Much remains unknown about the etiology of compulsion-like alcohol drinking, where consumption persists despite adverse consequences. The role of the anterior insula (AIC) in emotion, motivation, and interoception makes this brain region a likely candidate to drive challenge-resistant behavior, including compulsive drinking. Indeed, subcortical projections from the AIC promote compulsion-like intake in rats and are recruited in heavy-drinking humans during compulsion for alcohol, highlighting the importance of and need for more information about AIC activity patterns that support aversion-resistant responding. Single-unit activity was recorded in the AIC from 15 male rats during alcohol-only and compulsion-like consumption. We found three sustained firing phenotypes, sustained-increase, sustained-decrease, and drinking-onset cells, as well as several firing patterns synchronized with licking. While many AIC neurons had session-long activity changes, only neurons with firing increases at drinking onset had greater activity under compulsion-like conditions. Further, only cells with persistent firing increases maintained activity during pauses in licking, suggesting roles in maintaining drive for alcohol during breaks. AIC firing was not elevated during saccharin drinking, similar to lack of effect of AIC inhibition on sweet fluid intake in many studies. In addition, we observed subsecond changes in AIC neural activity tightly entrained to licking. One lick-synched firing pattern (determined for all licks in a session) predicted compulsion-like drinking, while a separate lick-associated pattern correlated with greater consumption across alcohol intake conditions. Collectively, these data provide a more integrated model for the role of AIC firing in compulsion-like drinking, with important relevance for how the AIC promotes sustained motivated responding more generally.

Alcohol Use Disorders (AUD) is characterized by compulsion-like alcohol drinking (CLAD), where intake despite negative consequences can be a major clinical obstacle. With few treatment options available for AUD, there is a significant need for novel therapies. The noradrenergic system is an important hub for regulating stress responses and maladaptive drives for alcohol. Studies have shown that drugs targeting alpha 1 adrenenergic receptors (ARs) may represent a pharmacological treatment for pathological drinking. However, the involvement of 3 ARs for treating human drinking has received scant investigation, and thus we sought to provide pre-clinical validation for possible AR utility for CLAD by analyzing whether 3 AR antagonists propranolol (31/2), betaxolol (31), and ICI, 118,551 (32) impacted CLAD and alcohol-only drinking (AOD) in male Wistar rats. We found that the highest dose of propranolol tested systemically (10 mg/kg) reduced alcohol drinking, while 5 mg/kg propranolol reduced drinking with a trend to impact CLAD more than AOD, and with no effects of 2.5 mg/kg. Betaxolol (2.5 mg/kg) also decreased drinking, while ICI 118.551 had no effects. Also, while AR compounds might have utility for AUD, they can also lead to undesirable side effects. Here, a combination of ineffective doses of propranolol and prazosin reduced both CLAD and AOD. Finally, we investigated the effect of propranolol and betaxolol in two brain areas related to pathological drinking, the anterior insula (aINS) and medial prefrontal cortex (mPFC). Surprisingly, propranolol (1-10 mu g) in aINS or mPFC did not affect CLAD or AOD. Together, our findings provide new pharmacological insights into noradrenergic regulation of alcohol consumption, which may inform AUD therapy.

ABSTRACT Compulsive-like alcohol drinking (CLAD), where intake persists despite adverse consequences, is often a core facet of alcohol use disorder. Recent work sheds light on underlying mechanisms, but much remains unknown about CLAD etiology. Previously, we showed that projections from anterior insula (aINS), a central mediator of emotion, motivation, and interoception, promote CLAD in rodents, and heavy human drinkers exhibit similar insula-circuit recruitment under compulsion-like conditions. However, global aINS inhibition also reduces alcohol-only drinking (AOD), and one major obstacle is the lack of information on aINS firing patterns that could promote different aspects of intake. Here, we recorded single-unit activity in right aINS from 15 rats during AOD or CLAD (10mg/L or 60mg/L quinine in alcohol). Neurons with a sustained-increase or sustained-decrease phenotype (SIP, SDP) showed no firing differences across drinking conditions. In contrast, aINS neurons with a phenotype of strong firing increase at initiation of responding (IRP) showed significantly greater activity across the rest of licking during CLAD versus AOD, concurring with our previous behavioral findings suggesting quick evaluation and response strategy adjustment under CLAD. There were also no condition-related differences in firing-phenotype abundance. Further, total responding only correlated with abundance of SDP cells, but SDP firing returned to baseline during pauses in licking, while IRP and SIP sustained responding through pauses in licking. Thus, only aINS cells with a particular strong firing at licking onset showd greater sustained responding under compulsion-like conditions, while other cells likely promoted drinking more generally, providing critical new information about how aINS activity could promote alcohol consumption under different drinking conditions.

With the substantial social and medical burden of addiction, there is considerable interest in understanding risk factors that increase the development of addiction. A key feature of alcohol use disorder (AUD) is compulsive alcohol (EtOH) drinking, where EtOH drinking becomes “inflexible” after chronic intake, and animals, such as humans with AUD, continue drinking despite aversive consequences. Further, since there is a heritable component to AUD risk, some work has focused on genetically-selected, EtOH-preferring rodents, which could help uncover critical mechanisms driving pathological intake. In this regard, aversion-resistant drinking (ARD) takes >1 month to develop in outbred Wistar rats (and perhaps Sardinian-P EtOH-preferring rats). However, ARD has received limited study in Indiana P-rats, which were selected for high EtOH preference and exhibit factors that could parallel human AUD (including front-loading and impulsivity). Here, we show that P-rats rapidly developed compulsion-like responses for EtOH; 0.4 g/L quinine in EtOH significantly reduced female and male intake on the first day of exposure but had no effect after one week of EtOH drinking (15% EtOH, 24 h free-choice paradigm). Further, after 4–5 weeks of EtOH drinking, males but not females showed resistance to even higher quinine (0.5 g/L). Thus, P-rats rapidly developed ARD for EtOH, but only males developed even stronger ARD with further intake. Finally, rats strongly reduced intake of quinine-adulterated water after 1 or 5 weeks of EtOH drinking, suggesting no changes in basic quinine sensitivity. Thus, modeling ARD in P-rats may provide insight into mechanisms underlying genetic predispositions for compulsive drinking and lead to new treatments for AUDs.

Alcohol Use Disorders (AUD) is characterized by compulsion-like alcohol drinking (CLAD), and this intake despite negative consequences can be a major clinical obstacle. With the quite limited treatment options available for AUD, there is a significant and critical unmet need for novel therapies. The noradrenergic system is an important hub for the stress response as well as maladaptive drives for alcohol, and pre-clinical (including our own) and clinical studies have shown that drugs targeting the α1 adrenenergic receptors (ARs) may represent a pharmacological treatment for pathological drinking. However, the involvement of β ARs for treating human drinking AUD has received somewhat scant investigation, and we sought to provide pre-clinical validation for possible AR utility for CLAD. Thus, we first examined whether β AR antagonist propranolol, betaxolol (β1), and ICI, 118 551 (β2) impacted compulsion-like intake and alcohol-only drinking (AOD) in male Wistar rats through systemic injections. The systemic highest dose of propranolol (10mg/kg) reduced both AOD and CLAD. 5mg/kg propranolol affected CLAD more than AOD, with no effects of 2.5mg/kg. Similar to propranolol, betaxolol also only decreased CLAD at the lower dose (2.5mg/kg). ICI 118.551 had no effects, suggesting propranolol regulates alcohol intake through β1. Also, while AR compounds might have utility for AUD, these compounds can also lead to undesirable cardiovascular system side effects; thus, any strategy incorporating lower doses of these compounds to reduce drinking could have broad utility. Importantly, here we found that a combination of ineffective doses of propranolol and prazosin administrated together did reduce both CLAD and AOD. Finally, we investigated the effect of propranolol and betaxolol into two brain areas related to pathological drinking, the anterior insula (aINS) and medial prefrontal cortex (mPFC). Surprisingly, propranolol (1-10μg) in aINS or mPFC did not affect CLAD or AOD (although with a trend for aINS betaxolol to impact CLAD), suggesting propranolol regulation of alcohol drinking through a target other than aINS and mPFC. Together, our findings provide new pharmacological insights into noradrenergic regulation of alcohol consumption, which may inform AUD therapy.

Despite the rising prevalence of methadone treatment in pregnant women with opioid use disorder, the effects of methadone on neurobehavioral development remain unclear. We developed a translational mouse model of prenatal methadone exposure (PME) that resembles the typical pattern of opioid use by pregnant women who first use oxycodone then switch to methadone maintenance pharmacotherapy, and subsequently become pregnant while maintained on methadone. We investigated the effects of PME on physical development, sensorimotor behavior, and motor neuron properties using a multidisciplinary approach of physical, biochemical, and behavioral assessments along with brain slice electrophysiology and in vivo magnetic resonance imaging. Methadone accumulated in the placenta and fetal brain, but methadone levels in offspring dropped rapidly at birth which was associated with symptoms and behaviors consistent with neonatal opioid withdrawal. PME produced substantial impairments in offspring physical growth, activity in an open field, and sensorimotor milestone acquisition. Furthermore, these behavioral alterations were associated with reduced neuronal density in the motor cortex and a disruption in motor neuron intrinsic properties and local circuit connectivity. The present study adds to the limited body of work examining PME by providing a comprehensive, translationally relevant characterization of how PME disrupts offspring physical and neurobehavioral development.

Rationale: The rate of cannabinoid intake by those with alcohol use disorder (AUD) exceeds that of the general public. The high prevalence of co-abuse of alcohol and cannabis has been postulated to be predicated upon both a common predisposing genetic factor and the interaction of the drugs within the organism. The current experiments examined the effects of cannabinoids in an animal model of AUD. Objectives: The present study assessed the reinforcing properties of a cannabinoid receptor 1 (CB1) agonist self-administered directly into the nucleus accumbens shell (AcbSh) in female Wistar and alcohol-preferring (P) rats. Methods: Following guide cannulae surgery aimed at AcbSh, subjects were placed in an operant box equipped with an 'active lever' (fixed ratio 1; FR1) that caused the delivery of the infusate and an 'inactive lever' that did not. Subjects were arbitrarily assigned to one of seven groups that self-administered either artificial cerebrospinal fluid (aCSF), or 3.125, 6.25, 12.5, or 25 pmol/100 nl of O-1057, a water-soluble CB1 agonist, dissolved in aCSF. The first four sessions of acquisition are followed by aCSF only infusates in sessions 5 and 6 during extinction, and finally the acquisition dose of infusate during session 7 as reinstatement. Results: The CB1 agonist was self-administered directly into the AcbSh. P rats self-administered the CB1 agonist at lower concentrations and at higher rates compared to Wistar rats. Conclusions: Overall, the data indicate selective breeding for high alcohol preference has produced rats divergent in response to cannabinoids within the brain reward pathway. The data support the hypothesis that there can be common genetic factors influencing drug addiction.

Alcohol use disorders (AUDs) cause serious problems in society and few effective treatments are available. Caenorhabditis elegans (C. elegans) is an excellent invertebrate model to study the neurobiological basis of human behavior with a conserved, fully tractable genome, and a short generation time for fast generation of data at a fraction of the cost of other organisms. C. elegans demonstrate movement toward, and concentration-dependent self-exposure to various psychoactive drugs. The discovery of opioid receptors in C. elegans provided the impetus to test the hypothesis that C. elegans may be used as a medications screen to identify new AUD treatments. We tested the effects of naltrexone, an opioid antagonist and effective treatment for AUDs, on EtOH preference in C. elegans. Six-well agar test plates were prepared with EtOH placed in a target zone on one side and water in the opposite target zone of each well. Worms were treated with naltrexone before EtOH preference testing and then placed in the center of each well. Wild-type worms exhibited a concentration-dependent preference for 50, 70 and 95% EtOH. Naltrexone blocked acute EtOH preference, but had no effect on attraction to food or benzaldehyde in wild-type worms. Npr-17 opioid receptor knockout mutants did not display a preference for EtOH. In contrast, npr-17 opioid receptor rescue mutants exhibited significant EtOH preference behavior, which was attenuated by naltrexone. Chronic EtOH exposure induced treatment resistance and compulsive-like behavior. These data indicate that C. elegans can serve as a model system to identify compounds to treat AUDs.

The nematode Caenorhabditis elegans (C. elegans) is a popular invertebrate model organism to study neurobiological disease states. This is due in part to the intricate mapping of all neurons and synapses of the entire animal, the wide availability of mutant strains, and the genetic and molecular tools that can be used to manipulate the genome and gene expression. We have shown that, C. elegans develops a conditioned preference for cues that had previously been paired with either cocaine or methamphetamine exposure that is dependent on dopamine neurotransmission, similar to findings using place conditioning with rats and mice. In the current study, we show C. elegans also display a preference for, and self-exposure to, cocaine and nicotine. This substance of abuse (SOA) preference response can be selectively blocked by pretreatment with naltrexone and is consistent with the recent discovery of an opioid receptor system in C. elegans. In addition, pre-exposure to the smoking cessation treatment varenicline also inhibits self-exposure to nicotine. Exposure to concentrations of treatments that inhibit SOA preference/self-exposure did not induce any significant inhibition of locomotor activity or affect food or benzaldehyde chemotaxis. These data provide predictive validity for the development of high-throughput C. elegans behavioral medication screens. These screens could enable fast and accurate generation of data to identify compounds that may be effective in treating human addiction. The successful development and validation of such models would introduce powerful and novel tools in the search for new pharmacological treatments for substance use disorders, and provide a platform to study the mechanisms that underlie addictions.

Drug addiction takes a massive toll on society. Novel animal models are needed to test new treatments and understand the basic mechanisms underlying addiction. Rodent models have identified the neurocircuitry involved in addictive behavior and indicate that rodents possess some of the same neurobiologic mechanisms that mediate addiction in humans. Recent studies indicate that addiction is mechanistically and phylogenetically ancient and many mechanisms that underlie human addiction are also present in invertebrates. The nematode Caenorhabditis elegans has conserved neurobiologic systems with powerful molecular and genetic tools and a rapid rate of development that enables cost-effective translational discovery. Emerging evidence suggests that C. elegans is an excellent model to identify molecular mechanisms that mediate drug-induced behavior and potential targets for medications development for various addictive compounds. C. elegans emit many behaviors that can be easily quantitated including some that involve interactions with the environment. Ethanol (EtOH) is the best-studied drug-of-abuse in C. elegans and at least 50 different genes/targets have been identified as mediating EtOH's effects and polymorphisms in some orthologs in humans are associated with alcohol use disorders. C. elegans has also been shown to display dopamine and cholinergic system-dependent attraction to nicotine and demonstrate preference for cues previously associated with nicotine. Cocaine and methamphetamine have been found to produce dopamine-dependent reward-like behaviors in C. elegans. These behavioral tests in combination with genetic/molecular manipulations have led to the identification of dozens of target genes/systems in C. elegans that mediate drug effects. The one target/gene identified as essential for drug-induced behavioral responses across all drugs of abuse was the cat-2 gene coding for tyrosine hydroxylase, which is consistent with the role of dopamine neurotransmission in human addiction. Overall, C. elegans can be used to model aspects of drug addiction and identify systems and molecular mechanisms that mediate drug effects. The findings are surprisingly consistent with analogous findings in higher-level organisms. Further, model refinement is warranted to improve model validity and increase utility for medications development.

Methamphetamine (MAP) addiction is substantially prevalent in today's society, resulting in thousands of deaths and costing billions of dollars annually. Despite the potential deleterious consequences, few studies have examined the long-term effects of embryonic MAP exposure. Using the invertebrate nematode Caenorhabditis elegans allows for a controlled analysis of behavioral and neurochemical changes due to early developmental drug exposure. The objective of the current study was to determine the long-term behavioral and neurochemical effects of embryonic exposure to MAP in C. elegans. In addition, we sought to improve our conditioning and testing procedures by utilizing liquid filtration, as opposed to agar, and smaller, 6-well testing plates to increase throughput. Wild-type N2 C. elegans were embryonically exposed to 50 μM MAP. Using classical conditioning, adult-stage C. elegans were conditioned to MAP (17 and 500 μM) in the presence of either sodium ions (Na+) or chloride ions (Cl-) as conditioned stimuli (CS+/CS-). Following conditioning, a preference test was performed by placing worms in 6-well test plates spotted with the CS+ and CS- at opposite ends of each well. A preference index was determined by counting the number of worms in the CS+ target zone divided by the total number of worms in the CS+ and CS- target zones. A food conditioning experiment was also performed in order to determine whether embryonic MAP exposure affected food conditioning behavior. For the neurochemical experiments, adult worms that were embryonically exposed to MAP were analyzed for dopamine (DA) content using high-performance liquid chromatography. The liquid filtration conditioning procedure employed here in combination with the use of 6-well test plates significantly decreased the time required to perform these experiments and ultimately increased throughput. The MAP conditioning data found that pairing an ion with MAP at 17 or 500 μM significantly increased the preference for that ion (CS+) in worms that were not pre-exposed to MAP. However, worms embryonically exposed to MAP did not exhibit significant drug cue conditioning. The inability of MAP-exposed worms to condition to MAP was not associated with deficits in food conditioning, as MAP-exposed worms exhibited a significant cue preference associated with food. Furthermore, our results found that embryonic MAP exposure reduced DA levels in adult C. elegans, which could be a key mechanism contributing to the long-term effects of embryonic MAP exposure. It is possible that embryonic MAP exposure may be impairing the ability of C. elegans to learn associations between MAP and the CS+ or inhibiting the reinforcing properties of MAP. However, our food conditioning data suggest that MAP-exposed animals can form associations between cues and food. The depletion of DA levels during embryonic exposure to MAP could be responsible for driving either of these processes during adulthood.

Nicotine and ethanol are commonly coabused drugs, and nicotine-laced ethanol products are growing in popularity. However, little is known about time-course changes in extracellular nicotine and cotinine levels in rat models of ethanol and nicotine coabuse. The objective of the present study was to determine the time-course changes in brain levels of nicotine and cotinine following subcutaneous (SC) and intragastric (IG) nicotine administration in alcohol-preferring (P) and Wistar rats. In vivo microdialysis was used to collect dialysate samples from the nucleus accumbens shell (NACsh) for nicotine and cotinine determinations, following SC administration of (-)-nicotine (0.18, 0.35, and 0.70 mg/kg) in female P and Wistar rats or IG administration of (-)-nicotine (0.35 and 0.70 mg/kg) in 15 % (v/v) ethanol or water in female P rats. SC nicotine produced nicotine and cotinine dialysate levels as high as 51 and 14 ng/ml, respectively. IG administration of 15 % EtOH + 0.70 mg/kg nicotine in P rats resulted in maximal nicotine and cotinine dialysate levels of 19 and 14 ng/ml, respectively, whereas administration of 0.70 mg/kg nicotine in water resulted in maximal nicotine and cotinine levels of 21 and 25 ng/ml, respectively. Nicotine and cotinine levels were detectable within the first 15 and 45 min, respectively, after IG administration. Overall, the results of this study suggest that nicotine is rapidly adsorbed and produces relevant extracellular brain concentrations of nicotine and its pharmacologically active metabolite, cotinine. The persisting high brain concentrations of cotinine may contribute to nicotine addiction.

Clinical evidence indicates a frequent co-morbidity of nicotine and alcohol abuse and dependence. The posterior ventral tegmental area (pVTA) appears to support the reinforcing and dopamine-stimulating effects of both drugs. The current study tested the hypothesis that repeated exposure of the pVTA to one drug would increase the sensitivity of local dopamine neurons to the stimulating effects of the other drug. Female Wistar rats received repeated daily microinjections of either 100 μM nicotine or vehicle directly into the pVTA for 7 days. On the 8th day, rats received microinjections of either vehicle or ethanol (100 or 200 mg%) into the pVTA while extracellular dopamine samples were collected from the ipsilateral nucleus accumbens shell (NACsh) with microdialysis. Another experiment tested the effects of challenge microinjections of 200 μM nicotine in the pVTA on extracellular dopamine levels in the NACsh following 7 daily pretreatments with 200 mg% ethanol in the pVTA. Nicotine pretreatments increased the dopamine-stimulating effects of ethanol in the pVTA (100 mg% ethanol: 115% vs 160% of baseline in the vehicle and nicotine groups, respectively, p < 0.05; 200 mg% ethanol: 145% vs 190% of baseline in the vehicle and nicotine groups, respectively, p < 0.05). In contrast, ethanol pretreatments did not alter the stimulating effects of nicotine in the pVTA. The results suggest that repeated exposure of the pVTA to nicotine increased the response of local dopamine neurons to the stimulating effects of ethanol, whereas repeated exposure of the pVTA to ethanol did not alter the responses of pVTA dopamine neurons to nicotine.

BACKGROUND:Alcohol abuse is frequently associated with nicotine (Nic) use. The current experiments were conducted to establish an oral operant ethanol + Nic (EtOH + Nic) co-use model and to characterize some aspects of EtOH + Nic co-use. METHODS:Rats were allowed to choose between EtOH alone or EtOH + Nic solutions. Additionally, alcohol-preferring (P) rats were allowed to concurrently self-administer 3 distinct EtOH solutions (10, 20, and 30%) with varying amounts of Nic (0.07, 0.14, or 0.21 mg/ml) under operant conditions. P rats were also allowed to concurrently self-administer 2 distinct amounts of Nic (0.07 and 0.14 mg/ml) added to saccharin (Sacc; 0.025%) solutions. RESULTS:During acquisition, P rats responded for the EtOH + Nic solutions at the same level as for EtOH alone, and responding for EtOH + Nic solutions was present throughout all drinking conditions. P rats also readily maintained stable self-administration behaviors for Nic + Sacc solutions. The results demonstrated that P rats readily acquired and maintained stable self-administration behaviors for EtOH + 0.07 and EtOH + 0.14 mg/ml Nic solutions. Self-administration of EtOH + 0.21 mg/ml Nic was established in only 50% of the subjects. P rats readily expressed seeking behaviors for the EtOH + Nic solutions and reacquired EtOH + Nic self-administration during relapse testing. In addition, tail blood samples indicated that EtOH + Nic co-use resulted in pharmacologically relevant levels of both EtOH and Nic in the blood. CONCLUSIONS:Overall, the results indicate that P rats readily consume EtOH + Nic solutions concurrently in the presence of EtOH alone, express drug-seeking behaviors, and will concurrently consume physiologically relevant levels of both drugs. These results support the idea that this oral operant EtOH + Nic co-use model would be suitable for studying the development of co-abuse and the consequences of long-term chronic co-abuse.