Acute nicotine activates orectic and inhibits anorectic brain regions in rats exposed to chronic nicotine.

Nicotine use produces psychoactive effects, and chronic use is associated with physiological and psychological symptoms of addiction. However, chronic nicotine use is known to decrease food intake and body weight gain, suggesting that nicotine also affects central metabolic and appetite regulation. We recently showed that acute nicotine self-administration in nicotine-dependent animals produces a short-term increase in food intake, contrary to its long-term decrease of feeding behavior. As feeding behavior is regulated by complex neural signaling mechanisms, this study aimed to test the hypothesis that nicotine intake in animals exposed to chronic nicotine may increase activation of pro-feeding regions and decrease activation of pro-satiety regions to produce the acute increase in feeding behavior. FOS immunohistochemistry revealed that acute nicotine intake in nicotine self-administering animals increased activation of the pro-feeding arcuate and lateral hypothalamic nuclei and decreased activation of the pro-satiety parabrachial nucleus. Regional correlational analysis also showed that acute nicotine changes the functional connectivity of the hunger/satiety network. Further dissection of the role of the arcuate nucleus using electrophysiology found that putative POMC neurons in animals given chronic nicotine exhibited decreased firing following acute nicotine application. These brain-wide central signaling changes may contribute to the acute increase in feeding behavior we see in rats after acute nicotine and provide new areas of focus for studying both nicotine addiction and metabolic regulation.

Acute nicotine intake increases feeding behavior through decreasing glucagon signaling in dependent male and female rats.

Chronic use of nicotine is known to dysregulate metabolic signaling through altering circulating levels of feeding-related hormones, contributing to the onset of disorders like type 2 diabetes. However, little is known about the acute effects of nicotine on hormonal signaling. We previously identified an acute increase in food intake following acute nicotine, and we sought to determine whether this behavior was due to a change in hormone levels. We first identified that acute nicotine injection produces an increase in feeding behavior in dependent rats, but not nondependent rats. We confirmed that chronic nicotine use increases circulating levels of insulin, leptin, and ghrelin, and these correlate with rats' body weight and food intake. Acute nicotine injection in dependent animals decreased circulating GLP-1 and glucagon levels, and administration of glucagon prior to acute nicotine injection prevented the acute increase in feeding behavior. Thus, acute nicotine injection increases feeding behavior in dependent rats by decreasing glucagon signaling.

Directed evolution unlocks oxygen reactivity for a nicotine-degrading flavoenzyme.

The flavoenzyme nicotine oxidoreductase (NicA2) is a promising injectable treatment to aid in the cessation of smoking, a behavior responsible for one in ten deaths worldwide. NicA2 acts by degrading nicotine in the bloodstream before it reaches the brain. Clinical use of NicA2 is limited by its poor catalytic activity in the absence of its natural electron acceptor CycN. Without CycN, NicA2 is instead oxidized slowly by dioxygen (O2), necessitating unfeasibly large doses in a therapeutic setting. Here, we report a genetic selection strategy that directly links CycN-independent activity of NicA2 to growth of Pseudomonas putida S16. This selection enabled us to evolve NicA2 variants with substantial improvement in their rate of oxidation by O2. The encoded mutations cluster around a putative O2 tunnel, increasing flexibility and accessibility to O2 in this region. These mutations further confer desirable clinical properties. A variant form of NicA2 is tenfold more effective than the wild type at degrading nicotine in the bloodstream of rats.

Leptin Protects Against the Development and Expression of Cocaine Addiction-Like Behavior in Heterogeneous Stock Rats.

Cocaine affects food intake, metabolism and bodyweight. It has been hypothesized that feeding hormones like leptin play a role in this process. Preclinical studies have shown a mutually inhibitory relationship between leptin and cocaine, with leptin also decreasing the rewarding effects of cocaine intake. But prior studies have used relatively small sample sizes and did not investigate individual differences in genetically heterogeneous populations. Here, we examined whether the role of individual differences in bodyweight and blood leptin level are associated with high or low vulnerability to addiction-like behaviors using data from 306 heterogeneous stock rats given extended access to intravenous self-administration of cocaine and 120 blood samples from 60 of these animals, that were stored in the Cocaine Biobank. Finally, we tested a separate cohort to evaluate the causal effect of exogenous leptin administration on cocaine seeking. Bodyweight was reduced due to cocaine self-administration in males during withdrawal and abstinence, but was increased in females during abstinence. However, bodyweight was not correlated with addiction-like behavior vulnerability. Blood leptin levels after ∼6 weeks of cocaine self-administration did not correlate with addiction-like behaviors, however, baseline blood leptin levels before any access to cocaine negatively predicted addiction-like behaviors 6 weeks later. Finally, leptin administration in a separate cohort of 59 animals reduced cocaine seeking in acute withdrawal and after 7 weeks of protracted abstinence. These results demonstrate that high blood leptin level before access to cocaine may be a protective factor against the development of cocaine addiction-like behavior and that exogenous leptin reduces the motivation to take and seek cocaine. On the other hand, these results also show that blood leptin level and bodyweight changes in current users are not relevant biomarkers for addiction-like behaviors.

Microstructural meal pattern analysis reveals a paradoxical acute increase in food intake after nicotine despite its long-term anorexigenic effects.

RATIONALE: Nicotine consumption in both human and animal studies has been strongly associated with changes in feeding-related behaviors and metabolism. The current dogma is that nicotine is an anorexic agent that decreases food intake and increases metabolism, leading to decreased body weight gain. However, there are conflicting reports about the acute effects of nicotine on hunger in humans. No study has reported nicotine-induced decreases in food intake within minutes of consumption, suggesting that our understanding of the pharmacological effects of nicotine on appetite and feeding may be incorrect. OBJECTIVES: The aim of this study was to elucidate effects of acute nicotine intake on feeding and drinking behavior. METHODS: Adult male Wistar rats were trained to intravenously self-administer nicotine. Microstructural and macrostructural behavioral analyses were employed to look at changes in food and water intake at different timescales. RESULTS: At the macrostructural level (hours to days), nicotine decreased body weight gain, decreased feeding, and was associated with increases in feeding and body weight gain during abstinence. At the microstructural level (seconds to minutes), nicotine increased feeding and drinking behavior during the first 5 min after nicotine self-administration. This effect was also observed in animals that passively received nicotine, but the effect was not observed in animals that self-administered saline or passively received saline. CONCLUSIONS: These results challenge the notion that the initial pharmacological effect of nicotine is anorexigenic and paradoxically suggest that an acute increase in food intake minutes after exposure to nicotine may contribute to the long-term anorexigenic effects of nicotine.

The Cocaine and Oxycodone Biobanks, Two Repositories from Genetically Diverse and Behaviorally Characterized Rats for the Study of Addiction.

The rat oxycodone and cocaine biobanks contain samples that vary by genotypes (by using genetically diverse genotyped HS rats), phenotypes (by measuring addiction-like behaviors in an advanced SA model), timepoints (samples are collected longitudinally before, during, and after SA, and terminally at three different timepoints in the addiction cycle: intoxication, withdrawal, and abstinence or without exposure to drugs through age-matched naive rats), samples collected (organs, cells, biofluids, feces), preservation (paraformaldehyde-fixed, snap-frozen, or cryopreserved) and application (proteomics, transcriptomics, microbiomics, metabolomics, epigenetics, anatomy, circuitry analysis, biomarker discovery, etc.Substance use disorders (SUDs) are pervasive in our society and have substantial personal and socioeconomical costs. A critical hurdle in identifying biomarkers and novel targets for medication development is the lack of resources for obtaining biological samples with a detailed behavioral characterization of SUD. Moreover, it is nearly impossible to find longitudinal samples. As part of two ongoing large-scale behavioral genetic studies in heterogeneous stock (HS) rats, we have created two preclinical biobanks using well-validated long access (LgA) models of intravenous cocaine and oxycodone self-administration (SA) and comprehensive characterization of addiction-related behaviors. The genetic diversity in HS rats mimics diversity in the human population and includes individuals that are vulnerable or resilient to compulsive-like responding for cocaine or oxycodone. Longitudinal samples are collected throughout the experiment, before exposure to the drug, during intoxication, acute withdrawal, and protracted abstinence, and include naive, age-matched controls. Samples include, but are not limited to, blood plasma, feces and urine, whole brains, brain slices and punches, kidney, liver, spleen, ovary, testis, and adrenal glands. Three preservation methods (fixed in formaldehyde, snap-frozen, or cryopreserved) are used to facilitate diverse downstream applications such as proteomics, metabolomics, transcriptomics, epigenomics, microbiomics, neuroanatomy, biomarker discovery, and other cellular and molecular approaches. To date, >20,000 samples have been collected from over 1000 unique animals and made available free of charge to non-profit institutions through https://www.cocainebiobank.org/ and https://www.oxycodonebiobank.org/.

Metabolic adaptation to calorie restriction.

Calorie restriction (CR) enhances health span (the length of time that an organism remains healthy) and increases longevity across species. In mice, these beneficial effects are partly mediated by the lowering of core body temperature that occurs during CR. Conversely, the favorable effects of CR on health span are mitigated by elevating ambient temperature to thermoneutrality (30°C), a condition in which hypothermia is blunted. In this study, we compared the global metabolic response to CR of mice housed at 22°C (the standard housing temperature) or at 30°C and found that thermoneutrality reverted 39 and 78% of total systemic or hypothalamic metabolic variations caused by CR, respectively. Systemic changes included pathways that control fuel use and energy expenditure during CR. Cognitive computing-assisted analysis of these metabolomics results helped to prioritize potential active metabolites that modulated the hypothermic response to CR. Last, we demonstrated with pharmacological approaches that nitric oxide (NO) produced through the citrulline-NO pathway promotes CR-triggered hypothermia and that leucine enkephalin directly controls core body temperature when exogenously injected into the hypothalamus. Because thermoneutrality counteracts CR-enhanced health span, the multiple metabolites and pathways altered by thermoneutrality may represent targets for mimicking CR-associated effects.

Validation of a nicotine vapor self-administration model in rats with relevance to electronic cigarette use.

The debate about electronic cigarettes is dividing healthcare professionals, policymakers, manufacturers, and communities. A key limitation in our understanding of the cause and consequences of vaping is the lack of animal models of nicotine vapor self-administration. Here, we developed a novel model of voluntary electronic cigarette use in rats using operant behavior. We found that rats voluntarily exposed themselves to nicotine vapor to the point of reaching blood nicotine levels that are similar to humans. The level of responding on the active (nicotine) lever was similar to the inactive (air) lever and lower than the active lever that was associated with vehicle (polypropylene glycol/glycerol) vapor, suggesting low positive reinforcing effects and low nicotine vapor discrimination. Lever pressing behavior with nicotine vapor was pharmacologically prevented by the α4ß2 nicotinic acetylcholine receptor partial agonist and α7 receptor full agonist varenicline in rats that self-administered nicotine but not vehicle vapor. Moreover, 3 weeks of daily (1 h) nicotine vapor self-administration produced addiction-like behaviors, including somatic signs of withdrawal, allodynia, anxiety-like behavior, and relapse-like behavior after 3 weeks of abstinence. Finally, 3 weeks of daily (1 h) nicotine vapor self-administration produced cardiopulmonary abnormalities and changes in α4, α3, and ß2 nicotinic acetylcholine receptor subunit mRNA levels in the nucleus accumbens and medial prefrontal cortex. These findings validate a novel animal model of nicotine vapor self-administration in rodents with relevance to electronic cigarette use in humans and highlight the potential addictive properties and harmful effects of chronic nicotine vapor self-administration.

Depletion of the Microbiome Alters the Recruitment of Neuronal Ensembles of Oxycodone Intoxication and Withdrawal.

Substance use disorders have a complex etiology. Genetics, the environment, and behavior all play a role in the initiation, escalation, and relapse of drug use. Recently, opioid use disorder has become a national health crisis. One aspect of opioid addiction that has yet to be fully examined is the effects of alterations of the microbiome and gut-brain axis signaling on central nervous system activity during opioid intoxication and withdrawal. The effect of microbiome depletion on the activation of neuronal ensembles was measured by detecting Fos-positive (Fos+) neuron activation during intoxication and withdrawal using a rat model of oxycodone dependence. Daily oxycodone administration (2 mg/kg) increased pain thresholds and increased Fos+ neurons in the basolateral amygdala (BLA) during intoxication, with a decrease in pain thresholds and increase in Fos+ neurons in the periaqueductal gray (PAG), central nucleus of the amygdala (CeA), locus coeruleus (LC), paraventricular nucleus of the thalamus (PVT), agranular insular cortex (AI), bed nucleus of the stria terminalis (BNST), and lateral habenula medial parvocellular region during withdrawal. Microbiome depletion produced widespread but region- and state-specific changes in neuronal ensemble activation. Oxycodone intoxication and withdrawal also increased functional connectivity among brain regions. Microbiome depletion resulted in a decorrelation of this functional network. These data indicate that microbiome depletion by antibiotics produces widespread changes in the recruitment of neuronal ensembles that are activated by oxycodone intoxication and withdrawal, suggesting that the gut microbiome may play a role in opioid use and dependence. Future studies are needed to better understand the molecular, neurobiological, and behavioral effects of microbiome depletion on addiction-like behaviors.

Role of corticotropin-releasing factor in alcohol and nicotine addiction.

The two most prevalent substance use disorders involve alcohol and nicotine, which are often co-abused. Robust preclinical and translational evidence indicates that individuals initiate drug use for the acute rewarding effects of the substance. The development of negative emotional states is key for the transition from recreational use to substance use disorders as subjects seek the substance to obtain relief from the negative emotional states of acute withdrawal and protracted abstinence. The neuropeptide corticotropin-releasing factor (CRF) is a major regulator of the brain stress system and key in the development of negative affective states. The present review examines the role of CRF in preclinical models of alcohol and nicotine abuse and explores links between CRF and anxiety-like, dysphoria-like, and other negative affective states. Finally, the present review discusses preclinical models of nicotine and alcohol use with regard to the CRF system, advances in molecular and genetic manipulations of CRF, and the importance of examining both males and females in this field of research.

Activation of Kappa Opioid Receptor Regulates the Hypothermic Response to Calorie Restriction and Limits Body Weight Loss.

Mammals maintain a nearly constant core body temperature (Tb) by balancing heat production and heat dissipation. This comes at a high metabolic cost that is sustainable if adequate calorie intake is maintained. When nutrients are scarce or experimentally reduced such as during calorie restriction (CR), endotherms can reduce energy expenditure by lowering Tb [1-6]. This adaptive response conserves energy, limiting the loss of body weight due to low calorie intake [7-10]. Here we show that this response is regulated by the kappa opioid receptor (KOR). CR is associated with increased hypothalamic levels of the endogenous opioid Leu-enkephalin, which is derived from the KOR agonist precursor dynorphin [11]. Pharmacological inhibition of KOR, but not of the delta or the mu opioid receptor subtypes, fully blocked CR-induced hypothermia and increased weight loss during CR independent of calorie intake. Similar results were seen with DIO mice subjected to CR. In contrast, inhibiting KOR did not change Tb in animals fed ad libitum (AL). Chemogenetic inhibition of KOR neurons in the hypothalamic preoptic area reduced the CR-induced hypothermia, whereas chemogenetic activation of prodynorphin-expressing neurons in the arcuate or the parabrachial nucleus lowered Tb. These data indicate that KOR signaling is a pivotal regulator of energy homeostasis and can affect body weight during dieting by modulating Tb and energy expenditure.

Gonadal hormones influence core body temperature during calorie restriction.

During calorie restriction (CR), endotherms adjust several physiological processes including the decrease of core body temperature (Tb) and reduction of energy expenditure. We recently found that CR-induced hypothermia is regulated in a sex-dependent manner in mice with lowered central insulin-like growth factor receptor signaling. Here, we describe the contribution of sex hormones to CR-induced hypothermia in wild type C57BL6 mice by measuring Tb of female and male mice following bilateral gonadectomy and hormonal replacement. Specifically, we evaluated the effects of progesterone (P4), 17-ß estradiol (E2), a combination of both (P4 + E2) in females and of 5-α dihydrotestosterone (5-α DHT) in males. Gonadectomy resulted in an earlier and stronger CR-induced hypothermia in both sexes. These effects were fully antagonized in females by E2 replacement, but not by P4, which had only minor and partial effects when used alone and did not prevent the action of E2 during CR when both hormones were given in combination. 5-α-DHT had only minor and transient effects on preventing the reduction of Tb during CR on gonadectomized male mice. These findings indicate that gonadal hormones contribute to sex-specific regulation of Tb and energy expenditure when nutrient availability is scarce. Abbreviations: AL: ad libitum; ANOVA: analysis of variance; CR: calorie restriction; E2: 17-ß estradiol; GNX: gonadectomy or gonadectomized; IGF-1R: insulin-like growth factor 1 receptor; POA: preoptic area; P4: progesterone; RM: repeated measures; SD: standard deviation; SEM: standard error of mean; Tb: core body temperature; WT: wildtype; 5-α DHT: 5-α dihydrotestosterone.