Noelia Granado

Persistent MDMA-induced dopaminergic neurotoxicity in the striatum and substantia nigra of mice

Acute administration of repeated doses of 3,4‐methylenedioxymethamphetamine (MDMA, ecstasy) dramatically reduces striatal dopamine (DA) content, tyrosine hydroxylase (TH), and DA transporter‐immunoreactivity in mice. In this study, we show for the first time the spatiotemporal pattern of dopaminergic damage and related molecular events produced by MDMA administration in mice. Our results include the novel finding that MDMA produces a significant decrease in the number of TH‐immunoreactive neurons in the substantia nigra (SN). This decrease appears 1 day after injection, remains stable for at least 30 days, and is accompanied by a dose‐dependent long‐lasting decrease in TH‐ and DA transporter‐immunoreactivity in the striatum, which peaked 1 day after treatment and persisted for at least 30 days, however, some recovery was evident from day 3 onwards, evidencing sprouting of TH fibers. No change is observed in the NAc indicating that MDMA causes selective destruction of DA‐containing neurons in the nigrostriatal pathway, sparing the mesolimbic pathway. The expression of Mac‐1 increased 1 day after MDMA treatment and glial fibrillary acidic protein increased 3 days post‐treatment in the striatum and SN but not in the NAc, in strict anatomical correlation with dopaminergic damage. These data provide the first evidence that MDMA causes persistent loss of dopaminergic cell bodies in the SN.

Dopamine D2-receptor knockout mice are protected against dopaminergic neurotoxicity induced by methamphetamine or MDMA

Methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA), amphetamine derivatives widely used as recreational drugs, induce similar neurotoxic effects in mice, including a marked loss of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the striatum. Although the role of dopamine in these neurotoxic effects is well established and pharmacological studies suggest involvement of a dopamine D2-like receptor, the specific dopamine receptor subtype involved has not been determined. In this study, we used dopamine D2 receptor knock-out mice (D2R(-/-)) to determine whether D2R is involved in METH- and MDMA-induced hyperthermia and neurotoxicity. In wild type animals, both drugs induced marked hyperthermia, decreased striatal dopamine content and TH- and DAT-immunoreactivity and increased striatal GFAP and Mac-1 expression as well as iNOS and interleukin 15 at 1 and 7days after drug exposure. They also caused dopaminergic cell loss in the SNpc. Inactivation of D2R blocked all these effects. Remarkably, D2R inactivation prevented METH-induced loss of dopaminergic neurons in the SNpc. In addition, striatal dopamine overflow, measured by fast scan cyclic voltammetry in the presence of METH, was significantly reduced in D2R(-/-) mice. Pre-treatment with reserpine indicated that the neuroprotective effect of D2R inactivation cannot be explained solely by its ability to prevent METH-induced hyperthermia: reserpine lowered body temperature in both genotypes, and potentiated METH toxicity in WT, but not D2R(-/-) mice. Our results demonstrate that the D2R is necessary for METH and MDMA neurotoxicity and that the neuroprotective effect of D2R inactivation is independent of its effect on body temperature.

Methamphetamine Causes Degeneration of Dopamine Cell Bodies and Terminals of the Nigrostriatal Pathway Evidenced by Silver Staining

Methamphetamine is a widely abused illicit drug. Recent epidemiological studies showed that methamphetamine increases the risk for developing Parkinson’s disease (PD) in agreement with animal studies showing dopaminergic neurotoxicity. We examined the effect of repeated low and medium doses vs single high dose of methamphetamine on degeneration of dopaminergic terminals and cell bodies. Mice were given methamphetamine in one of the following paradigms: three injections of 5 or 10 mg/kg at 3 h intervals or a single 30 mg/kg injection. The integrity of dopaminergic fibers and cell bodies was assessed at different time points after methamphetamine by tyrosine hydroxylase immunohistochemistry and silver staining. The 3 × 10 protocol yielded the highest loss of striatal dopaminergic terminals, followed by the 3 × 5 and 1 × 30. Some degenerating axons could be followed from the striatum to the substantia nigra pars compacta (SNpc). All protocols induced similar significant degeneration of dopaminergic neurons in the SNpc, evidenced by amino-cupric-silver-stained dopaminergic neurons. These neurons died by necrosis and apoptosis. Methamphetamine also killed striatal neurons. By using D1-Tmt/D2-GFP BAC transgenic mice, we observed that degenerating striatal neurons were equally distributed between direct and indirect medium spiny neurons. Despite the reduced number of dopaminergic neurons in the SNpc at 30 days after treatment, there was a partial time-dependent recovery of dopamine terminals beginning 3 days after treatment. Locomotor activity and motor coordination were robustly decreased 1–3 days after treatment, but recovered at later times along with dopaminergic terminals. These data provide direct evidence that methamphetamine causes long-lasting loss/degeneration of dopaminergic cell bodies in the SNpc, along with destruction of dopaminergic terminals in the striatum.

Dopamine D 1 receptor deletion strongly reduces neurotoxic effects of methamphetamine

Methamphetamine (METH) is a potent, highly addictive psychostimulant consumed worldwide. In humans and experimental animals, repeated exposure to this drug induces persistent neurodegenerative changes. Damage occurs primarily to dopaminergic neurons, accompanied by gliosis. The toxic effects of METH involve excessive dopamine (DA) release, thus DA receptors are highly likely to play a role in this process. To define the role of D(1) receptors in the neurotoxic effects of METH we used D(1) receptor knock-out mice (D(1)R(-/-)) and their WT littermates. Inactivation of D(1)R prevented METH-induced dopamine fibre loss and hyperthermia, and increases in gliosis and pro-inflammatory molecules such as iNOS in the striatum. In addition, D(1)R inactivation prevented METH-induced loss of dopaminergic neurons in the substantia nigra. To explore the relationship between hyperthermia and neurotoxicity, METH was given at high ambient temperature (29 °C). In this condition, D(1)R(-/-) mice developed hyperthermia following drug delivery and the neuroprotection provided by D(1)R inactivation at 23 °C was no longer observed. However, reserpine, which empties vesicular dopamine stores, blocked hyperthermia and strongly potentiated dopamine toxicity in D(1)R(-/-) mice, suggesting that the protection afforded by D(1)R inactivation is due to both hypothermia and higher stored vesicular dopamine. Moreover, electrical stimulation evoked higher DA overflow in D(1)R(-/-) mice as demonstrated by fast scan cyclic voltammetry despite their lower basal DA content, suggesting higher vesicular DA content in D(1)R(-/-) than in WT mice. Altogether, these results indicate that the D(1)R plays a significant role in METH-induced neurotoxicity by mediating drug-induced hyperthermia and increasing the releasable cytosolic DA pool.

The role of dopamine receptors in the neurotoxicity of methamphetamine.

Methamphetamine is a synthetic drug consumed by millions of users despite its neurotoxic effects in the brain, leading to loss of dopaminergic fibres and cell bodies. Moreover, clinical reports suggest that methamphetamine abusers are predisposed to Parkinsons disease. Therefore, it is important to elucidate the mechanisms involved in methamphetamine‐induced neurotoxicity. Dopamine receptors may be a plausible target to prevent this neurotoxicity. Genetic inactivation of dopamine D1 or D2 receptors protects against the loss of dopaminergic fibres in the striatum and loss of dopaminergic neurons in the substantia nigra. Protection by D1 receptor inactivation is due to blockade of hypothermia, reduced dopamine content and turnover and increased stored vesicular dopamine in D1R−/− mice. However, the neuroprotective impact of D2 receptor inactivation is partially dependent on an effect on body temperature, as well as on the blockade of dopamine reuptake by decreased dopamine transporter activity, which results in reduced intracytosolic dopamine levels in D2R−/− mice.

Nrf2 deficiency potentiates methamphetamine‐induced dopaminergic axonal damage and gliosis in the striatum

Oxidative stress that correlates with damage to nigrostriatal dopaminergic neurons and reactive gliosis in the basal ganglia is a hallmark of methamphetamine (METH) toxicity. In this study, we analyzed the protective role of the transcription factor Nrf2 (nuclear factor‐erythroid 2‐related factor 2), a master regulator of redox homeostasis, in METH‐induced neurotoxicity. We found that Nrf2 deficiency exacerbated METH‐induced damage to dopamine neurons, shown by an increase in loss of tyrosine hydroxylase (TH)‐ and dopamine transporter (DAT)‐containing fibers in striatum. Consistent with these effects, Nrf2 deficiency potentiated glial activation, indicated by increased striatal expression of markers for microglia (Mac‐1 and Iba‐1) and astroglia (GFAP) one day after METH administration. At the same time, Nrf2 inactivation dramatically potentiated the increase in TNFα mRNA and IL‐15 protein expression in GFAP+ cells in the striatum. In sharp contrast to the potentiation of striatal damage, Nrf2 deficiency did not affect METH‐induced dopaminergic neuron death or expression of glial markers or proinflammatory molecules in the substantia nigra. This study uncovers a new role for Nrf2 in protection against METH‐induced inflammatory and oxidative stress and striatal degeneration.

Amphetamine-related drugs neurotoxicity in humans and in experimental animals: Main mechanisms.

HIGHLIGHTSMDMA damages serotonergic system in primates and rats and dopaminergic system in mice.METH damages the dopaminergic system in all animal species including humans.The nigrostriatal system is more vulnerable than the mesolimbic system.Within the striatum the striosomes are more vulnerable than the matrix.METH kills dopamine neurons as demonstrated by silver‐staining in rodents.METH reduces DAT binding sites and motor skills in human addicts. ABSTRACT Amphetamine‐related drugs, such as 3,4‐methylenedioxymethamphetamine (MDMA) and methamphetamine (METH), are popular recreational psychostimulants. Several preclinical studies have demonstrated that, besides having the potential for abuse, amphetamine‐related drugs may also elicit neurotoxic and neuroinflammatory effects. The neurotoxic potentials of MDMA and METH to dopaminergic and serotonergic neurons have been clearly demonstrated in both rodents and non‐human primates. This review summarizes the species‐specific cellular and molecular mechanisms involved in MDMA and METH‐mediated neurotoxic and neuroinflammatory effects, along with the most important behavioral changes elicited by these substances in experimental animals and humans. Emphasis is placed on the neuropsychological and neurological consequences associated with the neuronal damage. Moreover, we point out the gap in our knowledge and the need for developing appropriate therapeutic strategies to manage the neurological problems associated with amphetamine‐related drug abuse.

Methamphetamine and Parkinson's Disease

Parkinsons disease (PD) is a neurodegenerative disorder predominantly affecting the elderly. The aetiology of the disease is not known, but age and environmental factors play an important role. Although more than a dozen gene mutations associated with familial forms of Parkinsons disease have been described, fewer than 10% of all cases can be explained by genetic abnormalities. The molecular basis of Parkinsons disease is the loss of dopamine in the basal ganglia (caudate/putamen) due to the degeneration of dopaminergic neurons in the substantia nigra, which leads to the motor impairment characteristic of the disease. Methamphetamine is the second most widely used illicit drug in the world. In rodents, methamphetamine exposure damages dopaminergic neurons in the substantia nigra, resulting in a significant loss of dopamine in the striatum. Biochemical and neuroimaging studies in human methamphetamine users have shown decreased levels of dopamine and dopamine transporter as well as prominent microglial activation in the striatum and other areas of the brain, changes similar to those observed in PD patients. Consistent with these similarities, recent epidemiological studies have shown that methamphetamine users are almost twice as likely as non-users to develop PD, despite the fact that methamphetamine abuse and PD have distinct symptomatic profiles.

Metabolic interactions between glutamatergic and dopaminergic neurotransmitter systems are mediated through D1 dopamine receptors

Interactions between the dopaminergic and glutamatergic neurotransmission systems were investigated in the adult brain of wild‐type (WT) and transgenic mice lacking the dopamine D1 or D2 receptor subtypes. Activity of the glutamine cycle was evaluated by using 13C NMR spectroscopy, and striatal activity was assessed by c‐Fos expression and motor coordination. Brain extracts from (1,2‐13C2) acetate‐infused mice were prepared and analyzed by 13C NMR to determine the incorporation of the label into the C4 and C5 carbons of glutamate and glutamine. D1R−/− mice showed a significantly higher concentration of cerebral (4,5‐13C2) glutamine, consistent with an increased activity of the glutamate‐glutamine cycle and of glutamatergic neurotransmission. Conversely, D2R−/− mice did not show any significant changes in (4,5‐13C2) glutamate or (4,5‐13C2) glutamine, suggesting that alterations in glutamine metabolism are mediated through D1 receptors. This was confirmed with D1R−/− and WT mice treated with reserpine, a dopamine‐depleting drug, or with reserpine followed by L‐DOPA, a dopamine precursor. Exposure to reserpine increased (4,5‐13C2) glutamine in WT to levels similar to those found in untreated D1R−/− mice. These values were the same as those reached in the reserpine‐treated D1R−/− mice. Treatment of WT animals with L‐DOPA returned (4,5‐13C2) glutamine levels to normal, but this was not verified in D1R−/− animals. Reserpine impaired motor coordination and decreased c‐Fos expression, whereas L‐DOPA restored both variables to normal values in WT but not in D1R−/−. Together, our results reveal novel neurometabolic interactions between glutamatergic and dopaminergic systems that are mediated through the D1, but not the D2, dopamine receptor subtype.

Aging-related dysregulation of dopamine and angiotensin receptor interaction

It is not known whether the aging-related decrease in dopaminergic function leads to the aging-related higher vulnerability of dopaminergic neurons and risk for Parkinsons disease. The renin-angiotensin system (RAS) plays a major role in the inflammatory response, neuronal oxidative stress, and dopaminergic vulnerability via type 1 (AT1) receptors. In the present study, we observed a counterregulatory interaction between dopamine and angiotensin receptors. We observed overexpression of AT1 receptors in the striatum and substantia nigra of young adult dopamine D1 and D2 receptor-deficient mice and young dopamine-depleted rats, together with compensatory overexpression of AT2 receptors or compensatory downregulation of angiotensinogen and/or angiotensin. In aged rats, we observed downregulation of dopamine and dopamine receptors and overexpression of AT1 receptors in aged rats, without compensatory changes observed in young animals. L-Dopa therapy inhibited RAS overactivity in young dopamine-depleted rats, but was ineffective in aged rats. The results suggest that dopamine may play an important role in modulating oxidative stress and inflammation in the substantia nigra and striatum via the RAS, which is impaired by aging.