Methamphetamine induces long-term changes in GABAA receptor alpha2 subunit and GAD67 expression

The present study investigated whether GABA(A) receptor alpha2 subunit and GAD(67) are involved in chronic high dose methamphetamine (METH)-induced sensitization and neurotoxicity. The METH sensitization was established in rats by 7-day pump infusion plus daily injection (25mg/kg/day) and a subsequent 28-day withdrawal period. Behavioral sensitization was assessed by behavioral ratings after challenge with METH (0.5mg/kg). The neurotoxicity was evaluated by the expression of glial fibrillary acidic protein (GFAP). Western blot assay showed that METH sensitization decreases GABA(A) alpha2 subunit and GAD(67) protein levels in the nucleus accumbens (NAc) core and shell, and conversely, these proteins were increased in the caudate. An upregulation of GFAP expression was observed in the caudate, but not in the NAc core and shell. These data suggest that inhibition of GABA transmission in the NAc is related to METH behavioral sensitization, whereas activation of GABA transmission in the caudate is associated with METH-induced neurotoxicity.     

l-Tetrahydropalmatine inhibits methamphetamine-induced locomotor activity via regulation of 5-HT neuronal activity and dopamine D3 receptor expression

Methamphetamine (METH) is a psychomotor stimulant that produces hyperlocomotion in rodents. l-tetrahydropalmatine (l-THP) is an active ingredient found in Corydalis ternata which has been used as a traditional herbal preparation in Asian countries for centuries, however, the effect of l-THP on METH-induced phenotypes largely unknown. In this study, to evaluate the effect of l-THP on METH-induced psychotropic effects, rats were pretreated with l-THP (10 and 15 mg/kg) before acute METH injection, following which the total distance the rats moved in an hour was measured. To clarify a possible mechanism underlying the effect of l-THP on METH-induced behavioral changes, dopamine receptor mRNA expression levels in the striatum of the rats was measured following the locomotor activity study. In addition, the effect of l-THP (10 and 15 mg/kg) on serotonergic (5-HTergic) neuronal pathway activation was studied by measurement of 5-HT (80 μg/10 μl/mouse)-induced head twitch response (HTR) in mice. l-THP administration significantly inhibited both hyperlocomotion in rats and HTR in mice. l-THP inhibited climbing behavior-induced by dopaminergic (DAergic) neuronal activation in mice. Furthermore, l-THP attenuated the decrease in dopamine D3 receptor mRNA expression levels in the striatum of the rats induced by METH. These results suggest that l-THP can ameliorate behavioral phenotype induced by METH through regulation of 5-HT neuronal activity and dopamine D3 receptor expression.     

Role of serine racemase in behavioral sensitization in mice after repeated administration of methamphetamine

Background

The N-methyl-D-aspartate (NMDA) receptors play a role in behavioral abnormalities observed after administration of the psychostimulant, methamphetamine (METH). Serine racemase (SRR) is an enzyme which synthesizes D-serine, an endogenous co-agonist of NMDA receptors. Using Srr knock-out (KO) mice, we investigated the role of SRR on METH-induced behavioral abnormalities in mice.

Methodology/Principal Findings

Evaluations of behavior in acute hyperlocomotion, behavioral sensitization, and conditioned place preference (CPP) were performed. The role of SRR on the release of dopamine (DA) in the nucleus accumbens after administration of METH was examined using in vivo microdialysis technique. Additionally, phosphorylation levels of ERK1/2 proteins in the striatum, frontal cortex and hippocampus were examined using Western blot analysis. Acute hyperlocomotion after a single administration of METH (3 mg/kg) was comparable between wild-type (WT) and Srr-KO mice. However, repeated administration of METH (3 mg/kg/day, once daily for 5 days) resulted in behavioral sensitization in WT, but not Srr-KO mice. Pretreatment with D-serine (900 mg/kg, 30 min prior to each METH treatment) did not affect the development of behavioral sensitization after repeated METH administration. In the CPP paradigm, METH-induced rewarding effects were demonstrable in both WT and Srr-KO mice. In vivo microdialysis study showed that METH (1 mg/kg)-induced DA release in the nucleus accumbens of Srr-KO mice previously treated with METH was significantly lower than that of the WT mice previously treated with METH. Interestingly, a single administration of METH (3 mg/kg) significantly increased the phosphorylation status of ERK1/2 in the striatum of WT, but not Srr-KO mice.

Conclusions/Significance

These findings suggest first, that SRR plays a role in the development of behavioral sensitization in mice after repeated administration of METH, and second that phosphorylation of ERK1/2 by METH may contribute to the development of this sensitization as seen in WT but not Srr-KO mice.     

Reduction of methamphetamine-induced sensitization and reward in matrix metalloproteinase-2 and -9-deficient mice

Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) function to remodel the pericellular environment. Their activation and regulation are associated with synaptic physiology and pathology. Here, we investigated whether MMP-2 and MMP-9 are involved in the rewarding effects of and sensitization to methamphetamine (METH) in animals, in which the remodelling of neural circuits may play a crucial role. Repeated METH treatment induced behavioural sensitization, which was accompanied by an increase in MMP-2 and MMP-9 activity in the brain. In MMP-2- and MMP-9-deficient mice [MMP-2-(-/-) and MMP-9-(-/-)], METH-induced behavioural sensitization and conditioned place preference, a measure of the rewarding effect, as well as METH-increased dopamine release in the nucleus accumbens (NAc) were attenuated compared with those in wild-type mice. In contrast, infusion of purified human MMP-2 into the NAc significantly potentiated the METH-increased dopamine release. The [(3)H]dopamine uptake into striatal synaptosomes was reduced in wild-type mice after repeated METH treatment, but METH-induced changes in [(3)H]dopamine uptake were significantly attenuated in MMP-2-(-/-) and MMP-9-(-/-) mice. These results suggest that both MMP-2 and MMP-9 play a crucial role in METH-induced behavioural sensitization and reward by regulating METH-induced dopamine release and uptake in the NAc.     

Role of tissue plasminogen activator in the sensitization of methamphetamine-induced dopamine release in the nucleus accumbens

We have previously demonstrated that repeated, but not acute, methamphetamine (METH) treatment increases tissue plasminogen activator (tPA) activity in the brain, which is associated with the development of behavioral sensitization to METH. In this study, we investigated whether the tPA-plasmin system is involved in the development of sensitization in METH-induced dopamine release in the nucleus accumbens (NAc). There was no difference in acute METH-induced increase in extracellular dopamine levels in the NAc between wild-type and tPA-deficient (tPA−/−) mice. Repeated METH treatment resulted in a significant enhancement of METH- induced dopamine release in wild-type mice, but not tPA−/− mice. Microinjection of exogenous tPA or plasmin into the NAc of wild-type mice significantly potentiated acute METH- induced dopamine release. Degradation of laminin was evident in brain tissues incubated with tPA plus plasminogen or plasmin in vitro although tPA or plasminogen alone had no effect. Immunohistochemical analysis revealed that microinjection of plasmin into the NAc reduced laminin immunoreactivity without neuronal damage. Our findings suggest that the tPA-plasmin system participates in the development of behavioral sensitization induced by repeated METH treatment, by regulating the processes underlying the sensitization of METH-induced dopamine release in the NAc, in which degradation of laminin by plasmin may play a role.     

Evaluation of the effects of alpha-phenyl-N-tert-butyl nitrone pretreatment on the neurobehavioral effects of methamphetamine

A relationship between formation of reactive oxygen species (ROS) and energy depletion has been proposed to play an important role in mediating methamphetamine (METH)-induced neurotoxicity. To evaluate this relationship, we examined the effect of the spin-trap agent, alpha-phenyl-N-tert-butyl nitrone (PBN) on hyperthermia and self-injurious behavior (SIB) and striatal dopamine (DA) depletion produced by METH (4 injections of 4 mg/kg, 2 hr intervals, s.c.) in BALB/c mice. Repeated administration of METH induced hyperthermia, incidence of SIB and striatal DA depletion (84% after 3 days). Pretreatment with PBN (4 injections of 60 or 120 mg/kg, i.p.) reduced METH-induced hyperthermia, but did not significantly attenuate METH-induced SIB or the striatal DA depletion. On the other hand, pretreatment with high doses of PBN (4 injections of 180 or 240 mg/kg, i.p.) protected against METH-induced hyperthermia and SIB, and PBN (180 mg/kg) also completely protected against the acute striatal DA depletion 60 min after the last injection of the drug. However, the long-lasting striatal DA depletion was only attenuated by 52 or 56%, respectively. These results indicate that METH-induced hyperthermia contributes to, but is not solely responsible for METH-induced neurotoxicity, and supports a role for formation of ROS and other mechanisms in the generation of METH-induced striatal dopaminergic neurotoxicity. In addition, the difference in the efficacy of PBN to protect against the acute or long-lasting striatal DA depletion induced by METH may indicate that both ROS formation and other mechanisms are required for METH-induced neurotoxicity to develop.     

Role of matrix metalloproteinase and tissue inhibitor of MMP in methamphetamine-induced behavioral sensitization and reward: implications for dopamine receptor down-regulation and dopamine release

Matrix metalloproteinases (MMPs) and its inhibitors (TIMPs) function to remodel the pericellular environment. We have demonstrated that methamphetamine (METH)-induced behavioral sensitization and reward were markedly attenuated in MMP-2- and MMP-9 deficient [MMP-2-(-/-) and MMP-9-(-/-)] mice compared with those in wild-type mice, suggesting that METH-induced expression of MMP-2 and MMP-9 in the brain plays a role in the development of METH-induced sensitization and reward. In the present study, we investigated the changes in TIMP-2 expression in the brain after repeated METH treatment. Furthermore, we studied a role of MMP/TIMP system in METH-induced behavioral changes and dopamine neurotransmission. Repeated METH treatment induced behavioral sensitization, which was accompanied by an increase in TIMP-2 expression. Antisense TIMP-2 oligonucleotide (TIMP-AS) treatment enhanced the sensitization, which was associated with the potentiation of METH-induced dopamine release in the nucleus accumbens (NAc). On the other hand, MMP-2/-9 inhibitors blocked the METH-induced behavioral sensitization and conditioned place preference, a measure of the rewarding effect, and reduced the METH-increased dopamine release in the NAc. Dopamine receptor agonist-stimulated [(35)S]GTPgammaS binding was reduced in the frontal cortex of sensitized rats. TIMP-AS treatment potentiated, while MMP-2/-9 inhibitor attenuated, the reduction of dopamine D2 receptor agonist-stimulated [(35)S]GTPgammaS binding. Repeated METH treatment also reduced dopamine D2 receptor agonist-stimulated [(35)S]GTPgammaS binding in wild-type mice, but such changes were significantly attenuated in MMP-2-(-/-) and MMP-9-(-/-) mice. These results suggest that the MMP/TIMP system is involved in METH-induced behavioral sensitization and reward, by regulating dopamine release and receptor signaling.     

The Involvement of Oxytocin in the Subthalamic Nucleus on Relapse to Methamphetamine-Seeking Behaviour

The psychostimulant methamphetamine (METH) is an addictive drug of abuse. The neuropeptide oxytocin has been shown to modulate METH-related reward and METH-seeking behaviour. Recent findings implicated the subthalamic nucleus (STh) as a key brain region in oxytocin modulation of METH-induced reward. However, it is unclear if oxytocin acts in this region to attenuate relapse to METH-seeking behaviour, and if this action is through the oxytocin receptor. We aimed to determine whether oxytocin pretreatment administered into the STh would reduce reinstatement to METH use in rats experienced at METH self-administration, and if this could be reversed by the co-administration of the oxytocin receptor antagonist desGly-NH2,d(CH2)5[D-Tyr2,Thr4]OVT. Male Sprague Dawley rats underwent surgery to implant an intravenous jugular vein catheter and bilateral microinjection cannulae into the STh under isoflourane anaesthesia. Rats were then trained to self-administer intravenous METH (0.1 mg/kg/infusion) by lever press during 2-hour sessions under a fixed ratio 1 schedule for 20 days. Following extinction of lever press activity, the effect of microinjecting saline, oxytocin (0.2 pmol, 0.6 pmol, 1.8 pmol, 3.6 pmol) or co-administration of oxytocin (3.6 pmol) and desGly-NH₂,d(CH₂)₅[D-Tyr²,Thr⁴]OVT (3 nmol) into the STh (200 nl/side) was examined on METH-primed reinstatement (1 mg/kg; i.p.). We found that local administration of the highest oxytocin dose (3.6 pmol) into the STh decreased METH-induced reinstatement and desGly-NH2,d(CH2)5[D-Tyr2,Thr4]OVT had a non-specific effect on lever press activity. These findings highlight that oxytocin modulation of the STh is an important modulator of relapse to METH abuse.     

Rapid ATP Loss Caused by Methamphetamine in the Mouse Striatum: Relationship Between Energy Impairment and Dopaminergic Neurotoxicity

Abstract: To study the relationship between energy impairment and the effects of α-methamphetamine (METH) on dopaminergic neurons, ATP and dopamine levels were measured in the brain of C57BL/6 mice treated with either a single or four injections of METH (10 mg/kg, i.p.) at 2-h intervals. Neither striatal ATP nor dopamine concentrations changed after a single injection of METH, but both were significantly decreased 1.5 h after the multiple-dose regimen. The effects of METH on ATP levels appear to be selective for the striatum, as ATP concentrations were not affected in the cerebellar cortex and hippocampus after either a single or multiple injections of METH. In a second set of experiments, an intraperitoneal injection of 2-deoxyglucose (2-DG; 1 g/kg), an inhibitor of glucose uptake and utilization, was given 30 min before the third and fourth injections of METH. 2-DG significantly potentiated METH-induced striatal ATP loss at 1.5 h and dopamine depletions at 1.5 h and 1 week. These results indicate that a toxic regimen of METH selectively causes striatal energy impairment and raise the possibility that perturbations of energy metabolism play a role in METH-induced dopaminergic neurotoxicity.     

Δ9-Tetrahydrocannabinol Prevents Methamphetamine-Induced Neurotoxicity

Methamphetamine (METH) is a potent psychostimulant with neurotoxic properties. Heavy use increases the activation of neuronal nitric oxide synthase (nNOS), production of peroxynitrites, microglia stimulation, and induces hyperthermia and anorectic effects. Most METH recreational users also consume cannabis. Preclinical studies have shown that natural (Δ9-tetrahydrocannabinol, Δ9-THC) and synthetic cannabinoid CB1 and CB2 receptor agonists exert neuroprotective effects on different models of cerebral damage. Here, we investigated the neuroprotective effect of Δ9-THC on METH-induced neurotoxicity by examining its ability to reduce astrocyte activation and nNOS overexpression in selected brain areas. Rats exposed to a METH neurotoxic regimen (4×10 mg/kg, 2 hours apart) were pre- or post-treated with Δ9-THC (1 or 3 mg/kg) and sacrificed 3 days after the last METH administration. Semi-quantitative immunohistochemistry was performed using antibodies against nNOS and Glial Fibrillary Acidic Protein (GFAP). Results showed that, as compared to corresponding controls (i) METH-induced nNOS overexpression in the caudate-putamen (CPu) was significantly attenuated by pre- and post-treatment with both doses of Δ9-THC (−19% and −28% for 1 mg/kg pre- and post-treated animals; −25% and −21% for 3 mg/kg pre- and post-treated animals); (ii) METH-induced GFAP-immunoreactivity (IR) was significantly reduced in the CPu by post-treatment with 1 mg/kg Δ9-THC1 (−50%) and by pre-treatment with 3 mg/kg Δ9-THC (−53%); (iii) METH-induced GFAP-IR was significantly decreased in the prefrontal cortex (PFC) by pre- and post-treatment with both doses of Δ9-THC (−34% and −47% for 1 mg/kg pre- and post-treated animals; −37% and −29% for 3 mg/kg pre- and post-treated animals). The cannabinoid CB1 receptor antagonist SR141716A attenuated METH-induced nNOS overexpression in the CPu, but failed to counteract the Δ9-THC-mediated reduction of METH-induced GFAP-IR both in the PFC and CPu. Our results indicate that Δ9-THC reduces METH-induced brain damage via inhibition of nNOS expression and astrocyte activation through CB1-dependent and independent mechanisms, respectively.     

The neurokinin-1 receptor modulates the methamphetamine-induced striatal apoptosis and nitric oxide formation in mice

In a previous study we showed that pharmacological blockade of the neurokinin-1 receptors attenuated the methamphetamine (METH)-induced toxicity of the striatal dopamine terminals. In the present study we examined the role of the neurokinin-1 receptors on the METH-induced apoptosis of some striatal neurons. To that end, we administered a single injection of METH (30 mg/kg, i.p.) to male mice. METH induced the apoptosis (terminal deoxyncleotidyl transferase-mediated dUTP nick end labeling) of approximately 20% of striatal neurons. This percentage of METH-induced apoptosis was significantly attenuated by either a single injection of the neurokinin-1 receptor antagonist, 17-β-hydroxy-17-a-ethynyl-5-a-androstano[3,2-β]pyrimido[1,2-a]benzimidazole (WIN-51,708) (5 mg/kg, i.p.), or the ablation of the striatal interneurons expressing the neurokinin-1 receptors (cholinergic and somatostatin) with the selective neurotoxin [Sar⁹,Met(O₂)¹¹] substance P–saporin. Next we assessed the levels of striatal 3-nitrotyrosine (3-NT) by HPLC and immunohistochemistry. METH increased the levels of striatal 3-NT and this increase was attenuated by pre-treatment with WIN-51,708. Our data support the hypothesis that METH-induced striatal apoptosis occurs via a mechanism involving the neurokinin-1 receptors and the activation of nitric oxide synthesis. Our findings are relevant for the treatment of METH abuse and may be relevant to certain neurological disorders involving the dopaminergic circuitry of the basal ganglia.     

Monoclonal Antibody Targeting of Fibroblast Growth Factor Receptor 1c Ameliorates Obesity and Glucose Intolerance via Central Mechanisms

We have generated a novel monoclonal antibody targeting human FGFR1c (R1c mAb) that caused profound body weight and body fat loss in diet-induced obese mice due to decreased food intake (with energy expenditure unaltered), in turn improving glucose control. R1c mAb also caused weight loss in leptin-deficient ob/ob mice, leptin receptor-mutant db/db mice, and in mice lacking either the melanocortin 4 receptor or the melanin-concentrating hormone receptor 1. In addition, R1c mAb did not change hypothalamic mRNA expression levels of Agrp, Cart, Pomc, Npy, Crh, Mch, or Orexin, suggesting that R1c mAb could cause food intake inhibition and body weight loss via other mechanisms in the brain. Interestingly, peripherally administered R1c mAb accumulated in the median eminence, adjacent arcuate nucleus and in the circumventricular organs where it activated the early response gene c-Fos. As a plausible mechanism and coinciding with the initiation of food intake suppression, R1c mAb induced hypothalamic expression levels of the cytokines Monocyte chemoattractant protein 1 and 3 and ERK1/2 and p70 S6 kinase 1 activation.     

Differential neurochemical consequences of an escalating dose-binge regimen followed by single-day multiple-dose methamphetamine challenges

Chronic intake of methamphetamine (METH) causes tolerance to its behavioral and subjective effects. To better mimic human patterns of drug abuse, the present study used a rodent model that took into account various facets of human drug administration and measured METH-induced effects on brain monoamine levels. Adult male Sprague–Dawley rats were injected with METH or saline according to an escalating dose schedule for 2 weeks. This was followed by a challenge regimen of either saline or one of two doses of METH (3 × 10 mg/kg every 2 h or 6 × 5 mg/kg given every hour, both given within a single day). Both challenge doses of METH caused significant degrees of depletion of dopamine in the striatum and norepinephrine and serotonin in the striatum, cortex, and hippocampus. Animals pre-treated with METH showed significant attenuation of METH-induced striatal dopamine depletion but not consistent attenuation of norepinephrine and serotonin depletion. Unexpectedly, METH pre-treated animals that received the 3 × 10 mg/kg challenge showed less increases in tympanic temperatures than saline pre-treated rats whereas METH pre-treated animals that received the 6 × 5 mg/kg METH challenge showed comparable increases in temperatures to saline pre-treated rats. Therefore, pre-treatment-induced partial protection against monoamine depletion is probably not because of attenuated METH-induced hyperthermia in those rats.     

Effects of VMAT2 inhibitors lobeline and GZ‐793A on methamphetamine‐induced changes in dopamine release,metabolism and synthesis in vivo

Vesicular monoamine transporter‐2 (VMAT2) inhibitors reduce methamphetamine (METH) reward in rats. The current study determined the effects of VMAT2 inhibitors lobeline (LOB; 1 or 3 mg/kg) and N‐(1,2R‐dihydroxylpropyl)‐2,6‐cis‐di(4‐methoxyphenethyl)piperidine hydrochloride (GZ‐793A; 15 or 30 mg/kg) on METH‐induced (0.5 mg/kg, SC) changes in extracellular dopamine (DA) and its metabolite dihydroxyphenylacetic acid (DOPAC) in the reward‐relevant nucleus accumbens (NAc) shell using in vivo microdialysis. The effect of GZ‐793A (15 mg/kg) on DA synthesis in tissue also was investigated in NAc, striatum, medial prefrontal cortex and orbitofrontal cortex. In NAc shell, METH produced a time‐dependent increase in extracellular DA and decrease in DOPAC. Neither LOB nor GZ‐793A alone altered extracellular DA; however, both drugs increased extracellular DOPAC. In combination with METH, LOB did not alter the effects of METH on DA; however, GZ‐793A, which has greater selectivity than LOB for inhibiting VMAT2, reduced the duration of the METH‐induced increase in extracellular DA. Both LOB and GZ‐793A enhanced the duration of the METH‐induced decrease in extracellular DOPAC. METH also increased tissue DA synthesis in NAc and striatum, whereas GZ‐793A decreased synthesis; no effect of METH or GZ‐793A on DA synthesis was found in medial prefrontal cortex or orbitofrontal cortex. These results suggest that selective inhibition of VMAT2 produces a time‐dependent decrease in DA release in NAc shell as a result of alterations in tyrosine hydroxylase activity, which may play a role in the ability of GZ‐793A to decrease METH reward.

     

Nupr1/Chop signal axis is involved in mitochondrion-related endothelial cell apoptosis induced by methamphetamine

Methamphetamine (METH) abuse has been a serious global public health problem for decades. Previous studies have shown that METH causes detrimental effects on the nervous and cardiovascular systems. METH-induced cardiovascular toxicity has been, in part, attributed to its destructive effect on vascular endothelial cells. However, the underlying mechanism of METH-caused endothelium disruption has not been investigated systematically. In this study, we identified a novel pathway involved in endothelial cell apoptosis induced by METH. We demonstrated that exposure to METH caused mitochondrial apoptosis in human umbilical vein endothelial cells and rat cardiac microvascular endothelial cells in vitro as well as in rat cardiac endothelial cells in vivo. We found that METH mediated endothelial cell apoptosis through Nupr1–Chop/P53–PUMA/Beclin1 signaling pathway. Specifically, METH exposure increased the expression of Nupr1, Chop, P53 and PUMA. Elevated p53 expression raised up PUMA expression, which initiated mitochondrial apoptosis by downregulating antiapoptotic Bcl-2, followed by upregulation of proapoptotic Bax, resulting in translocation of cytochrome c (cyto c), an apoptogenic factor, from the mitochondria to cytoplasm and activation of caspase-dependent pathways. Interestingly, increased Beclin1, upregulated by Chop, formed a ternary complex with Bcl-2, thereby decreasing the dissociative Bcl-2. As a result, the ratio of dissociative Bcl-2 to Bax was also significantly decreased, which led to translocation of cyto c and initiated more drastic apoptosis. These findings were supported by data showing METH-induced apoptosis was significantly inhibited by silencing Nupr1, Chop or P53, or by PUMA or Beclin1 knockdown. Based on the present data, a novel mechanistic model of METH-induced endothelial cell toxicity is proposed. Collectively, these results highlight that the Nupr1–Chop/P53–PUMA/Beclin1 pathway is essential for mitochondrion-related METH-induced endothelial cell apoptosis and may be a potential therapeutic target for METH-caused cardiovascular toxicity. Future studies using knockout animal models are warranted to substantiate the present findings.Methamphetamine (METH) is a widely used addictive stimulant with high potential of abuse. METH exposure damages both the nervous and cardiovascular systems.^{1, 2, 3, 4, 5} In particular, METH has been associated with a myriad of adverse effects on the circulatory system, including cardiomyopathy, hypertension, arrhythmia, myocardial ischemia, acute coronary syndrome, cardiac failure and sudden death.^{6, 7, 8} In METH abusers with acute aortic dissection or coronary syndrome, vascular structural alterations have been found in the myocardium in a number of clinical cases, indicating that METH can cause toxic effects on the blood vessels.^{9, 10} The above studies suggest that vascular endothelial cells may be a key target in METH-caused cardiovascular pathophysiologic alterations. Recent studies have shown that METH exposure causes endothelial cell apoptosis,^{11, 12, 13} but the underlying mechanisms remain to be elucidated.Endoplasmic reticulum stress (ERS) pathway is a classical apoptotic pathway following the discovery of death receptor signaling and mitochondrial pathways.^{14, 15} In the present study, we hypothesized that Chop (as an ERS marker protein) is involved in endothelial cell apoptosis induced by METH. Chop (encoded by the DDIT3 gene), is the key apoptosis inducer in the proteotoxic stress response.^{16, 17} Chop has been shown to be pro- and antiapoptotic depending on cell and stress context.¹⁸ Increased expression of the DDIT3 gene or microinjections of the Chop protein led to dissipation of the mitochondrial transmembrane potential (MMP), generation of reactive oxygen species and apoptotic cell death.¹⁹Recently, it was reported that increased expression of Chop and induction of apoptosis in response to ERS can be directly induced by nuclear protein 1 (Nupr1) in PANC-1 human pancreatic carcinoma cells.^{20, 21} It is known that Nupr1 (also named as p8 or com1) expression is upregulated in response to stress and thus influenced by the host microenvironment. Decreased Nupr1 expression is accompanied by suppression of cancer cell growth in vitro and in vivo.^{22, 23} However, increased Nupr1 mRNA also accompanies apoptotic changes in cancer cells.²⁴ While Nupr1 gene expression is induced in response to a variety of stress factors, DDIT3 gene is specifically related to the ERS response.²⁵The objective of this study was to investigate the role of ERS and Nupr1 in METH-caused apoptosis in vascular endothelial cells. We determined METH-induced changes of Nupr1 expression and cellular apoptosis level in vitro using human umbilical vein endothelial cells (HUVECs) and rat cardiac microvascular endothelial cells (CMECs), as well as in vivo using vascular endothelium from Sprague–Dawley rats exposed to METH. Our results indicate that ERS induced by Nupr1 plays a crucial role in METH-induced vascular endothelial cell apoptosis and the Nupr1–Chop/P53–PUMA/Beclin1 pathway may be a potential therapeutic target of METH-induced cardiovascular toxicity.     

Ifenprodil Attenuates Methamphetamine-Induced Behavioral Sensitization and Activation of Ras-ERK-∆FosB Pathway in the Caudate Putamen

Addiction is a debilitating, chronic psychiatric disorder that is difficult to cure completely owing to the high rate of relapse. Behavioral sensitization is considered to may underlie behavioral changes, such as relapse, caused by chronic abuse of psychomotor stimulants. Thus, its animal models have been widely used to explore the etiology of addiction. Recently, increasing evidence has demonstrated that N-methyl-d-aspartate receptors (NMDARs) play an important role in addiction to psychomotor stimulants. However, the role of GluN2B-containing receptors and their downstream signaling pathway(s) in behavioral sensitization induced by methamphetamine (METH) have not been investigated yet. In this study, we used different doses of ifenprodil (2.5, 5, 10 mg/kg), a selective antagonist of the GluN2B subunit, to investigate the role of GluN2B-containing NMDARs in METH-induced behavioral sensitization. We then examined changes in the levels of Ras, phosphorylated extracellular signal-regulated kinase (pERK)/ERK, and ?FosB in the caudate putamen (CPu) by western blot. We found that 2.5 or 10 mg/kg ifenprodil significantly attenuated METH-induced behavioral sensitization, whereas the mice treated with a moderate dose of ifenprodil (5 mg/kg) displayed no significant changes. Further results of western blot experiments showed that repeated administration of METH caused the increases in the levels of Ras, pERK/ERK and ?FosB in the CPu, and these changes were inhibited by only the 2.5 mg/kg dose of ifenprodil. In conclusion, these results demonstrated that 2.5 mg/kg ifenprodil could attenuate METH-induced behavioral sensitization. Moreover, GluN2B-containing NMDARs and their downstream Ras-ERK-?FosB signaling pathway in the CPu might be involved in METH-induced behavioral sensitization.     

Methamphetamine-induced hyperactivity and behavioral sensitization in PACAP deficient mice

Mice lacking the PACAP gene (PACAP(-/-)) display psychomotor abnormalities such as novelty-induced hyperactivity and jumping behavior, and they show different responses to amphetamine, a typical psychostimulant. The present study examined the possible role of endogenous PACAP in methamphetamine (METH)-induced hyperactivity and behavioral sensitization. The locomotor activity of hyperactive PACAP(-/-) mice was measured using the infrared photocell beam detection system, Acti-Track, after a habituation period. Single administration of METH (1 and 2mg/kg) caused a robust increase in locomotor activity of mice, but this effect did not differ between wild-type and PACAP(-/-) mice. Repeated administration of METH (1mg/kg) for 7 days enhanced METH-induced hyperactivity, and this sensitization was observed even when withdrawn for 7 days. There was no difference in the degree of development and expression of METH-induced behavioral sensitization between wild-type and PACAP(-/-) mice. In addition, there was no difference in METH-induced increases in extracellular serotonin and dopamine levels in the prefrontal cortex of the normal and sensitized mice between the two groups. These results suggest that endogenous PACAP is not involved in the locomotor stimulant activity of acute METH and repeated METH-induced behavioral and neurochemical sensitization.     

Psychoproteomic analysis of rat cortex following acute methamphetamine exposure

Methamphetamine (METH) is recognized as one of the most abused psychostimulants in the United States. METH is an illicit drug that is known to exert neurotoxic effects on both dopaminergic and serotonergic neural systems both in vivo and in vitro. Our laboratory and others have been studying the biochemical mechanisms underlying METH-induced neurotoxicity. Here, we applied a novel psychoproteomic approach to evaluate METH-induced neurotoxicity following acute METH administration (4x10 mg/kg, ip injections every 1 h). Samples of cortical tissue collected 24 h post METH treatment were pooled, processed and analyzed via a selective psychoproteomic platform. Protein separation was performed using our previously established offline tandem cation-anion exchange chromatography-SDS-1D-PAGE platform (CAX-PAGE). Gel bands exhibiting 2 or more fold changes were extracted, trypsinized and subjected to reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) analyses for protein identification. Differential changes of the selected proteins were further confirmed by quantitative immunoblotting. We identified 82 differentially expressed proteins, 40 of which were downregulated and 42 of which were upregulated following acute METH treatment. Proteins that decreased in abundance included collapsin response mediator protein-2 (CRMP-2), superoxide dismutase 1 (SOD 1), phosphatidylethanolamine-binding protein-1 (PEBP-1) and mitogen activated kinase kinase-1 (MKK-1). Proteins that increased in abundance included authophagy-linked microtubule-associated protein light chain 3 (LC3), synapsin-1, and Parkinsonism linked ubiquitin carboxy-terminal hydroxylase-L1 (UCH-L1). Lastly, we used these differentially expressed protein subsets to construct a "psychoproteomic" spectrum map in an effort to uncover potential protein interactions relevant to acute METH neurotoxicity.