Beatriz Martínez-Villayandre

Neural Plasticity and Proliferation in the Generation of Antidepressant Effects: Hippocampal Implication

It is widely accepted that changes underlying depression and antidepressant-like effects involve not only alterations in the levels of neurotransmitters as monoamines and their receptors in the brain, but also structural and functional changes far beyond. During the last two decades, emerging theories are providing new explanations about the neurobiology of depression and the mechanism of action of antidepressant strategies based on cellular changes at the CNS level. The neurotrophic/plasticity hypothesis of depression, proposed more than a decade ago, is now supported by multiple basic and clinical studies focused on the role of intracellular-signalling cascades that govern neural proliferation and plasticity. Herein, we review the state-of-the-art of the changes in these signalling pathways which appear to underlie both depressive disorders and antidepressant actions. We will especially focus on the hippocampal cellularity and plasticity modulation by serotonin, trophic factors as brain-derived neurotrophic factor (BDNF), and vascular endothelial growth factor (VEGF) through intracellular signalling pathways—cAMP, Wnt/β-catenin, and mTOR. Connecting the classic monoaminergic hypothesis with proliferation/neuroplasticity-related evidence is an appealing and comprehensive attempt for improving our knowledge about the neurobiological events leading to depression and associated to antidepressant therapies.

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.

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.

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.

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.

AMPA receptor downregulation induced by ischaemia/reperfusion is attenuated by age and blocked by meloxicam

S. Montori, S. Dos_Anjos, M. A. Ríos‐Granja, C. C. Pérez‐García, A. Fernández‐López and B. Martínez‐Villayandre (2010) Neuropathology and Applied Neurobiology36, 436–447
AMPA receptor downregulation induced by ischaemia/reperfusion is attenuated by age and blocked by meloxicam

Age-dependent modifications in the mRNA levels of the rat excitatory amino acid transporters (EAATs) at 48 hour reperfusion following global ischemia

This study reports the mRNA levels of some excitatory amino acid transporters (EAATs) in response to ischemia-reperfusion (I/R) in rat hippocampus and cerebral cortex. The study was performed in 3-month-old and 18-month-old animals to analyze the possible role of age in the I/R response of these transporters. The I/R resulted in a reduced transcription of both the neuronal EAAC1 (excitatory amino acid carrier-1) and the neuronal and glial GLT-1 (glial glutamate transporter 1), while the glial GLAST1a (l-glutamate/l-aspartate transporter 1a) transcription increased following I/R. The changes observed were more striking in 3-month-old animals than in 18-month-old animals. We hypothesize that increases in the GLAST1a mRNA levels following I/R insult can be explained by increases in glial cells, while the GLT-1 response to I/R mirrors neuronal changes. GLAST1a transcription increases in 3-month-old animals support the hypothesis that this transporter would be the main mechanism for extracellular glutamate clearance after I/R. Decreases in EAAC1 and GLT-1 mRNA levels would represent either neuronal changes due to the delayed neuronal death or a putative protective down-regulation of these transporters to decrease the amount of glutamate inside the neurons, which would decrease their glutamate release. This study also reports how the treatment with the anti-inflammatory agent meloxicam attenuates the transcriptional response to I/R in 3-month-old rats and decreases the survival of the I/R-injured animals.

Quantitative gene expression analysis in a brain slice model: Influence of temperature and incubation media

We describe the RNA integrity (28S/18S ratio) and the messenger RNA (mRNA) expression of genes encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH), microtubule-associated serine/threonine kinase 2 (Mast2), and beta-actin in cortical brain slices incubated for up to 24h in Ringers solution and Dulbeccos modified Eagles medium (DMEM) at 25 and 37 degrees C. Our data reveal an optimal temporal working window between 1 and 6h when slices are incubated in Ringers solution at 25 degrees C that allows experiments related to gene expression dynamics to be performed more suitably than those carried out at 37 degrees C. In addition, we show that reference gene expression may be modified in dynamic experiments and may compromise studies of gene expression.

Effect of δ-aminolevulinic acid and vitamin E treatments on the N-methyl-d-aspartate receptor at different ages in the striatum of rat brain

We report the effects of the chronic treatments with the oxidant agent delta-aminolevulinic acid (ALA) and with the antioxidant vitamin E on the N-methyl-D-aspartate (NMDA) receptors in the striatum of 4-, 12- and 24-month-old male Wistar rats. ALA and vitamin E were administered daily for 15 days (40 mg/kg i.p. and 20 mg/kg i.p. respectively). NMDA receptors were labeled by membrane homogenate binding, using tritiated dizocilpine ([3H]MK-801). [3H]MK-801 binding in the striatum was significantly decreased at all ages in ALA-treated rats with respect to their controls, and in contrast, was significantly increased at all ages when rats received the treatment with vitamin E. Western blot assays were performed using antibodies against the NR2A subunit, a NMDA receptor subunit widely distributed in the brain. We did not find significant differences in the amounts of NR2A in rats treated with either ALA or vitamin E with respect to those rats not treated. We conclude that the NMDA receptor densities in the rat striatum are modified by the chronic treatment with oxidants and antioxidants in an age-independent way, at least until 24 months. Also, our results support the notion that NR2A is not involved in these modifications.