Incubation of methamphetamine craving in punishment-resistant individuals is associated with activation of specific gene networks in the rat dorsal striatum.

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.

Biochemical Neuroadaptations in the Rat Striatal Dopaminergic System after Prolonged Exposure to Methamphetamine Self-Administration.

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.

Footshock-Induced Abstinence from Compulsive Methamphetamine Self-administration in Rat Model Is Accompanied by Increased Hippocampal Expression of Cannabinoid Receptors (CB1 and CB2).

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.

Epigenetic Regulatory Dynamics in Models of Methamphetamine-Use Disorder.

Methamphetamine (METH)-use disorder (MUD) is a very serious, potentially lethal, biopsychosocial disease. Exposure to METH causes long-term changes to brain regions involved in reward processing and motivation, leading vulnerable individuals to engage in pathological drug-seeking and drug-taking behavior that can remain a lifelong struggle. It is crucial to elucidate underlying mechanisms by which exposure to METH leads to molecular neuroadaptive changes at transcriptional and translational levels. Changes in gene expression are controlled by post-translational modifications via chromatin remodeling. This review article focuses on the brain-region specific combinatorial or distinct epigenetic modifications that lead to METH-induced changes in gene expression.

Oxycodone self-administration activates the mitogen-activated protein kinase/ mitogen- and stress-activated protein kinase (MAPK-MSK) signaling pathway in the rat dorsal striatum.

To identify signaling pathways activated by oxycodone self-administration (SA), Sprague-Dawley rats self-administered oxycodone for 20 days using short-(ShA, 3 h) and long-access (LgA, 9 h) paradigms. Animals were euthanized 2 h after SA cessation and dorsal striata were used in post-mortem molecular analyses. LgA rats escalated their oxycodone intake and separated into lower (LgA-L) or higher (LgA-H) oxycodone takers. LgA-H rats showed increased striatal protein phosphorylation of ERK1/2 and MSK1/2. Histone H3, phosphorylated at serine 10 and acetylated at lysine 14 (H3S10pK14Ac), a MSK1/2 target, showed increased abundance only in LgA-H rats. RT-qPCR analyses revealed increased AMPA receptor subunits, GluA2 and GluA3 mRNAs, in the LgA-H rats. GluA3, but not GluA2, mRNA expression correlated positively with changes in pMSK1/2 and H3S10pK14Ac. These findings suggest that escalated oxycodone SA results in MSK1/2-dependent histone phosphorylation and increases in striatal gene expression. These observations offer potential avenues for interventions against oxycodone addiction.

Potassium Channels and Their Potential Roles in Substance Use Disorders.

Substance use disorders (SUDs) are ubiquitous throughout the world. However, much remains to be done to develop pharmacotherapies that are very efficacious because the focus has been mostly on using dopaminergic agents or opioid agonists. Herein we discuss the potential of using potassium channel activators in SUD treatment because evidence has accumulated to support a role of these channels in the effects of rewarding drugs. Potassium channels regulate neuronal action potential via effects on threshold, burst firing, and firing frequency. They are located in brain regions identified as important for the behavioral responses to rewarding drugs. In addition, their expression profiles are influenced by administration of rewarding substances. Genetic studies have also implicated variants in genes that encode potassium channels. Importantly, administration of potassium agonists have been shown to reduce alcohol intake and to augment the behavioral effects of opioid drugs. Potassium channel expression is also increased in animals with reduced intake of methamphetamine. Together, these results support the idea of further investing in studies that focus on elucidating the role of potassium channels as targets for therapeutic interventions against SUDs.

Sex Differences in Escalated Methamphetamine Self-Administration and Altered Gene Expression Associated With Incubation of Methamphetamine Seeking.

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.

Escalated Oxycodone Self-Administration Causes Differential Striatal mRNA Expression of FGFs and IEGs Following Abstinence-Associated Incubation of Oxycodone Craving.

Addiction to prescribed opioids including oxycodone has reached tragic levels. Herein, we investigated the relevance of fibroblast growth factors (FGFs) and immediate early genes (IEGs) to withdrawal-induced incubation of drug craving following escalated oxycodone self-administration (SA). Rats were trained to self-administer oxycodone for 4 weeks. Seeking tests were performed at various intervals during 1 month of drug withdrawal. Rats were euthanized 1 day after the last test and nucleus accumbens and dorsal striata were dissected for use in PCR analyses. Rats given long access (LgA, 9 h), but not short access (ShA, 3 h) to drug escalated their oxycodone intake and exhibited incubation of oxycodone seeking during withdrawal. These rats exhibited dose-dependent increases in fgf2 expression in the dorsal striatum. Fgfr2 expression was also significantly increased in the striatum in LgA, but not ShA groups. Similarly, striatal c-fos and junB mRNA levels showed greater increases in LgA rats. The observations that fgf mRNA levels were more altered in the dorsal striatum than in the NAc of LgA rats suggest that changes in striatal FGF expression may be more salient to incubation of oxycodone craving than alterations in the NAc. Targeting FGF signaling pathways might offer novel strategies against opioid addiction.

Escalated Oxycodone Self-Administration and Punishment: Differential Expression of Opioid Receptors and Immediate Early Genes in the Rat Dorsal Striatum and Prefrontal Cortex.

Opioid use disorder (OUD) is characterized by compulsive drug taking despite adverse life consequences. Here, we sought to identify neurobiological consequences associated with the behavioral effects of contingent footshocks administered after escalation of oxycodone self-administration. To reach these goals, Sprague-Dawley rats were trained to self-administer oxycodone for 4 weeks and were then exposed to contingent electric footshocks. This paradigm helped to dichotomize rats into two distinct behavioral phenotypes: (1) those that reduce lever pressing (shock-sensitive) and (2) others that continue lever pressing (shock-resistant) for oxycodone during contingent punishment. The rats were euthanized at 2-h after the last oxycodone plus footshock session. The dorsal striata and prefrontal cortices were dissected for use in western blot and RT-qPCR analyses. All oxycodone self-administration rats showed significant decreased expression of Mu and Kappa opioid receptor proteins only in the dorsal striatum. However, expression of Delta opioid receptor protein was decreased in both brain regions. RT-qPCR analyses documented significant decreases in the expression of c-fos, fosB, fra2, junB, egr1, and egr2 mRNAs in the dorsal striatum (but not in PFC) of the shock-sensitive rats. In the PFC, junD expression was reduced in both phenotypes. However, egr3 mRNA expression was increased in the PFC of only shock-resistant rats. These results reveal that, similar to psychostimulants and alcohol, footshocks can dichotomize rats that escalated their intake of oxycodone into two distinct behavioral phenotypes. These animals also show significant differences in the mRNA expression of immediate early genes, mainly, in the dorsal striatum. The increases in PFC egr3 expression in the shock-resistant rats suggest that Egr3 might be involved in the persistence of oxycodone-associated memory under aversive conditions. This punishment-driven model may help to identify neurobiological substrates of persistent oxycodone taking and abstinence in the presence of adverse consequences.

Molecular Adaptations in the Rat Dorsal Striatum and Hippocampus Following Abstinence-Induced Incubation of Drug Seeking After Escalated Oxycodone Self-Administration.

Repeated exposure to the opioid agonist, oxycodone, can lead to addiction. Here, we sought to identify potential neurobiological consequences of withdrawal from escalated and non-escalated oxycodone self-administration in rats. To reach these goals, we used short-access (ShA) (3 h) and long-access (LgA) (9 h) exposure to oxycodone self-administration followed by protracted forced abstinence. After 31 days of withdrawal, we quantified mRNA and protein levels of opioid receptors in the rat dorsal striatum and hippocampus. Rats in the LgA, but not the ShA, group exhibited escalation of oxycodone SA, with distinction of two behavioral phenotypes of relatively lower (LgA-L) and higher (LgA-H) oxycodone takers. Both LgA, but not ShA, phenotypes showed time-dependent increases in oxycodone seeking during the 31 days of forced abstinence. Rats from both LgA-L and LgA-H groups also exhibited decreased levels of striatal mu opioid receptor protein levels in comparison to saline and ShA rats. In contrast, mu opioid receptor mRNA expression was increased in the dorsal striatum of LgA-H rats. Moreover, hippocampal mu and kappa receptor protein levels were both increased in the LgA-H phenotype. Nevertheless, hippocampal mu receptor mRNA levels were decreased in the two LgA groups whereas kappa receptor mRNA expression was decreased in ShA and LgA oxycodone groups. Decreases in striatal mu opioid receptor protein expression in the LgA rats may serve as substrates for relapse to drug seeking because these changes occur in rats that showed incubation of oxycodone seeking.

Methamphetamine Induces TET1- and TET3-Dependent DNA Hydroxymethylation of Crh and Avp Genes in the Rat Nucleus Accumbens.

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.

Selective Activation of Striatal NGF-TrkA/p75NTR/MAPK Intracellular Signaling in Rats That Show Suppression of Methamphetamine Intake 30 Days following Drug Abstinence.

Background: The continuing epidemic of methamphetamine addiction has prompted research aimed at understanding striatal dysfunctions potentially associated with long-term methamphetamine use. Methods: Here, we investigated transcriptional and translational alterations in the expression of neurotrophic factors in the rat striatum at 30 days following methamphetamine self-administration and footshock punishment. Male Sprague-Dawley rats were trained to self-administer methamphetamine (0.1 mg/kg/injection, i.v.) or saline during twenty-two 9-hour sessions. Subsequently, rats were subjected to incremental footshocks for 13 additional methamphetamine self-administration sessions. This paradigm led to the identification of rats with shock-resistant and shock-sensitive phenotypes. Thirty days following the last footshock session, the dorsal striatum was dissected and processed for gene expression and protein analyses. Results: PCR arrays revealed significant differences in neurotrophins and their receptors between the 2 phenotypes. Brain-derived neurotrophic factor and nerve growth factor protein levels were increased in the dorsal striatum of both shock-resistant and shock-sensitive rats. However, neurotrophic receptor tyrosine kinase 1 phosphorylation and nerve growth factor receptor protein expression were increased only in the shock-sensitive phenotype. Moreover, shock-sensitive rats showed increased abundance of several phosphorylated proteins known to participate in Ras/Raf/MEK/ERK signaling cascade including cRaf, ERK1/2, MSK1, and CREB. Conclusions: These findings support the notion that animals with distinct phenotypes for methamphetamine intake in the presence of adverse consequences also display differential changes in an intracellular signaling cascade activated by nerve growth factor-TrkA/p75NTR interactions. Thus, the development of pharmacological agents that can activate nerve growth factor-dependent pathways may be a promising therapeutic approach to combat methamphetamine addiction.

Compulsive methamphetamine taking in the presence of punishment is associated with increased oxytocin expression in the nucleus accumbens of rats.

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.

Compulsive methamphetamine taking under punishment is associated with greater cue-induced drug seeking in rats.

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.

Increased expression of proenkephalin and prodynorphin mRNAs in the nucleus accumbens of compulsive methamphetamine taking rats.

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.

An Acute Methamphetamine Injection Downregulates the Expression of Several Histone Deacetylases (HDACs) in the Mouse Nucleus Accumbens: Potential Regulatory Role of HDAC2 Expression.

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.

CAMKII-conditional deletion of histone deacetylase 2 potentiates acute methamphetamine-induced expression of immediate early genes in the mouse nucleus accumbens.

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.

Differential effects of binge methamphetamine injections on the mRNA expression of histone deacetylases (HDACs) in the rat striatum.

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.

Incubation of methamphetamine and palatable food craving after punishment-induced abstinence.

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.

Enhanced upregulation of CRH mRNA expression in the nucleus accumbens of male rats after a second injection of methamphetamine given thirty days later.

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.