SECTION ON MOLECULAR PATHOPHYSIOLOGY? ? Medications development: Target discovery and validation.? ? Our group utilizes rodent models, molecular studies and pharmacological approaches to identifying novel mechanisms and treatment targets, and validating them for human development. ? ? Two categories of models for target discovery? ? Pre-existing genetic susceptibility factors? ? Genetic selection for high alcohol preference is a well established approach to search for alleles that contribute to high alcohol consumption. Our group has over the years collaborated with two laboratories where such lines have been developed, that of Hyytia (Helsinki, Finland: Alko Alcohol preferring, or AA line ) and of Ciccocioppo (Camerino, Italy: marchigian-sardinian preferring, or msP line). Although selected for high alcohol preference, AA and msP lines are phenotypically very different, illustrating the multitude of factors that contribute to excessive voluntary alcohol intake. Behavioral characterization by our laboratory in collaboration with the Finnish and Italian groups has previously shown that the AA line has a high degree of behavioral disinhibition (impulsivity), while recent work has shown msP rats to be anxious and stress-sensitive.? ? Long-term neuroadaptations? ? In recent years there has been a realization that a shift in motivational and neural mechanisms that occurs as dependence evolves, and the development of animal models that allow a study of these neuroadaptive processes. Our group has been at the forefront of this development. We've had previously demonstrated that prolonged exposure of the rat brain to repeated cycles of intoxication and mild withdrawal results in a long-term up-regulation of voluntary alcohol intake, encoded by long-term changes in gene expression patterns (Rimondini et al. 2002). We have also established that animals with a history of dependence have a long-lasting sensitizations of stress responses; and respond to stress with increased voluntary alcohol consumption (Sommer et al, 2008; for review see Heilig and Koob 2007). Most recently, we identified two distinct neuronal networks involved in the neuroadaptations of the dependent state, and demonstrated a role for MAPK signalling in these changes (Hansson et al. 2008)? ? Discovery and validation of novel targets: focus on stress and negative affect? ? Stress is a major factor triggering relapse both in alcoholics and in animal models. Our recent findings point to several stress-related neuropeptide systems as a key category of targets, which are selectively sensitive in the post-dependent state. ? ? Corticotropin-Releasing Hormone (CRH) and its CRH1 receptor? ? We have found that both the elevated self-administration of alcohol and the increased behavioral sensitivity to stress in the post-dependent state is in large part mediated by an up-regulation of the CRH1 subtype of CRH receptors in the amygdala (Sommer et al. 2008). This converges with prior findings of a stress-sensitive / anxious behavioral phenotype in the msP rat, driven by an innate up-regulation of CRH1 receptors in the amygdala and several other brain regions in this line (Hansson et al. 2006). The elevated innate CRH1 expression of msP rats is rescued by voluntary alcohohol consumption. We have identified a series of CRH antagonists with suitable properties for clinical development (Gehlert et al. 2007; Thorsell et al, unpublished data).? ? Neuropeptide Y (NPY)? ? NPY is a potent endogenous anti-stress compound and counteracts the behavioral stress effects of CRH. We have recently showed that the Y1 receptor mediates the anti-stress actions of NPY (Karlsson et al. 2008). Agonism on NPY-Y1 receptors, or blockade of presynaptic Y2 autoreceptors, also suppresses post-dependent drinking, while leaving basal intake of alcohol in non-dependent animals unaffected (reviewed in Thorsell et al. 2006). Under a collaborative agreement with Johnson and Johnson Pharmaceuticals, we have evaluated non-peptide compounds targeting the Y2 receptor in order to identify molecules that could be developed for clinical use. This work is ongoing.? ? Substance P and its NK1 receptor? ? The prototypical member of the neurokinin family, Substance P, and its preferred neurokinin-1 (NK1) receptor are expressed throughout the fear-processing pathways of the brain, Blockade of NK1 receptors suppresses behavioral stress responses in experimental animals. Available NK1 antagonists have only low affinity for the rat and mouse NK1 receptor. We have therefore studied genetically deficient NK1R-/- mice with regard to ethanol sensitivity and intake. We have found that NK1R mutants have markedly decreased alcohol preference and increased alcohol sensitivity (George et al 2008). Conditioned place preference for alcohol is also eliminated in these animals, as is escalation in a recently developed paradigm (Thorsell et al. in preparation). These data parallel a clinical study in our clinical section, where an NK1 antagonist has been shown to suppress alcohol cravings (George et al. 2008).? ? Nociceptin? ? Nociceptin is the endogenous ligand for a GPCR, NOP, that belongs to the opioid recepto family but does not bind classical opioid peptides. NOP activation has both anti-opioid and anti-stress effects, making this receptor a candidate alcoholism treatment target. We collaborate around this target with the lab of Dr. Roberto Ciccocioppo. Recent work demonstrated that in the msP rat, a disruption of NOP signalling in central amygdala contributes to excessive alcohol intake, and can be rescued by local microinjections into this structure (Economidou et al. 2008). We are currently evaluating small molecules for their ability to reproduce the effects of nociceptin in alcohol paradigms. Support to a relevance of this target is provided by finding that a marker at the nociceptin gene locus is associated with alcohol dependence (Huang et al. 2008).? ? Glutamatergic genes? ? A hyperglutamatergic state is thought to evolve in alcohol dependence. GLAST, essential for removal of extracellular glutamate, is up-regulated in the post-dependent state. We have studied null-mutants for the GLAST-gene, with the hypothesis that these might provide a model of the neuroadapted dependent state. Unexpectedly, we have found that GLAST null mutants, rather than displaying excessive alcohol intake, have markedly decreased intake levels, and decreased conditioned place preference for alcohol (Karlsson et al in preparation). These animals also display hyperlocomotion, which is rescued both by conventional antipsychotics as well as agonists at presynaptic mGluR2/3 autoreceptors that inhibit release of glutamate (Karlsson et al, in press). ? ? Beta arrestin 2 ? ? A DNA-microarray screen in the preferring AA line identified an over-expression of beta arrestin 2 (BARR2). We subsequently found that this is due to the presence of a unique genetic variant (haplotype) of this gene in the AA rats. Using null-mutants for the BARR2 gene, we found that BARR2 appears involved in signalling that mediates reinforcing properties of alcohol, as a disruption of this gene markedly suppresses voluntary consumption (Bjrk et al. 2008).? ? Relevance? ? These studies identify targets for novel alcoholism treatments.
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