Neuregulin-1 isoform induces mitogenesis, KCa and Ca2+ currents in PC12 cells. A comparison with sciatic nerve conditioned medium.

Neuregulin-1 (NRG-1) is an active component found in sciatic nerve conditioned medium (CM). NRG-1 is a growth and differentiation factor shown to have an effect on neuritogenesis and survival of neural cells. PC12 cells chronically treated with NRG-1 (beta1 isoform) show an increase in proliferation under low-serum condition (2.5% fetal bovine serum and 1.25% horse serum) and serum deprivation, without visible morphological changes. NRG-1 and CM treatments of PC12 cells induced an increase of voltage-activated Ca2+ currents and large-conductance calcium-activated K+ currents (KCa). AG825, a specific inhibitor for erbB2 receptor, abolishes KCa current, though Ca2+ currents were not inhibited. These results showed that NRG-1 is capable of inducing functional changes but is not sufficient on its own to have an effect on cell morphology.

Neuron-like differentiation of PC12 cells treated with media conditioned by either sciatic nerves, optic nerves, or Schwann cells.

In previous works we reported the finding of neurotrophic activity in a serum-free Dulbecco's modified Eagle's medium conditioned by rat sciatic nerves, previously maintained in culture for 11 days. This medium produces rapid neuron-like differentiation of cultured PC12 cells, as revealed by an increase in the size of the cell body and by the extension of short and/or long neurites by most of the cells. Neuregulin present in the conditioned medium was demonstrated to play a key role in the observed differentiation. In the present work, taking into consideration those latter results, the neurotrophic activity of conditioned media prepared with sciatic and optic nerves cultured during days 1-4 and 9-12 were studied. Evaluation of the trophic activities of those media revealed an opposite timing in the activities of sciatic and optic nerves conditioned media. The activity of the sciatic nerve was not observed in the 1-4-day period, increasing then up to the 9-12-day period. On the contrary, the optic nerve conditioned medium was active in the 1-4-day period, decreasing down to the 9-12-day period. These results led us to explore the contribution of the different cellular constituents of those nerves to their neurotrophic properties. As a first step in that direction we also investigated the neurotrophic activity of media conditioned during 12 days by cultured Schwann cells isolated from rat sciatic nerves. The Schwann cell conditioned media did produce a rapid differentiation of the PC12 cells similar to that caused by the sciatic nerve conditioned medium, though of a lower magnitude. Variations in the trophic activities of the conditioned media used in the present work is discussed taking into consideration the production of trophic and inhibitory factors by the peripheral and central glial cells. The role played by the optic nerve glia and myelin is being investigated at present.

Role of glypican-1 in the trophic activity on PC12 cells induced by cultured sciatic nerve conditioned medium: identification of a glypican-1-neuregulin complex.

Glypican-1 is an extracellular matrix component found by microsequencing in a medium conditioned by cultured rat-sciatic nerves (CM). This CM was concentrated by ultrafiltration and fractionated by quaternary ammonium chromatography, followed by Hi-Trap blue affinity chromatography to obtain the active fraction B1.2. Previously, we have reported a 54 kDa neuregulin (NRG) in the same B1.2 fraction [Villegas et al., Brain Res. 852 (2001) 304]. The effect of Glypican-1 on the neuron-like differentiation of PC12 cells was investigated by immunoprecipitation, Western blot and cellular image analysis. Removal of glypican-1 by immunoprecipitation with increasing concentrations of specific antibodies revealed a gradual decrease of the differentiation activity of fraction B1.2, which paralleled the results obtained by removal of the 54 kDa NRG protein. Colorless native electrophoresis and Western blot analysis was used to identify a glypican-1-NRG protein complex, which could be afterwards separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis into its individual components. Additionally, it was demonstrated that glypican-1, in cooperation with the 54 kDa NRG, is involved in the neuronal-like differentiation of PC12 cells and could play an important role on the regeneration responses of peripheral nerves.

Microscopia electrónica de fibras nerviosas gigantes / Electron microscopy of giant nerve fibers

El presente trabajo es una revisión breve sobre los detalles ultraestructurales de las fibras nerviosas gigantes y su posible correlación con hallazgos fisiológicos hechos en el axón gigante del calamar. Tanto en secciones finas, como en réplicas obtenidas por crío-fractura, el aspecto más llamativo ha sido la gran cantidad de membranas apareadas que llenan la capa de Schwann. Estas membranas limitan hendiduras intercelulares permeables que conectan la superficie axónica con el espacio extracelular del endoneuro, dejando, así, al axolema como la única barrera continua interpuesta entre el axoplasma y el exterior de la neurona. Se ha observado, tanto en las secciones como en las réplicas, zonas de adosamiento íntimo del axón y de su célula de Schwann a nivel d elos llamados complejos estructurales, en los cuales aparecen involucradas las membranas plasmáticas de ambas células adyacentes. Estas zonas podrían constituir la expresión morfológica del acoplamiento funcional dado a conocer en la misma preparación. Además, la célula de Shwann parece ser muy activa a juzgar por la cantidad de perfiles de exo-endocitosis observados en todas sus superficies de fractura. Finalmente, las células del endoneuro aparecen diferentes en los varios tipos de fibras gigantes estudiados: en el calamar, ellas se muestran como células esponjosas, mientras que en la langosta, ellas exhiben una cantidad extraordinaria de imágenes de exo-endocitosis entremezcladas con algunas uniones epiteliales de baja resistencia y con uniones estrechas incompletas. Por último, en el camarón de río, la célula endoneurales muestran el mismo aspecto que el de la glía adaxónica (AU)

Microscopia electrónica de fibras nerviosas gigantes / Electron microscopy of giant nerve fibers

El presente trabajo es una revisión breve sobre los detalles ultraestructurales de las fibras nerviosas gigantes y su posible correlación con hallazgos fisiológicos hechos en el axón gigante del calamar. Tanto en secciones finas, como en réplicas obtenidas por crío-fractura, el aspecto más llamativo ha sido la gran cantidad de membranas apareadas que llenan la capa de Schwann. Estas membranas limitan hendiduras intercelulares permeables que conectan la superficie axónica con el espacio extracelular del endoneuro, dejando, así, al axolema como la única barrera continua interpuesta entre el axoplasma y el exterior de la neurona. Se ha observado, tanto en las secciones como en las réplicas, zonas de adosamiento íntimo del axón y de su célula de Schwann a nivel d elos llamados complejos estructurales, en los cuales aparecen involucradas las membranas plasmáticas de ambas células adyacentes. Estas zonas podrían constituir la expresión morfológica del acoplamiento funcional dado a conocer en la misma preparación. Además, la célula de Shwann parece ser muy activa a juzgar por la cantidad de perfiles de exo-endocitosis observados en todas sus superficies de fractura. Finalmente, las células del endoneuro aparecen diferentes en los varios tipos de fibras gigantes estudiados: en el calamar, ellas se muestran como células esponjosas, mientras que en la langosta, ellas exhiben una cantidad extraordinaria de imágenes de exo-endocitosis entremezcladas con algunas uniones epiteliales de baja resistencia y con uniones estrechas incompletas. Por último, en el camarón de río, la célula endoneurales muestran el mismo aspecto que el de la glía adaxónica