Georges Tocco

BDNF mRNA expression in the developing rat brain following kainic acid-induced seizure activity

Brain-derived neurotrophic factor (BDNF) mRNA expression was studied in the hippocampus at various developmental stages in normal rats and following kainic acid (KA)-induced seizure activity. Systemic administration of KA strongly elevated BDNF mRNA levels in all hippocampal subregions after postnatal day 21. In contrast, even though KA induced intense behavioral seizure activity at postnatal day 8, the seizures were not associated with elevations of BDNF mRNA levels, indicating a clear dissociation between behavioral seizures and increases in BDNF mRNA levels and contradicting the view that BDNF mRNA expression is principally regulated by neuronal activity. In the dentate gyrus at postnatal day 13, intense BDNF mRNA expression was limited to a defined area at the border between granule cell and molecular layers, suggesting the possibility that segregation of BDNF mRNA into defined subcellular compartments may play a role in establishing the well-delineated patterns of innervation in the hippocampus.

Maturational Regulation and Regional Induction of Cyclooxygenase-2 in Rat Brain: Implications for Alzheimer's Disease

We explored the constitutive expression, maturational regulation, and relation to kainic-acid-induced apoptosis of cyclooxygenase (COX)-2 mRNA in rat brain. In adult rats, COX-2 mRNA was expressed primarily in limbic structures. Constitutive COX-2 mRNA expression increased markedly between Postnatal Day 7 (P7) and P14, reaching adult levels by P21. Despite intense KA-induced seizures, no COX-2 mRNA induction was found before P14 in any brain region examined. During response to KA-induced seizures in adult brain, COX-2 mRNA induction paralleled temporally and overlapped anatomically the appearance of cellular morphological features of apoptosis in subsets of cells of the pyramidal neuron layer of the hippocampal formation, amygdaloid complex, and pyriform cortex. While COX-2 mRNA showed KA-induced elevation in the granule cell layer of the dentate gyrus, no detectable morphological features of apoptosis were found in this region. Finally, monotypic culture of rat corticohippocampal neurons confirmed the neuronal expression of COX-2 in vitro and revealed that COX-2 is induced during response to glutamate treatment, leading to neuron death. These studies may provide novel insights into the role of COX-2 and mechanisms of action of nonsteroidal anti-inflammatory drugs in Alzheimers disease.

Regionally specific and rapid increases in brain-derived neurotrophic factor messenger RNA in the adult rat brain following seizures induced by systemic administration of kainic acid

In situ hybridization techniques were used to analyse the spatiotemporal pattern of brain-derived neurotrophic factor messenger RNA elevation associated with kainic acid-induced seizure activity in the rat. Pronounced increases in hippocampal brain-derived neurotrophic factor messenger RNA levels were observed as early as 30 min following the onset of behavioral seizures. The greatest increase (10-fold) occurred in the dentate granule cell layer, while pyramidal layers CA1, CA3, and CA4 exhibited increases of two- to six-fold. Peak elevation of brain-derived neurotrophic factor messenger RNA in CA1 hippocampal region was evident at 4 h in CA3, and in the dentate granule layer at 30 min postseizure. Elevations persisted in the dentate and hilar regions to four days, while the increases in CA1 and CA3 returned to control levels by 16 h following seizure. Significant increases in brain-derived neurotrophic factor messenger RNA were also observed in the superficial layers of cortex (II and III) and in the piriform cortex which reached peak elevations by 8 h. No detectable changes were observed in the dorsomedial thalamus. Although histologically defined pyramidal and granule cell layers displayed relatively uniform increases in brain-derived neurotrophic factor messenger RNA in response to kainate, a closer examination of the labeling patterns using emulsion autoradiography revealed discrete areas of high grain densities overlapping uniform, moderate hybridization densities in the dentate granule cell layer and CA3, suggesting that the capacity to upregulate brain-derived neurotrophic factor messenger RNA in these regions may differ among individual neurons. In conclusion, our studies revealed that brain-derived neurotrophic factor messenger RNA induction in response to systemic kainate administration differs in hippocampal and cortical areas, in magnitude, time of onset and duration. The observed temperospatial pattern does not correspond in a simple way to increases in metabolic or electrical activity associated with seizures or neuronal vulnerability coincident with the seizures.

Abasic translesion synthesis by DNA polymerase beta violates the "A-rule". Novel types of nucleotide incorporation by human DNA polymerase beta at an abasic lesion in different sequence contexts.

The “A-rule” reflects the preferred incorporation of dAMP opposite abasic lesions in Escherichia coli in vivo. DNA polymerases (pol) from procaryotic and eucaryotic organisms incorporate nucleotides opposite abasic lesions in accordance with the A-rule. However, recent in vivo data demonstrate that A is not preferentially incorporated opposite abasic lesions in eucaryotes. Purified human DNA polymerases β and α are used to measure the specificity of nucleotide incorporation at a site-directed tetrahydrofuran abasic lesion, in 8-sequence contexts, varying upstream and downstream bases adjacent to the lesion. Extension past the lesion is measured in 4 sequence contexts, varying the downstream template base. Pol α strongly favors incorporation of dAMP directly opposite the lesion. In marked contrast, pol β violates the A-rule for incorporation directly opposite the lesion. In addition to incorporation taking place directly opposite the lesion, we also analyze misalignment incorporation directed by a template base downstream from the lesion. Lesion bypass by pol β occurs predominantly by “skipping over” the lesion, by insertion of a nucleotide complementary to an adjacent downstream template site. Misalignment incorporation for pol β occurs by a novel “dNTP-stabilized” mechanism resulting in both deletion and base substitution errors. In contrast, pol α shows no propensity for this type of synthesis. The misaligned DNA structures generated during dNTP-stabilized lesion bypass do not conform to misaligned structures reported previously.

Long-term potentiation is associated with increased [3H]AMPA binding in rat hippocampus

The location and nature of the changes underlying long-term potentiation (LTP) remain controversial issues. In this study, we tested the possibility that changes in binding properties of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA/quisqualate and N-methyl-D-aspartate (NMDA) subtype of glutamate receptors are associated with LTP. LTP was elicited in vivo by stimulation of the perforant pathway in anesthetized rats. One hour following stimulation the animals were sacrificed. We performed quantitative ligand binding autoradiography on frozen brain sections using [3H]AMPA and [3H]N-(1-(2-thienyl)cyclohexyl)-3,4-piperidine ([3H]TCP) to label the AMPA/quisqualate and the NMDA receptors, respectively. No changes in [3H]TCP binding were detected in any of the treatment groups. However, increases in [3H]AMPA binding were observed only in animals that exhibited LTP. These increases were bilateral and present in several subfields of the hippocampus and cortical areas. Administration of the NMDA receptor antagonist, ketamine, prior to tetanic stimulation prevented both the increase in binding and the induction of LTP. These results suggest that changes in the characteristics of AMPA/quisqualate receptors are a biochemical correlate of LTP.

Transforming growth factor β1 and fibronectin messenger RNA in rat brain: Responses to injury and cell-type localization

Transforming growth factor-beta 1 rapidly increases in adult rat brain in response to experimental lesions. This study characterized the schedule of changes, regional distribution, and cellular localization of striatal transforming growth factor-beta 1 messenger RNA and fibronectin messenger RNA following partial striatal deafferentation by frontal cortex ablation. Frontal cortex ablation induced striatal transforming growth factor-beta 1 messenger RNA elevations that coincided temporally and overlapped anatomically with the course of degeneration of cortico-striatal afferent fibers. Within three days post-lesioning, transforming growth factor-beta 1 messenger RNA was localized at the cortical wound. By 10 days, the anatomical site of transforming growth factor-beta 1 messenger RNA expression shifted to the dorsal half of the deafferented striatum and co-localized with OX-42+ immunostained microglia-macrophage at the site of degenerating afferent terminals. Similarly, fibronectin messenger RNA also shifted from the cortical wound to the deafferented striatum by 10 days post-lesioning. Fibronectin messenger RNA was localized to glial fibrillary acidic protein+ immunostained astrocytes surrounding degenerating corticostriatal afferents. Infusion of transforming growth factor-beta 1 peptide elevated striatal and cortical fibronectin messenger RNA. These findings suggest that microglia-macrophage associated with degenerating afferent fibres can upregulate transforming growth factor-beta 1 messenger RNA and may influence fibronectin messenger RNA synthesis in reactive astrocytes. This study suggests that transforming growth factor-beta 1 has a role in controlling extracellular matrix synthesis following brain injury, which is analogous to that in peripheral wound healing.

Cycloheximide prevents kainate-induced neuronal death and c-fos expression in adult rat brain

The present study was directed at evaluating the possible involvement of protein synthesis in excitotoxin-induced neuronal damage and prolonged expression of the proto-oncogene, c-fos. Kainic acid-induced seizure activity elicited varying degrees of neuronal damage and cell loss in selectively vulnerable regions of the adult rat limbic system. Pretreatment with cycloheximide, a protein synthesis inhibitor, did not alter behavioral seizure characteristics, but markedly attenuated damage to susceptible neuronal populations. A prolonged increase in c-fos mRNA was observed byin situ hybridization up to 16 h after the onset of seizures in regions exhibiting neuronal death. Pretreatment with cycloheximide did not affect the transient induction of c-fos observed in numerous structures, but significantly reduced the prolonged expression of c-fos mRNA in kainatevulnerable regions. Despite producing massive seizure activity, systemic kainic acid administration during the early postnatal period did not induce any neuronal death, and did not result in prolonged c-fos expression in any brain structures. The developmental onset of selective neuronal vulnerability coincided with that of prolonged c-fos expression in susceptible neuronal populations. In adult rats, seizure activity induced by pentylenetetrazole did not produce neuronal damage nor did it produce prolonged c-fos expression. These results not only demonstrate that kainate-induced neurotoxicity and the prolonged expression of c-fos are both prevented by cycloheximide, but also strengthen the idea that prolonged c-fos expression is a marker of neuronal death.

Activation of immediate early genes after acute stress.

We used in-situ hybridization to study the effect of acute stress on induction of the immediate early genes (IEGs), c-fos and zif/268, in the rat brain. After one hour of restraint plus intermittent tail shock, messenger RNA (mRNA) levels for both genes were significantly increased bilaterally in the neocortex, particularly in layers IV, V and VI, and in the CA1 region of the hippocampus. This regionally-specific response suggests that IEGs may have a role in the mediation of acute stress responses in the central nervous system.

Changes in expression of neuronal and glial glutamate transporters in rat hippocampus following kainate-induced seizure activity.

The expression of excitatory amino acid transporters (EAATs) in rat hippocampus was studied following kainic acid-induced seizure activity in vivo and in hippocampal slice cultures. Protein and mRNA levels of the glial (EAAT2) and neuronal (EAAT3) transporters were determined with affinity-purified antibodies and oligonucleotide probes, respectively. Kainate treatment decreased EAAT3 immunoreactivity in stratum lacunosum moleculare within 4 h of seizure onset. Upon pyramidal cell death (5 days after kainate treatment), EAAT3 immunoreactivity in stratum pyramidale of CA1 and in stratum lacunosum moleculare was almost completely eliminated. The rapid effect of kainate on EAAT3 expression was confirmed by in situ hybridization; EAAT3 mRNA levels were decreased in CA1 and CA3 regions within 4-8 h of seizure onset. Kainate treatment had an opposite effect on levels and expression of EAAT2. Developmental studies indicated that the rapid regulation of transporter expression was not observed in rats younger than 21 days, an observation congruent with previous reports regarding the resistance of young rats to kainate. In hippocampal organotypic cultures, which lack a major excitatory input from the entorhinal cortex, kainate produced a slow decrease in [3H]d-aspartate uptake. This study indicates that an early effect of kainate treatment consists of down-regulation of the neuronal transporter EAAT3 in restricted hippocampal regions, together with a modest increase in the expression of the glial transporter EAAT2. Differential regulation of neuronal and glial glutamate transporters may thus play a role in kainate-induced seizure, neurotoxicity and neuronal plasticity.

KID-1, a Protein Kinase Induced by Depolarization in Brain

Membrane depolarization leads to changes in gene expression that modulate neuronal plasticity. Using representational difference analysis, we have identified a previously undiscovered cDNA, KID-1 (kinase induced bydepolarization), that is induced by membrane depolarization or forskolin, but not by neurotrophins or growth factors, in PC12 pheochromocytoma cells. KID-1 is an immediate early gene that shares a high degree of sequence similarity with the family of PIM-1 serine/threonine protein kinases. Recombinant KID-1 fusion protein is able to catalyze both histone phosphorylation and autophosphorylation.KID-1 mRNA is present in a number of unstimulated tissues, including brain. In response to kainic acid and electroconvulsive shock-induced seizures, KID-1 is induced in specific regions of the hippocampus and cortex.