Michael S. Cowen

The orexin system regulates alcohol-seeking in rats

1 Orexin‐containing neurons have been implicated in feeding, sleep–wake cycles and more recently in drug‐seeking behaviour. 2 Pretreatment of alcohol‐preferring (iP) rats with an orexin1 receptor antagonist (SB‐334867, 20 mg kg−1, intraperitoneally) completely abolished an olfactory cue‐induced reinstatement of alcohol‐seeking behaviour, and also attenuated alcohol responding under an operant fixed ratio regimen without affecting water responding. 3 The mRNA encoding orexin within the hypothalamus was expressed at a similar density in iP and non‐preferring (NP) rats; chronic consumption of ethanol in iP rats did not significantly regulate the density of this expression, but did increase the area of expression within the lateral, but not medial, hypothalamus. 4 These data indicate that while orexin may not be implicated in the development of an alcohol preference, re‐exposure of cues previously associated with alcohol availability is sufficient and adequate to activate orexin‐containing neurons and drive reinstatement of alcohol‐seeking.

The GABAB receptor allosteric modulator CGP7930, like baclofen, reduces operant self-administration of ethanol in alcohol-preferring rats

Abstract GABA systems have been implicated as targets for ethanol at the cellular, molecular and behavioural level. The present study was designed to further examine the potential of the GABAB receptor as a target for regulating operant alcohol responding. Given that the prototypic agonist, baclofen, reduces the self-administration of alcohol, we hypothesized that the GABAB receptor allosteric modulator, CGP7930, might have similar actions but a reduced side-effect profile. In this context, inbred alcohol-preferring (iP) rats were trained to respond for 10% v/v ethanol in a fixed ratio paradigm; all drug testing was performed under an FR3 schedule. Both baclofen and CGP7930 independently reduced voluntary responding for 10% ethanol in a dose-related manner. Neither drug impacted upon responding for water. A combination of subthreshold doses of baclofen and CGP7930 was also able to reduce operant responding for ethanol, suggesting that CGP7930 is indeed acting to facilitate GABAB receptor-mediated signalling in this paradigm. These data demonstrate the potential of positive allosteric modulators of metabotropic GABAB receptors to regulate alcohol responding.

The acute anti‐craving effect of acamprosate in alcohol‐preferring rats is associated with modulation of the mesolimbic dopamine system

Acamprosate (Campral ?) is a drug used clinically for the treatment of alcoholism. In order to examine further the time‐course and mechanism of action of acamprosate, the effect of acute and repeated acamprosate administration was examined on (i) operant ethanol self‐administration and (ii) voluntary home cage ethanol consumption by alcohol‐preferring Fawn‐Hooded, iP and Alko Alcohol (AA) rats. Acutely, acamprosate was shown to cause a significant decrease in operant ethanol self‐administration by Fawn‐Hooded and alcohol‐preferring iP rats in part by decreasing the motivational relevance of a specific ethanol cue; however, repeated injection of acamprosate led to tolerance to this effect. Voluntary alcohol consumption in the home cage in Fawn‐Hooded and AA rats was also reduced by an acute acamprosate injection; however, again tolerance developed to repeated injections. In a separate experiment, the effect of acamprosate on markers of the dopaminergic system was examined. Interestingly, acute acamprosate was also shown to cause increased dopamine transporter density and decreased dopamine D2‐like receptor density within the nucleus accumbens but not in the caudate‐putamen, suggesting a link between the decreased motivational salience of the ethanol cue and altered dopaminergic signalling within the nucleus accumbens. With repeated injections of acamprosate, markers of the dopaminergic system returned to steady state levels with a similar temporal profile to the development of tolerance in the behavioural studies. Along with previous studies, our findings indicate that acamprosate modulates the mesolimbic dopaminergic system and may thereby decrease ethanol reinforcement processes; however, these effects undergo tolerance in alcohol‐preferring rats and may in part explain the fact why some subjects are non‐responders to chronic acamprosate treatment.

Combined antagonism of glutamate mGlu5 and adenosine A2A receptors interact to regulate alcohol-seeking in rats

Adenosine and glutamate have been implicated as mediators involved in the self-administration of alcohol. In the present study we sought to determine whether adenosine receptors could interact with metabotropic glutamate receptors to regulate operant responding for alcohol and also the integration of the salience of alcohol-paired cues. Alcohol-preferring (iP) rats were trained to self-administer alcohol under operant conditions. The availability of alcohol was paired with an olfactory cue plus a stimulus light. Rats were examined under fixed ratio responding and also following extinction under a cue-induced reinstatement paradigm. Administration of the selective adenosine A2A receptor antagonist, SCH 58261, reduced fixed ratio responding for alcohol in iP rats in a dose-related manner. Furthermore, the combination of a subthreshold dose of SCH 58261 with a subthreshold dose of the mGlu5 receptor antagonist MTEP also reduced alcohol self-administration and increased the latency to the first reinforced response, suggesting a pre-ingestive effect. Moreover, this combination of SCH 58261 and MTEP also prevented the conditioned reinstatement of alcohol-seeking elicited by the re-presentation of cues previously paired with alcohol availability. In contrast, combinations of the selective adenosine A1 receptor antagonist, DPCPX, with either SCH 58261 or MTEP had no effect on alcohol responding. Collectively, these data suggest a functional interaction between adenosine A2A and mGlu5 receptors in relation to alcohol-seeking and the integration of the drug-related cues.

A differential role for the adenosine A2A receptor in opiate reinforcement vs opiate-seeking behavior.

The adenosine A2A receptor is specifically enriched in the medium spiny neurons that make up the ‘indirect’ output pathway from the ventral striatum, a structure known to have a crucial, integrative role in processes such as reward, motivation, and drug-seeking behavior. In the present study we investigated the impact of adenosine A2A receptor deletion on behavioral responses to morphine in a number of reward-related paradigms. The acute, rewarding effects of morphine were evaluated using the conditioned place preference paradigm. Operant self-administration of morphine on both fixed and progressive ratio schedules as well as cue-induced drug-seeking was assessed. In addition, the acute locomotor response to morphine as well as sensitization to morphine was evaluated. Decreased morphine self-administration and breakpoint in A2A knockout mice was observed. These data support a decrease in motivation to consume the drug, perhaps reflecting diminished rewarding effects of morphine in A2A knockout mice. In support of this finding, a place preference to morphine was not observed in A2A knockout mice but was present in wild-type mice. In contrast, robust cue-induced morphine-seeking behavior was exhibited by both A2A knockout and wild-type mice after a period of withdrawal. The acute locomotor response to morphine in the A2A knockout was similar to wild-type mice, yet A2A knockout mice did not display tolerance to chronic morphine under the present paradigm. Both genotypes display locomotor sensitization to morphine, implying a lack of a role for the A2A receptor in the drug-induced plasticity necessary for the development or expression of sensitization. Collectively, these data suggest a differential role for adenosine A2A receptors in opiate reinforcement compared to opiate-seeking.

Importance of NO/cGMP signalling via cGMP-dependent protein kinase II for controlling emotionality and neurobehavioural effects of alcohol

Cyclic GMP is a second messenger for nitric oxide (NO) that acts as a mediator for many different physiological functions. The cGMP‐dependent protein kinases (cGKs) mediate cellular signalling induced by NO and cGMP. Here, we explored the localization of cGMP‐dependent protein kinase type II (cGKII) in the mouse brain. In situ hybridization revealed high levels of cGKII mRNA in cerebral cortex, thalamic nuclei, hypothalamic nuclei, and in several basal forebrain regions including medial septum, striatum and amygdala. The close link to NO and the distribution pattern of cGKII suggested that this enzyme might be involved in emotional reactions and responses to drugs of abuse. Therefore, cGKII knockout animals (cGKII–/–) were compared with littermate controls in behavioural tests (i) for emotion‐linked and (ii) for acute and chronic ethanol responses. Deletion of cGKII did not influence aggressive behaviour but led to enhanced anxiety‐like behaviour. In terms of acute responses to ethanol, cGKII–/– mice were hyposensitive to hypnotic doses of ethanol as measured by the loss of righting reflex, without an alteration in their blood alcohol elimination. In a two‐bottle free choice test, cGKII–/– mice showed elevated alcohol consumption. No taste differences to sweet solutions were observed compared to control animals. In summary, our data show that cGKII activity modulates anxiety‐like behaviour and neurobehavioural effects of alcohol.

Assessing appetitive and consummatory phases of ethanol self-administration in C57BL/6J mice under operant conditions: regulation by mGlu5 receptor antagonism

RationaleThe development of mouse models of ethanol consumption and ethanol-seeking behavior is of particular importance in understanding the underlying mechanisms of drug abuse because these models can enable an analysis of an effect of specific genotype on drug-seeking behavior and the interaction of potential therapeutics with genotype. However, there are some limitations with present models, notably the inability to examine appetitive and consummatory behavior separately.Materials and methodsIn the present study, C57BL/6 mice were trained to self-administer 10% ethanol in a modified operant protocol that allowed a clear delineation of consummatory and appetitive phases. The utility of this procedure was confirmed with the use of the metabotropic glutamate 5 (mGlu5) receptor antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (MTEP).ResultsLimited-access consumption during the dark phase of the light–dark cycle with intermittent access (every second or third day) led to a high level of consumption by the mice. MTEP caused a dose-dependent decrease in both the consumption of ethanol and the appetitive response for ethanol. Furthermore, this effect was unrelated to any effect of MTEP on locomotor activity.ConclusionsThe model provides a useful paradigm for examining both the appetitive and consummatory phases of ethanol consumption in mice; furthermore, the data indicate mGlu5 receptors are involved in both phases.

Neuropeptides: Implications for alcoholism

The role of neuromodulatory peptides in the aetiology of alcoholism has been relatively under‐explored; however, the development of selective ligands for neuropeptide receptors, the characterization and cloning of receptors, and the development of transgenic mouse models have greatly facilitated this analysis. The present review considers the most recent preclinical evidence obtained from animal models for the role of two of the opioid peptides, namely b‐endorphin and enkephalin; corticotropin‐releasing factor (CRF), urocortin I and neuropeptide Y (NPY) in deleterious and excessive alcohol consumption, focussing on specific brain regions, in particular the central nucleus of the amygdala, that appear to be implicated in the pathophysiology of alcoholism. The review also outlines potential directions for further research to clarify neuropeptide involvement in neuromodulation within discrete brain nuclei pertinent to behavioural patterns.

Role of Fyn Tyrosine Kinase in Ethanol Consumption by Mice

BACKGROUND Mice deficient for the intracellular protein Fyn tyrosine kinase (fynZ/fynZ mice) have been reported to show increased alcohol sensitivity and lack of tolerance to the effects of ethanol. To further study the involvement of Fyn in neurobehavioral effects of alcohol, we examined ethanol consumption and relapse drinking behavior in fynZ/fynZ mice. METHODS FynZ/fynZ and wild-type mice were given a free choice between water and increasing concentrations of ethanol (2-16%). Once a stable baseline of 16% ethanol consumption was established, access to ethanol was withdrawn for 2 weeks and then reinstated, to measure the alcohol deprivation effect (ADE). Forced swim stress was performed thereafter on 2 consecutive days. In a final experiment we studied alcohol sensitivity by measuring ethanol-induced loss of righting reflex (LORR). RESULTS The concentration of available ethanol had a significant effect on ethanol consumption and preference; however, there was no significant effect of genotype on these measures. Deprivation from ethanol led to a significant increase in ethanol consumption by all mice with no significant impact of genotype on ethanol consumption or water consumption during the ADE. Two consecutive days of forced swim stress led to a significant increase in ethanol consumption; again however, genotype had no effect on stress-associated ethanol consumption. Surprisingly, however, FynZ/fynZ mice showed no differences in alcohol sensitivity when compared to wild-type animals, in contrast to previously reported results ( Miyakawa et al., 1997). CONCLUSIONS Deletion of the Fyn tyrosine kinase gene may be involved in ethanol sensitivity but this effect may depend on a gene-environment interaction. Fyn does not influence ethanol consumption, neither under basal conditions nor following a deprivation period or stress. This finding indicates that phosphorylation and activation of N-methyl-D-aspartate (NMDA) receptors through Fyn is not a critical mechanism in alcohol drinking or relapse behavior.

Association analysis of HTR6 and HTR2A polymorphisms in sporadic Alzheimer's disease

Summary. In order to identify gene variants related to the serotonergic neurotransmitter system that possibly represent a hereditary risk factor for sporadic Alzheimers disease (AD), patients suffering from AD and non-demented psychiatric inpatients without symptoms of dementia were genotyped for polymorphisms of HTR6 (267C/T) and HTR2A (−1438G/A). Although there was a tendency toward an increased number of the genotype TT of the 5-HT6 receptor polymorphism in AD patients when compared to controls (2.8% vs. 1.3%), neither this nor the 5-HT2A promoter polymorphism showed significant differences in their genotypic or allelic distribution among patients and controls. These polymorphisms probably do not represent major genetic risk factors of AD. However, further studies including other genetic variants of the serotonergic neurotransmitter system are needed in order to elucidate their role in AD.