Publications

2010
David N. Stephens, Theodora Duka, Hans S. Crombag, Christopher L. Cunningham, Markus Heilig, and John C. Crabbe. “Reward sensitivity: issues of measurement, and achieving consilience between human and animal phenotypes.” Addiction Biology, 15, 2, Pp. 145–168. Abstract
Reward is a concept fundamental to discussions of drug abuse and addiction. The idea that altered sensitivity to either drug-reward, or to rewards in general, contributes to, or results from, drug-taking is a common theme in several theories of addiction. However, the concept of reward is problematic in that it is used to refer to apparently different behavioural phenomena, and even to diverse neurobiological processes (reward pathways). Whether these different phenomena are different behavioural expressions of a common underlying process is not established, and much research suggests that there may be only loose relationships among different aspects of reward. Measures of rewarding effects of drugs in humans often depend upon subjective reports. In animal studies, such insights are not available, and behavioural measures must be relied upon to infer rewarding effects of drugs or other events. In such animal studies, but also in many human methods established to objectify measures of reward, many other factors contribute to the behaviour being studied. For that reason, studying the biological (including genetic) bases of performance of tasks that ostensibly measure reward cannot provide unequivocal answers. The current overview outlines the strengths and weaknesses of current approaches that hinder the conciliation of cross-species studies of the genetics of reward sensitivity and the dysregulation of reward processes by drugs of abuse. Some suggestions are made as to how human and animal studies may be made to address more closely homologous behaviours, even if those processes are only partly able to isolate 'reward' from other factors contributing to behavioural output.
Jennifer B. Treweek, Amanda J. Roberts, and Kim D. Janda. “Superadditive effects of ethanol and flunitrazepam: implications of using immunopharmacotherapy as a therapeutic.” Molecular Pharmaceutics, 7, 6, Pp. 2056–2068. Abstract
While benzodiazepine intoxication alone may elicit sedative and antianxiety effects, alcohol coingestion greatly amplifies this central nervous system depression. As a result, this drug combination gained notoriety for its role in cases of facilitated sexual assault and fatal overdose. We previously validated the ability of the novel antiflunitrazepam monoclonal antibody (mAb) RCA3A3 to bind flunitrazepam (FLU) in vivo and block FLU-induced impairment of locomotion and memory. A therapeutically relevant application of this high affinity mAb (K(d,app) = 200 nM), however, is to the more tenuous indication of flunitrazepam (FLU) and alcohol cointoxication. Employing a murine behavioral model, passive immunization with mAb RCA3A3 before injection of ethanol (EtOH: low-dose, 1 g/kg, or high-dose, 1.5 g/kg), FLU (0.06 mg/kg), or a cocktail of both drugs offered partial to full restoration of motor activity levels in co-drug treated and FLU-treated mouse groups (n = 12), respectively. Whereas all drug treatments left contextual learning intact, auditory cued learning was severely disrupted. Prophylactic administration of mAb RCA3A3 prevented this deficit in cued learning in FLU-treated mice but not in the FLU- and EtOH-treated mice, in which co-drug exposure exacerbated the impairment in cued fear conditioning. To substantiate this finding, a dose-response study was performed, and the changes in locomotor activity incurred by different FLU (low-dose, 0.06 mg/kg, or high-dose, 0.09 mg/kg), EtOH (1.0 g/kg, 1.5 g/kg), and mAb RCA3A3 (14.5 mg/kg, 21.8 mg/kg) dose combinations illustrated the potentiation in motor effects by concomitant exposure to FLU and EtOH. Thus, motor activity and fear conditioning results demonstrated that both the amount of FLU left unbound by antibody and the pharmacological additivity between FLU and EtOH, a GABA mimetic, were limiting factors in the therapeutic efficacy of mAb RCA3A3. In sum, our study highlights the complex nature of psychomotor impairment upon co-drug versus singular drug exposure, which may pose a unique challenge to therapeutic treatment.
Yi-Fen Yen, Patrick Le Roux, Dirk Mayer, Randy King, Daniel Spielman, James Tropp, Kim Butts Pauly, Adolf Pfefferbaum, Shreyas Vasanawala, and Ralph Hurd. “T2 relaxation times of 13C metabolites in a rat hepatocellular carcinoma model measured in vivo using 13C-MRS of hyperpolarized [1-13C]pyruvate.” NMR in biomedicine, 23, 4, Pp. 414–423. Publisher's Version Abstract
A single-voxel Carr-Purcell-Meibloom-Gill sequence was developed to measure localized T2 relaxation times of 13C-labeled metabolites in vivo for the first time. Following hyperpolarized [1-13C]pyruvate injections, pyruvate and its metabolic products, alanine and lactate, were observed in the liver of five rats with hepatocellular carcinoma and five healthy control rats. The T2 relaxation times of alanine and lactate were both significantly longer in HCC tumors than in normal livers (p \textless 0.002). The HCC tumors also showed significantly higher alanine signal relative to the total 13C signal than normal livers (p \textless 0.006). The intra- and inter-subject variations of the alanine T2 relaxation time were 11% and 13%, respectively. The intra- and inter-subject variations of the lactate T2 relaxation time were 6% and 7%, respectively. The intra-subject variability of alanine to total carbon ratio was 16% and the inter-subject variability 28%. The intra-subject variability of lactate to total carbon ratio was 14% and the inter-subject variability 20%. The study results show that the signal level and relaxivity of [1-13C]alanine may be promising biomarkers for HCC tumors. Its diagnostic values in HCC staging and treatment monitoring are yet to be explored.
Sergey P. Pronko, Laura M. Saba, Paula L. Hoffman, and Boris Tabakoff. “Type 7 adenylyl cyclase-mediated hypothalamic-pituitary-adrenal axis responsiveness: influence of ethanol and sex.” The Journal of Pharmacology and Experimental Therapeutics, 334, 1, Pp. 44–52. Abstract
Although ethanol has been considered to be an anxiolytic agent, consumption of ethanol has also been shown to increase plasma adrenocorticotropin and glucocorticoids. The corticotrophin-releasing factor (CRF) receptor 1alpha (CRF-R1) is a G protein-coupled receptor that activates adenylyl cyclase (AC), leading to adrenocorticotropin (and subsequently glucocorticoid) release into the circulation. There are nine members of the membrane-bound AC family, and the type 7 AC (AC7) is most sensitive to ethanol, which enhances the responsiveness of AC7 to G protein-coupled receptor activation. We determined the time course of ethanol's effect on plasma adrenocorticotropin and corticosterone levels in male and female AC7 transgenic (Adcy7(huTG)) mice (in which AC7 is overexpressed in neural tissue) and AC7 heterozygous knockdown [Adcy7(+/-)] mice (in which AC7 is underexpressed in neural tissue), and their respective littermate controls [wild type (WT)]. CRF-R1 mRNA and mRNA and protein for different forms of ACs were measured by using gene expression arrays, quantitative reverse transcription-polymerase chain reaction, and immunoblotting in pituitaries of all animals. Our results demonstrated increased levels of AC7 in pituitary of Adcy7(huTG) mice and decreased levels in pituitary of Adcy7(+/-) mice compared with WT animals. Male and female Adcy7(huTG) mice displayed higher plasma adrenocorticotropin and corticosterone levels than WT and/or Adcy7(+/-) mice after ethanol injection. Female mice displayed higher adrenocorticotropin and corticosterone levels after ethanol injection than males, regardless of genotype. The data provide evidence for an integral role of AC7 in the increase of plasma adrenocorticotropin and corticosterone levels during alcohol intoxication.
Pamela Metten, Michelle L. Sorensen, Andy J. Cameron, Chia-Hua Yu, and John C. Crabbe. “Withdrawal severity after chronic intermittent ethanol in inbred mouse strains.” Alcoholism, Clinical and Experimental Research, 34, 9, Pp. 1552–1564. Abstract
BACKGROUND: To study withdrawal, ethanol is usually administered chronically without interruption. However, interest has recurred in models of episodic exposure. Increasing evidence suggests that chronic intermittent exposure to ethanol leads to a sensitization effect in both withdrawal severity and ethanol consumption. The goal of the present study was to examine mouse inbred strain differences in withdrawal severity following chronic intermittent exposure using the handling-induced convulsion as the behavioral endpoint. We also sought to compare the withdrawal responses of inbred strains across acute, chronic continuous, and chronic intermittent exposure regimens. METHODS: Male mice from 15 standard inbred strains were exposed to ethanol vapor for 16 hours each day for 3 days and removed to an air chamber during the intervening 8 hours. Mice in the control groups were handled the same, except that they were exposed only to air. Daily blood ethanol concentrations were averaged for each mouse to estimate total dose of ethanol experienced. RESULTS: Across strains, mice had an average daily blood ethanol concentration (BEC) of 1.45 +/- 0.02 mg/ml and we restricted the range of this value to 1.00-2.00 mg/ml. To evaluate strain differences, we divided data into two dose groups based on BEC, low dose (1.29 +/- 0.1 mg/ml) and high dose (1.71 +/- 0.02 mg/ml). After the third inhalation exposure, ethanol-exposed and air-exposed groups were tested hourly for handling-induced convulsions for 10 hour and at hour 24 and 25. Strains differed markedly in the severity of withdrawal (after subtraction of air control values) in both dose groups. CONCLUSION: The chronic intermittent exposure paradigm is sufficient to elicit differential withdrawal responses across nearly all strains. Data from the high-dose groups correlated well with withdrawal data derived from prior acute (single high dose) and chronic continuous (for 72 hours) ethanol withdrawal studies, supporting the influence of common genes on all three responses.
2009
Recent studies have indicated a role for the endocannabinoid system in the behavioral and physiological effects of alcohol (ethanol), particularly ethanol seeking behaviors. However, its role in modulating binge-like intake and/or the mechanism by which it may exert these effects remain poorly understood. The current study used a newly developed strain-specific animal model of binge drinking, dubbed 'Drinking In the Dark' (DID), to determine if facilitation of the endocannabinoid system with the synthetic cannabinoid agonist WIN 55-212,2 (WIN) modulates binge-like ethanol intake in male C57BL/6J (B6) mice. Based on the results of these systemic (i.p.) manipulations, and evidence in support of the involvement of subregions of the Ventral Tegmental Area (VTA) in governing self-administration of ethanol (Rodd-Henricks et al., (2000) Psychopharmacology (Berl) 149(3):217-224) as well as binge-like intake using the DID model (Moore & Boehm, (2009 Behav Neurosci 123(3):555-563), we extended these findings to evaluate the role of the endocannabinoid system within the anterior and posterior sub regions of the VTA using site-specific microinjections. Consistent with previous research, the lowest systemic dose of WIN (0.5 mg/kg) significantly increased ethanol intake in the first 30 minutes of access whereas the two highest doses (1 and 2 mg/kg) decreased ethanol intake within this time interval. Intra-posterior ventral tegmental area (pVTA) (but not aVTA (anterior ventral tegmental area) microinjections elicited time-dependent and dose-dependent increases (0.25 and 0.5 mug/side) and decreases (2.5 mug/side) in ethanol intake. Importantly, follow-up studies revealed that in some cases alterations in fluid consumption may have been influenced by competing locomotor activity (or inactivity). The present data are consistent with previous research in that agonism of the endocannabinoid system increases ethanol intake in rodents and implicate the pVTA in the modulation of drinking to intoxication. Moreover, the dose-dependent alterations in locomotor activity emphasize the importance of directly assessing multiple (possibly competing) behaviors when evaluating drug effects on voluntary consumption.
Dirk Mayer, Yi-Fen Yen, James Tropp, Adolf Pfefferbaum, Ralph E. Hurd, and Daniel M. Spielman. “Application of subsecond spiral chemical shift imaging to real-time multislice metabolic imaging of the rat in vivo after injection of hyperpolarized 13C1-pyruvate.” Magnetic Resonance in Medicine, 62, 3, Pp. 557–564. Abstract
Dynamic nuclear polarization can create hyperpolarized compounds with MR signal-to-noise ratio enhancements on the order of 10,000-fold. Both exogenous and normally occurring endogenous compounds can be polarized, and their initial concentration and downstream metabolic products can be assessed using MR spectroscopy. Given the transient nature of the hyperpolarized signal enhancement, fast imaging techniques are a critical requirement for real-time metabolic imaging. We report on the development of an ultrafast, multislice, spiral chemical shift imaging sequence, with subsecond acquisition time, achieved on a clinical MR scanner. The technique was used for dynamic metabolic imaging in rats, with measurement of time-resolved spatial distributions of hyperpolarized (13)C(1)-pyruvate and metabolic products (13)C(1)-lactate and (13)C(1)-alanine, with a temporal resolution of as fast as 1 s. Metabolic imaging revealed different signal time courses in liver from kidney. These results demonstrate the feasibility of real-time, hyperpolarized metabolic imaging and highlight its potential in assessing organ-specific kinetic parameters.
Jennifer L. Trujillo, Amanda J. Roberts, and Michael R. Gorman. “Circadian timing of ethanol exposure exerts enduring effects on subsequent ad libitum consumption in C57 mice.” Alcoholism, Clinical and Experimental Research, 33, 7, Pp. 1286–1293. Abstract
BACKGROUND: There is a daily rhythm in the voluntary intake of ethanol in mice, with greatest consumption in the early night and lowest intake during the day. The role of daily timing of ethanol exposure on the development and control of long-term ethanol self-administration has been neglected. The present study examines these issues using C57BL/6J mice. METHODS: Mice were repeatedly exposed to 10% ethanol for 2 hours early in the night or day for several weeks. Subsequently, ethanol was available at the opposite time (Expt 1) or 24 hours daily (Expts 1 and 2). Lick sensors recorded the patterns of drinking activity in Experiment 2. RESULTS: Mice exposed to ethanol during the night drink more than mice exposed during the day. Prior history did not affect ethanol intake when the schedule was reversed. Under 24-hour exposure conditions, mice with a history of drinking during the night consumed significantly more than mice drinking during the day. The circadian patterns of drinking were not altered. CONCLUSIONS: These results demonstrate that the daily timing of ethanol exposure exerts enduring effects of self-administration of ethanol in mice. Understanding how circadian rhythms regulate ethanol consumption may be valuable for modifying subsequent intake.
Joannalee C. Campbell, Karen K. Szumlinski, and Tod E. Kippin. “Contribution of early environmental stress to alcoholism vulnerability.” Alcohol (Fayetteville, N.Y.), 43, 7, Pp. 547–554. Abstract
The most problematic aspects of alcohol abuse disorder are excessive alcohol consumption and the inability to refrain from alcohol consumption during attempted abstinence. The root causes that predispose certain individuals to these problems are poorly understood but are believed to be produced by a combination of genetic and environmental factors. Early environmental trauma alters neurodevelopmental trajectories that can predispose an individual to a number of neuropsychiatric disorders, including substance abuse. Prenatal stress (PNS) is a well-established protocol that produces perturbations in nervous system development, resulting in behavioral alterations that include hyperresponsiveness to stress, novelty, and psychomotor stimulant drugs (e.g., cocaine, amphetamine). Moreover, PNS animals exhibit enduring alterations in basal and cocaine-induced changes in dopamine and glutamate transmission within limbic structures, which exhibit pathology in drug addiction and alcoholism, suggesting that these alterations may contribute to an increased propensity to self-administer large amounts of drugs of abuse or to relapse after periods of drug withdrawal. Given that cocaine and alcohol have actions on common limbic neural substrates (albeit by different mechanisms), we hypothesized that PNS would elevate the motivation for, and consumption of, alcohol. Accordingly, we have found that male C57BL/6J mice subject to PNS exhibit higher operant responding and consume more alcohol during alcohol reinforcement as adults. Alterations in glutamate and dopamine neurotransmission within the forebrain structures appear to contribute to the PNS-induced predisposition to high alcohol intake and are induced by excessive alcohol intake. Accordingly, we are exploring the interactions between neurochemical changes produced by PNS and changes induced by consumption of alcohol in adulthood to model the biological bases of high vulnerability to alcohol abuse.
Y. A. Blednov and R. A. Harris. “Deletion of vanilloid receptor (TRPV1) in mice alters behavioral effects of ethanol.” Neuropharmacology, 56, 4, Pp. 814–820. Abstract
The vanilloid receptor TRPV1 is activated by ethanol and this may be important for some of the central and peripheral actions of ethanol. To determine if this receptor has a role in ethanol-mediated behaviors, we studied null mutant mice in which the Trpv1 gene was deleted. Mice lacking this gene showed significantly higher preference for ethanol and consumed more ethanol in a two-bottle choice test as compared with wild type littermates. Null mutant mice showed shorter duration of loss of righting reflex induced by low doses of ethanol (3.2 and 3.4 g/kg) and faster recovery from motor incoordination induced by ethanol (2 g/kg). However, there were no differences between null mutant and wild type mice in severity of ethanol-induced acute withdrawal (4 g/kg) or conditioned taste aversion to ethanol (2.5 g/kg). Two behavioral phenotypes (decreased sensitivity to ethanol-induced sedation and faster recovery from ethanol-induced motor incoordination) seen in null mutant mice were reproduced in wild type mice by injection of a TRPV1 antagonist, capsazepine (10 mg/kg). These two ethanol behaviors were changed in the opposite direction after injection of capsaicin, a selective TRPV1 agonist, in wild type mice. The studies provide the first evidence that TRPV1 is important for specific behavioral actions of ethanol.
R. Hitzemann, S. Edmunds, W. Wu, B. Malmanger, N. Walter, J. Belknap, P. Darakjian, and S. McWeeney. “Detection of reciprocal quantitative trait loci for acute ethanol withdrawal and ethanol consumption in heterogeneous stock mice.” Psychopharmacology, 203, 4, Pp. 713–722. Publisher's Version Abstract
RationalePrevious studies have suggested that there is an inverse genetic relationship between ethanol consumption (two-bottle choice, continuous access) and ethanol withdrawal (e.g., Metten et al., Behav Brain Res 95:113–122, 1998a).ObjectivesThe current study used short-term selective breeding from heterogeneous stock (HS) animals to examine this relationship. The primary goal of the current study was to determine if reciprocal quantitative trait loci (QTLs) could be found in the selectively bred lines. The advantage of detecting QTLs in HS animals is that it is possible to extract a haplotype signature for the QTL, which in turn can be used to narrow the number of candidate genes generated from gene expression and sequence databases (see, e.g., Hitzemann et al., Mamm Genome 14:733–747, 2003).ResultsSeven reciprocal QTLs were detected on chromosomes (Chr) 1 (two), 3, 6, 11, 16, and 17 that exceeded the nominal LOD threshold of 10; genetic drift, which occurs during selection, dramatically increases the LOD threshold. The proximal Chr 1 QTL was examined in some detail. The haplotype structure of the QTL was such that the LP/J allele was associated with low withdrawal and high consumption. The QTL appears to be located in a gene-poor region between 170 and 173 Mbp. Based on available sequence data, two plausible candidate genes emerge—Nos1ap and Atf6α.ConclusionsThe data presented here confirm some aspects of the negative genetic relationship between acute ethanol withdrawal and ethanol consumption. The QTL data point to the potential involvement of NO signaling and/or the unfolded protein response.
Gang Chen, Matthew T. Reilly, Laura B. Kozell, Robert Hitzemann, and Kari J. Buck. “Differential activation of limbic circuitry associated with chronic ethanol withdrawal in DBA/2J and C57BL/6J mice.” Alcohol (Fayetteville, N.Y.), 43, 6, Pp. 411–420. Abstract
Although no animal model exactly duplicates clinically defined alcoholism, models for specific factors, such as the withdrawal syndrome, are useful for identifying potential neural determinants of liability in humans. The well-documented difference in withdrawal severity following chronic ethanol exposure, between the DBA/2J and C57BL/6J mouse strains, provides an excellent starting point for dissecting the neural circuitry affecting predisposition to physical dependence on ethanol. To induce physical dependence, we used a paradigm in which mice were continuously exposed to ethanol vapor for 72h. Ethanol-exposed and air-exposed (control) mice received daily injections of pyrazole hydrochloride, an alcohol dehydrogenase inhibitor, to stabilize blood ethanol levels. Ethanol-dependent and air-exposed mice were killed 7h after removal from the inhalation chambers. This time point corresponds to the time of peak ethanol withdrawal severity. The brains were processed to assess neural activation associated with ethanol withdrawal indexed by c-Fos immunostaining. Ethanol-withdrawn DBA/2J mice showed significantly (P\textless.05) greater neural activation than ethanol-withdrawn C57BL/6J mice in the dentate gyrus, hippocampus CA3, lateral septum, basolateral and central nuclei of the amygdala, and prelimbic cortex. Taken together with results using an acute model, our data suggest that progression from acute ethanol withdrawal to the more severe withdrawal associated with physical dependence following chronic ethanol exposure involves recruitment of neurons in the hippocampal formation, amygdala, and prelimbic cortex. To our knowledge, these are the first studies to use c-Fos to identify the brain regions and neurocircuitry that distinguish between chronic and acute ethanol withdrawal severity using informative animal models.
Ilona Obara, Richard L. Bell, Scott P. Goulding, Cindy M. Reyes, Lindsay A. Larson, Alexis W. Ary, William A. Truitt, and Karen K. Szumlinski. “Differential Effects of Chronic Ethanol Consumption and Withdrawal on Homer/Glutamate Receptor Expression in Subregions of the Accumbens and Amygdala of P Rats.” Alcoholism, clinical and experimental research, 33, 11, Pp. 1924–1934. Publisher's Version Abstract
Background Homer proteins are constituents of scaffolding complexes that regulate the trafficking and function of central Group1 metabotropic glutamate receptors (mGluRs) and N-methyl-D-aspartate (NMDA) receptors. Research supports the involvement of these proteins in ethanol-induced neuroplasticity in mouse. In this study, we examined the effects of short versus long-term withdrawal from chronic ethanol consumption on Homer and glutamate receptor protein expression within striatal and amygdala subregions of selectively bred, alcohol-preferring P rats. Methods For 6 months, male P rats had concurrent access to 15% and 30% ethanol solutions under intermittent (IA: 4 d/wk) or continuous (CA: 7 d/wk) access conditions in their home cage. Rats were killed 24 hours (short withdrawal: SW) or 4 weeks (long withdrawal: LW) after termination of ethanol access, subregions of interest were micropunched and tissue processed for detection of Group1 mGluRs, NR2 subunits of the NMDA receptor and Homer protein expression. Results Within the nucleus accumbens (NAC), limited changes in NR2a and NR2b expression were detected in the shell (NACsh), whereas substantial changes were observed for Homer2a/b, mGluRs as well as NR2a and NR2b subunits in the core (NACc). Within the amygdala, no changes were detected in the basolateral subregion, whereas substantial changes, many paralleling those observed in the NACc, were detected in the central nucleus (CeA) subregion. In addition, most of the changes observed in the CeA, but not NACc, were present in both SW and LW rats. Conclusions Overall, these subregion specific, ethanol-induced increases in mGluR/Homer2/NR2 expression within the NAC and amygdala suggest changes in glutamatergic plasticity had taken place. This may be a result of learning and subsequent memory formation of ethanol’s rewarding effects in these brain structures, which may, in part, mediate the chronic relapsing nature of alcohol abuse.
William J. McBride, Jonathan A. Schultz, Mark W. Kimpel, Jeanette N. McClintick, Mu Wang, Jinsam You, and Zachary A. Rodd. “Differential effects of ethanol in the nucleus accumbens shell of alcohol-preferring (P), alcohol-non-preferring (NP) and Wistar rats: a proteomics study.” Pharmacology, Biochemistry, and Behavior, 92, 2, Pp. 304–313. Abstract
The objective of this study was to determine the effects of ethanol injections on protein expression in the nucleus accumbens shell (ACB-sh) of alcohol-preferring (P), alcohol-non-preferring (NP) and Wistar (W) rats. Rats were injected for 5 consecutive days with either saline or 1 g/kg ethanol; 24 h after the last injection, rats were killed and brains obtained. Micro-punch samples of the ACB-sh were homogenized; extracted proteins were subjected to trypsin digestion and analyzed with a liquid chromatography-mass spectrometer procedure. Ethanol changed expression levels (1.15-fold or higher) of 128 proteins in NP rats, 22 proteins in P, and 28 proteins in W rats. Few of the changes observed with ethanol treatment for NP rats were observed for P and W rats. Many of the changes occurred in calcium-calmodulin signaling systems, G-protein signaling systems, synaptic structure and histones. Approximately half the changes observed in the ACB-sh of P rats were also observed for W rats. Overall, the results indicate a unique response to ethanol of the ACB-sh of NP rats compared to P and W rats; this unique response may reflect changes in neuronal function in the ACB-sh that could contribute to the low alcohol drinking behavior of the NP line.
E. Spangler, D. M. Cote, A. M. J. Anacker, G. P. Mark, and A. E. Ryabinin. “Differential sensitivity of the perioculomotor urocortin-containing neurons to ethanol, psychostimulants and stress in mice and rats.” Neuroscience, 160, 1, Pp. 115–125. Abstract
The perioculomotor urocortin-containing population of neurons (pIIIu: otherwise known as the non-preganglionic Edinger-Westphal nucleus) is sensitive to alcohol and is involved in the regulation of alcohol intake. A recent study indicated that this brain region is also sensitive to psychostimulants. Since pIIIu has been shown to respond to stress, we investigated how psychostimulant-induced pIIIu activation compares to stress- and ethanol-induced activation, and whether it is independent from a generalized stress response. Several experiments were performed to test how the pIIIu responds to psychostimulants by quantifying the number of Fos immunoreactive nuclei after acute i.p. injections of saline, 10-30 mg/kg cocaine, 5 mg/kg methamphetamine, 5 mg/kg amphetamine, 2.5 g/kg ethanol, 2 h of restraint stress, 10 min of swim stress, or six applications of mild foot shock in male C57BL/6 J mice. We also compared Fos immunoreactivity in pIIIu after acute (20 mg/kg cocaine) and repeated cocaine exposure (7 days of 20 mg/kg cocaine) injections in male C57BL/6 J mice in order to investigate the potential habituation of this response. Finally, we quantified the number of Fos immunoreactive nuclei in pIIIu after administration of saline, 2.5 g/kg ethanol, 20 mg/kg cocaine, or 2 h of restraint stress in male Sprague-Dawley rats. We found that exposure to psychostimulants and ethanol induced significantly higher Fos levels in pIIIu compared to stress in mice. Furthermore, repeated cocaine injections did not decrease Fos immunoreactivity as would be expected if this response were due to stress. In rats, exposure to ethanol, psychostimulant and restraint stress all induced pIIIu Fos immunoreactivity compared to saline-injected controls. In both mice and rats, ethanol- and cocaine-induced Fos immunoreactivity occurred exclusively in urocortin 1-positive, but not in tyrosine hydroxylase-positive, cells. These results provide evidence that the pIIIu Fos-response to psychostimulants is independent of a generalized stress in mice, but not rats. They additionally show that the pIIIu response to stress differs significantly between species.
Zachary A. Rodd, Richard L. Bell, Kelly A. Kuc, James M. Murphy, Lawrence Lumeng, and William J. McBride. “Effects of concurrent access to multiple ethanol concentrations and repeated deprivations on alcohol intake of high-alcohol-drinking (HAD) rats.” Addiction Biology, 14, 2, Pp. 152–164. Abstract
High-alcohol-drinking rats, given access to 10% ethanol, expressed an alcohol deprivation effect (ADE) only after multiple deprivations. In alcohol-preferring (P) rats, concurrent access to multiple ethanol concentrations combined with repeated cycles of EtOH access and deprivation produced excessive ethanol drinking. The current study was undertaken to examine the effects of repeated alcohol deprivations with concurrent access to multiple concentrations of ethanol on ethanol intake of HAD replicate lines of rats. HAD-1 and HAD-2 rats received access to 10, 20 and 30% (v/v) ethanol for 6 weeks. Rats from each replicate line were assigned to: (1) a non-deprived group; (2) a group initially deprived of ethanol for 2 weeks; or (3) a group initially deprived for 8 weeks. Following the restoration of the ethanol solutions, cycle of 2 weeks of ethanol exposure and 2 weeks of alcohol deprivation was repeated three times for a total of four deprivations. Following the initial ethanol deprivation period, deprived groups significantly increased ethanol intakes during the initial 24-hour re-exposure period. Multiple deprivations increased ethanol intakes, shifted preference to higher ethanol concentrations and prolonged the duration of the elevated ethanol intakes for up to 5 days. In addition, repeated deprivations increased ethanol intake in the first 2-hour re-exposure period as high as 5-7 g/kg (which are equivalent to amounts consumed in 24 hours by HAD rats), and produced blood ethanol levels in excess of 150 mg%. The results indicate that HAD rats exhibit 'loss-of-control' of alcohol drinking with repeated deprivations when multiple ethanol concentrations are available.
Ronnie Dhaher, Deborah A. Finn, Denesa L. Oberbeck, Naomi Yoneyama, Christopher C. Snelling, Weiran Wu, and Robert J. Hitzemann. “Electrolytic lesions of the medial nucleus accumbens shell selectively decrease ethanol consumption without altering preference in a limited access procedure in C57BL/6J mice.” Pharmacology Biochemistry and Behavior, 92, 2, Pp. 335–342. Publisher's Version Abstract
The central extended amygdala (cExtA) is a limbic region proposed to play a key role in drug and alcohol addiction and to contain the medial nucleus accumbens shell (MNAc shell). The aim of this study was to examine the involvement of the MNAc shell in ethanol and sucrose consumption in a limited and free access procedure in the C57BL/6J (B6) mouse. Separate groups of mice received bilateral electrolytic lesions of the MNAc shell or sham surgery, and following recovery from surgery, were allowed to voluntarily consume ethanol (15% v/v) in a 2 h limited access 2-bottle-choice procedure. Following 1 week of limited access ethanol consumption, mice were given 1 week of limited access sucrose consumption. A separate group of lesioned and sham mice were given free access (24 h) to ethanol in a 2-bottle choice procedure and were run in parallel to the mice receiving limited access consumption. Electrolytic lesions of the MNAc shell decreased ethanol (but not sucrose) consumption in a limited access procedure, but did not alter free access ethanol consumption. These results suggest that the MNAc shell is a component of the underlying neural circuitry contributing to limited access alcohol consumption in the B6 mouse.
Richard L. Bell, Mark W. Kimpel, Jeanette N. McClintick, Wendy N. Strother, Lucinda G. Carr, Tiebing Liang, Zachary A. Rodd, R. Dayne Mayfield, Howard J. Edenberg, and William J. McBride. “Gene expression changes in the nucleus accumbens of alcohol-preferring rats following chronic ethanol consumption.” Pharmacology, biochemistry, and behavior, 94, 1, Pp. 131–147. Publisher's Version Abstract
The objective of this study was to determine the effects of binge-like alcohol drinking on gene expression changes in the nucleus accumbens (ACB) of alcohol-preferring (P) rats. Adult male P rats were given ethanol under multiple scheduled access (MSA; three 1-hr dark-cycle sessions/day) conditions for 8 weeks. For comparison purposes, a second ethanol drinking group was given continuous/daily alcohol access (CA; 24 hr/day). A third group was ethanol-naïve (W group). Average ethanol intakes for the CA and MSA groups were approximately 9.5 and 6.5 g/kg/day, respectively. Fifteen hr after the last drinking episode, rats were euthanized, the brains extracted, and the ACB dissected. RNA was extracted and purified for microarray analysis. The only significant differences were between the CA and W groups (p \textless 0.01; Storey false discovery rate = 0.15); there were 374 differences in named genes between these 2 groups. There were 20 significant Gene Ontology (GO) categories, which included negative regulation of protein kinase activity, anti-apoptosis, and regulation of G-protein-coupled receptor signaling. Ingenuity® analysis indicated a network of transcription factors, involving oncogenes (Fos, Jun, Junb had higher expression in the ACB of the CA group), suggesting increased neuronal activity. There were 43 genes located within rat QTLs for alcohol consumption and preference; 4 of these genes (Tgfa, Hspa5, Mtus1 and Creb3l2) are involved in anti-apoptosis and increased transcription, suggesting that they may be contributing to cellular protection and maintaining high alcohol intakes. Overall, these findings suggest that chronic CA drinking results in genomic changes that can be observed during the early acute phase of ethanol withdrawal. Conversely, chronic MSA drinking, with its associated protracted withdrawal periods, results in genomic changes that may be masked by tight regulation of these genes following repeated experiences of ethanol withdrawal.
Dayne R Mayfield and Adron R Harris. “Gene expression profiling in blood: new diagnostics in alcoholism and addiction?.” Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology, 34, 1, Pp. 250–251. Publisher's Version
Boris Tabakoff, Laura Saba, Morton Printz, Pam Flodman, Colin Hodgkinson, David Goldman, George Koob, Heather N. Richardson, Katerina Kechris, Richard L. Bell, Norbert Hübner, Matthias Heinig, Michal Pravenec, Jonathan Mangion, Lucie LeGault, Maurice Dongier, Katherine M. Conigrave, John B. Whitfield, John Saunders, Bridget Grant, and Paula L. Hoffman. “Genetical genomic determinants of alcohol consumption in rats and humans.” BMC Biology, 7, Pp. 70. Publisher's Version Abstract
We have used a genetical genomic approach, in conjunction with phenotypic analysis of alcohol consumption, to identify candidate genes that predispose to varying levels of alcohol intake by HXB/BXH recombinant inbred rat strains. In addition, in two populations of humans, we assessed genetic polymorphisms associated with alcohol consumption using a custom genotyping array for 1,350 single nucleotide polymorphisms (SNPs). Our goal was to ascertain whether our approach, which relies on statistical and informatics techniques, and non-human animal models of alcohol drinking behavior, could inform interpretation of genetic association studies with human populations.

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