Focally evoked calcium waves in astrocyte cultures have been thought to propagate by gap-junction-mediated intercellular passage of chemical signal(s). In contrast to this mechanism we observed isolated astrocytes, which had no physical contact with other astrocytes in the culture, participating in a calcium wave. This observation requires an extracellular route of astrocyte signaling. To directly test for extracellular signaling we made cell-free lanes 10-300 microns wide in confluent cultures by deleting astrocytes with a glass pipette. After 4-8 hr of recovery, regions of confluent astrocytes separated by lanes devoid of cells were easily located. Electrical stimulation was used to initiate calcium waves. Waves crossed narrow (< 120 microns) cell-free lanes in 15 of 36 cases, but failed to cross lanes wider than 120 microns in eight of eight cases. The probability of crossing narrow lanes was not correlated with the distance from the stimulation site, suggesting that cells along the path of the calcium wave release the extracellular messenger(s). Calculated velocity across the acellular lanes was not significantly different from velocity through regions of confluent astrocytes. Focal superfusion altered both the extent and the direction of calcium waves in confluent regions. These data indicate that extracellular signals may play a role in astrocyte-astrocyte communication in situ.

Communication from astrocytes to neurons has recently been reported by two laboratories, but different mechanisms were though to underlie glial calcium wave activation of associated neurons. Neuronal calcium elevation by glia observed in the present report is similar to that reported previously, where an increase in neuronal calcium was demonstrated in response to glial stimulation. In the present study hippocampal neurons plated on a confluent glial monolayer displayed a transient increase in intracellular calcium following a short delay after the passage of a wave of increased calcium in underlying glia. Activated cells displayed action potentials in response to glial waves and showed antineurofilament immunoreactivity. Finally, the N-methyl-D-aspartate glutamate receptor antagonist DL-2-amino-5-phosphonovaleric acid and the non-NMDA glutamate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione significantly reduced the responsiveness of neurons to glial calcium waves. Our results indicate that hippocampal neurons growing on hippocampal or cortical astrocytes respond to glial calcium waves with elevations in calcium and increased electrical activity. Furthermore, we show that in most cases this communication appears to be mediated by ionotropic glutamate receptor channels.

Previous work has demonstrated that white matter in the adult mammalian CNS inhibits cell adhesion and neurite outgrowth. This phenomenon has been investigated most recently by culturing neurons on cryostat sections of the adult CNS. Employing this same technique, we have found, in accord with others, that neurons seldom adhere to or grow on central nervous system white matter but will attach and grow on gray matter. In the experiments presented here, embryonic rat hippocampal neurons were grown on cryostat sections from the adult rat CNS, in the presence of brain derived glial cocultures. It was found that the white matter in cryostat sections can be modified by interaction with medium conditioned by brain-derived glial cells. Neurons plated on sections pretreated by such media show significant increases in both attachment and neurite outgrowth. The activity contained in glial conditioned medium is likely complex in nature. While the majority of the activity can be eliminated by heat treatment and trypsinization, neural adhesion but not neurite initiation is affected by protease treatment. Therefore, cell attachment and neurite outgrowth may be regulated by different factors in the conditioned media. (C) 1994 Wiley-Liss, Inc.

Proteoglycans have been implicated in neuronal pathfinding during development, yet related second messenger and signaling systems are unknown. We have used the calcium indicator fura-2/AM to monitor cytoplasmic calcium ion concentration ([Ca2+]i) in chick dorsal root ganglion (DRG) neuronal growth cones elongating on laminin during contact with chondroitin sulfate proteoglycan (CSPG): (1) to determine whether there is a change in [Ca2+]i in neurons that contact CSPG, and (2) to determine whether changes in [Ca2+]i are necessary for inhibition of growth cone migration. The majority of DRG neurons responded to CSPG contact with a transient rise in [Ca2+]i (mean Δ[Ca2+]i above resting level was 554 ± 109 nM; P < 0.0001). The effect of CSPG contact was concentration dependent and required the carbohydrate moiety of CSPG. Addition of soluble CSPG did not elevate [Ca2+]. Treatment with reagents that blocked plasma membrane calcium channels, or that perturbed intracellular Ca2+ stores, indicated that extracellular Ca2+ was the major source of the [Ca2+]i elevation, and that Ca2+ entry occurred through non-voltage-gated calcium channels. Although general Ca2+ channel blockers abolished the CSPG-induced [Ca2+]i rise, they did not abolish growth cone avoidance of surface-bound CSPG in these assays. We conclude: (1) that DRG neurons elevate [Ca2+]i in response to CSPG contact to levels that can modify cytoskeletal mechanisms of growth cone migration, and (2) that avoidance of substratum-bound CSPG may not be dependent upon elevated [Ca2+]i.

A myelin-associated protein from the central nervous system, the neurite growth inhibitor NI-35, inhibits regeneration of lesioned neuronal fiber tracts in vivo and growth of neurites in vitro. Growth cones of cultured rat dorsal root ganglion neurons arrested their growth and collapsed when exposed to liposomes containing NI-35. Before morphological changes, the concentration of free intracellular calcium ([Ca2+]i) showed a rapid and large increase in growth cones exposed to liposomes containing NI-35. Neither an increase in [Ca2+]i nor collapse of growth cones was detected in the presence of antibodies to NI-35. Dantrolene, an inhibitor of calcium release from caffeine-sensitive intracellular calcium stores, protected growth cones from collapse evoked by NI-35. Depletion of these caffeine-sensitive intracellular calcium stores prevented the increase in [Ca2+]i evoked by NI-35. The NI-35-evoked cascade of intracellular messengers that mediates collapse of growth cones includes the crucial step of calcium release from intracellular stores.