Polymorphisms of steroid 5-alpha-reductase type I (SRD5A1) gene are associated to peripheral arterial disease.

Although animal studies support the hypothesis that androgenic biological actions may affect experimental atherosclerosis progression, evidence for a relationship between androgen effects and peripheral arterial disease (PAD), a common clinical form of atherosclerosis, is weak or contradictory. Testosterone, the main androgen hormone, is converted in a 5alpha-reduced form by enzymatic activities in the target cells and some specific actions are mediated by such metabolites. Steroid 5-alpha reductase isoenzymes (SRD5A1 and SRD5A2) catalyze the conversion to the bioactive potent androgen dihydrotestosterone and other reduced metabolites and represent relevant regulators of local hormonal actions. In the present study we tested for the association of selected single nucleotide polymorphisms (SNP) of SRD5A1 and SRD5A2 with symptomatic PAD patients. Two different SNP in the SRD5A1 were significantly associated which the PAD phenotype (p<0.03, odds ratio 1.73), while no association was found between PAD phenotypes and SRD5A2. Since the examined SRDA1 gene variant was previously associated with a low enzymatic activity, we suggest that a decreased local enzymatic conversion of testosterone may contribute to PAD genetic susceptibility.

Functional properties of channels formed by the neuronal gap junction protein connexin36.

The expression and functional properties of connexin36 (Cx36) were examined in two communication-deficient cell lines (N2A-neuroblastoma and PC-12 cells) transfected with Cx36 and in hippocampal neurons that express the connexin endogenously. Transfected cells expressed the expected 2.9 kb Cx36 transcript and Cx36 immunoreactivity, whereas nontransfected cells were devoid of Cx36. The relationship between steady-state junctional conductance (g(j)) and transjunctional voltage was well described by a two-state Boltzmann equation. The half-inactivation voltage (V(0)), the ratio of minimal to maximal g(j) (g(min)/g(max)), and the equivalent gating charge were +/- 75 mV, 0.55, and 1.75, respectively, indicating that Cx36 exhibits very low voltage sensitivity. Conductance of single Cx36 channels measured with patch pipettes containing 130 mM CsCl was 10-15 pS (n = 15 cell pairs); despite this low unitary conductance, Cx36 channels were permeable to the dye Lucifer yellow. Hippocampal neurons expressed Cx36 both in vivo and in culture. The electrophysiological properties of channels in cultured hippocampal neurons were similar to those of the channels expressed by the transfected cell lines, and the neuronal channels were similarly permeable to Lucifer yellow. The unique combination of weak voltage sensitivity, small unitary conductance, and permeation by anions as large as second messenger molecules endows Cx36 gap junction channels with properties well suited for mediating flexible electrical and biochemical interactions between neurons.

Cx36 preferentially connects beta-cells within pancreatic islets.

Previous studies have provided evidence for the transcripts of Cx43 and Cx45 within pancreatic islets. As of yet, however, it has proven difficult to unambiguously demonstrate the expression of these proteins by islet cells. We have investigated whether Cx36, a new connexin species recently identified in mammalian brain and retina, may also be expressed in pancreatic islets. Using probes that permitted the original identification of Cx36 in the central nervous system, we show that a transcript for Cx36 is clearly detectable in rat pancreatic islets. Using novel and affinity-purified polyclonal antibodies, we have found that Cx36 is actually expressed in pancreatic islets. Both in situ hybridization and immunolabeling indicated that this connexin is abundant in the centrally located insulin-producing beta-cells and is expressed much less, if at all, by the other endocrine cell types. This differential expression was further confirmed on fluorescence-activated cell sorter-purified preparations enriched in either beta- or non-beta-cells. The finding of a differential distribution of Cx36 within distinct regions of pancreatic islets creates the possibility that this connexin may provide the establishment of selective pathways of communication between the different types of endocrine cells comprising the pancreatic islet.

Activation of metabotropic glutamate receptors coupled to inositol phospholipid hydrolysis amplifies NMDA-induced neuronal degeneration in cultured cortical cells.

We have studied the influence of class I metabotropic glutamate receptors (mGluRs) on excitotoxic neuronal degeneration in cultured murine cortical neurons grown on a monolayer of astrocytes. These cultures expressed high levels of mGluR5 mRNA, which were comparable to those found in RNA extracts from cerebral cortex. Cortical neurons in mixed cultures were heavily stained with antibodies raised against mGluR5 and were also stained--albeit to a much lower extent--with mGluR1a but not with mGluR1b or c antibodies. Preferential agonists of class I mGluRs, such as quisqualate, 3,5-dihydroxyphenylglycine (DHPG), and trans-azetidine-2,4-dicarboxylic acid (t-ADA), as well as the mixed mGluR agonist, 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) all stimulated PPI hydrolysis in cultured cortical cells. The potency of N-methyl-D-aspartate (NMDA) in inducing neuronal degeneration was substantially enhanced when the drug was coincubated with quisqualate, DHPG or t-ADA during a 10-min pulse (paradigm of "fast" toxicity). None of the mGluR agonists influenced neuronal viability by itself. The amplification of NMDA toxicity by quisqualate or DHPG was attenuated by a series of protein kinase C (PKC) inhibitors, suggesting that class I mGluRs operate, at least in part, through activation of PKC. Quisqualate and, in particular, DHPG enhanced excitoxic neuronal degeneration even when applied after the toxic pulse with NMDA. This action is likely to occur early in the maturation of excitotoxic damage, because the functional activity of class I mGluRs was substantially reduced at 2 or 3 hr after the NMDA pulse. These results suggest that activation of class I mGluRs enhances NMDA-receptor mediated neuronal toxicity and encourage the search for selective antagonists for the experimental therapy of acute or chronic neurodegenerative diseases.

An enhanced expression of the immediate early gene, Egr-1, is associated with neuronal apoptosis in culture.

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.

Expression of metabotropic glutamate receptors in the rat and human testis.

The G protein-coupled receptor kinase type 4 mediates the homologous desensitisation of type-1 metabotropic glutamate (mGlu1) receptors and is predominantly expressed in the testis. Hence, we searched for the expression of mGlu1 or other mGlu receptor subtypes in rat and human testes. RT-PCR analysis showed the presence of mGlu1, -4 and -5 (but not -2 or -3) receptor mRNA in the rat testis. The presence of mGlu1 and -5 (but not mGlu2/3) receptor proteins was also demonstrated by Western blot analysis. In the rat testis, both mGlu1a and -5 receptors were highly expressed in cells of the germinal line. It is likely that these receptors are functional, because the agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid, was able to stimulate inositol phospholipid hydrolysis in slices prepared from rat testes. Immunocytochemical analysis of bioptic samples from human testes showed a high expression of mGlu5 receptors inside the seminiferous tubuli, whereas mGlu1a immunoreactivity was restricted to intertubular spaces. mGlu5 receptors were also present in mature spermatozoa, where they were localised in the mid-piece and tail. This localisation coincided with that of beta-arrestin, a protein that is critically involved in the homologous desensitisation and internalisation of G protein-coupled receptors. Taken collectively, these results offer the first evidence for the expression of any glutamate receptor in testes, and suggest that at least mGlu5 receptors are present and functionally active in mature human sperm.

Identification and functional expression of HCx31.9, a novel gap junction gene.

By combining in silico and bench molecular biology methods we have identified a novel human gap junction gene that encodes a protein designated HCx31.9. We have determined its human chromosomal location and gene structure, and we have identified a putative mouse ortholog, mCx30.2. We have observed the presence of HCx31.9 in human cerebral cortex, liver, heart, spleen, lung, and kidney and the presence of mCx30.2 in mouse cerebral cortex, liver and lung. Moreover, preliminary data on the electrophysiological properties of HCx31.9 have been obtained by functional expression in paired Xenopus oocytes and in transfected N2A cells.

Cx36 and the function of endocrine pancreas.

The secretory, duct, connective and vascular cells of pancreas are connected by gap junctions, made of different connexins. The insulin-producing beta-cells, which form the bulk of endocrine pancreatic islets, express predominantly Cx36. To assess the function of this connexin, we have first studied its expression in rats, during sequential changes of pancreatic function which were induced by the implantation of a secreting insulinoma. We observed that changes in beta-cell function were paralleled by changes in Cx36 expression. We have also begun to investigate mutant mice lacking Cx36. The absence of this protein did not affect the development and differentiation of beta-cells but appeared to alter their secretion. We have studied this effect in MIN6 cells which spontaneously express Cx36. After stable transfection of a construct that markedly reduced the expression of this connexin, we observed that MIN6 cells were no more able to secrete insulin, in contrast to wild type controls, and differentially displayed a series of still unknown genes. The data provide evidence that Cx36-dependent signaling contributes to regulate the function of native and tumoral insulin-producing cells.

Shortcuts in genome-scale cancer pharmacology research from multivariate analysis of the National Cancer Institute gene expression database.

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.

Seizures increase trkC mRNA expression in the dentate gyrus of rat hippocampus. Role of glutamate receptor activation.

In this study we have shown, by in situ hybridization and RNase protection assay, a significant trkC mRNA increase confined to the dentate gyrus of hippocampus, both after seizures induced by intracerebroventricular injection of kainic acid and bicuculline. Moreover, after bicuculline treatment we observed an earlier increase of trkC mRNA level, which peaked after 3 h and returned back to normal levels by 12 h. In contrast, the kainic acid treatment produced a delayed increase of trkC mRNA, which initiated after 6 h, peaked at 12 h, and returned to normal levels at 24 h. This increase, which involves also trkC mRNA receptor with tyrosine kinase activity, was mediated by non-NMDA receptors and counteracted by GABA potentiating agent diazepam. Using embryonic neuronal cultures from cerebral hemispheres, including hippocampus, we found that glutamate receptor agonists, including glutamate, kainate, NMDA, and t-ACPD, increase trkC mRNA levels with the following rank order of efficacy: NMDA > t-ACPD > kainic acid > glutamate. In conclusion, our data show that trkC mRNA expression in granule cells of the hippocampus dentate gyrus is increased during seizure activity and that it is mediated by non-NMDA receptors.

Neurotrophins and their trk receptors in cultured cells of the glial lineage and in white matter of the central nervous system.

Previous studies have analyzed the expression of different members of the neurotrophin family and their trk receptors in glial cultures composed mainly or exclusively of type-1 astrocytes, whereas only partial data have been published on other cultured glial types. In this article we compare the mRNA levels for neurotrophins (NGF, BDNF, NT-3, NT-4) and their high-affinity receptors (trkA, trkB, trkC) in cultures enriched in specific glial types, such as microglia, type-1 astroglia, and cells of the O/2A lineage (type-2 astroglia and oligodendroglia). Relatively high levels of NGF mRNA (comparable to those observed in adult rat cerebral cortex) are present in all types of cultured glial cells, except for a low level of expression in cultures enriched in microglial cells. In contrast, BDNF mRNA is undetectable in all cultures examined. NT-3 and NT-4 mRNA molecules, at a level equal to that observed in adult rat cerebral cortex, are easily detected in type-1 astrocyte cultures, whereas their hybridization signals are undetectable in cells of the O/2A lineage and in microglial cultures. The analysis of neurotrophin receptor mRNAs confirms the absence of trkA mRNA, the presence of relatively high levels of trkB mRNA (70-100% of cerebral cortex values), and low levels of trkC mRNA (10-18% of cerebral cortex values) in both cultured astroglial and oligodendroglial cells. Only very low levels of trkB and trkC mRNAs are observed in microglial cultures. Although cultured glial cells express mainly mRNAs encoding for the truncated form of trkB and trkC, a low level of mRNA encoding for the full-length catalytic form of these receptors is detected by the sensitive ribonuclease protection assay.

Inducible and constitutive transcription factor NF-kappa B-like DNA binding activities in rat brain cells cultured in vitro.

Description of constitutive and inducible transcription factors in brain cells is a prerequisite for understanding phenomena of long-term neuronal plasticity. In this report we report that basal levels of NF-kappa B-like transcription factor DNA binding activity (measured by electrophoretic mobility shift assay) are observed in two kinds of cultured rat brain cells, namely neurons of fetal cerebral hemispheres and astroglia derived from newborn brains. In the latter culture, phorbol ester induces additional forms of DNA binding activity, whereas L-glutamate fails to do so. In neurons, neither treatment is effective in inducing NF-kappa B-like DNA binding activity over the basal level. These results are in contrast to the fact that L-glutamate in both neurons and glia elevates DNA binding activity of AP-1, another transcription factor. These data, besides describing behavior of NF-kappa B-like DNA binding activities, also provide some evidence that L-glutamate exerts its modulatory functions on neurons and glia through specific transcription factors like AP-1.

NMDA receptor-dependent and -independent immediate early gene expression induced by focal mechanical brain injury.

In the present study we analysed, by in situ hybridization, the effects of an extremely localized mechanical brain injury, obtained by the simple needle insertion (30 g) in rat hippocampus or cortex, on the expression of several immediate early genes (c-fos, fosB, c-jun, junB, junD, zif/268). When the needle is deepened into the hippocampus through the cortex, a simultaneous ipsilateral activation of all examined IEGs is observed in both the cerebral cortex and in the dentate gyrus of hippocampus. Maximal effects are detected between 30 and 60 min with the following rank order of induction: zif/268 > c-fos- > junB > fosB > c-jun > junD. On the other hand, when the penetration of the needle is limited to the cerebral cortex the activation of the IEGs (c-fos, fosB, junB and zif/268) spreads throughout the ipsilateral cortex but does not involve the hippocampal region. Systemic administration of ketamine, a non-competitive antagonist of N-methyl-D-aspartate (NMDA) receptors, blocks IEG expression induced by brain injury in the cerebral cortex and in the hippocampal dentate gyrus. Pretreatment with the anticonvulsant diazepam, the anaesthetic urethane, or the muscarinic receptor antagonist scopolamine do not affect the injury-induced genomic response. An important regional difference in the sensitivity to the blocking effect of ketamine can be observed analysing the results regarding the zif/268 gene expression in the hippocampus. A clear induction of this gene by needle insertion can be detected both in the dentate gyrus and in the hippocampal layers. However, the dentate gyrus induction is completely blocked by the ketamine pretreatment, while the induction in the hippocampal layers is not affected by this NMDA antagonist. The zif/268 induction in the hippocampal layers is not blocked even if the intracerebroventricular administration of a non-NMDA glutamate receptor antagonist is associated to the systemic pretreatment with ketamine. This result represents the first observation of injury-induced neuronal genomic responses that are not critically dependent on the NMDA receptor activity.

Platelet-activating factor and its methoxy-analogue ET-18-OCH3 stimulate immediate early gene expression in rat astroglial cultures.

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.

Changes in gene expression of AMPA-selective glutamate receptor subunits induced by status epilepticus in rat brain.

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.

Differential regulation of BDNF and NT-3 mRNA levels in primary cultures of rat cerebellar neurons.

Reciprocal developmental patterns of expression for BDNF and NT-3 have been observed in several neuronal types, including cerebellar granule neurons: NT3 mRNA level decreased and BDNF mRNA increased in granule cells concomitantly with their migration and maturation. In the present study we analysed cultured cerebellar granule neurons prepared from postnatal rat cerebellum, a model system widely used for studies on the maturation and survival of these neurons. We show that chronic depolarization, induced by 25 mM K+ in the culture medium, is able to sustain a persistent increase of BDNF expression in cerebellar granule neurons. It has been suggested that chronic depolarization in vitro mimics the effect of the earliest afferent inputs received by granule cells in vivo: on this basis we suggest that the beginning of neuronal activity in differentiated granule neurons may represent one of the signals that trigger the developmental increase in BDNF expression. Interestingly, we observed that up-regulation of BDNF expression in vitro is accompanied by a dramatic decrease of NT-3 expression: a differential regulation that is highly reminiscent of the reciprocal developmental patterns of expression observed in vivo for BDNF and NT-3. Another point raised by the present results is the possible role of BDNF, acting in an autocrine or paracrine manner, in the trophic effect of high potassium concentration. Indeed, repeated additions of BDNF to the culture medium have a trophic effect on cerebellar granule neurons but reproduce only partially the survival effect observed with 25 mM K+ conditions, suggesting that the increased expression of BDNF is not the only mechanism responsible for the trophic effects of high potassium. In conclusion we show the existence of a reciprocal regulation of BDNF and NT-3 expression in cultured cerebellar granule neurons and we propose that this culture system could represent an in vitro model for the study of the molecular mechanisms underlying the developmental regulation of these neurotrophins in cerebellum.

Comparative effects of estrogens and prolactin on nigral and striatal GAD activity.

The comparative effects of a 10 day estrogen treatment and estrogen independent hyperprolactinemia on nigral and striatal glutamic acid decarboxylase (GAD, EC 4.1.1.15) activity were investigated in male rats. Data obtained show that estrogen treatment decreases GAD activity in substantia nigra, while an increase was observed in conditions of hyperprolactinemia induced by adenohypophysis homograft or acute and chronic sulpiride injection. The possibility of an opposite modulation of strio-nigral GABAergic system by estrogens and prolactin is suggested.

Expression of connexin36 in the adult and developing rat brain.

The distribution of connexin36 (Cx36) in the adult rat brain and retina has been analysed at the protein (immunofluorescence) and mRNA (in situ hybridization) level. Cx36 immunoreactivity, consisting primarily of round or elongated puncta, is highly enriched in specific brain regions (inferior olive and the olfactory bulb), in the retina, in the anterior pituitary and in the pineal gland, in agreement with the high levels of Cx36 mRNA in the same regions. A lower density of immunoreactive puncta can be observed in several brain regions, where only scattered subpopulations of cells express Cx36 mRNA. By combining in situ hybridization for Cx36 mRNA with immunohistochemistry for a general neuronal marker (NeuN), we found that neuronal cells are responsible for the expression of Cx36 mRNA in inferior olive, cerebellum, striatum, hippocampus and cerebral cortex. Cx36 mRNA was also demonstrated in parvalbumin-containing GABAergic interneurons of cerebral cortex, striatum, hippocampus and cerebellar cortex. Analysis of developing brain further revealed that Cx36 reaches a peak of expression in the first two weeks of postnatal life, and decreases sharply during the third week. Moreover, in these early stages of postnatal development Cx36 is detectable in neuronal populations that are devoid of Cx36 mRNA at the adult stage. The developmental changes of Cx36 expression suggest a participation of this connexin in the extensive interneuronal coupling which takes place in several regions of the early postnatal brain.

Characterization of metabotropic glutamate receptors negatively linked to adenylyl cyclase in brain slices.

We have characterized the pharmacological profile of activation of metabotropic glutamate receptors negatively linked to adenylyl cyclase (mGluR decreases cAMP) in brain slices. Among the putative mGluR agonists, (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), were the most potent inhibitors of forskolin-stimulated cAMP formation in hippocampal slices, followed by ibotenate, L-2-amino-3-phosphonopropionate (AP3), quisqualate, L-glutamate and beta-N-methylamino-L-alanine (BMAA). Inhibition of forskolin-stimulated cAMP formation by DL-2-amino-4-phosphonobutanoate (AP4) was biphasic, suggesting that the drug interacts with more than one mGluR decreases cAMP subtype. Both L-AP4 and L-serine-O-phosphate (a restricted analogue of AP4) were much more effective in inhibiting forskolin-stimulated cAMP formation than their D-isomers, indicating that interaction of these drugs with the mGluR decreases cAMP is stereoselective. Despite the fact that DCG-IV and ibotenate behave as NMDA receptor agonists, their effect was insensitive to MK-801. The regional pattern of expression of mGluR decreases cAMPS, as estimated by using 1S,3R-ACPD as an agonist, did not correlate with the steady-state levels of mGluR2 mRNA. Thus, 1S,3R-ACPD inhibited forskolin-stimulated cAMP in slices from hippocampus, cerebral cortex, corpus striatum, olfactory tubercle or hypothalamus, but not in slices from olfactory bulb or cerebellum; in contrast, mGluR2 mRNA levels were high in the olfactory bulb and very low in the corpus striatum. 1S,3R-ACPD also inhibited forskolin-stimulated cAMP formation in cortical membranes, excluding the involvement of trans-synaptic mechanisms in the activity of mGluR decreases cAMPS.(ABSTRACT TRUNCATED AT 250 WORDS)

GFAP gene methylation in different neural cell types from rat brain.

It is generally believed that specific demethylation processes take place in the promoter of tissue-specific genes during development. It has been suggested that hypomethylation of the -1500/-1100 domain of the 5' flanking regulatory region of the rat glial fibrillary acidic protein gene may be specific for neuroectodermal derivatives such as neurons and astrocytes. In the present work the methylation status of one of those seven CG sites (the -1176) of the 'neuroectoderm-specific domain' was analyzed. In agreement with the neuroectoderm hypothesis, the -1176 site is highly demethylated in astroglial, oligodendroglial and neuronal cells, but heavily methylated in microglial and fibroblast cells. The three different glial population are derived from the same tissue (cerebral hemispheres of newborn rats) but have a different embryological origin: oligodendrocytes and astrocytes originate from neuroectoderm, while microglia is of mesodermal origin. It is not clear if GFAP-negative neuronal cells maintain such demethylation in the advanced stage of maturation or if they undergo a second phase of de novo methylation. In order to clarify this point we used a subcellular fractionation method which allowed us to separate two different nuclear populations from adult rat cerebral hemispheres: one enriched in neuronal nuclei (called N1) and the other enriched in glial nuclei (N2). A higher methylation level of the -1176 site was detected in the N1 fraction, suggesting the GFAP gene undergo a de novo methylation process during neuronal maturation. This observation is in agreement with recent results showing a de novo methylation of the -1176 site during postnatal brain development. We hypothesize that a DNA demethylation process takes place in neuroectodermal precursor cells and that the -1176 site persists demethylated at the earlier stages of neuronal differentiation (immature neurons) and becomes fully methylated at more advanced stages of differentiation.