title: Modeling chronic brain exposure to amphetamines using primary rat neuronal cortical cultures.
abstract: Amphetamine-type psychostimulants (ATS) are used worldwide by millions of patients for several psychiatric disorders. Amphetamine (AMPH) and "ecstasy" (3,4-methylenedioxymethamphetamine or MDMA) are common drugs of abuse. The impact of chronic ATS exposure to neurons and brain aging is still undisclosed. Current neuronal culture paradigms are designed to access acute ATS toxicity. We report for the first time a model of chronic exposure to AMPH and MDMA using long-term rat cortical cultures. In two paradigms, ATS were applied to neurons at day 1 in vitro (DIV) (0, 1, 10 and 100 μM of each drug) up to 28 days (200 μM was applied to cultures up to 14 DIV). Our reincubation protocol assured no decrease in the neuronal media's drug concentration. Chronic exposure of neurons to concentrations equal to or above 100 μM of ATS up to 28 DIV promoted significant mitochondrial dysfunction and elicited neuronal death, which was not prevented by glutamate receptor antagonists at 14 DIV. ATS failed to promote accelerated senescence as no increase in β-galactosidase activity at 21 DIV was found. In younger cultures (4 or 8 DIV), AMPH promoted mitochondrial dysfunction and neuronal death earlier than MDMA. Overall, AMPH proved more toxic and was the only drug that decreased intraneuronal glutathione levels. Meanwhile, caspase 3 activity increased for either drug at 200 μM in younger cultures at 8 DIV, but not at 14 DIV. At 8 DIV, ATS promoted a significant change in the percentage of neurons and astroglia present in culture, promoting a global decrease in the number of both cells. Importantly, concentrations equal to or below 10 μM of either drug did not promote neuronal death or oxidative stress. Our paradigm of neuronal cultures long-term exposure to low micromolar concentrations of ATS closely reproduces the in vivo scenario, being valuable to study the chronic impact of ATS.
authors.affiliation: REQUIMTE (Rede de Química e Tecnologia), Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
authors.forename: T B
authors.initials: TB
authors.lastname: Nogueira
authors.affiliation: REQUIMTE (Rede de Química e Tecnologia), Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
authors.forename: S
authors.initials: S
authors.lastname: da Costa Araújo
authors.affiliation: REQUIMTE (Rede de Química e Tecnologia), Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
authors.forename: F
authors.initials: F
authors.lastname: Carvalho
authors.affiliation: Farmacologia e Terapêutica Experimental, Instituto Biomédico de Investigação da Luz e Imagem (IBILI), Faculdade de Medicina, Universidade de Coimbra, 3000-548 Coimbra, Portugal.
authors.forename: F C
authors.initials: FC
authors.lastname: Pereira
authors.affiliation: REQUIMTE (Rede de Química e Tecnologia), Laboratório de Química Aplicada, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
authors.forename: E
authors.initials: E
authors.lastname: Fernandes
authors.affiliation: REQUIMTE (Rede de Química e Tecnologia), Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
authors.forename: M L
authors.initials: ML
authors.lastname: Bastos
authors.affiliation: REQUIMTE (Rede de Química e Tecnologia), Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
authors.forename: V M
authors.initials: VM
authors.lastname: Costa
authors.affiliation: REQUIMTE (Rede de Química e Tecnologia), Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal; Faculdade de Ciências da Saúde, Universidade Fernando Pessoa, Rua Carlos da Maia, 296, 4200-150 Porto, Portugal. Electronic address: joaocapela@ff.up.pt.
authors.forename: J P
authors.initials: JP
authors.lastname: Capela
mesh_terms: D018759:Adrenergic Uptake Inhibitors / Q000494:pharmacology*; D000661:Amphetamine / Q000494:pharmacology*; D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000473:pathology / Q000502:physiology; D053148:Caspase 3 / Q000378:metabolism; D002452:Cell Count; D018929:Cell Culture Techniques; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects* / Q000473:pathology / Q000503:physiopathology; D004305:Dose-Response Relationship, Drug; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D018817:N-Methyl-3,4-methylenedioxyamphetamine / Q000494:pharmacology*; D009474:Neurons / Q000187:drug effects* / Q000473:pathology / Q000502:physiology; D017208:Rats, Wistar; D017470:Receptors, Glutamate / Q000378:metabolism; D013997:Time Factors
chemical_list: D018759:Adrenergic Uptake Inhibitors; D018691:Excitatory Amino Acid Antagonists; D017470:Receptors, Glutamate; D000661:Amphetamine; C505410:Casp3 protein, rat; D053148:Caspase 3; D005978:Glutathione; D018817:N-Methyl-3,4-methylenedioxyamphetamine
keywords: amphetamines; chronic exposure; cortical neurons; long-term neuronal cultures; neurotoxicity; “ecstasy”