Recent studies confirm that astrocytes and neurons are associated with the synaptic transmission, particularly with the regulation of glutamate (Glu) levels. Therefore, they have the capacity to modulate the Glu released from neurons into the extracellular space. It has also been demonstrated an intense astrocytic and microglia response to physical or chemical lesions of the central nervous system. However, the persistence of the response of the glial cells in adult brain had not been previously reported, after the excitotoxic damage caused by neonatal dosage of monosodium glutamate (MSG) to newborn rats. In this study, 4 mg/g body weight of MSG were administered to newborn rats at 1, 3, 5, and 7 days after birth, at the age of 60 days the astrocytes and the microglia cells were analyzed with immunohistochemical methods in the fronto-parietal cortex. Double labeling to glial fibrillary acidic protein (GFAP) and BrdU, or isolectin-B(4) and BrdU identified astrocytes or microglia cells that proliferated; immunoblotting and immunoreactivity to vimentin served for assess immaturity of astrocytic intermediate filaments. The results show that the neonatal administration of MSG-induced reactivity of astrocytes and microglia cells in the fronto-parietal cortex, which was characterized by hyperplasia; an increased number of astrocytes and microglia cells that proliferated, hypertrophy; increased complexity of the cytoplasm extension of both glial cells and expression of RNAm to vimentin, with the presence of vimentin-positive astrocytes. This glial response to neuroexcitotoxic stimulus of Glu on the immature brain, which persisted to adulthood, suggests that the neurotransmitter Glu could trigger neuro-degenerative illnesses.

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Monosodium glutamate (MSG) administered to neonatal rats during the first week of life induces a neurodegenerative process, which is represented by several neurochemical alterations of surviving neurons in the brain, where signalling mediated by GABA is essential for excitation threshold maintenance. GABA-positive cells, [(3)H]-GABA uptake, expression of mRNA for GABA transporters GAT-1 and GAT-3, and expression of mRNA and protein for two main GABA synthesizing enzymes, GAD(65) and GAD(67), were measured at postnatal day 60, after MSG neonatal treatment in two critical cerebral regions, cerebral cortex and hippocampus. GABA-positive cells, [(3)H]-GABA uptake, and mRNA for GAT-1, were significantly diminished in both cerebral regions. In the cerebral cortex, MSG neonatal treatment also decreased the mRNA for GAD(67) and protein for GAD(65) without significant changes in its corresponding protein and mRNA, respectively. Moreover in the hippocampus, mRNA and protein for GAD(65) were increased, whilst GAD(67) protein was elevated without significant changes in its mRNA. Clearly these results confirm the GABA cells loss after MSG neonatal treatment in both cerebral regions. As most of the GABAergic markers measured were reduced in the cerebral cortex, this region seems to be more sensitive than hippocampus, where interesting compensatory changes over GAD(65) and GAD(67) proteins were observed. However, it is possible that others neurotransmission systems are also compensating the GABA-positive cells loss in the cerebral cortex, and that elevations in two main forms of GAD in the hippocampus are not sufficient to maintain the neural excitation threshold for this region.

BackgroundGlutamate has been measured using different methods to determine its role under normal and pathological conditions. Although microdialysis coupled with HPLC is the preferred method to study glutamate, this technique exhibits poor temporal resolution and is time consuming. The concentration of glutamate in dialysis samples can be measured via glutamate oxidase using the Amplex Red method.MethodsA new device has been designed and constructed to rapidly deposit dialysis samples onto a polycarbonate plate at Cartesian coordinates (every five seconds). The samples were added to an enzymatic reaction that generates hydrogen peroxide from glutamate, which was quantified using fluorescence detection. Fluorescence emission was induced by laser excitation, stimulating each spot automatically, in addition to controlling the humidity, temperature and incubation time of the enzymatic reaction.ResultsThe measurement of standard glutamate concentrations was linear and could be performed in dialysis samples. This approach was used to determine the effect of the convulsant drugs bicuculline and 4-aminopyridine on the extracellular glutamate concentration. Seizure activity was associated with a considerable increase in glutamate that correlated with altered EEG patterns for both drugs.ConclusionsThese results indicate that this method is able to read samples with high temporal resolution, and it is easy to use compared with classical methods such as high-performance liquid chromatography, with the advantage that a large number of samples can be measured in a single experimental series. This method provides an alternative approach to determine the concentrations of neurotransmitters or other compounds that generate hydrogen peroxide as a reaction product.

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To understand better the cerebral functions, several methods have been developed to study the brain activity, they could be related with morphological, electrophysiological, molecular and neurochemical techniques. Monitoring neurotransmitter concentration is a key role to know better how the brain works during normal or pathological conditions, as well as for studying the changes in neurotransmitter concentration with the use of several drugs that could affect or reestablish the normal brain activity. Immediate response of the brain to environmental conditions is related with the release of the fast acting neurotransmission by glutamate (Glu), γ-aminobutyric acid (GABA) and acetylcholine (ACh) through the opening of ligand-operated ion channels. Neurotransmitter release is mainly determined by the classical microdialysis technique, this is generally coupled to high performance liquid chromatography (HPLC). Detection of neurotransmitters can be done by fluorescence, optical density, electrochemistry or other detection systems more sophisticated. Although the microdialysis method is the golden technique to monitor the brain neurotransmitters, it has a poor temporal resolution. Recently, with the use of biosensor the drawback of temporal resolution has been improved considerably, however other inconveniences have merged, such as stability, reproducibility and the lack of reliable biosensors mainly for GABA. The aim of this review is to show the important advances in the different ways to measure neurotransmitter concentrations; both with the use of classic techniques as well as with the novel methods and alternant approaches to improve the temporal resolution.