Using specific antisera to methionine-enkephalin and leucine-enkephalin, we have visualized apparent enkephalin-containing neuronal fibers and terminals throughout the central nervous system of the rat. Immunoreactive enkephalin displays sharply defined localizations. Regions of highest immunofluorescent density include the laminae I and II of the spinal cord, the substantia gelatinosa of the caudal nucleus of nerve V, the vagal nuclei of the medulla, the periventricular and periaqueductal areas of the upper medulla and midbrain, dorsomedial thalamic regions, specific hypothalamic nuclei, the basal ganglia, particularly the globus pallidus and the central nucleus of the amygdala, and the lateral septum. In certain regions enkephalin immunofluorescence corresponds closely with the distribution of autoradiographic opiate receptor grains.The opiate-like pentapeptides methionine-enkephalin (Metenk) and leucine-enkephalin (Leu-enk) (1, 2) appear to be endogenous ligands for the opiate receptor. The regional localization of enkephalin in mammalian brain, determined biochemically, resembles that of opiate receptor binding (3-6). In subcellular fractionation studies, enkephalin is localized to synaptosomal fractions that contain nerve terminals (7). Autoradiographic studies of the opiate receptor reveal sharply defined localizations to structures mediating functions affected by opiates, such as pain perception (8)(9)(10). If the enkephalins are neurotransmitters or neuromodulators associated with opiate receptors, one might expect enkephalin to be localized microscopically to neuronal systems impinging on opiate receptors. Preliminary immunohistochemical studies show immunoreactive enkephalin fluorescence in nerve fibers and terminals with highest densities in areas enriched in opiate receptors (11). We now report a detailed mapping of the rat central nervous system for immunoreactive enkephalin.MATERIALS AND METHODS Antisera Preparation. Met-enk or Leu-enk (20 mg) was coupled to keyhole limpet hemocyanin (10 mg) by incubation for 30 min in distilled water at room temperature with 150 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The material was dialyzed extensively against distilled water, lyophilized, suspended in 3 ml of distilled water, and stored. Guinea pigs were immunized with 1 mg of the hemocyanincoupled enkephalin diluted 1:10 in saline and mixed 1:1 with Freund's complete adjuvant. Rabbits were injected with 1.6 mg of this conjugate. The immunization was repeated three to four times at 3 to 4 week intervals using incomplete Freund's adjuvant. The guinea pigs and rabbits were bled 7-10 days after the third and subsequent immunizations and the sera were tested for enkephalin binding in radioimmunoassays (ref. 12; Abbreviations: Met-enk, methionine-enkephalin; Leu-enk, leucineenkephalin.* To whom reprint requests should be addressed.Simantov, Childers, and Snyder, unpublished data). Enkephalin binding by these antisera was not displaced by high concentrations of substance P, glucagon, insulin...

Cultured neurons from rat embryo striatum were found to contain two structurally distinct forms of pp60Jcsrc. The 60-kilodalton (kDa) form appeared similar to pp6Ocsrc from cultured rat fibroblasts or astrocytes.The 61-kDa form was specific to neurons and differed in the NH2-terminal 18 kDa of the molecule. In undifferentiated neurons the predominant phosphorylated species of pp60csrc was the fibroblast form. Upon differentiation, a second phosphorylated form of pp60Csrc was detected. This form had two or more additional sites of serine phosphorylation within the NH2-terminal 18-kDa region of the molecule, one of which was Ser-12. The specific protein-tyrosine kinase activity of the total pp60csl`population increased 14-fold, as measured by autophosphorylation, or 7-fold, as measured by phosphorylation of an exogenous substrate, as striatal neurons differentiated. This elevation in protein kinase activity occurred without a detectable decrease in Tyr-527 phosphorylation or increase in Tyr-416 phosphorylation. Our results support the idea that the expression of the neuron-specific form of pp60CSrC and the increase in specific protein kinase activity may be important for neuronal differentiation.Cellular genes homologous to retroviral oncogenes are present in the genomes of all vertebrates. These cellular proto-oncogenes have been highly conserved throughout evolution, suggesting that they are essential to the organism. Although their function remains largely unknown, evidence is accumulating that some may be important for normal cell differentiation or growth.The cellular gene c-src is homologous to the transforming gene of Rous sarcoma virus (59). It is highly conserved phylogenetically, being present in the genome of such widely divergent species as humans, Drosophila melanogaster (35,61,62), and the freshwater sponge Spongilla lacustris (3, 55). The c-src gene encodes a 60-kilodalton (kDa) membraneassociated phosphoprotein, pp6Oc-src, which is a proteintyrosine kinase (14-17, 24, 36, 38, 49, 52, 59). Evidence is emerging that pp60C-src is the product of a developmentally regulated gene that may participate in cell differentiation. High levels of pp60csrc are expressed in brain and other neural tissues of both chickens and humans during embryogenesis (22,42,45). Expression of pp60-src in the developing chick neural retina (64) and cerebellum (29) coincides with the onset of neuronal differentiation. Post mitotic neurons from the central nervous system of rat embryos express high levels of a structurally distinct, enzymatically activated form of pp60C-src (9). Similarly, embryonal carcinoma cells induced to differentiate into neuronlike cells have elevated levels of a slower-migrating form of pp60c-src (44). There are other nonproliferating cells, for example, platelets (31) and myeloid cells (2, 30), which contain increased pp6Oc-src kinase activity. The presence of high levels of pp6Oc-src protein-tyrosine kinase activity in * Corresponding author. these nondividing cells suggests that pp60csrc may be importan...

No abstract

No abstract

The nuclear transcription factor Nurr1 is involved in the development and maintenance of the midbrain dopaminergic (DA) neuronal phenotype. We analysed the cellular and biological effects of Nurr1 during embryonic stem (ES) cell differentiation using the ROSA26-engineered Tetinducible ES cell line J1-rtTA that does not express transgenes in mature neurons. Induction of Nurr1 at nestin-positive precursor and later stages of ES cell differentiation produced a non-neuronal DA cell type including functional DA transporters. In these cells, we found a clear correlation between Nurr1 and TH gene expression and specific midbrain DA cellular markers such as AADC, AHD2 and calbindin. Nurr1 did not alter gene expression of non-DA neuronal phenotypes and did not influence other midbrain developmental transcription factors, such as Otx1, Otx2, En-1, GBX2, Pitx3 and Imx1 b. In addition, Nurr1 expression was required for maintenance of the DA phenotype and mediated up-regulation of the tyrosine kinase Ret and associated trophic factor GDNF-family receptors a 1, 2, and 4. This demonstrates that Nurr1 is sufficient to induce and maintain a midbrainlike DA biochemical and functional cellular phenotype independent of neurogenesis.