Arsenio Fernández-López

Transient global ischemia in rat brain promotes different NMDA receptor regulation depending on the brain structure studied.

The mRNA expression of the major subunits of N-methyl-d-aspartate receptors (NR1, NR2A and NR2B) following ischemia-reperfusion was studied in structures with different vulnerabilities to ischemic insult in the rat brain. The study was performed using quantitative real-time PCR on samples from 3-month-old male Sprague-Dawley rats after global transient forebrain ischemia followed by 48h of reperfusion. Expression of NMDA receptor subunits mRNAs decreased significantly in all structures studied in the injured animals as compared to the sham-operated ones. The hippocampal subfields (CA1, CA3 and dentate gyrus) as well as the caudate-putamen, both reported to be highly ischemic-vulnerable structures, showed outstandingly lower mRNA levels of NMDA receptor subunits than the cerebral cortex, which is considered a more ischemic-resistant structure. The ratios of the mRNA levels of the different subunits were analyzed as a measure of the NMDA receptor expression pattern for each structure studied. Hippocampal areas showed changes in NMDA receptor expression after the insult, with significant decreases in the NR2A with respect to the NR1 and NR2B subunits. Thus, the NR1:NR2A:NR2B (1:1:2) ratios observed in the sham-operated animals became (2:1:4) in insulted animals. This modified expression pattern was similar in CA1, CA3 and the dentate gyrus, in spite of the different vulnerabilities reported for these hippocampal areas. In contrast, no significant differences in the expression pattern were observed in the caudate-putamen or cerebral cortex on comparing the sham-operated animals with the ischemia-reperfused rats. Our results support the notion that the regulation of NMDA receptor gene expression is dependent on the brain structure rather than on the higher or lower vulnerability of the area studied.

Global ischemia-induced modifications in the expression of AMPA receptors and inflammation in rat brain

Alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPAR) and inflammatory processes have been related to ischemia-induced damage, but there are few studies addressing their response in different brain areas. Here we compare AMPAR expression after ischemia in several brain areas (hippocampus, cerebral cortex and caudate-putamen) in an attempt to correlate it with their different vulnerabilities. We found outstanding decreases in GluR1 and GluR2 mRNA levels after global ischemia and 48 h reperfusion (I/R) in all the areas studied, however, protein levels maintained in some areas such as CA3, suggesting different post-transcriptional control in different areas of the brain. To characterize the inflammatory response in these areas, we measured the mRNA levels of CD11b/CD18 membrane integrin (a reactive microglia marker), which showed an important but similar up-regulation in all brain areas studied, which was confirmed by immunohistochemistry. We conclude that the down-regulation of AMPAR gene expression following I/R does not explain differences in the vulnerability of different areas. Additionally, our data indicate that the level of inflammation is independent of the vulnerability of the different brain areas and does not explain differences in the AMPAR expression observed in the brain areas studied.

Effects of chronic treatment with ethanol and withdrawal of ethanol on levels of dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid in the striatum of the rat. Influence of benzodiazepines, barbiturate and somatostatin

Administration of ethanol for 40 days, at 10.53 +/- 0.25 g/kg/day did not modify levels of dopamine (DA) or 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum of the rat; however, the concentration of homovanillic acid (HVA) and the ratio of turnover were increased in a statistically significant way (P < 0.05). Twenty-four hours after withdrawal of ethanol appears as the central time of the ethanol-induced abstinence syndrome, showing noticeable decreases in levels of DA (P < 0.05) and DOPAC (P < 0.05), with respect to control and chronically ethanol-treated groups. The concentrations of DA, DOPAC and HVA and ratio of turnover values showed a tendency to return to control normal levels of 48 hr after ethanol withdrawal, although the differences still showed statistical significance (P < 0.05). The intraperitoneal injection of saline, the water soluble benzodiazepine midazolam, the barbiturate thiopental and somatostatin, in single doses, resulted in a noticeable increase in levels of DA, DOPAC and HVA and ratio of turnover values. The intraperitoneal injection of midazolam produced statistically significant decreases in levels of DOPAC and ratio of turnover values (P < 0.01) in rats 48 hr after withdrawal of ethanol, with respect to control and chronically ethanol-treated animals, in contrast to the absence of changes produced when injecting thiopental or somatostatin.

Distribution of the GABAA receptor complex β2/3 subunits in the brain of the frog Rana pipiens

This report describes the distribution of labeling of the monoclonal antibody bd-17 against the beta 2/3 subunits of the mammalian GABAA receptor complex throughout the brain of the frog Rana pipiens. The distribution matches quite closely those in homologous brain regions as previously described for this antibody in fishes, birds, and mammals, indicating that this antibody also labels beta 2/3 subunits of frog. A semiquantitative analysis of the distribution of labeling throughout the brain is based upon relative optical densities with respect to the structure showing maximal optical density in each brain, using standard illumination conditions. Comparison with distributions in birds and mammals suggests that these GABAA receptor complex subunits are strongly conserved in vertebrate evolution and play an important role in the visual, auditory, olfactory and motor systems.

Age and meloxicam attenuate the ischemia/reperfusion-induced down-regulation in the NMDA receptor genes

This study describes the effect of global brain ischemia followed by 48 h reperfusion, when delayed neuronal death can be already observed. We quantified the mRNA levels of the N-methyl-D-aspartate receptor (NMDAR) subunits and those of the astroglia (glial fibrilar acidic protein, GFAP) and microglia (CD11b) markers using real time PCR on the cerebral cortex and hippocampus of 3- and 18-month-old Sprague-Dawley rats. Data show an ischemia/reperfusion-induced decrease in the mRNA levels of the NMDAR NR1, NR2A and NR2B subunits genes, which contrasts with the increase in the CD11b and GFAP mRNA levels. These effects are attenuated in all the genes studied in 18-month-old animals, suggesting that this mechanism of response is less efficient in aged animals. Western blot assays of NR1, NR2A and NR2B show parallels with the real time PCR data, indicating that the down-regulation of these genes is controlled at the transcriptional level. We suggest that a decrease in the efficiency in the control of the NMDAR transcription could account for the higher vulnerability in aged animals, but it cannot explain by itself differences in the vulnerability to ischemia in different areas of the brain. In the assays of ischemia/reperfusion followed by a treatment with the anti-inflammatory agent meloxicam, we observed that ischemic insult was unable to elicit changes in the NMDAR transcription, thus suggesting that inflammation plays a crucial role in the transcriptional control of these genes.

The autoradiographic perspective of central benzodiazepine receptors: A short review

1. We reviewed studies performed to characterize central benzodiazepine binding sites. 2. An overview of the different radioligands used to characterize BZ1 and BZ2 binding sites and a mapping of these central benzodiazepine sites are described. 3. Saturation studies carried out by autoradiogram quantification also are reviewed. 4. The specific use of the autoradiographic technique to carry out studies on ontogeny, development, and phylogeny is discussed, as well as studies performed using this technique on some diseases and experimental conditions, such as drug treatments or chemical and mechanical lesions.

Autoradiographic localization of α2-adrenoceptors in chick brain

Abstract α 2 -Adrenoceptors were localized in the chick brain by ‘in vitro’ receptor autoradiography using [ 3 H]UK 14304 as a ligand. High or very high densities of binding sites were found in the hyperstriatum, tuberculum olfactorium, hypothalamic nuclei, tectum opticum and some medullary nuclei. Comparatively, intermediate densities were observed over the thalamic nuclei and locus ceruleus, among others. Low densities of α 2 -adrenoceptors were detected in the paleostriatum, hippocampus and cerebellum. Our data indicate that α 2 -adrenoceptors in the chick brain present similar properties and homologous anatomical distribution to those reported in mammalian brain.

Unfolded protein response to global ischemia following 48 h of reperfusion in the rat brain: the effect of age and meloxicam

The unfolded protein response (UPR) in the hippocampal regions Cornu Ammonis 1 hippocampal region, Cornu Ammonis 3 hippocampal region, and dentate gyrus, as well as in the cerebral cortex of 3‐month‐old and 18‐month‐old rats were studied in a model of 15 min of global cerebral ischemia followed by 48 h of reperfusion. UPR was measured by quantifying the protein disulfide isomerase (PDI), C/EBP‐homologous protein (CHOP), GRP78 and GRP94 transcripts using qPCR and the amounts of PDI and GRP78 by western blot. The study shows how the mRNA levels of these genes were similar in 3‐month‐old and 18‐month‐old sham‐operated animals, but the ischemic insult elicited a noticeable increase in the expression of these genes in young animals that was scarcely appreciable in older animals. The striking increase in the mRNA levels of these genes in 3‐month‐old animals was abolished or even reverted by treatment with meloxicam, an anti‐inflammatory agent. Western blot assays showed that the UPR was still detectable 48 h after ischemia in some of the studied areas, and provided evidence that the UPR is different between young and older animals. Western blot assays carried out in young animals also showed that meloxicam elicited different effects on the levels of PDI and GRP78 in the cerebral cortex and the hippocampus. We conclude that the UPR response to ischemic/reperfusion insult is age‐ and probably inflammation‐dependent and could play an important role in ischemic vulnerability. The UPR appears to be strongly decreased in aged animals, suggesting a reduced ability for cell survival.

Pre- and post-hatching developmental changes in β-adrenoceptor subtypes in chick brain

This study used [3H]CGP 12177 as a radioligand to determine the beta1 and beta2-adrenoceptor changes from the pre-hatching E17 stage, where the beta2 subtype is first detected, to the post-hatching P30 stage. While beta1-adrenoceptors were found to be present from E18 and were limited to cerebellum and hyperstriatum in all stages studied, beta2-adrenoceptors showed a wider distribution throughout the brain. In most of the structures analysed both beta1- and beta2-adrenoceptor binding values reached a maximum in the P2 stage, followed by a decrease over the following days. A second increase in both subtypes was detected again in the P15 and P30 stages. These results support the notion of a specific role for beta-adrenoceptors in neural plasticity in the first week after hatching and suggest that the beta2 subtype is the main adrenoceptor in chick brain throughout its development.

EARLY MODIFICATIONS IN N-METHYL-D-ASPARTATE RECEPTOR SUBUNIT mRNA LEVELS IN AN OXYGEN AND GLUCOSE DEPRIVATION MODEL USING RAT HIPPOCAMPAL BRAIN SLICES

Glutamatergic N-methyl-d-aspartate NMDA receptors (NMDAR) are considered to play a key role in ischemia-induced damage. Long-term (hours) changes in their expression upon ischemia have been shown. Here we report short-term changes in the mRNA levels of the major hippocampal NMDAR subunits (NR1, NR2A and NR2B), as well as c-fos, in an ex vivo ischemia model using hippocampal slices. This effect can be observed also in a calcium free incubation solution. Striking early decreases in the NMDAR subunit mRNA levels were observed after 30 min of oxygen and glucose deprivation (OGD) as well as a partial recovery when the tissues were returned to the balanced salt solution (reperfusion-like period) for 3 h. Since OGD-induced damage has been reported to be a consequence of the increase in OGD-related glutamate release, we also analyzed NMDAR mRNA levels following increased glutamate levels in hippocampal sections in which no significant effects on NMDAR subunit mRNA levels were detected. Furthermore, we describe that the presence of MK-801 (a selective NMDAR antagonist), CNQX (a selective AMPA/kainate receptor antagonist) or their combined action in the incubation solution is able to induce a significant decrease in NMDAR expression but in these conditions the OGD does not induce further decreases in mRNA levels. We suggest that the mechanisms triggered during OGD to downregulate mRNA levels of NMDAR subunits could be the same than those induced by glutamate receptor antagonists.