Basic fibroblast growth factor regulates the ability of astrocytes to support hypothalamic neuronal survival in vitro.

The putative neurotrophic effects of basic fibroblast growth factor (bFGF) were tested on embryonic hypothalamic neurons in dissociated cell culture. Basic FGF dramatically increased the survival of embryonic hypothalamic astrocytes plated on a poly-L-lysine (PLL) substrate. Basic FGF treatment also increased the number of hypothalamic neurons surviving in vitro; however, no neurotrophic effects were observed when astrocyte proliferation was prevented by using serum-free N2 medium or by using the mitotic inhibitor cytosine arabinoside. In contrast to effects when PLL was used as a substrate, bFGF reduced the survival of hypothalamic neurons plated on a confluent, contact-inhibited monolayer of astrocytes. This effect appears to be due to the direct actions of bFGF on astrocytes: treatment of confluent astrocytes with 5 ng/ml bFGF caused the protoplasmic astrocytes to develop a fibrillar morphology and reduced the ability of the astrocyte monolayer to promote neuronal survival after a further 24 hr in bFGF-free medium. It is concluded that in addition to its mitogenic effects, bFGF acts as a differentiation factor for protoplasmic astrocytes in vitro, and these morphological and functional changes may reflect the process of normal astrocytic development and response to brain injury in vivo.

Both survival and development of spontaneously active rat hypothalamic neurons in dissociated culture are dependent on membrane depolarization.

Suppression of endogenous electrical activity was found to have an adverse effect on the survival and bioelectric development of dissociated, embryonic rat hypothalamic neurons in long-term culture. Cultures were treated during the first two weeks in vitro with tetrodotoxin (TTX), a selective blocker of voltage-gated sodium channels, alone and in combination with high extracellular KCl ([K+]o), a membrane depolarizer. Neuron survival was assessed through cell counting experiments, while the development of spontaneous electrical activity was examined with extracellular, patch-electrode recordings. TTX caused both a decrease in cell survival and a decrease in spontaneously active cells; concurrent treatment with K+ protected cells from the adverse effects of TTX. K+ treatment alone increased the fraction of spontaneously active neurons without significantly affecting cell survival. When taken together, these results suggest that the long-term survival of active cells depends on continual membrane depolarization. From these observations, we conclude that there exists two populations of neurons: the electrically active population, whose survival is sensitive to electrical activity, and the quiescent population, whose survival is not.

Astrocyte topography and tenascin cytotactin expression: correlation with the ability to support neuritic outgrowth.

We have observed a heterogeneity in the ability of a monolayer of cultured rat astrocytes to support the attachment and growth of dissociated embryonic hypothalamic neurons in culture. Areas of the monolayer which have an uneven surface ('rocky' astrocytes) are poor substrates for neuronal attachment and neuritic outgrowth, while surrounding areas of the glial monolayer ('flat' astrocytes) support extensive neuronal growth. Astrocytes obtained from both neonatal cerebral cortex or hypothalamus displayed 'rocky' morphology. We utilized immunocytochemical techniques with antibodies directed against putative adhesion molecules to investigate the source of this heterogeneity. Antibodies against tenascin/cytotacin, fibronectin, laminin, N-CAM, thrombospondin, heparan sulfate proteoglycan, and the p185 protein product of the neu oncogene were employed in indirect-immunofluorescence experiments. We found that the difference in the surface properties of astrocytes appears to be correlated with the expression of the extracellular matrix molecule tenascin/cytotacin, but not with any of the other molecules we tested. Our data suggest that tenascin/cytotactin is inhibitory to neuronal attachment and process outgrowth in the developing nervous system.

Development of spontaneous electrical activity by rat hypothalamic neurons in dissociated culture.

The development of spontaneous electrical activity by embryonic rat hypothalamic neurons in dissociated culture was monitored using an extracellular, patch electrode recording method. Embryonic day 17 hypothalamic neurons were plated onto a feeder layer of astrocytes obtained from neonatal rat cerebral cortex, and extracellular electrical activity was monitored beginning the day after plating. The rates and patterns of spontaneous discharge were examined using interpike interval histograms. The percentage of spontaneously active neurons increased steadily with time in culture, from 13% in the first week to 56% during the sixth week in vitro. Although the percentage of spontaneously active cells increased, average firing rates did not change with time in culture. The pattern of electrical discharge was primarily random at all time points, with a small number of cells displaying regular activity while 4 cells were classified as phasic/bursty. In general, spontaneous action potential discharge was not dependent on synaptic transmission, as activity persisted after perfusion with bath solution containing either low Ca2+/high Mg2+ or kynurenic acid. Tetrodotoxin consistently and reversibly abolished spontaneous firing, regardless of culture age. We conclude that spontaneous activity in low density hypothalamic culture develops gradually though steadily, and is generated through an endogenous mechanism, independent of synaptic excitation.