Once bound to methylated CpG sites, methyl-CpG-binding protein 2 (MeCP2) is thought to silence transcription of downstream genes by recruiting a histone deacetylase (HDAC). Mutations within the MeCP2 gene have been found to cause Rett syndrome, a disorder of arrested neuronal development. Using immunohistochemistry, we found that Mecp2, as well as the methyl-CpG-binding protein MBD1, were significantly induced in normal adult rat brain after repeated injections of fluoxetine or cocaine for 10 days (one injection per day). Mecp2 was not induced by repeated injections of 1-(2-bis(4-fluorphenyl)-methoxy)-ethyl)-4-(3-phenyl-propyl)piperazine (GBR-12909) or nortriptyline. Together, the data indicate that the serotonergic system is predominantly involved. Using real-time reverse transcription-polymerase chain reaction experiments, MBD1 mRNA and both Mecp2_e1 and Mecp2_e2 transcripts were found to be induced by fluoxetine. Induction of the methylbinding proteins was accompanied with enhanced HDAC2 labeling intensity and mRNA synthesis in response to fluoxetine. In tandem, acetylated forms of histone H3 were found to be decreased. The effect was characterized in three serotonin projection areas, the caudate-putamen, the frontal cortex, and the dentate gyrus subregion of hippocampus. Our data highlight GABAergic neurons as major target cells expressing Mecp2 in response to the serotonin-elevating agents and suggest that serotonin signaling enhances gene silencing in postmitotic neurons.

Neuronal cells from cerebral hemispheres of 13-day-old rat embryos were grown in a serum-free culture medium for 48 h, 4 or 8 days. The neuronal precursor cells proliferate for 5 days. The addition of bovine brain basic fibroblast growth factor stimulates their proliferation as determined by measurement of [1zsIj-iod~eoxyu~dine inco~oration and by autoradio~aphic analysis after ~~thymidine incorporation. The proliferating responsive cells were characterized as neurons by immunostai~ng against neurofilament proteins. Five other growth factors tested were without effect on the proliferative activity of these neuronal cells. The present results show that bFGF is mitogenic in vitro for rat neuronal precursor cells of the central nervous system.Fibroblast growth factor; Proliferation; Stimulator effect; (Rat neuronal cell)

Intraperitoneal injection of epidermal growth factor (EGF) into mice resulted in the phosphorylation of liver nuclei phospholipase Cgamma1 (PLCgamma1) at the tyrosine, coincident with the time course of nuclear membrane epidermal growth factor receptor (EGFR) activation. The function of PLCgamma1 in mice liver nuclei was attributed to a 120 kDa protein fragment. This 120 kDa protein was immunoprecipitated with the isozyme specific PLCgamma1 antibody and was found to be sensitive to a PLCgamma1 specific blocking peptide. The 10-partial sequence analysis revealed that the 120 kDa protein contains the PELCQVSLSE sequence at its N-terminal end and the RTRVNGDNRL sequence at its C-terminal end, which reveals that this protein is a major fragment of PLCgamma1 devoid of an amino acid portion at the N-terminal end. The tyrosine-phosphorylated 120 kDa protein interacts with activated EGFR, binds phosphatidylinositol-3-OH-kinase enhancer (PIKE), enhances nuclear phosphatidylinositol-3-OH-kinase (PI[3]K) activity, and generates diacylglycerol (DAG) in response to the EGF signal to the nucleus in vivo. The immunoprecipitated 120 kDa protein fragment displayed phosphatidylinositol (PI) hydrolysis activity. These results establish the capacity of EGF-triggered nuclear signaling which is mediated by EGFR itself, located on the inner nuclear membrane. This is the first report identifying a 120 kDa PLCgamma1 fragment generated in vivo in the nucleus and capable of discharging the function of nuclear PLCgamma1.

Neuronal cells were obtained by dissociating cells from the cerebral hemispheres of rat embryos (10 to 17-day-old), either cleaned entirely or only partially of their meningeal membranes. These cells were seeded on poly-lysine-coated Petri dishes in serum-containing medium. The cultures most enriched in neuronal cells were obtained from brains of 13- to 15-day-old embryos and after 2 h, the culture medium was switched to Dulbecco's modified Eagle's medium, without serum, supplemented with the N1 supplements as described by Bottenstein et al. (1980). The proliferation of neuroblasts from 13-day-old embryos in the presence or absence of meningeal cells was studied by using a combination of tritiated thymidine autoradiography and immuno-staining against neurofilament proteins. The neuroblasts seem to proliferate during the first 3 days. The proliferative activity was further enhanced in the presence of meningeal cells. The glioblasts multiply only after a period of one week in culture conditions as observed here. The subsequent development of the neuroblasts was followed over a period of 4 weeks and the ultrastructural appearance of these cells was investigated at 2 and 3 weeks. In the presence of meningeal cells, many neurons, intensely stained for neurofilament proteins, survived for 21 days, while in control cultures they underwent massive degeneration after 2 weeks. Synapses with numerous clear vesicles were abundant in cultures grown under the influence of meningeal cells; they were rare and possessed few vesicles in control cultures. The data indicate that meningeal cells affect the proliferation and maturation of rat neuroblasts in culture.

Studies over the past ten years have revealed that neuronal precursors from the central nervous system of chick, rat and mouse embryos are able to divide in culture and that their proliferation is enhanced by several nervous tissue extracts as well as by growth factors, hormones and various other molecules. In this article we present an overview of this subject. It has been found that neuronal precursors from chick embryo cerebral hemispheres proliferate in culture during the first week and that those from 6 day-old chick embryos possess the highest proliferative activity. Neuronal precursors from fetal rat cerebral cortex and spinal cord can also proliferate in vitro. The highest proliferative activity was observed between 24 and 48 h. Brain and meningeal extracts have been shown to stimulate the proliferation of chick neuroblasts. Moreover, RNAs, purine nucleotides, purine bases and transferrin present in these extracts are able to reinduce the proliferation of these cells. Other investigations have indicated that several hormones and growth factors stimulate the proliferation of rat and mouse neuronal precursors. Acidic and basic fibroblast growth factors are potent mitogens for these cells. Nerve growth factor, epidermal growth factor and insulin-like growth factor also affect the growth of the neuroblasts. The reported in vitro observations are discussed in relation to the physiological role of these molecules during neuronal proliferation in brain development.

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