Tripartite synapses: glia, the unacknowledged partner.

According to the classical view of the nervous system, the numerically superior glial cells have inferior roles in that they provide an ideal environment for neuronal-cell function. However, there is a wave of new information suggesting that glia are intimately involved in the active control of neuronal activity and synaptic neurotransmission. Recent evidence shows that glia respond to neuronal activity with an elevation of their internal Ca2+ concentration, which triggers the release of chemical transmitters from glia themselves and, in turn, causes feedback regulation of neuronal activity and synaptic strength. In view of these new insights, this article suggests that perisynaptic Schwann cells and synaptically associated astrocytes should be viewed as integral modulatory elements of tripartite synapses.

Prostaglandin E(2) stimulates glutamate receptor-dependent astrocyte neuromodulation in cultured hippocampal cells.

Recent Ca(2+) imaging studies in cell culture and in situ have shown that Ca(2+) elevations in astrocytes stimulate glutamate release and increase neuronal Ca(2+) levels, and that this astrocyte-neuron signaling can be stimulated by prostaglandin E(2) (PGE(2)). We investigated the electrophysiological consequences of the PGE(2)-mediated astrocyte-neuron signaling using whole-cell recordings on cultured rat hippocampal cells. Focal application of PGE(2) to astrocytes evoked a Ca(2+) elevation in the stimulated cell by mobilizing internal Ca(2+) stores, which further propagated as a Ca(2+) wave to neighboring astrocytes. Whole-cell recordings from neurons revealed that PGE(2) evoked a slow inward current in neurons adjacent to astrocytes. This neuronal response required the presence of an astrocyte Ca(2+) wave and was mediated through both N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors. Taken together with previous studies, these data demonstrate that PGE(2)-evoked Ca(2+) elevations in astrocyte cause the release of glutamate which activates neuronal ionotropic receptors.

Glutamate-dependent astrocyte modulation of synaptic transmission between cultured hippocampal neurons.

The idea that astrocytes merely provide structural and trophic support for neurons has been challenged by the demonstration that astrocytes can regulate neuronal calcium levels. However, the physiological consequences of astrocyte-neuron signalling are unknown. Using mixed cultures of rat hippocampal astrocytes and neurons we have determined functional consequences of elevating astrocyte calcium levels on co-cultured neurons. Electrical or mechanical stimulation of astrocytes to increase their calcium level caused a glutamate-dependent slow inward current (SIC) in associated neurons. Microinjection of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) into astrocytes to prevent the stimulus-dependent increase in astrocyte calcium level, blocks the appearance of the neuronal SIC. Pharmacological manipulations indicate that this astrocyte-dependent SIC is mediated by extracellular glutamate acting on N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors. Additionally, stimulation of astrocytes reduced the magnitude of action potential-evoked excitatory and inhibitory postsynaptic currents through the activation of metabotropic glutamate receptors. The demonstration that astrocytes modulate neuronal currents and synaptic transmission raises the possibility that astrocytes play a neuromodulatory role by controlling the extracellular level of glutamate.

Calcium elevation in astrocytes causes an NMDA receptor-dependent increase in the frequency of miniature synaptic currents in cultured hippocampal neurons.

Astrocytes exhibit a form of excitability and communication on the basis of intracellular Ca2+ variations (Cornell-Bell et al., 1990; Charles et al., 1991) that can be initiated by neuronal activity (Dani et al., 1992; Porter and McCarthy, 1996). A Ca2+ elevation in astrocytes induces the release of glutamate (Parpura et al., 1994; Pasti et al., 1997; Araque et al., 1998;Bezzi et al., 1998), which evokes a slow inward current in neurons and modulates action potential-evoked synaptic transmission between cultured hippocampal cells (Araque et al., 1998), suggesting that astrocytes and neurons may function as a network with bidirectional communication. Here we show that a Ca2+ elevation in astrocytes increases the frequency of excitatory as well as inhibitory miniature postsynaptic currents (mPSCs), without modifying their amplitudes. Thapsigargin incubation, microinjection of the Ca2+ chelator BAPTA, and photolysis of the Ca2+ cage NP-EGTA demonstrate that a Ca2+ elevation in astrocytes is both necessary and sufficient to modulate spontaneous transmitter release. This Ca2+-dependent release of glutamate from astrocytes enhances mPSC frequency by acting on NMDA glutamate receptors, because it is antagonized by D-2-amino-5-phosphonopentanoic acid (AP5) or extracellular Mg2+. These NMDA receptors are located extrasynaptically, because blockage specifically of synaptic NMDA receptors by synaptic activation in the presence of the open channel blocker MK-801 did not impair the AP5-sensitive astrocyte-induced increase of mPSC frequency. Therefore, astrocytes modulate spontaneous excitatory and inhibitory synaptic transmission by increasing the probability of transmitter release via the activation of NMDA receptors.

Astrocyte-induced modulation of synaptic transmission.

The idea that astrocytes simply provide structural and trophic support to neurons has been challenged by recent evidence demonstrating that astrocytes exhibit a form of excitability and communication based on intracellular Ca2+ variations and intercellular Ca2+ waves, which can be initiated by neuronal activity. These astrocyte Ca2+ variations have now been shown to induce glutamate-dependent Ca2+ elevations and slow inward currents in neurons. More recently, it has been demonstrated that synaptic transmission between cultured hippocampal neurons can be directly modulated by astrocytes. We have reported that astrocyte stimulation can increase the frequency of miniature synaptic currents. Furthermore, we also have demonstrated that an elevation in the intracellular Ca2+ in astrocytes induces a reduction in both excitatory and inhibitory evoked synaptic transmission through the activation of selective presynaptic metabotropic glutamate receptors.

Site directed mutagenesis of DNA polymerase I (Klenow) from Escherichia coli. The significance of Arg682 in catalysis.

We have reported that a domain containing Arg682 in the Klenow fragment of Escherichia coli DNA polymerase I (pol I) is important for the template-dependent dNTP-binding function [Pandey, V.N., Kaushik, N. A., Pradhan, D. S. & Modak, M. J. (1990) J. Biol. Chem. 265, 3679-3884]. In order to further define the role of Arg682 in the catalytic process, we have performed site-directed mutagenesis of this residue. For this purpose the Klenow-coding region of the DNA-pol-I gene was selectively amplified from the genomic DNA of E. coli and was cloned in an expression vector, pET-3a. This clone under appropriate conditions overproduces the Klenow fragment in E. coli. Using this clone (pET-3a-K) as the template, two mutant polymerase clones were constructed in which arginine has been replaced with either alanine, [R682A] pol I, or lysine [R682K] pol I. Both mutant enzymes showed significantly lower specific activity as compared to the wild-type enzyme. The kinetic analyses of the mutant enzymes indicated a 3-4-fold increase in the Km for the substrate dNTP, a 20-25-fold decrease in the Vmax and an overall decrease in the processive nature of DNA synthesis in both the mutant enzymes. The reverse mutation of Ala682 to the wild-type form Arg682 fully restored the processive nature and the polymerase activity of the enzyme. These observations suggest that the positively charged guanidino group in the side chain of Arg682 is catalytically important but not absolutely essential for synthesis of DNA. Furthermore it appears to maintain high processivity of the DNA synthesis catalyzed by the enzyme.

Modular structural elements in the replication origin region of Tetrahymena rDNA.

Computer analyses of the DNA replication origin region in the amplified rRNA genes of Tetrahymena thermophila identified a potential initiation zone in the 5'NTS [Dobbs, Shaiu and Benbow (1994), Nucleic Acids Res. 22, 2479-2489]. This region consists of a putative DNA unwinding element (DUE) aligned with predicted bent DNA segments, nuclear matrix or scaffold associated region (MAR/SAR) consensus sequences, and other common modular sequence elements previously shown to be clustered in eukaryotic chromosomal origin regions. In this study, two mung bean nuclease-hypersensitive sites in super-coiled plasmid DNA were localized within the major DUE-like element predicted by thermodynamic analyses. Three restriction fragments of the 5'NTS region predicted to contain bent DNA segments exhibited anomalous migration characteristic of bent DNA during electrophoresis on polyacrylamide gels. Restriction fragments containing the 5'NTS region bound Tetrahymena nuclear matrices in an in vitro binding assay, consistent with an association of the replication origin region with the nuclear matrix in vivo. The direct demonstration in a protozoan origin region of elements previously identified in Drosophila, chick and mammalian origin regions suggests that clusters of modular structural elements may be a conserved feature of eukaryotic chromosomal origins of replication.