of seizure initiation and propagation remain poorly understood. We performed in vivo two-photon calcium imaging in an experimental model of Dravet syndrome (Scn1a+/- mice)-a severe neurodevelopmental disorder defined by temperature-sensitive, treatment-resistant epilepsy-and record activity of putative excitatory neurons and parvalbumin-positive GABAergic neocortical interneurons (PV-INs) during naturalistic seizures induced by increased core body temperature. PV-IN activity was higher in Scn1a+/- relative to wild-type controls during quiet wakefulness. However, wild-type PV-INs showed progressive synchronization in response to temperature elevation that was absent in PV-INs from Scn1a+/- mice before seizure onset. Hence, impaired PV-IN synchronization may contribute to transition to seizure in Dravet syndrome.

Natural and Drug Rewards Engage Distinct Pathways that Converge on Coordinated Hypothalamic and Reward Circuits.

Motivated behavior is influenced by neural networks that integrate physiological needs. Here, we describe coordinated regulation of hypothalamic feeding and midbrain reward circuits in awake behaving mice. We find that alcohol and other non-nutritive drugs inhibit activity in hypothalamic feeding neurons. Interestingly, nutrients and drugs utilize different pathways for the inhibition of hypothalamic neuron activity, as alcohol signals hypothalamic neurons in a vagal-independent manner, while fat and satiation signals require the vagus nerve. Concomitantly, nutrients, alcohol, and drugs also increase midbrain dopamine signaling. We provide evidence that these changes are interdependent, as modulation of either hypothalamic neurons or midbrain dopamine signaling influences reward-evoked activity changes in the other population. Taken together, our results demonstrate that (1) food and drugs can engage at least two peripheral→central pathways to influence hypothalamic neuron activity, and (2) hypothalamic and dopamine circuits interact in response to rewards.

Cholinergic modulation of hippocampal calcium activity across the sleep-wake cycle.

Calcium is a critical second messenger in neurons that contributes to learning and memory, but how the coordination of action potentials of neuronal ensembles with the hippocampal local field potential (LFP) is reflected in dynamic calcium activity remains unclear. Here, we recorded hippocampal calcium activity with endoscopic imaging of the genetically encoded fluorophore GCaMP6 with concomitant LFP in freely behaving mice. Dynamic calcium activity was greater in exploratory behavior and REM sleep than in quiet wakefulness and slow wave sleep, behavioral states that differ with respect to theta and septal cholinergic activity, and modulated at sharp wave ripples (SWRs). Chemogenetic activation of septal cholinergic neurons expressing the excitatory hM3Dq DREADD increased calcium activity and reduced SWRs. Furthermore, inhibition of muscarinic acetylcholine receptors (mAChRs) reduced calcium activity while increasing SWRs. These results demonstrate that hippocampal dynamic calcium activity depends on behavioral and theta state as well as endogenous mAChR activation.

Hypothalamic Neurons that Regulate Feeding Can Influence Sleep/Wake States Based on Homeostatic Need.

Eating and sleeping represent two mutually exclusive behaviors that satisfy distinct homeostatic needs. Because an animal cannot eat and sleep at the same time, brain systems that regulate energy homeostasis are likely to influence sleep/wake behavior. Indeed, previous studies indicate that animals adjust sleep cycles around periods of food need and availability. Furthermore, hormones that affect energy homeostasis also affect sleep/wake states: the orexigenic hormone ghrelin promotes wakefulness, and the anorexigenic hormones leptin and insulin increase the duration of slow-wave sleep. However, whether neural populations that regulate feeding can influence sleep/wake states is unknown. The hypothalamic arcuate nucleus contains two neuronal populations that exert opposing effects on energy homeostasis: agouti-related protein (AgRP)-expressing neurons detect caloric need and orchestrate food-seeking behavior, whereas activity in pro-opiomelanocortin (POMC)-expressing neurons induces satiety. We tested the hypotheses that AgRP neurons affect sleep homeostasis by promoting states of wakefulness, whereas POMC neurons promote states of sleep. Indeed, optogenetic or chemogenetic stimulation of AgRP neurons in mice promoted wakefulness while decreasing the quantity and integrity of sleep. Inhibition of AgRP neurons rescued sleep integrity in food-deprived mice, highlighting the physiological importance of AgRP neuron activity for the suppression of sleep by hunger. Conversely, stimulation of POMC neurons promoted sleep states and decreased sleep fragmentation in food-deprived mice. Interestingly, we also found that sleep deprivation attenuated the effects of AgRP neuron activity on food intake and wakefulness. These results indicate that homeostatic feeding neurons can hierarchically affect behavioral outcomes, depending on homeostatic need.