INTRODUCTION

Endoplasmic reticulum (ER)¹ Ca²⁺-binding proteins provide a high capacity buffering mechanism which results in the lowering of [Ca²⁺]_free in the ER, and thus a reduction in the gradient against which pumps must transport cytoplasmic Ca²⁺ into the store. They are also thought to be important in localizing Ca²⁺ to sites of release, and in modulating release activity, via protein-protein interactions with release channels (1-4). The best described of these Ca²⁺-binding proteins are calsequestrin and calreticulin. In many cells, calreticulin is the major calcium-binding protein of the ER lumen (2, 5). Three subtypes of inositol 1,4,5-trisphosphate receptor (InsP₃R) are now known (see Ref. 6 for review), which can have different modulatory properties and discrete functions even when expressed together in the same cell (7-9). Localization of calreticulin to InsP₃R-containing membrane vesicles has been reported in some cell types using density gradient techniques (2, 10). The function of this coexpression, however, has remained controversial. Recent reports have indicated that calreticulin may play a role in regulating Ca²⁺ signals, including perhaps serving as a luminal sensor for Ca²⁺ store depletion (4, 11).

InsP₃-mediated Ca²⁺ waves in several cell types propagate over long distances by regenerative Ca²⁺ release at specialized cellular domains (12-15). In glial cells, these specialized wave amplification sites are characterized by significantly higher amplitude in local Ca²⁺ signals and steeper rate of rise of the signals (12, 14, 16). Ca²⁺ waves typically travel in complex nonlinear paths through three-dimensional space (12, 15, 17-19), making analysis of their mechanisms of propagation problematical. In recent studies, however, we have investigated Ca²⁺ waves in cultured cortical oligodendrocytes which because of their long, thin, relatively linear processes, allow for the analysis of Ca²⁺ waves as one dimensional propagatory entities. Oligodendrocytes express a variety of receptors coupled to the hydrolysis of phosphoinositides and consequent mobilization of Ca²⁺ from InsP₃R-expressing intracellular stores. These include _1A adrenoreceptors, M1 muscarinic cholinoceptors, and bradykinin receptors (20-23). We have previously demonstrated that oligodendrocytes respond to the muscarinic receptor agonist methacholine (MCh) by the induction of Ca²⁺ waves initiating in several distinct regions of oligodendrocyte processes. These waves travel along each process and into the cell body via multiple amplification sites (14). One or more mitochondria are closely associated with each of these specialized Ca²⁺ wave amplification sites in oligodendrocytes, and inhibition of mitochondrial activity markedly affects methacholine-evoked Ca²⁺ responses (14).

The present study was undertaken to investigate whether specializations in ER protein distribution as well as mitochondrial location would underlie the specialized Ca²⁺ release sites found during InsP₃-evoked Ca²⁺ waves. Our results indicate that type 2 InsP₃Rs (InsP₃R2), and calreticulin, are expressed in much higher intensity at Ca²⁺ wave amplification sites along oligodendrocyte processes compared with other regions. Furthermore, in the processes stationary mitochondria were found only at these specialized Ca²⁺ release sites in close association with high density of ER proteins. These findings suggest that wave propagation in glia may be modulated by specialized microdomains of Ca²⁺ release involving both mitochondria and ER proteins.

EXPERIMENTAL PROCEDURES

(±)-Norepinephrine hydrochloride and acetyl--methylcholine chloride were obtained from Sigma. Bradykinin, fura 2-AM and fluo 3-AM were obtained from Research Biochemicals International. MitoTracker CMXRos was from Molecular Probes. PA3-900 was from Affinity Bioreagents Inc. AP42 was a gift from Dr. A. Sharp (Johns Hopkins University, Baltimore, MD).

Oligodendrocytes were prepared from 2-day-old rat pups as described previously (14, 24). Briefly, cortices were removed and manually dissociated, and cells cultured in plastic flasks (25). After 8 days in vitro, the flasks were vigorously shaken overnight. The supernatant was repeatedly plated onto plastic dishes, to which endothelial cells, microglia, and fibroblasts quickly attach. Non-adherent cells were then replated onto glass coverslips coated with 0.1 mg/ml polyornithine. Cells were cultured in DME-N1 containing 0.5% fetal bovine serum for 24 h, and thereafter in DME-N1 containing 2% fetal bovine serum, and maintained in 10% CO₂, 90% air, under which conditions the bipotential cells developed into oligodendrocytes. Cells were >85% positive for the oligodendrocyte marker galactocerebroside. Culture medium was replaced every 3 days, and all cells were used 4-8 days after replating.

For the study of Ca²⁺ wave propagation in glial cell processes, cells were incubated with 5 µM fluo 3-AM for 20 min at room temperature as described previously (12, 26). Experiments were performed in a Leiden coverslip chamber continuously perfused with balanced salt solution. The perfusion chamber was positioned on the stage of an inverted microscope, and fluorescence images acquired at 495 nm excitation (510 nm emission) wavelength through a microchannel plate intensifier with a CCD camera (12). Images were digitized and averaged (2 frames at each wavelength) in a Trapix 55/4256 image processor. Cells were divided for analysis into 0.8-2.0-µm-wide regions sequentially along the longitudinal axis of the cell (14). Fluorescence intensity values in the nonzero pixels within each slice were averaged (F) and plotted as normalized fluorescence intensities (F/F) against time, where F was calculated as the difference between the average value of the first 20 data points prior to stimulation of the cell and F.

Oligodendrocyte membranes were prepared and Western blots performed according to previously published methods (7). Membrane proteins were separated in 7.5% SDS-polyacrylamide gels using the Phastgel system (Pharmacia Biotech Inc.). Proteins were electrophoretically transferred to nitrocellulose and blots were incubated overnight at 4 °C in Tris-buffered saline-Tween 20 containing 5% nonfat dried milk, followed by incubation in primary antibodies (1:1000 dilution). After incubation in peroxidase-coupled secondary antibodies (Amersham Corp.), blots were developed using enhanced chemiluminescence reagents. Rabbit polyclonal antibody AP42, raised against a peptide sequence corresponding to the C-terminal region of mouse InsP₃R subtype 2 (GSNTPHENHHMPPH) (27), was a gift from Dr. A. Sharp. This sequence is unconserved, i.e. it is not present in the other known subtypes of InsP₃R or in any other known oligodendrocyte protein. PA3-900, a rabbit polyclonal antibody raised against recombinant human calreticulin produced in the baculovirus insect cell system (28), was from Affinity Bioreagents.

Immunocytochemistry on either naive cells or cells in which Ca²⁺ waves were first measured was performed as described previously (14). Cy3- or fluorescein isothiocyanate-labeled anti-rabbit secondary antibodies (Jackson Immunocytochemical Laboratories) were used as appropriate. Briefly, cells were fixed in 100% methanol at 20 °C for 3 min, washed three times in phosphate-buffered saline (PBS) (pH 7.1), then incubated in primary antibody (1:200 for anti-InsP₃R antibodies, 1:300 for anti-calreticulin) overnight at 4 °C. After further PBS washes, cell were incubated for 1-2 h in secondary antibody (1:400 dilution in 10% goat serum). Cells were then washed three times, and the coverslips were mounted on a glass microscope slide using Mowiol. Controls consisted of substitution of the primary antibody with normal serum from the same host species, used at a dilution equal to that of the primary antibody, and consistently showed negligible fluorescence levels. For the study of mitochondrial distribution, living cells were incubated with MitoTracker Red CMXRos (500 nM, 30 min, Molecular Probes) at 37 °C, then washed in prewarmed PBS. Cells were then imaged using a Cy3 filter set in a fluorescence microscope. To examine the spatial relationship between mitochondria and ER markers, MitoTracker-loaded cells were fixed in 2% paraformaldehyde (4 min, 4 °C) and 100% methanol (20 °C, 3 min), washed, and developed for immunocytochemistry as above. Use of fluorescein isothiocyanate-labeled secondary antibodies enabled good resolution and separation of antibody fluorescence from that of MitoTracker. Cells previously subjected to [Ca²⁺]_i measurements were processed for immunocytochemistry or organelle labeling on the microscope stage following the same procedures described for naive cells.

For comparison of immunofluorescence or mitochondrial staining with Ca²⁺ release kinetics, cells were imaged in the Ca²⁺ imaging system using a Cy3 filter set (Chroma Technologies, Inc., Brattleboro, VT). For analysis of subcellular immunofluorescence with high resolution, a digital confocal microscopy technique was employed (14). For this, cells were imaged with a cooled CCD camera (Photometrics, Inc., Tucson, AZ) using the Cellscan software environment (Scanalytics, Inc., Billerica, MA). The Cellscan environment allows for acquisition of wide angle fluorescence microscope images at all the focal planes through cells (z-series). Images are then restored using a deconvolution procedure (extensive photon reassignment protocol, EPR) into confocal images by removing out-of-focus light. The software system is based on the algorithm developed by Dr. Fay and co-workers (14, 29). The algorithm used the point-spread function of the microscope obtained by acquiring a z-series images of a subresolution polystyrene bead 200 nm in diameter filled with fluorophore. This data set was used by the algorithm to reassign out-of-focus plane light, which causes blurring of confocal images (29). Under our measurement conditions, the z-resolution by our optics was 0.48 µm (measured as full width at half-maximum intensity).

The spatial patterns of local Ca²⁺ release kinetics measured in 0.83-µm-wide subregions of oligodendrocyte processes and of mitochondrial distribution measured by fluorescently tagging mitochondria in the same cells were compared using a cross-correlation function as a quantitative test for similarity (14, 30). Cross-correlation function is derived from the fast Fourier transform of the two data sets being compared. For this analysis the mean values of the amplitudes in the patterns were subtracted out and the resulting zero mean waves were embedded in surrounding zeros, to eliminate circular correlations, i.e. between the beginning and end of the data sets, due to the periodic nature of Fourier series (30). The data were then analyzed via a Fast Fourier Transform algorithm, using standard functions in Mathematica (Wolfram Research Inc.). Cross-spectra were formed as a product of one data set with the complex conjugate transform of a second data set. The cross-correlation function was produced by inverse Fourier transformation of the cross-spectrum. Performing these operations on a single data set produced the power density spectrum and the auto-correlation function. Results are presented as mean ± S.D. The validity of this quantitative analysis technique was evaluated in two different control experiments. In one, we compared synthesized noisy sine waves of identical frequency but slightly out of phase with each other (Fig. 4A, bottom traces). The cross-correlation of these waveforms (Fig. 4B, solid circles) is a wave pattern of the same frequency as the original waves, with peak correlation shifted from phase to a position equivalent to the delay between the two signals. The noise experimentally inserted into the two sine waves (see figure legend) has minimal effect on the outcome of the cross-correlation. In the second control experiment, a real data set was compared with a scrambled data set (Fig. 4A, top traces) to determine if chance alone would cause high cross-correlation. The method used was a random permutation in the order of the data points, generated by the program Random Permutation in the Discrete package of Mathematica. By this method non-correlational statistics (mean, variance, and all other parameters of the distribution) were kept constant, but spatial correlations were deleted via a random permutation in the order of the data points. The bottom patterns in Fig. 4A are equivalent to the data presented in Fig. 4C (see below), except that one of the patterns has been scrambled with respect to position. The resultant cross-correlation (Fig. 4B, open circles) is a somewhat noisy, relatively flat function that does not greatly deviate from zero correlation at any delay, unlike the cross-correlation function of the original data sets shown in Fig. 4D. These results are consistent with cross-correlation analysis conservatively detecting genuine but not coincidental correlations within related data sets.

RESULTS

Initial experiments characterized Ca²⁺ waves in oligodendrocytes evoked by activation of different phosphoinositide-coupled receptors. Oligodendrocytes stimulated with norepinephrine (200 nM) or bradykinin (200 µM) responded with propagating Ca²⁺ wavefronts. High resolution spatiotemporal analysis revealed that these waves initiated at discrete cellular regions and propagated along the processes and the cell body. In Fig. 1 (A and B), offset plots show typical time course of the local changes in [Ca²⁺] observed in successive 2.0-µm-wide sections of the same cell receiving the two stimuli. Ca²⁺ waves initiated at four distinct sites where the wave reaches 50% of maximum amplitude sooner than surrounding regions. These wave initiation sites (marked with asterisks) are identifiable as the local "minima" in Fig. 1C. Three are located in cellular processes and one in the cell body. In contrast wave amplification occurs at other more numerous loci. At these wave amplification sites (marked with arrows), the magnitude and rate of Ca²⁺ rise were substantially higher than in surrounding regions (Fig. 1, D and E).

We have previously described similar distinct initiation and propagation domains in oligodendrocytes responding to methacholine (14) and in astrocytes responding to norepinephrine (12, 13). Applying the cross-correlation analysis also used in the previous work, we have now examined the distribution of sites associated with responses to norepinephrine and bradykinin. Local Ca²⁺ peak amplitudes had a maximal cross-correlation coefficient of 0.81 at 0.0 µm from phase. Similarly, the comparison between the half-rise time and the rates of Ca²⁺ rise for norepinephrine and bradykinin responses gave peak coefficients of 0.79 and 0.75 at 2.0 µm from phase. Such analysis also confirms that wave initiation and amplification sites are indeed distinct. For example, comparison of half-rise time for bradykinin with norepinephrine local Ca²⁺ peak amplitudes had a maximal coefficient of 0.08 at 0.0 µm. Other control analyses (see "Experimental Procedures") established that this analytical protocol does not readily identify false positive relationships.

The expression of InsP₃R2 and calreticulin in oligodendrocytes was investigated using Western blotting and immunocytochemical analyses. The antibody AP42, raised against an unique sequence in the C-terminal region of InsP₃R2, detected a single band of approximately 250 kDa in oligodendrocyte membranes (Fig. 2A), consistent with the expected size of this protein from previous reports (8, 27). Previous experiments clearly showed that this antibody does not cross-react with either InsP₃R1 or InsP₃R3. Antibody PA3-900, raised against recombinant human calreticulin, reacted against a single band at approximately 60 kDa (Fig. 2B) (28), indicating the expression of calreticulin in or associated with oligodendrocyte membranes. These antibodies were then used to investigate the distribution of InsP₃R2 and calreticulin in oligodendrocytes using standard immunocytochemical techniques and high resolution fluorescence microscopy (see "Experimental Procedures") (Fig. 3). InsP₃R2 immunofluorescence (Fig. 3A) was found distributed in a variegated manner throughout the cell body, except the nucleus, and along the length of the cell processes. Similarly, calreticulin immunofluorescence was also found in the cell body and in a punctate pattern along oligodendrocyte processes (Fig. 3B). The size of these clusters of immunofluorescence varied between different cells, being typically longer and more graded in thick processes, but small and highly punctate in thin processes (see also Fig. 5).

Since, in oligodendrocyte processes, mitochondria appear to be always associated with Ca²⁺ wave amplification sites (14), experiments were performed to investigate if mitochondria are mobile or stationary in these processes. For this, we incubated oligodendrocytes with MitoTracker CMXRos (MitoTracker), a mitochondria-specific fluorescent dye, and imaged mitochondria with high spatial resolution in cellular processes over time. Cells were imaged every 2 s over a period of up to 15 min. Neither rapid movement nor slow migration of mitochondria were observed during this period of time under either resting (Fig. 3C) or agonist-stimulated (data not shown) conditions.

We have previously demonstrated that the distribution of ER in oligodendrocyte processes appears approximately uniform (14). This finding, however, does not preclude the possibility of specializations in the distribution of ER proteins. To examine whether the sites of high density ER protein distribution were related to the sites of high Ca²⁺ release kinetics, we performed immunocytochemistry in cells after measurement of wave kinetics in the same cells. We stimulated cells with InsP₃-generating agonists, and measured the kinetics of the resulting wave in serial x, y sections of the cell along its axis, as in data shown in Fig. 1. We then fixed the cell on the microscope stage and incubated with appropriate primary antibody and fluorescent secondary antibodies. The pattern of immunofluorescence was then imaged, and the intensities were measured within the same serial sections of the cell in which the Ca²⁺ wave kinetics were measured. The resultant profile was then compared with the profile of Ca²⁺ wave kinetics using cross-correlation analysis.

Fig. 4 (C-F) shows the results of two experiments where the local kinetics of Ca²⁺ release (half-rise time, local peak Ca²⁺ amplitudes, and rates of Ca²⁺ rise) were measured as described in the legend to Fig. 1. A plot of the local Ca²⁺ amplitudes against the length of the process, together with the intensity of calreticulin staining measured in the same cellular sites, showed that the regions with high intensity calreticulin immunofluorescence corresponded closely with the regions of the process where the local peak Ca²⁺ amplitudes were highest (Fig. 4C). Local peak Ca²⁺ amplitudes are found either coincident with or within 1-4 µm of peaks in calreticulin immunofluorescence. Cross-correlation coefficient was high and was similar to the auto-correlation function of the calreticulin fluorescence pattern (Fig. 4D). In parallel experiments, the intensity of InsP₃R2 immunofluorescence in oligodendrocyte processes was similarly compared with the pattern of local Ca²⁺ peak amplitudes. In the cell shown in Fig. 4E, high intensity InsP₃R2 fluorescence was found close to sites where high local peak Ca²⁺ amplitudes were measured and comparison yielded high degree of correlation coefficients in phase (Fig. 4F).

In several experiments, we consistently found similar high cross-correlation values between the patterns of local Ca²⁺ release amplitudes and the patterns of ER protein (calreticulin and InsP₃R2) distribution (Table I). In particular, cell regions with elevated levels of InsP₃R2 and calreticulin consistently displayed significantly higher amplitude local Ca²⁺ release signals than were found in surrounding regions (Table I). The pattern of rates of rise of the responses (i.e. slopes), however, showed more modest cross-correlation values (Table I), probably reflecting the noise level inherent in that measurement.

Table I. Correlation between distribution of ER proteins and Ca2+ wave propagation characteristics Results of cross-correlation analysis of Ca2+ wave patterns evoked by InsP3-generating agonists and fluorescence of labeled antibodies (AP42, PA3-900) against InsP3R2 and calreticulin. Values are peak cross-correlation (mean ± S.D.). High correlation is found between location of high protein expression and two parameters of high Ca2+ release kinetics, peak (amplitude of Ca2+ response) and slope (rate of rise of Ca2+ response). Correlation occurs very close to phase, i.e. the locations of maxima in these kinetic parameters are very close to maxima in antibody fluorescence. Comparison Peak correlation Location of peak correlation n µm Peak vs. InsP3R2 0.57  ± 0.25 1.88  ± 1.04 4 Peak vs. calreticulin 0.64  ± 0.25 1.66  ± 1.04 6 Slope vs. InsP3R2 0.47  ± 0.27 0.85  ± 0.98 4 Slope vs. calreticulin 0.23  ± 0.54 1.26  ± 1.57 6

In another series of experiments, we compared the distribution pattern of ER proteins with the location of mitochondria in oligodendrocyte processes. We have shown previously that, in oligodendrocyte processes, mitochondria are distributed singly or in groups along processes only at sites of wave amplification (14). Since the specialized wave amplification sites in these processes also contain accumulation of calreticulin and InsP₃R2, we wanted to investigate the spatial relationship between the ER proteins and mitochondria. In these experiments, we again used MitoTracker to selectively stain mitochondria. MitoTracker reacts with accessible thiol groups to form an aldehyde-fixable conjugate and so, unlike other mitochondrial dyes, is retained within mitochondria after fixation and permeabilization (31, 32). Cells were incubated with MitoTracker, washed, fixed, and then incubated with antibodies against InsP₃R2 or calreticulin. High resolution analysis of the results of these experiments demonstrated that, in oligodendrocyte processes, high concentrations of the ER proteins were always found in close apposition to mitochondria (Figs. 5 and 6). Calreticulin immunofluorescence (green) was found predominantly in several intense patches with little fluorescence in between (Fig. 5A) (cell shown was typical of 4 cells analyzed by digital confocal microscopy). In this cell, rendered from from multiple z-plane images in three-dimensional voxel, MitoTracker staining (red) closely corresponded with the regions of high calreticulin immunofluorescence (Fig. 5A). A side-on view of this process (Fig. 5B) illustrates that calreticulin and mitochondria were found in closely apposed z-planes, consistent with a close spatial correspondence between ER and mitochondria. A zoomed-in, single optical plane view of the area within the box in Fig. 5A reveals that even within a single 0.09-µm z-dimensional plane, calreticulin fluorescence entwines closely around a group of mitochondria (Fig. 5C). Only occasionally was a strongly staining bead of calreticulin found without a nearby mitochondrion.

Fig. 6 shows a three-dimensional voxel rendering of multiple z-plane images of a cell in which mitochondria were stained with MitoTracker, and InsP₃R2 was identified by AP42 immunofluorescence. InsP₃R2 fluorescence extended throughout the ER in these cells with significant perinuclear staining (Fig. 6A) (cell shown was typical of 3 cells using digital confocal microscopy). In processes, however, InsP₃R2 immunofluorescence consistently appeared in high density patches (Fig. 6A). These regions of high density InsP₃R2 staining in processes were closely associated with mitochondria, as shown by MitoTracker fluorescence in the same regions of the cell (Fig. 6B). A zoomed-in view of one such site (Fig. 6C) illustrates a convoluted group of mitochondria (red) such as are typically found in thicker oligodendrocyte processes (see also Fig. 5C and Ref. 14), surrounded by multiple InsP₃R2 "hot-spots" (green) located within ~1 µm of the mitochondria. A side-on view of the region in C is shown in Fig. 6D. Mitochondria and InsP₃R2 are found to be in similar z-planes to each other (Fig. 6D), consistent with a close spatial apposition in three-dimensional space. Numerous mitochondria, relatively short and rounded in shape, were observed in the cell body (Fig. 6B). Unlike the close similarity in the patterns of distribution between the ER proteins and mitochondria along processes in the cell body, both calreticulin (data not shown) and InsP₃R2 distribution did not closely mirror the distribution of mitochondria (Fig. 6, A and B).

These results demonstrate that both calreticulin and InsP₃R2 are expressed in accumulated patches in a close spatial relationship with one or more mitochondria along oligodendrocyte processes. This finding is consistent with our previous observation that mitochondria are always found at regions of high Ca²⁺ release kinetics (14), and indicates that ER specializations occur at sites close enough to single or a cluster of mitochondria to potentially permit complex functional interactions between mitochondria and Ca²⁺ stores during intracellular Ca²⁺ signaling.

DISCUSSION

We have investigated the structural specializations that underlie enhanced Ca²⁺ release sites in oligodendrocyte processes that support long distance propagation of Ca²⁺ waves. Our results demonstrate that in oligodendrocytes wave initiation and amplification sites remain invariant during Ca²⁺ waves activated by different InsP₃-generating agonists in the same cell. This is in contrast to a report that different InsP₃-coupled agonists evoke Ca²⁺ waves that initiate in different subcellular domains within pancreatic acinar cells (33). The molecular basis of wave initiation sites in oligodendrocytes remains undetermined, although locally elevated resting Ca²⁺ levels have been suggested to underlie the sites of wave initiation in astrocytes (12, 13, 16), and spatial compartmentalization of Ca²⁺ signaling complexes to determine wave initiation sites in pancreatic acinar cells (33) (for review, see Ref. 34).

Our results are consistent with the existence of cellular specializations supporting enhanced Ca²⁺ release function at discrete wave amplification sites, whereas no such specializations were identified at wave initiation sites. When we compared local Ca²⁺ release kinetics during agonist-induced Ca²⁺ waves with distribution of ER proteins and mitochondria, we found that a number of cellular factors appear to colocalize with wave amplification sites. These include high density patches of InsP₃R2, accumulation of calreticulin, and the presence of mitochondria singly or in convoluted groups. It appears, therefore, that in oligodendrocyte processes, multiple cellular specializations may underlie enhanced Ca²⁺ release sites, and mitochondria may function together with the ER to generate the local cytosolic Ca²⁺ signals that support wave propagation.

A number of previous studies have shown that interactions occur between mitochondria and ER-dependent Ca²⁺ signals in various types of cells (18, 19, 35-37). Our present study demonstrates an intimate and regionally specialized relationship between ER and mitochondria, which has apparent functional consequences for wave propagation. Previous findings suggest that the ER membrane system extends approximately uniformly throughout oligodendrocytes (14). However, regions of ER near mitochondria display accumulations of the Ca²⁺-binding protein, calreticulin, and a high density of InsP₃R2 Ca²⁺ release channels in the membrane. These regions display larger amplitude and more rapidly rising Ca²⁺ responses and regeneratively support wave propagation. Mitochondria take up Ca²⁺ during cytosolic Ca²⁺ waves in oligodendrocytes (14). The localization of mitochondria in intimate association with Ca²⁺ release sites (InsP₃Rs) could thus regulate the Ca²⁺-dependent gating kinetics of the InsP₃R Ca²⁺ release channels. Indeed, inhibition of mitochondrial activity using p-trifluoromethoxyphenyl hydrazone or antimycin often inhibits or abolishes cytosolic Ca²⁺ responses (14). While the precise mechanisms involved remain to be elucidated, the functional consequence of the ER/mitochondria specialization in regions of oligodendrocyte processes may be amplification of local Ca²⁺ release kinetics, an important feature of long distance Ca²⁺ wave propagation.

The finding of calreticulin and InsP₃R2 together in the ER in the present study is consistent with findings in several other cell types (2, 10). Previous studies have also shown that overexpression of calreticulin in Xenopus oocytes reduced the frequency of InsP₃-mediated Ca²⁺ waves (4), whereas overexpression in HeLa cells decreased the rate of return to basal [Ca²⁺] after InsP₃-evoked Ca²⁺ release (11). In addition, decreasing calreticulin levels has been reported to lower the amplitude of the Ca²⁺ response to bradykinin in NG-108-15 neuroblastoma cells (38). Although calreticulin is believed to have a variety of functions, including an apparently complex role in regulating Ca²⁺ responses (2, 34, 39), the precise molecular role it plays in signaling is not understood. Our observation that calreticulin is found in high concentrations near InsP₃Rs in oligodendrocyte processes is suggestive of a role in localizing Ca²⁺ to release site domains, thereby contributing to enhanced release kinetics at specialized Ca²⁺ release sites. A physical interaction between activated InsP₃Rs and calreticulin, which results in a conformation change in calreticulin, thus releasing bound Ca²⁺, has been previously suggested (4).

Recent experiments show that oligodendrocytes in both brain and spinal cord express at least type 1 InsP₃Rs, with the expression in brain oligodendrocytes being transient during development while in the spinal cord it is more persistent (40). The expression of type 2 and type 3 InsP₃Rs in oligodendrocytes, however, has not previously been studied. We show here that, in culture, oligodendrocytes express InsP₃R2 in the cell body and throughout the process arborizations, and that they are found in high density at specialized Ca²⁺ release sites. Type 1 and type 3 InsP₃Rs are also expressed in oligodendrocytes but are only found in the perinuclear region of the cell, suggesting that they are unlikely to be important for Ca²⁺ wave propagation along processes in these cells.² Such spatially discrete functional roles for InsP₃R subtypes has been suggested recently in Xenopus oocytes (9).

The results presented here show that in oligodendrocyte processes, Ca²⁺ wave propagation is supported by specialized Ca²⁺ release sites with enhanced Ca²⁺ release kinetics. These sites are identified by accumulation of the ER proteins, calreticulin and InsP₃R2, and the presence of mitochondria, which may modulate the Ca²⁺ release process. A combination of ER proteins and intimate mitochondrial involvement may facilitate large localized changes in Ca²⁺ response characteristics and support long distance signaling. While several aspects of these structural specializations have now been identified, the precise molecular mechanisms and the interactions between the individual components that underlie the enhanced kinetic behavior of the local machinery remain to be investigated.