A novel method has been developed for the preparation of nearly pure separate cultures of astrocytes and oligodendrocytes . The method is based on (a) the absence of viable neurons in cultures prepared from postnatal rat cerebra, (b) the stratification of astrocytes and oligodendrocytes in culture, and (c) the selective detachment of the overlying oligodendrocytes when exposed to sheer forces generated by shaking the cultures on an orbital shaker for 15-18 h at 37°C. Preparations appear >98% pure and contain -1-2 x 10' viable cells (20-40 mg of cell protein). Three methods were used to characterize these two culture types. First, electron microscopic examination was used to identify the cells in each preparation (mixed and separated cultures of astrocytes and oligodendrocytes) and to assess the purity of each preparation. Second, two oligodendroglial cell markers, 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3 .1 .4.37) and glycerol phosphate dehydrogenase (EC 1 .1 .1 .8) were monitored. Third, the regulation of cyclic AMP accumulation in each culture type was examined. In addition to these studies, we examined the influence of brain extract and dibutyryl cAMP on the gross morphology and ultrastructure of each preparation. These agents induced astroglial process formation without any apparent morphological effect on oligodendrocytes. Collectively, the results indicate that essentially pure cultures of astrocytes and of oligodendrocytes can be prepared and maintained . These preparations should significantly aid in efforts to examine the biochemistry, physiology, and pharmacology of these two major classes of central nervous system cells.The cellular heterogeneity of the central nervous system has largely prevented investigators from describing the biochemical characteristics of welldefined cell populations within this system . To circumvent this problem, we and others have directed our efforts toward developing new tech-890 niques that could be used to prepare homogeneous populations of neurons, oligodendrocytes, or astrocytes (1,4,7,13,15,20,25). The separation of cells in primary culture has a number of advantages over other methods often used to purify brain cells (20) . As a result, we have concentrated J . CELL BIOLOGY

To investigate the role of astrocytes in regulating synaptic transmission, we generated inducible transgenic mice that express a dominant-negative SNARE domain selectively in astrocytes to block the release of transmitters from these glial cells. By releasing adenosine triphosphate, which accumulates as adenosine, astrocytes tonically suppressed synaptic transmission, thereby enhancing the dynamic range for long-term potentiation and mediated activity-dependent, heterosynaptic depression. These results indicate that astrocytes are intricately linked in the regulation of synaptic strength and plasticity and provide a pathway for synaptic cross-talk.

During neuronal activity, extracellular potassium concentration ([Kϩ] out ) becomes elevated and, if uncorrected, causes neuronal depolarization, hyperexcitability, and seizures. Clearance of K ϩ from the extracellular space, termed K ϩ spatial buffering, is considered to be an important function of astrocytes. Results from a number of studies suggest that maintenance of [K ϩ ] out by astrocytes is mediated by K ϩ uptake through the inward-rectifying K ir 4.1 channels. To study the role of this channel in astrocyte physiology and neuronal excitability, we generated a conditional knock-out (cKO) of K ir 4.1 directed to astrocytes via the human glial fibrillary acidic protein promoter gfa2. K ir 4.1 cKO mice die prematurely and display severe ataxia and stress-induced seizures. Electrophysiological recordings revealed severe depolarization of both passive astrocytes and complex glia in K ir 4.1 cKO hippocampal slices. Complex cell depolarization appears to be a direct consequence of K ir 4.1 removal, whereas passive astrocyte depolarization seems to arise from an indirect developmental process. Furthermore, we observed a significant loss of complex glia, suggestive of a role for K ir 4.1 in astrocyte development. K ir 4.1 cKO passive astrocytes displayed a marked impairment of both K ϩ and glutamate uptake. Surprisingly, membrane and action potential properties of CA1 pyramidal neurons, as well as basal synaptic transmission in the CA1 stratum radiatum appeared unaffected, whereas spontaneous neuronal activity was reduced in the K ir 4.1 cKO. However, high-frequency stimulation revealed greatly elevated posttetanic potentiation and short-term potentiation in K ir 4.1 cKO hippocampus. Our findings implicate a role for glial K ir 4.1 channel subunit in the modulation of synaptic strength.

A long-standing question in neurobiology is whether astrocytes respond to the neuronal release of neurotransmitters in vivo. To address this question, acutely isolated hippocampal slices were loaded with the calcium-sensitive dye Calcium Green-1 and the responses of the astrocytes to electrical stimulation of the Schaffer collaterals were monitored by confocal microscopy. To confirm that the responsive cells were astrocytes, the slices were immunostained for the astrocytic marker glial fibrillary acidic protein. Stimulation of the Schaffer collaterals (50 Hz, 2 sec) resulted in increases in the concentration of intracellular calcium ([Ca 2ϩ ] i ) in the astrocytes located in the stratum radiatum of CA1. The astrocytic responses were blocked by the sodium channel blocker tetrodotoxin, the voltage-dependent calcium channel blocker -conotoxin-MVIIC, and the selective metabotropic glutamate receptor antagonist ␣-methyl-4-carboxyphenylglycine (MCPG). These results suggest that the astrocytic responses were induced by stimulation of metabotropic glutamate receptors on the astrocytes by neuronally released glutamate. The astrocytic responses to neuronal stimulation were enhanced in the presence of the K ϩ channel antagonist 4-aminopyridine (4-AP). Inhibition of the astrocytic responses in the presence of 4-AP required the presence of both MCPG and the ionotropic glutamate receptor antagonist kynurenic acid. These results suggest that higher levels of neuronal activity result in stimulation of both metabotropic and ionotropic glutamate receptors on the astrocytes. Overall, the results indicate that hippocampal astrocytes in situ are able to respond to the neuronal release of the neurotransmitter glutamate with increases in [Ca 2ϩ ] i .

Astrocytes comprise approximately half of the volume of the adult mammalian brain and are the primary neuronal structural and trophic supportive elements. Astrocytes are organized into distinct nonoverlapping domains and extend elaborate and dense fine processes that interact intimately with synapses and cerebrovasculature. The recognition in the mid 1990s that astrocytes undergo elevations in intracellular calcium concentration following activation of G protein-coupled receptors by synaptically released neurotransmitters demonstrated not only that astrocytes display a form of excitability but also that astrocytes may be active participants in brain information processing. The roles that astrocytic calcium elevations play in neurophysiology and especially in modulation of neuronal activity have been intensely researched in recent years. This review will summarize the current understanding of the function of astrocytic calcium signaling in neurophysiological processes and discuss areas where the role of astrocytes remains controversial and will therefore benefit from further study.

The concept that astrocytes release neuroactive molecules (gliotransmitters) to affect synaptic transmission has been a paradigm shift in neuroscience research over the past decade. This concept suggests that astrocytes, together with pre- and postsynaptic neuronal elements, make up a functional synapse. Astrocyte release of gliotransmitters (for example, glutamate and adenosine triphosphate) is generally accepted to be a Ca2+-dependent process. We used two mouse lines to either selectively increase or obliterate astrocytic Gq G protein-coupled receptor Ca2+ signaling to further test the hypothesis that astrocytes release gliotransmitters in a Ca2+-dependent manner to affect synaptic transmission. Neither increasing nor obliterating astrocytic Ca2+ fluxes affects spontaneous and evoked excitatory synaptic transmission or synaptic plasticity. Our findings suggest that, at least in the hippocampus, the mechanisms of gliotransmission need to be reconsidered.

Spontaneous neurotransmitter release and activation of group I metabotropic glutamate receptors (mGluRs) each play a role in the plasticity of neuronal synapses. Astrocytes may contribute to short-and long-term synaptic changes by signaling to neurons via these processes. Spontaneous whole-cell AMPA receptor (AMPAR) currents were recorded in CA1 pyramidal cells in situ while evoking Ca 2ϩ increases in the adjacent stratum radiatum astrocytes by uncaging IP 3 . Whole-cell patch clamp was used to deliver caged IP 3 and the Ca 2ϩ indicator dye Oregon green BAPTA-1 to astrocytes. Neurons were patch-clamped and filled with Alexa 568 hydrazide dye to visualize their morphological relationship to the astrocyte. On uncaging of IP 3 , astrocyte Ca 2ϩ responses reliably propagated as a wave into the very fine distal processes, synchronizing Ca 2ϩ activity within astrocyte microdomains. The intracellular astrocyte Ca 2ϩ wave coincided with a significant increase in the frequency of AMPA spontaneous EPSCs, but with no change in their kinetics. AMPAR current amplitudes were increased as well, but not significantly ( p ϭ 0.06). The increased frequency of AMPAR currents was sensitive to the group I mGluR antagonists LY367385 and 2-methyl-6-(phenylethynyl)-pyridine, suggesting that (1) astrocytes released glutamate in response to IP 3 uncaging, and (2) glutamate released by astrocytes activated group I mGluRs to facilitate the release of glutamate from excitatory neuronal presynaptic boutons. These results extend previous studies, which have shown astrocyte modulation of neuronal activity in vitro and suggest that astrocyte-to-neuron signaling in intact tissue may contribute to synaptic plasticity.

Astrocytes are considered the third component of the synapse, responding to neurotransmitter release from synaptic terminals and releasing gliotransmitters--including glutamate--in a Ca(2+)-dependent manner to affect neuronal synaptic activity. Many studies reporting astrocyte-driven neuronal activity have evoked astrocyte Ca(2+) increases by application of endogenous ligands that directly activate neuronal receptors, making astrocyte contribution to neuronal effect(s) difficult to determine. We have made transgenic mice that express a Gq-coupled receptor only in astrocytes to evoke astrocyte Ca(2+) increases using an agonist that does not bind endogenous receptors in brain. By recording from CA1 pyramidal cells in acute hippocampal slices from these mice, we demonstrate that widespread Ca(2+) elevations in 80%-90% of stratum radiatum astrocytes do not increase neuronal Ca(2+), produce neuronal slow inward currents, or affect excitatory synaptic activity. Our findings call into question the developing consensus that Ca(2+)-dependent glutamate release by astrocytes directly affects neuronal synaptic activity in situ.