Gabriela Beatriz Acosta

Effects of glutamate transport substrates and glutamate receptor ligands on the activity of Na+/K+-ATPase in brain tissue in vitro

1. It has been suggested that Na+/K+‐ATPase and Na+‐dependent glutamate transport (GluT) are tightly linked in brain tissue. In the present study, we have investigated Na+/K+‐ATPase activity using Rb+ uptake by ‘minislices’ (prisms) of the cerebral cortex. This preparation preserves the morphology of neurons, synapses and astrocytes and is known to possess potent GluT that has been well characterized. Uptake of Rb+ was determined by estimating Rb+ in aqueous extracts of the minislices, using atomic absorption spectroscopy.

Long-term effects of early life stress exposure: Role of epigenetic mechanisms

Stress is an adaptive response to demands of the environment and thus essential for survival. Exposure to stress during the first years of life has been shown to have profound effects on the growth and development of an adult individual. There are evidences demonstrating that stressful experiences during gestation or in early life can lead to enhanced susceptibility to mental disorders. Early-life stress triggers hypothalamic-pituitary-adrenocortical (HPA) axis activation and the associated neurochemical reactions following glucocorticoid release are accompanied by a rapid physiological response. An excessive response may affect the developing brain resulting in neurobehavioral and neurochemical changes later in life. This article reviews the data from experimental studies aimed to investigate hormonal, functional, molecular and epigenetic mechanisms involved in the stress response during early-life programming. We think these studies might prove useful for the identification of novel pharmacological targets for more effective treatments of mental disorders.

A possible interaction between CCKergic and GABAergic systems in the rat brain

Cholecystokinin sulfated octapeptide (CCK-8S) was given to rats i.p. at single doses of 10 and 100 nmol/kg, respectively. It produced a modification in GABA levels in several areas of the rat brain. After 30 min of injection, the lower dose (10 nmol/kg) increased GABA levels in striatum by 31% (P<0.05). The higher dose (100 nmol/kg) enhanced GABA levels either in hippocampus by 78% (P<0.05) or in frontal cerebral cortex by 81% (P<0.05) and decreased in olfactory bulbs by 57% (P<0.01). Thus, these results show that systemic injection of CCK-8S, produced regional specific changes on GABA levels in brain, and these effects were dose-dependent. Systemic pretreatment with the CCK(B) receptor antagonist, PD 135,158, 1 mg/kg i.p., on the endogenous levels of GABA in certain regions was also studied. The selective CCK(B) receptor antagonist, PD 135,158, did not have an effect per se on the endogenous levels of GABA but prevents the action induced by the neuropeptide. We suggest that the action of CCK may be mediated via a selective action on the CCK(B) receptor subtypes.

Chronic postnatal stress induces voluntary alcohol intake and modifies glutamate transporters in adolescent rats.

Abstract Postnatal stress alters stress responses for life, with serious consequences on the central nervous system (CNS), involving glutamatergic neurotransmission and development of voluntary alcohol intake. Several drugs of abuse, including alcohol and cocaine, alter glutamate transport (GluT). Here, we evaluated effects of chronic postnatal stress (CPS) on alcohol intake and brain glutamate uptake and transporters in male adolescent Wistar rats. For CPS from postnatal day (PD) 7, pups were separated from their mothers and exposed to cold stress (4 °C) for 1 h daily for 20 days; controls remained with their mothers. Then they were exposed to either voluntary ethanol (6%) or dextrose (1%) intake for 7 days (5–7 rats per group), then killed. CPS: (1) increased voluntary ethanol intake, (2) did not affect body weight gain or produce signs of toxicity with alcohol exposure, (3) increased glutamate uptake by hippocampal synaptosomes in vitro and (4) reduced protein levels (Western measurements) in hippocampus and frontal cortex of glial glutamate transporter-1 (GLT-1) and excitatory amino-acid transporter-3 (EAAT-3) but increased glutamate aspartate transporter (GLAST) levels. We propose that CPS-induced decrements in GLT-1 and EAAT-3 expression levels are opposed by activation of a compensatory mechanism to prevent excitotoxicity. A greater role for GLAST in total glutamate uptake to prevent enlarged extracellular glutamate levels is inferred. Although CPS strongly increased intake of ethanol, this had little impact on effects of CPS on brain glutamate uptake or transporters. However, the impact of early life adverse events on glutamatergic neurotransmission may underlie increased alcohol consumption in adulthood.

Rottlerin Inhibits (Na+, K+)-ATPase Activity in Brain Tissue and Alters d-Aspartate Dependent Redistribution of Glutamate Transporter GLAST in Cultured Astrocytes

The naturally occurring toxin rottlerin has been used by other laboratories as a specific inhibitor of protein kinase C-delta (PKC-δ) to obtain evidence that the activity-dependent distribution of glutamate transporter GLAST is regulated by PKC-δ mediated phosphorylation. Using immunofluorescence labelling for GLAST and deconvolution microscopy we have observed that d-aspartate-induced redistribution of GLAST towards the plasma membranes of cultured astrocytes was abolished by rottlerin. In brain tissue in vitro, rottlerin reduced apparent activity of (Na+, K+)-dependent ATPase (Na+, K+-ATPase) and increased oxygen consumption in accordance with its known activity as an uncoupler of oxidative phosphorylation (“metabolic poison”). Rottlerin also inhibited Na+, K+-ATPase in cultured astrocytes. As the glutamate transport critically depends on energy metabolism and on the activity of Na+, K+-ATPase in particular, we suggest that the metabolic toxicity of rottlerin and/or the decreased activity of the Na+, K+-ATPase could explain both the glutamate transport inhibition and altered GLAST distribution caused by rottlerin even without any involvement of PKC-δ-catalysed phosphorylation in the process.

Myosin VI reduces proliferation, but not differentiation, in pluripotent P19 cells.

Background We have previously shown marked upregulation of the mRNA and corresponding protein for the cellular motor molecule myosin VI (Myo6) after an extremely traumatic stress experience, along with a delayed decrease in 5-bromo-2′-deoxyuridine incorporation in the murine hippocampus, a brain structure believed to undergo adult neurogenesis. In this study, we investigated the role of Myo6 in both proliferation and differentiation in pluripotent P19 cells by using stable transfection and RNA interference techniques. Methodology/Principal Findings Stable overexpression of Myo6 not only led to significant inhibition of the reducing activity of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and the size of clustered aggregates in P19 cells, but also resulted in selectively decreased mRNA expression of the repressor type proneural gene Hes5 without affecting the expression of neuronal and astroglial marker proteins. In P19 cells transfected with Myo6 siRNA, by contrast, a significant increase was found in the size of aggregate and MTT reduction along with increased Sox2 protein levels, in addition to marked depletion of the endogenous Myo6 protein. In C6 glioma cells, however, introduction of Myo6 siRNA induced a drastic decrease in endogenous Myo6 protein levels without significantly affecting MTT reduction. The Ca2+ ionophore A23187 drastically increased the luciferase activity in P19 cells transfected with a Myo6 promoter reporter plasmid, but not in HEK293, Neuro2A and C6 glioma cells transfected with the same reporter. Conclusions/Significance These results suggest that Myo6 may play a predominant pivotal role in the mechanism underlying proliferation without affecting differentiation to progeny lineages in pluripotent P19 cells.

The Response to Postnatal Stress: Amino Acids Transporters and PKC Activity

It is well known that animals exposed to stressful stimuli during their early life develop different neurological disorders when they become adults. In this study, we evaluated the effect of acute cold stress on γ-aminobutyric acid (GABA) and L-Serine (L-Ser) transporters in vitro, using the uptake of [3H]-GABA and [3H]L-Ser by synaptosomes-enriched fractions isolated from rat cerebral cortex during postnatal development. GABA and L-Ser uptake studies in vitro will be used in this investigation as a colateral evidence of changes in the expression of transporters of GABA and L-Ser. We observed that the maximum velocity (Vmax) in L-Ser and GABA uptake after stress session increased in all stages studied. In contrast, Km values of L-Ser uptake enhancent in almost age calculated, excluding at PD21 after cold stress during development, at the same time as Km (uptake affinity) values of GABA increased in just about age considered but not at PD5 compared with the control group. Finally we investigated the mechanism by which cells regulate the substrate affinity of L-Ser and GABA transporters. We demonstrated a significantly increase in total PKC activity to PD5 from PD21. Pretreatment with PKC inhibitor: staurosporine (SP) led to a restoration of control uptake in several postnatal-days suggesting a relationship between amino acids system and PKC activation. These findings suggest that a single exposure to postnatal cold stress at different periods after birth modifies both GABA and L-Ser transporters and the related increase in total PKC activity could be intracellular events that participate in neuronal plasticity by early life stress, which could be relevant to function of transporters in the adult rat brain.

In Search of the Mysterious Alzheimer's Disease

This article present the complexity of Alzheimer’s Disease (AD) improves, the biological bases underlying its pathogenesis are gradually being disclosed, and we can expect that new therapeutic targets will emerge. AD is characterized behaviorally by progressive memory loss and cognitive decline and physiologically by the presence of beta-amyloid peptide (Aβ) and Neurofibrillary Tangles (NFT) in the brain. The aim is to prevent or at least slow down the progression towards clinical impairment. The pathological mechanisms implicated the actions of β-amyloid, the accumulation of aggregates, the inflammatory cascade, oxidative neuronal damage, tau protein alterations and the formation of NFT, synaptic failure and neurotransmitter depletion. Several of these events are common to many slowly progressive neurodegenerative disorders. The familial forms of Alzheimer’s, secondary to inherited mutations have provided an insight into the molecular mechanisms implicated in disease pathogenesis. The underlying cause of AD, as well as its treatment, is still under investigation. A number of valuable diagnostic tools have been developed and continue to be improved. Risk factors for AD include age, genetic predisposition, environmental factors, cardiovascular diseases, diabetes and diet.

Voluntary alcohol intake after noise exposure in adolescent rats: Hippocampal-related behavioral alterations

Different physical or chemical agents, such as noise or alcohol, can induce diverse behavioral and biochemical alterations. Considering the high probability of young people to undergo consecutive or simultaneous exposures, the aim of the present work was to investigate in an animal model if noise exposure at early adolescence could induce hippocampal-related behavioral changes that might be modified after alcohol intake. Male Wistar rats (28-days-old) were exposed to noise (95-97 dB, 2 h). Afterwards, animals were allowed to voluntarily drink alcohol (10% ethanol in tap water) for three consecutive days, using the two-bottle free choice paradigm. After that, hippocampal-related memory and anxiety-like behavior tests were performed. Results show that whereas noise-exposed rats presented deficits in habituation memory, those who drank alcohol exhibited impairments in associative memory and anxiety-like behaviors. In contrast, exposure to noise followed by alcohol intake showed increases in exploratory and locomotor activities as well as in anxiety-like behaviors, unlike what was observed using each agent separately. Finally, lower levels of alcohol intake were measured in these animals when compared with those that drank alcohol and were not exposed to noise. Present findings demonstrate that exposure to physical and chemical challenges during early adolescence might induce behavioral alterations that could differ depending on the schedule used, suggesting a high vulnerability of rat developing brain to these socially relevant agents.

Nicotine-induced molecular alterations are modulated by GABAB receptor activity

It has been demonstrated that GABAB receptors modulate nicotine (NIC) reward effect; nevertheless, the mechanism implicated is not well known. In this regard, we evaluated the involvement of GABAB receptors on the behavioral, neurochemical, biochemical and molecular alterations associated with the rewarding effects induced by NIC in mice, from a pharmacological and genetic approach. NIC‐induced rewarding properties (0.5 mg/kg, subcutaneously, sc) were evaluated by conditioned place preference (CPP) paradigm. CPP has three phases: preconditioning, conditioning and postconditioning. GABAB receptor antagonist 2‐hydroxysaclofen (0.25, 0.5 and 1 mg/kg; intraperitoneally, ip) or the GABAB receptor agonist baclofen (3 mg/kg; ip) was injected before NIC during the conditioning phase. GABAB1 knockout (GABAB1KO) mice received NIC during the conditioning phase. Vehicle and wild‐type controls were employed. Neurochemical (dopamine, serotonin and their metabolites), biochemical (nicotinic receptor α4β2, α4β2nAChRs) and molecular (c‐Fos) alterations induced by NIC were analyzed after the postconditioning phase by high‐performance liquid chromatography (HPLC), receptor‐ligand binding assays and immunohistochemistry, respectively, in nucleus accumbens (Acb), prefrontal cortex (PFC) and ventral tegmental area (VTA). NIC induced rewarding effects in the CPP paradigm and increased dopamine levels in Acb and PFC, α4β2nAChRs density in VTA and c‐Fos expression in Acb shell (AcbSh), VTA and PFC. We showed that behavioral, neurochemical, biochemical and molecular alterations induced by NIC were prevented by baclofen. However, in 2‐hydroxysaclofen pretreated and GABAB1KO mice, these alterations were potentiated, suggesting that GABAB receptor activity is necessary to control alterations induced by NIC‐induced rewarding effects. Therefore, the present findings provided important contributions to the mechanisms implicated in NIC‐induced rewarding effects.