a-Synuclein expression in the brain and blood during abstinence from chronic alcohol drinking in mice Barbara Ziolkowska a , Agnieszka Gieryk a , Agnieszka Wawrzczak-Bargiela a , Tomasz Krowka a , Dorota Kaminska a , Agnieszka Korkosz b , Przemyslaw Bienkowski b , Ryszard Przewlocki a, * a Department of Molecular Neuropharmacology, Institute of Pharmacology, Polish Academy of Sciences, Sm˛ etna 12, 31-343 Krakow, Poland b Department of Pharmacology and Physiology of the Nervous System, Institute of Psychiatry and Neurology, Sobieskiego 9, 02-957 Warsaw, Poland article info Article history: Received 9 October 2007 Received in revised form 27 March 2008 Accepted 1 April 2008 Keywords: a-Synuclein Alcohol drinking Two-bottle choice Abstinence Amygdala Craving abstract a-Synuclein is a presynaptic protein proposed to serve as a negative regulator of dopaminergic neuro- transmission. Recent research has implicated a-synuclein in chronic neuroadaptations produced by psychostimulant and opiate use, as well as in genetically determined susceptibility to alcoholism in humans. The aim of our study was to characterize the changes in a-synuclein expression after short-term abstinence from chronic alcohol drinking in mice. Male C57BL/6J mice were allowed to drink increasing concentrations of alcohol in the two-bottle choice procedure. Then the mice were given constant access to an 8% alcohol solution and water for 32 days, and were sacrificed 2 h, 24 h or 48 h after alcohol withdrawal. RT-PCR, in situ hybridization and Western blotting techniques were used to measure a-synuclein mRNA and protein levels in the brain and blood. a-Synuclein protein levels were elevated by up to 80% in the amygdala of mice withdrawn from alcohol for 24 h or 48 h. No changes in a-synuclein levels were found in the mesencephalon or striatum/ accumbens. The levels of a-synuclein mRNA remained unchanged in all brain regions examined (the striatum, nucleus accumbens, amygdala, substantia nigra, ventral tegmental area). a-Synuclein mRNA was up-regulated in the whole blood 48 h after alcohol withdrawal. The accumulation of a-synuclein in the amygdala, observed in this study, seems to be a common feature of alcohol and opiate abstinence. This finding suggests a role of a-synuclein in common neuroadaptations produced by long-term alcohol and drug use. Although a-synuclein expression in the blood seems un- related to that in the brain, it may serve as a peripheral biomarker of chronic alcohol consumption. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction a-Synuclein is a protein abundantly expressed by neurons, and is present almost exclusively in presynaptic terminals (Iwai et al., 1995; Clayton and George, 1998, 1999). It is best known for its in- volvement in neuropathology, especially Parkinson’s disease and other synucleinopathies, whereas its physiological roles remain unclear (Lotharius and Brundin, 2002; Marti et al., 2003; Vekrellis et al., 2004). Its proposed physiological functions include the regu- lation of neurotransmitter release and reuptake, presynaptic vesicle recycling and the role of a molecular chaperone (reviewed by Vekrellis et al., 2004). Reports focusing on brain dopaminergic neurons, which express a-synuclein at high levels, have suggested that the protein might negatively regulate the function of dopami- nergic cells by several mechanisms. a-Synuclein was found to inhibit activity or expression of enzymes involved in dopamine synthesis (Perez et al., 2002; Baptista et al., 2003), affect function of the dopamine transporter (Lee et al., 2001;Wersinger and Sidhu, 2003) and inhibit dopamine release in response to repetitive stimulation (Abeliovich et al., 2000; Yavich et al., 2004; Larsen et al., 2006). This suggests participation of a-synuclein in the regulation of reward and reinforcement mechanisms mediated by the mesocorticolimbic dopaminergic system, a conclusion supported by the observation of an altered rate of intracranial self-stimulation of medial forebrain bundle in a-synuclein knockout mice (Oksman et al., 2006). Several recent studies have also implicated a-synuclein in sub- stance dependence and abuse, processes known to involve abnor- malities in dopaminergic function. Elevated levels of the protein were found in the brains of cocaine addicts (Mash et al., 2003; Qin et al., 2005) and in rodents treated with psychostimulants (Brenz Verca et al., 2003; Fornai et al., 2005). Moreover, we previously reported a long-lasting accumulation of a-synuclein in the mouse striatum and amygdala following withdrawal from chronic mor- phine treatment (Ziolkowska et al., 2005). Taking into account the inhibitory influence of a-synuclein on dopaminergic neurotrans- mission, we proposed that the up-regulation of a-synuclein * Corresponding author. Tel.: þ48 12 66 23 218; fax: þ48 12 637 45 00. E-mail address: nfprzewl@cyf-kr.edu.pl (R. Przewlocki). Contents lists available at ScienceDirect Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm Neuropharmacology 54 (2008) 1239–1246 Contents lists available at ScienceDirect Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm 0028-3908/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2008.04.001