Project description:Methamphetamine (METH) is a widely abused illicit amphetamine that causes acute biochemical and molecular changes in the brain. However, there are very few studies that have investigated the long-term effects of a single METH exposure in adult animals. The purpose of this study was thus to determine the consequences of a single injection of METH (10) mg/kg) on the biochemical and transcriptional effects of a second challenge injection of the drug (2.5 mg/kg) given one month later. Towards that end, we measured monoamine levels and gene expression by microarray and quantitative PCR analyses in the nucleus accumbens (NAc) of 4 groups of rats: saline-pretreated and saline-challenged (SS); saline-pretreated and METH-challenged (SM); METH-pretreated and saline-challenged (MS); and METH-pretreated and METH-challenged (MM). The rats were euthanized 2 hours after the acute challenge injection and NAc samples were harvested. The acute METH challenge caused increases in NAc DA and HVA levels in both the SM and MM groups. Microarray analyses revealed that an injection of METH (2.5 mg/kg) produced acute changes (1.8-fold; p < 0.01) in the expression of 418 (358 up-, 60 downregulated) genes including several immediate early genes (IEGs) such as Arc, c-fos, and fosB in the SM group. Several neuropeptides including Cart, Crf (Crh), and vasopressin were upregulated in that group. Injection of the higher dose of METH (10 mg/kg) did not influence monoamine levels but caused changes in 510 (345 up-, 165 downregulated) transcripts measured one month later (MS group). This list included Cart and Crf but not any IEGs. The MM group showed changes in 774 (572 up-, 202 downregulated) genes that also included vasopressin, Cart, and Crf whose expression was enhanced by the second METH injection. Quantitative PCR confirmed the METH-induced changes in the expression of the neuropeptides. We also confirmed the METH-induced potentiation of Crf expression in the METH-pretreated group. These observations suggest that a single injection of a moderate dose of METH can produce long-lasting changes in gene expression in the rodent NAc. The long-term increases in Crf expression also suggest that a single injection of a moderate METH dose can cause prolonged dysregulation in the endogenous stress system in the rat brain. 24 samples. SS (4), SM (6), MS (7), MM (7)

Project description:Methamphetamine (METH) addiction is associated with a plethora of neuropsychiatric symptoms. In rodents, administration of the drug is accompanied by complex changes in gene expression in the dorsal striatum. Very little is known about the effects of the drug on gene expression and epigenetic modifications in the nucleus accumbens (NAc). The present study was conducted in order to investigate the effects of a single injection of METH on gene expression in that structure. We also queried whether changes in transcript levels were accompanied by alterations in histone acetylation as well as in the expression of the histone acetyltransferase (HAT), ATF2, and of the histone deacetylases (HDACs), HDAC1 and HDAC2. Microarray analyses revealed that METH caused significant time-dependent increases in the expression of several genes that had previously been implicated in the acute and longterm effects of psychostimulants in the brain. These include several immediate early genes (c-fos, Egrs, c-jun, and Nurr1) and corticotropin-releasing factor (Crf). There were also increases in the levels of neuromedin U, Tnf-alpha, and Kcnk18, among others. In contrast, the METH injection caused decreases in the expression of many genes including Npas4 and cholecystokin (Cck). Pathway analyses showed that differentially affected genes participate in behavioral performance, cell-to-cell signaling and interactions, and regulation of gene expression. Other differentially affected genes are known to be involved in cellular compromise and death pathways. PCR analyses were used to confirm the changes in the expression of c-fos, fosB, c-jun, junB, Crf, NmU, Cck, and Npas4 transcripts. Western blot analyses also identified METH-mediated decreases in the acetylation of histone H3 at lysine 9 (H3K9) and lysine 18 (H3K18) as well as in histone 4 at lysine 16 (H4K16). In contrast, the METH injection caused time-dependent increases in the acetylation of H4K8 and H4K12. The changes in histone acetylation were also accompanied by decreased expression of HDAC1 but increased expression of ATF2 and of HDAC2. These results suggest that METH-induced alterations in global gene expression might be due, in part, to diverse effects of METH-induced histone acetylation secondary to changes in HAT and HDAC expression. At the indicated time after the METH or saline injections, rats (n = 5 per group) were euthanized and the NAc was dissected and immediately put on dry ice. The tissues were kept at -70 oC until RNA extraction. Total RNA was isolated from the nucleus accumbens according to the manufacturer’s manual using Qiagen RNeasy mini kit (Qiagen, Valencia, CA, USA). RNA integrity was detected using an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA).

Project description:Methamphetamine (METH) addiction is associated with a plethora of neuropsychiatric symptoms. In rodents, administration of the drug is accompanied by complex changes in gene expression in the dorsal striatum. Very little is known about the effects of the drug on gene expression and epigenetic modifications in the nucleus accumbens (NAc). The present study was conducted in order to investigate the effects of a single injection of METH on gene expression in that structure. We also queried whether changes in transcript levels were accompanied by alterations in histone acetylation as well as in the expression of the histone acetyltransferase (HAT), ATF2, and of the histone deacetylases (HDACs), HDAC1 and HDAC2. Microarray analyses revealed that METH caused significant time-dependent increases in the expression of several genes that had previously been implicated in the acute and longterm effects of psychostimulants in the brain. These include several immediate early genes (c-fos, Egrs, c-jun, and Nurr1) and corticotropin-releasing factor (Crf). There were also increases in the levels of neuromedin U, Tnf-alpha, and Kcnk18, among others. In contrast, the METH injection caused decreases in the expression of many genes including Npas4 and cholecystokin (Cck). Pathway analyses showed that differentially affected genes participate in behavioral performance, cell-to-cell signaling and interactions, and regulation of gene expression. Other differentially affected genes are known to be involved in cellular compromise and death pathways. PCR analyses were used to confirm the changes in the expression of c-fos, fosB, c-jun, junB, Crf, NmU, Cck, and Npas4 transcripts. Western blot analyses also identified METH-mediated decreases in the acetylation of histone H3 at lysine 9 (H3K9) and lysine 18 (H3K18) as well as in histone 4 at lysine 16 (H4K16). In contrast, the METH injection caused time-dependent increases in the acetylation of H4K8 and H4K12. The changes in histone acetylation were also accompanied by decreased expression of HDAC1 but increased expression of ATF2 and of HDAC2. These results suggest that METH-induced alterations in global gene expression might be due, in part, to diverse effects of METH-induced histone acetylation secondary to changes in HAT and HDAC expression.

Project description:The anti-epileptic drug zonisamide is reported to exert beneficial effects in patients with Parkinson's disease. To elucidate the pathophysiological mechanisms underlying the anti-parkinsonism effects of zonisamide, we examined the effect of zonisamide co-administered with levodopa in the striata of rats with 6-hydoroxydopamine hemiparkinsonism by using a DNA microarray for genome-wide gene expression profiling. We found that the expression of some genes related to metabolism and nervous system development and function were upregulated by zonisamide; expression of these genes was downregulated by levodopa. Furthermore, many genes related to the immune system and inflammation were downregulated by zonisamide, and their expression was upregulated by levodopa. These results indicate that zonisamide has a protective effect when co-administered with levodopa. Rats were anesthetized with pentobarbital 240 mg/kg, i.p. and unilateral lesions of the left medial forebrain bundle were made via injection of 8 mg 6-OHDA hydrobromide in 4 ml of sterile saline containing 0.01% ascorbic acid. Motor disturbance was assessed by counting the number of full rotations per min in a cylindrical container of M-OM-^F30 cm diameter at 10-min intervals for the first 60 min after METH (3 mg/kg, i.p.) administration [3]. Behavioral screening was carried out after 2-3 weeks of recovery and animals that performed at least 7 turns/min during METH challenge were certified as hemiparkinsonism rats and included in the study. Seven days after METH challenge, hemiparkinsonism rats were treated with saline, L-DOPA (100 mg/kg, i.p.), or L-DOPA (100 mg/kg, i.p.) and ZNS (50 mg/kg, i.p.) once per day for 7 days. L-DOPA was injected 30 min after benserazide hydrochloride (a peripheral decarboxylase inhibitor). ZNS was injected 30 min after L-DOPA injection. The 4 experimental groups were classified as follows: the non-lesioned side of saline-treated 6-OHDA-injected rats (NL), the lesioned side of saline-treated 6-OHDA-injected rats (SL), the lesioned side of L-DOPA-treated 6-OHDA-injected rats (DL), and the lesioned side of L-DOPA and ZNS-treated 6-OHDA-injected rats (ZDL). Three animals were assigned to each group.

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Enhanced upregulation of CRH mRNA expression in the rat nucleus accumbens after a delayed second injection of methamphetamine

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