Marcelo Ganzella

NMDA preconditioning protects against seizures and hippocampal neurotoxicity induced by quinolinic acid in mice.

Summary:  Purpose: N‐methyl d‐aspartate (NMDA) preconditioning has been used to prevent cellular death induced by glutamate or NMDA in cultured neurons. Quinolinic acid (QA)‐induced seizures are used to average NMDA receptors–evoked neurotoxicity in animal models. The purpose of this study was to investigate the potential neuroprotective effects of NMDA preconditioning against QA‐induced seizures and hippocampal damage in vivo.

Omega-3 fatty acids deprivation affects ontogeny of glutamatergic synapses in rats: relevance for behavior alterations.

Essential omega-3 polyunsaturated fatty acids (omega3) are crucial to brain development and function, being relevant for behavioral performance. In the present study we examined the influence of dietary omega3 in the development of the glutamatergic system and on behavior parameters in rats. Female rats received isocaloric diets, either with omega3 (omega3 group) or a omega3 deficient diet (D group). In ontogeny experiments of their litters, hippocampal immunocontent of ionotropic NMDA and AMPA glutamatergic receptors subunits (NR2 A\B and GluR1, respectively) and the alpha isoform of the calcium-calmodulin protein kinase type II (alphaCaMKII) were evaluated. Additionally, hippocampal [(3)H]glutamate binding and uptake were assessed. Behavioral performance was evaluated when the litters were adult (60 days old), through the open-field, plus-maze, inhibitory avoidance and flinch-jump tasks. The D group showed decreased immunocontent of all proteins analyzed at 02 days of life (P2) in comparison with the omega3 group, although the difference disappeared at 21 days of life (except for alphaCaMKII, which content normalized at 60 days old). The same pattern was found for [(3)H]glutamate binding, whereas [(3)H]glutamate uptake was not affected. The D group also showed memory deficits in the inhibitory avoidance, increased in the exploratory pattern in open-field, and anxiety-like behavior in plus-maze. Taken together, our results suggest that dietary omega3 content is relevant for glutamatergic system development and for behavioral performance in adulthood. The putative correlation among the neurochemical and behavioral alterations caused by dietary omega3 deficiency is discussed.

Time course of oxidative events in the hippocampus following intracerebroventricular infusion of quinolinic acid in mice.

The excitotoxicity induced by QA has been related to its ability to increase free radical content and oxidative stress. In order to investigate the time course of toxicity and oxidative profile in the mice hippocampus following seizures induced by QA infusion (36.8 nM, i.c.v.), we evaluated the cellular damage (PI uptake assay), content of ROS formation (DCF assay) and the total radical antioxidant potential (TRAP) and reactivity (TAR) levels. The present results showed that a cellular damage occurred as early as 4 h after QA infusion coincident with an increase in the ROS contents, which returned to control levels after 24 h, while the cellular damage persisted for 72 h. There was a marked increased in the total antioxidant capacity at 8 h after QA infusion in both reactivity and potential levels. By 72 h post-treatment, the TRAP levels decreased, but the TAR levels remained augmented. Therefore, the delayed and persistent increase in the antioxidant capacity after QA insult may be a cellular adaptative response, probably contributing to decrease the ROS levels in order to prevent the spreading of the cellular damage. Therefore, the increase in the QA level in the brain ventricle may induce oxidative stress, which is followed by a persistent response in the antioxidant system in the hippocampus. The present study may, therefore, contribute to elucidate the mechanism of the brain dysfunction in patients with several neurological disorders involving elevation of QA in the CSF.

Effects of chronic guanosine treatment on hippocampal damage and cognitive impairment of rats submitted to chronic cerebral hypoperfusion

Chronic cerebral hypoperfusion contributes to a cognitive decline related to brain disorders. Its experimental model in rats is a permanent bilateral common carotid artery occlusion (2VO). Overstimulation of the glutamatergic system excitotoxicity due to brain energetic disturbance in 2VO animals seems to play a pivotal role as a mechanism of cerebral damage. The nucleoside guanosine (GUO) exerts extracellular effects including antagonism of glutamatergic activity. Accordingly, our group demonstrated several neuroprotective effects of GUO against glutamatergic excitotoxicity. Therefore, in this study, we evaluated a chronic GUO treatment effects in rats submitted to 2VO. We evaluated the animals performance in the Morris water maze and hippocampal damage by neurons and astrocytes immunohistochemistry. In addition, we investigated the cerebrospinal fluid (CSF) brain derived neurotrophic factor (BDNF) and serum S100B levels. Additionally, the purine CSF and plasma levels were determined. GUO treatment did not prevent the cognitive impairment promoted by 2VO. However, none of the 2VO animals treated with GUO showed differences in the hippocampal regions compared to control, while 20% of 2VO rats not treated with GUO presented loss of pyramidal neurons and increased glial labeling cells in CA1 hippocampal region. In addition, we did not observe differences in CSF BDNF nor serum S100B levels among the groups. Of note, both the 2VO surgery and GUO treatment changed the purine CSF and plasma profile. In conclusion, GUO treatment did not prevent the cognitive impairment observed in 2VO animals, but our data suggest that GUO could be neuroprotective against hippocampal damage induced by 2VO.

Systemic administration of GMP induces anxiolytic-like behavior in rats.

The glutamatergic system has received considerable attention over the last few years as potential target to develop anxiolytic drugs. Guanine based purines (GBPs) play an important neurmodulatory effect in the glutamatergic system. Several studies have shown the ability of the GBPs to reduce glutamatergic activity. In the present study, we investigated the anxiolytic effect of GBPs - by Guanosina Monophosphate (GMP) administration - in rodents. Adult male Wistar rats were pretreated with GMP (10, 25, 50, 100 and 150mg/kg: i.p.); or saline (NaCl 0.9%; i.p.) (control); or, diazepam (2mg/kg: i.p.) (positive control). One hour after the injection, the anxiety-related behaviors for each animal was evaluated in the light/dark, elevated plus-maze, and open field tasks. Additionally, purines concentration in the cerebrospinal fluid (CSF) was verified. The administration of 25 and 50mg/kg GMP was able to promote anxiolytic-like behavior, in the light/dark and elevated plus-maze task, similar to diazepam effect. However, no changes in the open field task, or CSF purines concentration were found for either GMP or diazepam treated animals, when compared with saline group. Thus, this study suggests that acute administration of GMP was able to decrease the levels of anxiety in classical behavioral tasks.

Intracerebroventricular administration of inosine is anticonvulsant against quinolinic acid-induced seizures in mice: an effect independent of benzodiazepine and adenosine receptors.

Inosine (INO) has an anticonvulsant effect against seizures induced by antagonists of GABAergic system. Quinolinic acid (QA) is an agonist NMDA receptors implicated in the neurobiology of seizures. In the present study, we investigated the anticonvulsant effect of intracerebroventricular (i.c.v.) INO administration against QA-induced seizures in adult mice. We also investigated whether the benzodiazepines (BZ) or adenosine (ADO) receptors were involved in the INO effects. Animals were pretreated with an i.c.v. injection of either vehicle or INO before an i.c.v. administration of 4 μl QA (36.8 nmol). All animals pretreated with vehicle followed by QA presented seizures. INO protected against QA-induced seizures in a time and dose dependent manner (up to 60% at 400 nmol, 5 min before QA injection). Diazepam (DZ) and ADO (i.c.v.) also exhibited anticonvulsant effect against QA induced seizures. Additionally, i.p. administration of either flumazenil, a BZ receptor antagonist, or caffeine, an ADO receptor antagonist, did not change the anticonvulsant potency of INO i.c.v. injection, but completely abolished the DZ and ADO anticonvulsant effects, respectively. In conclusion, this study demonstrated that INO exert anticonvulsant effect against hyperactivity of the glutamatergic system independently of BZ or ADO receptors activation.

The effects of JM-20 on the glutamatergic system in synaptic vesicles, synaptosomes and neural cells cultured from rat brain

JM-20 (3-ethoxycarbonyl-2-methyl-4-(2-nitrophenyl)-4,11-dihydro-1H-pyrido[2,3-b][1,5]benzodiazepine) is a novel benzodiazepine dihydropyridine hybrid molecule, which has been shown to be a neuroprotective agent in brain disorders involving glutamate receptors. However, the effect of JM-20 on the functionality of the glutamatergic system has not been investigated. In this study, by using different in vitro preparations, we investigated the effects of JM-20 on (i) rat brain synaptic vesicles (L-[(3)H]-glutamate uptake, proton gradient built-up and bafilomycin-sensitive H(+)-ATPase activity), (ii) rat brain synaptosomes (glutamate release) and (iii) primary cultures of rat cortical neurons, astrocytes and astrocyte-neuron co-cultures (L-[(3)H]-glutamate uptake and glutamate release). We observed here that JM-20 impairs H(+)-ATPase activity and consequently reduces vesicular glutamate uptake. This molecule also inhibits glutamate release from brain synaptosomes and markedly increases glutamate uptake in astrocytes alone, and co-cultured neurons and astrocytes. The impairment of vesicular glutamate uptake by inhibition of the H(+)-ATPase caused by JM-20 could decrease the amount of the transmitter stored in synaptic vesicles, increase the cytosolic levels of glutamate, and will thus down-regulate neurotransmitter release. Together, these results contribute to explain the anti-excitotoxic effect of JM-20 and its strong neuroprotective effect observed in different in vitro and in vivo models of brain ischemia.

Original mechanisms of antipsychotic action by the indole alkaloid alstonine (Picralima nitida)

Alstonine is the major component of plant based remedies that traditional psychiatrists use in Nigeria. Alstonine is an indole alkaloid that has an antipsychotic experimental profile comparable with that of clozapine and is compatible with the alleged effects in mental patients. Representing a desirable innovation in the pharmacodynamics of antipsychotic medications, the evidence indicates that alstonine does not bind to D2 dopamine receptors (D2R) and differentially regulates dopamine in the cortical and limbic areas. The purpose of this study was to further investigate the effects of alstonine on D2R binding in specific brain regions using quantitative autoradiography (QAR) and its effects on dopamine (DA) uptake in mouse striatal synaptosomes. The effects of alstonine on D2R binding were determined in the nucleus accumbens and caudate-putamen using QAR in mice treated with alstonine doses that have antipsychotic effects. The effects of alstonine [3H]DA uptake were assessed in synaptosomes prepared from striatal tissue obtained from mice treated acutely or for 7 days with alstonine. Alstonine did not change the D2R binding densities in the studied regions. DA uptake was increased after acute (but not after 7 days) treatment with alstonine. Consistent with the alstonine behavioral profile, these results indicate that alstonine indirectly modulates DA receptors, specifically by modulating DA uptake. This unique mechanism for DA transmission modulation contributes to the antipsychotic-like effects of alstonine and is compatible with its behavioral profile in mice and alleged effects in patients. These results may represent an innovation in the antipsychotic development field.

Impairment of GABAergic system contributes to epileptogenesis in glutaric acidemia type I

Glutaric acidemia type I (GA‐I) is an inherited neurometabolic disorder caused by deficiency of glutaryl‐CoA dehydrogenase (GCDH) and characterized by increased levels of glutaric, 3‐OH‐glutaric, and glutaconic acids in the brain parenchyma. The increment of these organic acids inhibits glutamate decarboxylase (GAD) and consequently lowers the γ‐aminobutyric acid (GABA) synthesis. Untreated patients exhibit severe neurologic deficits during development, including epilepsy, especially following an acute encephalopathy outbreak. In this work, we evaluated the role of the GABAergic system on epileptogenesis in GA‐I using the Gcdh−/− mice exposed to a high lysine diet (Gcdh−/−‐Lys).