Regina Célia Cussa Kubrusly

L-DOPA supply to the neuro retina activates dopaminergic communication at the early stages of embryonic development

DOPA decarboxylase (DDC; aromatic‐l‐amino acid decarboxylase; EC 4.1.1.28) is absent in retinas from 6‐day‐old chicken embryos (E6) but is expressed in retina of E8 embryos, in the presumptive outer plexiform layer. Thereafter, DDC appears in cell bodies of presumptive amacrine cells. The dopamine (DA) content of E9/10 and E15/16 retinas, pre‐incubated with l‐DOPA for 1 h, increased 250‐ and 600‐fold, respectively, showing that DDC is active since early in development. Intercellular communication, measured by endogenous cyclic AMP accumulation, was observed when retinas from E9/10 to E15/16 were pre‐incubated for 1 h with 1 mm l‐DOPA, washed and followed by incubation in the presence of 0.5 mm 3‐isobutyl‐1‐methylxanthine, a phosphodiesterase inhibitor. Cyclic AMP accumulation was prevented when pre‐incubation with l‐DOPA was carried out in the presence of carbidopa. Moreover, the accumulation of cyclic AMP was inhibited by SCH 23390 (2 µm). The incubation of retinas in medium previously conditioned by retina‐pigmented epithelium (RPE) also increased its cyclic AMP content with the characteristics described for l‐DOPA. Our results show that dopaminergic communication takes place in the embryonic retina, before tyrosine hydroxylase expression, provided l‐DOPA is supplied to the tissue. It also shows that RPE is a potential source of l‐DOPA early in development.

Hippocampal biomarkers of fear memory in an animal model of generalized anxiety disorder

Generalized anxiety disorder (GAD) is highly prevalent and incapacitating. Here we used the Carioca High-Conditioned Freezing (CHF) rats, a previously validated animal model for GAD, to identify biomarkers and structural changes in the hippocampus that could be part of the underlying mechanisms of their high-anxiety profile. Spatial and fear memory was assessed in the Morris water maze and passive avoidance test. Serum corticosterone levels, immunofluorescence for glucocorticoid receptors (GR) in the dentate gyrus (DG), and western blotting for hippocampal brain derived neurotrophic factor (BDNF) were performed. Immunohistochemistry for markers of cell proliferation (bromodeoxiuridine/Ki-67), neuroblasts (doublecortin), and cell survival were undertaken in the DG, along with spine staining (Golgi) and dendritic arborization tracing. Hippocampal GABA release was assessed by neurochemical assay. Fear memory was higher among CHF rats whilst spatial learning was preserved. Serum corticosterone levels were increased, with decreased GR expression. No differences were observed in hippocampal cell proliferation/survival, but the number of newborn neurons was decreased, along with their number and length of tertiary dendrites. Increased expression of proBDNF and dendritic spines was observed; lower ratio of GABA release in the hippocampus was also verified. These findings suggest that generalized anxiety/fear could be associated with different hippocampal biomarkers, such as increased spine density, possibly as a compensatory mechanism for the decreased hippocampal number of neuroblasts and dendritic arborization triggered by high corticosterone. Disruption of GABAergic signaling and BDNF impairment are also proposed as part of the hippocampal mechanisms possibly underlying the anxious phenotype of this model.

Acute administration of vinpocetine, a phosphodiesterase type 1 inhibitor, ameliorates hyperactivity in a mice model of fetal alcohol spectrum disorder

BACKGROUND Maternal alcohol use during pregnancy causes a continuum of long-lasting disabilities in the offspring, commonly referred to as fetal alcohol spectrum disorder (FASD). Attention-deficit/hyperactivity disorder (ADHD) is possibly the most common behavioral problem in children with FASD and devising strategies that ameliorate this condition has great clinical relevance. Studies in rodent models of ADHD and FASD suggest that impairments in the cAMP signaling cascade contribute to the hyperactivity phenotype. In this work, we investigated whether the cAMP levels are affected in a long-lasting manner by ethanol exposure during the third trimester equivalent period of human gestation and whether the acute administration of the PDE1 inhibitor vinpocetine ameliorates the ethanol-induced hyperactivity. METHODS From postnatal day (P) 2 to P8, Swiss mice either received ethanol (5g/kg i.p.) or saline every other day. At P30, the animals either received vinpocetine (20mg/kg or 10mg/kg i.p.) or vehicle 4h before being tested in the open field. After the test, frontal cerebral cortices and hippocampi were dissected and collected for assessment of cAMP levels. RESULTS Early alcohol exposure significantly increased locomotor activity in the open field and reduced cAMP levels in the hippocampus. The acute treatment of ethanol-exposed animals with 20mg/kg of vinpocetine restored both their locomotor activity and cAMP levels to control levels. CONCLUSIONS These data lend support to the idea that cAMP signaling system contribute to the hyperactivity induced by developmental alcohol exposure and provide evidence for the potential therapeutic use of vinpocetine in FASD.

Exposure to tobacco smoke containing either high or low levels of nicotine during adolescence: Differential effects on choline uptake in the cerebral cortex and hippocampus

INTRODUCTION There is a lack of experimental studies that investigate the effects of tobacco smoke exposure during adolescence. Here, we investigated the effects of tobacco smoke generated from cigarettes containing either high or low levels of nicotine on the cholinergic system. METHODS From postnatal day (PN) 30 to 45, 18 C57BL/6 (inbred) and 16 Swiss (outbred) mice of both sexes were exposed to tobacco smoke (whole body exposure for 8 hr/day and 7 days/week) generated from one of two reference research cigarettes: type 3R4F (HighNIC group-nicotine = 0.73 mg/cigarette) or type 4A1 (LowNIC group-nicotine = 0.14 mg/cigarette). Control mice (CT) were exposed to air. On PN 45, cotinine (nicotine metabolite) serum levels and [(3)H]choline uptake in the cerebral cortex and hippocampus were assessed. RESULTS Cotinine serum levels were eight times higher in HighNIC mice (C57BL/6:142.0 +/- 16.7 ng/ml and Swiss: 197.6 +/- 11.1 ng/ml) when compared with LowNIC ones (C57BL/6:17.4 +/- 7.4 ng/ml and Swiss: 24.6 +/- 2.2 ng/ml). Only HighNIC mice presented a significant increase in [(3)H]choline uptake in the hippocampus (C57BL/6: HighNIC > CT and HighNIC > LowNIC, p < .001 and Swiss: HighNIC > CT and HighNIC > LowNIC, p < .001), whereas in the cerebral cortex, both HighNIC and LowNIC mice presented increased [(3)H]choline uptake (C57BL/6: HighNIC > CT and LowNIC > CT, p < .05 and Swiss: HighNIC > CT and LowNIC > CT, p < .001). DISCUSSION Our results indicate that tobacco smoke exposure during adolescence increases [(3)H]choline uptake. However, the effects are dependent on the type of cigarette and on the brain region.

Atypical effect of dopamine in modulating the functional inhibition of NMDA receptors of cultured retina cells.

Cultured retina cells released accumulated [3H]GABA (gamma-aminobutyric acid) when stimulated by L-glutamate, N-methyl-D-aspartate (NMDA) and kainate. In the absence of Mg2+, dopamine at 200 microM (IC50 60 microM), inhibited in more than 50% the release of [3H]GABA induced by L-glutamate and NMDA, but not by kainate. This effect was not blocked by the D1-like dopamine receptor antagonist, R-(+)-7-chloro-8-hydroxy-3-methyl- -phenyl-2,3,4,5-tetrahydro- H-3-benzazepine hydrochloride (SCH 23390), neither by haloperidol nor spiroperidol (dopamine D2-like receptor antagonists). The dopamine D1-like receptor agonist R(+)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,diol hydrochloride (SKF 38393) at 50 microM, but not its enantiomer, also inhibited the release of [3H]GABA induced by NMDA, but not by kainate; an effect that was not prevented by the antagonists mentioned above. (+/-)-6-Chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepin e hydrobromide (SKF 812497) had no effect. Neither 8BrcAMP (5 mM) nor forskolin (10 microM) inhibited the release of [3H]GABA. Our results suggest that dopamine and (+)-SKF 38393 inhibit the glutamate and NMDA-evoked [3H]GABA release through mechanisms that seem not to involve known dopaminergic receptor systems.

Caffeine potentiates the release of GABA mediated by NMDA receptor activation: Involvement of A1 adenosine receptors.

Caffeine, a methylated derivative of xanthine and widely consumed psychoactive substance, acts in several targets in the nervous system. We investigated its role in retinal explants of chick embryo analyzing the role of purinergic receptors in [(3)H]-GABA release induced by d-aspartate (d-asp). d-Asp increases GABA-release 4.5-fold when compared to basal levels from 13-day-old chick embryo retinal explants. Caffeine 500μM elevated d-asp-induced GABA release in 60%. The release was inhibited in the presence of NNC-711, a GABA transporter-1 (GAT-1) blocker or by MK-801, an N-methyl-d-aspartate receptor (NMDAR) antagonist. Caffeine did not modify [(3)H]-GABA uptake carried out for 5, 10, 30 and 60min and did not increase the release of d-asp or glutamate at basal or stimulated conditions. The caffeine effect was mimicked by the adenosine A1 receptor antagonist DPCPX and by the adenylyl cyclase (AC) activator forskolin. It was also blocked by the protein kinase A (PKA) inhibitor H-89, tyrosine kinase inhibitor genistein or by the src family kinase (SFK) inhibitor PP1. Forskolin-stimulated cyclic adenosine monophosphate (cAMP) levels were reduced in the presence of the A1 receptor agonist CHA. Western blot analysis revealed that 500μM caffeine increased phosphoGluN2B expression levels in approximately 60% when compared to total GluN2B levels in embryonic E13 retina. The GluN2B subunit-containing NMDAR antagonist ifenprodil inhibited the caffeine effect. Our results suggest that caffeine potentiates d-asp-induced GABA release, which is mediated by GAT-1, via inhibition of adenosine A1 receptor and activation of the PKA pathway. Regulation of NMDAR by phosphorylation of GluN2B subunit by a SFK may also be involved in the effect promoted by caffeine.

Ethanol increases GABA release in the embryonic avian retina

Several mechanisms underlying ethanol action in GABAergic synapses have been proposed, one of these mechanisms is on GABA release. Here, we report that in ovo exposure to ethanol induces an increase on GABA release in the embryonic chick retina. Eleven‐day‐old chick embryos (E11) received an injection of either phosphate buffer saline (PBS) or ethanol (10%, v/v, diluted in PBS), and were allowed to develop until E16. A single glutamate stimulus (2 mM) showed approximately a 40% increase on GABA release in E16 retinas when compared to controls. The effect was dependent on NMDA receptors and GAD65 mRNA levels, which were increased following the ethanol treatment. However, the numbers of GABA‐, GAD‐, and NR1‐immunoreactive cells, and the expression levels of these proteins, were not affected. We conclude that ethanol treatment at a time point when synapses are being formed during development selectively increases GABA release in the retina via a NMDA receptor‐dependent process.

Glutathione-Induced Calcium Shifts in Chick Retinal Glial Cells.

Neuroglia interactions are essential for the nervous system and in the retina Müller cells interact with most of the neurons in a symbiotic manner. Glutathione (GSH) is a low-molecular weight compound that undertakes major antioxidant roles in neurons and glia, however, whether this compound could act as a signaling molecule in neurons and/or glia is currently unknown. Here we used embryonic avian retina to obtain mixed retinal cells or purified Müller glia cells in culture to evaluate calcium shifts induced by GSH. A dose response curve (0.1–10mM) showed that 5–10mM GSH, induced calcium shifts exclusively in glial cells (later labeled and identified as 2M6 positive cells), while neurons responded to 50mM KCl (labeled as βIII tubulin positive cells). BBG 100nM, a P2X7 blocker, inhibited the effects of GSH on Müller glia. However, addition of DNQX 70μM and MK-801 20μM, non-NMDA and NMDA blockers, had no effect on GSH calcium induced shift. Oxidized glutathione (GSSG) at 5mM failed to induce calcium mobilization in glia cells, indicating that the antioxidant and/or structural features of GSH are essential to promote elevations in cytoplasmic calcium levels. Indeed, a short GSH pulse (60s) protects Müller glia from oxidative damage after 30 min of incubation with 0.1% H2O2. Finally, GSH induced GABA release from chick embryonic retina, mixed neuron-glia or from Müller cell cultures, which were inhibited by BBG or in the absence of sodium. GSH also induced propidium iodide uptake in Müller cells in culture in a P2X7 receptor dependent manner. Our data suggest that GSH, in addition to antioxidant effects, could act signaling calcium shifts at the millimolar range particularly in Müller glia, and could regulate the release of GABA, with additional protective effects on retinal neuron-glial circuit.

Long Withdrawal of Methylphenidate Induces a Differential Response of the Dopaminergic System and Increases Sensitivity to Cocaine in the Prefrontal Cortex of Spontaneously Hypertensive Rats

Methylphenidate (MPD) is one of the most prescribed drugs for alleviating the symptoms of Attention Deficit/Hyperactivity Disorder (ADHD). However, changes in the molecular mechanisms related to MPD withdrawal and susceptibility to consumption of other psychostimulants in normal individuals or individuals with ADHD phenotype are not completely understood. The aims of the present study were: (i) to characterize the molecular differences in the prefrontal dopaminergic system of SHR and Wistar strains, (ii) to establish the neurochemical consequences of short- (24 hours) and long-term (10 days) MPD withdrawal after a subchronic treatment (30 days) with Ritalin® (Methylphenidate Hydrochloride; 2.5 mg/kg orally), (iii) to investigate the dopaminergic synaptic functionality after a cocaine challenge in adult MPD-withdrawn SHR and Wistar rats. Our results indicate that SHR rats present reduced [3H]-Dopamine uptake and cAMP accumulation in the prefrontal cortex (PFC) and are not responsive to dopaminergic stimuli in when compared to Wistar rats. After a 24-hour withdrawal of MPD, SHR did not present any alterations in [3H]-Dopamine Uptake, [3H]-SCH 23390 binding and cAMP production; nonetheless, after a 10-day MPD withdrawal, the results showed a significant increase of [3H]-Dopamine uptake, of the quantity of [3H]-SCH 23390 binding sites and of cAMP levels in these animals. Finally, SHR that underwent a 10-day MPD withdrawal and were challenged with cocaine (10 mg/kg i.p.) presented reduced [3H]-Dopamine uptake and increased cAMP production. Wistar rats were affected by the 10-day withdrawal of MPD in [3H]-dopamine uptake but not in cAMP accumulation; in addition, cocaine was unable to induce significant modifications in [3H]-dopamine uptake and in cAMP levels after the 10-day withdrawal of MPD. These results indicate a mechanism that could explain the high comorbidity between ADHD adolescent patients under methylphenidate treatment and substance abuse in adult life.

Single exposure to cocaine impairs aspartate uptake in the pre-frontal cortex via dopamine D1-receptor dependent mechanisms.

Dopamine and glutamate play critical roles in the reinforcing effects of cocaine. We demonstrated that a single intraperitoneal administration of cocaine induces a significant decrease in [(3)H]-d-aspartate uptake in the pre-frontal cortex (PFC). This decrease is associated with elevated dopamine levels, and requires dopamine D1-receptor signaling (D1R) and adenylyl cyclase activation. The effect was observed within 10min of cocaine administration and lasted for up to 30min. This rapid response is related to D1R-mediated cAMP-mediated activation of PKA and phosphorylation of the excitatory amino acid transporters EAAT1, EAAT2 and EAAT3. We also demonstrated that cocaine exposure increases extracellular d-aspartate, l-glutamate and d-serine in the PFC. Our data suggest that cocaine activates dopamine D1 receptor signaling and PKA pathway to regulate EAATs function and extracellular EAA level in the PFC.