Neural Activity in the Anterior Insula at Drinking Onset and Licking Relates to Compulsion-Like Alcohol Consumption.

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.

Embryonic Methamphetamine Exposure Inhibits Methamphetamine Cue Conditioning and Reduces Dopamine Concentrations in Adult N2 Caenorhabditis elegans.

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.

The role of beta- and alpha-adrenergic receptors on alcohol drinking.

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 α1 adrenenergic receptors (ARs) may represent a pharmacological treatment for pathological drinking. However, the involvement of ß 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 ß AR antagonists propranolol (ß1/2), betaxolol (ß1), and ICI, 118,551 (ß2) 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 µg) 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.

Development of an oral operant nicotine/ethanol co-use model in alcohol-preferring (p) rats.

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.

Modeling Aversion Resistant Alcohol Intake in Indiana Alcohol-Preferring (P) Rats.

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.

Prenatal methadone exposure disrupts behavioral development and alters motor neuron intrinsic properties and local circuitry.

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.

The far-reaching opioid crisis extends to babies born to mothers who take prescription or illicit opioids during pregnancy. Opioids such as oxycodone and methadone can freely cross the placenta from mother to baby. With the rising misuse of and addiction to opioids, the number of babies born physically dependent on opioids has risen sharply over the last decade. Although these infants are only passively exposed to opioids in the womb, they can still experience withdrawal symptoms at birth. This withdrawal is characterized by irritability, excessive crying, body shakes, problems with feeding, fevers and diarrhea. While considerable attention has been given to treating opioid withdrawal in newborn babies, little is known about how these children develop in their first years of life. This is, in part, because it is difficult for researchers to separate drug-related effects from other factors in a child's home environment that can also disrupt their development. In addition, the biological mechanisms underpinning opioid-related impairments to infant development also remain unclear. Animal models have been used to study the effects of opioid exposure during pregnancy (termed prenatal exposure) on infants. These models, however, could be improved to better replicate the typical pattern of opioid use among pregnant women. Recognizing this gap, Grecco et al. have developed a mouse model of prenatal methadone exposure where female mice that were previously dependent on oxycodone were treated with methadone throughout their pregnancy. Methadone is an opioid drug commonly prescribed for treating opioid use disorder in pregnant women and was found to accumulate at high levels in the fetal brain of mice, which fell quickly after birth. The offspring also experienced withdrawal symptoms. Grecco et al. then examined the physical, behavioral and brain development of mice born to opioid-treated mothers. These included assessments of the animals' motor skills, sensory reflexes and behavior in their first four weeks of life. Additional experiments tested the properties of nerve cells in the brain to examine cell-level changes. The assessments showed that methadone exposure in the womb impaired the physical growth of offspring and this persisted into 'adolescence'. Prenatal methadone exposure also delayed progress towards key developmental milestones and led to hyperactivity in three-week-old mice. Moreover, Grecco et al. found that these mice had reduced neuron density and cell-to-cell connectivity in the part of the brain which controls movement. These findings shed light on the potential consequences of prenatal methadone exposure on physical, behavioral and brain development in infants. This model could also be used to study new potential treatments or intervention strategies for offspring exposed to opioids during pregnancy.

Selective breeding for high alcohol preference is associated with increased sensitivity to cannabinoid reward within the nucleus accumbens shell.

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.

Caenorhabditis elegans as a model system to identify therapeutics for alcohol use disorders.

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.

Caenorhabditis elegans Show Preference for Stimulants and Potential as a Model Organism for Medications Screening.

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.

Impact of ambient temperature on hyperthermia induced by (+/-)3,4-methylenedioxymethamphetamine in rhesus macaques.

The ambient temperature (T(A)) under which rodents are exposed to (+/-)3,4-methylenedioxymethamphetamine (MDMA) affects the direction and magnitude of the body temperature response, and the degree of hypo/hyperthermia generated in subjects can modify the severity of lasting brain changes in 'neurotoxicity' models. The thermoregulatory effects of MDMA have not been well described in non-human primates and it is unknown if T(A) has the potential to affect acute hyperthermia and therefore other lasting consequences of MDMA. The objective of this study was to determine if the temperature alteration produced by MDMA in nonhuman primates depends on T(A) as it does in rats and mice. Body temperature and spontaneous home cage activity were monitored continuously in six male rhesus monkeys via radiotelemetry. The subjects were challenged intramuscularly with 0.56-2.4 mg/kg (+/-)MDMA under each of three T(A) conditions (18, 24, and 30 degrees C) in a randomized order. The temperature was significantly elevated following injection with all doses of MDMA under each ambient temperature. The magnitude of mean temperature change was approximately 1 degrees C in most conditions suggesting a closely controlled thermoregulatory response in monkeys across a range of doses and ambient temperatures. Activity levels were generally suppressed by MDMA; however, a 50% increase over vehicle was observed after 0.56 MDMA under the 30 degrees C condition. It is concluded that MDMA produces a similar degree of hyperthermia in rhesus monkeys across a range of T(A) conditions that result in hypothermia or exaggerated hyperthermia in rodents. Monkey temperature responses to MDMA appear to be more similar to humans than to rodents and therefore the monkey may offer an improved model of effects related to MDMA-induced hyperthermia.

Robust and stable drinking behavior following long-term oral alcohol intake in rhesus macaques.

Face validity in animal models of alcohol abuse and dependence is often at odds with robust demonstrations of ethanol-seeking. This study determined the relative influence of ethanol and a flavorant in maintaining ethanol intake in a nonhuman primate model of "cocktail" drinking. Four-year-old male monkeys were maintained on a 6% ethanol/6% Tang solution made available in daily (M-F) 1-h sessions. Experiments determined the effect of (1) a second daily access session, (2) concurrent presentation of the Tang vehicle, (3) sequential presentation of the vehicle in the first daily session and the ethanol solution in the second session, (4) altering the Tang concentration, (5) altering the ethanol concentration, and (6) removal of the flavorant. Mean daily intake (2.7+/-0.2 g/kg/day) was stable over 7 months. Simultaneous availability of a large, but not a low-moderate, volume of the vehicle reduced ethanol intake by about 50%. Decreasing the concentration of Tang in the first daily session reduced ethanol intake, whereas intake of the standard solution was increased in the second session. Ethanol consumption was decreased by only 27% when the flavorant was removed. In summary, alterations that reduced intake in the first daily session resulted in compensatory increases in ethanol intake in the second session, suggesting that animals sought a specific level of ethanol intake per day. It is concluded that models with excellent face validity (flavored beverages) can produce reliable ethanol intake in patterns that are highly consistent with ethanol-seeking behavior.

Differential contributions of dopaminergic D1- and D2-like receptors to cognitive function in rhesus monkeys.

RATIONALE: Dopaminergic neurotransmission is critically involved in many aspects of complex behavior and cognition beyond reward/reinforcement and motor function. Mental and behavioral disorders associated with major disruptions of dopamine neurotransmission, including schizophrenia, attention deficit/hyperactivity disorder, Parkinson's disease, Huntington's disease, and substance abuse produce constellations of neuropsychological deficits in learning, memory, and attention in addition to other defining symptoms. OBJECTIVE: To delineate the role dopaminergic D1- and D2-like receptor subtypes play in complex brain functions. MATERIALS AND METHODS: Monkeys (N = 6) were trained on cognitive tests adapted from a human neuropsychological assessment battery (CAmbridge Neuropsychological Test Automated Battery). The battery included tests of spatial working memory (self-ordered spatial search task), visuo-spatial associative memory and learning (visuo-spatial paired associates learning task, vsPAL) and motivation (progressive ratio task, PR). Tests of motor function (bimanual motor skill task, BMS; rotating turntable task, RTT) were also included. The effects of the dopamine D2-like antagonist raclopride (10-56 microg/kg, i.m.) and the D1-like antagonist SCH23390 (SCH, 3.2-56 microg/kg, i.m.) on cognitive performance were then determined. RESULTS: Deficits on PR, RTT, and BMS performance were observed after both raclopride and SCH23390. Spatial working memory accuracy was reduced to a greater extent by raclopride than by SCH, which was unexpected, given prior reports on the involvement of D1 signaling for spatial working memory in monkeys. Deficits were observed on vsPAL performance after raclopride, but not after SCH23390. CONCLUSIONS: The intriguing results suggest a greater contribution of D2- over D1-like receptors to both spatial working memory and object-location associative memory.

Hyperthermia induced by 3,4-methylenedioxymethamphetamine in unrestrained rhesus monkeys.

BACKGROUND: Exposure to (+/-)3,4-methylenedioxymethamphetamine ((+/-)MDMA) results in lasting reductions of many markers for serotonin terminals in a range of species. In rodents, the severity of insult depends in large part on the generation of hyperthermia in the subject. (+/-)MDMA can produce either hyperthermia or hypothermia in rodents depending on the ambient temperature and these effects may be limited to the S(+) enantiomer. Limited prior evidence suggests (+/-)MDMA does not produce hyperthermia in chair-restrained monkeys [Bowyer, J.F., Young, J.F., Slikker, W., Itzak, Y., Mayorga, A.J., Newport, G.D., Ali, S.F., Frederick, D.L., Paule, M.G., 2003. Plasma levels of parent compound and metabolites after doses of either d-fenfluramine or d-3,4-methylenedioxymethamphetamine (MDMA) that produce long-term serotonergic alterations. Neurotoxicology 24, 379-390]. This study was therefore conducted to determine if racemic MDMA and its enantiomers induce hyperthermia and increase spontaneous locomotor activity in unrestrained rhesus monkeys. METHODS: Body temperature and spontaneous home cage activity were monitored continuously in four monkeys via radiotelemetric devices. The subjects were challenged with 1.7 mg/kg, i.m., (+/-)MDMA, S(+)MDMA and R(-)MDMA in pseudorandomized order. RESULTS: Maximum and average temperature in the 4h interval post-dosing was elevated 0.7-0.9 degrees C by (+/-)MDMA and each enantiomer. Reductions in locomotor activity following dosing did not reliably differ from vehicle effects. CONCLUSIONS: MDMA produces an acute hyperthermia in unrestrained rhesus monkeys, much as it does with rats, mice, pigs, rabbits and humans. Hyperthermia occurs despite no increase in locomotor activity thus the effect does not depend on motor activation. Each enantiomer appears to be equivalently active thus primates may differ from rodents in thermoregulatory sensitivity to the R(-) enantiomer. Significant differences in outcome between this and a prior study in monkeys indicate a need for additional study of the thermoregulatory impact of MDMA in nonhuman primates.

Caenorhabditis elegans as a Model to Study the Molecular and Genetic Mechanisms of Drug Addiction.

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.

Modeling human methamphetamine exposure in nonhuman primates: chronic dosing in the rhesus macaque leads to behavioral and physiological abnormalities.

Acute high dose methamphetamine (METH) dosing regimens are frequently used in animal studies, however, these regimens can lead to considerable toxicity and even death in experimental animals. Acute high dosing regimens are quite distinct from the chronic usage patterns found in many human METH abusers. Furthermore, such doses, especially in nonhuman primates, can result in unexpected death, which is unacceptable, especially when such deaths fail to accurately model effects of human usage. As a model of chronic human METH abuse we have developed a nonlethal chronic METH administration procedure for the rhesus macaque that utilizes an escalating dose protocol. This protocol slowly increases the METH dosage from 0.1 to 0.7 mg/kg b.i.d. over a period of 4 weeks, followed by a period of chronic METH administration at 0.75 mg/kg b.i.d. (= total daily METH administration of 1.5 mg/kg). In parallel to human usage patterns, METH injections were given 20-23 times a month. This regimen produced a number of behavioral and physiological effects including decreased food intake and a significant increase in urinary cortisol excretion.

Time-course of extracellular nicotine and cotinine levels in rat brain following administration of nicotine: effects of route and ethanol coadministration.

RATIONALE: 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. OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Effects of nicotine and mecamylamine on cognition in rhesus monkeys.

RATIONALE: Nicotine and other agonists of nicotinic cholinergic receptors (nAChR) have been shown to improve performance in specific memory domains in rodents and monkeys. Such beneficial effects are observed in preclinical models of age-related cognitive decline, stimulating interest in nAChR ligands as possible therapeutics. Prior work has typically focused on assays of spatial working memory in rodent studies and visual recognition memory in monkey studies. OBJECTIVE: The current study was conducted to determine the role played by nAChRs in multiple types of memory in monkeys. METHODS: Rhesus monkeys (n=6) were trained to perform a battery of six behavioral tasks and then serially challenged with acute doses of nicotine (3.2-56 microg/kg, i.m.) and the nAChR antagonist mecamylamine (0.32-1.78 mg/kg, i.m.). RESULTS: Nicotine improved performance on tests designed to assay visual recognition memory, spatial working memory and visuo-spatial associative memory, while mecamylamine impaired visuo-spatial associative memory. Ballistic and fine motor performance was not significantly improved by nicotine but fine motor performance was impaired by mecamylamine. CONCLUSIONS: Although nicotine may improve performance in multiple domains, effects on visuo-spatial associative memory is the most specifically attributable to nAChR signaling.

EEG and ERP profiles in the high alcohol preferring (HAP) and low alcohol preferring (LAP) mice: relationship to ethanol preference.

Neurophysiological measures, such as decreased P300 amplitude and altered EEG alpha activity, have been associated with increased alcoholism risk. The purpose of the present study was to extend the assessment of the neurophysiological indices associated with alcohol consumption to a recently developed mouse model of high ethanol consumption, the first replicate line of high alcohol preferring (HAP-1) and low alcohol preferring (LAP-1) mice. Male HAP-1, LAP-1, and HS mice from the Institute for Behavioral Genetics at the University of Colorado Health Science Center (i.e., HS/Ibg mice) were implanted with cortical electrodes. EEG activity, and event related potentials (ERPs) were then examined. Following electrophysiological assessment, ethanol preference was assessed to examine the relationship between neurophysiological indices and ethanol consumption. EEG analyses revealed that HAPs and HS/Ibgs had greater peak frequency in the 2-4-Hz band and lower peak frequency in the 6-8- and 1-50-Hz bands of the cortical EEG compared to LAPs. Compared to HAPs, LAPs and HS/Ibgs had decreased peak EEG frequency in the 8-16-Hz band. Decreased parietal cortical power from 8 to 50 Hz was associated with high initial ethanol preference in HAP mice. In regards to ERPs, P1 amplitude was greater in HAPs compared to both LAPs and HS/Ibgs and the P3 latency in LAPs was decreased compared to both HAPs and HS/Ibgs. As expected, HAPs consumed more ethanol and had higher ethanol preference than LAPs and HS/Ibgs. There were no significant differences in ethanol intake or preference between HS/Ibgs and LAPs. These data indicate that selective breeding of the HAP and LAP lines has resulted in the divergence of EEG and ERP phenotypes. The differences observed suggest that increased cortical P1 amplitude and altered cortical EEG activity in the 8-50-Hz frequency range may be neurophysiological 'risk factors' associated with high ethanol consumption in mice. Decreased P3 latency in LAPs compared to HAPs and HS/Ibgs mice may be a 'protective factor'.

Neuropeptide Y administration into the amygdala does not affect ethanol consumption.

Evidence seems to indicate that the anxiolytic effects of centrally administered neuropeptide Y (NPY) are mediated by the central nucleus of the amygdala. Because findings seem to indicate that ethanol may be self-administered partially for its anxiolytic effects, it was hypothesized that NPY, microinjected into the central nucleus of the amygdala, would decrease ethanol intake. In this study, we examined the effects of NPY, administered into the central nucleus of the amygdala, on ethanol, sucrose, and food consumption, as well as the concomitant effects of NPY on cortical electroencephalographic activity. Wistar rats were implanted with cortical recording electrodes and cannulae above the central amygdaloid nuclei, after use of a sucrose-substitution procedure, to establish ethanol self-administration. Neuropeptide Y (0-250 pmol/0.5 micro l) was infused into the amygdala before drinking sessions, when 10% ethanol (10 E), 2% sucrose (2S), or food was available. Consumption, locomotor activity, and cortical electroencephalographic activity were then monitored concurrently. Neuropeptide Y had no effect on the intake of 10 E, 2S, or food, nor on the cortical electroencephalographic or locomotor activity. However, as reported previously, distinct changes in the electroencephalogram were associated with consumption of ethanol and sucrose. Cortical power in the 6-8 Hz frequency range was significantly increased during the beginning of the sucrose and ethanol sessions, with greater increases observed during the sucrose session. Overall, these findings support the suggestion that NPY administration into the central nucleus of the amygdala does not alter consumption of 10 E, 2S, or food, nor the cortical electroencephalographic or locomotor activity.

Animal models of motivation for drinking in rodents with a focus on opioid receptor neuropharmacology.

Ethanol, like other drugs of abuse, has motivating properties that can be developed as animal models of self-administration. A major strength of the operant approach where an animal must work to obtain ethanol is that it reduces confounds due to palatability and controls for nonspecific malaise-inducing effects. In the domain of opioid peptide systems, limited access paradigms have good predictive validity. In addition, animal models of excessive drinking-either environmentally or genetically induced-also appear sensitive to blockade or inactivation of opioid peptide receptors. Ethanol availability can be predicted by cues associated with positive reinforcement, and these models are sensitive to the administration of opioid antagonists. Perhaps most exciting are the recent results suggesting that the key element in opioid peptide systems that is important for the positive reinforcing effects of ethanol is the mu-opioid receptor. How exactly ethanol modulates mu-receptor function will be a major challenge of future research. Nevertheless, the apparently critical role of the mu receptor in ethanol reinforcement refocuses the neuropharmacology of ethanol reinforcement in the opioid peptide domain and opens a novel avenue for exploring medications for treating alcoholism.