Annika Thorsell

A genetic determinant of the striatal dopamine response to alcohol in men

Excessive alcohol use, a major cause of morbidity and mortality, is less well understood than other addictive disorders. Dopamine release in ventral striatum is a common element of drug reward, but alcohol has an unusually complex pharmacology, and humans vary greatly in their alcohol responses. This variation is related to genetic susceptibility for alcoholism, which contributes more than half of alcoholism risk. Here, we report that a functional OPRM1 A118G polymorphism is a major determinant of striatal dopamine responses to alcohol. Social drinkers recruited based on OPRM1 genotype were challenged in separate sessions with alcohol and placebo under pharmacokinetically controlled conditions, and examined for striatal dopamine release using positron emission tomography and [11C]-raclopride displacement. A striatal dopamine response to alcohol was restricted to carriers of the minor 118G allele. To directly establish the causal role of OPRM1 A118G variation, we generated two humanized mouse lines, carrying the respective human sequence variant. Brain microdialysis showed a fourfold greater peak dopamine response to an alcohol challenge in h/mOPRM1-118GG than in h/mOPRM1-118AA mice. OPRM1 A118G variation is a genetic determinant of dopamine responses to alcohol, a mechanism by which it likely modulates alcohol reward.

3-(4-Chloro-2-Morpholin-4-yl-Thiazol-5-yl)-8-(1-Ethylpropyl)-2,6-Dimethyl-Imidazo[1,2-b]Pyridazine: A Novel Brain-Penetrant, Orally Available Corticotropin-Releasing Factor Receptor 1 Antagonist with Efficacy in Animal Models of Alcoholism

We describe a novel corticotropin-releasing factor receptor 1 (CRF1) antagonist with advantageous properties for clinical development, and its in vivo activity in preclinical alcoholism models. 3-(4-Chloro-2-morpholin-4-yl-thiazol-5-yl)-8-(1-ethylpropyl)-2,6-dimethyl-imidazo[1,2-b]pyridazine (MTIP) inhibited 125I-sauvagine binding to rat pituitary membranes and cloned human CRF1 with subnanomolar affinities, with no detectable activity at the CRF2 receptor or other common drug targets. After oral administration to rats, MTIP inhibited 125I-sauvagine binding to rat cerebellar membranes ex vivo with an ED50 of ∼1.3 mg/kg and an oral bioavailability of 91.1%. Compared with R121919 (2,5-dimethyl-3-(6-dimethyl-4-methylpyridin-3-yl)-7-dipropylamino-pyrazolo[1,5-a]pyrimidine) and CP154526 (N-butyl-N-ethyl-4,9-dimethyl-7-(2,4,6-trimethylphenyl)-3,5,7-triazabicyclo[4.3.0]nona-2,4,8,10-tetraen-2-amine), MTIP had a markedly reduced volume of distribution and clearance. Neither open-field activity nor baseline exploration of an elevated plus-maze was affected by MTIP (1–10 mg/kg). In contrast, MTIP dose-dependently reversed anxiogenic effects of withdrawal from a 3 g/kg alcohol dose. Similarly, MTIP blocked excessive alcohol self-administration in Wistar rats with a history of dependence, and in a genetic model of high alcohol preference, the msP rat, at doses that had no effect in nondependent Wistar rats. Also, MTIP blocked reinstatement of stress-induced alcohol seeking both in postdependent and in genetically selected msP animals, again at doses that were ineffective in nondependent Wistar rats. Based on these findings, MTIP is a promising candidate for treatment of alcohol dependence.

Behavioral and endocrine adaptation, and up-regulation of NPY expression in rat amygdala following repeated restraint stress.

A single 1 h restraint increases experimental anxiety in the elevated plus-maze through actions within the amygdala, while intra-amygdala administration of neuropeptide Y (NPY) has the opposite effect. Endogenous amygdala NPY expression is suppressed by single restraint, providing a possible mechanism for the anxiety-promoting action of this stressor. Here, we examined whether repeated stressor exposure might lead to an adaptation (habituation or sensitization) with regard to plus-maze behavior and glucocorticoid response, and whether this might be accompanied by altered effects of the stressor on NPY expression. Following repeated restraint (1 h/day, 9-10 days), neither an anxiogenic-like effect of the stressor nor a glucocorticoid response were present. This behavioral and endocrine adaptation was accompanied by an up-regulation of prepro-NPY mRNA and NPY peptide in amygdala but not in hypothalamic or cortical extracts, an effect opposite to that previously seen after a single restraint session. Thus, an up-regulation of NPY expression in the amygdala complex may be an adaptive mechanism recruited to cope with a repeated stressor.

Diverse functions of neuropeptide Y revealed using genetically modified animals.

Neuropeptide Y (NPY), a peptide abundantly expressed in the mammalian nervous system, has been extensively studied using traditional pharmacological and behavioral models. Central administration of NPY or synthetic ligands for its receptors has indicated a role of NPY in anxiety-related behaviors, feeding, regulation of blood pressure, circadian rhythm and other functions. Some limitations inherent in pharmacological approaches, such as lack of selectivity of receptor antagonists, can be elegantly circumvented using genetically modified animals. For NPY, mice lacking NPY, the Y1, the Y2 or the Y5 receptors have been generated. In addition, both mice and rats overexpressing NPY in the central nervous system are available. Here, we review the research carried out so far in the NPY-field using genetically modified animals. Together, these models indicate that stress-related behaviors and regulation of voluntary alcohol intake perhaps are among the most important functions of central NPY, and may provide attractive targets for developing novel therapies in depression, anxiety disorders and alcohol dependence.

Decreased cerebrospinal fluid neuropeptide Y (NPY) in patients with treatment refractory unipolar major depression: preliminary evidence for association with preproNPY gene polymorphism

Extensive animal studies suggest neuropeptide Y (NPY) to be involved in coping with a wide range of stressors, and that impaired central NPY signalling could be involved in the pathophysiology of anxiety and depression. Human studies of central NPY levels in depression have, however, been inconclusive. Here, we examined levels of NPY-like immunoreactivity (NPY-LI) in the cerebrospinal fluid (CSF) of medication-free subjects with treatment refractory unipolar depression. Patients were admitted to a research inpatient unit, examined under standardized conditions, and compared with a sample of volunteers in whom psychiatric morbidity was excluded. A robust suppression of NPY levels in patient CSF was found, while other putative CSF markers (monoamine metabolites, somatostatin) did not differ between the groups. We then explored whether this finding might be related to a recently described T1128C coding polymorphism which results in a Leu7-> Pro7 substitution of the signal peptide, and a previously not described T -399C polymorphism in the promoter region of the preproNPY gene. Preliminary evidence was found for an association of both markers with a diagnosis of depression, indicating the possibility of an underlying haplotype influencing the vulnerability for developing depressive illness. Our present findings are in line with an extensive animal literature, and further support the notion that impaired NPY function could contribute to depressive illness.

Anxiogenic-Like Action of Galanin after Intra-Amygdala Administration in the Rat

The neuropeptide galanin is expressed in brain structures implicated in regulation of emotionality. The amygdala is known to play a central role in mechanisms of fear and anxiety. We therefore examined the effects of galanin (0.2 and 0.6 nmol/side) on experimental anxiety upon microinjection into the amygdala. Two established animal models of anxiety were used: a punished drinking test, and the elevated plus-maze. Punished responding was dose dependently suppressed by intra-amygdala galanin, whereas unpunished responding, drinking motivation, locomotor activity, and shock thresholds were unaffected. No effects on experimental anxiety were seen in the plus- maze following galanin injection. When injected into parietal cortex, no anxiety promoting properties of galanin were detected. These data suggest that activation of galanin receptors in amygdala modulates neurotransmission involved in fear and experimental anxiety.

Brain Neuropeptide Υ (NPY) in Stress and Alcohol Dependence

Neuropeptide Y (NPY), the prototypical member of the NPY-like peptide family, antagonizes behavioral consequences of stress through actions within the brain. This was initially indicated by microinjection studies with NPY receptor ligands, suggesting that NPY Y1 receptors mediate the anti-stress effects of NPY. Behavioral anti-stress actions of NPY are note-worthy in that 1) their magnitude surpasses that of other endogenous compounds; 2) they are produced across a wide range of animal models, normally thought to reflect different aspects of emotionality. These findings suggest that NPY acts with a high potency on a common core mechanism of emotionality and behavioral stress responses. This hypothesis is supported by behavioral studies in genetically modified animals. Increased emotionality, as well as increased alcohol intake, has been reported in mice with a homologous recombination knockout of the preproNPY gene. More detailed studies have been made possible by a transgenic rat system, in which NPY is selectively overexpressed within the hippocampus. These subjects display no overt phenotype under baseline conditions and have a normal endocrine stress response, but lack behavioral responses to stress. These findings point to the potential of the NPY system for developing novel pharmacological treatments of stress-related disorders, including anxiety and depression. Recent data additionally point to a role of NPY in the regulation of alcohol intake, and alcohol dependence emerges as a novel potential indication for compounds targeting the NPY system.

Translating the neuroscience of alcoholism into clinical treatments : from blocking the buzz to curing the blues

Understanding the pathophysiology of addictive disorders is critical for development of new treatments. A major focus of addiction research has for a long time been on systems that mediate acute positively reinforcing effects of addictive drugs, most prominently the mesolimbic dopaminergic (DA) system and its connections. This research line has been successful in shedding light on the physiology of both natural and drug reward, but has not led to therapeutic breakthroughs. The role of classical reward systems is perhaps least clear in alcohol addiction. Here, recent work is summarized that points to some clinically important conclusions. First, important pharmacogenetic differences exist with regard to positively reinforcing effects of alcohol and the ability of this drug to activate classical reward pathways. This offers an opportunity for personalized treatment approaches in alcoholism. Second, brain stress and fear systems become pathologically activated in later stages of alcoholism and their activation is a major influence in escalation of alcohol intake, sensitization of stress responses, and susceptibility to relapse. These findings offer a new category of treatment mechanisms. Corticotropin-releasing hormone (CRH) signaling through CRH1 receptors is a major candidate target in this category, but recent data indicate that antagonists for substance P (SP) neurokinin 1 (NK1) receptors may have a similar potential.

Suppressed neuropeptide Y (NPY) mRNA in rat amygdala following restraint stress.

We have previously demonstrated that NPY produces anxiolytic-like effects through actions in the amygdala, and that anxiogenic-like effects of restraint stress are mediated through this structure. Here, we examined the effects of restraint stress on NPY mRNA levels in amygdala and several other brain regions. A sensitive solution hybridization-RNase protection assay (RPA) was developed, employing a combination of internal and external standards, which allowed absolute quantitation of NPY mRNA in tissue-samples of less than 10 mg. NPY mRNA levels were determined, following a 1-h restraint stress, in homogenates of tissue from the amygdala, neocortex, striatum and hypothalamus, and the time course of these effects was examined. A highly significant decrease in NPY-mRNA levels was seen in the amygdala at 1 h and 2 h following restraint, with levels returning to normal within 10 h. A similar effect was seen in the neocortex, but was less pronounced and slower in onset. Striatal and hypothalamic NPY expression was not significantly affected. Tissue levels of NPY-peptide were modestly decreased in the amygdala at 1 h following restraint and had returned to normal within 4 h. The present findings support the hypothesis that anxiety related behavioral effects of stress may in part be mediated through modulation of NPY function in the amygdala.

Blockade of central neuropeptide Y (NPY) Y2 receptors reduces ethanol self-administration in rats

Activation of central neuropeptide Y (NPY) receptors is known to produce several behavioral effects, including feeding, modulation of memory and antagonism of behavioral effects of stress. In addition, experiments in knock-out and transgenic mice have suggested a possible role of NPY regulation of voluntary ethanol intake. NPY receptors involved in this action are not known. Here, we examined the effects of a selective NPY-Y2 receptor antagonist, BIIE0246, on operant responding for ethanol in a sweetened solution, or the sweetened solution without ethanol, during 30 min sessions of free choice between the two. BIIE0246 produced a robust suppression of responding for ethanol (40% reduction, P=0.013) at an intracerebroventricular dose of 1.0 nmol, but not 0.3 nmol. Responding for the saccharin solution was not significantly affected. The dose range examined was selected since preliminary experiments with doses of 3 nmol and higher indicated sedative effects, but such effects were absent up to 1.0 nmol, as shown by unaffected exploratory locomotor activity. In summary, antagonism at central NPY-Y2 receptors seems to selectively suppress operant self-administration of ethanol. This suggests that Y2 receptors might be candidate targets for developing novel pharmacological treatments of alcoholism.