The finding that astrocytes possess glutamate-sensitive ion channels hinted at a previously unrecognized signaling role for these cells. Now it is reported that cultured hippocampal astrocytes can respond to glutamate with a prompt and oscillatory elevation of cytoplasmic free calcium, visible through use of the fluorescent calcium indicator fluo-3. Two types of glutamate receptor--one preferring quisqualate and releasing calcium from intracellular stores and the other preferring kainate and promoting surface-membrane calcium influx--appear to be involved. Moreover, glutamate-induced increases in cytoplasmic free calcium frequently propagate as waves within the cytoplasm of individual astrocytes and between adjacent astrocytes in confluent cultures. These propagating waves of calcium suggest that networks of astrocytes may constitute a long-range signaling system within the brain.
Can neurons induce surrounding glia to provide a more favorable microenvironment? Synapses and nerve growth cones have been shown to release neurotransmitters (Hume et al. Nature 1983;305:632-634; Kater et al. Trends Neurosci. 1988;11:315-321; Young and Poo Nature 1983;305:634-637) providing a possible mechanism for this type of control. The excitatory neurotransmitter glutamate induces an increase in the number of filopodia on the surface of astrocytes cultured from the neonatal rat hippocampus. This seems to be associated with a receptor-mediated event that is activated to a lesser degree by the quisqualate and kainate, but not NMDA receptors. In addition, time-lapse video recordings have revealed a rapid extension of filopodia from the apical margins of cells treated with glutamate. The apical margins of glutamate-treated cells studied with electron microscopy contained dense cortical actin networks that are devoid of microtubules. Coated pits are often seen to invaginate from the apical membrane in the vicinity of filopodia. A receptor-binding step may be followed by a rapid reorganization of cortical actin resulting in actin-containing filopodia. This process may be mediated by inositol lipid hydrolysis. Pyramidal neurons settled on glial cultures induced filopodia to form around the entire margin of growth cones and neurite tips suggesting that these events might occur in situ.
Astrocytes respond to the excitatory neurotransmitter glutamate with dynamic spatio-temporal changes in intracellular calcium [Ca2+]i. Although they share a common wave-like appearance, the different [Ca2+]i changes--an initial spike, sustained elevation, oscillatory intracellular waves, and regenerative intercellular waves--are actually separate and distinct phenomena. These separate components of the astrocytic Ca2+ response appear to be generated by two different signal transduction pathways. The metabotropic response evokes an initial spatial Ca2+ spike that can propagate rapidly from cell to cell and appears to involve IP3. The metabotropic response can also produce oscillatory intracellular waves of various amplitudes and frequencies that propagate within cells and are sustained only in the presence of external Ca2+. The ionotropic response, however, evokes a sustained elevation in [Ca2+]i associated with receptor-mediated Na+ and Ca2+ influx, depolarization, and voltage-dependent Ca2+ influx. In addition, the ionotropic response can lead to regenerative intercellular waves that propagate smoothly and nondecrementally from cell to cell, possibly involving Na+/Ca2+ exchange. All these astrocytic [Ca2+]i changes tend to appear wave-like, traveling from region to region as a transient rise in [Ca2+]i. Nevertheless, as our understanding of the cellular events that underlie these [Ca2+]i changes grows, it becomes increasingly clear that glutamate-induced Ca2+ signaling is a composite of separate and distinct phenomena, which may be distinguished not based on appearance alone, but rather on their underlying mechanisms.
Astrocytes are coupled to each other via gap-junctions both in vivo and in vitro. Gap-junction coupling is essential to a number of astrocyte functions including the spatial buffering of extracellular K+ and the propagation of Ca2+ waves. Using fluorescence recovery after photo-bleach, we quantitatively assayed and compared the coupling of astrocytes cultured from six different central nervous system (CNS) regions in the rat: spinal cord, cortex, hypothalamus, hippocampus, optic nerve, and cerebellum. The degree of fluorescence recovery (% recovery) and time constant of recovery (tau) served as quantitative indicators of coupling strength. Gap-junction coupling differed markedly between CNS regions. Coupling was weakest in astrocytes derived from spinal cord (43% recovery, tau approximately 400 s) and strongest in astrocytes from optic nerve (91% recovery, tau approximately 226 s) and cerebellum (95% recovery, tau approximately 100 s). As indicated by the degree of recovery, coupling strength among CNS regions could be ranked as follows: spinal cord < cortex < hypothalamus < hippocampus = optic nerve = cerebellum. Gap-junction coupling also differed between CNS regions with respect to its sensitivity to inhibition by the uncoupling agent octanol. Kd values for 50% inhibition by octanol ranged from 188 microM in spinal cord astrocytes to 654 microM in hippocampal astrocytes. Sensitivity of gap-junctions to octanol could be ranked as follows: spinal cord = cortex = hypothalamus > cerebellum > optic nerve > hippocampus. The observed differences in coupling indicate differences in the number of gap-junction connections in astrocytes cultured from the six CNS regions. These differences may reflect the adaptation of astrocytes to varying functional requirements in different CNS regions.
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