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Methamphetamine use disorder (MUD) is characterized by loss of control over compulsive drug use. Here, we used a self-administration (SA) model to investigate transcriptional changes associated with the development of early and late compulsivity during contingent footshocks. Punishment initially separated methamphetamine taking rats into always shock-resistant (ASR) rats that continued active lever pressing and shock-sensitive (SS) rats that reduced their lever pressing. At the end of the punishment phase, rats underwent 15 days of forced abstinence at the end of which they were re-introduced to the SA paradigm followed by SA plus contingent shocks. Interestingly, 36 percent of the initial SS rats developed delayed shock-resistance (DSR). Of translational relevance, ASR rats showed more incubation of methamphetamine craving than DSR and always sensitive (AS) rats. RNA sequencing revealed increased striatal Rab37 and Dipk2b mRNA levels that correlated with incubation of methamphetamine craving. Interestingly, Bdnf mRNA levels showed HDAC2-dependent decreased expression in the AS rats. The present SA paradigm should help to elucidate the molecular substrates of early and late addiction-like behaviors., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Perturbations in striatal dopamine (DA) homeostasis might underlie the behavioral and pathobiological consequences of METH use disorder in humans. To identify potential consequences of long-term METH exposure, we modeled the adverse consequence DSM criterion of substance use disorders by giving footshocks to rats that had escalated their intake of METH during a drug self-administration procedure. Next, DA D1 receptor antagonist, SCH23390 was injected. Thereafter, rats were euthanized to measure several indices of the striatal dopaminergic system. Footshocks split the METH rats into two phenotypes: (i) shock-sensitive that decreased their METH-intake and (ii) shock-resistant that continued their METH intake. SCH23390 caused substantial dose-dependent reduction of METH taking in both groups. Stopping SCH23390 caused re-emergence of compulsive METH taking in shock-resistant rats. Compulsive METH takers also exhibited greater incubation of METH seeking than non-compulsive rats during withdrawal from METH SA. Analyses of DA metabolism revealed non-significant decreases (about 35%) in DA levels in resistant and sensitive rats. However, striatal contents of the deaminated metabolites, DOPAL and DOPAC, were significantly increased in sensitive rats. VMAT2 and DAT protein levels were decreased in both phenotypes. Moreover, protein expression levels of the D1-like DA receptor, D5R, and D2-like DA receptors, D3R and D4R, were significantly decreased in the compulsive METH takers. Our results parallel findings in post-mortem striatal tissues of human METH users who develop Parkinsonism after long-term METH intake and support the use of this model to investigate potential therapeutic interventions for METH use disorder.

Methamphetamine (METH) use disorder (MUD) is characterized by compulsive and repeated drug taking despite negative life consequences. Large intake of METH in humans and animals is accompanied by dysfunctions in learning and memory processes. The endocannabinoid system (ECS) is known to modulate synaptic plasticity and cognitive functions. In addition, the ECS has been implicated in some of the manifestations of substance use disorders (SUDs). We therefore sought to identify potential changes in the expression of various enzymes and of the receptors (CB1 and CB2) that are members of that system. Herein, we used a model of METH self-administration (SA) that includes a punishment phase (footshocks) that helps to separate rats into a compulsive METH phenotype (compulsive) that continues to take METH and a non-compulsive METH (abstinent) group that suppressed or stopped taking METH. Animals were euthanized 2 h after the last METH SA session and their hippocampi were used to measure mRNA levels of cannabinoid receptors (CB/Cnr), as well as those of synthesizing (DAGL-A, DAGL-B, NAPEPLD) and metabolizing (MGLL, FAAH, PTGS2) enzymes of the endocannabinoid cascade. Non-compulsive rats exhibited significant increased hippocampal expression of CB1/Cnr1 and CB2/Cnr2 mRNAs. mRNA levels of the synthesizing enzyme, DAGL-A, and of the metabolic enzymes, MGLL and FAAH, were also increased. Non-compulsive rats also exhibited a significant decrease in hippocampal Ptgs2 mRNA levels. Taken together, these observations implicate the hippocampal endocannabinoid system in the suppression of METH intake in the presence of adverse consequences., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Rationale: Abuse of the psychostimulant methamphetamine (METH) can cause long-lasting damage to brain monoaminergic systems and is associated with profound mental health problems for users, including lasting cognitive impairments. Animal models of METH exposure have been useful in dissecting the molecular effects of the drug on cognition, but many studies use acute, non-contingent "binge" administrations of METH which do not adequately approximate human METH use. Long-term METH exposure via long-access (LgA) self-administration paradigms has been proposed to more closely reflect human use and induce cognitive impairments., Objective: To better understand the role of contingency and patterns of exposure in METH-induced cognitive impairments, we analyzed behavioral and neurochemical outcomes in adult male rats, comparing non-contingent "binge" METH administration with contingent (LgA) METH self-administration and non-contingent yoked partners., Results: Binge METH (40 mg/kg, i.p., over 1 day) dramatically altered striatal and hippocampal dopamine, DOPAC, 5-HT, 5-HIAA, BDNF, and TrkB 75 days after drug exposure. In contrast, 6-h LgA METH self-administration (cumulative 24.8-48.9 mg METH, i.v., over 16 days) altered hippocampal BDNF in both contingent and yoked animals but reduced striatal 5-HIAA in only contingent animals. Neurochemical alterations following binge METH administration were not accompanied by cognitive deficits in Morris water maze, novel object recognition, or Y-maze tests. However, contingent LgA METH self-administration resulted in impaired spatial memory in the water maze., Conclusions: Overall, substantial differences in neurochemical markers between METH exposure and self-administration paradigms did not consistently translate to deficits in cognitive tasks, highlighting the complexity of correlating METH-induced neurochemical changes with cognitive outcomes.

The transition from occasional to escalated psychostimulant use is accelerated by prior drug exposure. These behavioral observations may be related to long-lasting transcriptional and/or epigenetic changes induced by the drug pre-exposure. Herein, we investigated if a single methamphetamine (METH) injection would enhance METH self-administration (SA) and impact any METH SA-induced epigenetic or transcriptional alterations. We thus injected a single METH dose (10 mg/kg) or saline to rats before training them to self-administer METH or saline. There were three experimental groups in SA experiments: (1) a single saline injection followed by saline SA (SS); (2) a single saline injection followed by METH SA (SM); and (3) a single METH injection followed by METH SA (MM). METH-pretreated rats escalated METH SA earlier and took more METH than saline-pretreated animals. Both groups showed similar incubation of cue-induced METH craving. Because compulsive METH takers and METH-abstinent rats show differences in potassium (K + ) channel mRNA levels in their nucleus accumbens (NAc), we wondered if K + channel expression might also help to distinguish between SM and MM groups. We found increases in mRNA and protein expression of shaker-related voltage-gated K + channels (Kv1: Kcna1, Kcna3, and Kcna6) and calcium-activated K + channels (Kcnn1) in the SM compared to MM rats. SM rats also showed decreased DNA methylation at the CpG-rich sites near the promoter region of Kcna1, Kcna3 and Kcnn1 genes in comparison to MM rats. Together, these results provide additional evidence for potentially using K + channels as therapeutic targets against METH use disorder.

Background: Methamphetamine (METH) use disorder is prevalent worldwide. There are reports of sex differences in quantities of drug used and relapses to drug use among individuals with METH use disorder. However, the molecular neurobiology of these potential sex differences remains unknown., Methods: We trained rats to self-administer METH (0. 1 mg/kg/infusion, i.v.) on an fixed-ratio-1 schedule for 20 days using two 3-hour daily METH sessions separated by 30-minute breaks. At the end of self-administration training, rats underwent tests of cue-induced METH seeking on withdrawal days 3 and 30. Twenty-four hours later, nucleus accumbens was dissected and then used to measure neuropeptide mRNA levels., Results: Behavioral results show that male rats increased the number of METH infusions earlier during self-administration training and took more METH than females. Both male and female rats could be further divided into 2 phenotypes labeled high and low takers based on the degree of escalation that they exhibited during the course of the METH self-administration experiment. Both males and females exhibited incubation of METH seeking after 30 days of forced withdrawal. Females had higher basal mRNA levels of dynorphin and hypocretin/orexin receptors than males, whereas males expressed higher vasopressin mRNA levels than females under saline and METH conditions. Unexpectedly, only males showed increased expression of nucleus accumbens dynorphin after METH self-administration. Moreover, there were significant correlations between nucleus accumbens Hcrtr1, Hcrtr2, Crhr2, and Avpr1b mRNA levels and cue-induced METH seeking only in female rats., Conclusion: Our results identify some behavioral and molecular differences between male and female rats that had self-administered METH. Sexual dimorphism in responses to METH exposure should be considered when developing potential therapeutic agents against METH use disorder., (Published by Oxford University Press on behalf of CINP 2019. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Methamphetamine (METH) addiction is a biopsychosocial disorder that is accompanied by multiple relapses even after prolonged abstinence, suggesting the possibilities of long-lasting maladaptive epigenetic changes in the brain. Here, we show that METH administration produced time-dependent increases in the expression of corticotropin-releasing hormone (Crh/Crf), arginine vasopressin (Avp), and cocaine- and amphetamine-regulated transcript prepropeptide (Cartpt) mRNAs in the rat nucleus accumbens (NAc). Chromatin immunoprecipitation (ChIP) assays revealed that METH increased the abundance of phosphorylated CREB (pCREB) at the promoter of Cartpt but not at Avp or Crh DNA sequences. In contrast, METH produced DNA hypomethylation at sites near the Crh transcription start site (TSS) and at intragenic Avp sequences. METH also increased DNA hydroxymethylation at the Crh TSS and at intragenic Avp sites. In addition, METH increased the protein expression of ten-eleven-translocation enzymes that catalyze DNA hydroxymethylation. Importantly, METH increased TET1 binding at the Crh promoter and increased TET3 binding at Avp intragenic regions. We further tested the role of TET enzymes in METH-induced changes in gene expression by using the TET inhibitor, 1,5-isoquinolinediol (IQD), and found that IQD blocked METH-induced increases in Crh and Avp mRNA expression. Together, these results indicate that METH produced changes in neuropeptide transcription by both activation of the cAMP/CREB pathway and stimulation of TET-dependent DNA hydroxymethylation. These results provide molecular evidence for epigenetic controls of METH-induced changes in the expression of neuropeptides.

Methamphetamine addiction is mimicked in rats that self-administer the drug. However, these self-administration (SA) models do not include adverse consequences that are necessary to reach a diagnosis of addiction in humans. Herein, we measured genome-wide transcriptional consequences of methamphetamine SA and footshocks in the rat brain. We trained rats to self-administer methamphetamine for 20 days. Thereafter, lever-presses for methamphetamine were punished by mild footshocks for 5 days. Response-contingent punishment significantly reduced methamphetamine taking in some rats (shock-sensitive, SS) but not in others (shock-resistant, SR). Rats also underwent extinction test at one day and 30 days after the last shock session. Rats were euthanized one day after the second extinction test and the nucleus accumbens (NAc) and dorsal striatum were collected to measure gene expression with microarray analysis. In the NAc, there were changes in the expression of 13 genes in the SRvsControl and 9 genes in the SRvsSS comparison. In the striatum, there were 9 (6 up, 3 down) affected genes in the SRvsSS comparison. Among the upregulated genes was oxytocin in the NAc and CARTpt in the striatum of SR rats. These observations support a regional role of neuropeptides in the brain after a long withdrawal interval when animals show incubation of methamphetamine craving.

Methamphetamine (METH) addicts lose control over drug consumption despite suffering multiple adverse medicolegal consequences. To mimic the negative events associated with drug addiction in humans, we recently introduced a rat model of self-administration (SA) with response-contingent punishment on METH intake. These procedures allowed us to distinguish between two addiction-like phenotypes in rats, those that sustained METH taking despite negative consequences (shock-resistant, SR) and rats that significantly reduced their METH intake (shock-sensitive, SS). Here, we further developed our adverse consequence model and examined incubation of METH craving by measuring cue-induced drug seeking in SR and SS rats. Male Sprague-Dawley rats were trained to self-administer METH (0.1mg/kg/injection) or saline intravenously (i.v.) during twenty-two 9-h sessions that consisted of 3 separate 3-h sessions separated by 30min. Subsequently, rats were subjected to incremental footshocks during thirteen additional 9-h METH SA sessions performed in a fashion identical to the training phase. Cue-induced drug craving was then assessed at 2 and 21days after the footshock phase. All rats escalated their intake of METH, with both phenotypes showing similar drug taking patterns during SA training. In addition, rats that continued their METH intake despite negative consequences showed even greater cue-induced drug craving following withdrawal than the rats that reduced METH intake following negative consequences. Taken together, our adverse consequence-based model highlights the possibility of identifying rats by addiction-like phenotypes and subsequent vulnerability to relapse-like behaviors. The use of similar SA models should help in the development of better therapeutic approaches to treat different stages of METH addiction., (Published by Elsevier B.V.)

Addiction is associated with neuroadaptive changes in the brain. In the present paper, we used a model of methamphetamine self-administration during which we used footshocks to divide rats into animals that continue to press a lever to get methamphetamine (shock-resistant) and those that significantly reduce pressing the lever (shock-sensitive) despite the shocks. We trained male Sprague-Dawley rats to self-administer methamphetamine (0.1 mg/kg/infusion) for 9 hours daily for 20 days. Control group self-administered saline. Subsequently, methamphetamine self-administration rats were punished by mild electric footshocks for 10 days with gradual increases in shock intensity. Two hours after stopping behavioral experiments, we euthanized rats and isolated nucleus accumbens (NAc) samples. Affymetrix Array experiments revealed 24 differentially expressed genes between the shock-resistant and shock-sensitive rats, with 15 up- and 9 downregulated transcripts. Ingenuity pathway analysis showed that these transcripts belong to classes of genes involved in nervous system function, behavior, and disorders of the basal ganglia. These genes included prodynorphin (PDYN) and proenkephalin (PENK), among others. Because PDYN and PENK are expressed in dopamine D1- and D2-containing NAc neurons, respectively, these findings suggest that mechanisms, which impact both cell types may play a role in the regulation of compulsive methamphetamine taking by rats.

Methamphetamine (METH) administration alters gene expression in the nucleus accumbens (NAc). We recently demonstrated that an acute METH injection produced prolonged increases in the expression of immediate early genes in the NAc of HDAC2-deficient mice, suggesting that HDAC2 might be an important regulator of gene expression in the rodent brain. Here, we tested the possibility that HDAC2 deletion might also impact METH-induced changes in the expression of various HDAC classes in the NAc. Wild-type (WT) and HDAC2 knockout (KO) mice were given a METH (20 mg/kg) injection, and NAc tissue was collected at 1, 2, and 8 h post treatment. We found that METH decreased HDAC3, HDAC4, HDAC7, HDAC8, and HDAC11 mRNA expression but increased HDAC6 mRNA levels in the NAc of WT mice. In contrast, the METH injection increased HDAC3, HDAC4, HDAC7, HDAC8, and HDAC11 mRNA levels in HDAC2KO mice. These observations suggest that METH may induce large-scale transcriptional changes in the NAc by regulating the expression of several HDACs, in part, via HDAC2-dependent mechanisms since some of the HDACs showed differential responses between the two genotypes. Our findings further implicate HDACs as potential novel therapeutic targets for neurotoxic complications associated with the abuse of certain psychostimulants.

Methamphetamine (METH) produces increases in the expression of immediate early genes (IEGs) and of histone deacetylase 2 (HDAC2) in the rat nucleus accumbens (NAc). Here, we tested whether HDAC2 deletion influenced the effects of METH on IEG expression in the NAc. Microarray analyses showed no baseline differences in IEG expression between wild-type (WT) and HDAC2 knockout (KO) mice. Quantitative-PCR analysis shows that an acute METH injection produced time-dependent increases in mRNA levels of several IEGs in both genotypes. Interestingly, HDAC2KO mice displayed greater METH-induced increases in Egr1 and Egr2 mRNA levels measured at one hour post-injection. The levels of Fosb, Fra2, Egr1, and Egr3 mRNAs stayed elevated in the HDAC2KO mice 2 hours after the METH injection whereas these mRNAs had normalized in the WT mice. In WT mice, METH caused increased HDAC2 recruitment to the promoters some IEGs at 2 hours post injection. METH-induced prolonged increases in Fosb, Fra2, Egr1, and Egr3 mRNA levels in HDAC2KO mice were associated with increased enrichment of phosphorylated CREB (pCREB) on the promoters of these genes. Based on our observations, we hypothesize that HDAC2 may regulate the expression of these genes, in part, by prolonging the actions of pCREB in the mouse NAc.

Methamphetamine use disorder is characterized by recurrent binge episodes. Humans addicted to methamphetamine experience various degrees of cognitive deficits and show evidence of neurodegenerative processes in the brain. Binge injections of METH to rodents also cause significant toxic changes in the brain. In addition, this pattern of METH injections can alter gene expression in the dorsal striatum. Gene expression is regulated, in part, by histone deacetylation. We thus tested the possibility that METH toxic doses might cause changes in the mRNA levels of histone deacetylases (HDACs). We found that METH did produce significant decreases in the mRNA expression of HDAC8, which is a class I HDAC. METH also decreased expression of HDAC6, HDAC9, and HDAC10 that are class II HDACs. The expression of the class IV HDAC, HDAC11, was also suppressed by METH. The expression of Sirt2, Sirt5, and Sirt6 that are members of class III HDACs was also downregulated by METH injections. Our findings implicate changes in HDAC expression may be an early indicator of impending METH-induced neurotoxicity in the striatum. This idea is consistent with the accumulated evidence that some HDACs are involved in neurodegenerative processes in the brain., (Published by Elsevier B.V.)

In a rat model of drug craving and relapse, cue-induced drug seeking progressively increases after withdrawal from methamphetamine and other drugs, a phenomenon termed 'incubation of drug craving'. However, current experimental procedures used to study incubation of drug craving do not incorporate negative consequences of drug use, which is a common factor promoting abstinence in humans. Here, we studied whether incubation of methamphetamine craving is observed after suppression of drug seeking by adverse consequences (punishment). We trained rats to self-administer methamphetamine or palatable food for 9 h per day for 14 days; reward delivery was paired with a tone-light cue. Subsequently, for one group within each reward type, 50% of the lever-presses were punished by mild footshock for 9-10 days, whereas for the other group lever-presses were not punished. Shock intensity was gradually increased over time. Next, we assessed cue-induced reward seeking in 1-h extinction sessions on withdrawal days 2 and 21. Response-contingent punishment suppressed extended-access methamphetamine or food self-administration; surprisingly, food-trained rats showed greater resistance to punishment than methamphetamine-trained rats. During the relapse tests, both punished and unpunished methamphetamine- and food-trained rats showed significantly higher cue-induced reward seeking on withdrawal day 21 than on day 2. These results demonstrate that incubation of both methamphetamine and food craving occur after punishment-induced suppression of methamphetamine or palatable food self-administration. Our procedure can be used to investigate mechanisms of relapse to drug and palatable food seeking under conditions that more closely approximate the human condition.

Background: Chronic methamphetamine (METH) exposure causes neuroadaptations at glutamatergic synapses., Methods: To identify the METH-induced epigenetic underpinnings of these neuroadaptations, we injected increasing METH doses to rats for 2 weeks and measured striatal glutamate receptor expression. We then quantified the effects of METH exposure on histone acetylation. We also measured METH-induced changes in DNA methylation and DNA hydroxymethylation., Results: Chronic METH decreased transcript and protein expression of GluA1 and GluA2 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) and GluN1 N-methyl-D-aspartate receptor subunits. These changes were associated with altered electrophysiological glutamatergic responses in striatal neurons. Chromatin immunoprecipitation-polymerase chain reaction revealed that METH decreased enrichment of acetylated histone H4 on GluA1, GluA2, and GluN1 promoters. Methamphetamine exposure also increased repressor element-1 silencing transcription factor (REST) corepressor 1, methylated CpG binding protein 2, and histone deacetylase 2 enrichment, but not of sirtuin 1 or sirtuin 2, onto GluA1 and GluA2 gene sequences. Moreover, METH caused interactions of REST corepressor 1 and methylated CpG binding protein 2 with histone deacetylase 2 and of REST with histone deacetylase 1. Surprisingly, methylated DNA immunoprecipitation and hydroxymethylated DNA immunoprecipitation-polymerase chain reaction revealed METH-induced decreased enrichment of 5-methylcytosine and 5-hydroxymethylcytosine at GluA1 and GluA2 promoter sequences. Importantly, the histone deacetylase inhibitor, valproic acid, blocked METH-induced decreased expression of AMPAR and N-methyl-D-aspartate receptor subunits. Finally, valproic acid also attenuated METH-induced decrease H4K16Ac recruitment on AMPAR gene sequences., Conclusions: These observations suggest that histone H4 hypoacetylation may be the main determinant of METH-induced decreased striatal glutamate receptor expression., (Published by Society of Biological Psychiatry on behalf of Society of Biological Psychiatry.)

Methamphetamine (METH) is a widely abused amphetamine analog. Few studies have investigated the molecular effects of METH exposure in adult animals. Herein, we determined the consequences of an injection of METH (10 mg/kg) on transcriptional effects of a second METH (2.5 mg/kg) injection given one month later. We thus measured gene expression by microarray analyses in the nucleus accumbens (NAc) of 4 groups of rats euthanized 2 hours after the second injection: saline-pretreated followed by saline-challenged (SS) or METH-challenged (SM); and METH-pretreated followed by saline-challenged (MS) or METH-challenged (MM). Microarray analyses revealed that METH (2.5 mg/kg) produced acute changes (1.8-fold; P<0.01) in the expression of 412 (352 upregulated, 60 down-regulated) transcripts including cocaine and amphetamine regulated transcript, corticotropin-releasing hormone (Crh), oxytocin (Oxt), and vasopressin (Avp) that were upregulated. Injection of METH (10 mg/kg) altered the expression of 503 (338 upregulated, 165 down-regulated) transcripts measured one month later (MS group). These genes also included Cart and Crh. The MM group showed altered expression of 766 (565 upregulated, 201 down-regulated) transcripts including Avp, Cart, and Crh. The METH-induced increased Crh expression was enhanced in the MM group in comparison to SM and MS groups. Quantitative PCR confirmed the METH-induced changes in mRNA levels. Therefore, a single injection of METH produced long-lasting changes in gene expression in the rodent NAc. The long-term increases in Crh, Cart, and Avp mRNA expression suggest that METH exposure produced prolonged activation of the endogenous stress system. The METH-induced changes in oxytocin expression also suggest the possibility that this neuropeptide might play a significant role in the neuroplastic and affiliative effects of this drug.

Neuroplastic changes in the dorsal striatum participate in the transition from casual to habitual drug use and might play a critical role in the development of methamphetamine (METH) addiction. We examined the influence of METH self-administration on gene and protein expression that may form substrates for METH-induced neuronal plasticity in the dorsal striatum. Male Sprague-Dawley rats self-administered METH (0.1mg/kg/injection, i.v.) or received yoked saline infusions during eight 15-h sessions and were euthanized 2h, 24h, or 1month after cessation of METH exposure. Changes in gene and protein expression were assessed using microarray analysis, RT-PCR and Western blots. Chromatin immunoprecipitation (ChIP) followed by PCR was used to examine epigenetic regulation of METH-induced transcription. METH self-administration caused increases in mRNA expression of the transcription factors, c-fos and fosb, the neurotrophic factor, Bdnf, and the synaptic protein, synaptophysin (Syp) in the dorsal striatum. METH also caused changes in ΔFosB, BDNF and TrkB protein levels, with increases after 2 and 24h, but decreases after 1month of drug abstinence. Importantly, ChIP-PCR showed that METH self-administration caused enrichment of phosphorylated CREB (pCREB), but not of histone H3 trimethylated at lysine 4 (H3K4me3), on promoters of c-fos, fosb, Bdnf and Syp at 2h after cessation of drug intake. These findings show that METH-induced changes in gene expression are mediated, in part, by pCREB-dependent epigenetic phenomena. Thus, METH self-administration might trigger epigenetic changes that mediate alterations in expression of genes and proteins serving as substrates for addiction-related synaptic plasticity., (Published by Elsevier Inc.)

Background: METH is an illicit drug of abuse that influences gene expression in the rat striatum. Histone modifications regulate gene transcription., Methods: We therefore used microarray analysis and genome-scale approaches to examine potential relationships between the effects of METH on gene expression and on DNA binding of histone H4 acetylated at lysine 4 (H4K5Ac) in the rat dorsal striatum of METH-naïve and METH-pretreated rats., Results: Acute and chronic METH administration caused differential changes in striatal gene expression. METH also increased H4K5Ac binding around the transcriptional start sites (TSSs) of genes in the rat striatum. In order to relate gene expression to histone acetylation, we binned genes of similar expression into groups of 100 genes and proceeded to relate gene expression to H4K5Ac binding. We found a positive correlation between gene expression and H4K5Ac binding in the striatum of control rats. Similar correlations were observed in METH-treated rats. Genes that showed acute METH-induced increased expression in saline-pretreated rats also showed METH-induced increased H4K5Ac binding. The acute METH injection caused similar increases in H4K5Ac binding in METH-pretreated rats, without affecting gene expression to the same degree. Finally, genes that showed METH-induced decreased expression exhibited either decreases or no changes in H4K5Ac binding., Conclusion: Acute METH injections caused increased gene expression of genes that showed increased H4K5Ac binding near their transcription start sites.

In the present study, we analyzed mice with a targeted deletion of β-catenin in DA neurons (DA-βcat KO mice) to address the functional significance of this molecule in the shaping of synaptic responses associated with motor learning and following exposure to drugs of abuse. Relative to controls, DA-βcat KO mice showed significant deficits in their ability to form long-term memories and displayed reduced expression of methamphetamine-induced behavioral sensitization after subsequent challenge doses with this drug, suggesting that motor learning and drug-induced learning plasticity are altered in these mice. Morphological analyses showed no changes in the number or distribution of tyrosine hydroxylase-labeled neurons in the ventral midbrain. While electrochemical measurements in the striatum determined no changes in acute DA release and uptake, a small but significant decrease in DA release was detected in mutant animals after prolonged repetitive stimulation, suggesting a possible deficit in the DA neurotransmitter vesicle reserve pool. However, electron microscopy analyses did not reveal significant differences in the content of synaptic vesicles per terminal, and striatal DA levels were unchanged in DA-βcat KO animals. In contrast, striatal mRNA levels for several markers known to regulate synaptic plasticity and DA neurotransmission were altered in DA-βcat KO mice. This study demonstrates that ablation of β-catenin in DA neurons leads to alterations of motor and reward-associated memories and to adaptations of the DA neurotransmitter system and suggests that β-catenin signaling in DA neurons is required to facilitate the synaptic remodeling underlying the consolidation of long-term memories.

Methamphetamine (METH) addiction is associated with several neuropsychiatric symptoms. Little is known about the effects of METH on gene expression and epigenetic modifications in the rat nucleus accumbens (NAC). Our study investigated the effects of a non-toxic METH injection (20 mg/kg) on gene expression, histone acetylation, and the expression of the histone acetyltransferase (HAT), ATF2, and of the histone deacetylases (HDACs), HDAC1 and HDAC2, in that structure. Microarray analyses done at 1, 8, 16 and 24 hrs after the METH injection identified METH-induced changes in the expression of genes previously implicated in the acute and longterm effects of psychostimulants, including immediate early genes and corticotropin-releasing factor (Crf). In contrast, the METH injection caused time-dependent decreases in the expression of other genes including Npas4 and cholecystokinin (Cck). Pathway analyses showed that genes with altered expression participated in behavioral performance, cell-to-cell signaling, and regulation of gene expression. PCR analyses confirmed the changes in the expression of c-fos, fosB, Crf, Cck, and Npas4 transcripts. To determine if the METH injection caused post-translational changes in histone markers, we used western blot analyses and identified METH-mediated decreases in histone H3 acetylated at lysine 9 (H3K9ac) and lysine 18 (H3K18ac) in nuclear sub-fractions. In contrast, the METH injection caused time-dependent increases in acetylated H4K5 and H4K8. The changes in histone acetylation were accompanied by decreased expression of HDAC1 but increased expression of HDAC2 protein levels. The histone acetyltransferase, ATF2, showed significant METH-induced increased in protein expression. These results suggest that METH-induced alterations in global gene expression seen in rat NAC might be related, in part, to METH-induced changes in histone acetylation secondary to changes in HAT and HDAC expression. The causal role that HATs and HDACs might play in METH-induced gene expression needs to be investigated further.

Rationale: Repeated injections of cocaine cause blunted responses to acute cocaine challenge-induced increases in the expression of immediate early genes (IEGs)., Objectives: The aim of this study was to test if chronic methamphetamine (METH) exposure might cause similar blunting of acute METH-induced increases in IEG expression., Results: Repeated saline or METH injections were given to rats over 14 days. After 1 day of withdrawal, they received a single injection of saline or METH (5 mg/kg). Acute injection of METH increased c-fos, fosB, fra2, junB, Egr1-3, Nr4a1 (Nur77), and Nr4a3 (Nor-1) mRNA levels in the striatum of saline-pretreated rats. Chronic METH treatment alone reduced the expression of AP1, Erg1-3, and Nr4a1 transcription factors below control levels. Acute METH challenge normalized these values in METH-pretreated rats. Unexpectedly, acute METH challenge to METH-pretreated animals caused further decreases in Nr4a2 (Nurr1) mRNA levels. In contrast, the METH challenge caused significant but blunted increases in Nr4a3 and Arc expression in METH-pretreated rats. There were also chronic METH-associated decreases in the expression of cAMP responsive element binding protein (CREB) which modulates IEG expression via activation of the cAMP/PKA/CREB signal transduction pathway. Chronic METH exposure also caused significant decreases in preprotachykinin, but not in prodynorphin, mRNA levels., Conclusions: These results support the accumulated evidence that chronic administration of psychostimulants is associated with blunting of their acute stimulatory effects on IEG expression. The METH-induced renormalization of the expression of several IEGs in rats chronically exposed to METH hints to a potential molecular explanation for the recurrent self-administration of the drug by human addicts.

Methamphetamine (METH) is an addictive and neurotoxic psychostimulant widely abused in the USA and throughout the world. When administered in large doses, METH can cause depletion of striatal dopamine terminals, with preservation of midbrain dopaminergic neurons. Because alterations in the expression of transcription factors that regulate the development of dopaminergic neurons might be involved in protecting these neurons after toxic insults, we tested the possibility that their expression might be affected by toxic doses of METH in the adult brain. Male Sprague-Dawley rats pretreated with saline or increasing doses of METH were challenged with toxic doses of the drug and euthanized two weeks later. Animals that received toxic METH challenges showed decreases in dopamine levels and reductions in tyrosine hydroxylase protein concentration in the striatum. METH pretreatment protected against loss of striatal dopamine and tyrosine hydroxylase. In contrast, METH challenges caused decreases in dopamine transporters in both saline- and METH-pretreated animals. Interestingly, METH challenges elicited increases in dopamine transporter mRNA levels in the midbrain in the presence but not in the absence of METH pretreatment. Moreover, toxic METH doses caused decreases in the expression of the dopamine developmental factors, Shh, Lmx1b, and Nurr1, but not in the levels of Otx2 and Pitx3, in saline-pretreated rats. METH pretreatment followed by METH challenges also decreased Nurr1 but increased Otx2 and Pitx3 expression in the midbrain. These findings suggest that, in adult animals, toxic doses of METH can differentially influence the expression of transcription factors involved in the developmental regulation of dopamine neurons. The combined increases in Otx2 and Pitx3 expression after METH preconditioning might represent, in part, some of the mechanisms that served to protect against METH-induced striatal dopamine depletion observed after METH preconditioning.

Single large doses of methamphetamine (METH) cause endoplasmic reticulum (ER) stress and mitochondrial dysfunctions in rodent striata. The dopamine D(1) receptor appears to be involved in these METH-mediated stresses. The purpose of this study was to investigate if dopamine D(1) and D(2) receptors are involved in ER and mitochondrial stresses caused by single-day METH binges in the rat striatum. Male Sprague-Dawley rats received 4 injections of 10 mg/kg of METH alone or in combination with a putative D(1) or D(2) receptor antagonist, SCH23390 or raclopride, respectively, given 30 min prior to each METH injection. Rats were euthanized at various timepoints afterwards. Striatal tissues were used in quantitative RT-PCR and western blot analyses. We found that binge METH injections caused increased expression of the pro-survival genes, BiP/GRP-78 and P58(IPK), in a SCH23390-sensitive manner. METH also caused up-regulation of ER-stress genes, Atf2, Atf3, Atf4, CHOP/Gadd153 and Gadd34. The expression of heat shock proteins (HSPs) was increased after METH injections. SCH23390 completely blocked induction in all analyzed ER stress-related proteins that included ATF3, ATF4, CHOP/Gadd153, HSPs and caspase-12. The dopamine D(2)-like antagonist, raclopride, exerted small to moderate inhibitory influence on some METH-induced changes in ER stress proteins. Importantly, METH caused decreases in the mitochondrial anti-apoptotic protein, Bcl-2, but increases in the pro-apoptotic proteins, Bax, Bad and cytochrome c, in a SCH23390-sensitive fashion. In contrast, raclopride provided only small inhibition of METH-induced changes in mitochondrial proteins. These findings indicate that METH-induced activation of striatal ER and mitochondrial stress pathways might be more related to activation of SCH23390-sensitive receptors.

Unilateral injections of 6-hydroxydopamine into the medial forebrain bundle are used extensively as a model of Parkinson's disease. The present experiments sought to identify genes that were affected in the dopamine (DA)-denervated striatum after 6-hydroxydopamine-induced destruction of the nigrostriatal dopaminergic pathway in the rat. We also examined whether a single injection of methamphetamine (METH) (2.5 mg/kg) known to cause changes in gene expression in the normally DA-innervated striatum could still influence striatal gene expression in the absence of DA. Unilateral injections of 6-hydroxydopamine into the medial forebrain bundle resulted in METH-induced rotational behaviors ipsilateral to the lesioned side and total striatal DA depletion on the lesioned side. This injection also caused decrease in striatal serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) levels. DA depletion was associated with increases in 5-HIAA/5-HT ratios that were potentiated by the METH injection. Microarray analyses revealed changes (±1.7-fold, p<0.025) in the expression of 67 genes on the lesioned side in comparison to the intact side of the saline-treated hemiparkinsonian animals. These include follistatin, neuromedin U, and tachykinin 2 which were up-regulated. METH administration caused increases in the expression of c-fos, Egr1, and Nor-1 on the intact side. On the DA-depleted side, METH administration also increased the expression of 61 genes including Pdgf-d and Cox-2. There were METH-induced changes in 16 genes that were common in the DA-innervated and DA-depleted sides. These include c-fos and Nor-1 which show greater changes on the normal DA side. Thus, the present study documents, for the first time, that METH mediated DA-independent changes in the levels of transcripts of several genes in the DA-denervated striatum. Our results also implicate 5-HT as a potential player in these METH-induced alterations in gene expression because the METH injection also caused significant increases in 5-HIAA/5-HT ratios on the DA-depleted side.

Methamphetamine (METH) is a psychostimulant that can cause long-lasting neurodegenerative effects in humans and animals. These toxic effects appear to occur, in part, via activation of dopamine (DA) D1 receptors. This paper assessed the possibility that the DA D1 receptor antagonist, SCH23390, might inhibit METH-induced changes in the expression of several members of immediate early genes (IEGs) which are known to control more delayed expression of other genes. We found that injections of METH (4x10 mg/kg, given at 2 h intervals) caused significant increases in c-fos and fra-2 expression which lasted from 30 min to 4 h. Pre-treatment with SCH23390, given 30 min before each METH injection, completely blocked METH-induced expression of c-fos, but only partially inhibited fra-2 mRNA expression. These results were confirmed by Western blot analysis which showed METH-induced changes in c-Fos protein expression that were blocked by pretreatment with SCH23390. There were also delayed METH-induced DA D1 receptor-dependent effects on fosB mRNA expression. Even though fra-1 expression was not affected by pretreatment with METH alone, the repeated injections of SCH23390 caused substantial decreases in fra-1 mRNA expression in both the presence and absence of METH. The repeated injections of METH caused no changes in the mRNAs for c-jun, junB or junD. However, there were significant increases in the phosphorylation of c-Jun protein (ser63). Phosphorylation of c-Jun occurred in a delayed fashion (16 and 24 h after the last METH injections) and was attenuated by SCH23390 pretreatment. Interestingly, SCH23390 given alone caused significant decreases in phospho-c-Jun at all time-points. The METH injections also caused delayed induction in the expression of members of the Egr family of transcription factors in a DA D1 receptor-dependent fashion. Repeated injections of SCH23390 caused substantial suppression of basal striatal egr-1 and egr-2 mRNA expression but not of that of egr-3. Both crem and arc mRNA levels were induced by METH in a SCH23390-sensitive fashion. Moreover, multiple injections of SCH23390 given alone caused marked inhibition of basal arc expression. These results show that multiple injections of METH can differentially affect the expression of several IEGs, some of which occurred in a DA D1 receptor dependent fashion. The SCH23390-mediated suppression of basal fra-1, egr-1, and egr-2 mRNA levels suggests that their basal expression in the striatum might be dependent on tonic stimulation of the DA D1 receptor., (Published by Elsevier B.V.)

Methamphetamine (meth) is an illicit psychostimulant that is abused throughout the world. Repeated passive injections of the drug given in a single day or over a few days cause significant and long-term depletion of dopamine and serotonin in the mammalian brain. Because meth self-administration may better mimic some aspects of human drug-taking behaviors, we examined to what extent this pattern of drug treatment might also result in damage to monoaminergic systems in the brain. Rats were allowed to intravenously self-administer meth (yoked control rats received vehicle) 15 hours per day for 8 days before being euthanized at either 24 hours or at 7 and 14 days after cessation of drug taking. Meth self-administration by the rats was associated with a progressive escalation of daily drug intake to 14 mg/kg per day. Animals that self-administered meth exhibited dose-dependent decreases in striatal dopamine levels during the period of observation. In addition, there were significant reductions in the levels of striatal dopamine transporter and tyrosine hydroxylase proteins. There were also significant decreases in the levels of dopamine, dopamine transporter, and tyrosine hydroxylase in the cortex. In contrast, meth self-administration caused only transient decreases in norepinephrine and serotonin levels in the two brain regions, with these values returning to normal at seven days after cessation of drug taking. Importantly, meth self-administration was associated with significant dose-dependent increases in glial fibrillary acidic protein in both striatum and cortex, with these changes being of greater magnitude in the striatum. These results suggest that meth self-administration by rats is associated with long-term biochemical changes that are reminiscent of those observed in post-mortem brain tissues of chronic meth abusers.

Methamphetamine (METH) is an illicit drug which is neurotoxic to the mammalian brain. Numerous studies have revealed significant decreases in dopamine and serotonin levels in the brains of animals exposed to moderate-to-large METH doses given within short intervals of time. In contrast, repeated injections of small nontoxic doses of the drug followed by a challenge with toxic METH doses afford significant protection against monoamine depletion. The present study was undertaken to test the possibility that repeated injections of the drug might be accompanied by transcriptional changes involved in rendering the nigrostriatal dopaminergic system refractory to METH toxicity. Our results confirm that METH preconditioning can provide significant protection against METH-induced striatal dopamine depletion. In addition, the presence and absence of METH preconditioning were associated with substantial differences in the identity of the genes whose expression was affected by a toxic METH challenge. Quantitative PCR confirmed METH-induced changes in genes of interest and identified additional genes that were differentially impacted by the toxic METH challenge in the presence of METH preconditioning. These genes include small heat shock 27 kD 27 protein 2 (HspB2), thyrotropin-releasing hormone (TRH), brain derived neurotrophic factor (BDNF), c-fos, and some encoding antioxidant proteins including CuZn superoxide dismutase (CuZnSOD), glutathione peroxidase (GPx)-1, and heme oxygenase-1 (Hmox-1). These observations are consistent, in part, with the transcriptional alterations reported in models of lethal ischemic injuries which are preceded by ischemic or pharmacological preconditioning. Our findings suggest that multiple molecular pathways might work in tandem to protect the nigrostriatal dopaminergic pathway against the deleterious effects of the toxic psychostimulant. Further analysis of the molecular and cellular pathways regulated by these genes should help to provide some insight into the neuroadaptive potentials of the brain when repeatedly exposed to drugs of abuse.

Methamphetamine (METH) is a psychostimulant that causes damage to dopamine (DA) axons and to non-monoaminergic neurons in the brain. The aim of the present study was to investigate short- and long-term effects of neurotoxic METH treatment on novelty-induced locomotor activity in mice. Male BALB/c mice, 12-14 weeks old, were injected with saline or METH (i.p., 7.5 mg/kg x 4 times, every 2 h). Behavior and neurotoxic effects were assessed at 10 days, 3 and 5 months following drug treatment. METH administration caused marked decreases in DA levels in the mouse striatum and cortex at 10 days post-drug. However, METH did not induce any changes in novelty-induced locomotor activity. At 3 and 5 months after treatment METH-exposed mice showed significant recovery of DA levels in the striatum and cortex. In contrast, these animals demonstrated significant decreases in locomotor activity at 5 months in comparison to aged-matched control mice. Further assessment of METH toxicity using TUNEL staining showed that the drug induced increased cell death in the striatum and cortex at 3 days after administration. Taken together, these data suggest that delayed deficits in novelty-induced locomotor activity observed in METH-exposed animals are not due to neurodegeneration of DA terminals but to combined effects of METH and age-dependent dysfunction of non-DA intrinsic striatal and/or corticostriatal neurons.

Methamphetamine (METH) is an illicit toxic psychostimulant which is widely abused. Its toxic effects depend on the release of excessive levels of dopamine (DA) that activates striatal DA receptors. Inhibition of DA-mediated neurotransmission by the DA D1 receptor antagonist, SCH23390, protects against METH-induced neuronal apoptosis. The initial purpose of the present study was to investigate, using microarray analyses, the influence of SCH23390 on transcriptional responses in the rat striatum caused by a single METH injection at 2 and 4 hours after drug administration. We identified 545 out of a total of 22,227 genes as METH-responsive. These include genes which are involved in apoptotic pathways, endoplasmic reticulum (ER) stress, and in transcription regulation, among others. Of these, a total of 172 genes showed SCH23390-induced inhibition of METH-mediated changes. Among these SCH23390-responsive genes were several genes that are regulated during ER stress, namely ATF3, HSP27, Hmox1, HSP40, and CHOP/Gadd153. The secondary goal of the study was to investigate the role of DA D1 receptor stimulation on the expression of genes that participate in ER stress-mediated molecular events. We thus used quantitative PCR to confirm changes in the METH-responsive ER genes identified by the microarray analyses. We also measured the expression of these genes and of ATF4, ATF6, BiP/GRP78, and of GADD34 over a more extended time course. SCH23390 attenuated or blocked METH-induced increases in the expression of the majority of these genes. Western blot analysis revealed METH-induced increases in the expression of the antioxidant protein, Hmox1, which lasted for about 24 hours after the METH injection. Additionally, METH caused DA D1 receptor-dependent transit of the Hmox1 regulator protein, Nrf2, from cytosolic into nuclear fractions where the protein exerts its regulatory functions. When taken together, these findings indicate that SCH23390 can provide protection against neuronal apoptosis by inhibiting METH-mediated DA D1 receptor-mediated ER stress in the rat striatum. Our data also suggest that METH-induced toxicity might be a useful model to dissect molecular mechanisms involved in ER stress-dependent events in the rodent brain.

Pretreatment with methamphetamine (METH) can attenuate toxicity due to acute METH challenges. The majority of previous reports have focused mainly on the effects of the drug on the striatal dopaminergic system. In the present study, we used a regimen that involves gradual increases in METH administration to rats in order to mimic progressively larger doses of the drug used by some human METH addicts. We found that this METH preconditioning was associated with complete protection against dopamine depletion caused by a METH challenge (5 mg/kg x 6 injections given 1 h apart) in the striatum and cortex. In contrast, there was no preconditioning-mediated protection against METH-induced serotonin depletion in the striatum and hippocampus, with some protection being observed in the cortex. There was also no protection against METH-induced norepinephrine (NE) depletion in the hippocampus. These results indicate that, in contrast to the present dogmas, there might be differences in the mechanisms involved in METH toxicity on monoaminergic systems in the rodent brain. Thus, chronic injections of METH might activate programs that protect against dopamine toxicity without influencing drug-induced pathological changes in serotoninergic systems. Further studies will need to evaluate the cellular and molecular bases for these differential responses.

Background: Methamphetamine (METH) is an addictive drug that can cause neurological and psychiatric disorders. In the rodent brain, toxic doses of METH cause damage of dopaminergic terminals and apoptosis of nondopaminergic neurons. The olfactory bulb (OB) is a brain region that is rich with dopaminergic neurons and terminals., Methods: Rats were given a single injection of METH (40 mg/kg) and sacrificed at various time points afterward. The toxic effects of this injection on the OB were assessed by measuring monoamine levels, tyrosine hydroxylase (TH) immunocytochemistry, terminal deoxynucleotidyl transferase-mediated deoxyribonucleotide triphosphate (dNTP) nick end labeling (TUNEL) histochemistry, and caspase-3 immunochemistry., Results: Methamphetamine administration caused marked decreases in dopamine (DA) levels and TH-like immunostaining in the mouse OB. The drug also caused increases in TUNEL-labeled OB neurons, some of which were also positive for TH expression. Moreover, there was METH-induced expression of activated caspase-3 in TH-positive cells. Finally, the METH injection was associated with increased expression of the proapoptotic proteins, Bax and Bid, but with decreased expression of the antideath protein, Bcl2., Conclusions: These observations show, for the first time, that METH can cause loss of OB DA terminals and death of DA neurons, in part, via mechanisms that are akin to an apoptotic process.

Methamphetamine (METH), a highly addictive drug, can cause degeneration of monoaminergic terminals and neuronal apoptosis in the mammalian brain. In the present article, we have used serial analysis of gene expression (SAGE) to investigate patterns of gene expression in the striata of rats that were given a neurotoxic dose of the drug. SAGE libraries were generated from animals treated with either saline or METH (40 mg/kg) and sacrificed 2 h later. A total of 315 transcripts were differentially expressed between the two libraries (P < 0.05), with 179 (56%) being upregulated and 136 (44%) being downregulated by the METH injection. Of these, CAATT enhancer-binding protein homologous protein (CHOP)/GADD153 (growth arrest- and DNA damage-inducible gene 153) was found to be upregulated by about threefold. Analysis of the expression of genes downstream of CHOP (DOCs) revealed significant METH-induced increases in their expression. Because DOC1 is an analog of carbonic anhydrase (CA) which is involved in the interconversion between carbon dioxide and the bicarbonate ion, we also measured the effects of METH on the expression of several CAs. These were significantly upregulated by METH in a time-dependent fashion. These results indicate that METH toxicity is mediated, in part, by drug-induced perturbations of physiological processes that are dependent on normal pH and CO(2) homeostasis.

Methamphetamine (METH) is an illicit drug that causes neuronal apoptosis in the mouse striatum, in a manner similar to the neuronal loss observed in neurodegenerative diseases. In the present study, injections of METH to mice were found to cause the death of enkephalin-positive projection neurons but not the death of neuropeptide Y (NPY)/nitric oxide synthase-positive striatal interneurons. In addition, these METH injections were associated with increased expression of neuropeptide Y mRNA and changes in the expression of the NPY receptors Y1 and Y2. Administration of NPY in the cerebral ventricles blocked METH-induced apoptosis, an effect that was mediated mainly by stimulation of NPY Y2 receptors and, to a lesser extent, of NPY Y1 receptors. Finally, we also found that neuropeptide Y knock-out mice were more sensitive than wild-type mice to METH-induced neuronal apoptosis of both enkephalin- and nitric oxide synthase-containing neurons, suggesting that NPY plays a general neuroprotective role within the striatum. Together, our results demonstrate that neuropeptide Y belongs to the class of factors that maintain neuronal integrity during cellular stresses. Given the similarity between the cell death patterns induced by METH and by disorders such as Huntington's disease, our results suggest that NPY analogs might be useful therapeutic agents against some neurodegenerative processes.

Methamphetamine [METH ("speed")] is an abused psychostimulant that can cause psychotic, cognitive, and psychomotor impairment in humans. These signs and symptoms are thought to be related to dysfunctions in basal ganglionic structures of the brain. To identify possible molecular bases for these clinical manifestations, we first used cDNA microarray technology to measure METH-induced transcriptional responses in the striatum of rats treated with an apoptosis-inducing dose of the drug. METH injection resulted in increased expression of members of the Jun, Egr, and Nur77 subfamilies of transcription factors (TFs), changes that were confirmed by quantitative PCR. Because pathways linked to these factors are involved in the up-regulation of Fas ligand (FasL), FasL mRNA was quantified and found to be increased. Immunohistochemical studies also revealed METH-induced increased FasL protein expression in striatal GABAergic neurons that express enkephalin. Moreover, there were METH-mediated increases in calcineurin, as well as shuttling of nuclear factor of activated T cells (NFAT)c3 and NFATc4 from the cytosol to the nucleus of METH-treated rats, mechanisms also known to be involved in FasL regulation. Furthermore, METH induced cleavage of caspase-3 in FasL- and Fas-containing neurons. Finally, the METH-induced changes in the FasL-Fas death pathway were attenuated by pretreatment with the dopamine D1 receptor antagonist, SCH23390, which also caused attenuation of METH-induced apoptosis. These observations indicate that METH causes some of its neurodegenerative effects, in part, via stimulation of the Fas-mediated cell death pathway consequent to FasL up-regulation mediated by activation of multiple TFs.

Methamphetamine (METH) is an illicit drug that causes neurodegenerative effects in humans. In rodents, METH induces apoptosis of striatal glutamic acid decarboxylase (GAD) -containing neurons. This paper provides evidence that METH-induced cell death occurs consequent to interactions of ER stress and mitochondrial death pathways. Specifically, injections of METH are followed by an almost immediate activation of proteases calpain and caspase-12, events consistent with drug-induced ER stress. Involvement of ER stress was further supported by observations of increases in the expression of GRP78/BiP and CHOP. Participation of the mitochondrial pathway was demonstrated by the transition of AIF, smac/DIABLO, and cytochrome c from mitochondrial into cytoplasmic fractions. These changes occur before the apoptosome-associated pro-caspase-9 cleavage. Effector caspases-3 and -6, but not -7, were cleaved with the initial time of caspase-3 activation occurring before caspase 9 cleavage; this suggests possible earlier cleavage of caspase-3 by caspase-12. These events preceded proteolysis of the caspase substrates DFF-45, lamin A, and PARP in nuclear fractions. These findings indicate that METH causes neuronal apoptosis in part via cross-talks between ER- and mitochondria-generated processes, which cause activation of both caspase-dependent and -independent pathways.

The regional distribution of c-Jun expression and of the number of apoptotic cells was compared in various brain areas after methamphetamine administration to mice. Our results showed that there was methamphetamine-induced overexpression of c-Jun in the cortex and striatum but not in the cerebellar cortex. There was an almost totally similar regional appearance of methamphetamine-induced apoptotic cells in the mouse brain; no apoptosis was present in the cerebellum. Additionally, in the neocortical area, more positive signals for c-Jun immunoreactivity were observed in the piriform cortex, an area that also showed more positive terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) signals than the frontal and parietal cortices. These observations suggested that c-Jun might be involved in methamphetamine-induced apoptosis. This idea was confirmed by using heterozygous c-Jun knockout mice that showed much less apoptosis than wild-type controls. In addition, we found that the majority of TUNEL-positive cells were also positive for c-Jun-like immunoreactivity in both genotypes. Moreover, methamphetamine-induced caspase-3 activity and PARP cleavage were also reduced in c-Jun heterozygous knockout mice. In contrast, methamphetamine-induced hyperthermia was essentially identical in the two genotypes. When taken together, our data support the hypothesis that c-Jun is involved in methamphetamine-induced apoptosis.

Methamphetamine (METH) is a drug of abuse which can cause apoptosis and degeneration of monoaminergic terminals in the mammalian brain. c-fos appears to play a protective role against METH-induced damage because METH toxicity is exacerbated in c-fos heterozygous knockout mice. In the present study, we used the comprehensive technique of cDNA array to test the idea that heterozygous c-fos knockout mice might show differential METH-induced molecular responses in comparison to wild-type (WT) animals. Of 1,176 genes examined, the expression of 195 genes in either of the two groups of mice was affected by at least 2-fold at 2 or 12 h after METH treatment. More genes were either up- or downregulated in the WT mice. Moreover, there were substantial differences in the pattern of responses between the two genotypes, with more genes involved in DNA repair and protective processes being upregulated in WT mice after METH administration. These results support the idea that the c-fos knockout genotype may render the animals unable to trigger multicomponent responses in order to protect against the multifaceted toxic effects of this illicit neurotoxin., (Copyright 2002 Wiley-Liss, Inc.)

The clinical abuse of methamphetamine (METH) is a major concern because it can cause long-lasting neurodegenerative effects in humans. Current concepts of the molecular mechanisms underlying these complications have centered on the formation of reactive oxygen species. Herein, we provide cDNA microarray evidence that METH administration caused the induction of c-Jun and of other members involved in the pathway leading to c-Jun activation [stress-activated protein kinase/Jun N-terminal kinase (JNK3), Crk-associated substrate-Cas and c-Src] after environmental stresses or cytokine stimulation. Reverse transcription-polymerase chain reaction analysis confirmed these increases and also showed that the expression of JNK1 and JNK3 but not JNK2 was also increased in the METH-treated mice. Western blot analysis showed that METH increased the expression of c-Jun phosphorylated at serine-63 and serine-73 residues. Other upstream members of the JNK pathway, including phosphorylated JNKs, mitogen-activated protein kinase kinase 4, mitogen-activated protein kinase kinase 7, Crk II, Cas, and c-Src were also increased at the protein level. These values returned to baseline by 1 week after drug treatment. These results are discussed in terms of their support for a possible role of the activation of the JNK/Jun pathway in the pathophysiological effects of METH.

Multidrug resistance protein 1a (MDR1a) potentiated methylenedioxymethamphetamine (MDMA)-induced decreases of dopamine (DA) and dopamine transport protein in mouse brain one week after MDMA administration. In the present study, we examined if mdr1a wild-type (mdr1a +/+) and knock-out (mdr1a −/−) mice differentially handle the acute effects of MDMA on the nigrostriatal DA system 0–24 h following a single drug injection. 3-way ANOVA revealed significant 2-way interactions of strain × time ( F5,152 = 32.4, P < 0.001) and strain × dose ( F3,152 = 25.8, P < 0.001) on 3,4-dihydroxyphenylacetic acid (DOPAC)/DA ratios in mdr1a +/+ and −/− mice. 0.3–3 h after 10 mg/kg MDMA, DOPAC/DA ratios were increased in mdr1a +/+ mice, but decreased 0.3–1 h after MDMA in mdr1a −/− mice. Twenty-four hours after 10 mg/kg MDMA, DOPAC/DA ratios were increased 600% in mdr1a +/+ mice compared to saline-treated control mice, while in mdr1a −/− mice DOPAC/DA ratios were unchanged. Striatal MDMA and its metabolite, methylenedioxyamphetamine, concentrations by gas chromatography–mass spectrometry were similar in both strains 0.3–4 h after MDMA, discounting the role of MDR1a-facilitated MDMA transport in observed inter-strain differences. Increased DOPAC/DA turnover in mdr1a +/+ mice following MDMA is consistent with the previous report that MDMA neurotoxicity is increased in mdr1a +/+ mice. Increased DA turnover via monoamine oxidase in mdr1a +/+ vs −/− mice might increase exposure to neurotoxic reactive oxygen species. [ABSTRACT FROM AUTHOR]

Methamphetamine (METH) is a neurotoxic psychostimulant that causes damage to striatal dopamine (DA) terminals and to non-monoaminergic cells in the striatum (STR) and cortex of rodents. The aim of the present study was to investigate short- and long-term consequences of neurotoxic METH doses on mice behaviors. Male BALB/c mice, 12-14 weeks old, were injected with dl-METH (i.p., 7.5 mg/kg x 4 times, every 2 hours) or saline. Behaviors were accessed at 10 days after drug treatment. METH administration caused significant decreases in responding to object displacement using an Open Field Object Recognition test. In contrast, METH caused increased response to object novelty. There were no drug-induced effects on locomotor activity. Moreover, repeated METH injections resulted in significant decreases in dopamine (DA) levels in the STR, frontal cortex (FC) and olfactory bulb (OB) in mice sacrificed 10 days after drug treatment. Furthermore, as previously reported for the cortex and striatum, METH administration caused marked increases in the number of cells that were positive for TUNEL staining in the OB at 3 days, observations that are indicative of drug-induced enhancement of cell death in that structure. When taken together, these results suggest that METH caused impairments in spatial learning and/or memory as a consequence of its neurodegenerative effects. [ABSTRACT FROM AUTHOR]

Methamphetamine (METH) is a toxic drug of abuse, which can cause significant decreases in the levels of monoamines in various brain regions. However, animals treated with progressively increasing doses of METH over several weeks are protected against the toxic effects of the drug. In the present study, we tested the possibility that this pattern of METH injections might be associated with transcriptional changes in the rat striatum, an area of the brain which is known to be very sensitive to METH toxicity and which is protected by METH preconditioning. We found that the presence and absence of preconditioning followed by injection of large doses of METH caused differential expression in different sets of striatal genes. Quantitative PCR confirmed METH-induced changes in some genes of interest. These include small heat shock 27 kD proteins 1 and 2 (HspB1 and HspB2), brain derived neurotrophic factor (BDNF), and heme oxygenase-1 (Hmox-1). Our observations are consistent with previous studies which have reported that ischemic or pharmacological preconditioning can cause reprogramming of gene expression after lethal ischemic insults. These studies add to the growing literature on the effects of preconditioning on the brain transcriptome. [ABSTRACT FROM AUTHOR]