Existence of muscarinic acetylcholine receptor (mAChR) and fibroblast growth factor receptor (FGFR) heteroreceptor complexes and their enhancement of neurite outgrowth in neural hippocampal cultures.

BACKGROUND: Recently, it was demonstrated that G-protein-coupled receptors (GPCRs) can transactivate tyrosine kinase receptors in absence of their ligands. In this work, driven by the observation that mAChRs and fibroblast growth factor receptors (FGFRs) share signalling pathways and regulation of brain functions, it was decided to explore whether mAChRs activation may transactivate FGFRs and, if so, to characterize the related trophic effects in cultured hippocampal neurons. METHODS: Oxotremorine-M transactivation of FGFRs and related trophic effects were tested in primary hippocampal neurons. Western blotting and in situ proximity ligation assay (PLA) were used to detect FGFR phosphorylation (pFGFR) levels and M1R-FGFR1 heteroreceptor complexes, respectively. RESULTS: Oxotremorine-M, a non-selective mAChRs agonist, was able to transactivate FGFR and this transactivation was blocked by Src inhibitors. Oxotremorine-M treatment produced a significant increase in the primary neurite outgrowth that was blocked by pre-treatment with the pFGFR inhibitor SU5402 and Src inhibitors. This trophic effect was almost similar to that induced by fibroblast growth factor-2 (FGF-2). By using atropine as nonselective mAChRs or pirenzepine as selective antagonist for M1 receptor (M1R) we could show that mAChRs are involved in modulating the pFGFRs. Using PLA, M1R-FGFR1 heteroreceptor complexes were identified in the hippocampus and cerebral cortex. CONCLUSION: The current findings, by showing functional mAChR-FGFR interactions, will contribute to advance the understanding of the mechanisms involved in the actions of cholinergic drugs on neuronal plasticity. GENERAL SIGNIFICANT: Data may help to develop novel therapeutic strategies not only for neurodegenerative diseases but also for depression-induced atrophy of hippocampal neurons.

Recovery of damaged skeletal muscle in mdx mice through low-intensity endurance exercise.

The lack of dystrophin in mdx mice leads to cycles of muscle degeneration and regeneration processes. Various strategies have been proposed in order to reduce the muscle-wasting component of muscular dystrophy, including implementation of an exercise programme. The aim of this study was to examine how low-intensity endurance exercise affects the degeneration-regeneration process in dystrophic muscle of male mdx mice. Mice were subjected to low-intensity endurance exercise by running on a motorized Rota-Rod for 5 days/week for 6 weeks. Histomorphological analysis showed a significant reduction of measured inflammatory-necrotic areas in both gastrocnemius and quadriceps muscle of exercised mdx mice as compared to matched sedentary mdx mice. The degenerative-regenerative process was also evaluated by examining the protein levels of connexin 39 (Cx39), a specific gene expressed in injured muscles. Cx39 was not detected in sedentary wild type mice, whereas it was found markedly increased in sedentary mdx mice, revealing active muscle degeneration-regeneration process. These Cx39 protein levels were significantly reduced in muscles of mdx mice exercised for 30 and 40 days, revealing together with histomorphological analysis a strong reduction of degeneration process in mice subjected to low-intensity endurance exercise. Muscles of exercised mdx mice did not show significant changes in force and fatigue resistance as compared to sedentary mdx mice. Overall in this study we found that specific low-intensity endurance exercise induces a beneficial effect probably by reducing the degeneration of dystrophic muscle.

The FGF-2/FGFRs neurotrophic system promotes neurogenesis in the adult brain.

Neurogenesis occurs in two regions of the adult brain, namely, the subventricular zone (SVZ) throughout the wall of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG) in hippocampal formation. Adult neurogenesis requires several neurotrophic factors to sustain and regulate the proliferation and differentiation of the adult stem cell population. In the present review, we examine the cellular and functional aspects of a trophic system mediated by fibroblast growth factor-2 (FGF-2) and its receptors (FGFRs) related to neurogenesis in the SVZ and SGZ of the adult rat brain. In the SVZ, FGF-2 is expressed in GFAP-positive cells of SVZ but is not present in proliferating precursor cells, which instead express FGFR-1 and FGFR-2, but not FGFR-3 mRNA, although expressed in the SVZ, and FGFR-4. Therefore, it seems that in the SVZ FGF-2 may be released by GFAP-positive cells, different from the precursor cell lineage, and via volume transmission it reaches the proliferating precursor cells. FGFR-1 mRNA is also expressed in the SGZ and is localized in BrdU-labeled precursor cells, whereas FGFR-2 and FGFR-3 mRNA, although expressed in the SGZ, are not located within proliferating precursor cells. An aged-related decline of proliferating precursor cells in the SVZ and DG of old rats has been well documented, and there is the suggestion that in part it could be the consequence of alterations in growth factor expression levels. Thus, the old precursors may respond to growth factors, suggesting that during aging the basic components for neuronal precursor cell proliferation are retained and the capacity to increase neurogenesis after appropriate stimulation is still preserved. In conclusion, the trophic system mediated by FGF-2 and its receptors contributes to create an important micro-environmental niche that promotes neurogenesis in the adult and aged brain.

Acute intermittent nicotine treatment induces fibroblast growth factor-2 in the subventricular zone of the adult rat brain and enhances neuronal precursor cell proliferation.

Over the past years, evidence has accumulated that stem cells are present in the adult brain, and generate neurons and/or glia from two active germinal zones: the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus of the hippocampus. This study shows that acute intermittent nicotine treatment significantly enhances neuronal precursor cell proliferation in the SVZ of adult rat brain, but not in the SGZ of the hippocampus, and pre-treatment with mecamylamine, a nonselective nAChR antagonist, blocks the enhanced precursor proliferation by nicotine. This effect is supported by up-regulation of fibroblast growth factor-2 (FGF-2) mRNA in the SVZ and the expression of its receptor FGFR-1 in cells of SVZ showing precursor cells profile. It is also demonstrated that the nicotine effect on neuronal precursor proliferation is mediated by FGF-2 via fibroblast growth factor receptor 1 (FGFR-1) activation by showing that i.c.v. pre-treatment with anti-FGF-2 antibodies or with FGFR-1 inhibitor 3-[(3-(2-carboxyethyl)-4-methylpyrrol-2-yl)methylene]-2-indolinone (SU5402) blocks nicotine-induced precursor cell proliferation. This nicotine enhancement of neuronal precursor cell proliferation was not accompanied by an increase in the number of apoptotic cells. Taken together the present findings revealed the existence in the SVZ of the adult rat brain of a trophic mechanism mediated by FGF-2 and its receptor and regulated by nAchR activation. This possibility of in vivo regulation of neurogenesis in the adult brain by exogenous factors may aid to develop treatments stimulating neurogenesis with potential therapeutic implications.

Transcription factor gene expression profiling after acute intermittent nicotine treatment in the rat cerebral cortex.

Several studies in different in vitro and in vivo models have demonstrated neuroprotective effects of nicotinic receptor agonists and indirect trophic actions of nicotine on brain are suggested from observations describing nicotine as a cognitive enhancer by increasing vigilance and improving learning and memory. While an increasing number of studies have given evidence of neuroprotective and neurotrophic effects of nicotine treatment, the molecular mechanism mediating the neurotrophic effects of nicotine are not fully understood. Previously in an analysis of several neurotrophic factors as possible mediators of nicotine-induced neuroprotection and/or neurotrophic effects we could reveal that an acute intermittent nicotine treatment increases fibroblast growth factor-2 mRNA and protein in several brain regions of rat brain. Even if other studies have demonstrated in different paradigms that nicotine administration modulates expression level of a variety of genes, there is still a lack of indication which candidate genes, involved in neuroprotective responses are modulated by nicotine. In the present work we have used a microarray assay to further find and characterize new genes responsive to acute intermittent nicotine treatment and linked to neuroprotection. Therefore, we used Rat Genome U34A Affymetrix GeneChip arrays containing about 8800 probe sets to characterize transcriptional responses in the rat parietal cortex after acute intermittent nicotine treatment. We focused our attention to expression of transcription factors and several of them were up- or down-regulated by nicotine, among these Nr4a1 (Nurr77), Egr-1 and Egr-2. In situ hybridization was used to corroborate the microarray data and to reveal further spatial and temporal patterns of these nicotine induced genes. Taken together the present results identified several novel candidate genes modified by acute intermittent nicotine exposure and as such potentially involved in neuroprotective-neurotrophic actions.

Comparative localization of fibroblast growth factor receptor-1, -2, and -3 mRNAs in the rat brain: in situ hybridization analysis.

The present study provides a detailed comparative description in the adult rat brain of areas that express mRNAs coding for the fibroblast growth factor subtype receptors 1-3 (FGFR1-3). One observation in this analysis was a widespread expression in the brain of all three FGFR mRNAs, according to the following rank order: FGFR1, diencephalon < telencephalon < mesencephalon and metencephalon < myelencephalon; FGFR2 and FGFR3, telencephalon < diencephalon < mesencephalon and metencephalon < myelencephalon. Another observation was an apparent cellular specificity in their basal expression. Thus, the FGFR1 mRNA was expressed mainly in large and weakly stained cells, whereas FGFR2 transcripts were expressed primarily in small and strongly stained cells and in cells of brain regions devoid of neuronal cells, such as the white matter. FGFR3 mRNA was always detected in small and strongly stained cells with scattered distribution and was not expressed in the white matter. However, FGFR2 mRNA was weakly expressed also in large cells localized in some nuclei of the lower brainstem, in the diagonal band, and in the septum. Furthermore, in the medial habenula and in the nuclei of the pons, there exists a high density of cells expressing both FGFR1 and FGFR2 (60-100%). With neurotoxic lesions involving 6-hydroxydopamine microinjections in the substantia nigra, reactive glial cells in the lesioned area and surrounding the cannula tract showed an increase in the expression of both FGFR1 and FGFR2 mRNAs, whereas no increased expression was found for FGFR3 mRNA. Taken together, these findings showed that these three FGF receptors exist in all subtypes of cells of each brain region. Their apparent cellular specificity suggests that these receptor subtypes can have a differential trophic role in the brain, reflecting the various biological activities shown by the ligands of the FGF family.

Acute intermittent nicotine treatment produces regional increases of basic fibroblast growth factor messenger RNA and protein in the tel- and diencephalon of the rat.

Several findings show a neuroprotective effect of nicotine treatment in different experimental models, and a negative correlation has been observed between cigarette smoking and the incidence of Parkinson's disease. It seems possible that nicotine may in part exert its neuroprotective actions by favouring the synthesis of neurotrophic factors. The aim of this study was to determine whether the nicotine treatment could be associated with the induction of a neurotrophic factor in brain regions with nicotinic receptors. Thus, we analysed by in situ hybridization and RNAse protection assay the effects of (-)nicotine on basic fibroblast growth factor messenger RNA and by immunocytochemistry fibroblast growth factor-2 protein in the tel- and diencephalon of rats following single or acute intermittent (-)nicotine treatment. The present results showed that acute intermittent (-)nicotine treatment (four i.p. injections at intervals of 30 min), but not single injections, lead to a substantial and dose-related (0.1-2 mg/kg) up-regulation of fibroblast growth factor-2 messenger RNA levels in the cerebral cortex, in the hippocampus, in the striatum and ventral midbrain. This induction of fibroblast growth factor-2 expression peaked 4 h after the first injection and returned to normal levels within 24 h. The change of fibroblast growth factor-2 messenger RNA levels was associated with increased fibroblast growth factor-2 immunoreactivity mainly localized to nerve cells. The treatment was effective also when repeated in the same animals three or five days after the first injection. The pre-treatment with the non-competitive (-)nicotine receptor antagonist mecamylamine blocked the (-)nicotine effects on fibroblast growth factor-2 messenger RNA levels. In the above areas, no changes were observed in the fibroblast growth factor-1, 2 and 3 receptor messenger RNA levels nor in brain-derived neurotrophic factor messenger RNA levels. The present data indicate an ability of intermittent (-)nicotine to increase fibroblast growth factor-2 in many tel- and diencephalic areas. In view of the trophic function of fibroblast growth factor-2, the previously observed neuroprotective effects of (-)nicotine may at least in part involve an activation of the neuronal fibroblast growth factor-2 signalling, and open up new avenues for treatment of Parkinson's disease and Alzheimer's disease based on the existence of nicotinic receptor subtypes enhancing fibroblast growth factor-2 signalling in many regions of the tel- and diencephalon.

Chronic continuous infusion of (-)nicotine reduces basic fibroblast growth factor messenger RNA levels in the ventral midbrain of the intact but not of the 6-hydroxydopamine-lesioned rat.

A negative correlation has been found between smoking and Parkinson's disease. There is evidence to suggest that this correlation appears to be associated with a neuroprotective role of nicotine for dopamine neurons at least in relation to mechanical injury. However, 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP) neurotoxicity to dopamine neurons is enhanced by chronic continuous (-)nicotine. More recently, basic fibroblast growth factor has been found to possess neurotrophic activities for many nerve cells including the dopamine cells in vivo and in vitro. Therefore, it is of interest to explore a possible effect of nicotine on basic fibroblast growth factor expression in the ventral midbrain of intact and 6-hydroxydopamine-lesioned rats and how treatment with nicotine can alter the 6-hydroxydopamine-induced injury of the nigral dopamine nerve cells as evaluated by dopamine biochemistry. In the present paper, an analysis of the effects of chronic continuous infusion of (-)nicotine via minipumps was carried out on basic fibroblast growth factor expression in the vental midbrain of the intact male rat and of the 6-hydroxydopamine lesioned rat. A quantitative messenger RNA protection assay analysis was used as well as an immunocytochemical analysis in the substantia nigra. Our findings give evidence that a two-week continuous infusion with (-)nicotine in the intact rat leads to substantial and dose-related (0.03-0.3 mg/kg per h) reductions of basic fibroblast growth factor messenger RNA levels in the ventral midbrain. These changes are not associated with changes in neuronal and glial basic fibroblast growth factor immunoreactivity in this region with the antibodies used. However, a one-week continuous infusion with (-)nicotine (0.125 mg/kg per h) failed to significantly alter the basic fibroblast growth factor messenger RNA levels in the ventral midbrain of solvent and 6-hydroxydopamine-injected rats and thus also the 6-hydroxydopamine-induced increase of basic fibroblast growth factor messenger RNA levels in the ventral midbrain of the lesioned side observed at this time-interval and known to be of astroglial origin [Chadi G. et al. (1994) Neuroscience 61, 891-910]. In agreement, the 6-hydroxydopamine-induced depletion of dopamine in the neostriatum was unaltered by the nicotine treatment (0.125 mg/kg per h). Thus, chronic continuous (-)nicotine treatment may lead to a reduced basic fibroblast growth factor trophic tone in the ventral midbrain of the intact but not of the 6-hydroxydopamine-lesioned rat neither on the lesioned nor on the unlesioned side of the ventral midbrain. It seems possible that chronic nicotine treatment mainly reduces basic fibroblast growth factor messenger RNA levels of neuronal origin, since the astroglial messenger RNA levels dominate after the 6-hydroxydopamine-induced lesions.

Increased expression of trkB and trkC messenger RNAs in the rat forebrain after focal mechanical injury.

Tyrosine protein kinases trkA, trkB and trkC are signal transduction receptors for a family of neurotrophic factors known as the neurotrophins. Here we report on changes in the expression of messenger RNAs for trkA, trkB and trkC in the brain following an injury caused by insertion of a 30-gauge needle into adult rat hippocampus or neocortex. Quantitative in situ hybridization revealed no change in the level of trkA messenger RNAs in any brain region following this insult. In contrast, increased levels of trkB messenger RNA compared to untreated animals were seen in the granule cell layer of the dentate gyrus ipsilateral to the injury already 30 min after the injury. The increase reached maximal levels (four-fold) between 2 and 4 h, but returned to control levels 8 h after the injury. No change was seen in the contralateral dentate gyrus. The levels of trkC messenger RNA increased in the same brain regions as trkB messenger RNA, though with a delayed response, reaching a maximal increase of 3.3-fold 4 h after the injury. As for trkB messenger RNA, the level of trkC messenger RNA then tapered off and reached control levels 8 h after the injury. However, 4 h after the injury, a 1.7-fold increase of trkB and trkC messenger RNAs were seen in the ipsilateral piriform cortex. The increases of trkB and trkC messenger RNAs were confirmed using a nuclease protection assay. Increases of both trkB and trkC messenger RNAs were also seen in the piriform cortex, but not in the hippocampus, following needle insertion into the neocortex. Pretreatment of the animals with the non-competitive N-methyl-D-aspartate antagonist ketamine completely prevented the increases of trkB and trkC messenger RNAs, suggesting that the brain injury caused a release of glutamate with subsequent activation of N-methyl-D-aspartate receptors. In contrast, the anticonvulsive drug diazepam, the muscarinic antagonist atropine and the calcium-channel antagonist nimodipine had no effect on the increases of trkB and trkC messenger RNAs. Combined with previous data on the expression of neurotrophin messenger RNAs following similar injuries, our results support the hypothesis that increased levels of neurotrophins and their receptors could protect against neuronal damage following a brain insult.

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.

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.

The nicotinic acetylcholine receptor agonist (+/-)-epibatidine increases FGF-2 mRNA and protein levels in the rat brain.

In a previous work, we showed that acute intermittent nicotine treatment up-regulates the level of fibroblast growth factor-2 (FGF-2) mRNA in brain regions of tel- and mesencephalon of rats suggesting that neuroprotective effect of (-)nicotine may, at least in part, involve an activation of the neuronal FGF-2 signalling. The present experiments were designed to extend the study on the nicotinic receptor mediated up-regulation of FGF-2 mRNA levels to the use of the potent nicotinic acetylcholine receptor (nAChR) agonist (+/-)-epibatidine. The (+/-)-epibatidine treatment led to a strong and long lasting up-regulation of FGF-2 mRNA expression in the cerebral cortex, in the hippocampal formation, in the striatum and in the substantia nigra. This FGF-2 mRNA induction, already statistically significant at 4 h, peaked at 12 h from treatment and was only partially returned towards normal levels at 48 h, the last time point examined. Using Western blot analysis it was found that the epibatidine-induced upregulation of FGF-mRNA is accompaned by an increase of FGF-2 protein level at the 20-h time-interval. These (+/-)-epibatidine effects on FGF-2 expression were antagonized by the non-competitive nAChR antagonist mecamylamine, indicating an involvement of nicotinic receptors. In the same brain areas examined, no changes were observed in the fibroblast growth factor receptor-1 (FGFR-1) mRNA levels, in brain-derived neurotrophic factor (BDNF) and in glial cell line-derived neurotrophic factor (GDNF) mRNA levels. In view of the neurotrophic function of FGF-2, these results, together with previous ones, could further help to understand the molecular mechanisms mediating the previously observed neuroprotective effects of (-)nicotine.

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.

Expression of mRNAs for neurotrophins and their receptors in the rat choroid plexus and dura mater.

The presence of the neurotrophin, nerve growth factor, brain derived neurotrophic factor, neurotrophin-3 and neurotrophin-4 (NGF, BDNF, NT-3 and NT-4) and their receptors of the tyrosine kinase family (trkA, trkB and trkC) have been investigated in the choroid plexus and dura mater of the adult rat by ribonuclease protection assay. The choroid plexus contained high levels of mRNAs for NGF and NT-4, and low levels of NT-3 and BDNF mRNA; and high levels of trkB mRNA, and undetectable levels of trkA and trkC mRNA. In the dura mater there were high levels of NT-3 and NGF, and low levels of BDNF and NT-4 mRNAs; and high levels of trkC mRNA, and relatively high amount of trkB mRNA, while levels of trkA mRNA was undetectable. The present analysis revealed a different distribution of neurotrophins and their related receptors in the choroid plexus and dura mater.

The nicotinic acetylcholine receptor agonist ABT-594 increases FGF-2 expression in various rat brain regions.

The present experiments were designed to extend previous work showing that acute intermittent (-)nicotine treatment upregulates the level of fibroblast growth factor-2 (FGF2) mRNA in several rat brain regions, by the use of the nicotinic acetylcholine receptor (nAChR) agonist ABT-594 with preferential selectivity for the alpha4beta2 nAChR subtype. ABT594 treatment led to a well-defined temporal and regional upregulation of FGF-2 mRNA. A double labelling analysis showed that the up-regulation of FGF-2 mRNA involves both neuronal and non-neuronal cells. The effects of ABT-594 on FGF-2 expression were antagonized by the preferential alpha4beta2 antagonist dihydrobetaerythroidine (DHbetaE), but not by alpha7 antagonist methyllycaconitine (MLA). In conclusion, FGF-2 mRNA levels can be increased in several brain regions upon alpha4beta2 nAChR activation, suggesting a therapeutic significance in neurodegenerative disorders.

Central nicotinic receptors, neurotrophic factors and neuroprotection.

The multiple combinations of nAChR subunits identified in central nervous structures possess distinct pharmacological and physiological properties. A growing number of data have shown that compounds interacting with neuronal nAChRs have, both in vivo and in vitro, the potential to be neuroprotective and that treatment with nAChR agonists elicit long-lasting improving of cognitive performance in a variety of behavioural tests in rats, monkeys and humans. Epidemiological and clinical studies suggested also a potential neuroprotective/trophic role of (-)-nicotine in neurodegenerative disease, such as Alzheimer's and Parkinson's disease. Taken together experimental and clinical data largely indicate a neuroprotective/trophic role of nAChR activation involving mainly alpha7 and alpha4beta2 nAChR subtypes, as evidenced using selective nAChR antagonists, and by potent nAChR agonists recently found displaying efficacy and/or larger selective affinities than (-)-nicotine for neuronal nAChR subtypes. A neurotrophic factor gene regulation by nAChR signalling has been taken into consideration as possible mechanism involved in neuroprotective/trophic effects by nAChR activation and has evidenced an involvement of the fibroblast growth factor (FGF-2) gene as a target of nAChR signalling. These findings suggested that FGF-2 could be involved, according to the FGF-2 neurotrophic functions, in nAChR mechanisms mediating the neuronal survival, trophism and plasticity.