FABEL: Forecasting Animal Behavioral Events with Deep Learning-Based Computer Vision.

Behavioral neuroscience aims to provide a connection between neural phenomena and emergent organism-level behaviors. This requires perturbing the nervous system and observing behavioral outcomes, and comparing observed post-perturbation behavior with predicted counterfactual behavior and therefore accurate behavioral forecasts. In this study we present FABEL, a deep learning method for forecasting future animal behaviors and locomotion trajectories from historical locomotion alone. We train an offline pose estimation network to predict animal body-part locations in behavioral video; then sequences of pose vectors are input to deep learning time-series forecasting models. Specifically, we train an LSTM network that predicts a future food interaction event in a specified time window, and a Temporal Fusion Transformer that predicts future trajectories of animal body-parts, which are then converted into probabilistic label forecasts. Importantly, accurate prediction of food interaction provides a basis for neurobehavioral intervention in the context of compulsive eating. We show promising results on forecasting tasks between 100 milliseconds and 5 seconds timescales. Because the model takes only behavioral video as input, it can be adapted to any behavioral task and does not require specific physiological readouts. Simultaneously, these deep learning models may serve as extensible modules that can accommodate diverse signals, such as in-vivo fluorescence imaging and electrophysiology, which may improve behavior forecasts and elucidate invervention targets for desired behavioral change.

Hypothalamic CRF neurons facilitate brain reward function.

Aversive stimuli activate corticotropin-releasing factor (CRF)-expressing neurons in the paraventricular nucleus of hypothalamus (PVNCRF neurons) and other brain stress systems to facilitate avoidance behaviors. Appetitive stimuli also engage the brain stress systems, but their contributions to reward-related behaviors are less well understood. Here, we show that mice work vigorously to optically activate PVNCRF neurons in an operant chamber, indicating a reinforcing nature of these neurons. The reinforcing property of these neurons is not mediated by activation of the hypothalamic-pituitary-adrenal (HPA) axis. We found that PVNCRF neurons send direct projections to the ventral tegmental area (VTA), and selective activation of these projections induced robust self-stimulation behaviors, without activation of the HPA axis. Similar to the PVNCRF cell bodies, self-stimulation of PVNCRF-VTA projection was dramatically attenuated by systemic pretreatment of CRF receptor 1 or dopamine D1 receptor (D1R) antagonist and augmented by corticosterone synthesis inhibitor metyrapone, but not altered by dopamine D2 receptor (D2R) antagonist. Furthermore, we found that activation of PVNCRF-VTA projections increased c-Fos expression in the VTA dopamine neurons and rapidly triggered dopamine release in the nucleus accumbens (NAc), and microinfusion of D1R or D2R antagonist into the NAc decreased the self-stimulation of these projections. Together, our findings reveal an unappreciated role of PVNCRF neurons and their VTA projections in driving reward-related behaviors, independent of their core neuroendocrine functions. As activation of PVNCRF neurons is the final common path for many stress systems, our study suggests a novel mechanism underlying the positive reinforcing effect of stressful stimuli.

Precise 3D Localization of Intracerebral Implants with a simple Brain Clearing Method.

Determining the localization of intracerebral implants in rodent brain stands as a critical final step in most physiological and behaviroral studies, especially when targeting deep brain nuclei. Conventional histological approaches, reliant on manual estimation through sectioning and slice examination, are error-prone, potentially complicating data interpretation. Leveraging recent advances in tissue-clearing techniques and light-sheet fluorescence microscopy, we introduce a method enabling virtual brain slicing in any orientation, offering precise implant localization without the limitations of traditional tissue sectioning. To illustrate the method's utility, we present findings from the implantation of linear silicon probes into the midbrain interpeduncular nucleus (IPN) of anesthetized transgenic mice expressing chanelrhodopsin-2 and enhanced yellow fluorescent protein under the choline acetyltransferase (ChAT) promoter/enhancer regions (ChAT-Chr2-EYFP mice). Utilizing a fluorescent dye applied to the electrode surface, we visualized both the targeted area and the precise localization, enabling enhanced inter-subject comparisons. Three dimensional (3D) brain renderings, presented effortlessly in video format across various orientations, showcase the versatility of this approach.

Shared and divergent transcriptomic regulation in nucleus accumbens D1 and D2 medium spiny neurons by cocaine and morphine.

Substance use disorders (SUDs) induce widespread molecular dysregulation in the nucleus accumbens (NAc), a brain region pivotal for coordinating motivation and reward. These molecular changes are thought to support lasting neural and behavioral disturbances that promote drug-seeking in addiction. However, different drug classes exert unique influences on neural circuits, cell types, physiology, and gene expression despite the overlapping symptomatology of SUDs. To better understand common and divergent molecular mechanisms governing SUD pathology, our goal was to survey cell-type-specific restructuring of the NAc transcriptional landscape in after psychostimulant or opioid exposure. We combined fluorescence-activated nuclei sorting and RNA sequencing to profile NAc D1 and D2 medium spiny neurons (MSNs) across cocaine and morphine exposure paradigms, including initial exposure, prolonged withdrawal after repeated exposure, and re-exposure post-withdrawal. Our analyses reveal that D1 MSNs display many convergent transcriptional responses across drug classes during exposure, whereas D2 MSNs manifest mostly divergent responses between cocaine and morphine, with morphine causing more adaptations in this cell type. Utilizing multiscale embedded gene co-expression network analysis (MEGENA), we discerned transcriptional regulatory networks subserving biological functions shared between cocaine and morphine. We observed largely integrative engagement of overlapping gene networks across drug classes in D1 MSNs, but opposite regulation of key D2 networks, highlighting potential therapeutic gene network targets within MSNs. These studies establish a landmark, cell-type-specific atlas of transcriptional regulation induced by cocaine and by morphine that can serve as a foundation for future studies towards mechanistic understanding of SUDs. Our findings, and future work leveraging this dataset, will pave the way for the development of targeted therapeutic interventions, addressing the urgent need for more effective treatments for cocaine use disorder and enhancing the existing strategies for opioid use disorder.

Central and peripheral actions of nicotine that influence blood glucose homeostasis and the development of diabetes.

Cigarette smoking has long been recognized as a risk factor for type 2 diabetes (T2D), although the precise causal mechanisms underlying this relationship remain poorly understood. Recent evidence suggests that nicotine, the primary reinforcing component in tobacco, may play a pivotal role in connecting cigarette smoking and T2D. Extensive research conducted in both humans and animals has demonstrated that nicotine can elevate blood glucose levels, disrupt glucose homeostasis, and induce insulin resistance. The review aims to elucidate the genetic variants of nicotinic acetylcholine receptors associated with diabetes risk and provide a comprehensive overview of the available data on the mechanisms through which nicotine influences blood glucose homeostasis and the development of diabetes. Here we emphasize the central and peripheral actions of nicotine on the release of glucoregulatory hormones, as well as its effects on glucose tolerance and insulin sensitivity. Notably, the central actions of nicotine within the brain, which encompass both insulin-dependent and independent mechanisms, are highlighted as potential targets for intervention strategies in diabetes management.

New medications development for smoking cessation.

Diseases associated with nicotine dependence in the form of habitual tobacco use are a major cause of premature death in the United States. The majority of tobacco smokers will relapse within the first month of attempted abstinence. Smoking cessation agents increase the likelihood that smokers can achieve long-term abstinence. Nevertheless, currently available smoking cessation agents have limited utility and fail to prevent relapse in the majority of smokers. Pharmacotherapy is therefore an effective strategy to aid smoking cessation efforts but considerable risk of relapse persists even when the most efficacious medications currently available are used. The past decade has seen major breakthroughs in our understanding of the molecular, cellular, and systems-level actions of nicotine in the brain that contribute to the development and maintenance of habitual tobacco use. In parallel, large-scale human genetics studies have revealed allelic variants that influence vulnerability to tobacco use disorder. These advances have revealed targets for the development of novel smoking cessation agents. Here, we summarize current efforts to develop smoking cessation therapeutics and highlight opportunities for future efforts.

The Neurobiology of Eating Behavior in Obesity: Mechanisms and Therapeutic Targets: A Report from the 23rd Annual Harvard Nutrition Obesity Symposium.

Obesity is increasing at an alarming rate. The effectiveness of currently available strategies for the treatment of obesity (including pharmacologic, surgical, and behavioral interventions) is limited. Understanding the neurobiology of appetite and the important drivers of energy intake (EI) can lead to the development of more effective strategies for the prevention and treatment of obesity. Appetite regulation is complex and is influenced by genetic, social, and environmental factors. It is intricately regulated by a complex interplay of endocrine, gastrointestinal, and neural systems. Hormonal and neural signals generated in response to the energy state of the organism and the quality of food eaten are communicated by paracrine, endocrine, and gastrointestinal signals to the nervous system. The central nervous system integrates homeostatic and hedonic signals to regulate appetite. Although there has been an enormous amount of research over many decades regarding the regulation of EI and body weight, research is only now yielding potentially effective treatment strategies for obesity. The purpose of this article is to summarize the key findings presented in June 2022 at the 23rd annual Harvard Nutrition Obesity Symposium entitled "The Neurobiology of Eating Behavior in Obesity: Mechanisms and Therapeutic Targets." Findings presented at the symposium, sponsored by NIH P30 Nutrition Obesity Research Center at Harvard, enhance our current understanding of appetite biology, including innovative techniques used to assess and systematically manipulate critical hedonic processes, which will shape future research and the development of therapeutics for obesity prevention and treatment.

Hedgehog-interacting protein acts in the habenula to regulate nicotine intake.

Hedgehog-interacting protein (HHIP) sequesters Hedgehog ligands to repress Smoothened (SMO)-mediated recruitment of the GLI family of transcription factors. Allelic variation in HHIP confers risk of chronic obstructive pulmonary disease and other smoking-related lung diseases, but underlying mechanisms are unclear. Using single-cell and cell-type-specific translational profiling, we show that HHIP expression is highly enriched in medial habenula (MHb) neurons, particularly MHb cholinergic neurons that regulate aversive behavioral responses to nicotine. HHIP deficiency dysregulated the expression of genes involved in cholinergic signaling in the MHb and disrupted the function of nicotinic acetylcholine receptors (nAChRs) through a PTCH-1/cholesterol-dependent mechanism. Further, CRISPR/Cas9-mediated genomic cleavage of the Hhip gene in MHb neurons enhanced the motivational properties of nicotine in mice. These findings suggest that HHIP influences vulnerability to smoking-related lung diseases in part by regulating the actions of nicotine on habenular aversion circuits.

Control of fear by discrete prefrontal GABAergic populations encoding valence-specific information.

Neurons activated by learning have been ascribed the unique potential to encode memory, but the functional contribution of discrete cell types remains poorly understood. In particular, it is unclear whether learning engages specific GABAergic interneurons and, if so, whether they differ functionally from interneurons recruited by other experiences. Here, we show that fear conditioning activates a heterogeneous neuronal population in the medial prefrontal cortex (mPFC) that is largely comprised of somatostatin-expressing interneurons (SST-INs). Using intersectional genetic approaches, we demonstrate that fear-learning-activated SST-INs exhibit distinct circuit properties and are selectively reactivated to mediate cue-evoked memory expression. In contrast, an orthogonal population of SST-INs activated by morphine experience exerts opposing control over fear and supports reward-like motivational effects. These results outline an important role for discrete subsets of GABAergic cells in emotional learning and point to an unappreciated capacity for functional specialization among SST-INs.

Utility of 'substance use disorder' as a heuristic for understanding overeating and obesity.

Rates of obesity and obesity-associated diseases have increased dramatically in countries with developed economies. Substance use disorders (SUDs) are characterized by the persistent use of the substance despite negative consequences. It has been hypothesized that overconsumption of palatable energy dense food can elicit SUD-like maladaptive behaviors that contribute to persistent caloric intake beyond homeostatic need even in the face of negative consequences. Palatable food and drugs of abuse act on many of the same motivation-related circuits in the brain, and can induce, at least superficially, similar molecular, cellular, and physiological adaptations on these circuits. As such, applying knowledge about the neurobiological mechanisms of SUDs may serve as useful heuristic to better understand the persistent overconsumption of palatable food that contributes to obesity. However, many important differences exist between the actions of drugs of abuse and palatable food in the brain. This warrants caution when attributing weight gain and obesity to the manifestation of a putative SUD-related behavioral disorder. Here, we describe similarities and differences between compulsive drug use in SUDs and overconsumption in obesity and consider the merit of the concept of "food addiction".

Neurobiological Mechanisms of Nicotine Reward and Aversion.

Neuronal nicotinic acetylcholine receptors (nAChRs) regulate the rewarding actions of nicotine contained in tobacco that establish and maintain the smoking habit. nAChRs also regulate the aversive properties of nicotine, sensitivity to which decreases tobacco use and protects against tobacco use disorder. These opposing behavioral actions of nicotine reflect nAChR expression in brain reward and aversion circuits. nAChRs containing α4 and ß2 subunits are responsible for the high-affinity nicotine binding sites in the brain and are densely expressed by reward-relevant neurons, most notably dopaminergic, GABAergic, and glutamatergic neurons in the ventral tegmental area. High-affinity nAChRs can incorporate additional subunits, including ß3, α6, or α5 subunits, with the resulting nAChR subtypes playing discrete and dissociable roles in the stimulatory actions of nicotine on brain dopamine transmission. nAChRs in brain dopamine circuits also participate in aversive reactions to nicotine and the negative affective state experienced during nicotine withdrawal. nAChRs containing α3 and ß4 subunits are responsible for the low-affinity nicotine binding sites in the brain and are enriched in brain sites involved in aversion, including the medial habenula, interpeduncular nucleus, and nucleus of the solitary tract, brain sites in which α5 nAChR subunits are also expressed. These aversion-related brain sites regulate nicotine avoidance behaviors, and genetic variation that modifies the function of nAChRs in these sites increases vulnerability to tobacco dependence and smoking-related diseases. Here, we review the molecular, cellular, and circuit-level mechanisms through which nicotine elicits reward and aversion and the adaptations in these processes that drive the development of nicotine dependence. SIGNIFICANCE STATEMENT: Tobacco use disorder in the form of habitual cigarette smoking or regular use of other tobacco-related products is a major cause of death and disease worldwide. This article reviews the actions of nicotine in the brain that contribute to tobacco use disorder.

Structure and function differences in the prelimbic cortex to basolateral amygdala circuit mediate trait vulnerability in a novel model of acute social defeat stress in male mice.

Stressful life events are ubiquitous and well-known to negatively impact mental health. However, in both humans and animal models, there is large individual variability in how individuals respond to stress, with some but not all experiencing long-term adverse consequences. While there is growing understanding of the neurobiological underpinnings of the stress response, much less is known about how neurocircuits shaped by lifetime experiences are activated during an initial stressor and contribute to this selective vulnerability versus resilience. We developed a model of acute social defeat stress (ASDS) that allows classification of male mice into "susceptible" (socially avoidant) versus "resilient" (expressing control-level social approach) one hour after exposure to six minutes of social stress. Using circuit tracing and high-resolution confocal imaging, we explored differences in activation and dendritic spine density and morphology in the prelimbic cortex to basolateral amygdala (PL→BLA) circuit in resilient versus susceptible mice. Susceptible mice had greater PL→BLA recruitment during ASDS and activated PL→BLA neurons from susceptible mice had more and larger mushroom spines compared to resilient mice. We hypothesized identified structure/function differences indicate an overactive PL→BLA response in susceptible mice and used an intersectional chemogenetic approach to inhibit the PL→BLA circuit during or prior to ASDS. We found in both cases that this blocked ASDS-induced social avoidance. Overall, we show PL→BLA structure/function differences mediate divergent behavioral responses to ASDS in male mice. These results support PL→BLA circuit overactivity during stress as a biomarker of trait vulnerability and potential target for prevention of stress-induced psychopathology.

Networks of habenula-projecting cortical neurons regulate cocaine seeking.

How neurons in the medial prefrontal cortex broadcast stress-relevant information to subcortical brain sites to regulate cocaine relapse remains unclear. The lateral habenula (LHb) serves as a "hub" to filter and propagate stress- and aversion-relevant information in the brain. Here, we show that chemogenetic inhibition of cortical inputs to LHb attenuates relapse-like reinstatement of extinguished cocaine seeking in mice. Using an RNA sequencing­based brain mapping procedure with single-cell resolution, we identify networks of cortical neurons that project to LHb and then preferentially innervate different downstream brain sites, including the ventral tegmental area, median raphe nucleus, and locus coeruleus (LC). By using an intersectional chemogenetics approach, we show that inhibition of cortico-habenular neurons that project to LC, but not to other sites, blocks reinstatement of cocaine seeking. These findings highlight the remarkable complexity of descending cortical inputs to the habenula and identify a cortico-habenulo-hindbrain circuit that regulates cocaine seeking.

Gene splicing SETs the scene for cocaine addiction.

Cocaine triggers gene splicing in brain reward circuits, but the mechanisms and importance of this response are unclear. In this issue of Neuron, Xu et al. (2021) show that the histone modification H3K36me3 marks genes spliced in response to cocaine and, using epigenome editing, establish a causal relationship between gene splicing and addiction-related behavioral responses.

Opposing roles for striatonigral and striatopallidal neurons in dorsolateral striatum in consolidating new instrumental actions.

Comparatively little is known about how new instrumental actions are encoded in the brain. Using whole-brain c-Fos mapping, we show that neural activity is increased in the anterior dorsolateral striatum (aDLS) of mice that successfully learn a new lever-press response to earn food rewards. Post-learning chemogenetic inhibition of aDLS disrupts consolidation of the new instrumental response. Similarly, post-learning infusion of the protein synthesis inhibitor anisomycin into the aDLS disrupts consolidation of the new response. Activity of D1 receptor-expressing medium spiny neurons (D1-MSNs) increases and D2-MSNs activity decreases in the aDLS during consolidation. Chemogenetic inhibition of D1-MSNs in aDLS disrupts the consolidation process whereas D2-MSN inhibition strengthens consolidation but blocks the expression of previously learned habit-like responses. These findings suggest that D1-MSNs in the aDLS encode new instrumental actions whereas D2-MSNs oppose this new learning and instead promote expression of habitual actions.

Smoking status links habenular volume to glycated hemoglobin: Findings from the Human Connectome Project-Young Adult.

BACKGROUND: The habenula-pancreas axis regulates the stimulatory effects of nicotine on blood glucose levels and may participate in the emergence of type 2 diabetes in human tobacco smokers. This secondary analysis of young adults from the Human Connectome Project (HCP-YA) evaluated whether smoking status links the relationship between habenular volume and glycated hemoglobin (HbA1c), a marker of long-term glycemic control. METHODS: Habenula segmentation was performed using a fully-automated myelin content-based approach in HCP-YA participants and the results were inspected visually (n = 693; aged 22-37 years). A linear regression analysis was used with habenular volume as the dependent variable, the smoking-by-HbA1c interaction as the independent variable of interest, and age, gender, race, ethnicity, education, income, employment status, body mass index, and total gray matter volume as covariates. RESULTS: Habenula volume and HbA1c were similar in smokers and nonsmokers. There was a significant interaction effect (F(1, 673)= 5.03, p = 0.025) indicating that habenular volume was related to HbA1c in a manner that depended on smoking status. Among participants who were smokers (n = 120), higher HbA1c was associated with apparently larger habenular volume (ß = 6.74, standard error=2.36, p = 0.005). No such association between habenular volume and HbA1c was noted among participants who were nonsmokers (n = 573). DISCUSSION: Blood glucose levels over an extended time period, reflected by HbA1c, were correlated with habenular volume in smokers, consistent with a relationship between the habenula and blood glucose homeostasis in smokers. Future studies are needed to evaluate how habenular function relates to glycemic control in smokers and nonsmokers.

Addiction-related neuroadaptations following chronic nicotine exposure.

The addiction-relevant molecular, cellular, and behavioral actions of nicotine are derived from its stimulatory effects on neuronal nicotinic acetylcholine receptors (nAChRs) in the central nervous system. nAChRs expressed by dopamine-containing neurons in the ventral midbrain, most notably in the ventral tegmental area (VTA), contribute to the reward-enhancing properties of nicotine that motivate the use of tobacco products. nAChRs are also expressed by neurons in brain circuits that regulate aversion. In particular, nAChRs expressed by neurons in the medial habenula (mHb) and the interpeduncular nucleus (IPn) to which the mHb almost exclusively projects regulate the "set-point" for nicotine aversion and control nicotine intake. Different nAChR subtypes are expressed in brain reward and aversion circuits and nicotine intake is titrated to maximally engage reward-enhancing nAChRs while minimizing the recruitment of aversion-promoting nAChRs. With repeated exposure to nicotine, reward- and aversion-related nAChRs and the brain circuits in which they are expressed undergo adaptations that influence whether tobacco use will transition from occasional to habitual. Genetic variation that influences the sensitivity of addiction-relevant brain circuits to the actions of nicotine also influence the propensity to develop habitual tobacco use. Here, we review some of the key advances in our understanding of the mechanisms by which nicotine acts on brain reward and aversion circuits and the adaptations that occur in these circuits that may drive addiction to nicotine-containing tobacco products.

Mechanisms of Nicotine Addiction.

Tobacco smoking results in more than five million deaths each year and accounts for ∼90% of all deaths from lung cancer.3 Nicotine, the major reinforcing component of tobacco smoke, acts in the brain through the neuronal nicotinic acetylcholine receptors (nAChRs). The nAChRs are allosterically regulated, ligand-gated ion channels consisting of five membrane-spanning subunits. Twelve mammalian α subunits (α2-α10) and three ß subunits (ß2-ß4) have been cloned. The predominant nAChR subtypes in mammalian brain are those containing α4 and ß2 subunits (denoted as α4ß2* nAChRs). The α4ß2* nAChRs mediate many behaviors related to nicotine addiction and are the primary targets for currently approved smoking cessation agents. Considering the large number of nAChR subunits in the brain, it is likely that nAChRs containing subunits in addition to α4 and ß2 also play a role in tobacco smoking. Indeed, genetic variation in the CHRNA5-CHRNA3-CHRNB4 gene cluster, encoding the α5, α3, and ß4 nAChR subunits, respectively, has been shown to increase vulnerability to tobacco dependence and smoking-associated diseases including lung cancer. Moreover, mice, in which expression of α5 or ß4 subunits has been genetically modified, have profoundly altered patterns of nicotine consumption. In addition to the reinforcing properties of nicotine, the effects of nicotine on appetite, attention, and mood are also thought to contribute to establishment and maintenance of the tobacco smoking habit. Here, we review recent insights into the behavioral actions of nicotine, and the nAChR subtypes involved, which likely contribute to the development of tobacco dependence in smokers.