Yossef Itzhak

Methamphetamine-Induced Dopaminergic Neurotoxicity: Role of Peroxynitrite and Neuroprotective Role of Antioxidants and Peroxynitrite Decomposition Catalysts

Abstract: Oxidative stress, reactive oxygen (ROS), and nitrogen (RNS) species have been known to be involved in a multitude of neurodegenerative disorders such as Parkinsons disease (PD), Alzheimers disease (AD), and amyotrophic lateral sclerosis (ALS). Both ROS and RNS have very short half‐lives, thereby making their identification very difficult as a specific cause of neurodegeneration. Recently, we have developed a high performance liquid chromatography/electrochemical detection (HPLC/EC) method to identify 3‐nitrotyrosine (3‐NT), an in vitro and in vivo biomarker of peroxynitrite production, in cell cultures and brain to evaluate if an agent‐driven neurotoxicity is produced by the generation of peroxynitrite. We show that a single or multiple injections of methamphetamine (METH) produced a significant increase in the formation of 3‐NT in the striatum. This formation of 3‐NT correlated with the striatal dopamine depletion caused by METH administration. We also show that PC12 cells treated with METH has significantly increased formation of 3‐NT and dopamine depletion. Furthermore, we report that pretreatment with antioxidants such as selenium and melatonin can completely protect against the formation of 3‐NT and depletion of striatal dopamine. We also report that pretreatment with peroxynitrite decomposition catalysts such as 5, 10,15,20‐tetrakis(N‐methyl‐4′‐pyridyl)porphyrinato iron III (FeTMPyP) and 5, 10, 15, 20‐tetrakis (2,4,6‐trimethyl‐3,5‐sulfonatophenyl) porphinato iron III (FETPPS) significantly protect against METH‐induced 3‐NT formation and striatal dopamine depletion. We used two different approaches, pharmacological manipulation and transgenic animal models, in order to further investigate the role of peroxynitrite. We show that a selective neuronal nitric oxide synthase (nNOS) inhibitor, 7‐nitroindazole (7‐NI), significantly protect against the formation of 3‐NT as well as striatal dopamine depletion. Similar results were observed with nNOS knockout and copper zinc superoxide dismutase (CuZnSOD)‐overexpressed transgenic mice models. Finally, using the protein data bank crystal structure of tyrosine hydroxylase, we postulate the possible nitration of specific tyrosine moiety in the enzyme that can be responsible for dopaminergic neurotoxicity. Together, these data clearly support the hypothesis that the reactive nitrogen species, peroxynitrite, plays a major role in METH‐induced dopaminergic neurotoxicity and that selective antioxidants and peroxynitrite decomposition catalysts can protect against METH‐induced neurotoxicity. These antioxidants and decomposition catalysts may have therapeutic potential in the treatment of psychostimulant addictions.

Cocaine-induced Conditioned Place Preference in Mice: Induction, Extinction and Reinstatement by Related Psychostimulants☆

Reinstatement of drug-seeking behavior in animals is relevant to drug relapse in humans. In the present study, we employed the conditioned place preference (CPP) paradigm to investigate the extinction and reinstatement of the place-conditioned response, a model that is consistent with drug-seeking behavior. Cocaine-induced CPP was rendered in Swiss Webster mice and then extinguished after repeated saline injections (8 days) in both the previously cocaine-paired compartment and the saline-paired compartment. Following the extinction phase, the reinstatement of CPP was investigated. Cocaine-experienced mice were challenged with one of the following psychostimulants, cocaine (15 mg/kg), methamphetamine (METH; 0.5 mg/kg), methylphenidate (MPD; 20 mg/kg) and phencyclidine (PCP; 5 mg/kg). The priming injection of cocaine, METH and MPD, unlike PCP, induced a marked preference for the previously cocaine-paired compartment. This finding suggests that all three psychostimulants reinstated the CPP response, and METH and MPD substituted for the reinforcing cue of cocaine. A challenge injection of cocaine administered two and four weeks after the reinstatement of CPP indicated that CPP was maintained up to two weeks. The finding that METH and MPD but not PCP reinstated and supported cocaine-induced CPP suggests that the CPP paradigm may be a useful tool for drug discrimination studies and the reinstatement of drug-seeking behavior.

Peroxynitrite plays a role in methamphetamine-induced dopaminergic neurotoxicity: evidence from mice lacking neuronal nitric oxide synthase gene or overexpressing copper-zinc superoxide dismutase

The use of methamphetamine (METH) leads to neurotoxic effects in mammals. These neurotoxic effects appear to be related to the production of free radicals. To assess the role of peroxynitrite in METH‐induced dopaminergic, we investigated the production of 3‐nitrotyrosine (3‐NT) in the mouse striatum. The levels of 3‐NT increased in the striatum of wild‐type mice treated with multiple doses of METH (4 × 10 mg/kg, 2 h interval) as compared with the controls. However, no significant production of 3‐NT was observed either in the striata of neuronal nitric oxide synthase knockout mice (nNOS –/–) or copper–zinc superoxide dismutase overexpressed transgenic mice (SOD‐Tg) treated with similar doses of METH. The dopaminergic damage induced by METH treatment was also attenuated in nNOS–/– or SOD‐Tg mice. These data further confirm that METH causes its neurotoxic effects via the production of peroxynitrite.

Long-term memory of cocaine-associated context: disruption and reinstatement

Long-term memory of cocaine-associated context was established by conditioned place preference learning. After 1 week, exposure to context in the absence of cocaine (memory retrieval) was paired with one of the following treatments: saline, scopolamine (muscarinic acetylcholine receptor antagonist), dizocilpine (MK-801; noncompetitive N-methyl-D-aspartate antagonist) or D-cycloserine (partial N-methyl-D-aspartate agonist). In subsequent conditioned place preference tests, place preference was suppressed in the drug-treated groups but not saline-treated groups. Results suggest that the amnesic agents, scopolamine and MK-801, disrupted reconsolidation of cocaine-associated contextual memory. In contrast, the mnemonic agent D-cycloserine might have facilitated extinction learning during context exposure in the absence of cocaine. Challenge administration of cocaine reinstated place preference in all groups except the MK-801 group, suggesting that suppression of conditioned response may or may not suppress memory evoked by drug-context reexposure.

Methylphenidate and MDMA adolescent exposure in mice: Long-lasting consequences on cocaine-induced reward and psychomotor stimulation in adulthood

Pre-exposure to psychostimulants enhances the rewarding and psychomotor stimulating effects of subsequent drug exposure. Currently, there is a prevalence of adolescent exposure to the psychostimulants methylphenidate (MPD) and 3,4-methylenedioxymethamphetamine (MDMA). However, there is a paucity of investigation concerning the long-term behavioral consequences of exposure to these stimulants during adolescence. The aim of the present study was to investigate the effect of MPD and MDMA exposure in adolescence on cocaine-induced reward and psychomotor stimulation in adulthood. Adolescent Swiss-Webster mice received intraperitoneal injections of saline, MPD (10 mg/kg) or MDMA (10 mg/kg) from PD 26 to PD 32. Animal weights were monitored during and after drug administration. One month later, cocaine-induced conditioned place preference (CPP) and locomotor activity (LMA) were investigated. MPD and MDMA inhibited weight increase from PD 28 to PD 39 compared to the saline group, but weights amongst the three groups equalized by PD 46. MDMA exposure resulted in the same magnitude of cocaine (20 mg/kg)-induced CPP as saline exposure; however, MPD exposure caused significantly less CPP. Two weeks following extinction of CPP and withdrawal from cocaine, a priming injection of cocaine (5 mg/kg) reinstated significantly higher CPP in the MPD and MDMA groups than in the saline group. In the LMA experiments, cocaine (15 mg/kg) was administered for 5 consecutive days. On days 1 and 5, cocaine-induced hyperlocomotion in the MPD group was significantly higher than in the saline and MDMA groups. After a 2-week withdrawal period, cocaine (5 mg/kg) evoked significantly higher LMA responses in the MPD and MDMA groups compared to the saline group. Results suggest that exposure of mice to both MPD and MDMA during adolescence involves long-lasting neural adaptations, manifested as sensitized responses to cocaine-induced reward and psychomotor stimulation following cocaine withdrawal.

The role of neuronal nitric oxide synthase in cocaine-induced conditioned place preference

PREVIOUS studies suggested the involvement of the neuronal nitric oxide synthase (nNOS) in the development of sensitization to psychostimulants. In the present study we investigated the role of nNOS in the rewarding properties of cocaine. Swiss Webster mice treated with cocaine (20 mg/kg) and saline every other day for 8 days (four drug and four saline sessions) developed conditioned place preference (CPP) for the drug-paired compartment of the cage. Pretreatment with the nNOS inhibitor, 7-nitroindazole (7-NI; 25 mg/kg), completely blocked cocaine-induced CPP. Mice deficient for the nNOS gene (homozygote nNOS(−/−) mice) were resistant to cocaine-induced CPP, while wild-type nNOS(+/+) mice developed a marked CPP following cocaine administration. Both, the pharmacological and genetic manipulations of nNOS suggest that nitric oxide (NO) is involved in the rewarding properties of cocaine.

Acute liver failure and hyperammonemia increase peripheral-type benzodiazepine receptor binding and pregnenolone synthesis in mouse brain

We investigated the role of brain peripheral-type benzodiazepine receptors (PBRs) and pregnenolone (a product of PBRs activation) in hepatic encephalopathy (HE)/hyperammonemia. Administration of the hepatotoxin, thioacetamide, or ammonium acetate to mice for 3 days significantly increased the number of brain PBRs (138-146% of control) and the affinity of the ligands for these receptors (2-fold). The total content of pregnenolone and its rate of synthesis in brain of the experimental animals were significantly increased. Our results suggest a novel integrated mechanism by which ammonia-induced activation of PBRs leads to elevated levels of pregnenolone-derived neurosteroids which are known to enhance GABA-ergic neurotransmission. This mechanism may play a pivotal role in pathogenesis of HE.

nNOS inhibitors attenuate methamphetamine-induced dopaminergic neurotoxicity but not hyperthermia in mice

Methamphetamine (METH)-induced dopaminergic neurotoxicity is associated with hyperthermia. We investigated the effect of several neuronal nitric oxide synthase (nNOS) inhibitors on METH-induced hyperthermia and striatal dopaminergic neurotoxicity. Administration of METH (5 mg/kg; q. 3 h × 3) to Swiss Webster mice produced marked hyperthermia and 50-60% depletion of striatal dopaminergic markers 72 h after METH administration. Pretreatment with the nNOS inhibitors S-methylthiocitrulline (SMTC; 10 mg/kg) or 3-bromo-7-nitroindazole (3-Br-7-Nl; 20 mg/kg) before each METH injection did not affect the persistent hyperthermia produced by METH, but afforded protection against the depletion of dopaminergic markers. A low dose (25 mg/kg) of the nNOS inhibitor 7-nitroindazole (7-Nl) did not affect METH-induced hyperthermia, but a high dose (50 mg/kg) produced significant hypothermia. These findings indicate that low dose of selective nNOS inhibitors protect against METH-induced neurotoxicity with no effect on body temperature and support the hypothesis that nitric oxide (NO) and peroxynitrite have a major role in METH-induced dopaminergic neurotoxicity.

The Peripheral Benzodiazepine Receptor and Neurosteroids in Hepatic Encephalopathy

Hepatic encephalopathy (HE) represents one of the most common events in terminal liver failure. Acute HE (fulminant hepatic failure, FHF) presents with the abrupt onset of delirium, seizures, and coma, and has a mortality rate of about 90%. The principal cause of death in FHF is brain edema associated with increased intracranial pressure. Chronic HE, sometimes referred to as portal-systemic encephalopathy, most often occurs in the setting of cirrhosis and is characterized by change in personality, altered mood and behavior, diminished intellectual capacity, abnormal muscle tone and tremor (asterixis), stupor and coma. For reviews, see (Rothstein and Herlong, 1989; Lockwood. 1992).

Repeated administration of gamma-hydroxybutyric acid (GHB) to mice: Assessment of the sedative and rewarding effects of GHB

Abstract: Because of the sedative/hypnotic and euphoric effects of gamma‐hydroxybutyric acid (GHB), the recreational use of the drug has increased significantly. In the current study we investigated the sedative and rewarding effects of GHB in Swiss Webster mice. Although the acute administration of GHB (200 mg/kg) caused marked hypolocomotion, repeated administration of the drug for 6 or 14 days produced tolerance to this effect. In addition, the administration of GHB 300 mg/kg to naive mice caused catalepsy, which dissipated in mice pre‐exposed to GHB (200 mg/kg). Consequently, after repeated treatment with GHB, tolerance developed to both the hypolocomotion and cataleptic effects of the drug. The administration of GHB or its precursor gamma‐butyrolactone for 14 days increased the striatal content of dopamine. The sedative effects of GHB may be due to hypodopaminergic activity from inhibition of dopamine release and a subsequent increase in the intraneuronal dopamine level. The rewarding effect of GHB was assessed in the conditioned place preference paradigm. Mice treated repeatedly with 250 mg/kg for 7 days developed conditioned preference for the GHB‐paired compartment of the cage, suggesting that the GHB cue is rewarding. The development of tolerance to the sedative effects of GHB coupled with the rewarding properties of the drug support the abuse potential of GHB. Further studies are necessary to determine the mechanism underlying the development of tolerance to GHB and the rewarding effect of the drug.