Glutamatergic supramammillary nucleus neurons respond to threatening stressors and promote active coping.

Threat-response neural circuits are conserved across species and play roles in normal behavior and psychiatric diseases. Maladaptive changes in these neural circuits contribute to stress, mood, and anxiety disorders. Active coping in response to stressors is a psychosocial factor associated with resilience against stress-induced mood and anxiety disorders. The neural circuitry underlying active coping is poorly understood, but the functioning of these circuits could be key for overcoming anxiety and related disorders. The supramammillary nucleus (SuM) has been suggested to be engaged by threat. SuM has many projections and a poorly understood diversity of neural populations. In studies using mice, we identified a unique population of glutamatergic SuM neurons (SuMVGLUT2+::POA) based on projection to the preoptic area of the hypothalamus (POA) and found SuMVGLUT2+::POA neurons have extensive arborizations. SuMVGLUT2+::POA neurons project to brain areas that mediate features of the stress and threat responses including the paraventricular nucleus thalamus (PVT), periaqueductal gray (PAG), and habenula (Hb). Thus, SuMVGLUT2+::POA neurons are positioned as a hub, connecting to areas implicated in regulating stress responses. Here we report SuMVGLUT2+::POA neurons are recruited by diverse threatening stressors, and recruitment correlated with active coping behaviors. We found that selective photoactivation of the SuMVGLUT2+::POA population drove aversion but not anxiety like behaviors. Activation of SuMVGLUT2+::POA neurons in the absence of acute stressors evoked active coping like behaviors and drove instrumental behavior. Also, activation of SuMVGLUT2+::POA neurons was sufficient to convert passive coping strategies to active behaviors during acute stress. In contrast, we found activation of GABAergic (VGAT+) SuM neurons (SuMVGAT+) neurons did not alter drive aversion or active coping, but termination of photostimulation was followed by increased mobility in the forced swim test. These findings establish a new node in stress response circuitry that has projections to many brain areas and evokes flexible active coping behaviors.

MouseVUER: video based open-source system for laboratory mouse home-cage monitoring.

Video monitoring of mice in the home-cage reveals behavior profiles without the disruptions caused by specialized test setups and makes it possible to quantify changes in behavior patterns continually over long time frames. Several commercial home-cage monitoring systems are available with varying costs and capabilities; however there are currently no open-source systems for home-cage monitoring. We present an open-source system for top-down video monitoring of research mice in a slightly modified home-cage. The system is designed for integration with Allentown NexGen ventilated racks and allows unobstructed view of up to three mice, but can also be operated outside the rack. The system has an easy to duplicate and assemble home-cage design along with a video acquisition solution. The system utilizes a depth video camera, and we demonstrate the robustness of depth video for home-cage mice monitoring. For researchers without access to Allentown NexGen ventilated racks, we provide designs and assembly instructions for a standalone non-ventilated rack solution that holds three systems for more compact and efficient housing. We make all the design files, along with detailed assembly and installation instructions, available on the project webpage ( https://github.com/NIH-CIT-OIR-SPIS/MouseVUER ).

Impact of Δ9-Tetrahydrocannabinol and oxycodone co-administration on measures of antinociception, dependence, circadian activity, and reward in mice.

Oxycodone is commonly prescribed for moderate to severe pain disorders. While efficacious, long-term use can result in tolerance, physical dependence, and the development of opioid use disorder. Cannabis and its derivatives such as Δ9-Tetrahydrocannabinol (Δ9-THC) have been reported to enhance oxycodone analgesia in animal models and in humans. However, it remains unclear if Δ9-THC may facilitate unwanted aspects of oxycodone intake, such as tolerance, dependence, and reward at analgesic doses. This study sought to evaluate the impact of co-administration of Δ9-THC and oxycodone across behavioral measures related to antinociception, dependence, circadian activity, and reward in both male and female mice. Oxycodone and Δ9-THC produced dose-dependent antinociceptive effects in the hotplate assay that were similar between sexes. Repeated treatment (twice daily for 5 days) resulted in antinociceptive tolerance. Combination treatment of oxycodone and Δ9-THC produced a greater antinociceptive effect than either administered alone, and delayed the development of antinociceptive tolerance. Repeated treatment with oxycodone produced physical dependence and alterations in circadian activity, neither of which were exacerbated by co-treatment with Δ9-THC. Combination treatment of oxycodone and Δ9-THC produced CPP when co-administered at doses that did not produce preference when administered alone. These data indicate that Δ9-THC may facilitate oxycodone-induced antinociception without augmenting certain unwanted features of opioid intake (e.g. dependence, circadian rhythm alterations). However, our findings also indicate that Δ9-THC may facilitate rewarding properties of oxycodone at therapeutically relevant doses which warrant consideration when evaluating this combination for its potential therapeutic utility.

TMEM135 links peroxisomes to the regulation of brown fat mitochondrial fission and energy homeostasis.

Mitochondrial morphology, which is controlled by mitochondrial fission and fusion, is an important regulator of the thermogenic capacity of brown adipocytes. Adipose-specific peroxisome deficiency impairs thermogenesis by inhibiting cold-induced mitochondrial fission due to decreased mitochondrial membrane content of the peroxisome-derived lipids called plasmalogens. Here, we identify TMEM135 as a critical mediator of the peroxisomal regulation of mitochondrial fission and thermogenesis. Adipose-specific TMEM135 knockout in mice blocks mitochondrial fission, impairs thermogenesis, and increases diet-induced obesity and insulin resistance. Conversely, TMEM135 overexpression promotes mitochondrial division, counteracts obesity and insulin resistance, and rescues thermogenesis in peroxisome-deficient mice. Mechanistically, thermogenic stimuli promote association between peroxisomes and mitochondria and plasmalogen-dependent localization of TMEM135 in mitochondria, where it mediates PKA-dependent phosphorylation and mitochondrial retention of the fission factor Drp1. Together, these results reveal a previously unrecognized inter-organelle communication regulating mitochondrial fission and energy homeostasis and identify TMEM135 as a potential target for therapeutic activation of BAT.

Kappa opioid receptor activation increases thermogenic energy expenditure which drives increased feeding.

Opioid receptors, including the kappa opioid receptor (KOR), exert control over thermoregulation and feeding behavior. Notably, activation of KOR stimulates food intake, leading to postulation that KOR signaling plays a central role in managing energy intake. KOR has also been proposed as a target for treating obesity. Herein, we report studies examining how roles for KOR signaling in regulating thermogenesis, feeding, and energy balance may be interrelated using pharmacological interventions, genetic tools, quantitative thermal imaging, and metabolic profiling. Our findings demonstrate that activation of KOR in the central nervous system causes increased energy expenditure via brown adipose tissue activation. Importantly, pharmacologic, or genetic inhibition of brown adipose tissue thermogenesis prevented the elevated food intake triggered by KOR activation. Furthermore, our data reveal that KOR-mediated thermogenesis elevation is reversibly disrupted by chronic high-fat diet, implicating KOR signaling as a potential mediator in high-fat diet-induced weight gain.

Toggling between food-seeking and self-preservation behaviors via hypothalamic response networks.

Motivated behaviors are often studied in isolation to assess labeled lines of neural connections underlying innate actions. However, in nature, multiple systems compete for expression of goal-directed behaviors via complex neural networks. Here, we examined flexible survival decisions in animals tasked with food seeking under predation threat. We found that predator exposure rapidly induced physiological, neuronal, and behavioral adaptations in mice highlighted by reduced food seeking and consumption contingent on current threat level. Diminishing conflict via internal state or external environment perturbations shifted feeding strategies. Predator introduction and/or selective manipulation of danger-responsive cholecystokinin (Cck) cells of the dorsal premammilary nucleus (PMd) suppressed hunger-sensitive Agouti-related peptide (AgRP) neurons, providing a mechanism for threat-evoked hypophagia. Increased caloric need enhanced food seeking under duress through AgRP pathways to the bed nucleus of the stria terminalis (BNST) and/or lateral hypothalamus (LH). Our results suggest oscillating interactions between systems underlying self-preservation and food seeking to promote optimal behavior.

Induction of a torpor-like hypothermic and hypometabolic state in rodents by ultrasound.

Torpor is an energy-conserving state in which animals dramatically decrease their metabolic rate and body temperature to survive harsh environmental conditions. Here, we report the noninvasive, precise and safe induction of a torpor-like hypothermic and hypometabolic state in rodents by remote transcranial ultrasound stimulation at the hypothalamus preoptic area (POA). We achieve a long-lasting (>24 h) torpor-like state in mice via closed-loop feedback control of ultrasound stimulation with automated detection of body temperature. Ultrasound-induced hypothermia and hypometabolism (UIH) is triggered by activation of POA neurons, involves the dorsomedial hypothalamus as a downstream brain region and subsequent inhibition of thermogenic brown adipose tissue. Single-nucleus RNA-sequencing of POA neurons reveals TRPM2 as an ultrasound-sensitive ion channel, the knockdown of which suppresses UIH. We also demonstrate that UIH is feasible in a non-torpid animal, the rat. Our findings establish UIH as a promising technology for the noninvasive and safe induction of a torpor-like state.

Fused fiber couplers for fiber photometry.

In this issue of Cell Reports Methods, Formozov et al. present an innovative fiber photometry system that uses a fused fiber coupler (FFC) instead of a dichroic mirror to split the excitation and emission light. The FFC-based photometry system is highly flexible and can be easily reconfigured to record from different biosensors.

Dopaminergic innervation at the central nucleus of the amygdala reveals distinct topographically segregated regions.

The central nucleus of the amygdala (CeA) is involved in the expression of fear and anxiety disorders. Anatomically, it is divided into medial (CeM), lateral (CeL), and capsular (CeC) divisions. The CeA is densely innervated by dopaminergic projections that originate in the ventral periaqueductal gray/dorsal raphe (vPAG/DR) and the ventral tegmental area (VTA). However, whether dopamine (DA) exerts a homogenous control over the CeA or differentially regulates the various CeA subdivisions is still unknown. Here, we performed a neuroanatomical analysis of the mouse CeA and found that DAergic innervations from the PAG/DR and VTA constitute distinct, non-overlapping, pathways differing also in the relative expression of the dopamine transporter. By quantifying the distribution of DAergic fibers and the origin of DA inputs we identified two distinct regions in the CeL: a frontal region innervated by the VTA and vPAG/DR, a caudal region innervated only by the vPAG/DR, and three distinct regions in the CeC: fronto-dorsal innervated only by the VTA, fronto-ventral with sparse DAergic innervation, and a caudal region with low innervation from the vPAG/DR. In addition, we found that each region displays a distinct pattern of c-Fos activation following the administration of various DAeric drugs such as cocaine, SKF 38,393, quinpirole or haloperidol. In summary, we revealed unique properties of the DAergic pathways innervating the CeA, distinguishing six topographically segregated and functionally distinct regions. This unanticipated level of heterogeneity calls for more precise neuroanatomical specificity in future functional studies of the CeA.

Nucleus Accumbens D1 Receptor-Expressing Spiny Projection Neurons Control Food Motivation and Obesity.

BACKGROUND: Obesity is a chronic relapsing disorder that is caused by an excess of caloric intake relative to energy expenditure. There is growing recognition that food motivation is altered in people with obesity. However, it remains unclear how brain circuits that control food motivation are altered in obese animals. METHODS: Using a novel behavioral assay that quantifies work during food seeking, in vivo and ex vivo cell-specific recordings, and a synaptic blocking technique, we tested the hypothesis that activity of circuits promoting appetitive behavior in the core of the nucleus accumbens (NAc) is enhanced in the obese state, particularly during food seeking. RESULTS: We first confirmed that mice made obese with ad libitum exposure to a high fat diet work harder than lean mice to obtain food, consistent with an increase in food motivation in obese mice. We observed greater activation of D1 receptor-expressing NAc spiny projection neurons (NAc D1SPNs) during food seeking in obese mice relative to lean mice. This enhanced activity was not observed in D2 receptor-expressing neurons (D2SPNs). Consistent with these in vivo findings, both intrinsic excitability and excitatory drive onto D1SPNs were enhanced in obese mice relative to lean mice, and these measures were selective for D1SPNs. Finally, blocking synaptic transmission from D1SPNs, but not D2SPNs, in the NAc core decreased physical work during food seeking and, critically, attenuated high fat diet-induced weight gain. CONCLUSIONS: These experiments demonstrate the necessity of NAc core D1SPNs in food motivation and the development of diet-induced obesity, establishing these neurons as a potential therapeutic target for preventing obesity.

Oral oxycodone self-administration leads to features of opioid misuse in male and female mice.

Use of prescription opioids, particularly oxycodone, is an initiating factor driving the current opioid epidemic. There are several challenges with modelling oxycodone abuse. First, prescription opioids including oxycodone are orally self-administered and have different pharmacokinetics and dynamics than morphine or fentanyl, which have been more commonly used in rodent research. This oral route of administration determines the pharmacokinetic profile, which then influences the establishment of drug-reinforcement associations in animals. Moreover, the pattern of intake and the environment in which addictive drugs are self-administered are critical determinants of the levels of drug intake, of behavioural sensitization and of propensity to relapse behaviour. These are all important considerations when modelling prescription opioid use, which is characterized by continuous drug access in familiar environments. Thus, to model features of prescription opioid use and the transition to abuse, we designed an oral, homecage-based oxycodone self-administration paradigm. Mice voluntarily self-administer oxycodone in this paradigm without any taste modification such as sweeteners, and the majority exhibit preference for oxycodone, escalation of intake, physical signs of dependence and reinstatement of seeking after withdrawal. In addition, a subset of animals demonstrate drug taking that is resistant to aversive consequences. This model is therefore translationally relevant and useful for studying the neurobiological substrates of prescription opioid abuse.

Identification of a Stress-Sensitive Anorexigenic Neurocircuit From Medial Prefrontal Cortex to Lateral Hypothalamus.

BACKGROUND: A greater understanding of how the brain controls appetite is fundamental to developing new approaches for treating diseases characterized by dysfunctional feeding behavior, such as obesity and anorexia nervosa. METHODS: By modeling neural network dynamics related to homeostatic state and body mass index, we identified a novel pathway projecting from the medial prefrontal cortex (mPFC) to the lateral hypothalamus (LH) in humans (n = 53). We then assessed the physiological role and dissected the function of this mPFC-LH circuit in mice. RESULTS: In vivo recordings of population calcium activity revealed that this glutamatergic mPFC-LH pathway is activated in response to acute stressors and inhibited during food consumption, suggesting a role in stress-related control over food intake. Consistent with this role, inhibition of this circuit increased feeding and sucrose seeking during mild stressors, but not under nonstressful conditions. Finally, chemogenetic or optogenetic activation of the mPFC-LH pathway is sufficient to suppress food intake and sucrose seeking in mice. CONCLUSIONS: These studies identify a glutamatergic mPFC-LH circuit as a novel stress-sensitive anorexigenic neural pathway involved in the cortical control of food intake.

Fiber photometry in striatum reflects primarily nonsomatic changes in calcium.

Fiber photometry enables recording of population neuronal calcium dynamics in awake mice. While the popularity of fiber photometry has grown in recent years, it remains unclear whether photometry reflects changes in action potential firing (that is, 'spiking') or other changes in neuronal calcium. In microscope-based calcium imaging, optical and analytical approaches can help differentiate somatic from neuropil calcium. However, these approaches cannot be readily applied to fiber photometry. As such, it remains unclear whether the fiber photometry signal reflects changes in somatic calcium, changes in nonsomatic calcium or a combination of the two. Here, using simultaneous in vivo extracellular electrophysiology and fiber photometry, along with in vivo endoscopic one-photon and two-photon calcium imaging, we determined that the striatal fiber photometry does not reflect spiking-related changes in calcium and instead primarily reflects nonsomatic changes in calcium.

Natural locus coeruleus dynamics during feeding.

Recent data demonstrate that noradrenergic neurons of the locus coeruleus (LC-NE) are required for fear-induced suppression of feeding, but the role of endogenous LC-NE activity in natural, homeostatic feeding remains unclear. Here, we found that LC-NE activity was suppressed during food consumption, and the magnitude of this neural response was attenuated as mice consumed more pellets throughout the session, suggesting that LC responses to food are modulated by satiety state. Visual-evoked LC-NE activity was also attenuated in sated mice, suggesting that satiety state modulates LC-NE encoding of multiple behavioral states. We also found that food intake could be attenuated by brief or longer durations of LC-NE activation. Last, we found that activation of the LC to the lateral hypothalamus pathway suppresses feeding and enhances avoidance and anxiety-like responding. Our findings suggest that LC-NE neurons modulate feeding by integrating both external cues (e.g., anxiogenic environmental cues) and internal drives (e.g., satiety).

Partial Ablation of Postsynaptic Dopamine D2 Receptors in the Central Nucleus of the Amygdala Increases Risk Avoidance in Exploratory Tasks.

The central nucleus of the amygdala (CeA) is involved in the expression of fear and has been implicated in several anxiety disorders. This structure is densely innervated by DAergic projections that impinge on amygdalar neurons expressing various dopamine (DA) receptor subtypes, including D2 receptors (D2Rs). Although various pharmacological approaches have assessed the role of D2Rs in the CeA, the actual participation of postsynaptic D2Rs in the CeA to defensive behaviors remains unclear. Here, we investigated the distribution of D2Rs in the CeA and their role in modifying neuronal activity and fear related behaviors in mice. First, using the mouse reporter strain D2R-EGFP, we verified that D2Rs are present both in neurons of the CeA and in A10 dorsocaudal (A10dc) DAergic neurons that innervate the CeA. Moreover, we showed that pharmacological stimulation of D2Rs increases the activity of protein kinase C (PKC)δ cells present in the CeA, a type of neuron previously associated with reduced defensive behaviors. Finally, using a molecular genetics approach that discriminates postsynaptic D2Rs from presynaptic D2 autoreceptors, we demonstrated that mice carrying targeted deletions of postsynaptic D2Rs in the CeA display increased risk avoidance in exploratory tasks. Together, our results indicate that postsynaptic D2Rs in the CeA attenuate behavioral reactions to potential environmental threats.

Metabolic sensing in AgRP neurons integrates homeostatic state with dopamine signalling in the striatum.

Agouti-related peptide (AgRP) neurons increase motivation for food, however, whether metabolic sensing of homeostatic state in AgRP neurons potentiates motivation by interacting with dopamine reward systems is unexplored. As a model of impaired metabolic-sensing, we used the AgRP-specific deletion of carnitine acetyltransferase (Crat) in mice. We hypothesised that metabolic sensing in AgRP neurons is required to increase motivation for food reward by modulating accumbal or striatal dopamine release. Studies confirmed that Crat deletion in AgRP neurons (KO) impaired ex vivo glucose-sensing, as well as in vivo responses to peripheral glucose injection or repeated palatable food presentation and consumption. Impaired metabolic-sensing in AgPP neurons reduced acute dopamine release (seconds) to palatable food consumption and during operant responding, as assessed by GRAB-DA photometry in the nucleus accumbens, but not the dorsal striatum. Impaired metabolic-sensing in AgRP neurons suppressed radiolabelled 18F-fDOPA accumulation after ~30 min in the dorsal striatum but not the nucleus accumbens. Impaired metabolic sensing in AgRP neurons suppressed motivated operant responding for sucrose rewards during fasting. Thus, metabolic-sensing in AgRP neurons is required for the appropriate temporal integration and transmission of homeostatic hunger-sensing to dopamine signalling in the striatum.

Scalable and modular wireless-network infrastructure for large-scale behavioural neuroscience.

The use of rodents to acquire understanding of the function of neural circuits and of the physiological, genetic and developmental underpinnings of behaviour has been constrained by limitations in the scalability, automation and high-throughput operation of implanted wireless neural devices. Here we report scalable and modular hardware and software infrastructure for setting up and operating remotely programmable miniaturized wireless networks leveraging Bluetooth Low Energy for the study of the long-term behaviour of large groups of rodents. The integrated system allows for automated, scheduled and real-time experimentation via the simultaneous and independent use of multiple neural devices and equipment within and across laboratories. By measuring the locomotion, feeding, arousal and social behaviours of groups of mice or rats, we show that the system allows for bidirectional data transfer from readily available hardware, and that it can be used with programmable pharmacological or optogenetic stimulation. Scalable and modular wireless-network infrastructure should facilitate the remote operation of fully automated large-scale and long-term closed-loop experiments for the study of neural circuits and animal behaviour.

An open-source device for measuring food intake and operant behavior in rodent home-cages.

Feeding is critical for survival, and disruption in the mechanisms that govern food intake underlies disorders such as obesity and anorexia nervosa. It is important to understand both food intake and food motivation to reveal mechanisms underlying feeding disorders. Operant behavioral testing can be used to measure the motivational component to feeding, but most food intake monitoring systems do not measure operant behavior. Here, we present a new solution for monitoring both food intake and motivation in rodent home-cages: the Feeding Experimentation Device version 3 (FED3). FED3 measures food intake and operant behavior in rodent home-cages, enabling longitudinal studies of feeding behavior with minimal experimenter intervention. It has a programmable output for synchronizing behavior with optogenetic stimulation or neural recordings. Finally, FED3 design files are open-source and freely available, allowing researchers to modify FED3 to suit their needs.

Obesity and anorexia nervosa are two health conditions related to food intake. Researchers studying these disorders in animal models need to both measure food intake and assess behavioural factors: that is, why animals seek and consume food. Measuring an animal's food intake is usually done by weighing food containers. However, this can be inaccurate due to the small amount of food that rodents eat. As for studying feeding motivation, this can involve calculating the number of times an animal presses a lever to receive a food pellet. These tests are typically conducted in hour-long sessions in temporary testing cages, called operant boxes. Yet, these tests only measure a brief period of a rodent's life. In addition, it takes rodents time to adjust to these foreign environments, which can introduce stress and may alter their feeding behaviour. To address this, Matikainen-Ankney, Earnest, Ali et al. developed a device for monitoring food intake and feeding behaviours around the clock in rodent home cages with minimal experimenter intervention. This 'Feeding Experimentation Device' (FED3) features a pellet dispenser and two 'nose-poke' sensors to measure total food intake, as well as motivation for and learning about food rewards. The battery-powered, wire-free device fits in standard home cages, enabling long-term studies of feeding behaviour with minimal intervention from investigators and less stress on the animals. This means researchers can relate data to circadian rhythms and meal patterns, as Matikainen-Ankney did here. Moreover, the device software is open-source so researchers can customise it to suit their experimental needs. It can also be programmed to synchronise with other instruments used in animal experiments, or across labs running the same behavioural tasks for multi-site studies. Used in this way, it could help improve reproducibility and reliability of results from such studies. In summary, Matikainen-Ankney et al. have presented a new practical solution for studying food-related behaviours in mice and rats. Not only could the device be useful to researchers, it may also be suitable to use in educational settings such as teaching labs and classrooms.