Acetyl-L-carnitine provides effective in vivo neuroprotection over 3,4-methylenedioximethamphetamine-induced mitochondrial neurotoxicity in the adolescent rat brain.

3,4-Methylenedioximethamphetamine (MDMA, ecstasy) is a worldwide abused stimulant drug, with persistent neurotoxic effects and high prevalence among adolescents. The massive release of 5-HT from pre-synaptic storage vesicles induced by MDMA followed by monoamine oxidase B (MAO-B) metabolism, significantly increases oxidative stress at the mitochondrial level. l-Carnitine and its ester, acetyl-l-carnitine (ALC), facilitate the transport of long chain free fatty acids across the mitochondrial membrane enhancing neuronal anti-oxidative defense. Here, we show the potential of ALC against the neurotoxic effects of MDMA exposure. Adolescent male Wistar rats were assigned to four groups: control saline solution, isovolumetric to the MDMA solution, administered i.p.; MDMA (4x10 mg/kg MDMA, i.p.); ALC/MDMA (100 mg/kg 30 min of ALC prior to MDMA, i.p.) and ALC (100 mg/kg, i.p.). Rats were killed 2 weeks after exposure and brains were analyzed for lipid peroxidation, carbonyl formation, mitochondrial DNA (mtDNA) deletion and altered expression of the DNA-encoded subunits of the mitochondrial complexes I (NADH dehydrogenase, NDII) and IV (cytochrome c oxidase, COXI) from the respiratory chain. Levels of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) were also assessed. The present work is the first to successfully demonstrate that pretreatment with ALC exerts effective neuroprotection against the MDMA-induced neurotoxicity at the mitochondrial level, reducing carbonyl formation, decreasing mtDNA deletion, improving the expression of the respiratory chain components and preventing the decrease of 5-HT levels in several regions of the rat brain. These results indicate potential benefits of ALC application in the prevention and treatment of neurodegenerative disorders.

Neurodevelopment milestone abnormalities in rats exposed to stress in early life.

Manipulation of the corticosteroid milieu by interfering with the mother-newborn relationship has received much attention because of its potential bearing on psychopathology later in life. In the present study, infant rats that were deprived of maternal contact between the 2nd and the 15th postnatal days (MS2-15) for 6 h/day were subjected to a systematic assessment of neurodevelopmental milestones between postnatal days 2 and 21. The analyses included measurements of physical growth and maturation and evaluation of neurological reflexes. Although some somatic milestones (e.g. eye opening) were anticipated, MS2-15 animals showed retardation in the acquisition of postural reflex, air righting and surface righting reflexes, and in the wire suspension test; the latter two abnormalities were only found in males. A gender effect was also observed in negative geotaxis, with retardation being observed in females but not males. To better understand the delay of neurological maturation in MS2-15 rats, we determined the levels of various monoamines in different regions of the brain stem, including the vestibular area, the substantia nigra, ventral tegmental area and dorsal raphe nuclei. In the vestibular region of MS2-15 rats the levels of 5-HT were reduced, while 5-HT turnover was increased. There was also a significant increase of the 5-HT turnover in MS2-15 animals in the raphe nuclei, mainly due to increased 5-hydroxyindoleacetic acid (5-HIAA) levels, and an increase of 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the ventral tegmental area (VTA) of stressed females. No significant differences were found in the immunohistochemical sections for tyrosine and tryptophan hydroxylase in these regions of the brain stem. In conclusion, the present results show that postnatal stress induces signs of neurological pathology that may contribute to the genesis of behavioral abnormalities later in life.

Acetyl-L-Carnitine Prevents Methamphetamine-Induced Structural Damage on Endothelial Cells via ILK-Related MMP-9 Activity.

Methamphetamine (METH) is a potent psychostimulant highly used worldwide. Recent studies evidenced the involvement of METH in the breakdown of the blood-brain-barrier (BBB) integrity leading to compromised function. The involvement of the matrix metalloproteinases (MMPs) in the degradation of the neurovascular matrix components and tight junctions (TJs) is one of the most recent findings in METH-induced toxicity. As BBB dysfunction is a pathological feature of many neurological conditions, unveiling new protective agents in this field is of major relevance. Acetyl-L-carnitine (ALC) has been described to protect the BBB function in different paradigms, but the mechanisms underling its action remain mostly unknown. Here, the immortalized bEnd.3 cell line was used to evaluate the neuroprotective features of ALC in METH-induced damage. Cells were exposed to ranging concentrations of METH, and the protective effect of ALC 1 mM was assessed 24 h after treatment. F-actin rearrangement, TJ expression and distribution, and MMPs activity were evaluated. Integrin-linked kinase (ILK) knockdown cells were used to assess role of ALC in ILK mediated METH-triggered MMPs' activity. Our results show that METH led to disruption of the actin filaments concomitant with claudin-5 translocation to the cytoplasm. These events were mediated by MMP-9 activation in association with ILK overexpression. Pretreatment with ALC prevented METH-induced activation of MMP-9, preserving claudin-5 location and the structural arrangement of the actin filaments. The present results support the potential of ALC in preserving BBB integrity, highlighting ILK as a new target for the ALC therapeutic use.

Methamphetamine promotes α-tubulin deacetylation in endothelial cells: the protective role of acetyl-l-carnitine.

Methamphetamine (METH) is a powerful psychostimulant drug used worldwide for its reinforcing properties. In addition to the classic long-lasting monoaminergic-disrupting effects extensively described in the literature, METH has been consistently reported to increase blood brain barrier (BBB) permeability, both in vivo and in vitro, as a result of tight junction and cytoskeleton disarrangement. Microtubules play a critical role in cell stability, which relies on post-translational modifications such as α-tubulin acetylation. As there is evidence that psychostimulants drugs modulate the expression of histone deacetylases (HDACs), we hypothesized that in endothelial cells METH-mediation of cytoplasmatic HDAC6 activity could affect tubulin acetylation and further contribute to BBB dysfunction. To validate our hypothesis, we exposed the bEnd.3 endothelial cells to increasing doses of METH and verified that it leads to an extensive α-tubulin deacetylation mediated by HDACs activation. Furthermore, since we recently reported that acetyl-l-carnitine (ALC), a natural occurring compound, prevents BBB structural loss in a context of METH exposure, we reasoned that ALC could also preserve the acetylation of microtubules under METH action. The present results confirm that ALC is able to prevent METH-induced deacetylation providing effective protection on microtubule acetylation. Although further investigation is still needed, HDACs regulation may become a new therapeutic target for ALC.

Effects of neonatal exposure to cocaine in the development of the neurotransmitters retinal systems: an immunocytochemical and neurochemical study.

The visual system of rodents is affected if exposure to drugs, e.g., cocaine, occurs during prenatal or early postnatal development. This study aims to evaluate, in an experimental model of neonatal exposure to cocaine in the rat, the immunocytochemical expression of tyrosine hydroxylase in the retina and the levels of different neurotransmitters and its metabolites. Male Wistar rats were given 15 mg cocaine hydrochloride/kg body weight/day, subcutaneously, in two daily doses, from the day after birth (PND1) to PND6, 13, and 29. Controls were given 0.9% saline. Groups of rats were perfused at PND7, 14, and 30 with fixative, and the retinas were processed as wholemounts, and immunostained with the antibody anti-TH. Other groups were decapitated, and the retinas were dissected and processed by high performance liquid chromatography with electrochemical detection (HPLC-EC) for determination of dopamine and metabolites (DOPAC and HVA). A reduction in the retinal surface area was detected in the PND30 cocaine group, and a decrease in the density of the small TH-IR cells was found in the PND14 cocaine group although not reaching significant levels. The other quantitative parameters did not differ between the control and cocaine groups. The levels of neurotransmitters did not significantly differ between the groups at any age. These results show a differential vulnerability of the dopaminergic system of rats exposed neonatally to cocaine when compared with the effects found after prenatal exposure to the same drug.

Long-term effects of chronic cocaine exposure throughout adolescence on anxiety and stress responsivity in a Wistar rat model.

Adolescents display increased vulnerability to engage in drug experimentation. This is often considered a risk factor for later drug abuse. In this scenario, the permanent effects of cocaine exposure during adolescence on anxiety levels and stress responsivity, which may result in behavioral phenotypes prone to addiction, are now starting to be unveiled. Thus, the purpose of the present study was to evaluate the long-lasting effects of chronic cocaine administration during adolescence, on anxiety-like behavior and on stress response. Adolescent male Wistar rats were daily administered 45-mg cocaine/kg of body weight in three equal intraperitoneal doses with 1-h interval, from postnatal day (PND) 35 to 50. The effects of cocaine administration on anxiety levels, assessed in the Elevated Plus Maze (EPM), and on social stress response, assessed in the resident-intruder paradigm (R/I), were evaluated 10 days after withdrawal, when rats were reaching the adulthood. The underlying dopaminergic activity, and the corticosterone and testosterone levels were determined. Our results showed that cocaine induced long-lasting alterations in the hypothalamus-pituitary-adrenals (HPA) axis function and in testosterone levels. Such alterations resulted in significant and enduring changes in behavioral responses to environmental challenges, such as the EPM and R/I, including the evaluation of potential threats that may lead to high-risk behavior and low-benefit choices. This was further supported by an altered dopaminergic function in the amygdala and hippocampus. The present findings provide new insights into how the use of cocaine during adolescent development may modulate emotional behavior later in life. Compromised ability to recognize and deal with potential threats is an important risk factor to perpetuate compulsive drug seeking and relapse susceptibility.

Impaired spatial memory after ketamine administration in chronic low doses.

Ketamine is a noncompetitive antagonist of the NMDA-receptors, used as a dissociative anesthetic, presently included in the category of the psychoactive substances known as "club drugs". Ketamine administration was associated with impaired working memory and increased psychopathological symptoms, but there is a lack of information regarding the effects of chronic sub-anesthetic doses. Adult Wistar rats were administered ketamine, 5 and 10 mg/kg twice daily, subcutaneously for 14 days. One week later, rats were tested in an object recognition/object location task and in the open field arena. There was altered performance in both the object recognition/location and in the open field tests by the group chronically exposed to the lower dose of ketamine. These animals displayed a decreased discrimination index (p<0.05) in the object recognition task, were unable to recognize the displacement of a familiar object and displayed decreased activity across open filed sessions. Importantly, these alterations were not observed in animals administered a higher dose of ketamine. Collectively, these results consistently show that chronic administration of ketamine in sub-anesthetic doses may lead to decreased habituation and inability to update spatial representations.

Monoamine deficits in the brain of methyl-CpG binding protein 2 null mice suggest the involvement of the cerebral cortex in early stages of Rett syndrome.

Rett syndrome is a neurodevelopmental disorder caused by mutations in the methyl-CpG binding protein 2 gene (MECP2). Several neural systems are affected in Rett, resulting in an autonomic dysfunction, a movement disorder with characteristic loss of locomotor abilities and profound cognitive impairments. A deregulation of monoamines has been detected in the brain and cerebrospinal fluid of both Rett patients and a Rett syndrome murine model, the Mecp2 knock-out mouse. Our goal was to characterize the onset and progression of motor dysfunction in Mecp2(tm1.1Bird) knock-out mice and the possible neurochemical alterations in different brain regions potentially playing a role in Rett-like pathophysiology, at two different time-points, at weaning (3 weeks old) and in young adults when overt symptoms are observed (8 weeks old). Our results revealed significant age- and region-dependent impairments in these modulatory neurotransmitter systems that correspond well with the motor phenotype observed in these mice. At 3 weeks of age, male Mecp2 knock-out mice exhibited ataxia and delayed motor initiation. At this stage, noradrenergic and serotonergic transmission was mainly altered in the prefrontal and motor cortices, whereas during disease progression the neurochemical changes were also observed in hippocampus and cerebellum. Our data suggest that the deregulation of norepinephrine and serotonin systems in brain regions that participate in motor control are involved in the pathophysiology of Rett syndrome motor phenotypes. Moreover, we highlight the contribution of cortical regions along with the brainstem to be in the origin of the pathology and the role of hippocampus and cerebellum in the progression of the disease rather than in its establishment.