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Glial connexins (Cxs) make an extensively interconnected functional syncytium created by a network of gap junctions between astrocytes and oligodendrocytes. Among Cxs expressed in the brain, Cx30 is expressed in grey matter astrocytes, as shown at the protein level by immunoistochemistry. In the present study we aimed to perform a detailed study of the regional distribution of Cx30 mRNA in the adult and postnatal developing rat brain, analyzing its expression by in situ hybridization, and determining its cell type localization by double labeling. Recently, it has been suggested that neuronal activity may control the level of intercellular communication between astrocytes through gap junctions channels. Thus, a second aim of the present study was to investigate the short-term effects of kainate-induced seizures on Cx30 expression. The results showed that, in basal condition, Cx30 was expressed only in grey matter astrocytes with distinct regional patterns in developing and adult brain. Kainate treatment induced strong and region-specific changes of astroglial Cx30 mRNA levels and expression of Cx30 mRNA in neuronal cells undergoing cell death, suggesting a direct or indirect involvement of this connexin in the neuronal apoptotic process.

We have identified a novel gap junction gene by searching the human genome sequence database that encodes a protein designated as connexin31.9 (Cx31.9). Cx31.9 was most homologous to human Cx32.4 and did not cluster with either the purported alpha- or beta-connexin subfamilies. Expression of Cx31.9 was detected by RT-PCR in human mRNA from several tissues including cerebral cortex, heart, liver, lung, kidney, spleen, and testis. A partial Cx31.9 sequence was also represented in the human Expressed Sequence Tag database. Cx31.9 formed intercellular channels in both paired Xenopus oocytes and transfected neuroblastoma N2A cells that were distinguished by an apparent low unitary conductance (12-15 pS) and a remarkable insensitivity to transjunctional voltage. In contrast, Cx31.9 channels were gated by cytoplasmic acidification or exposure to halothane like other connexins. Cx31.9 was able to form heterotypic channels with the highly voltage-sensitive Xenopus Cx38 (XenCx38), which provides an opportunity to study gating in heterotypic channels formed by hemichannels (connexons) composed of connexins with widely divergent properties. Thus Cx31.9 is a novel human connexin that forms channels with unique functional properties.

Application of a soft multivariate statistical procedure, called PLS, partial least squares modelling in latent variables or projections to latent structures, allows extensive exploitation of the enormous amount of information embedded in the National Cancer Institute gene expression and antitumour screen databases. Interpretation of the statistical results provides new significant biological insights such as classification of human tumour cell lines based on their gene expression patterns, evaluation of the influence of gene transcripts on drug efficacy and assessment of their selectivity for classes of compounds which act by the same mechanism, and identification of uncharacterized gene expression targets involved in cancer chemotherapy. Among them, the transcripts GC11121, GC17689, and GC18564 (unknown gene products extremely selective for RNA/DNA antimetabolites) are indicated by the present work as deserving high priority in future molecular studies.

GFAPbeta mRNA is an alternative transcript of the glial fibrillary acidic protein (GFAP) gene, whose transcriptional start site is located 169 nucleotides upstream to the classical GFAPalpha mRNA. By an RT-PCR method with primers on separate exons, we were able to confirm the presence of GFAP transcripts with a longer 5' untranslated region in all the examined areas of rat brain and in primary cultures of astroglial cells. Northern blot analysis, using an oligoprobe specific for the 5' region of GFAPbeta, revealed a single hybridization band of 2.9 kb in all the brain regions examined and in primary cultures of astroglial cells. The availability of the quantitative Northern blot assay allowed further studies on the regulation of GFAPbeta expression in vivo. Since it is well-known that neuronal brain injury is one of the most powerful inducers of GFAP, we examined the expression of GFAPalpha and beta after a neurotoxic lesion in the rat hippocampus. Results obtained show a parallel increase in both GFAP transcripts with an identical time-course, suggesting that regulatory regions of the gene influence in similar way the rate of transcription at the two different start sites (alpha and beta) or that a similar post-transcriptional mechanism is involved in regulating both mRNA isoforms.

Cultured cerebellar granule cells grown in medium containing 10 mM K+ (K10) underwent apoptosis after four to five days in vitro, unless they were rescued by the addition of insulin-like growth factor-I. The few GABAergic neurons present in the cultures were more resistant to apoptotic degeneration, as indicated by double fluorescent staining with the chromatin dye Hoechst 33258 and with glutamate decarboxylase-67 antibodies. As compared with sister cultures grown in 25 mM K+, K10 cultures showed an increased expression of the Egr-1 protein and a reduced expression of the Fos protein. The increase in Egr-1 was more substantial in granule cells than in GABAergic neurons, and was not observed in K10 cultures chronically exposed to insulin-like growth factor-I. To examine the temporal relationship between the increase in Egr-1 and the development of programmed cell death, we induced apoptosis in K25 cultures at six days in vitro by replacing their medium with serum-free K10 medium. A substantial, but transient, increase in Egr- expression was observed in granule cells 6 h after switching the medium, a time that preceded the appearance of the phenotypical markers of apoptotic death. An early reduction in the Fos protein was observed after switching the medium from K25 into serum-free K10, but also after switching the medium into serum-free K25, a condition which was not associated with the development of apoptosis nor with the increase in Egr-1. We suggest that a transient induction of Egr-1 contributes to the chain of events leading to the execution phase of neuronal apoptosis in culture.

The levels of glial fibrillary acidic protein mRNA were analysed by in situ hybridization during the first 6 h in experimental models of status epilepticus in the rat. Two different models of status epilepticus were studied: one is produced by the administration of pilocarpine to lithium-treated rats and the other by the intracerebroventricular administration of kainate. Results obtained in the present study showed a very rapid (as early as 1.5 h in periventricular zones of hypothalamus, cerebral cortex, and hippocampal area) up-regulation of GFAP mRNA levels following the pharmacological induction of seizures. Several other areas showed a GFAP activation starting at 3 h such as septum, habenular nuclei, corpus callosum, and cingulum. The comparison of the results obtained in the two models of status epilepticus revealed interesting differences in some brain areas, such as cerebellum and striatum, which can be related to the specific neurotransmitter receptors and neurochemical pathways stimulated by the drugs. Interestingly, some brain areas whose neurons are strongly activated by pilocarpine and kainate (amygdala and CA3 hippocampal field) and that undergo neuronal degeneration did not show the early GFAP response. An interesting spatial feature was observed in several brain regions examined (striatum, septum, and hypothalamus): the response first appeared in the periventricular zones and then diffused to the rest of the brain area. In general GFAP responses in the periventricular zones were early and intense.

In the present investigation we address the question of whether one of the responses to increased neuronal activity is a modification of the expression of the different subunits of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-selective glutamate receptors (GluR-1, GluR-2, GluR-3). Thus, we used two different models of generalized status epilepticus, as widespread elevated neuronal activity, to study in vivo responses of the AMPA receptor mRNA expression in rat forebrain. By Northern blot analysis and in situ hybridization, we show that one of the delayed responses to LiCl/pilocarpine-induced status epilepticus is a dramatic change in the mRNA level of some subunits of AMPA-selective glutamate receptors. These effects, which appear between 6 and 12 h after the drug treatment, are subunit and brain region specific. The most striking example of differential expression of the three examined GluR mRNAs can be observed in the dentate gyrus of the hippocampus. In this specific brain subregion an increase of GluR-3 mRNA level is induced 12 h after LiCl/pilocarpine treatment, while a clear decrease in GluR-1 mRNA level and no significant change in GluR-2 mRNA level can be observed in the same area under these experimental conditions. Both the GluR-1 decrease and the GluR-3 increase are transient effects and a return to basal level can be observed after 48-72 h. In the CA1 layer of the hippocampus, a parallel decrease of both GluR-1 and GluR-3 expression is found 12-24 h after drug treatment, followed by a recovery of the expression to control values at 48 h. In kainate-induced epilepsy we could reproduce the late increase (12-24 h) in GluR-3 mRNA in the dentate gyrus; however, under this experimental condition, no clear decrease of GluR-1 expression can be observed in this area. A general decrease in mRNA level for the AMPA receptor subunits (GluR-1-3) in the hippocampal layers, in particular in CA3 and CA4 subfields, was also observed. In conclusion the results reported in the present paper reveal a specific regulation of GluR gene expression in the granule cells of the hippocampal dentate gyrus and stimulate further investigation on the functional role of the GluR-3 subunit in the receptor-channel complex.

By northern blot analysis and ribonuclease protection assay, we observed the presence of a high level of trkB mRNA in primary brain cultures devoid of neuronal cells and highly enriched in glial fibrillary acidic protein-positive astroglial cells prepared from newborn rat cerebral hemispheres, cerebral cortex, hippocampus, and striatum. In primary astroglial cultures, the more abundant trkB transcripts code for the truncated receptor without tyrosine kinase activity; probes specific for the full-length trkB mRNA did not detect any signal in northern blot analysis. By the sensitive ribonuclease protection assay, we could show the presence of trkC mRNA in cultured astrocytes, whereas no trkA mRNA was detected. We confirmed the presence of relatively high levels of nerve growth factor and neurotrophin-3 mRNA, and very low basal level of brain-derived neurotrophic factor mRNA. Moreover, we demonstrated that another member of the neurotrophin family, neurotrophin-4, is also expressed in cultured astroglial cells. In view of the fact that many functional receptors for conventional neurotransmitters or neuropeptides present on astroglial cells may act via the adenylate cyclase system, we studied also the effect of agents able to increase the intracellular cyclic AMP concentration. A sharp increase in the trkB mRNA level was observed after treatment of primary astroglial cultures with dibutyryl cyclic AMP, 8-bromo-cyclic AMP, or the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine. On the contrary, trkC mRNA levels were unaffected by treatment with cyclic AMP-elevating agents. All the neurotrophin mRNAs examined, except neutrophin-4, were increased by 3-isobutyl-1-methylxanthine treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

We have used primary neuronal cultures prepared from fetal cerebral hemispheres to investigate the effects of different glutamate receptor agonists and antagonists on the expression of transcription factor encoding genes, such as c-fos, fosB, c-jun, junB, junD, c-myc, and zif/268. The addition of glutamate (100 microM) to the culture medium rapidly activated c-fos, fosB, c-jun, junB and zif/268 gene expression, reaching the maximal level at 30-60 minutes for zif/268 and at 60 minutes for the other genes. The onset of fosB mRNA accumulation was slightly delayed in comparison to the other genes. No clear induction was found for junD and c-myc. Different glutamate receptor agonists, such as NMDA, kainate, quisqualate, trans-(+/-)-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) were able to increase c-fos, c-jun, and zif-268 mRNA levels with rapid and transient kinetics similar to those observed after glutamate treatment. Similar results were obtained for junB and fosB after kainate and quisqualate stimulation. Pretreatment with MK-801, a non competitive NMDA antagonist, produced an almost complete inhibition of glutamate-driven expression of transcription factor genes, thus suggesting that NMDA receptor plays a major role in glutamate induced-gene expression. On the contrary the kainate/AMPA receptor antagonist, DNQX, did not influence glutamate induced-gene expression. Under the conditions used in the present study, NMDA was effective in inducing the simultaneous activation of several IEGs even when added to the culture medium containing millimolar concentration of magnesium. When experiments were performed in Krebs solution, NMDA was effective in stimulating zif/268 and c-fos mRNAs only in the absence of Mg2+, while glutamate activated c-fos and zif/268 both in the presence and absence of magnesium ions. As expected, NMDA effect was fully inhibited by MK-801. The level of AP-1 DNA binding activity, as measured by electrophoretic mobility shift assay, increased after addition of glutamate and NMDA to cultured neurons and such increase was antagonized by the pretreatment with MK-801.

In the present paper we analyzed c-fos and zif/268 expression in rat primary astroglial cell cultures after treatment with Platelet-activating Factor (PAF) and its 2-O-methyl-analogue, 1-O-octadecyl-2-O-methoxy-glycero-3-phosphocholine (ET-18-OCH3). Both compounds, at a dose (2 microM) that did not produce toxic effects on astroglial cells, induced a rapid and transient increase of c-fos and zif/268 mRNA level. Pretreatment of astroglial cells with the PAF antagonist BN50730 (5 microM) 10 min prior to the addition of alkyl-phospholipids almost completely prevented the activation of the immediate early genes. On the contrary triazolam, another PAF inhibitor, did not block PAF induced gene expression when added to the medium at 5 microM concentration. ET-18-OCH3 effect on gene expression is blocked by the same antagonist (BN50730) which is effective in inhibiting PAF effect on astrocytes, suggesting that both substances act through the same binding site. Results obtained support the view that astroglial cells are a cellular target for this lipid mediator, and, like macrophages, respond to its methoxy-analogue.