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.

Chronic administration of a norepinephrine antagonist prevents and partially reverses escalation of cocaine self-administration.

Anxiety is a critical component of the development and maintenance of drug addiction; however, anti-anxiety medications such as benzodiazepines and beta-blockers (ß-adrenergic receptor antagonists) are not used for the treatment of substance use disorder, except for the management of acute withdrawal syndrome. Preclinical studies have shown that beta-blockers may reduce stress-induced relapse; however, the effect of beta blockers on the escalation and maintenance of drug intake has not been tested. To address this issue, we chronically administered the ß-adrenergic receptor antagonist propranolol during the escalation or maintenance of cocaine intake in a model of extended access (6 h) to cocaine self-administration (0.5 mg/kg). The behavioural specificity of propranolol was tested using a non-drug reward (saccharin). Daily administration of propranolol (15 mg/kg) prevented the development of escalation of cocaine self-administration and partially reversed self-administration after the establishment of escalation of intake. Moreover, propranolol dose-dependently decreased the motivation for cocaine tested under a progressive ratio schedule of reinforcement during the development of escalation and after maintenance. Finally, propranolol administration had no effect on the escalation and maintenance of saccharin self-administration. These results demonstrate that chronic treatment with propranolol provides therapeutic efficacy in reducing cocaine self-administration during the development and after the establishment of escalation of cocaine self-administration in an animal model relevant to cocaine use disorder. These results suggest that beta blockers should be further investigated as a target for medication development for the treatment of cocaine use disorder.

Nociceptin attenuates the escalation of oxycodone self-administration by normalizing CeA-GABA transmission in highly addicted rats.

Approximately 25% of patients who are prescribed opioids for chronic pain misuse them, and 5 to 10% develop an opioid use disorder. Although the neurobiological target of opioids is well known, the molecular mechanisms that are responsible for the development of addiction-like behaviors in some but not all individuals are poorly known. To address this issue, we used a unique outbred rat population (heterogeneous stock) that better models the behavioral and genetic diversity that is found in humans. We characterized individual differences in addiction-like behaviors using an addiction index that incorporates the key criteria of opioid use disorder: escalated intake, highly motivated responding, and hyperalgesia. Using in vitro electrophysiological recordings in the central nucleus of the amygdala (CeA), we found that rats with high addiction-like behaviors (HA) exhibited a significant increase in γ-aminobutyric acid (GABA) transmission compared with rats with low addiction-like behaviors (LA) and naive rats. The superfusion of CeA slices with nociceptin/orphanin FQ peptide (N/OFQ; 500 nM), an endogenous opioid-like peptide, normalized GABA transmission in HA rats. Intra-CeA levels of N/OFQ were lower in HA rats than in LA rats. Intra-CeA infusions of N/OFQ (1 µg per site) reversed the escalation of oxycodone self-administration in HA rats but not in LA rats. These results demonstrate that the downregulation of N/OFQ levels in the CeA may be responsible for hyper-GABAergic tone in the CeA that is observed in individuals who develop addiction-like behaviors. Based on these results, we hypothesize that small molecules that target the N/OFQ system might be useful for the treatment of opioid use disorder.

Tsix-Mecp2 female mouse model for Rett syndrome reveals that low-level MECP2 expression extends life and improves neuromotor function.

Rett syndrome (RTT) is a severe neurodevelopmental disorder caused by a mutation in the X-linked methyl-CpG-binding protein 2 (MECP2). There is currently no disease-specific treatment, but MECP2 restoration through reactivation of the inactive X (Xi) has been of considerable interest. Progress toward an Xi-reactivation therapy has been hampered by a lack of suitable female mouse models. Because of cellular mosaicism due to random X-chromosome inactivation (XCI), Mecp2+/- heterozygous females develop only mild RTT. Here, we create an improved female mouse model by introducing a mutation in Tsix, the antisense regulator of XCI allelic choice. Tsix-Mecp2 mice show reduced MECP2 mosaicism and closely phenocopy the severely affected Mecp2-null males. Tsix-Mecp2 females demonstrate shortened lifespan, motor weakness, tremors, and gait disturbance. Intriguingly, they also exhibit repetitive behaviors, as is often seen in human RTT, including excessive grooming and biting that result in self-injury. With a Tsix allelic series, we vary MECP2 levels in brain and demonstrate a direct, but nonlinear correlation between MECP2 levels and phenotypic improvement. As little as 5-10% MECP2 restoration improves neuromotor function and extends lifespan five- to eightfold. Our study thus guides future pharmacological strategies and suggests that partial MECP2 restoration could have disproportionate therapeutic benefit.

A mixed modality approach towards Xi reactivation for Rett syndrome and other X-linked disorders.

The X-chromosome harbors hundreds of disease genes whose associated diseases predominantly affect males. However, a subset, including neurodevelopmental disorders, Rett syndrome (RTT), fragile X syndrome, and CDKL5 syndrome, also affects females. These disorders lack disease-specific treatment. Because female cells carry two X chromosomes, an emerging treatment strategy has been to reawaken the healthy allele on the inactive X (Xi). Here, we focus on methyl-CpG binding protein 2 (MECP2) restoration for RTT and combinatorially target factors in the interactome of Xist, the noncoding RNA responsible for X inactivation. We identify a mixed modality approach combining an Xist antisense oligonucleotide and a small-molecule inhibitor of DNA methylation, which, together, achieve 30,000-fold MECP2 up-regulation from the Xi in cultured cells. Combining a brain-specific genetic Xist ablation with short-term 5-aza-2'-deoxycytidine (Aza) treatment models the synergy in vivo without evident toxicity. The Xi is selectively reactivated. These experiments provide proof of concept for a mixed modality approach for treating X-linked disorders in females.

Systemic Administration of the Cyclin-Dependent Kinase Inhibitor (S)-CR8 Selectively Reduces Escalated Ethanol Intake in Dependent Rats.

BACKGROUND: Chronic exposure to ethanol (EtOH) and other drugs of abuse can alter the expression and activity of cyclin-dependent kinase 5 (CDK5) and its cofactor p35, but the functional implication of CDK5 signaling in the regulation of EtOH-related behaviors remains unknown. In the present study, we sought to determine whether CDK5 activity plays a role in the escalation of EtOH self-administration triggered by dependence. METHODS: We tested the effect of systemically administered (S)-CR8, a nonselective CDK inhibitor, on operant responding for EtOH or saccharin, a highly palatable reinforcer, in adult male Wistar rats. Half of the rats were made EtOH-dependent via chronic intermittent EtOH inhalation (CIE). We then sought to identify a possible neuroanatomical locus for the behavioral effect of (S)-CR8 by quantifying protein levels of CDK5 and p35 in subregions of the extended amygdala and prefrontal cortex from EtOH-naïve, nondependent, and dependent rats at the expected time of EtOH self-administration. We also analyzed the phosphorylation of 4 CDK5 substrates and of the CDK substrate consensus motif. RESULTS: (S)-CR8 dose-dependently reduced EtOH self-administration in dependent rats. It had no effect on water or saccharin self-administration, nor in nondependent rats. The abundance of CDK5 or p35 was not altered in any of the brain regions analyzed. In the bed nucleus of the stria terminalis, CDK5 abundance was negatively correlated with intoxication levels during EtOH vapor exposure but there was no effect of dependence on the phosphorylation ratio of CDK5 substrates. In contrast, EtOH dependence increased the phosphorylation of low-molecular-weight CDK substrates in the basolateral amygdala (BLA). CONCLUSIONS: The selective effect of (S)-CR8 on excessive EtOH intake has potential therapeutic value for the treatment of alcohol use disorders. Our data do not support the hypothesis that this effect would be mediated by the inhibition of up-regulated CDK5 activity in the extended amygdala nor prefrontal cortex. However, increased activity of CDKs other than CDK5 in the BLA may contribute to excessive EtOH consumption in alcohol dependence. Other (S)-CR8 targets may also be implicated.

A high-throughput small molecule screen identifies synergism between DNA methylation and Aurora kinase pathways for X reactivation.

X-chromosome inactivation is a mechanism of dosage compensation in which one of the two X chromosomes in female mammals is transcriptionally silenced. Once established, silencing of the inactive X (Xi) is robust and difficult to reverse pharmacologically. However, the Xi is a reservoir of >1,000 functional genes that could be potentially tapped to treat X-linked disease. To identify compounds that could reactivate the Xi, here we screened ∼367,000 small molecules in an automated high-content screen using an Xi-linked GFP reporter in mouse fibroblasts. Given the robust nature of silencing, we sensitized the screen by "priming" cells with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5azadC). Compounds that elicited GFP activity include VX680, MLN8237, and 5azadC, which are known to target the Aurora kinase and DNA methylation pathways. We demonstrate that the combinations of VX680 and 5azadC, as well as MLN8237 and 5azadC, synergistically up-regulate genes on the Xi. Thus, our work identifies a synergism between the DNA methylation and Aurora kinase pathways as being one of interest for possible pharmacological reactivation of the Xi.

A Thiolactone Strategy for Straightforward Synthesis of Disulfide-Linked Side-Chain-to-Tail Cyclic Peptides Featuring an N-Terminal Modification Handle.

The development of straightforward and versatile peptide cyclisation methods is highly desired to meet the demand for more stable peptide-based drugs. Herein, a new method for the synthesis of side-chain-to-tail cyclic peptides with the simultaneous introduction of an N-terminal handle, based on the introduction of an N-terminal thiolactone building block, is described. A primary amine liberates a homocysteine analogue from the thiolactone building block, which further enables cyclisation of the peptide through disulfide-bond formation with a C-terminal cysteamine. Postcyclisation modification can be achieved by using small bifunctional amines. Alternatively, the synthesis of lipopeptides is demonstrated through direct thiolactone opening with long-chain alkyl amines.

Triplex crosslinking through furan oxidation requires perturbation of the structured triple-helix.

Short oligonucleotides can selectively recognize duplexes by binding in the major groove thereby forming triplexes. Based on the success of our recently developed strategy for furan-based crosslinking in DNA duplexes, we here investigated for the first time the use of the furan-oxidation crosslink methodology for the covalent locking of triplex structures by an interstrand crosslink. It was shown that in a triplex context, although crosslinking yields are surprisingly low (to nonexistent) when targeting fully complementary duplexes, selective crosslinking can be achieved towards mismatched duplex sites at the interface of triplex to duplex structures. We show the promising potential of furan-containing probes for the selective detection of single-stranded regions within nucleic acids containing a variety of structural motifs.

A synthetic oligonucleotide model for evaluating the oxidation and crosslinking propensities of natural furan-modified DNA.

We have previously developed a crosslinking methodology for oligonucleotides based on the incorporation of furan moieties, which can be selectively oxidised to reactive intermediates that will quickly react with the opposite bases in DNA, forming toxic interstrand crosslinks (ICLs). Furan moieties also occur in natural DNA, as a result of oxidative stress. Moreover, the furan-containing degradation product of this modified DNA-kinetin-has been found to display beneficial anti-ageing effects. To investigate the apparent discrepancy between the effects of the synthetic and the natural furan modifications in DNA, a quick and easy postsynthetic method providing access to the natural modification in short synthetic oligonucleotides was developed. On checking for potential crosslinking propensity, we found that the furan moiety does indeed undergo oxidation, in this way functioning as an important scavenger for oxidative stress. The reactive intermediate, however, was shown to degrade without producing toxic crosslinked products.

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.

Toxicity inspired cross-linking for probing DNA-peptide interactions.

A cross-linking methodology for the study of DNA-protein interactions is described. The method is inspired by the metabolic activation of furans causing toxic DNA damage, including DNA-protein cross-links (DPC). The furan moiety, representing a latent functionality, is easily incorporated into oligonucleotides, and can be activated on demand to release a reactive aldehyde. Reaction with nucleophilic lysine side chains is shown to be distance-sensitive and allows for site-selective DPC formation.

Hyperconnectivity of Two Separate Long-Range Cholinergic Systems Contributes to the Reorganization of the Brain Functional Connectivity during Nicotine Withdrawal in Male Mice.

Chronic nicotine results in dependence with withdrawal symptoms on discontinuation of use, through desensitization of nicotinic acetylcholine receptors and altered cholinergic neurotransmission. Nicotine withdrawal is associated with increased whole-brain functional connectivity and decreased network modularity; however, the role of cholinergic neurons in those changes is unknown. To identify the contribution of nicotinic receptors and cholinergic regions to changes in the functional network, we analyzed the contribution of the main cholinergic regions to brain-wide activation of the immediate early-gene Fos during withdrawal in male mice and correlated these changes with the expression of nicotinic receptor mRNA throughout the brain. We show that the main functional connectivity modules included the main long-range cholinergic regions, which were highly synchronized with the rest of the brain. However, despite this hyperconnectivity, they were organized into two anticorrelated networks that were separated into basal forebrain-projecting and brainstem-thalamic-projecting cholinergic regions, validating a long-standing hypothesis of the organization of the brain cholinergic systems. Moreover, baseline (without nicotine) expression of Chrna2, Chrna3, Chrna10, and Chrnd mRNA of each brain region correlated with withdrawal-induced changes in Fos expression. Finally, by mining the Allen Brain mRNA expression database, we were able to identify 1755 gene candidates and three pathways (Sox2-Oct4-Nanog, JAK-STAT, and MeCP2-GABA) that may contribute to nicotine withdrawal-induced Fos expression. These results identify the dual contribution of the basal forebrain and brainstem-thalamic cholinergic systems to whole-brain functional connectivity during withdrawal; and identify nicotinic receptors and novel cellular pathways that may be critical for the transition to nicotine dependence.

Hyperconnectivity of two separate long-range cholinergic systems contributes to the reorganization of the brain functional connectivity during nicotine withdrawal in male mice.

Chronic nicotine results in dependence with withdrawal symptoms upon discontinuation of use, through desensitization of nicotinic acetylcholine receptors and altered cholinergic neurotransmission. Nicotine withdrawal is associated with increased whole-brain functional connectivity and decreased network modularity, however, the role of cholinergic neurons in those changes is unknown. To identify the contribution of nicotinic receptors and cholinergic regions to changes in the functional network, we analyzed the contribution of the main cholinergic regions to brain-wide activation of the immediate early-gene FOS during withdrawal in male mice and correlated these changes with the expression of nicotinic receptor mRNA throughout the brain. We show that the main functional connectivity modules included the main long-range cholinergic regions, which were highly synchronized with the rest of the brain. However, despite this hyperconnectivity they were organized into two anticorrelated networks that were separated into basal forebrain projecting and brainstem-thalamic projecting cholinergic regions, validating a long-standing hypothesis of the organization of the brain cholinergic systems. Moreover, baseline (without nicotine) expression of Chrna2 , Chrna3 , Chrna10 , and Chrnd mRNA of each brain region correlated with withdrawal-induced changes in FOS expression. Finally, by mining the Allen Brain mRNA expression database, we were able to identify 1755 gene candidates and three pathways (Sox2-Oct4-Nanog, JAK-STAT, and MeCP2-GABA) that may contribute to nicotine withdrawal-induced FOS expression. These results identify the dual contribution of the basal forebrain and brainstem-thalamic cholinergic systems to whole-brain functional connectivity during withdrawal; and identify nicotinic receptors and novel cellular pathways that may be critical for the transition to nicotine dependence. Significance Statement: Discontinuation of nicotine use in dependent users is associated with increased whole-brain activation and functional connectivity and leads to withdrawal symptoms. Here we investigated the contribution of the nicotinic cholinergic receptors and main cholinergic projecting brain areas in the whole-brain changes associated with withdrawal. This not only allowed us to visualize and confirm the previously described duality of the cholinergic brain system using this novel methodology, but also identify nicotinic receptors together with 1751 other genes that contribute, and could thus be targets for treatments against, nicotine withdrawal and dependence.

Abstinence from Escalation of Cocaine Intake Changes the microRNA Landscape in the Cortico-Accumbal Pathway.

Cocaine administration alters the microRNA (miRNA) landscape in the cortico-accumbal pathway. These changes in miRNA can play a major role in the posttranscriptional regulation of gene expression during withdrawal. This study aimed to investigate the changes in microRNA expression in the cortico-accumbal pathway during acute withdrawal and protracted abstinence following escalated cocaine intake. Small RNA sequencing (sRNA-seq) was used to profile miRNA transcriptomic changes in the cortico-accumbal pathway [infralimbic- and prelimbic-prefrontal cortex (IL and PL) and nucleus accumbens (NAc)] of rats with extended access to cocaine self-administration followed by an 18-h withdrawal or a 4-week abstinence. An 18-h withdrawal led to differential expression (fold-change > 1.5 and p < 0.05) of 21 miRNAs in the IL, 18 miRNAs in the PL, and two miRNAs in the NAc. The mRNAs potentially targeted by these miRNAs were enriched in the following pathways: gap junctions, neurotrophin signaling, MAPK signaling, and cocaine addiction. Moreover, a 4-week abstinence led to differential expression (fold-change > 1.5 and p < 0.05) of 23 miRNAs in the IL, seven in the PL, and five miRNAs in the NAc. The mRNAs potentially targeted by these miRNAs were enriched in pathways including gap junctions, cocaine addiction, MAPK signaling, glutamatergic synapse, morphine addiction, and amphetamine addiction. Additionally, the expression levels of several miRNAs differentially expressed in either the IL or the NAc were significantly correlated with addiction behaviors. Our findings highlight the impact of acute and protracted abstinence from escalated cocaine intake on miRNA expression in the cortico-accumbal pathway, a key circuit in addiction, and suggest developing novel biomarkers and therapeutic approaches to prevent relapse by targeting abstinence-associated miRNAs and their regulated mRNAs.

Single-nucleus genomics in outbred rats with divergent cocaine addiction-like behaviors reveals changes in amygdala GABAergic inhibition.

The amygdala processes positive and negative valence and contributes to addiction, but the cell-type-specific gene regulatory programs involved are unknown. We generated an atlas of single-nucleus gene expression and chromatin accessibility in the amygdala of outbred rats with high and low cocaine addiction-like behaviors following prolonged abstinence. Differentially expressed genes between the high and low groups were enriched for energy metabolism across cell types. Rats with high addiction index (AI) showed increased relapse-like behaviors and GABAergic transmission in the amygdala. Both phenotypes were reversed by pharmacological inhibition of the glyoxalase 1 enzyme, which metabolizes methylglyoxal-a GABAA receptor agonist produced by glycolysis. Differences in chromatin accessibility between high and low AI rats implicated pioneer transcription factors in the basic helix-loop-helix, FOX, SOX and activator protein 1 families. We observed opposite regulation of chromatin accessibility across many cell types. Most notably, excitatory neurons had greater accessibility in high AI rats and inhibitory neurons had greater accessibility in low AI rats.

Glucocorticoid Receptor-Regulated Enhancers Play a Central Role in the Gene Regulatory Networks Underlying Drug Addiction.

Substance abuse and addiction represent a significant public health problem that impacts multiple dimensions of society, including healthcare, the economy, and the workforce. In 2021, over 100,000 drug overdose deaths were reported in the US, with an alarming increase in fatalities related to opioids and psychostimulants. Understanding the fundamental gene regulatory mechanisms underlying addiction and related behaviors could facilitate more effective treatments. To explore how repeated drug exposure alters gene regulatory networks in the brain, we combined capped small (cs)RNA-seq, which accurately captures nascent-like initiating transcripts from total RNA, with Hi-C and single nuclei (sn)ATAC-seq. We profiled initiating transcripts in two addiction-related brain regions, the prefrontal cortex (PFC) and the nucleus accumbens (NAc), from rats that were never exposed to drugs or were subjected to prolonged abstinence after oxycodone or cocaine intravenous self-administration (IVSA). Interrogating over 100,000 active transcription start regions (TSRs) revealed that most TSRs had hallmarks of bonafide enhancers and highlighted the KLF/SP1, RFX, and AP1 transcription factors families as central to establishing brain-specific gene regulatory programs. Analysis of rats with addiction-like behaviors versus controls identified addiction-associated repression of transcription at regulatory enhancers recognized by nuclear receptor subfamily 3 group C (NR3C) factors, including glucocorticoid receptors. Cell-type deconvolution analysis using snATAC-seq uncovered a potential role of glial cells in driving the gene regulatory programs associated with addiction-related phenotypes. These findings highlight the power of advanced transcriptomics methods to provide insight into how addiction perturbs gene regulatory programs in the brain.

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.

Cannabidiol reduces withdrawal symptoms in nicotine-dependent rats.

RATIONALE: Cannabidiol (CBD) reduces craving in animal models of alcohol and cocaine use and is known to modulate nicotinic receptor function, suggesting that it may alleviate symptoms of nicotine withdrawal. However, preclinical evaluation of its efficacy is still lacking. OBJECTIVES: The goal of this study was to test the preclinical efficacy of a chronic CBD treatment in reducing nicotine dependence using measures of withdrawal symptoms including somatic signs, hyperalgesia, and weight gain during acute and protracted abstinence. METHODS: Male and female Wistar rats were made dependent on nicotine using osmotic minipumps (3.15 mg/kg/day) for 2 weeks, after which minipumps were removed to induce spontaneous withdrawal. Three groups received CBD injections at doses of 7.5, 15, and 30 mg/kg/day for 2 weeks, starting 1 week into chronic nicotine infusion. The control groups included rats with nicotine minipumps that received vehicle injections of sesame oil instead of CBD; rats implanted with saline minipumps received sesame oil injections (double vehicle) or the highest dose of CBD 30 mg/kg/day. Throughout the experiment, serum was collected for determination of CBD and nicotine concentrations, mechanical sensitivity threshold and withdrawal scores were measured, and body weight was recorded. RESULTS: CBD prevented rats from exhibiting somatic signs of withdrawal and hyperalgesia during acute and protracted abstinence. There was no dose-response observed for CBD, suggesting a ceiling effect at the doses used and the potential for lower effective doses of CBD. The saline minipump group did not show either somatic signs of withdrawal or hyperalgesia during acute and protracted abstinence, and the highest dose of CBD used (30 mg/kg/day) did not alter these results. CONCLUSIONS: This preclinical study suggests that using CBD as a strategy to alleviate the withdrawal symptoms upon nicotine cessation may be beneficial.

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/.