Neuron,
Vol. 8, 429-440, March, 1992, Copyright
0 1992 by Cell Press
Neuronal Activity Triggers Calcium Waves in Hippocampal Astrocyte Networks John W. Dani, Alex Chernjavsky, and Stephen J Smith Department of Molecular and Cellular Beckman Center Stanford University School of Medicine Stanford, California 94305-5426
Physiology
Summary The recent discovery that the neurotransmitter glutamate can trigger actively propagating CaZ+ waves in the cytoplasm of cultured astrocytes suggests the possibility that synaptically released glutamate may trigger similar Ca*+ waves in brain astrocytes in situ. To explore this possibility, we used confocal microscopy and the Ca*+ indicator fluo-3 to study organotypically cultured slices of rat hippocampus, where astrocytic and neuronal networks are intermingled in their normal tissue relationships. We find that astrocytic Ca*+ waves are present under these circumstances and that these waves can be triggered by the firing of glutamatergic neuronal afferents with latencies as short as 2 s. The Ca*+ waves closely resemble those previously observed in cultured astrocytes: they propagate both within and between astrocytes at velocities of 7-27 wm/s at 21°C. The ability of tissue astrocyte networks to respond to neuronal network activity suggests that astrocytes may have a much more dynamic and active role in brain function than has been generally recognized. introduction Astrocytes of the cortical gray matter have elaborate dendritic morphologies, superficially similar to those of neurons, and their fine processes mingle intimately with those of neurons throughout the synaptic neuropil. These cells actually outnumber neurons in many brain regions and are interconnected by gap junctions into vast networks. While these structural features hintat signalingoreven information-processing functions, astrocytes have traditionally been assigned relatively passive, background roles in structural, metabolic, and trophic support of neurons. This view arose and persisted mainly because there has been no evidence in astrocytes for the key property underlying signaling by neurons, namely electrical excitability. Very recently, however, studies of cultured astrocytes have suggested that these cells may possess an alternative form of excitability based directly on intracellular Ca*+ dynamics, essentially independent of plasma membrane potential (Cornell-Bell et al., 1990; Cornell-Bell and Finkbeiner, 1991). Several lines of investigation point toward the inadequacy of viewing the astrocyte as a passive supporting cell. For instance, there is growing evidence that the astrocytic roles in K+ buffering and neurotransmit-
ter metabolism may be actively regulated (Karwoski et al., 1989; Marrero et al., 1989; Barbour et al., 1989; Nicholls and Attwell, 1990). In addition, astrocytes have been shown to possess functional receptors for a wide range of the same neuroactive agents that support intercellular signaling within neuronal networks (for reviews, see Dermietzel et al., 1991; Barres, 1989; Laming, 1989; Kimelberg and Norenberg, 1989; Vernadakis, 1988; Stewart and Rosenberg, 1979; see also Calambos, 1961). For instance, there have been numerous reports of responses by cultured astrocytes to the ubiquitous excitatory neurotransmitter glutamate (Pearce et al., 1986; Enkvist et al., 1989; Cornell-Bell et al., 1990; Glaum et al., 1990; Ahmed et al., 1990; Jensen and Chiu, 1990, 1991; Charles et al., 1991) and other neurotransmitters (Sugino et al., 1984; Schieren and MacDermott, 1988; Salm and McCarthy, 1990; lnagaki et al., 1991). Several of these studies have established that glutamate and other neurotransmitters can trigger both Ca*+oscillations and propagating Ca*+waves, indicative of a Ca*+-based form of excitability (Cornell-Bell et al., 1990; Jensen and Chui, 1990). The Ca*+-based excitability of cultured astrocytes appearsto be based primarily on the regulated release of Ca*+ ions from intracellular stores (Cornell-Bell and Finkbeiner, 1991). Such activity can take the form of waves of elevated cytosolic Ca*+ that propagate both within individual astrocytes and between cells within networks of astrocytes that have grown to confluence (Cornell-Bell et al., 1990). Ca2+waves resembling those observed in astrocytes have been observed in many other cell types (see Jaffe, 1983)and tissues (Sanderson et al., 1990). The underlying mechanisms are probably conserved across many of these examples (Berridge, 1990; Jacob, 1990; Meyer, 1991). Within astrocytes, intracellular and intercellular Ca*+ waves have similar velocities, ranging between 6 and 30 pm/s at 21% The numerousgap junctions that form between astrocytes in confluent culture (Dermietzel et al., 1991; Kettenmann and Ransom, 1988) provide a likely avenue for intercellular Ca*+ wave propagation. Astrocyte Ca*+ waves are often oscillatory, with periods in the range of IO-30 s, but the form of such oscillations is quite variable and depends upon how the cells are activated (Cornell-Bell et al., 1990; Cornell-Bell and Finkbeiner, 1991). As noted above, the amino acid glutamate has been identified as an effective stimulus for Ca*+ excitation in cultured astrocytes. Since glutamate is thought to be the major excitatory neurotransmitter within the brain and since astrocyte membranes juxtapose or ensheathe most synapses in the mammalian CNS (Peters et al., 1991), it seems possible that synaptically released glutamate might initiate Ca*+ waves within brain astrocytes in situ. Because astrocytes within the CNS, as in culture, are extensively coupled by gap junctions (Peters et al., 1991), there is also the possibil-
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ity that astrocytic Ca2+ waves might propagate for long distances through brain tissue. Finally, as cytoplasmic Ca’+ signals have come to be recognized as ubiquitous regulators of cellular function, one might expect that neuronally triggered Ca2’ signaling in brain astrocyte networks could have substantial functional significance. We therefore felt that it was important to determine whether Ca2+ waves, like those described in cultured astrocytes, actually do occur within brain astrocytes in situ and whether synaptically released neurotransmitters are sufficientto trigger such waves.
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Results We used organotypicallycultured hippocampal slices (Gahwiler, 1988,19&l, 1981) and a laser confocal microscope in order to obtain clear fluorescence images of live astrocytes within a synaptic neuropil environment. Neurons and astrocytes in these slices were readily loaded with the fluorescent Ca2+ indicator flue-3 using the acetoxymethyl ester derivative (Kao etal.,1989;Mintaetal.,1989;Tsien,1988).Theconfocal microscope was equipped for time-lapse video recording. With these methods, we were able to monitor the dynamics of cytosolic Ca*’ in both neurons and astrocytes deep within the organotypic slice. In preliminary work, we tried similar procedures using freshly prepared hippocampal slices from adult rats, but were unable to obtain useful optical signals. Flue-3 reports cytosolic Ca2+ increases by increases in fluorescence intensity. Thefluo-3 images presented here use the gray scale from black to white to encode either total fluorescence or increases over baseline fluorescence, as specified in the figure legends. With our methods, quantification of the flue3 signals in terms of cytosolic Ca2’ concentration would be both difficult and uncertain. Fortunately, such quantification was not necessary to interpret the Ca2+dynamics, which are the subject of the present study. Low Frequency Stimulation of Dentate Cyrus Elicits Both Neuronal and Astrocytic Responses within Area CA3 As diagrammed in Figure 1, current from an extracellular pipette electrode was used to stimulate electrically mossy fibers originating in dentate gyrus, while Ca2+ signals were visualized in cells within region CA3, at a site 1.5-3 m m distant from the stimulating electrode. This target area is rich in mossy fiber varicosities known to release glutamate at giant synapses onto CA3 pyramidal cells. All effects of electrical stimulation described below were found to be blocked by 1 uM tetrodotoxin, confirming that axonal impulse conduction between dentate and CA3 was necessary to elicit these responses. Low frequency stimulation (8 Hz) of dentate generated very prompt cytoplasmic Ca*+ increases within cells of region CA3: the earliest responses were evident within milliseconds of the first action potential volley. These early Ca2+ responses were observed in
Figure 2. Positioning of Stimulating within the Hippocampal Slice
Electrode
and Imaged Area
Diagram showing positions of stimulating electrode (Stim) and the imaged areas shown in (Figures 2A-2F) (Image) with respect to overall anatomy of a hippocampal slice and the course of a typical mossyfiherfmf)fromdentatethrough regionCA3.Within the rectangular imaged area, the loci of a typical CA3 pyramidal cell (p), the pyramidal cell body palisade (dotted outlines), and a typical gray matter astrocyte !a) are also indicated. Generally similar arrangements for stimulation and recording were employed in all experiments described in this paper. Drawing modified from Shepherd (1974).
axons and varicosities of the mossy fibers and in the cell bodies and dendrites of the CA3 pyramidal neurons,aswellasin numerousothersmall neuronalcells and processes (Figure 2). As sampled by our imaging system, the neuronal Ca*+ increases usually reached steady levels within a few seconds of sustained 8 Hz stimulation (e.g., see trace 6 in Figure 3). After several seconds of sustained dentate stimulation, a second population of cells began to exhibit large increases in Ca2+ (fluorescence increases ranged from 50%-200%, measured over areas like those indicated by the solid boxes in Figure 2B). It was this population of cells that were retrospectively identified as astrocytes by correlative glial fibrillary acidic protein (GFAP) staining (see Figure 2F). As shown in Figures 2E and 2F, the cell bodies identifiable as astrocytes were found mainly in strata outside the pyramidal layer (i.e., stratum radiatum, lucidum, and oriens), although a few astrocyte cell bodies were interspersed among those of the pyramidal neurons. This is precisely the anatomical distribution expected for hippocampal tissue (Del Rio et al., 1991; Faddis and Vijayan, 1988; Beach et al., 1982). Astrocytes Exhibit Ca2+ Oscillations in Response to Continuous Neuronal Stimulation With sustained neuronal stimulation, astrocyte cell bodies often exhibited irregularly oscillating cytoplasmic Ca2’- concentrations. In addition, oscillations
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Figure 2. Electrical CA3
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(A) Average fluorescence of the flue-3.loaded slice observed during 32 frames of electrical stimulation. (B) Baseline fluorescence of the same slice observed prior to stimulation (averaged over 32 frames). Dashed rectangles indicate subareas sampled for time series images in (C) and (D). Solid boxes represent areas over which fluorescence intensities expressed in Figure 3 were averaged. (C) Time sequence showing Ca2+ oscillations within a cell (arrow) identifiable by GFAP staining as an astrocyte. Each time point was numbered relative to the time of the last prestimulus frame and shows the change in fluorescence over baseline measurements. (D) Time sequence, as in (C), showing Ca* oscillations within two astrocytic processes (arrow and arrowhead). The cell body from which over this sequence. the process marked by the arrowhead emerges also exhibits Caz+ oscillation (E) A Nomarski DIC image taken from the same field as the previous fluorescence images. Notice the palisading organization of pyramidal cell bodies in the right half of the field and the clear boundary formed with stratum lucidum to the left. (F) GFAP immunocytochemical stain corresponding to the same field as in (B). Arrows and arrowhead point to CFAP-positive cells designated as astrocytes in (C) and (D). Bar, 20 urn.
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A few examples of such delayed soiitary spikes are evident on the decay tails between 600 and 900 s in traces 1-4 of Figure 3.
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Figure 3. Ca I+ Fluctuations Observed within Identified Cells of Area CA3 following Dentate Stimulation (Same Experiment as Figure 2) Fluorescence change signals measured over image areas indicated by boxes in Figure 26, before, during, and after an electrical stimulus delivered between times indicated.as O-600 s. For clarity, these curves are shifted arbitrarily along the ordinate axis. The first five curves show the irregularly oscillatory Ca”+ responses typical of astrocytes; the sixth curve shows the steadier plateau response typical of neurons. At the end of stimulation (600 s), Ca2+ concentrations return to resting levels over a period of about 300 s.
were observed within numerous CFAP-positive (astrocytic) processes located throughout the slice. These clearly discernible oscillations in astrocytic Ca2+ signals contrasted with the apparently steady Ca2+ increases observed in the vast majority of GFAPnegative (presumably mostly neuronal) processes. As one example, compare the oscillating Ca2+ levels evident in the astrocytic processes and cell body in the lower halves of the time series of images in Figure 2D with the steady growth and stabilization of fluorescence in the CFAP-negative (probably neuronal) process that zigzags across the middle of those frames. For another perspective on the differing dynamics of astrocytic and neuronal Ca2+ increase, compare traces l-5 in Figure 3, measured from astrocytes identified by retrospective immunohistochemistry, with trace 6, measured from a pyramidal cell body. Though the periods of such astrocytic Ca*+ oscillations (mean = 22 s, range 16-30 s; 22 cells, three experiments) were generally in the same range as the glutamate-induced oscillations observed in cultured astrocytes, periods of the oscillations in slices exhibited more variability from cell to cell, and the oscillations were much less regular (contrast Figure 3 with Figure 1 in Cornell-Bell et al., 1990). Upon termination of nerve fiber stimulation, both neuronal and astrocytic populations returned to resting Ca 2+ levels with time courses extending over a few minutes (see Figure 3). Astrocytes often exhibited a tendency toward persistent generation of solitary or repetitive CaZ’ spikes in the aftermath of stimulation.
Astrocytic Responses to Neuronal Stimulation Can Be Prompt Figure 4 demonstrates that the minimum latency between mossy fiber stimulation and astrocyte Ca2+ response can be less than 2 s. Figure 4A shows the resting level of flue-3 fluorescence apparent in one optical section. Figure 4B shows the retrospective immunostain used to identifyastrocyteswithin thissame optical section. A burst of high frequency dentate stimulation (50 Hz for 6 s) was begun after the time point indicated as 0 s in Figure4C. Traces l-5 in Figure 4C show Ca2’signals measured from image areas positioned over astrocyte cell bodies or processes, and trace 6 shows a Ca2’ signal measured over one of the pyramidal neuron cell bodies. As indicated bythedata plotted in trace 6, neuronal responses grew to nearly maximal levels during the 1 s period over which this frame was scanned. Figure 4D illustrates the appearance of the image scanned during the onset of neuronal stimulation. This image, consisting of 480 horizontal lines, was scanned at 500 lines per s over a total period of approximately 1 s, beginning at the top. This scanning process creates an implicit time axis extending from the top to the bottom of the frame. The scan line active at the precise time of stimulus onset (approximately 120 ms after beginning the scan) is indicated by the flanking horizontal arrows. The distinct boundary between dark and light just below this scan line reflects a Ca*+ increase in fine neuronal processes that occurs within 4-6 ms of stimulus onset. Figure 4E shows a similar scan beginning exactly 2 s after that shown in Figure 4D. By this time, large CaL+ increases are evident over the entire frame. Astrocyte cell bodies (arrows) and a process (arrowhead) identified from the GFAP stain shown in Figure 4B are indicated in Figures 4E and 4F. The large astrocytic Ca*’ signals evident in Figure 4E are a clear indication that astrocytes can respond to neuronal activity within 2 s or less. A similar conclusion can be drawn from traces l-5 in Figure 4C, with the caveat that the astrocyte traces l-5 surely include fluorescence increases from fine neuronai processes (see measurement boxes in Figure4A and the first poststimuius frame, Figure4D). Astrocytes Display Both Intracellular and intercellular Ca*+ Waves following Neuronal Stimulation Ca*‘waves propagating both within and between individual astrocytes were observed in most of our experiments. Figure 5A shows a time series of images illustratingwave propagation within an astrocytic process (or perhaps a small bundle of parallel processes), and Figure 5B shows measurements of fluorescence as a function of time at several points along the progress of the wave. The phase-shifted, but otherwise similar, nature of these waveforms demonstrates the wave-like nature of the underlying signal. At room
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Figure 4. High Frequency CA3 Astrocytes
Ca2+ Waves
(50 Hz) Electrical
Stimulation
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(A) Resting fluorescence observed within area CA3 after fluo-3 loading (average of 32 frames). Stratum pyramidale lies to the left; stratum lucidum lies to the extreme right. Solid boxes indicate areas where fluorescence intensity measurements were taken for (C). (6) CFAP immunofluorescence image showing the same field as (A). The arrows and arrowhead mark GFAP-positive cell bodies and a process, respectively, that lie within the measurement boxes l-5 shown in (A). (C) Flue-3 fluorescence measured over both astrocytes (traces l-5) and a neuron (trace 6) during CaZ+ responses to dentate stimulation. Traces are shifted arbitrarily along the ordinate axis for clarity. (D-F) Fluorescence changes observed at several time points following electrical stimulation. fD) The earliest response to electrical stimulation. The horizontal arrows indicate the horizontal scan line active at the time of stimulus onset. (See text for more details.) Numerous fine processes and pyramidal cell bodies exhibit Caz+ increases. (E) After 2 s more of stimulation (t = 4 s), the pyramidal cell bodies exhibit large cytosolic Ca*+ Increases, especially within their nuclei. Many GFAP-positive cell bodies and processes (e.g., arrows and arrowhead, same positions as in [B]) also now exhibit substantial cytosolic Ca2+ increases. (F) After 4 s of stimulation (t = 6 s), nearly every astrocyte within the field has responded. Again, the position of the arrow corresponds to the same arrow marking a GFAP-poshive cell body in (B). Bar, 20 Pm.
temperature (21°C), intracellular wave velocities ranged from 7-27 pm/s (mean = 15 vm/s; 5 cells; three experiments) and encompassed both cell bodies and processes. Such waves were observed to propagate through processes both away from and toward astrocytic cell bodies. Ca*+ waves were often observed to propagate from astrocyte to astrocyte. Such intercellular Ca*+ waves could be followed only over short distances (e.g., 2-3 astrocyte diameters) in most of our recordings, but longer range waves were sometimes observed with the most intense stimuli. While it was difficult to discern any patterning of the intercellular Ca*+ waves when viewed in real time, strikingly intricate and varied patterns of wave propagation were apparent when Ca2+ images were visualized by video playback of time-lapse recordings (time compression of lO:l-1OO:l). The intercellular wave propagation patterns are difficulttodescribeinanysuccinctway: they haveagenerally chaotic appearanceand seem to be highly variable both within and between individual slices.
Although electrical stimulation was able to elicit intercellular Ca*+ waves, this phenomenon was most notable when preparations were stimulated by bath application of N-methyl-o-aspartate (NMDA), as shown in Figure 6. It seems likely that such astrocytic Ca*+ waves are an indirect result of NMDA application, mediated by the release of glutamate from NMDAstimulated neurons. While NMDA is known for its potent excitatory effects on neurons (Wood et al., 1990; Thomson, 1989; Llano et al., 1988), no direct actions on isolated astrocytes have been described. This latter result is in spite of numerous reported attempts to demonstrate astrocytic NMDA responses (CornellBell et al., 1990; Cornell-Bell and Finkbeiner, 1991; Bevan, 1990; Pearce et al., 1986). In our experiments, NMDA was found to initiate prompt increases in neuronal Ca*+, which were invariably followed by the propagation of Ca*+ waves between cells identifiable as astrocytes. The intercellular Ca*+ waves observed after NMDA application often spread coherently throughout entire hippocampal ar-
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Seconds Frgure 5. Intracellular Ca 2+ Waves Are Observed CA3 Astrocytes during 8 Hz Electrical Stimulation
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(A)Time series showing the fluorescence changeswithin a single astrocyte or, perhaps, a small bundle of astrocytic processes. Each image represents the increase in f!uorescence over baseline. Beginning with t = 30 s after stimulus onset, the Ca” wave has not yet moved within the field of view. The Ca2’ wave first appears as a faint rise in fluorescence within several processes toward the right edge (t = 38 s). At t = 40 s, the position of the wave front has clearly moved toward ihe left. By t = 42 s, the wave, which has spread almost to the cell body, encompasses several more processes. In addition, the CaJ’ concentration has continued to rise, indicating that this is a nonuniform Caz+ wave. Finally at 44 s, the wave reaches the astrocyte cell body and initiates a Ca2+ spike. Boxes are drawn over the regions where fluorescence measurements were made for (B). (B) Measurements of fluorescence changes at the six positions indicated by numbered boxes (A). Each waveform is normalized to a constant peakamplitude. Waveformsareshiftedarbitrarilyalongtheordinate axis for clarity. The second set of Ca*+ peaks represents the Ca2+ wave shown in (A). The phase shifts among these curves (delay increases progressively by a total of about 4 s between curve 1 and curve 6) demonstrate wave-like propagation of this astrocytic Ca2+ signal. Other differences among these six waveforms (e.g., faster rise times in curve 6 than in curve 1) probably reflect the transition between elongated processes and cell body. Bar, 10 urn.
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Seconds Figure 6. Long-Distance(IntercelIular)AstrocyteCaz’ WavesAre Observed in Organotypic Hippocampal Slices following the Application of NMDA, an Agent Thought to Stimulate Neurons Selectively and Trigger Synaptic Glutamate Release (See Text) (A) Time series showing increases over baseline fluorescence in area CA3 following perfusion with 20 PM NMDA plus 2 PM glytine in Mg *+-free saline. Each image represents the increase in fluorescence over baseline measurements at 14 s, 26 s, 34 s, 52 s, and 64 s after NMDA application. The Ca2+ wave enters the field of view at the top of the image (t = 14 s) and spreads down and toward the left (t = 26-64 s). The boxes drawn in the last imageft = 64s)indicate15adjacentsubareasfromwhichfluorescent measurements were made for (B). (B) increases in flue-3 fluorescence at the 15 subareas indicated in (A). The topmost trace corresponds to the topmost box in (A) and so on. The progressive phase shifts evident in this sequence of traces indicate the propagation of awave from top toward bottom subareas at a velocity of approximately 9.6 bm/s. Bar, 100 Wm.
t = 64s
eas. The involvement of approximately 50 astrocytes was ascertained from inspection of the image sequence illustrated in Figure 6. At room temperature, intercellular wave velocities ranged from 8-10 pm/s for both electrical and NMDA stimulation (several hundred cells, four experiments). We saw no indications of a delay as waves propagated from one astrocyte to the next. Discussion A CaZ+-Based Form of Cellular Excitability We have shown that neuronal activity can trigger Ca2+ oscillations and waves in astrocytes and their networks within cultured hippocampal slices. While earlier studies have shown prompt changes in glial membrane potential in response to neuronal activity, these effects were identified as passive glial responses to neuronal K+ efflux (Orkand et al., 1966; Trachtenberg and Pollen, 1970). In contrast, the astrocytic Ca*+ responses described here are characterized by oscillations and nondecremental spatial propagation. These astrocytic Ca2+ responses can therefore be considered expressions of a distinctive form of cellular excitability. Mechanism of the Cal+ Wave Phenomenological similarities between the astrocytic Ca2+ signals described in this paper and those described in earlier studies of cultured astrocytes strongly suggest that the same underlying mechanisms are at work. Most notably, the astrocytic Ca*+ waves reported here propagate at velocities very similar to those described in astrocyte cultures. There are also similarities in the observed patterns of oscillation, although the oscillations in the hippocampal slices tend to be less regular than those observed in the astrocyte cultures. The relative irregularity of astroin the brain slice may be due cytic Ca2+ oscillation to the complexity of astrocytic networks within such tissues and/or due to perturbing neuronal influences. The basic mechanisms of Ca2+ excitability in cultured astrocytes and in many other cell types are currently under investigation by many laboratories (for reviews see Cornell-Bell and Finkbeiner, 1991; Meyer, 1991; see also Bezprozvanny et al., 1991; Finch et al., 1991). In brief, it appears that such Ca*+ excitability is primarily a consequence of the regenerative release of Ca2+ from endoplasmic reticulum. Coupling of Neuronal Activity to Astrocytic Ca2+ Responses While all of the presently available evidence supports our original notion that synaptically released glutamate might trigger astrocytic Ca2+ waves, we have not conclusively proven that this particular agent is the proximal stimulus tothe neuropil astrocyte. Certainly, however, glutamate is the primary neurotransmitter released by the mossy fiber afferents stimulated in our experiments, and there is indeed a very strong
resemblance between the Ca2+ waves reported here and those observed in cultured astrocytes exposed directly to glutamate. In addition, we have found that the glutamate receptor antagonist kynurenic acid (Robinson et al., 1985, 1984) at 3 mM blocks all astrocytic responses to neuronal stimulation (unpublished data). it is still remotely possible, however, that synaptic glutamate acts via some effect secondary to its activation of neurons intrinsic to area CA3. For instance, activation of these intrinsic neurons might stimulate astrocytes through release of K+, or some other neurotransmitter or coreleased factor such as ATP. Such indirect actions seem relatively unlikely, but more work will be necessary to evaluate critically this classof possibilities. We hasten to pointout nonetheless that the most intriguing functional questions raised by the present description of neuronal-gliai signaling are independent of the precise identity of the proximal chemical mediator involved. The Organotypic Slice As an Experimental Model The observations described here were made using organotypic hippocampal slices. As a model system for the study of neuronal-glial interactions, such slices have many advantages, including being in avery stable physiological condition and providing excellent access for optical recording methods. Acute brain slice preparations are substantially less attractive for optical study due to the presence of very large numbers of dead cells, especially near the cut surfaces, and due to a much greater difficulty in applying the fluo 3-AM loading procedure (see Yuste and Katz, 1991). Furthermore, the physiological condition of acute brain s!ices is far less stable: they typically die within 24 hr or so of initial preparation. On the other hand, the fact remains that neuronal or astrocytic properties may change during the period of organotypic culture stabilization. Although many aspects of normal synaptic function and neuronal and glial ultrastructure have been shown to be maintained for long periods in organotypic culture(Zimmer and CPhwiler, 1984; Zhabotinski et al., 1979; Del Rio et al., 1991), it remains to be determined whether astrocytic Ca2+ waves are stimulated by neuronal activity in vivo. To resolve this important question, it may be necessary to repeat experiments similar to those described here within acute slices and intact animals. Possible Effector Actions of the Caz+ Wave We can begin to ponder the possible physiologicaT actions of astrocytic Ca 2+ transients like those described here. One of the most intriguing possibilities is that neuronally induced astrocytic Ca*’ signals may feed back to influence neuronal excitability or regulate synaptic transmission. Such effects might be mediated through changes in extracellular ion concentrations (effects on extracellular K+ and Ca*’ are most likely to be of physiological importance; Barres et al., 1990; Quandt and MacVicar, 1986; O&and et al., 1966; Olson et al., 1990; Ciardo and Meldolesi, 1990; MacVic-
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ar and Tse, 1988; Benjamin, 1987), or through changes in neurotransmitter metabolism (Vernadakis, 1988; Laming, 1989). Astrocytic Ca2+ could exert such influences via Ca2+-dependent membrane transport mechanisms (i.e., ion channels or exchangers) or through actions of cytoplasmic metabolic or regulatory enzymes (Laming, 1989; Stryer, 1988; Alberts et al., 1989). Astrocytes are also capable of secreting neuroactive substances (e.g., peptides, amino acids, eicosanoids, and nitric oxide; Pasantes-Moraleset al., 1990; Murphy et al., 1988, 1990; Ehrenreich et al., 1991; Benveniste et al., 1990; Shinoda et al., 1989), and such secretion processes are likelytargets for Ca2+ regulation. In this context, it is also interesting to note that there have been indications that astrocytes may regulate synaptic plasticity (Sastry et al., 1990; Muller and Best, 1989). There is an additional range of possible targets for astrocyte Ca2+ signaling that might be somewhat less immediate but equally important in their impact on neuronal function. Astrocytes contain the brain’s major glycogen stores and are very likely to play a dynamic role in meeting neuronal energy demands (Vernadakis, 1988). There are reasons to believe that astrocytic energy metabolism may be regulated by intracellular Ca2+ (Pearce at al., 1988; Arbones et al., 1990). Astrocytes are also likely to exert profound effectsoncerebralvascularfunctionviatheirprominent and highly specialized contacts onto blood vessels (Peters et al., 1991; Raff and Lillien, 1988; Janzer and Raff, 1987; Bradbury, 1985). It is quite possible that the astrocytic-vascular interaction is somehow regulated by astrocytic Ca2+. Finally, cytoplasmic Ca2+ may regulate changes in astrocytic gene expression (Morgan and Curran, 1986, 1988; Arenander et al., 1989; Condorelli et al., 1989) and mitotic responses (Hart et al., 1989; Neary et al., 1987). Biological Significance The long-distance Ca2+ waves described here suggest that gray matter astrocytes may function as an active signaling network. At present we can only speculate on the possible function or informational content of astrocytic Ca2+ signals, but a general analogy between glial Ca2+ excitability and neuronal electrical excitability may be noted. The capability of neural networks to process information is thought to be fundamentally dependent on the neuron’s underlying electrical excitability. Astrocyte networks might possess similar capabilities as a consequence of Ca2+-based excitability. Thus, in spite of very different temporal and spatial domains of astrocytic and neuronal signaling, the possibility arises that astrocytic networks, like neuronal networks, may process information. The slowness of astrocytic Ca2+ signaling does not necessarily diminish a possible informational role: astrocytic Ca2+ responses are no slower than most actions of the wellknown biogenic amine and peptide neuromodulators. We have shown that neuronal activity can trigger active and elaborate astrocytic Ca2+ signals. If these astrocytic Ca2+ signals somehow feed back to
influence neuronal excitability or synaptic transmission, astrocytes could participate in brain information processing to an extent comparable with the participation of aminergic and peptidergic neurons. In other words, the current conception that neuronal networks alone provide the basis for the brain’s computational function may need to be expanded to encompass interacting neuronal and astrocytic networks. Experimental
Procedures
Preparation of Organotypic Hippocampal Slices Organotypic hippocampal slices were made from postnatal day 7 Sprague-Dawley rat pups according to established procedures (Clhwiler, 1988,1984,1981). Briefly, slices (300-400 Rm thickness) were cut from the middle two-thirds of the hippocampus and attached to rectangular coverslips (11 x 22 mm) using a plasma clot (chicken plasma and bovine thrombin). Coverslips were then transferred singly to sterile glass tubes having an internal diameter slightly larger than the width of the coverslips. One milliliter of rollertube medium (50% Earle’s minimal essential medium, 25% Hanks’ balanced salt solution, 25% horse serum, 10 m M HEPES, 6 mg/ml dextrose, 0.292 mglml t-glutamine) was added to each culture tube, which was then sealed with a plastic cap and placed in a rotating drum at 37X Slices were maintained for l-2 weeks in this manner prior to experimentation: by this time, slice thickness was typically reduced to about 100 pm. Flue 3-AM loading Organotypic slices were loaded with flue-3 by incubating for 2 hr in rollertube culture medium containing 110 PM fluo 3.AM (Kao et al., 1989; Minta et al., 1989; Tsien, 1988). Experiments were performed with coverslips containing the tissue slices mounted in a flow-through perfusion chamber. The standard saline used in these experiments was 137 m M NaCI, 5.3 m M KCI, 3 m M CaCl,, 1 m M MgCI,, 10 m M HEPES, and 25 m M dextrose, with modifications or drug additions as specifically noted. All experiments described here were carried out at room temperature fapproxlmately 21°C), but exploratory experiments indicated that effects qualitatively similar to those we describe occur at highertemperatures(upto37V). Highertemperatureswerenot used routinely because the Ca2+ signals observed became too rapid to characterize accurately with our instrumentation. Image Acquisition The histology of organotypically cultured hippocampal slices most closely resembles that of hippocampus in situ when examined midway between the topand bottom section planes. Confocal microscopy was used in this study to isolate these most favorable tissue planes by optical sectioning. All observations reported herewere made at optical sections approximatelyequidistant from the top and bottom slice surfaces (i.e., at a depth of approximately 60 wrn within a 120 pm thick slice). Images were acquired using a modified laser confocal microscope (Bio-Rad MRC-500) and stored at intervals of one frame every 2 s on an optical memorydiskvideo recorder (PanasonicTQ-2028F). Modifications to the MRC-500 included a faster shutter to eliminate unnecessary light exposure, high reflectance dielectric mirrors, an improved analog sampling filter, a motorized stage to relocate specific fields of interest, and a silicon photodiode to capture transmitted laser light for Nomarski DIC imaging. Image Processing The intracellular Ca2+ dynamics described in this report are extremely vivid when raw fluorescence image sequences are viewed bytime-lapsevideo playback. When the same raw images are displayed as stills, however, these inherently dynamic phenomena become far more difficult to visualize. It was therefore necessary to use digital image enhancement and analysis methods to render our image data for print publication. The images described in this paper as”fluorescence increases”werederived
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bydigitallysubtracting imagesofthe”resting”fluorescencefrom images collected at specified time points before, during, or after stimulation. To minimize noise in the processed images, the resting fluorescence images were usually derived by averaging 16 or 32 frames collected during the period of steady fluorescence priorto stimulation. To preserve dynamic information, no averaging was performed over the individual time point frames. The resulting difference images were then offset and scaled by arbitrary factors so as to make use of the entire gray scale available through the final print rendering. Arbitrary spatial low pass filtering was used to achieve a favorable trade-off between signal-to-noise ratio and fine image detail in the final renderings. Across any set of images presented as a time series (e.g., Figure 20, however, all image processing parameters were held constant. Upon request and payment for duplication and shipping, the authors will provide a VHS videocassette representing the primary observations in the more vivid time-lapse playback form. For several reasons, quantitative calibration of fluorescence images in terms of cytosolic free Ca2+ concentration was not attempted here. First and foremost, the subject of this report is an exploration of temporal and spatial dynamics, which are quite accessible without Cal’ calibration. We prefer to present our datawiththeminimum necessaryintermediateanalysis.Second, Ca’+ calibrations in this case would have required ratio imaging using a spectral-shift Ca*+ dye, such as fura- or indo-1. Unfortunately, all such dyes presently require excitation by UV light. The technology for confocal imaging of UV-excited dyes is just beginning to emerge and was not available at the time of these experiments. Third, the complex hippocampal tissue environments limit the usefulness of Cal+ ionophores and the other reagents that would be necessary to calibrate even approximately signals from a single-wavelength dye such as fluo-3. Finally, in spite of the excellent optical sectioning abilities of the confocal microscope, there is still substantial optical confusion of signals from adjacent and overlying cells. Such optical confusion would severely compromise Ca2‘ calibrations. Electrical Stimulation Monopolarelectrical stimulation was performed using a polyethylene pipette electrode, having an inner tip diameter of approximately 150 Wm. An isolated stimulator (World Precision Instruments lsostim A 320) was set to deliver trains of 1 ms, 0.2 mA current pulses between the pipette and a separate bath return electrode. The frequency of stimulation was varied from 50 Hz to as low as 2 Hz as described in the text. In some preparations, we found that high frequency electrical stimulation initiated recurrent, seizure-like activity within the slice (McBain et al., 1989; Fowler et al., 1986). Such slices provided for a fortuitous control experiment: astrocyte Ca*+ activity followed each spontaneous”seizure”discharge just as it did following electrically stimulated neuronal activity. This result helps argue against any possibility that our astrocyte responses were merely some direct effect of electrical stimulation, rather than being mediated by neuronal activation as we propose here. Correlative GFAP lmmunocytochemistry CFAP is an intermediate filament protein unique to astrocytes (Bignami et al., 1972). We developed procedures that allowed confocal visualization of fluorescent anti-CFAP immunostains at precisely the same field areas and optical sections visualized in our physiological experiments. Specimens were fixed in 4% formaldehyde at the end of each experiment, and any fixationinduced tissuedistortionwas recorded using Nomarski DICoptical sections. After the specimen position was recorded very precisely (using computer-controlled motors driving the microscope’s x, y, and z stage axes and a reference mark engraved on the culture coverslip), the specimen was removed for further processing. Actual GFAP immunocytochemistry was performed according to standard procedures. Briefly, the fixed slices were extracted with 0.3% Triton X, washed in 10 m M NH4CI, and blocked with 20% normal goat serum. The GFAP antibody that we used was a commercially available polyclonal antibody (diluted 1:25), and the secondary antibody was conjugated to fluo-
rescein (diluted l:lO).All incubation timeswereextrnded to faciiitate access into the rather thick (120 r.lm) whole-mounted slice. After staining, the specimen was returned to the confocal microscope, and the motorized stage was used to restore precisely the same specimen position as during the flue-3 experiment. Specimen repositioning was checked and, if necessary, refined using Nomarski DIC imaging of neuronal nucleoli as intrinsic reference marks. Fluorescence of the secondary antibody rag was then visualized by confocal fluorescence. Although almost every oscillating cell or process could be clearly identified by CFAP immunostaining, there were a small percentage (less than 10%) thatwere GFAP negative. This population may represent astrocytes of unusually poor GFAP epitope availability or other glial cell types such as oligodendrocytes, which do not express GFAP, or alternatively some interneuronal population. The clearly identifiable pyramidal neurons were never observed to oscillate. NMDA Stimulation NMDA (20 vM) was applied in the absence of MgZ+ and the presence of glycine (2 /LM) to activate maximally NMDA receptor-bearing neurons (Wood et al., 1990; Thomson, 1989). After a short baselinewas collected in an NMDA-free, Mg*+-free, glycine saline, an NMDA-containing, Mg2’-free glycine saline was perfused intothechamber, resulting inapromptneuronal response (
Vol. 8, 429-440, March, 1992, Copyright
0 1992 by Cell Press
Neuronal Activity Triggers Calcium Waves in Hippocampal Astrocyte Networks John W. Dani, Alex Chernjavsky, and Stephen J Smith Department of Molecular and Cellular Beckman Center Stanford University School of Medicine Stanford, California 94305-5426
Physiology
Summary The recent discovery that the neurotransmitter glutamate can trigger actively propagating CaZ+ waves in the cytoplasm of cultured astrocytes suggests the possibility that synaptically released glutamate may trigger similar Ca*+ waves in brain astrocytes in situ. To explore this possibility, we used confocal microscopy and the Ca*+ indicator fluo-3 to study organotypically cultured slices of rat hippocampus, where astrocytic and neuronal networks are intermingled in their normal tissue relationships. We find that astrocytic Ca*+ waves are present under these circumstances and that these waves can be triggered by the firing of glutamatergic neuronal afferents with latencies as short as 2 s. The Ca*+ waves closely resemble those previously observed in cultured astrocytes: they propagate both within and between astrocytes at velocities of 7-27 wm/s at 21°C. The ability of tissue astrocyte networks to respond to neuronal network activity suggests that astrocytes may have a much more dynamic and active role in brain function than has been generally recognized. introduction Astrocytes of the cortical gray matter have elaborate dendritic morphologies, superficially similar to those of neurons, and their fine processes mingle intimately with those of neurons throughout the synaptic neuropil. These cells actually outnumber neurons in many brain regions and are interconnected by gap junctions into vast networks. While these structural features hintat signalingoreven information-processing functions, astrocytes have traditionally been assigned relatively passive, background roles in structural, metabolic, and trophic support of neurons. This view arose and persisted mainly because there has been no evidence in astrocytes for the key property underlying signaling by neurons, namely electrical excitability. Very recently, however, studies of cultured astrocytes have suggested that these cells may possess an alternative form of excitability based directly on intracellular Ca*+ dynamics, essentially independent of plasma membrane potential (Cornell-Bell et al., 1990; Cornell-Bell and Finkbeiner, 1991). Several lines of investigation point toward the inadequacy of viewing the astrocyte as a passive supporting cell. For instance, there is growing evidence that the astrocytic roles in K+ buffering and neurotransmit-
ter metabolism may be actively regulated (Karwoski et al., 1989; Marrero et al., 1989; Barbour et al., 1989; Nicholls and Attwell, 1990). In addition, astrocytes have been shown to possess functional receptors for a wide range of the same neuroactive agents that support intercellular signaling within neuronal networks (for reviews, see Dermietzel et al., 1991; Barres, 1989; Laming, 1989; Kimelberg and Norenberg, 1989; Vernadakis, 1988; Stewart and Rosenberg, 1979; see also Calambos, 1961). For instance, there have been numerous reports of responses by cultured astrocytes to the ubiquitous excitatory neurotransmitter glutamate (Pearce et al., 1986; Enkvist et al., 1989; Cornell-Bell et al., 1990; Glaum et al., 1990; Ahmed et al., 1990; Jensen and Chiu, 1990, 1991; Charles et al., 1991) and other neurotransmitters (Sugino et al., 1984; Schieren and MacDermott, 1988; Salm and McCarthy, 1990; lnagaki et al., 1991). Several of these studies have established that glutamate and other neurotransmitters can trigger both Ca*+oscillations and propagating Ca*+waves, indicative of a Ca*+-based form of excitability (Cornell-Bell et al., 1990; Jensen and Chui, 1990). The Ca*+-based excitability of cultured astrocytes appearsto be based primarily on the regulated release of Ca*+ ions from intracellular stores (Cornell-Bell and Finkbeiner, 1991). Such activity can take the form of waves of elevated cytosolic Ca*+ that propagate both within individual astrocytes and between cells within networks of astrocytes that have grown to confluence (Cornell-Bell et al., 1990). Ca2+waves resembling those observed in astrocytes have been observed in many other cell types (see Jaffe, 1983)and tissues (Sanderson et al., 1990). The underlying mechanisms are probably conserved across many of these examples (Berridge, 1990; Jacob, 1990; Meyer, 1991). Within astrocytes, intracellular and intercellular Ca*+ waves have similar velocities, ranging between 6 and 30 pm/s at 21% The numerousgap junctions that form between astrocytes in confluent culture (Dermietzel et al., 1991; Kettenmann and Ransom, 1988) provide a likely avenue for intercellular Ca*+ wave propagation. Astrocyte Ca*+ waves are often oscillatory, with periods in the range of IO-30 s, but the form of such oscillations is quite variable and depends upon how the cells are activated (Cornell-Bell et al., 1990; Cornell-Bell and Finkbeiner, 1991). As noted above, the amino acid glutamate has been identified as an effective stimulus for Ca*+ excitation in cultured astrocytes. Since glutamate is thought to be the major excitatory neurotransmitter within the brain and since astrocyte membranes juxtapose or ensheathe most synapses in the mammalian CNS (Peters et al., 1991), it seems possible that synaptically released glutamate might initiate Ca*+ waves within brain astrocytes in situ. Because astrocytes within the CNS, as in culture, are extensively coupled by gap junctions (Peters et al., 1991), there is also the possibil-
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ity that astrocytic Ca2+ waves might propagate for long distances through brain tissue. Finally, as cytoplasmic Ca’+ signals have come to be recognized as ubiquitous regulators of cellular function, one might expect that neuronally triggered Ca2’ signaling in brain astrocyte networks could have substantial functional significance. We therefore felt that it was important to determine whether Ca2+ waves, like those described in cultured astrocytes, actually do occur within brain astrocytes in situ and whether synaptically released neurotransmitters are sufficientto trigger such waves.
/mf
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Results We used organotypicallycultured hippocampal slices (Gahwiler, 1988,19&l, 1981) and a laser confocal microscope in order to obtain clear fluorescence images of live astrocytes within a synaptic neuropil environment. Neurons and astrocytes in these slices were readily loaded with the fluorescent Ca2+ indicator flue-3 using the acetoxymethyl ester derivative (Kao etal.,1989;Mintaetal.,1989;Tsien,1988).Theconfocal microscope was equipped for time-lapse video recording. With these methods, we were able to monitor the dynamics of cytosolic Ca*’ in both neurons and astrocytes deep within the organotypic slice. In preliminary work, we tried similar procedures using freshly prepared hippocampal slices from adult rats, but were unable to obtain useful optical signals. Flue-3 reports cytosolic Ca2+ increases by increases in fluorescence intensity. Thefluo-3 images presented here use the gray scale from black to white to encode either total fluorescence or increases over baseline fluorescence, as specified in the figure legends. With our methods, quantification of the flue3 signals in terms of cytosolic Ca2’ concentration would be both difficult and uncertain. Fortunately, such quantification was not necessary to interpret the Ca2+dynamics, which are the subject of the present study. Low Frequency Stimulation of Dentate Cyrus Elicits Both Neuronal and Astrocytic Responses within Area CA3 As diagrammed in Figure 1, current from an extracellular pipette electrode was used to stimulate electrically mossy fibers originating in dentate gyrus, while Ca2+ signals were visualized in cells within region CA3, at a site 1.5-3 m m distant from the stimulating electrode. This target area is rich in mossy fiber varicosities known to release glutamate at giant synapses onto CA3 pyramidal cells. All effects of electrical stimulation described below were found to be blocked by 1 uM tetrodotoxin, confirming that axonal impulse conduction between dentate and CA3 was necessary to elicit these responses. Low frequency stimulation (8 Hz) of dentate generated very prompt cytoplasmic Ca*+ increases within cells of region CA3: the earliest responses were evident within milliseconds of the first action potential volley. These early Ca2+ responses were observed in
Figure 2. Positioning of Stimulating within the Hippocampal Slice
Electrode
and Imaged Area
Diagram showing positions of stimulating electrode (Stim) and the imaged areas shown in (Figures 2A-2F) (Image) with respect to overall anatomy of a hippocampal slice and the course of a typical mossyfiherfmf)fromdentatethrough regionCA3.Within the rectangular imaged area, the loci of a typical CA3 pyramidal cell (p), the pyramidal cell body palisade (dotted outlines), and a typical gray matter astrocyte !a) are also indicated. Generally similar arrangements for stimulation and recording were employed in all experiments described in this paper. Drawing modified from Shepherd (1974).
axons and varicosities of the mossy fibers and in the cell bodies and dendrites of the CA3 pyramidal neurons,aswellasin numerousothersmall neuronalcells and processes (Figure 2). As sampled by our imaging system, the neuronal Ca*+ increases usually reached steady levels within a few seconds of sustained 8 Hz stimulation (e.g., see trace 6 in Figure 3). After several seconds of sustained dentate stimulation, a second population of cells began to exhibit large increases in Ca2+ (fluorescence increases ranged from 50%-200%, measured over areas like those indicated by the solid boxes in Figure 2B). It was this population of cells that were retrospectively identified as astrocytes by correlative glial fibrillary acidic protein (GFAP) staining (see Figure 2F). As shown in Figures 2E and 2F, the cell bodies identifiable as astrocytes were found mainly in strata outside the pyramidal layer (i.e., stratum radiatum, lucidum, and oriens), although a few astrocyte cell bodies were interspersed among those of the pyramidal neurons. This is precisely the anatomical distribution expected for hippocampal tissue (Del Rio et al., 1991; Faddis and Vijayan, 1988; Beach et al., 1982). Astrocytes Exhibit Ca2+ Oscillations in Response to Continuous Neuronal Stimulation With sustained neuronal stimulation, astrocyte cell bodies often exhibited irregularly oscillating cytoplasmic Ca2’- concentrations. In addition, oscillations
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Figure 2. Electrical CA3
Stimulation
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(A) Average fluorescence of the flue-3.loaded slice observed during 32 frames of electrical stimulation. (B) Baseline fluorescence of the same slice observed prior to stimulation (averaged over 32 frames). Dashed rectangles indicate subareas sampled for time series images in (C) and (D). Solid boxes represent areas over which fluorescence intensities expressed in Figure 3 were averaged. (C) Time sequence showing Ca2+ oscillations within a cell (arrow) identifiable by GFAP staining as an astrocyte. Each time point was numbered relative to the time of the last prestimulus frame and shows the change in fluorescence over baseline measurements. (D) Time sequence, as in (C), showing Ca* oscillations within two astrocytic processes (arrow and arrowhead). The cell body from which over this sequence. the process marked by the arrowhead emerges also exhibits Caz+ oscillation (E) A Nomarski DIC image taken from the same field as the previous fluorescence images. Notice the palisading organization of pyramidal cell bodies in the right half of the field and the clear boundary formed with stratum lucidum to the left. (F) GFAP immunocytochemical stain corresponding to the same field as in (B). Arrows and arrowhead point to CFAP-positive cells designated as astrocytes in (C) and (D). Bar, 20 urn.
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A few examples of such delayed soiitary spikes are evident on the decay tails between 600 and 900 s in traces 1-4 of Figure 3.
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Figure 3. Ca I+ Fluctuations Observed within Identified Cells of Area CA3 following Dentate Stimulation (Same Experiment as Figure 2) Fluorescence change signals measured over image areas indicated by boxes in Figure 26, before, during, and after an electrical stimulus delivered between times indicated.as O-600 s. For clarity, these curves are shifted arbitrarily along the ordinate axis. The first five curves show the irregularly oscillatory Ca”+ responses typical of astrocytes; the sixth curve shows the steadier plateau response typical of neurons. At the end of stimulation (600 s), Ca2+ concentrations return to resting levels over a period of about 300 s.
were observed within numerous CFAP-positive (astrocytic) processes located throughout the slice. These clearly discernible oscillations in astrocytic Ca2+ signals contrasted with the apparently steady Ca2+ increases observed in the vast majority of GFAPnegative (presumably mostly neuronal) processes. As one example, compare the oscillating Ca2+ levels evident in the astrocytic processes and cell body in the lower halves of the time series of images in Figure 2D with the steady growth and stabilization of fluorescence in the CFAP-negative (probably neuronal) process that zigzags across the middle of those frames. For another perspective on the differing dynamics of astrocytic and neuronal Ca2+ increase, compare traces l-5 in Figure 3, measured from astrocytes identified by retrospective immunohistochemistry, with trace 6, measured from a pyramidal cell body. Though the periods of such astrocytic Ca*+ oscillations (mean = 22 s, range 16-30 s; 22 cells, three experiments) were generally in the same range as the glutamate-induced oscillations observed in cultured astrocytes, periods of the oscillations in slices exhibited more variability from cell to cell, and the oscillations were much less regular (contrast Figure 3 with Figure 1 in Cornell-Bell et al., 1990). Upon termination of nerve fiber stimulation, both neuronal and astrocytic populations returned to resting Ca 2+ levels with time courses extending over a few minutes (see Figure 3). Astrocytes often exhibited a tendency toward persistent generation of solitary or repetitive CaZ’ spikes in the aftermath of stimulation.
Astrocytic Responses to Neuronal Stimulation Can Be Prompt Figure 4 demonstrates that the minimum latency between mossy fiber stimulation and astrocyte Ca2+ response can be less than 2 s. Figure 4A shows the resting level of flue-3 fluorescence apparent in one optical section. Figure 4B shows the retrospective immunostain used to identifyastrocyteswithin thissame optical section. A burst of high frequency dentate stimulation (50 Hz for 6 s) was begun after the time point indicated as 0 s in Figure4C. Traces l-5 in Figure 4C show Ca2’signals measured from image areas positioned over astrocyte cell bodies or processes, and trace 6 shows a Ca2’ signal measured over one of the pyramidal neuron cell bodies. As indicated bythedata plotted in trace 6, neuronal responses grew to nearly maximal levels during the 1 s period over which this frame was scanned. Figure 4D illustrates the appearance of the image scanned during the onset of neuronal stimulation. This image, consisting of 480 horizontal lines, was scanned at 500 lines per s over a total period of approximately 1 s, beginning at the top. This scanning process creates an implicit time axis extending from the top to the bottom of the frame. The scan line active at the precise time of stimulus onset (approximately 120 ms after beginning the scan) is indicated by the flanking horizontal arrows. The distinct boundary between dark and light just below this scan line reflects a Ca*+ increase in fine neuronal processes that occurs within 4-6 ms of stimulus onset. Figure 4E shows a similar scan beginning exactly 2 s after that shown in Figure 4D. By this time, large CaL+ increases are evident over the entire frame. Astrocyte cell bodies (arrows) and a process (arrowhead) identified from the GFAP stain shown in Figure 4B are indicated in Figures 4E and 4F. The large astrocytic Ca*’ signals evident in Figure 4E are a clear indication that astrocytes can respond to neuronal activity within 2 s or less. A similar conclusion can be drawn from traces l-5 in Figure 4C, with the caveat that the astrocyte traces l-5 surely include fluorescence increases from fine neuronai processes (see measurement boxes in Figure4A and the first poststimuius frame, Figure4D). Astrocytes Display Both Intracellular and intercellular Ca*+ Waves following Neuronal Stimulation Ca*‘waves propagating both within and between individual astrocytes were observed in most of our experiments. Figure 5A shows a time series of images illustratingwave propagation within an astrocytic process (or perhaps a small bundle of parallel processes), and Figure 5B shows measurements of fluorescence as a function of time at several points along the progress of the wave. The phase-shifted, but otherwise similar, nature of these waveforms demonstrates the wave-like nature of the underlying signal. At room
Neuronal 433
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Figure 4. High Frequency CA3 Astrocytes
Ca2+ Waves
(50 Hz) Electrical
Stimulation
of the Dentate
Granule
Cells Elicits a Prompt
Intracellular
CaLi Response
in Area
(A) Resting fluorescence observed within area CA3 after fluo-3 loading (average of 32 frames). Stratum pyramidale lies to the left; stratum lucidum lies to the extreme right. Solid boxes indicate areas where fluorescence intensity measurements were taken for (C). (6) CFAP immunofluorescence image showing the same field as (A). The arrows and arrowhead mark GFAP-positive cell bodies and a process, respectively, that lie within the measurement boxes l-5 shown in (A). (C) Flue-3 fluorescence measured over both astrocytes (traces l-5) and a neuron (trace 6) during CaZ+ responses to dentate stimulation. Traces are shifted arbitrarily along the ordinate axis for clarity. (D-F) Fluorescence changes observed at several time points following electrical stimulation. fD) The earliest response to electrical stimulation. The horizontal arrows indicate the horizontal scan line active at the time of stimulus onset. (See text for more details.) Numerous fine processes and pyramidal cell bodies exhibit Caz+ increases. (E) After 2 s more of stimulation (t = 4 s), the pyramidal cell bodies exhibit large cytosolic Ca*+ Increases, especially within their nuclei. Many GFAP-positive cell bodies and processes (e.g., arrows and arrowhead, same positions as in [B]) also now exhibit substantial cytosolic Ca2+ increases. (F) After 4 s of stimulation (t = 6 s), nearly every astrocyte within the field has responded. Again, the position of the arrow corresponds to the same arrow marking a GFAP-poshive cell body in (B). Bar, 20 Pm.
temperature (21°C), intracellular wave velocities ranged from 7-27 pm/s (mean = 15 vm/s; 5 cells; three experiments) and encompassed both cell bodies and processes. Such waves were observed to propagate through processes both away from and toward astrocytic cell bodies. Ca*+ waves were often observed to propagate from astrocyte to astrocyte. Such intercellular Ca*+ waves could be followed only over short distances (e.g., 2-3 astrocyte diameters) in most of our recordings, but longer range waves were sometimes observed with the most intense stimuli. While it was difficult to discern any patterning of the intercellular Ca*+ waves when viewed in real time, strikingly intricate and varied patterns of wave propagation were apparent when Ca2+ images were visualized by video playback of time-lapse recordings (time compression of lO:l-1OO:l). The intercellular wave propagation patterns are difficulttodescribeinanysuccinctway: they haveagenerally chaotic appearanceand seem to be highly variable both within and between individual slices.
Although electrical stimulation was able to elicit intercellular Ca*+ waves, this phenomenon was most notable when preparations were stimulated by bath application of N-methyl-o-aspartate (NMDA), as shown in Figure 6. It seems likely that such astrocytic Ca*+ waves are an indirect result of NMDA application, mediated by the release of glutamate from NMDAstimulated neurons. While NMDA is known for its potent excitatory effects on neurons (Wood et al., 1990; Thomson, 1989; Llano et al., 1988), no direct actions on isolated astrocytes have been described. This latter result is in spite of numerous reported attempts to demonstrate astrocytic NMDA responses (CornellBell et al., 1990; Cornell-Bell and Finkbeiner, 1991; Bevan, 1990; Pearce et al., 1986). In our experiments, NMDA was found to initiate prompt increases in neuronal Ca*+, which were invariably followed by the propagation of Ca*+ waves between cells identifiable as astrocytes. The intercellular Ca*+ waves observed after NMDA application often spread coherently throughout entire hippocampal ar-
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20
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30
40
Seconds Frgure 5. Intracellular Ca 2+ Waves Are Observed CA3 Astrocytes during 8 Hz Electrical Stimulation
within Area of Dentate
(A)Time series showing the fluorescence changeswithin a single astrocyte or, perhaps, a small bundle of astrocytic processes. Each image represents the increase in f!uorescence over baseline. Beginning with t = 30 s after stimulus onset, the Ca” wave has not yet moved within the field of view. The Ca2’ wave first appears as a faint rise in fluorescence within several processes toward the right edge (t = 38 s). At t = 40 s, the position of the wave front has clearly moved toward ihe left. By t = 42 s, the wave, which has spread almost to the cell body, encompasses several more processes. In addition, the CaJ’ concentration has continued to rise, indicating that this is a nonuniform Caz+ wave. Finally at 44 s, the wave reaches the astrocyte cell body and initiates a Ca2+ spike. Boxes are drawn over the regions where fluorescence measurements were made for (B). (B) Measurements of fluorescence changes at the six positions indicated by numbered boxes (A). Each waveform is normalized to a constant peakamplitude. Waveformsareshiftedarbitrarilyalongtheordinate axis for clarity. The second set of Ca*+ peaks represents the Ca2+ wave shown in (A). The phase shifts among these curves (delay increases progressively by a total of about 4 s between curve 1 and curve 6) demonstrate wave-like propagation of this astrocytic Ca2+ signal. Other differences among these six waveforms (e.g., faster rise times in curve 6 than in curve 1) probably reflect the transition between elongated processes and cell body. Bar, 10 urn.
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Seconds Figure 6. Long-Distance(IntercelIular)AstrocyteCaz’ WavesAre Observed in Organotypic Hippocampal Slices following the Application of NMDA, an Agent Thought to Stimulate Neurons Selectively and Trigger Synaptic Glutamate Release (See Text) (A) Time series showing increases over baseline fluorescence in area CA3 following perfusion with 20 PM NMDA plus 2 PM glytine in Mg *+-free saline. Each image represents the increase in fluorescence over baseline measurements at 14 s, 26 s, 34 s, 52 s, and 64 s after NMDA application. The Ca2+ wave enters the field of view at the top of the image (t = 14 s) and spreads down and toward the left (t = 26-64 s). The boxes drawn in the last imageft = 64s)indicate15adjacentsubareasfromwhichfluorescent measurements were made for (B). (B) increases in flue-3 fluorescence at the 15 subareas indicated in (A). The topmost trace corresponds to the topmost box in (A) and so on. The progressive phase shifts evident in this sequence of traces indicate the propagation of awave from top toward bottom subareas at a velocity of approximately 9.6 bm/s. Bar, 100 Wm.
t = 64s
eas. The involvement of approximately 50 astrocytes was ascertained from inspection of the image sequence illustrated in Figure 6. At room temperature, intercellular wave velocities ranged from 8-10 pm/s for both electrical and NMDA stimulation (several hundred cells, four experiments). We saw no indications of a delay as waves propagated from one astrocyte to the next. Discussion A CaZ+-Based Form of Cellular Excitability We have shown that neuronal activity can trigger Ca2+ oscillations and waves in astrocytes and their networks within cultured hippocampal slices. While earlier studies have shown prompt changes in glial membrane potential in response to neuronal activity, these effects were identified as passive glial responses to neuronal K+ efflux (Orkand et al., 1966; Trachtenberg and Pollen, 1970). In contrast, the astrocytic Ca*+ responses described here are characterized by oscillations and nondecremental spatial propagation. These astrocytic Ca2+ responses can therefore be considered expressions of a distinctive form of cellular excitability. Mechanism of the Cal+ Wave Phenomenological similarities between the astrocytic Ca2+ signals described in this paper and those described in earlier studies of cultured astrocytes strongly suggest that the same underlying mechanisms are at work. Most notably, the astrocytic Ca*+ waves reported here propagate at velocities very similar to those described in astrocyte cultures. There are also similarities in the observed patterns of oscillation, although the oscillations in the hippocampal slices tend to be less regular than those observed in the astrocyte cultures. The relative irregularity of astroin the brain slice may be due cytic Ca2+ oscillation to the complexity of astrocytic networks within such tissues and/or due to perturbing neuronal influences. The basic mechanisms of Ca2+ excitability in cultured astrocytes and in many other cell types are currently under investigation by many laboratories (for reviews see Cornell-Bell and Finkbeiner, 1991; Meyer, 1991; see also Bezprozvanny et al., 1991; Finch et al., 1991). In brief, it appears that such Ca*+ excitability is primarily a consequence of the regenerative release of Ca2+ from endoplasmic reticulum. Coupling of Neuronal Activity to Astrocytic Ca2+ Responses While all of the presently available evidence supports our original notion that synaptically released glutamate might trigger astrocytic Ca2+ waves, we have not conclusively proven that this particular agent is the proximal stimulus tothe neuropil astrocyte. Certainly, however, glutamate is the primary neurotransmitter released by the mossy fiber afferents stimulated in our experiments, and there is indeed a very strong
resemblance between the Ca2+ waves reported here and those observed in cultured astrocytes exposed directly to glutamate. In addition, we have found that the glutamate receptor antagonist kynurenic acid (Robinson et al., 1985, 1984) at 3 mM blocks all astrocytic responses to neuronal stimulation (unpublished data). it is still remotely possible, however, that synaptic glutamate acts via some effect secondary to its activation of neurons intrinsic to area CA3. For instance, activation of these intrinsic neurons might stimulate astrocytes through release of K+, or some other neurotransmitter or coreleased factor such as ATP. Such indirect actions seem relatively unlikely, but more work will be necessary to evaluate critically this classof possibilities. We hasten to pointout nonetheless that the most intriguing functional questions raised by the present description of neuronal-gliai signaling are independent of the precise identity of the proximal chemical mediator involved. The Organotypic Slice As an Experimental Model The observations described here were made using organotypic hippocampal slices. As a model system for the study of neuronal-glial interactions, such slices have many advantages, including being in avery stable physiological condition and providing excellent access for optical recording methods. Acute brain slice preparations are substantially less attractive for optical study due to the presence of very large numbers of dead cells, especially near the cut surfaces, and due to a much greater difficulty in applying the fluo 3-AM loading procedure (see Yuste and Katz, 1991). Furthermore, the physiological condition of acute brain s!ices is far less stable: they typically die within 24 hr or so of initial preparation. On the other hand, the fact remains that neuronal or astrocytic properties may change during the period of organotypic culture stabilization. Although many aspects of normal synaptic function and neuronal and glial ultrastructure have been shown to be maintained for long periods in organotypic culture(Zimmer and CPhwiler, 1984; Zhabotinski et al., 1979; Del Rio et al., 1991), it remains to be determined whether astrocytic Ca2+ waves are stimulated by neuronal activity in vivo. To resolve this important question, it may be necessary to repeat experiments similar to those described here within acute slices and intact animals. Possible Effector Actions of the Caz+ Wave We can begin to ponder the possible physiologicaT actions of astrocytic Ca 2+ transients like those described here. One of the most intriguing possibilities is that neuronally induced astrocytic Ca*’ signals may feed back to influence neuronal excitability or regulate synaptic transmission. Such effects might be mediated through changes in extracellular ion concentrations (effects on extracellular K+ and Ca*’ are most likely to be of physiological importance; Barres et al., 1990; Quandt and MacVicar, 1986; O&and et al., 1966; Olson et al., 1990; Ciardo and Meldolesi, 1990; MacVic-
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Triggering
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Cal+ Waves
ar and Tse, 1988; Benjamin, 1987), or through changes in neurotransmitter metabolism (Vernadakis, 1988; Laming, 1989). Astrocytic Ca2+ could exert such influences via Ca2+-dependent membrane transport mechanisms (i.e., ion channels or exchangers) or through actions of cytoplasmic metabolic or regulatory enzymes (Laming, 1989; Stryer, 1988; Alberts et al., 1989). Astrocytes are also capable of secreting neuroactive substances (e.g., peptides, amino acids, eicosanoids, and nitric oxide; Pasantes-Moraleset al., 1990; Murphy et al., 1988, 1990; Ehrenreich et al., 1991; Benveniste et al., 1990; Shinoda et al., 1989), and such secretion processes are likelytargets for Ca2+ regulation. In this context, it is also interesting to note that there have been indications that astrocytes may regulate synaptic plasticity (Sastry et al., 1990; Muller and Best, 1989). There is an additional range of possible targets for astrocyte Ca2+ signaling that might be somewhat less immediate but equally important in their impact on neuronal function. Astrocytes contain the brain’s major glycogen stores and are very likely to play a dynamic role in meeting neuronal energy demands (Vernadakis, 1988). There are reasons to believe that astrocytic energy metabolism may be regulated by intracellular Ca2+ (Pearce at al., 1988; Arbones et al., 1990). Astrocytes are also likely to exert profound effectsoncerebralvascularfunctionviatheirprominent and highly specialized contacts onto blood vessels (Peters et al., 1991; Raff and Lillien, 1988; Janzer and Raff, 1987; Bradbury, 1985). It is quite possible that the astrocytic-vascular interaction is somehow regulated by astrocytic Ca2+. Finally, cytoplasmic Ca2+ may regulate changes in astrocytic gene expression (Morgan and Curran, 1986, 1988; Arenander et al., 1989; Condorelli et al., 1989) and mitotic responses (Hart et al., 1989; Neary et al., 1987). Biological Significance The long-distance Ca2+ waves described here suggest that gray matter astrocytes may function as an active signaling network. At present we can only speculate on the possible function or informational content of astrocytic Ca2+ signals, but a general analogy between glial Ca2+ excitability and neuronal electrical excitability may be noted. The capability of neural networks to process information is thought to be fundamentally dependent on the neuron’s underlying electrical excitability. Astrocyte networks might possess similar capabilities as a consequence of Ca2+-based excitability. Thus, in spite of very different temporal and spatial domains of astrocytic and neuronal signaling, the possibility arises that astrocytic networks, like neuronal networks, may process information. The slowness of astrocytic Ca2+ signaling does not necessarily diminish a possible informational role: astrocytic Ca2+ responses are no slower than most actions of the wellknown biogenic amine and peptide neuromodulators. We have shown that neuronal activity can trigger active and elaborate astrocytic Ca2+ signals. If these astrocytic Ca2+ signals somehow feed back to
influence neuronal excitability or synaptic transmission, astrocytes could participate in brain information processing to an extent comparable with the participation of aminergic and peptidergic neurons. In other words, the current conception that neuronal networks alone provide the basis for the brain’s computational function may need to be expanded to encompass interacting neuronal and astrocytic networks. Experimental
Procedures
Preparation of Organotypic Hippocampal Slices Organotypic hippocampal slices were made from postnatal day 7 Sprague-Dawley rat pups according to established procedures (Clhwiler, 1988,1984,1981). Briefly, slices (300-400 Rm thickness) were cut from the middle two-thirds of the hippocampus and attached to rectangular coverslips (11 x 22 mm) using a plasma clot (chicken plasma and bovine thrombin). Coverslips were then transferred singly to sterile glass tubes having an internal diameter slightly larger than the width of the coverslips. One milliliter of rollertube medium (50% Earle’s minimal essential medium, 25% Hanks’ balanced salt solution, 25% horse serum, 10 m M HEPES, 6 mg/ml dextrose, 0.292 mglml t-glutamine) was added to each culture tube, which was then sealed with a plastic cap and placed in a rotating drum at 37X Slices were maintained for l-2 weeks in this manner prior to experimentation: by this time, slice thickness was typically reduced to about 100 pm. Flue 3-AM loading Organotypic slices were loaded with flue-3 by incubating for 2 hr in rollertube culture medium containing 110 PM fluo 3.AM (Kao et al., 1989; Minta et al., 1989; Tsien, 1988). Experiments were performed with coverslips containing the tissue slices mounted in a flow-through perfusion chamber. The standard saline used in these experiments was 137 m M NaCI, 5.3 m M KCI, 3 m M CaCl,, 1 m M MgCI,, 10 m M HEPES, and 25 m M dextrose, with modifications or drug additions as specifically noted. All experiments described here were carried out at room temperature fapproxlmately 21°C), but exploratory experiments indicated that effects qualitatively similar to those we describe occur at highertemperatures(upto37V). Highertemperatureswerenot used routinely because the Ca2+ signals observed became too rapid to characterize accurately with our instrumentation. Image Acquisition The histology of organotypically cultured hippocampal slices most closely resembles that of hippocampus in situ when examined midway between the topand bottom section planes. Confocal microscopy was used in this study to isolate these most favorable tissue planes by optical sectioning. All observations reported herewere made at optical sections approximatelyequidistant from the top and bottom slice surfaces (i.e., at a depth of approximately 60 wrn within a 120 pm thick slice). Images were acquired using a modified laser confocal microscope (Bio-Rad MRC-500) and stored at intervals of one frame every 2 s on an optical memorydiskvideo recorder (PanasonicTQ-2028F). Modifications to the MRC-500 included a faster shutter to eliminate unnecessary light exposure, high reflectance dielectric mirrors, an improved analog sampling filter, a motorized stage to relocate specific fields of interest, and a silicon photodiode to capture transmitted laser light for Nomarski DIC imaging. Image Processing The intracellular Ca2+ dynamics described in this report are extremely vivid when raw fluorescence image sequences are viewed bytime-lapsevideo playback. When the same raw images are displayed as stills, however, these inherently dynamic phenomena become far more difficult to visualize. It was therefore necessary to use digital image enhancement and analysis methods to render our image data for print publication. The images described in this paper as”fluorescence increases”werederived
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bydigitallysubtracting imagesofthe”resting”fluorescencefrom images collected at specified time points before, during, or after stimulation. To minimize noise in the processed images, the resting fluorescence images were usually derived by averaging 16 or 32 frames collected during the period of steady fluorescence priorto stimulation. To preserve dynamic information, no averaging was performed over the individual time point frames. The resulting difference images were then offset and scaled by arbitrary factors so as to make use of the entire gray scale available through the final print rendering. Arbitrary spatial low pass filtering was used to achieve a favorable trade-off between signal-to-noise ratio and fine image detail in the final renderings. Across any set of images presented as a time series (e.g., Figure 20, however, all image processing parameters were held constant. Upon request and payment for duplication and shipping, the authors will provide a VHS videocassette representing the primary observations in the more vivid time-lapse playback form. For several reasons, quantitative calibration of fluorescence images in terms of cytosolic free Ca2+ concentration was not attempted here. First and foremost, the subject of this report is an exploration of temporal and spatial dynamics, which are quite accessible without Cal’ calibration. We prefer to present our datawiththeminimum necessaryintermediateanalysis.Second, Ca’+ calibrations in this case would have required ratio imaging using a spectral-shift Ca*+ dye, such as fura- or indo-1. Unfortunately, all such dyes presently require excitation by UV light. The technology for confocal imaging of UV-excited dyes is just beginning to emerge and was not available at the time of these experiments. Third, the complex hippocampal tissue environments limit the usefulness of Cal+ ionophores and the other reagents that would be necessary to calibrate even approximately signals from a single-wavelength dye such as fluo-3. Finally, in spite of the excellent optical sectioning abilities of the confocal microscope, there is still substantial optical confusion of signals from adjacent and overlying cells. Such optical confusion would severely compromise Ca2‘ calibrations. Electrical Stimulation Monopolarelectrical stimulation was performed using a polyethylene pipette electrode, having an inner tip diameter of approximately 150 Wm. An isolated stimulator (World Precision Instruments lsostim A 320) was set to deliver trains of 1 ms, 0.2 mA current pulses between the pipette and a separate bath return electrode. The frequency of stimulation was varied from 50 Hz to as low as 2 Hz as described in the text. In some preparations, we found that high frequency electrical stimulation initiated recurrent, seizure-like activity within the slice (McBain et al., 1989; Fowler et al., 1986). Such slices provided for a fortuitous control experiment: astrocyte Ca*+ activity followed each spontaneous”seizure”discharge just as it did following electrically stimulated neuronal activity. This result helps argue against any possibility that our astrocyte responses were merely some direct effect of electrical stimulation, rather than being mediated by neuronal activation as we propose here. Correlative GFAP lmmunocytochemistry CFAP is an intermediate filament protein unique to astrocytes (Bignami et al., 1972). We developed procedures that allowed confocal visualization of fluorescent anti-CFAP immunostains at precisely the same field areas and optical sections visualized in our physiological experiments. Specimens were fixed in 4% formaldehyde at the end of each experiment, and any fixationinduced tissuedistortionwas recorded using Nomarski DICoptical sections. After the specimen position was recorded very precisely (using computer-controlled motors driving the microscope’s x, y, and z stage axes and a reference mark engraved on the culture coverslip), the specimen was removed for further processing. Actual GFAP immunocytochemistry was performed according to standard procedures. Briefly, the fixed slices were extracted with 0.3% Triton X, washed in 10 m M NH4CI, and blocked with 20% normal goat serum. The GFAP antibody that we used was a commercially available polyclonal antibody (diluted 1:25), and the secondary antibody was conjugated to fluo-
rescein (diluted l:lO).All incubation timeswereextrnded to faciiitate access into the rather thick (120 r.lm) whole-mounted slice. After staining, the specimen was returned to the confocal microscope, and the motorized stage was used to restore precisely the same specimen position as during the flue-3 experiment. Specimen repositioning was checked and, if necessary, refined using Nomarski DIC imaging of neuronal nucleoli as intrinsic reference marks. Fluorescence of the secondary antibody rag was then visualized by confocal fluorescence. Although almost every oscillating cell or process could be clearly identified by CFAP immunostaining, there were a small percentage (less than 10%) thatwere GFAP negative. This population may represent astrocytes of unusually poor GFAP epitope availability or other glial cell types such as oligodendrocytes, which do not express GFAP, or alternatively some interneuronal population. The clearly identifiable pyramidal neurons were never observed to oscillate. NMDA Stimulation NMDA (20 vM) was applied in the absence of MgZ+ and the presence of glycine (2 /LM) to activate maximally NMDA receptor-bearing neurons (Wood et al., 1990; Thomson, 1989). After a short baselinewas collected in an NMDA-free, Mg*+-free, glycine saline, an NMDA-containing, Mg2’-free glycine saline was perfused intothechamber, resulting inapromptneuronal response (
