Effects of morphine on gene expression in the rat amygdala J. M. Rodriguez Parkitna,* ,1 W. Bilecki,* ,1 P. Mierzejewski, R. Stefanski, A. Ligeza,* A. Bargiela,* B. Ziolkowska,* W. Kostowski and R. Przewlocki* *Department of Molecular Neuropharmacology, Institute of Pharmacology PAN, Cracow, Poland  Department of Pharmacology and Physiology of the Nervous System, Institute of Psychiatry and Neurology, Warsaw, Poland Abstract Influence of morphine self-administration on gene expression in the rat amygdala was studied using rat genome DNA arrays U34A from Affymetrix. Animals were trained to self-administer morphine, each having two ‘yoked’ control animals, receiving passive injections of either morphine or saline. After 40 ses- sions of self-administration, amygdalae were removed, total RNA was isolated and used to prepare probes for Genechip Ò arrays. The treatment was found to significantly change abundance of 29 transcripts. Analysis by means of reverse transcription real-time PCR showed significant changes in abundance of five transcripts: c protein kinase C (PKC), upstream binding factor 2 (UBF2), lysozyme, noggin and heat shock protein 70 (hsp70). After 30 days of forced abstinence from morphine self-administration, abundance of hsp70 and lysozyme returned to basal levels. Changes in abundance of UBF2 persisted, and abundance of three additional genes, namely nuclear factor I/A, c 1 subunit of GABA A receptor and the neuronal calcium sensor 1, changed. Additionally, acute as well as chronic intraperitoneal morphine administration changed the abundance of PKC c, c 1 subunit of GABA A and hsp70 genes. Keywords: addiction, amygdala, DNA arrays, morphine self- administration. J. Neurochem. (2004) 91, 38–48. Addiction, a persistent state in which compulsive drug use escapes control, is the most serious consequence of repetitive drug taking. Although the number of known drugs that produce dependence is increasing, little is known regarding cellular and molecular mechanisms of addiction. It is believed that the development of addiction is associated with plastic changes and long-term potentiation in discrete parts of the central nervous system (Nestler 2001). Never- theless, molecular changes in gene expression underlying these phenomena are still poorly understood. Brain regions involved in reinforcing drug effects, devel- opment of addiction and drug craving comprise the meso- corticostriatal areas and the so-called extended amygdala (Alheid and Heimer 1988; Kreek and Koob 1998). The best known targets for drugs of abuse are the dopaminergic neurones that project from the ventral tegmental area of the midbrain to the nucleus accumbens, as well as to other forebrain sites, including amygdala. The amygdala has been characterized as a relay station between the input of emotionally significant stimuli and the output of behavioural responses (Dent et al. 2001). It processes an information about the emotional and motivational significance of envi- ronmental stimuli (Everitt et al. 1991; McDonald 1991; Hatfield et al. 1996). Animal models of drug craving and relapse are based largely on conditioned reinforcement. The neural substrates for such conditioned positive reinforcement appear to involve parts of the extended amygdala and afferent pathways from the basolateral amygdala (Everitt et al. 1991). Thus, the amygdala is implicated in conditioned reinforcement and may play a crucial role in conditioned and emotional aspects of addiction. Received December 8, 2003; revised manuscript received April 3, 2004; accepted May 26, 2004. Address correspondence and reprint requests to R. Przewlocki, Insti- tute of Pharmacology PAN, Smetna 12, 31–343 Cracow, Poland. E-mail: nfprzewl@cyf-kr.edu.pl 1 These authors equally contributed to this work. Abbreviations used: FR-5, fixed-ratio 5 schedule of drug injection; GAD, glutamic acid decarboxylase; GAPDH, glyceraldehyde 3-phos- phate dehydrogenase; GST, glutathione S-transferase; HPRT, hypoxan- thine phosphoribosyl transferase; hsp, heat shock protein; MAS, Microarray Analysis Suite; MBEI, model-based expression index; MM, mismatch probe; NCS-1, neuronal calcium sensor 1, frequenin homo- logue; NF, nuclear factor; PKC, protein kinase C; PM, perfect match probe; SAPKB, stress-associated protein kinase B; SVP2B, synaptic vesicle protein 2B; UBF2, upstream binding factor 2; Zfp, zinc finger protein. Journal of Neurochemistry , 2004, 91, 38–48 doi:10.1111/j.1471-4159.2004.02697.x 38 Ó 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 38–48