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Role of calcium influx in process extension of oligodendrocytes Yoo, Andrew 1998

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ROLE OF CALCIUM INFLUX IN PROCESS EXTENSION OF OLIGODENDROCYTES by Andrew Yoo B.Sc, McGill University, 1995 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Medicine, Neurology) We accept this thesis as conforming to/the required standard. THE UNIVERSITY OF BRITISH COLUMBIA August 1998 © Andrew Yoo, 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. c Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) Abstract The effects of phorbol ester, which activates protein kinase C (PKC), and free cytosolic Ca 2 + were studied on their role in process extension in cultured murine oligodendrocytes. Previous studies have shown that PKC activation by phorbol esters greatly enhances process formation in cultured oligodendrocytes (OLG), an important step for myelination. Because of the possible involvement of PKC and Ca 2 + in process extension in OLGs, we studied the effect of PKC activation on changes in intracellular free Ca 2 + concentration ([Ca2+]i). We found that PKC activation by phorbol esters induced a sustained rise in [Ca2+]i of OLGs. This rise was due to transmembrane influx of Ca 2 + since omission of extracellular Ca 2 + failed to trigger the rise in [Ca2+]i. Changes in [Ca2+]; were also produced by modifying the extracellular [Ca2+] where increasing extracellular [Ca2+] led to a rise in [Ca2+]i. In order to establish the relationship between Ca 2 + influx and OLG process formation, OLGs were incubated in media containing various [Ca2+] with or without phorbol ester treatment. After 72 hours, OLGs were immunostained using an antibody against proteolipid protein (PLP). The correlation between the process formation of OLGs and extracellular [Ca2+] was assessed by obtaining the percentage of OLGs with longer processes in the OLG population, the number of primary process per cell body and the area covered by PLP-positive OLG processes. Our results indicate that the degree of OLG process extension is related to the [Ca2+] present in the culture media. We found that increased extracellular [Ca2+] alone, without concurrent phorbol ester application, resulted in increased OLG process extension. When OLGs were treated with phorbol ester, positive correlations between increasing extracellular [Ca2+] and some aspects of OLG process extension were seen, as suggested by the results from analysis of covariance (ANCOVA). In addition, blocking the intracellular C a 2 + signalling by B A P T A as well as inhibiting P K C by R O - 31 leads to retraction o f membrane sheath o f O L G s . O u r results demonstrate that increasing [Ca 2 + ] j stimulates O L G process outgrowth and suggest that intracellular C a 2 + signalling fo l lowing either phorbol ester treatment or increasing extracellular [ C a 2 + ] may be an important mediator for O L G process extension. Table of Contents Abstract ii Table of Contents iv List of Figures vi List of Table vii List of Abbreviations viii Acknowledgements x Chapter 1 Introduction 1 1.1 Biology of oligodendrocytes 2 1.1.1 Myelination 2 1.1.2 The importance of OLG process outgrowth in demyelinating diseases 4 1.1.3 PKC isoforms in OLGs 6 1.2 Ca 2 + in neurite outgrowth and its relation to OLG process extension 7 1.3 Ca 2 + entry pathways in OLGs 9 1.3.1 Voltage-gated Ca 2 + channels in OLGs 9 1.3.2 Ligand-gated Ca 2 + channels in OLGs 11 1.3.3 Capacitative Ca 2 + entry in OLGs 12 1.3.4 Ca 2 + release from intracellular stores in OLGs 13 1.3.5 Other stimuli that cause [Ca2+], increases in OLGs 16 1.4 Objectives 19 Chapter 2 Materials and Methods 21 2.1 Materials 21 2.2 Tissue Culture 21 2.2.1 Murine oligodendrocytes 21 2.3 Measurement of intracellular free [Ca2+] 23 2.3.1 Preparation of buffers and cell loading with fura-2 23 2.3.2 Fura-2 Imaging 24 2.3.3 Calibration 24 2.3.4 Preparation of test solution 26 2.4 Culturing in different extracellular [Ca2+], immunostaining and cell morphology assessment 28 2.4.1 Culturing in different extracellular [Ca2+] 28 2.4.2 Immunocytochemistry 28 2.4.3 Cell Morphology Assessment 29 2.5 Statistics 31 V Chapter 3 Results 33 3.1 Fura-2 calibration 33 3.2 Effect of phorbol ester-induced protein kinase C activation on [Ca2+]i in OLG 33 3.2.1 Effect of PDB treatment of OLG in media with normal [Ca2+] (1.8 mM) 33 3.2.2 Effect of PDB treatment of OLG in Ca2+-free buffer 39 3.3 Effect of changing extracellular [Ca2+] on [Ca2+]; in OLGs 47 3.4 Effect of extracellular [Ca2+] on OLG process extension 52 3.4.1 Effect of extracellular [Ca2+] on the percentage of OLGs with long processes 55 3.4.2 Effect of extracellular [Ca2+] on the number of primary processes 58 3.4.3 Effect of extracellular [Ca2+] on areas covered by OLG processes 60 3.5 Effects of inhibitors on OLG process formation at high [Ca2+] 62 Chapter 4 Discussion 65 4.1 Fura-2 calibration 65 4.2 Effect of phorbol ester-induced PKC activation on [Ca2+]i in OLG 66 4.2 Effect of extracellular Ca 2 + on [Ca2+]; in OLGs 72 4.4 Effect of extracellular Ca 2 + on OLG process extension 75 References 85 vi List of Figures Fig. 1 Routes of Ca 2 + entry identified in oligodendrocytes (OLGs) 14 Fig.2.1 Experimental setup of fura-2 imaging system 25 Fig.2.2 Fura-2 imaging of oligodendrocytes 27 Fig.2.3 Measurement of oligodendrocyte process formation 30 Fig.3. la-b Calibration of fura-2 signal 34 Fig. 3.2.1 a-b Intracellular Ca 2 + response of OLGs to PDB 36 Fig.3.2. lc Ca 2 + response of OLGs pretreated with RO-31 (1 uM) to PDB treatment 37 Fig.3.2. Id Effect of nifedipine on PDB-induced [Ca2+]; increase 38 Fig.3.2.2a-b PDB exposure (1 uM) in Ca2+-free media 40 Fig.3.2.2c OLGs exposed to Ca2+-free medium and reestablishment of extracellular Ca 2 + in the absence of PDB 42 Fig.3.2.2d-e Capacitative Ca 2 + entry in OLGs 44 Fig.3.2.2f Effect of Ca2+ATPase inhibition on PDB-induced [Ca2+]i response 45 Fig.3.2.2g Effect of Ca2+ATPase inhibition during PDB-induced [Ca2+]i response 46 Fig.3.3a Exposure of OLGs to a Ca2+-free environment 48 Fig.3.3b-c Ca 2 + response of OLGs to changes in extracellular [Ca2+] 49 Fig.3.3d Effect of CPA on [Ca2+]; increase mediated by high extracellular [Ca2+] ....50 Fig.3.3e Effect of RO-31-8220 on [Ca2+]i increase mediated by high extracellular [Ca2+] 51 Fig.3.4a-c OLGs grown in various extracellular [Ca2+] 54 Fig.3.4.1 Percentages of cells with processes equal or longer than 3 times cell body diameter as an index of process extension of OLGs grown in various extracellular [Ca2+] 56 Fig.3.4.2 Number of primary processes per cell body as an index of process extension of OLGs grown in various extracellular [Ca2+] 59 Fig.3.4.3 Areas covered by OLG processes as an index of process extension of OLGs grown in various [Ca2+] 61 Fig.3.5a-b Effects of inhibiting PKC and intracellular Ca 2 + signalling on OLG process formation 63 V l l List of Table Table 1. Stimulus-evoked intracellular Ca2 + elevation in oligodendrocytes 2 List of Abbreviations AA Arachidonic acid ABC Adivin-biotin complex AMPA a-amino-3-hydroxy-5-methyl-4-isozaxole propionic acid AMPA DL-a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid ANCOVA Analysis of covariance ATP Adenosine triphosphate BAPTA Bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid bFGF Basic fibroblast growth factor BK Bradykinin BSA Bovine serum albumin [Ca2+]i Intracellular free Ca 2 + concentration cAMP Cyclic Adenosine monophosphate CCCP Carbonyl cyanide /w-chlorophenylhydrazone CICR Ca2+-induced Ca 2 + release CNP Cyclic nucleotide phosphodiesterase CNS Central nervous system CPA Cyclopiazonic acid CRAC Ca 2 + release-activated Ca 2 + (channels) DAB 3,3'-diaminobenzidine DAG Diacylglycerol DMEM Dulbecco's Modified Eagle Medium DMSO Dimethyl sulfoxide EGTA Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid FMAX(380nm) Maximum fluorescence intensity at 380 nm (under saturating [Ca2+]) FMiN(380nm) Minimum fluorescence intensity at 380 nm (under zero [Caz+]) Fura-2/AM Fura-2 acetoxymethyl ester Fura-2/K5 Fura-2 pentapotassium salt GAB A Gamma-aminobutyric acid GalC Galactocerebroside GluR Glutamate receptor HBSS HEPES buffered Hank's balanced salt solution IGF-I Insulin-like growth factor I InsP3 Inositol 1,4,5-triphosphate Kd Dissociation constant LGCC Ligand gated Ca 2 + channel mAb Monoclonal antibody MAG Myelin-associated glycoprotein MAPK Mitogen-activated protein kinase MBP Myelin basic protein MEM Minimal essential medium MGDG Monogalactosyl diglyceride MMP Matrix metalloproteinase mRNA Messenger ribonucleic acid NMDA N-methyl-D-Aspartate NMD A N-methyl-D-aspartate 0-2A oligodoendrocyte-type-2 astocyte (progenitor) OLG Oligodendrocyte PBS Phosphate-buffered saline PDB 4P-phorbol-12,13-dibutyrate PDD 4-a-phorbol-12,13-didecanoate PDGF Platelet derived growth factor PIP2 Phosphatidylionositol 4,5-bisphosphate PKA Protein kinase A PKC Protein kinase C PLC Phospholipase C PLP Proteolipid protein R Ratio Rmax Maximum ratio Rmin Minimum ratio ROC Receptor-operated channel rpm Revolutions per minute S.D. Standard deviation SEM Standard error of the mean SERCA Sarcoplasmic/Endoplasmic reticulum Ca 2 + ATPase SIT Silicone intensified target SP Substance P TG Thapsigargin VDCC Voltage-dependent Ca 2 + channel Acknowledgements I am grateful to my supervisors, Dr. Seung U . Kim and Dr. Charles Krieger, for their patience, encouragement and enthusiasm. Without their guidance, this study would not have been possible. I also wish to thank Dr. Michael Schulzer and Edwin Mak for their help with statistical analysis, and Yong Beom Lee for helpful discussions. My sincere gratitude also goes to Linda Kang for always giving me emotional supports and Dave Kang and my friends who make my life fruitful. My deepest gratitude goes to my family whose love and support is everlasting and to whom I will always be grateful. This work was supported by the Myelin Project of Canada. Chapter 1 Introduction The calcium ion (Ca2+) plays an important role in the function of cells. Various stimulators including hormones, neurotransmitters and electrical activity trigger Ca 2 + signalling by increasing the level of intracellular free Ca 2 + ([Ca2+]i). Ca 2 + acts as a second messenger in signal transduction cascades by binding as a co-factor, or activating Ca2+-dependent enzymes including protein kinase C and calcium-calmodulin kinase. The role of calcium in the central nervous system (CNS) has gained considerable attention in recent years, in particular its involvement in neurotransmitter release, intracellular communication, signal processing, neuronal excitotoxicity and neurite outgrowth. Much of the research has focused on Ca 2 + handling by neurons and only recently has Ca 2 + signalling been examined in glial cells. Extensive investigations of glial cell electrophysiology have shown that glial cells express the similar complex variety of ionic channels and neurotransmitter receptors as neurons do (Verkhratshy and Kettenmann, 1996). Glial cells show a wide pattern of expression of voltage-gated membrane channels selective for Na+, Ca 2 + , anions, as well as both ionotropic and metabotropic membrane receptors. Like other cell types, glial cells have cytoplasmic Ca 2 + stores, mainly in endoplasmic reticulum and mitochondria. It has been shown that oligodendrocytes (OLGs) increase their intracellular [Ca2+] level in response to various stimuli either via release from internal Ca 2 + stores or influx through Ca 2 + channels (Takeda et al., 1995). The current study is designed to elucidate one aspect of Ca 2 + signalling in OLGs in which Ca 2 + signalling might play an important role in the process outgrowth of OLGs. 2 1.1 Biology of oligodendrocytes ^ 1.1.1 Myelination Oligodendrocytes (OLGs) are the myelin-producing cells of the CNS (Norton, 1984). The myelin sheath allows saltatory conduction of nervous impulses, which enables transmission to occur at a greater velocity than unmyelinated axons. During myelination, OLG processes wrap around axons, and since each OLG process is able to myelinate each axon, a single OLG forms myelin sheaths around many axons. The myelin membrane is composed mainly of lipid, but also contains a number of structural proteins including proteolipid protein (PLP), myelin basic protein (MBP), galactocerebroside (GalC) as well as myelin-associated glycoprotein (MAG) and cyclic nucleotide phosphodiesterase (CNP). Antibodies against these proteins can be used as specific immunological markers for OLGs (Kim, 1985). OLGs isolated from adult human brains, for instance, were found to express these proteins (Kim et al., 1983; Kim et al., 1985). During myelination, the levels of major myelin proteins and their respective mRNAs, including MBP, PLP, MAG and CNP increase, and the time course of the mRNA and protein expressions coincides the production of mature myelin (Roach et al., 1983; Wiktorowicz and Roach, 1991; Zeller et al., 1985). The maturation of OLG has been shown to be under the control of growth factors in vitro. Several previous studies in primary cultures isolated from the neonatal rat optic nerve have identified glial precursor cells termed "oligodoendrocyte-type-2 astocyte (0-2A) progenitor" cells, which can differentiate into either OLGs or astrocytes depending on the presence of serum in the culture (Raff, 1989). Accumulating evidence suggests that a variety of soluble factors influence both proliferation and differentiation of these cells in which bipolar 02-A progenitor cells divide and differentiate into multipolar OLGs. Platelet-derived growth factor (PDGF) has been reported to be a potent mitogen for rat 0-2A progenitor cells (Noble et al., 1988; McKinnon et al., 1993), while insulin-like growth factor I (IGF-I) induces maturation of these cells (McMorris and Dubois-Dalcq, 1988). It is also believed that the expression of myelin-specific proteins increases with PDGF exposure in culture. PDGF treatment increases the rate of OLG precursor cell proliferation, the number of mature OLGs, as well as the levels of PLP and MBP mRNAs within 24 hours of treatment (Grinspan et al., 1993). The increase in myelin-specific protein mRNA expression is seen before any significant changes in OLG population or their precursors, suggesting that PDGF regulates myelin gene expression. Basic fibroblast growth factor (bFGF) has also been shown to promote OLG differentiation that treating OLGs with bFGF resulted in pronounced elongation of OLG processes, a characteristic property of mature OLGs (Oh et al., 1997). Insulin and insulin-like growth factor (IGF)-I have been demonstrated to promote differentiation of OLGs isolated from fetal human brains since insulin and IGF-I induced 3-fold increase in the number of OLGs immunoreactive to Ranscht-monoclonal antibody (R-mAb) and 04, other makers for mature OLGs (Satoh and Kim, 1994). Insulin and IGF-I did not, however, induce proliferation of these R-mAb+04+ cells, as evidenced by the lack of an increase in bromodeoxyuridine (BrdU) incorporation (Satoh and Kim, 1994). Recent studies have focused on the signal cascades during myelination. The prevailing finding is that the majority of myelin-specific proteins are phosphorylated during myelination through activation of protein kinases. MAG, which is expressed by the myelin-forming subpopulations of OLGs at the time of or after their first assocation with axons (Martini and Schachner, 1988; Bartsch et al., 1989; Owens and Bunge, 1989), has been shown to be phosphorylated by the 4 activation of protein kinase C (Kirchhoff et al., 1993). Fyn tyrosine kinase, one of the non-receptor-type tyrosine kinases of the Src family, has been suggested to be an important enzyme during myelinogenesis since Fyn kinase phosphorylates MAG on tyrosine residues (Umemori et al., 1994). CNP, an enzyme associated with myelin membranes and OLG cytoplasm (Kim et al, 1984; Sprinkle, 1989), has also been found to be phosphorylated by protein kinase A and protein kinase C (Agrawal et al., 1994). Forskolin and phorbol ester treatment of brain slices result in enhanced CNP phosphorylation (Agrawal et al., 1994). These findings suggest that myelination is a process having a complex cascade of signal transduction steps in which phosphorylation of various OLG-specific proteins occurs through the activation of tyrosine kinases, protein kinase C and protein kinase A. 1.1.2 The importance of OLG process outgrowth in demyelinating diseases Autoradiographic studies indicate that OLG turnover time is much slower than that of astrocytes in adult mice, and was calculated to be between one and two years (MacLaurin and Yong, 1995). Even though very slow, there is apparently a continuous remyelination of the CNS throughout normal adult life. However, in the case of demyelinating diseases such as multiple sclerosis, there is evidence that OLGs proliferation, differentiation and remyelination are increased (Rodrigues, 1992; Prineas et al., 1993). Multiple sclerosis (MS) is a common, disabling neurological disease of adults. It is a demyelinating disease of the human central nervous system, and is characterized by the loss of myelin and OLGs (Rodrigues, 1992; Prineas et al., 1993). Although the exact etiology of MS still remains to be established, the consensus of opinion regarding the pathogenesis of MS is that MS is an autoimmune disease in which major parts of myelin (including MBP and PLP) act as autoantigens targeted by cytotoxic T cells. While MS is a demyelinating disease where the inflammation results in a substantial loss of myelin and significantly reduced OLG population at the lesion center, OLG are found in substantial numbers at the edge of lesions. The presence of such OLGs, together with relative sparing of axons in MS, suggest the potential of remyelination by the remaining OLGs, and it has been shown that there is limited remyelination occurring in the lesions in MS (Raine et al., 1981; Raine and Wu, 1993). In order to improve remyelination in demyelinating diseases such as MS, one approach might be to stimulate the remaining OLGs in the lesions to promote process formation of OLG, an early even in myelin formation. Investigations to find stimuli which promote OLG process extension have revealed that activation of protein kinase C is required (Yong et al., 1988; Althaus et al., 1991; Yong et al, 1994). For instance, treating OLGs with the protein kinase C (PKC) stimulator, 4P-phorbol-12,13-dibutyrate (PDB), which results in a 400-500% increase in oligodendroglial PKC activity, significantly enhanced process formation in adult OLGs. In addition, the synthesis of myelin basic protein (MBP), a prerequisite component for myelinogenesis, was increased by 2-fold in PDB-treated OLGs. Further studies employing isoform-specific antagonists of PKC also indicated that the a-isoform of PKC is a major mediator of process outgrowth in OLGs (Yong et al., 1994). According to an earlier study when OLGs adhere to substratum in vitro, there is phosphorylation of MBP by the phorbol ester-mediated PKC activation (Vartanian et al., 1986). Monogalactosyl diglyceride (MGDG), a minor galactolipid enriched in myelin (Morell and Toews, 1984), can be found in relatively high amounts in brain. The highest concentration of MGDG as well as increase of MGDG synthetase activity was observed in the rat brain during the period of active myelination, which coincides with enhanced PKC activity (Hashimoto et al., 1988). In addition, MGDG, a marker for the onset of myelination, has been shown to activate PKC in OLGs (Schmidt-Schultz and Althaus, 1994). They also found that MGDG primarily stimulated PKC-a, a PKC isoform also reported by others to be important during OLG process elongation (Yong et al., 1994). 1.1.3 PKC isoforms in OLGs There is differential expression of PKC isozymes according to the developmental stages of rat OLGs and some Ca2+-independent PKC isozymes (PKC-8, -s and -Q are present in OLG progenitors while most of the Ca2+-dependent isozymes (PKC-a, -P and -y) are absent (Asorta and Macklin, 1994). As OLG progenitors develop into mature OLGs as defined by expression of the OLG-specific markers, PLP, MBP and galactocerebroside (GalC), Ca2+-dependent PKC isozymes appear and increase in expression until maturity. From these results, it has been suggested that Ca2+-independent PKC isozymes are involved in the proliferation and differentiation of OLG progenitors while Ca2+-dependent isozymes are involved in OLG differentiation and the synthesis of MBP and PLP (Asorta and Macklin, 1994). Treatment of OLGs with phorbol esters resulted in differential modulation of PKC isozyme activities. An eight hour exposure of mature OLGs to PDB caused a delayed downregulation of PKC-P, -5 and -8 isozymes with only minor or no effects on -a and -C, isozymes, suggesting specific roles for PKC-a and -C, during differentiation of OLGs (Asorta and Macklin, 1994). The notion that PKC-a activation is an important factor for process extension of OLGs has been strengthened by the studies of Schmidt-Schultz and Althaus (1994). They found that in isolated pig OLGs, MGDG, a glycolipid marker for myelination, primarily activates PKC-a and induces process extension much more potently than DAG which primarily activates PKC-y (Schmidt-Schultz 7 and Althaus, 1994). A correlation between the increased occurrence of MGDG-stimulated PKC activity and a period of maximal process formation of OLGs further supported the important role for both PKC-a and its endogenous activator, MGDG in OLGs process extension. PKC activation by phorbol esters has also been shown to have an effect on OLG progenitor development in culture. Phorbol ester treatment to OLG progenitor cells has been shown to enhance proliferation of the progenitors, and long term exposure (4 hrs) with phorbol ester induced the terminal differentiation of OLG progenitors to GalC and MBP-expressing mature OLGs (Avossa and Pfeiffer, 1993). The observations that OLGs lack Ca2+-dependent PKC isoforms while in immature stages of development and start expressing Ca2+-dependent PKC isoforms as they differentiate into mature OLGs, suggest that Ca 2 + might play an important role during the differentiation and process extension of OLGs. As bipolar OLGs differentiate into mature, multipolar OLGs, it can be speculated that Ca 2 + signalling as well as PKC activation somehow activates a signal transduction pathway that is involved in process outgrowth of OLGs. 1.2 Ca 2 + influx in neurite outgrowth and its relation to OLG process extension Accumulating evidence suggests that OLG process extension and neurite outgrowth may share similar mechanisms. Basic FGF and an astrocyte substratum, which have been shown to enhance OLG process extension (Oh and Yong, 1996), also promote axonal outgrowth (Williams et al., 1994a; Williams et al., 1994b; Doherty and Walsh, 1996), and this effect can be blocked by inhibiting Ca2+/Calmodulin kinase (Williams et al., 1995). Matrix metalloproteinase (MMP), a key effector of extracellular matrix remodeling whose expression 8 is under the influence of Ca 2 + influx (Kohn et al., 1994), has been reported to be an important enzyme for neurite outgrowth since inhibiting this enzyme resulted in the retraction of axonal outgrowth stimulated by bFGF (Muir, 1994). OLGs were also found to utilize MPP to extend processes (Uhm et al., 1997). The activation of mitogen-activated protein kinase (MAPK) whose inhibition resulted in the retardation of OLG process outgrowth (Stariha et al., 1997) has been reported to be a key enzyme during axonal outgrowth as well (Hundle et al., 1995). The activation of MAPK pathway in OLGs is dependent on PKC activity since there is an increased immunoreactivity to extracellular signal-regulated protein kianse (ERK) and increased ERK activity when OLGs are treated with phorbol esters (Stariha et al., 1997). PC12 cells stably transfected to overexpress PKC exhibited enhanced neurite outgrowth (Hundle et al., 1995). In the SH-SY5Y neuroblastoma cell line, the PKC-a isoform, a key isoform during OLG process elongation (Yong et al., 1994), has also been found to be enriched in growth cones of neurites during the differentiation as revealed by Western blot analysis (Parrow et al., 1995). Recently, the role of Ca 2 + during neurite outgrowth has been studied extensively. In PC 12 cells, Ca 2 + influx through voltage-dependent Ca 2 + channels causes neurite outgrowth, which is accompanied by MAP kinase activation and the subsequent induction of the immediate early gene NGFI-A (Rusanescu et al., 1995). Ca 2 + influx through both N- and L-type Ca 2 + channels, triggered by binding of Thy-1 antibodies to PC 12 cells, causes neurite outgrowth and this response can be blocked by antagonists of L- and N-type channels or by reducing extracellular Ca 2 + to 0.25 mM (Doherty et al., 1993). The importance of Ca 2 + influx has also been demonstrated in cerebellar neurons in which FGF treatment of the neurons resulted in increased [Ca2+],, as well as extensive neurite outgrowth (Williams et al., 1992). Basic FGF-treated hippocampal neurons displayed larger [Ca2+]i increases than untreated neurons in response to high K + induced depolarization with increased activities of L-type voltage-dependent Ca 2 + channels, demonstrating the Ca 2 + influx is an important factor for the process of neurite outgrowth (Shitaka et al., 1996). As Ca 2 + influx is important in neurite outgrowth, it might also play an important role during process extension in OLGs. 1.3 Ca 2 + entry pathways in OLGs 1.3.1 Voltage-gated Ca 2 + channels in OLGs Voltage-gated Ca 2 + channels (VGCC) are an important pathway for Ca 2 + entry into cells. Ca 2 + channels are integral membrane proteins composed of five subunits, each playing a distinct role in channel function. The functional heterogeneity of Ca 2 channels mainly comes from differences in ai-subunit proteins and at least six major subtypes of ai-subunit have been cloned and characterized (Hofmann et al. 1994). Depending on the physiological properties and pharmacological profile, Ca 2 + channels are classified as low-voltage-activated (T-type) channels and several types of high-voltage-activated channels (L, N, P, Q, and R types). Electrophysiological studies of glial cells have revealed that some glial cell types including precursor cells, astrocytes, and OLGs, exhibit the expression of voltage-gated Ca 2 + channels that were previously believed to be present only in electrically excitable cells (Barres et al., 1989; Barres et al., 1990; Kirischuck et al., 1995c). However, not all glia express Ca 2 + channels. For instance, Bergmann glial cells, microglia, and certain populations of astrocytes appear to lack voltage-dependent Ca 2 + channels (Imoto et al., 1996; Kirischuk et al, 1996; Verkhratsky and Kettenmann, 1996). Nevertheless, these glial cells are equipped with metabotropic glutamate receptors which control Ca 2 + release from internal stores (Steinhauser and Gallo, 1996). OLGs are heterogeneous with regard to the expression of voltage-gated Ca 2 + channels. Voltage-gated Ca 2 + currents were identified in cultured mouse OLGs precursors although the density of these channels was found to be very low (Verkhratsky et al., 1990). OLG precursors demonstrate both low-voltage (T-type) and high-voltage (presumably L-type) Ca 2 + currents, and both of these currents have also been identified in mature mouse OLGs (Von Blankenfeld et al., 1992). In contrast, voltage-clamp analysis of membrane currents in cultured OLGs isolated from rat optic nerve did not reveal any Ca 2 + currents (Barres et al., 1989). In situ recordings from OLGs in a white matter preparation also failed to detect any voltage-gated Ca 2 + currents (Berger et al., 1992). The pattern of expression of Ca 2 + channels undergoes considerable change during the development of OLGs. Utilizing developmental stage-specific antibodies against OLGs specific markers ("O" series antibodies, Sommer and Schachner, 1982), it was found that Ca 2 + channels were present in early OLGs precursor cells, absent in immature OLGs, but evident in mature OLGs in culture (Von Blakenfeld et al., 1992). OLG precursors exhibited both T- and L-type Ca 2 + currents whereas late OLG progenitors expressed only one component of Ca 2 + current resembling the L-type current (Verkhratsky et al., 1990). Ca 2 + currents were substantially smaller in immature OLGs and late precursors than in early precursors. Ca 2 + currents could not be detected in young OLGs, but were detectable in mature OLGs bearing multiple processes (Berger et al., 1992). Even though the amplitude of Ca 2 + currents in OLGs was small, depolarization of cultured OLG precursors and mature OLGs with KC1 revealed substantial increases in [Ca2+], that were sensitive to removal of extracellular Ca 2 + , blocked by Cd 2 + and verapamil, and potentiated by the agonist BAY K 8644 (Borges et al., 1995; Kirischuk et al., 1995c). It is also important to note that the depolarization-induced [Ca ]i increases in cultured OLGs were spatially heterogeneous, being more pronounced in OLG processes (Kirischuk et al., 1995c). A moderate depolarization of OLG precursors by 20 mM KC1 resulted in an increased [Ca2+]; only in the processes, and when the concentration of KC1 was increased, the [Ca2+], rise was demonstrated in the soma and the amplitude of [Ca2+], elevation in the processes was reduced (Kirischuk et al., 1995c). An uneven distribution of Ca 2 + channels was also observed in mature OLGs in which a depolarization-induced [Ca2+]; increase was only detectable in the processes of OLGs (Kirischuk et al., 1995c). 1.3.2 Ligand-gated Ca 2 + channels in OLGs Ligand-gated Ca 2 + channels (LGCC) or receptor-operated channels (ROCs) are comprised of multi-subunit proteins spanning the plasma membrane. Glutamate is the major excitatory neurotransmitter in the CNS and it acts upon ionotropic glutamate receptors (GluRs), ligand-gated cationic channels assembled from five subunits. Based on their pharmacological properties, GluRs are grouped into a-amino-3-hydroxy-5-methyl-4-isozaxole propionic acid (AMPA), kainate, and N-methyl-D-aspartate (NMDA) channels. The GluR subunits A, B, C and D (or 1-4) form AMPA-sensitive receptors whereas GluR5, -6 and -7, and KAI and KA2 subunits form kainate-sensitive GluRs (see review by Hollmann and Heinemann, 1994). Moreover, the NMDA-sensitive GluR is formed by NMD A Rl and NMD A R2A-D subunits (Monyer et al., 1994). When activated by binding with glutamate, some GluRs allow an influx of Ca 2 +. The permeability of Ca 2 + depends on the subunit structure of the GluRs. GluRs containing GluR B subunit.are almost impermeable to Ca 2 + whereas GluRs lacking GluR B subunit are highly Ca 2 + permeable (Geiger et al., 1995; Burnashev, 1996). OLG lineage cells have been shown to express GluRs B, C, and D, GluR6 and -7, and KAI and KA2 mRNAs (Patneau et al., 1994). The finding that these cells express high levels of GluR B subunit also implies a low Ca 2 + permeability of OLG GluRs. Borges et al. (1994) found that application of glutamate and its agonists to OLG precursors resulted in [Ca2+]i increases through the activation of AMPA/kainate receptors, and this increase was mainly due to Ca 2 + influx through voltage-gated channels as well as AMPA receptors (Holtzclaw et al., 1995). The expression of GluRs has been found to be developmentally regulated since mature OLGs lose their ability to respond to glutamate stimulation (Borges et al., 1994). 0-2A precursor cells isolated from adult rat optic nerve have also been found to lose their response to glutamate while differentiating into mature OLGs. Quisqualate-stimulated Co 2 + uptake (which is believed to reflect Co 2 + entry through Ca 2 + permeable AMPA receptors) is only observed in precursor cells and absent in mature OLGs (Fulton et al., 1992). In OLG precursor cells, activation of GAB A A receptors leads to a depolarization of the membrane potential, in contrast to mature neurons where GABA hyperpolarizes the membrane (Kirchhoff and Kettenmann, 1992). This difference is due to the presence of an inwardly directed CI" transport in OLG precursor cells. Activation of the GAB A A receptor-linked CI" channel leads to an efflux of CI" and thus results in depolarization of the membrane, which is sufficient to activate VGCC in OLGs (Kirchhoff and Kettenmann, 1992). 1.3.3 Capacitative Ca 2 + entry in OLGs Recently, the gating of Ca 2 + entry across the plasma membrane by depletion of intracellular Ca 2 + stores, known as capacitative Ca 2 + entry (Putney, 1990), has been a subject of intensive studies. One of the major signalling pathways for increasing [Ca2 +]j is the activation of phospholipase C (PLC) which produces inositol 1,4,5-triphosphate (InsP3) to release Ca from intracellular Ca 2 + stores. In most cell types, InsP3 binding to intracellular stores results in the transient release of Ca 2 + , followed by a more sustained response which is dependent on the presence of extracellular Ca 2 + , and is believed to result from Ca 2 + influx across the plasma membrane (see review by Putney, 1997). This Ca 2 + influx is known as capacitative Ca 2 + influx, or Ca 2 + release-activated Ca 2 + influx (CRAC), and is believed to occur upon the depletion of intracellular Ca 2 + stores provoked by activation of InsP3 receptors (Putney, 1997). The discovery of potent and selective inhibitors of endoplasmic reticulum (ER) Ca2+-ATPase such as thapsigargin and cyclopiazonic acid (CPA) has enabled investigators to deplete intracellular Ca 2 + stores and to trigger capacitative Ca 2 + influx in many cell types (Berridge, 1993; Puteny 1997). Capacitative Ca 2 + influx has been demonstrated in glial cells where treating the C6 glioma cell line with CPA and thapsigargin resulted in a prolonged [Ca2+]; increase, and the cytoplasmic Ca 2 + store depletion-mediated increase in [Ca2+]; could be blocked by La 3 + and Ni 2 + , suggesting the presence of capacitative Ca 2 + influx in this cell line (Wu et al., 1997). In cultured rat OLGs, it has been demonstrated that inhibition of ER Ca 2 +-ATPase by CPA resulted in Ca 2 + release from the intracellular stores followed by capacitative Ca 2 + entry (Simpson and Russell, 1997). In nominally Ca2+-free media, inhibition of ER Ca 2 + -ATPase resulted in only a small, transient increase of [Ca2+]i. Upon the reestablishment of extracellular Ca 2 + , OLGs showed a prolonged increase in [Ca2+]j, indicating the presence of capacitative Ca 2 + entry in OLGs (Simpson and Russell, 1997). 1.3.4 Ca 2 + release from intracellular stores in OLGs A major source of Ca 2 + ions necessary for an increase in [Ca2+]j is associated with intracellular structures which are able to accumulate, store and release Ca 2 + ions in response to appropriate stimuli (see Fig. 1). These structures include the distinct intracellular compartments 14 Fig. 1 Routes of Ca 2 + entry identified in oligodendrocytes (OLGs) Oligodendrocytes express ionotropic and metabotropic receptors which are coupled to Ca 2 + signalling. Activation of ionotropic receptors depolarizes the cell resulting in the opening of voltage-dependent Ca 2 + channels (VDCC). Activation of metabotropic receptors mediate InsP3-induced Ca 2 + release from intracellular Ca 2 + stores. Ca 2 + might also enter the cell via activation of capacitative Ca 2 + entry in OLGs. It is also important to note that the expression of VDCC in OLGs is developmentally regulated. OLGs have not been reported to display any Ca2+-induced Ca 2 + release mechanism. Abbreviations: BR, bradykinin receptor; GluR, glutamate receptor; HR, Wstarnine receptor; InsP3, inositol 1,4,5-triphosphate; M, muscarinic receptor; P 2 Y / r purinoreceptor; PDGFR, PDGF receptor; SPR, substance P receptor. formed within the endoplasmic reticulum (ER) and are equipped with Ca 2 + pumps [sarco(endo) plasmic reticulum Ca 2 + (SERCA) pumps]. The major mechanism for Ca 2 + release from internal stores involves the activation of InsP3-gated Ca 2 + release channels (Berridge, 1993). The production of InsP3 is coupled to the activation of metabotropic receptors including G protein-linked receptors and receptor tyrosine kinases. Binding of ligand to these receptors triggers activation of G proteins which in turn activates phospholipase C (PLC) which cleaves phosphatidylionositol 4,5-bisphosphate (PIP2) to InsP3 and 1,2-diacylglycerol (DAG). An earlier study has indicated that cultured rat OLGs express 3 isoforms of PLC, PLC-P, PLC-5 and PLC-y (Mizuguch et al., 1991). OLGs have been reported to transiently express InsP3 receptors for a brief period during the onset of myelination, suggesting that InP3-mediated Ca 2 + release might play a role during myelination (Dent et al., 1996). In OLGs, purinoreceptor activation by adenosine 5'-triphophate (ATP) application to cultured mouse OLGs resulted in Ca release from nVsensitive intracellular stores (Kirischuk et al., 1995b). OLGs acquire sensitivity to ATP during their development; ATP-evoked Ca 2 + elevation occurs only in late precursor cells and mature OLGs but not in early precursor cells (Kirischuk et al., 1995b). Similarly, in recordings from corpus callosum slices, ATP responses were present only after postnatal day 12, after maturation of OLGs was completed. ATP-induced Ca 2 + signalling was also detectable in Ca2+-free recording buffer, suggesting that the elevation of [Ca2+]j was due to Ca 2 + liberation from internal stores (Kirischuk et al., 1995b). Another type of intracellular Ca 2 + release channel, the ryanodine receptor, has been detected in several classes of glial cells, including periaxonal Schwann cells and Muller glial-cells isolated from salamander retina (Keirstead and Miller, 1995; Lev Ram and Ellisman, 1995). Activation of ryanodine receptors occurs following depolarization of the plasma membrane that leads to a small influx of extracellular Ca 2 + ions through voltage-gated channels. Binding of these Ca 2 + ions induces Ca 2 + release from the ER. This Ca 2 + liberation through a positive feedback loop is known as Ca2+-induced Ca 2 + release (CICR) and can be activated by ryanodine and caffeine. The presence of a high intracellular Ca 2 + level, however, inhibits channel operation, allowing for the control of Ca 2 + mobilization by negative feedback (see review by Meissner, 1994). In cultured rat OLGs, caffeine and ryanodine did not have an effect on [Ca2+]; suggesting that these receptors might be absent in OLGs although OLGs could still respond to thapsigargin treatment (Kirischuk et al., 1995c; Takeda et al., 1995). Mitochondria also serve as a site for intracellular Ca 2 + storage. The uptake of Ca 2 + into mitochondria is believed to occur through an electrophoretic uniporter by which the electrical gradient across the mitochondrial membrane acts as driving force for the Ca 2 + entry into mitochondria. The efflux of Ca ions from the mitochondria occurs via the Na7Ca2 + or FT7Ca2+ exchanger (see review by Gunter and Gunter, 1994). In OLGs, the collapse of the mitochondrial electrochemical gradient by protonophores, such as carbonyl caynide m-chlorophenylhydrazone (CCCP), resulted in a [Ca2+], increase (Kirischuk et al., 1995a, Kirischuk et al., 1995c). However, CCCP treatment did not have any effect on the depolarization-induced [Ca2+]i rise in OLGs, suggesting that mitochondrial Ca 2 + storage is not involved in Ca 2 + buffering in OLGs (Kirischuk et al., 1995c). 1.3.5 Other stimuli that cause [Ca2+]i increases in OLGs Recently, investigators have identified various agonists which are able to trigger Ca 2 + signalling by increasing intracellular Ca 2 + in OLGs (see table 1). Studies of the activation of muscarinic receptors of OLGs have shown that carbachol, an acetylcholine analog, produces an increase in [Ca2+]i> which are dependent both on the mobilization of internal Ca 2 + stores and transmembrane influx of extracellular Ca2+(Cohen and Almazan, 1993, 1994). Muscarinic activation by carbachol also leads to increased D?3 production and the level of cyclic AMP (cAMP). Cohen and Almazan (1994) postulated that muscarinic receptor activation might be important in OLGs development, since increased production of cAMP was related to OLG differentiation (Raible and McMorris, 1990) and carbachol or the endogenous ligand, acetylcholine, may serve as a regulator of OLG differentiation. Exposure of rat spinal cord OLGs to arachidonic acid (AA) elicited a concentration-dependent increase in [Ca2+]i that was derived mainly from extracellular Ca 2 + (Soliven et al., 1993). The AA-derived Ca 2 + influx was mediated by the activation of voltage-independent Ca 2 + channels since the AA response was not influenced by depolarization. PDGF, a potent mitogen for rat 0-2A progenitor cells (Noble et al., 1988; McKinnon et al., 1993), induced oscillatory Ca 2 + responses in the OLG cell lines, CG4 and CEINGE cl3 (Fatatis and Miller, 1996; Fatatis and Miller, 1997). It is believed that the intracellular sphingosine level plays an important role during PDGF-mediated Ca 2 + response since blocking of the enzyme, sphingosine kinase, results in an increase in intracellular sphingosine levels and an increase in the percentage of cells responding to PDGF treatment (Fatatis and Miller, 1996). It is also believed that the increase in [Ca2+]i can facilitate intercellular signalling between OLGs since gap junctional contacts between OLGs exist and permit limited intercellular progagation of mechanically stimulated Ca 2 + responses (Takeda et al., 1995). Both 0-2A progenitor cells and mature OLGs respond to ATP, histamine, bradykinin and substance P by releasing Ca 2 + from intracellular stores and the percentage of cells that responded to these neuroligands increases with differentiation of 0-2A progenitor cells into mature oligodendrocytes. This suggests that neuroligands whose actions depend on 18 developmentally regulated expressions of their respective receptors in OLGs, might play an important role during OLG differentiation (Kastritsis and McCarthy, 1993; Heath et al., 1994). Ca signalling in OLGs after activation of the immune system has also been studied by several investigators. Complement activation has been shown to produce transient increases in [Ca2+]i in rat optic nerve OLGs (Scolding et al., 1989). The increase in [Ca2+]i was from internal and external sources as evidenced by prior EGTA application to bath solution which produced half maximal responses to complement attack. T-cell perforin attack on rat OLGs also causes an increase in [Ca2+]; by the mechanism of transmembrane Ca 2 + influx through the perforin pore formed after the perforin attack (Jones et al., 1991). The effect of perforin attack ranged from small transient increases in [Ca2+]i to rapid cell death. The Ca 2 + ionophores, A23187 and ionomycin, mimicked both complement and perforin attack, causing OLG lysis at concentrations that do not lyse other glial cells (Scolding et al., 1992). Membrane vesiculation, the mechanism by which OLGs resist and recover from complement and perforin attack (Jones et al., 1991), was also induced by A23187, suggesting that changes in intracellular Ca 2 + level play dual roles dictating both susceptibility and cellular recovery. Injecting ionomycin into myelinated tracts of adult rat spinal cords induced demyelination, together with a variable degree of axonal degeneration in vivo (Smith and Hall, 1994). It was speculated that the demyelination induced by ionophore in vivo might occur as a result of activation of endogenous Ca2+-dependent enzymes. The detrimental effects of a rise in intracellular Ca 2 + level have also been reported by Benjamin and colleagues. Antibodies to GalC, a specific glycolipid marker for OLGs, caused a transmembrane influx of Ca 2 + in OLGs (Dyer and Benjamin, 1990). They showed that transmembrane signalling by plasma membrane-associated GalC participated in the opening of Ca 2 + channels, which causes a disruption of microtubules in the oligodendroglial membrane sheets. Treatment o f O L G s with thapsigargin, a sesquitepene lactone, which releases intracellular C a 2 + stores by inhibiting E R C a 2 + A T P a s e , resulted in retraction o f membrane sheets and cell death in mature mouse O L G s (Benjamin and Nedelkoska , 1996). These findings suggest that C a 2 + signalling is a process that should be tightly regulated in response to various stimuli. Depending on the source o f the stimuli and the pathway o f [ C a 2 + ] i increase, C a 2 + plays a various biological role in O L G s . 1.4 Objectives It is evident that O L G s exhibit a complexity o f C a 2 + s ignalling as do other cell types. In v iew o f the f inding that P K C activation (especially the a- isoform) results in an augmentation o f O L G process extension and that C a 2 + also plays an important role in O L G s differentiation, there might be a close relationship between P K C activation and C a 2 + s ignall ing in O L G process extension. T h e current study is designed to investigate whether C a 2 + s ignalling is an important factor during O L G process extension. Since P K C activation results in an augmentation o f O L G process outgrowth, the changes in [ C a 2 + ] i in response to phorbol ester treatment was measured by microfluormetric imaging o f [ C a 2 + ] ; using fura-2. In order to find out whether [ C a 2 + ] i is related to the extracellular concentrations o f C a 2 + , O L G s were exposed to different concentrations o f extracellular C a 2 + and the changes in [ C a 2 + ] i were measured. In order to find out h o w extracellular C a 2 + interacts with P K C activation and how it relates to the degree o f O L G process extension, O L G s were also grown in feeding media containing various extracellular concentrations o f C a 2 + with or without phorbol ester. 20 Table 1. Stimulus-evoked intracellular Ca elevation in oligodendrocytes Preparation Stimulus Mechanism of Ca2+ rise References VGCCs LGCCs Release from internal stores OUter Ca"+ influx Culture/mouse cortex, rabbit retina Depolarization (KC1) +++ Kirischuk et al. (1995), Borges et al. (1994) Culture/rat cortex Myelin extract +++ Moorman and Hume (1994) Culture/mouse/ precursors GABA +++ ' Kirchhoff and Kettenman (1992) Culture/mouse/ precursors Kainate +++ Borges et al. (1994) Culture/mouse cortex ATP +++ Kirischuk et al. (1995) Culture/mouse/ cerebrum Antibodies to GalC +++ Dyer and Benjamins (1990) Culture/rat/ optic nerve T-cell perforin +++ Jones et al. (1991) Culture/rat/ optic nerve Complement +++ Scolding et al. (1989) Culture/rat/ optic nerve A23187and ionomycin +++ Scolding et al. (1992) In Vivo/rat/ spinal cord Ionomycin +++ Smidi and Hall (1994) Culture/mouse/ cortex Thapsigargin +++ Benjamin and Nedelkoska (1996) Culture/rat AMPA ? +++ Borges et al. (1994) Holtzclaw et al. (1995) Culture/rat/ spinal cord Substance P +++ Headi et al. (1994) OLG cell line Neuroligands: SP, ATP, BK, histamine +++ Kastritsis and McCarthy (1993) Culture/rat/ spinal cord Arachidonic Acid +++ Soliven et al. (1993) OLG cell line PDGF -H-+ Fatitis and Miller (1996; 1997) Culture/rat/ cortex Carbachol +++ +++ Cohen and Almazan (1994) Chapter 2 Materials and Methods 2.1 Materials Fura-2/AM and 4-bromo-A23187 were purchased from Molecular Probes (Eugene, OR, USA). Ca2+-free-Dulbecco's Modified Eagle Medium (DMEM) was purchased from Stem Cell Technologies (Vancouver, BC). Percoll was purchased from Pharmacia (Piscataway, NJ, U.S.A.). 4P-phorbol-12,13-dibutyrate (PDB) and phorbol ester 4-a-phorbol-12,13-didecanoate (PDD), an inactive form of phorbol ester, were purchased from Sigma (St. Louis, M O , USA). PKC inhibitor, RO-31-8220 and intracellular C a 2 + chelator, B A P T A / A M were purchased from Calbiochem (San Diego, CA, USA). Matrigel was purchased from Collaborative Biomedical Products (Bedford, M A , USA). Adivin-biotin complex (ABC) kit for immunostaining was purchased from Vector laboratories (Mississauga, ON). AA3, a rat monoclonal antibody specific for PLP, was provided by Prof. K. Ikenaka of Okazaki, Japan. 2.2 Tissue Culture 2.2.1 Murine Oligodendrocytes Oligodendrocyte (OLG)-enriched cultures were prepared from brain tissue from 3-week old Balb/C and CD-I mice. The procedure used has been previously described for human brain (Kim et al., 1983, 1985). The brains were isolated, dissected into small cubes of approximately 3 mm3 and incubated at 37°C for 45 min in phosphate-buffered saline (PBS) containing 0.25% trypsin and 40 u.g/ml DNase. The dissociated cells were passed through a nylon filter having a pore size of 100 u.m, and the filtrate was then centrifuged in 30% Percoll for 30 min at 15,000 rpm. After removing the first and second layer that contained mainly cell debris and myelin, the third layer, containing an enriched population of OLGs, was collected and diluted with PBS and centrifuged for 10 min at 1,800 rpm to remove the Percoll. The cell pellet was subsequently washed with PBS and centrifuged for 10 min at 1,400 rpm. Finally, the cells were suspended in a feeding medium of Eagle's minimal essential medium (MEM) containing 5 % horse serum, 0.5% glucose, 20 ug/ml gentamicin, and 2 ug/ml amphotericin B (Fungizone) and centrifuged for 10 min at 1,200 rpm. The OLG pellet was then resuspended in feeding medium, and the cells are plated onto 10-cm-diameter plastic Petri dishes. Cells were then incubated at 37°C in a humidified atmosphere of 5% C02/95% air. Twenty-four hours later, most OLGs (as opposed to astrocytes, endothelial cells, or microglia) had not attached to the petri dish and suspended freely in the medium. These OLGs were collected and plated onto 12-mm-diameter round Aclar plastic coverslips, previously coated with Matrigel, a laminin-rich reconstituted basement membrane matrix, to coat the coverslips for attaching OLGs, for immunocytochemical studies and 25-mm-diameter round glass coverslips previously coated with 10 ug/ml poly-L-lysine for microfluormetric studies. Over 95% of cells isolated by this replating procedure were OLGs as determined by immunostaining with mouse anti-galactocerebroside (anti-GalC) monoclonal antibody (Kim et al., 1983, 1985). 23 2.3 Measurement of intracellular free [Ca2+] 2.3.1 Preparation of buffers and cell loading with fura-2 Oligodendrocytes were grown on 25mm-round glass coverslips at least for 24 hours before [Ca2+]i was measured. Buffers used in all experiments consisted of Hepes-buffered Hanks' balanced salt solution (HBSS, pH 7.4) containing (in mM) NaCl, 145; KC1, 2.5; MgCl2, 1.0; HEPES, 20 mM; glucose, 10; CaCl2 ) 1.8. When Ca 2 + free medium was required, Ca 2 + was omitted and 50 uM EGTA was added. [Ca2+]i was measured using the fluorescent Ca 2 + indicator fura-2 acetoxymethyl ester (fura-2/AM). Fura-2/AM was dissolved in dimethyl sulfoxide (DMSO) and further solubilized with Pluronic F-127 acid (0.2%) in HBSS containing 0.02% bovine serum albumin (BSA). Glucose was supplemented to HBSS since this appeared to enhance cell viability over extended periods. Cells incubated in the absence of glucose tended to lose their fluorescence signal faster than those in glucose do. Cells were incubated in HBSS containing fura-2/AM (5uM) at 37°C for 30 minutes. Fura-2/AM, an ester form, diffuses through membranes into the cytosol where fura-2/AM is hydrolyzed to fura-2, the membrane-impermeant polycarboxylate anion. Cells were then washed twice in HBSS and incubated in HBSS for an additional 30 minutes to ensure complete de-esterification of fura-2/AM to fura-2. Coverslips with fura-2-loaded cells were mounted in a perfusion chamber with a working volume of 0.3 ml and fitted into the stage of an inverted microscope. 2.3.2 Fura-2 Imaging Fluorescence signals by fura-2 was measured using a Nikon 40X fluorite epifluorescence objective fitted to a Nikon Diaphot TMD inverted microscope, equipped with a 100W Xenon light source (Osram, Germany). Emitted fluorescence was detected by a silicone intensified target (SIT) video camera (Hamamatsu C2400-08) and fed to an 80386-based desktop computer, equipped with image-detection software and an anlog-to-digital video digitizer interface (Image-l/FL, Universal Imaging Corp., West Chester, PA). Fura-2 was excited at the wavelength pair of 340 nm and 380 nm and the displayed images (images at 340 nm and 380 nm) as well as ratios (fluorescence intensity at 340 nm/at 380 nm) were stored in the computer. Ratios were obtained from 8-frame averages of pixel intensities (ranging from 0 to 255) at each of the excitation frequencies. The software package controlled all the main parameters of fluorescence data acquisition including background subtraction, filter wheel/shutter switch, sampling rate and camera sensitivity (see Fig.2.1). The stored results were transferred to a data spreadsheet (MS Excel) and ratios were converted to actual calcium concentrations using methods described by Grynkiewicz et al. (1985). Cells for imaging were selected on the basis of sufficient fluorescence intensity, stable baseline [Ca2+]i for at least 5 minutes, and morphology. 2.3.3 Calibration The imaging system was calibrated using fura-2 pentapotassium salt (fura-2/K5, 5uM) with a series of calibration buffers containing: KC1 100 mM, MOPS 10 mM and Ca 2 +-EGTA/EGTA in ratios to yield Ca 2 + concentrations between 0 and 39.8 uM (pH 7.2, Molecular Probes). Ratio values were obtained for all of the known Ca 2 + concentrations 25 Fig. 2.1 Experimental setup of fiira-2 imaging system A schematic representation of the digital imaging system used to measure and monitor intracellular [Ca2+] in fura-2 loaded cells Fura-2 Loaded Cell Data Storage Drive Computer Monitor Video Monitor of calibration buffer and plotted against log ([Ca2+]). Kd for fura-2/K5 was calculated according to the formula [Ca2+]; = Kd x P x (R-Rmin)/(Rmax-R), where P is the ratio of fluorescence intensities at 380 nm under zero and saturating Ca 2 + concentrations (Fmin^o/Fmax^so)), Rmaxis the maximum ratio available for the system and Rmin is the minimal ratio for the system. First, (R-RminyCRmax-R) was plotted against log [Ca2+]; values to show the saturation curve and the dependency of (R-R m i n ) / (R m a x -R) on [Ca2+]. In order to achieve a linear relationship between R and [Ca2+], log (R-Rmin)/(RmaX-R) was plotted against log [Ca2+] (in which log (R-Rmin)/(Rm a x-R) becomes the linear function of log [Ca2+]). The plot was best fitted with a straight line, and the X-axis intercept value where log (R-R m i n ) / (R m a x -R) became zero was obtained since this value corresponds to log (Kd x P) value. From the estimated log (Kd x P), Kd value was obtained by substituting the known P value into the equation. This Kd value was assumed to be similar to the Kd for intracellular fura-2, and was used for conversion of ratios into [Ca2+] in all the experiments (see Fig.2.2). Rmax and F m a x osonm) were obtained by the addition of the Ca2+-ionophore, 4-bromo-A23187 (10 uM) to the cells incubated in 1.8 mM HBSS. Rmin and Fmin(38onm) were obtained by the addition of EGTA (5 uM) to the recording bath. 2.3.4 Preparation of test solution Stock solutions of 4P-phorbol-12,13-dibutyrate (PDB) were prepared in dimethyl sulfoxide (DMSO) at 1 mM concentration, further solubilized by vigorous vortexing. The application of PDB was made in such a way that the final concentration of DMSO did not exceed 0.1%. In addition, changes of [Ca2+]; were also monitored when 0.1% DMSO was applied alone in order to ensure that DMSO had no effect on [Ca2+],. Fig.2.2 Fura-2 imaging of oligodendrocytes, a) Before OLGs are stimulated with phorbol ester, there is a stronger fura-2 signal at excitation wavelenth 380 nm than at 340 nm, resulting in low ratio (340nm/380nm) values, b) Aiter OLGs are stimulated with phorbol ester, the signal from excitation wavelength 340 nm increases whereas the signal at 380 nm decreases, resulting in an overall increase of ratio values, a 28 2.4 Culturing in various extracellular [Ca2+1 and cell morphology assessment 2.4.1 Culturing in various extracellular [Ca2+] After the preparation of OLG culture, the coverslips were transferred to feeding media containing various concentrations ranging from 0.01 mM to 7.2 mM of [Ca2+] in 0.5% HS. OLGs were then grown for 72 hrs before being immunostained. The feeding media containing various [Ca2+] was made from Ca2+-free DMEM (Stem Cell), later supplemented with the corresponding amount of Ca 2 +. There is growing evidence that the presence of other cell types (i.e. astrocytes) in OLG culture has a significant effect on OLG morphology (Oh and Yong, 1997). Since the proliferation rate of astrocytes is related to the amount of serum present in the media, the amount of serum in the feeding media was minimized (0.5%) in order to perform a better assessment of the effect of [Ca2+] alone on OLG morphology. The same feeding medium was also used for normal [Ca2+] (1.8 mM). 2.4.2 Immunocytochemistry After being cultured for 72 hr, cells were fixed with methanol for 10 min at -20°C and incubated with a rat monoclonal antibody against proteolipid protein (PLP, 1:4), a cell-type specific marker for OLG, at 4°C overnight. The antibody used in this study was AA3 (provided by Prof. K. Ikenaka) that is a rat monoclonal antibody (mAb) raised against bovine PLP (Yamamura et al., 1991). AA3 monoclonal antibody shows a reactivity to a major antigenic determinant located at the carboxy-terminal region of PLP (residues 264-276) (Yamamura et al., 1991). Cells were washed three times in PBS and incubated with a second antibody, biotinylated goat-anti-rat IgG (1:100) for 1 hr at 24°C. Cells were washed three times in PBS and incubated with ABC solution (Vector Laboratories, Mississauga, ON) which readily binds to biotinylated second antibody, for 1 hr at 24°C. Cells were washed three times in PBS and incubated for 5 min in 0.1 M Tris-HC1 buffer, pH 7.4, containing 0.5 mM 3,3'-diaminobenzidine (DAB) and 0.02% hydrogen peroxide. Coverslips were dehydrated in alcohol, cleaned in xylene and embedded on microscopic slides with permount. 2.4.3 Cell Morphology Assessment Three indices of OLG process formation were evaluated in different extracellular [Ca2+] (see Fig.2.3): 1) the percentage of cells with processes that were greater or equal to 3 times the diameter of cell body, 2) the number of primary processes per cell body, and 3) the areas covered by OLG processes. These methods have been previously used for monitoring morphological changes in OLGs as well as other cell types. Previous investigators have used the method of counting the cell percentage with processes greater than 3 times cell body diameter as an index of observing OLG process extension (Yong et al., 1989, 1991 and 1994, Oh et al, 1997). The rationale behind defining the criteria of OLG with enhanced process formations stemmed from the fact that OLGs meeting this criteria populated about 30-40% of the whole cell population, and once stimulated by phorbol ester to enhance the process formation, this percentage was critical. For measuring neurite outgrowth of neurons, the numbers of neurite per cell body are often counted for assessing neuronal differentiation (Strittmatter et al., 1994; Bellosta et al., Fig. 2.3 Measurement of oligodendrocyte process formation A schematic representation of various methods to assess the degree of PLP-stained oligodendrocyte process formation. Method 1. Obtaining the percentage of cells that have processes longer or equal to 3X cell body diameter. After whole cell population is counted in a given field, cells with long procesess are counted and the percentage of those cells are calculated. / \ : OLG with processes less than 3X cell body diameter : OLG with processes equal or greater than 3X cell body diameter Method 2. Counting the number of primary processes. Primary processes that project immediately from a given cell body are counted and averaged for many cells. 5 4 A 3 2 6 ™ 7 1 4 ^ 2 V '1 2 1 3 • — Primary ^ Processes — • Secondary Processes Method 3. Measuring the total areas covered by oligodendrocyte processes. A circle is drawn around the extremeties of processes and the area is calculated by measuring out the diameter D: Diameter of the circle R: Radius of the circle (D/2) Area = R X R X 3 . 1 4 1995), and the same measurement was taken to assess OLG process extension as well. For measuring areas, photographs were taken of the PLP-immunoreactive OLGs. Circles were drawn around the OLG processes so that the circles covered most of the PLP-immunoreactive areas. The areas were then calculated by measuring out the diameter of these circles. To obtain the percentage of cells with long processes, a field is chosen from a coverslip, all the PLP-positive cells are counted to get the whole cell population in the given field and the cells meeting the criteria of having processes 3 times cell body diameter are counted. The percentage was calculated by dividing this number by the whole cell population. The number of primary processes was obtained by counting the numbers of all the processes that project directly from a given cell body. Results are graphically plotted and error bars are presented as standard error means (SEM). 2.5 Statistics Results are presented as mean ± SEM unless otherwise noted. For fura-2 experiments, individual experiments were performed more than 3 times using different coverslips. The Vs" noted in graphs from imaging experiments refer to the number of cells within one individual experiment. Statistical comparison between different experimental groups (i.e. different [Ca2+] groups) is performed by analysis of covariance (ANCOVA). This analysis gives the average of the product of deviations of data points from their respective means. Covariance is a measure of the relationship between two ranges of data and covariance can be used to determine whether two ranges of data move together. Positive covariance value indicates that large values of one set are associated with large values of the other, while negative covariance indicates that small values of one set are associated with large values of the other or vice versa. If values in both sets are unrelated, the covariance value is near zero. In this study, the test was to determine whether increasing extracellular [Ca2+] was related to OLG process formation. Thus, the ANCOVA appeared to a valid choice to establish the dependency of OLG process formation on extracellular [Ca2+]. The strength of ANCOVA was expressed in terms of slopes and their respective probabilities when log [Ca2+] was plotted against log or square root of OLG process measurements. The means for OLG process formation was from 4 individual experiments, giving a total of 8 different coverslips. Approximately, 10 fields were chosen in each coverslip for cell counting. For inhibition studies, each control group (1.8 mM and 7.2 mM Ca 2 + without inhibitor treatment) was compared to each inhibition group (RO-31-8220 and BAPTA) of different concentrations by analysis of variance (ANOVA). ANOVA was performed to see how inhibiting PKC activity (by RO-31-8220) or inhibiting intracellular Ca 2 + signalling (by BAPTA) affects the process formation of OLGs grown in normal (1.8 mM) and high (7.2 mM) extracellular Ca 2 +. The significance of the analysis was expressed as probabilities (p values). In order to achieve a better distribution of data points, the values from the measurements of OLG process formation and the values for extracellular [Ca2+] were all transformed by either square rooting or logging. We used BMDP statistical software (University of California Press, Berkley, 1990) to perform ANCOVA and ANOVA. Chapter 3 Results 3.1 Fura-2 calibration The calibration of fura-2 was performed using fura-2 pentapotassium salt (fura-2/K5, 5uM) using methods as described in the materials and methods section. The ratio of the fluorescence intensity at 340 nm and 380 nm was plotted against the concentration of Ca 2 + in the buffer solution. The relationship between the fluorescence ratio and [Ca2+] indicates that as [Ca2+] increases, the ratio increases and saturates near a fluorescence ratio of 3.3 (Fig.3. la). The relation between [Ca2+] and the ratio measurements is given by [Ca2+]; = K d x P x ( R -Rmin)/ (Rmax-R) . The value for K d x P was obtained from the X-axis intercept of the log ( ( R -R m i n ) / ( R m a x - R ) ) vs. log[Ca2+] plot (see section 2.3.3 in Materials and Methods). The product of the apparent dissociation constant for fura-2 binding ( K d ) times the ratio of fluorescence at 380 nm under zero and saturating Ca 2 + conditions (P) was determined to be 5.4 nm (Fig. 3.1b), from which the K d value was calculated to be 221 nM. These constants were used to calculate [Ca2 +]i throughout all the fura-2 imaging experiments. 3.2 Effect of phorbol ester-induced protein kinase C activation on [Ca2+]i in OLG 3.2.1 Effect of PDB treatment of OLG in media with normal [Ca2+] (1.8 mM) Application of PDB (1 uM) to OLG in culture resulted in a 2-3 fold increase of [Cans from the basal [Ca2 +]i of 96 ± 6 nM to 244 ± 10 nM (Fig.3.2.1a). After exposure to PDB, [Ca2+]i increased for approximately 2 min before reaching a plateau and this increase was 34 Fig.3.1 Calibration of fura-2 signal, a) Relation between -log[Ca2+] and fluorescence ratio. As [Ca2+] increased, the fluorescence ratio increased and saturated at a ratio value of approximately 3.0. b) Plot of log [Ca2+] vs. log((R-Rmin)/(Rmax-R)). The K d x p value was obtained when log((R-Rmjn)/(Rmax-R)) became 0. The best fit line was drawn by eye to demonstrate the relation between log[Ca2+] and log((R-Rmin)/(Rma -R)). 35 sustained for at least 20 min after the treatment. When the [Ca2+]i reached a steady state level, OLGs were washed twice with the recording buffer; however, this did not have any effect on the sustained increase of [Ca2+]j. In order to evaluate the dose-dependence of PDB on the Ca response, OLGs were treated with 0.1 uM PDB. At this concentration, only a fraction of OLGs (approximately 40 %) responded to the PDB (0.1 uM) treatment. OLGs that responded had modest elevations of [Ca2+]i (up to 171 ± 8 nM) (Fig.3.2.1b). The stock PDB was prepared in DMSO and diluted in recording buffer before being applied. The final concentration of DMSO in the PDB solution did not exceed 0.1%. To ensure that DMSO had no effect on [Ca2+]i in OLGs, DMSO alone was applied in recording buffer, and at 0.1%, DMSO did not induce any changes in [Ca2+]i (data not shown). In order to verify that the changes in [Ca2+]i by PDB application were due to PKC activation, OLGs were treated with RO-31-8220, a specific PKC inhibitor, for 15 min and then exposed to PDB (luM). Under these conditions, OLGs failed to respond to PDB treatment, suggesting that the increase in [Ca2+]i was due to PKC activation (Fig.3.2. lc). The phorbol ester, 4-a-phorbol-12,13-didecanoate (PDD) which does not activate PKC, also had no effect on [Ca2+]i in OLGs, further suggesting the changes in [Ca2+]i resulted from PKC activation. In order to evaluate whether voltage-dependent Ca 2 + channels (VDCC) are involved in the rise of[Ca2+], various Ca 2 + channel blockers including nifedipine (100 uM), a specific L-type VDCC blocker, verapamil (25 uM), a specific T-type VDCC blocker and lanthanum (1.0 mM), non-selective Ca 2 + channel blocker, were applied to OLGs followed by PDB treatment. OLGs still responded to PDB treatment in the presence of various Ca 2 + channel blockers, suggesting that the rise in [Ca2+]i was not due to Ca 2 + influx through VDCC (Fig.3.2. Id). Fig.3.2.1a 1 5 0 300 250 H | 200 5 150 1 0 0 i j— 50 Fig.3.2.1b 350 300 250 200 u 150 100 50 W ash P D B (1 UM ) 10 T i m e (m in) Wash PDB (100 nM) + 10 Time (min) 1 5 15 20 •i 1 20 Fig.3.2.1a-b Intracellular Ca 2 + response of OLGs to PDB. a) Treatment of OLGs with PDB causes a 3-fold increase in [Ca2+]i which was sustained afterwards at least for another 15 min. After a stable baseline was achieved, cells were treated with PDB (1 u.M) followed by washing with recording buffer. The graph represents the average of 20 individual cells. In all cases, the final concentration of DMSO did not exceed 0.1%. b) OLGs treated with 100 nM PDB displayed modest elevations of [Ca2+]j. It is also important to note that only a fraction of OLGs responded (40 %). This figure represents [Ca2+]j changes only from the cells that responded. 37 Fig.3.2.1 c Ca 2 + response of OLGs pretreated with RO-31 -8220 (1 uM) and exposed to PDB treatment (1 uM). OLGs were pre-incubated with RO-31 for 10 minutes, then treated PDB. The fiira-2 measurement indicates that in the presence of PKC inhibitor, PDB treatment failed to trigger any [Ca2+]f increases in OLGs. 38 Fig. 3.2.1d Fig.3.2. Id Application of nifedipine did not have any effect on PDB-induced [Ca2+]f response in OLGs. Treating OLGs with PDB still resulted in a sustained increase in [Ca2+]f when nifedipine (10 uM) was present in the buffer. Similar ineffectiveness was also observed with other Ca 2 + channel blockers including verapamil (10 uM) and lanthanum (1.0 mM). 3.2.2 Effect of PDB treatment of OLG in Ca2+-free buffer To determine the mechanism by which PDB altered [Ca2+]i, we evaluated the effect of extracellular Ca 2 + on the PDB response. In Ca2+-free buffer, PDB application failed to induce [Ca2+]i changes in OLGs (Fig.3.2.2a). Although this effect could be interpreted as a requirement for extracellular Ca 2 + in the PDB-induced rise in [Ca2+]i, two confounding variables might be present. First, intracellular Ca 2 + stores could be depleted when OLGs were incubated with Ca2+-free media and if the PDB-mediated rise in [Ca2+]i was dependent on intracellular stores, no response would be observed. The second confounding factor is that changing extracellular [Ca2+] might have independent effects on [Ca2+]i. To explore these possibilities further, we employed the following paradigm. After a steady basal value of [Ca2+]i was reached in buffer containing 1.8 mM Ca 2 + , the recording chamber was perfused with Ca2+-free buffer. When OLGs were incubated in the absence of extracellular Ca 2 + , the [Ca ]i started to decline slowly. After 2 minutes, PDB was applied to the bath and OLGs failed to respond to PDB application in the absence of extracellular Ca 2 + (Fig.3.2.2a). Upon replacing Ca 2 + to buffer containing 1.8 mM Ca 2 + 3 min after PDB application, [Ca2+]i started to increase rapidly reaching a steady state level at approximately 241 ± 16 nM (Fig.3.2.2a). The failure of cells to respond to PDB in Ca2+-free medium and the rapid [Ca2+]i increase upon the reestablishment of extracellular Ca 2 + indicate that the rise of [Ca2+]i with PDB treatment was due to the transmembrane influx of Ca 2 +. In order to find out how OLGs behave when they are re-exposed to Ca2+-free environment after extracellular Ca 2 + was restored, OLGs were re-exposed to Ca2+-free medium after [Ca2+]i reached a steady state level 40 Fig.3.2.2a 350 300 250 S 200 c 5 iso 1 00 50 [ C a 2 ] 0 m M 10 Tim e (m in) 1 5 20 Fig.3.2.2b 350 -i 6 1 5 0 [ C a 2 + ] 0 m M 10 15 Time (min) 20 25 Fig.3.2.2 PDB exposure (1 pM) in Ca2+-free media, a) No response to PDB was seen under Ca2+-free conditions. Reestablishment of extracellular Ca 2 + led to a substantial increase in [Ca 2 +] r b) OLGs were then re-exposed to Ca2+1free environment, which caused the [Ca2+]. to return towards the basal level. upon PDB stimulation. [Ca2+]i started to decrease again towards the basal level upon the consecutive removal of extracellular Ca 2 + (Fig.3.2.2b). [Ca2+]i then reached a stable level after 10 min of the addition of Ca2+-free buffer. We also evaluated [Ca2+]i changes by removing and restoring the extracellular Ca 2 + without PDB treatment. [Ca2+]i decreased when the extracellular Ca 2 + was removed and increased towards the basal level when the extracellular Ca 2 + was restored (Fig.3.2.2c). However, the amplitude of the increase (up to 133 ± 7 nM) was smaller than that observed when PDB was present in the buffer (see Fig.3.2.2b). This observation further supports that the rise in [Ca2+]; in response to PDB treatment was due to transmembrane Ca 2 + influx. In order to further evaluate the mechanism by which Ca 2 + influx occurs, the presence of capacitative Ca 2 + entry or Ca 2 + released-activated Ca 2 + influx (CRAC) was evaluated in OLGs. Capacitative Ca 2 + influx normally follows discharge of intracellularly stored Ca 2 + by Ca 2 + store-depleting agents such as thapsigargin (TG) or cyclopiazonic acid (CPA), a specific inhibitor of the endoplasmic reticulum (ER) Ca 2 + ATPase pump (Berridge, 1993; Putney 1997). Treating OLGs with 5 uM CPA resulted in a 2-fold increase of [Ca2+]j from the basal level of 92.3 ± 6.7 nM and reached a peak at 200.2 ± 8.5 nM over a period of 2 minutes. The increased [Ca2+]i was then maintained for at least another 20 minutes (Fig.3.2.2d). The same experiment was repeated in the absence of extracellular Ca 2 +. OLGs were exposed to a Ca 2 + free environment for 5 minutes before being treated with CPA (5 uM). An initial decrease in [Ca2+]i was observed when OLGs were exposed to Ca2+-free buffer. Adding CPA led to a transient increase in [Ca2+]i due to Ca 2 + released from the intracellular store by CPA (Fig.3.2.2e). The observation that there was a transient increase in [Ca2+]j when OLGs were 42 Fig.3.2.2c OLGs exposed to Ca2+-free medium and reestablishment of extracellular Ga 2 + in the absence of PDB. Upon Ca 2 + being restored, [Ca2+\ increased near the basal level. Note that the increase after Ca 2 + reestablishment was smaller than that observed when PDB was present in the buffer. 43 treated with CPA after being exposed to a Ca2+-free environment for 5 min, also indicates that exposing OLGs to Ca2+-free buffer for 5 min does not deplete intracellular Ca 2 + stores in OLGs. When extracellular Ca 2 + was restored to 1.8 mM, there was a rapid increase in [Ca 2 +]i which reached a sustained plateau around 294.2 ± 36.5 nM, demonstrating the presence of capacitative Ca 2 + entry in mouse OLGs. The intracellular Ca 2 + stores were depleted in OLGs by pretreating the cells with 5 uM CPA, and OLGs were exposed to PDB (1 uM). OLGs pretreated with CPA were still able to increase [Ca 2 + ]i when treated with PDB, indicating that Ca 2 + mobilization from intracellular store does not play a role in PDB-induced [Ca2+]j increase. OLGs exhibited much higher increase of [Ca 2 +]i when exposed phorbol ester compared to OLGs not pretreated with CPA. This increase reached a peak at approximately 900 nM and the plateau phase was sustained afterwards (Fig.3.2.2f). OLGs pretreated with CPA also had slightly higher basal [Ca2+]i at near 117 ± 10 nM. The higher amplitude of [Ca2+]i increase in response to PDB in OLGs pretreated with CPA indicates that Ca 2 + influx is required for OLGs responding to PDB treatment and that intracellular Ca 2 + stores seem to buffer this [Ca2+] increase. In order to evaluate how OLGs maintain the sustained phase of the [Ca 2 +]i response, OLGs were treated with CPA (5 uM) after a plateau was reached upon PDB stimulation. The stable plateau seen after phorbol ester treatment was disrupted such that [Ca2+]i was increased from approximately 200 to 350 nM (Fig.3.2.2g), indicating that the Ca 2 + efflux into ER as well as Ca 2 + influx is involved during the plateau phase of [Ca 2 +]i increase in response to PDB treatment. 44 15 20 Time (min) 25 30 Fig.3.2.2d-e Capacitative Ca 2 + entry in OLGs. d) Treatment with 5 pM CPA resulted in [Ca2+]; increase which reached a plateau after 10 minutes. The sustained [Ca2+]; increase lasted at least for 25 minutes after the addition of 5 pM CPA. e) OLGs were exposed to Ca2+-free environment before being treated with 5 uM CPA. A transient increase in [Ca2+]; was observed in the absece of extracellular Ca 2 + . When the extracellular [Ca2+] was restored to 1.8 mM, there was a sustained increase in [Ca2+]j, demonstrating the presence of capacitative Ca 2 + influx in mouse OLGs. 45 Fig.3.2.2f 1200 1000 PDB (1 uM) 800 -2 e ^ 600 -+ cS CJ 400 -200 10 15 20 25 30 Time (min) 35 40 45 50 Fig.3.2.2f Effect of ER-specific Ca 2 + ATPase inhibtion on PDB-induced [Ca2+]( increase. OLGs were preincubated with CPA for 15 minutes before being exposed to PDB (1 uM). After the intracellular Ca 2 + stores were depleted by treatment with CPA, OLGs still responed to PDB treatment, indicating the [Ca2+]f increase was due to Ca 2 + influx. Note the amplitude of the [Ca2+]; increase was significantly larger than that observed in OLGs not pretreated with CPA. 46 Fig.3.2.2g In order to see whether Ca 2 + filling into ER via Ca 2 + pump was involved during the sustained phase of OLGs' response to PDB treatment, OLGs were treated with CPA (5 pM) after a stable level was achieved. Note that the sustained phase was disturbed by adding CPA which resulted in a further increase of [Ca2+]; from the sustained phase of the PDB response. 47 3.3 Effect of changing extracellular [Ca2+] on [Ca2+], in OLGs In view of OLGs' ability to respond to changes in extracellular [Ca2+], OLGs were exposed to buffer containing various extracellular Ca 2 + concentrations. After the basal [Ca2+]i achieved a steady level in buffer containing control [Ca2+] (1.8 mM), the extracellular [Ca2+] was changed by perfusing the recording chamber with a buffer containing higher [Ca2+]. Excluding the extracellular Ca 2 + in the buffer caused [Ca2+]i to decline in OLGs by approximately 4-fold (from 99 ± 6 nM to 26 ± 4 nM). [Ca2+]i reached the lowered steady state at approximately 10 minutes after OLGs were exposed to the Ca2+-free environment (Fig.3.3a). Exposure of OLGs to 3.6 mM of [Ca2+] resulted in a Ca 2 + rise by 4 fold (from 92 ± 8 nM to 414 ± 20 nM) which was sustained for approximately 10 minutes before returning towards the basal level (Fig.3.3b). [Ca2+]i did not, however, fully return to the basal level after 30 minutes. Incubating cells in 7.2 mM resulted in a higher amplitude of [Ca2+]j increase (up to 563 ± 37 nM) and a more transient pattern of [Ca2+]i rise where the plateau was sustained only for about 5 min before falling towards the basal level (Fig.3.3c). This amplitude was about 150 nM larger than the increase when cells were treated with 3.6 mM Ca 2 +. In order to evaluate whether Ca 2 + influx participates in [Ca2+]j increase in response to exposure to high extracellular [Ca2+], OLGs were depleted of intracellular Ca 2 + stores by pretreating OLGs with CPA, and OLGs were exposed to 7.2 mM extracellular [Ca2+]. OLGs still responded to 7.2 mM Ca 2 + by increasing [Ca2+]i (Fig.3.3d). This observation indicates that the [Ca2+]j increase in response to high extracellular [Ca2+] was due to Ca 2 + influx. The amplitude of the increase in [Ca2+]i was significantly larger (up to 880 + 57 nM) than that Fig.3.3a Exposure of OLGs to a Ca2+-free environment. When the extracellular Ca 2 + was removed from the buffer, [Ca2+]( started to decrease and reached a stable level at approximately 26 nM. 49 Fig.3.3b 1 0 0 0 -i 9 0 0 -I 8 0 0 H 7 0 0 -i [ C a 2 + ] 3.6 m M 0 H 1 1 1 1 , 1 , 0 5 10 15 20 25 30 35 T i m e (min ) Fig.3.3c 8 0 0 n [Ca2+] 7.2 m M 0 -I , • , , , , , , 1 0 5 10 15 20 25 30 35 40 45 T im e ( m i n ) Fig. 3.3b-c Ca 2 + response of OLGs to changes in extracellular [Ca2+] b) After stable baseline was achieved, the [Ca2+] in the recording buffer was changed from 1.8 mM to 3.6 mM. OLGs responded by increasing [Ca2+]j approximately 4-fold, c) OLGs also responded to changing extracellular [Ca2+] to 7.2 mM by increasing [Ca2+];. The amplitude of the [Ca2+]j increase was greater when OLGs were exposed to 7.2 mM than when exposed to 3.6 mM of extracellular Ca 2 + . 50 Fig.3.3d 1400 [Ca 2 + ] 7.2 mM 10 15 20 Time (min) 25 30 35 Fig.3.3d Effect of CPA on [Ca2+]; increase mediated by high extracellular [Ca2+]. OLGs were preincubated with CPA (5 pM) for 10 minutes before being exposed to high extracellular [Ca2+] (7.2 mM). Note the high amplitude of [Ca2+]j increase in response to high extracellular [Ca2+]. 51 Fig.3.3e Fig.3.3e Effect of RO-31-8220 on [Ca2+] increase induced by high extracellular [Ca2+]. OLGs were preincubated with RO-31-8220 (1 p.M) for 15 minutes before being exposed to 7.2 mM Ca 2 + . OLGs still responded to high extracellular [Ca2+] when PKC activity was inhibited. observed in OLGs not treated with CPA, and the transient pattern was no longer present. The results indicate that Ca 2 + filling into ER by Ca 2 + ATPase might be an important mechanism for OLGs to maintain [Ca2+]i homeostasis and drive the increased [Ca2+]i towards the basal level while exposed to high extracellular [Ca2+]j. OLGs were also preincubated with RO-31-8220 (1 pM) before being exposed to high [Ca2+] to see whether PKC activity was involved during the [Ca2+]i increase induced by high extracellular [Ca2+]. OLG with PKC inhibited by RO-31-8220 still responded to high extracellular [Ca2+] (7.2 mM) by increasing [Ca2+]i, suggesting that extracellular Ca2+-induced [Ca2+]j increase occurs through PKC independent pathway (Fig.3.3e). The amplitude of [Ca2+]; increase was higher than that observed in OLGs not pretreated with RO-31-8220 (up to 1240 ± 38 nM), suggesting that PKC activity might be important for Ca 2 + homeostasis after the Ca 2 + influx was induced. 3.4 Effect of extracellular [Ca2+] on OLG process extension Several parameters were measured to evaluate the effect of extracellular [Ca2+] on process extensions of OLGs in culture. Cells were fixed after being grown in culture for 72 hrs, immunostained by an antibody against PLP and evaluated for the degree of process formation. Cells with PLP-immunoreactivity displayed round cell bodies with multipolar, highly branched processes (Fig.3.4). As shown in Fig.3.4 a, b and c, the general pattern of OLG morphology was that with increasing [Ca2+], OLGs displayed longer and more branched processes (Fig.3.4a-c). The assessments made here for OLG process extension included: 1) counting the percentage of cells with processes having a length equal to or greater than 3 times the cell body diameter of the whole cell population, 2) counting the number of primary 53 Fig.3.4a Fig.3.4b 54 Fig.3.4 Oligodendrocytes grown in various extracellular [Ca2+]. a) 0.03 mM Ca 2 + , b) 1.8 mM Ca 2 + , and c) 7.2 mM Ca 2 + . In order to minimize the proliferation of other cell types including astrocytes and microglia, the feeding medium contained only 0.5% horse serum. With higher extracellular [Ca2+] present in the medium, oligodendrocytes displayed longer and more branched processes. processes per cell body and 3) calculating the area of circles covered by the OLG processes (see Materials and Methods 2.3.3). 3.4.1 Effect of extracellular [Ca2+] on the percentage of OLGs with long processes OLGs were grown in feeding media containing various [Ca2+] ranging from 0.01 mM to 7.2 mM. The percentage of OLGs with processes longer than 3 x cell body diameter was calculated and plotted against the [Ca2+] present in the feeding media. Fig.3.4.1 indicates that there was a positive correlation between extracellular [Ca2+] and the number of OLGs with processes longer than 3 times cell body diameter (Fig. 3.4.1). At 1.8 mM Ca 2 + , OLGs with long processes occupied approximately 50% of the whole cell population. As the extracellular [Ca2+] present in the medium was reduced, there were less cells with long processes. Reducing extracellular [Ca2+] to 0.01 mM resulted in only 25% of OLGs displaying long processes. Analysis of covariance (ANCOVA) was performed to test the significance of a relationship between extracellular [Ca2+] and OLG process extension (see Materials and Methods 2.4). Covariance is a measure of the relationship between two sets of data and can be used to determine whether two sets of data are correlated. To determine whether OLG process formation is related to extracellular Ca 2 + , we used ANCOVA to test the relationship between OLG process formation and extracellular [Ca2+], The strength of the test results was expressed in terms of the slope and the significance level of a logarithmic transformation of both the measurements of OLG process formation and extracellular [Ca2+]. The results from ANCOVA indicated that there was a statistically significant regression between log 56 Fig.3.4.1 Fig.3.4.1 Percentages of cells with processes equal or longer than 3 times cell body diameter as an index of process extension of OLGs grown in various extracellular [Ca2+]. Cells were grown in feeding medium containing various [Ca2+] ranging from 0.01 to 7.2 mM, immunostained by PLP antibody and counted. There was an approximately two-fold increase of OLG population with enhanced process formation when extracellular [Ca2+]j increased from 0.01 mM to 7.2 mM. Filled bars represent results from OLGs grown in various extracellular [Ca2+] in the presense of PDB (100 nM). 57 (percentage of OLGs with long processes) and log (extracellular [Ca2+]) with a slope of 0.14 ± 0.01 (p<0.001). Similar estimates of the percentage of OLGs having long processes were made from OLGs treated with PDB (100 nM) in various extracellular [Ca2+]. PDB treatment in various [Ca ] also resulted in a gradual increase in the measures of OLG process formation. The percentage of PDB-treated OLGs with long processes increased from approximately 33 % at 0.01 mM Ca 2 + to 61% at 7.2 mM Ca 2 + (Fig.3.4.1). As it has been shown that OLGs treated with PDB displayed an enhanced process formation (Yong et al., 1988; Althaus et al., 1991), we expected that PDB treated-OLGs would have more pronounced processes in high extracellular [Ca2+]. However, PDB treatment did not cause OLGs to have significantly higher percentages of cells with long processes in control (1.8 mM) or higher [Ca2+] (3.6 mM and 7.2 mM) compared to non-PDB treated OLGs (Fig.3.4.1). PDB treatment between 0.1 mM and 0.45 mM Ca2+resulted in slightly higher averages of percentages of cells (approximately 10% of the difference). The results from ANCOVA indicated the same positive association existed when PDB was applied in various extracellular [Ca ] (p<0.001). The regression line of log (percentage of OLGs with long processes) on log ([Ca2+] in the presence of PDB) did not, however, have a statistically different slope (p=0.260) nor a different level (p=0.098) from the corresponding regression line in the absence of PDB, suggesting that treating OLGs with PDB did not have statistically significant effect on the percentage OLGs with long processes in various extracellular [Ca2+]. 3.4.2 Effect of extracellular [Ca2+] on the number of primary processes of OLGs The number of primary processes was counted as another measure of OLG process formation. All cells with processes were selected in a given field and the processes protruding directly out of cell body were counted as the primary processes. The results indicate that there is a positive correlation between the number of primary processes and the extracellular [Ca2+] present in the feeding media. Fig.3.4.2 indicates that as extracellular [Ca ] increases, there is a tendency for OLGs to have a larger number of primary processes. The number of primary OLGs increased from approximately 3+0.01 processes at 0.01 mM to over 5 ± 0.01 processes per cell body at 7.2 mM Ca 2 +. In control [Ca2+] (1.8 mM), OLGs had an average of approximately 4 processes per cell body. Results from ANCOVA also indicated that there was a statistically significant regression between square root (number of primary processes) and log ([Ca2+]) and with a slope of 0.080 ± 0.0027 (p<0.001). The same (p=0.182) positive association existed when PDB was applied in various extracellular [Ca2+]. However, the regression line of square root (number of primary processes) on log ([Ca2+]) in the presence of PDB was statistically higher (p<0.001) than, but parallel (p=0.182) to the corresponding regression line in the absence of PDB. Fig.3.4.2 shows that there was a gradual increase of the number of primary processes for PDB-treated OLGs as extracellular [Ca ] increased. The number of primary OLGs increased from 3 ± 0.1 processes at 0.01 mM to over 5 ± 0.1 processes per cell body at 7.2 mM Ca 2 +. 59 Fig.3.4.2 Fig.3.4.2 Number of primary processes per cell body as an index of process extension of OLGs grown in various extracellular [Ca2+]. OLGs were grown in feeding medium containing various [Ca2+] ranging from 0.01 to 7.2 mM, immunostained by PLP antibody and the number of primary processes per cell body was counted. OLGs displayed a greater number of primary processes as extracellular [Ca2+] increased. Filled bars represent results from OLGs grown in various extracellular [Ca2+] in the presence of PDB (100 nM). 3.4.3 Effect of extracellular [Ca2+] on areas covered by OLG processes As an additional measure of OLG process formation, OLGs with long processes were selected on a given field and circles were drawn around them so that the circles covered most of the PLP-positive areas. The diameter was measured from these circles and the areas of the circles were calculated (see Methods and Materials 2.3.3). Fig.3.4.3 shows that there was a approximately 3-fold increase of the PLP-positive area (from 0.24 ± 0.012 pm2 to 0.80 ± 0.061 pm2) as extracellular [Ca2+] increased from 0.01 mM to 7.2 mM. (Fig.3.4.3). At the lowest [Ca2+], 0.01 mM, the average of the area was only half of the areas at 1.8 mM (0.46 ± 0.031 pm2). The results from ANCOVA indicated that there was a strong correlation between square root (area) and log (extracellular [Ca2+]) with a slope of 0.045 ± 0.0024 (p<0.001). A similar measurement was made from OLGs treated with PDB (100 nM) in various [Ca2+]. As extracellular [Ca2+] increased, the PLP-positive areas also increased from 0.46 + 0.051 pm2 at 0.01 mM to 1.17 ± 0.12 pm2 at 7.2 mM (Fig.3.4.3). When the PLP-positive areas observed from PDB-treated OLGs were compared to that from non-treated OLGs in a same extracellular [Ca2+], there was about 50 % increase in the PLP-positive areas in PDB-treated OLGs. For instance, at 1.8 mM Ca 2 + , the area increased from 0.46 pm2 to 0.72 pm2 when OLGs were treated with PDB. At 7.2 mM of Ca 2 + , the PLP-positive area increased from 0.80 ± 0.061 pm2 to 1.17 ± 0.12 pm2 in PDB-treated OLGs. The results from ANCOVA stated that the same regression line applied when PDB was present or absent (p=0.633), and the regression line in the presence of PDB was parallel (p=0.658) to the corresponding line in the absence of PDB. There was no systematic difference between the two regression lines in either slopes or levels. 61 Fig. 3.4.3 Fig.3.4.3 Areas covered by OLG processes as an index of process extension of OLGs grown in various [Ca2+]. Cell were grown in feeding medium containing various [Ca2+], immunostained and photographed. OLGs with processes were selected and circles were drawn so that they covered most of PLP-immunoreactive areas. OLGs exhibited larger PLP-immunoreactive areas as the extracellular [Ca2+] increased. Treating OLGs with PDB in various [Ca2+] also resulted in greater areas as extracellular [Ca2+] increased. At the same extracellular [Ca2+], PDB-treated OLGs displayed greater areas compared to non-treated OLGs. 62 3.5 Effects of inhibitors on OLG process formation at high [Ca2+] In view of the relationship between process outgrowth and extracellular [Ca2+], various inhibitors were applied to OLGs in culture at 1.8 mM and 7.2 mM Ca 2 +. OLGs were grown in culture with RO-31-8220 (1, 0.5 and 0.2 pM), a specific PKC inhibitor, in the presence of 1.8 mM and 7.2 mM Ca 2 +. Cells were immunostained with PLP antibody and the process formation was assessed by obtaining the percentage of cells with processes equal or longer than 3 times cell body diameter. The rationale for using this assay was based on the observation that the dependency of OLG process formation on [Ca2+] was strongest among the assays used in this study (see Results 3.3.1). ANOVA was performed to see significant differences between different experimental groups. The results indicate that PKC inhibition blocked the OLG process formation in both control (1.8 mM) and high (7.2 mM) [Ca2+] in a dose-dependent manner (Fig. 3.5a). At 1 pm of RO-31-8220 and 1.8 mM Ca 2 + , OLGs with long processes occupied approximately 25-30 % of the whole cell population, which was significantly reduced from the control (p<0.001). Reducing RO-31-8220 concentration to 0.5 pM still had significant inhibitory effect on the percentage of OLGs with long processes (p<0.001). At 0.2 pM, the inhibitory effect of RO-31-8220 was not significant. OLGs grown in 7.2 mM appeared to be somewhat more resistant to the PKC inhibition. Same doses of RO-31-8220 yielded slightly higher averages of cell percentage (by approximately 6 %) for OLGs grown in 7.2 mM than OLGs grown in 1.8 mM. For instance, for OLGs treated with 0.5 pM RO-31-8220 and grown in 7.2 mM Ca 2 +, the inhibitory effect of RO-31-8220 was not as significant (p<0.05) as that observed in OLGs grown in 1.8 mM (p<0.001). Increasing RO-31-8220 concentration to 1 pM resulted in a significant inhibition of process outgrowth for Fig. 3.5a Fig. 3.5b 90 a '•5 T J X A 80 70 60 50 40 H * 20 J a o 10 • 1.8 mM[Ca2+] • 7.2 mM [Ca2+] 0.2 0.5 RO-31 Concentration (uM) • 1.8 mM [Ca2+] • 7.2 mM [Ca2+] 10 B A P T A Concentration (jiM) Fig.3.5a-b Effects of inhibiting PKC and intracellular Ca 2 + signalling on OLG process formation, a) OLGs were treated with RO-31, a specific PKC inhibitor, and the effect of PKC inhibition on OLG process formation was assessed by obtaining percentages of cells with long processes in a whole cell population. RO-31 reduced the population of OLGs with long processes in a dose-dependent manner, b) OLGs were treated with BAPTA, an intracellular Ca 2 + chelator, and the effect of blocking intracellular Ca 2 + signalling on OLG process formation was assessed by counting cells with long processes. BAPTA also inhibited process formations of OLGs in a dose-dependent manner. ANOVA was performed to compare inhibition data at 1.8 mM and 7.2 mM Ca 2 + to the respective controls. *: p<0.05, **: PO.001. OLGs grown in 7.2 mM (p<0.001). At 0.2 pM RO-31-8220, there was not much of an inhibition of OLG process outgrowth. OLGs were also treated with BAPTA/AM, an intracellular Ca 2 + chelator and grown in normal and high [Ca2+]. Three dosages of BAPTA were tested: 20, 10 and 5 pM. 20 pM BAPTA caused cell deaths of OLGs (as noted by retraction of processes and shrinkage of cell bodies) and smaller concentrations of BAPTA (10 pM and 5 pM) resulted in overall inhibitions of OLG process formations for both normal and high extracellular [Ca2+] (Fig.3.5b). 10 pM BAPTA resulted in a significant inhibition of OLG process outgrowth grown in both 1.8 mM and 7.2 mM Ca 2 + (p<0.05). However, inhibition occurring at 5 pM BAPTA was not as significant (p<0.5) as inhibited by 10 pM BAPTA (p<0.05) for both 1.8 mM and 7.2 mM Ca 2 +. At the same concentrations of BAPTA, OLGs grown in 7.2 mM Ca 2 + appeared to be more resistant to BAPTA treatment than OLGs in normal [Ca2+]. For instance, OLGs grown in 7.2 mM yielded about 8% higher average of the cell percentage than OLGs in 1.8 mM when treated with 10 pM of BAPTA. Our results so far suggest that the enhancing effect of high extracellular [Ca2+] on OLG process formation occurs through the activation of PKC (as evidenced by the inhibition of PKC by RO-31-8220) as well as intracellular Ca 2 + signalling (as evidenced by disrupting intracellular Ca 2 + signalling by BAPTA). 65 Chapter 4 Discussion 4.1 Fura-2 calibration The value for the dissociation constant (Kd) of the fura-2 imaging system under the conditions used for these experiments, was determined to be 221 nM, which is similar to values reported elsewhere (224 nM, Grynkiewicz et al., 1985; 266 nM, Uto et al., 1991). The calibration curve obtained using a series of buffers with known [Ca2+] exhibited a sigmoidal relationship which was similar to other in vitro curves reported elsewhere using a similar imaging system (Williams et al., 1985; Uto et al., 1991). The fluorescence ratio showed a steep Ca 2 + dependence (over 10 fold), ranging from 0.25 at min to 3.5 at max (where the dye became saturated). The temperature at which our system (25°C) was calibrated, was different from the temperature used by other investigators (20°C). However, temperature was shown to have little effect on the Kj value (Uto et al., 1991). The imaging software I used incorporated a number of options to optimize the signal to noise ratio. In order to minimize the variability for each ratio value, multiple video frames were sequentially added to memory and averaged before yielding a final ratio. The background image, resulting from autofluorescence from the optics, tube dark current as well as a level setup voltage from the camera, was acquired when the UV shutter was closed and stored as a pair of complete images. The background was then subtracted from every subsequent image on a pixel-by-pixel basis. The shading image, an image showing the relative intensities of the two excitation wavelengths over the entire field of view, was not used in this study. The shading image is sometimes used to correct the subsequent ratio images for having weaker fluorescence intensities towards the periphery of 66 coverslips (Tsien and Harootunian, 1990) and is obtained by imaging the recording field with the dye uniformly distributed in the recording chamber. However, the cell density used on the coverslips employed in this study was high enough to select a considerable number of cells (20 to 30 cells) towards the center of the field of view, where the fluorescence intensity was even. This eliminated the necessity of using the shading correction. The intensity threshold, which controlled the minimum fluorescence intensity to be acquired at each wavelength, was adjusted such that the final ratio values were not altered by any non-cellular regions that would have an intensity of zero, making a ratio value inaccurate. The imaging system also utilized a neutral density filter that minimized the effects of photobleaching of fura-2-filled cells. Finally, in order to ensure that the ratio values were derived only from OLGs, imaging regions were only selected from cells having a characteristic OLG morphology including small, round cell bodies with multiple processes. 4.2 Effect of phorbol ester-induced PKC activation on [Ca2+]i in OLG OLGs had a mean resting [Ca2+]i value (94 ± 2 nM, n = 586 cells) which was similar to previously published [Ca2+]i in rat cultured OLGs (100 nM, Cohen and Almazan, 1994; Takeda et al., 1995; Tzeng et al., 1995). OLGs responded to phorbol ester treatment by increasing [Ca2+]; by 2-3 times the basal level to 240 ± 1 0 nM and the sustained increase lasted for at least 20 min. I did not evaluate changes in [Ca2+]i after longer duration. The concentration of PDB was kept at 1 pM throughout this study to evaluate [Ca2+]j changes with fura-2-imaging, since this concentration triggered Ca 2 + signalling from most of OLG population. At a concentration of 0.1 uM, PDB triggered responses from only a fraction of 67 the OLG population (40%). A similar Ca 2 + response was observed in human microglia where exposure to phorbol ester resulted in a sustained increase of [Ca2+]i (Yoo et al., 1997). In human microglia, [Ca2+]i rose from 87 ± 9 nM to 275 nM ±21 nM in response to phorbol ester treatment. The time course of the [Ca2+]i increase followed a similar pattern to that observed in murine OLGs, in which [Ca2+]i rose over a period of approximately 2 min before reaching a plateau. This prolonged pattern of [Ca2+]i increase in glial cells was different from the rapid transient elevation of [Ca2+]j seen in rat hippocampal neurons when treated with phorbol ester (Mironov and Hermann, 1996). The phorbol ester-mediated elevation of [Ca2+] in OLGs and microglia resembles that observed in cultured frog esophageal epithelial cells (Levin et al., 1997). In this cell type, applying 4a- 12-O-tetradecanoyl-phorbol-13-acetate (TPA) resulted in a sustained increase of [Ca2+]; which reached a plateau over 3 min and was similar to that seen in OLGs. Despite the sustained increase in [Ca2 +]i, esophageal epithelial cells displayed fluctuations in [Ca2+]i during the periods when [Ca2+]i was increasing as well as during the sustained phase after TPA treatment (Levin et al., 1997). OLGs pretreated with nifedipine, an inhibitor of L-type VDCC, still responded to phorbol ester treatment indicating that the Ca 2 + rise in response to PDB treatment was not due to Ca 2 + influx through L-type VGCCs. In addition, I could not detect any [Ca2+]i changes by fura-2 imaging when OLGs were depolarized with a high concentration of K + , suggesting VGCCs were present at a very low density or absent in mouse OLGs. Pretreating OLGs with RO-31-8220, a specific PKC inhibitor (Keller and Niggli, 1993), completely abolished the [Ca ]i response seen upon treating OLGs with PDB. In addition, the observation that PDD, an inactive phorbol ester, failed to trigger any Ca 2 + signalling further substantiates the view 68 that phorbol ester was activating PKC that in turn triggered Ca 2 + signalling in OLGs. When extracellular Ca 2 + was removed from the buffer, OLGs failed to respond to phorbol ester treatment, which indicates that the [Ca2+]i increase was due to transmembrane Ca 2 + influx. To avoid any possibility of depleting intracellular Ca 2 + stores upon removal of extracellular Ca 2 + , the incubation time of OLGs in Ca2+-free media was minimized to less than 5 minutes. The observation that treating OLGs with cyclopiazonic acid (CPA), a Ca2+-releasing agent, after perfusing OLGs with Ca2+-free buffer for 5 minutes, resulted in a transient [Ca2+]i increase, further confirmed that the intracellular Ca 2 + store was not depleted in OLGs. It has been also shown that OLGs responded to CPA by depleting intracellular Ca 2 + stores and increasing [Ca2+]i(Engel et al., 1994; Simpson and Russell, 1997). The [Ca2+]i increase after PDB treatment which was absent when the extracellular Ca 2 + was removed and restored upon the replenishment of extracellular Ca 2 + , further indicatedd that the [Ca2+]; increase was due to Ca2+influx. After the [Ca2+]; increase reached a plateau, OLGs were re-exposed to a Ca -free environment and the [Ca2+]i was reduced again. This observation indicated that the during the plateau phase of the [Ca2+]i increase, the presence of extracellular Ca 2 + was required to keep the [Ca2+]; at the elevated level. I also evaluated [Ca2+]j changes by removing and restoring extracellular Ca 2 + in the absence of phorbol ester treatment. After the reduction of [Ca2+]i which occurred in Ca2+-free buffer, [Ca2+]j returned towards the basal level upon the replenishment of Ca 2 +. However, the amplitude of the [Ca2+]j rise was smaller than when PDB was present in the buffer, further supporting the conclusion that the [Ca2+]i increase with PDB treatment was due to transmembrane Ca 2 + influx. 69 The exact mechanism of how OLGs mediate Ca 2 + influx in response to phorbol ester-induced PKC activation was not fully determined in this study. The observation that application of various Ca 2 + channel blockers failed to inhibit phorbol ester-induced [Ca2+]i increase, makes the possibility of any VDCC involvement in the PDB-mediated [Ca ]i increase unlikely. Another possible mechanism is that the Ca 2 + influx resulting in the sustained increase of [Ca2+]; might be due to capacitative Ca 2 + entry, since there is evidence that the capacitative Ca 2 + entry is closely related to PKC activity. Bode and Goke showed that PKC activation by phorbol ester enhanced capacitative Ca 2 + influx in a pancreatic cell line, PJNm5F (Bode and Goke, 1994). In that experiment, capacitative Ca 2 + entry was induced by incubating the cells with thapsigargin and this response was augmented when the cells were pretreated with phorbol ester, suggesting that PKC stimulation activates the capacitative calcium influx pathway of pancreatic cells. On the contrary, other studies demonstrate an inhibitory effect of PKC activation on capacitative Ca 2 + entry in other cell types. Capacitative Ca 2 + entry was induced by thapsigargin treatment in rat thymocytes and human neutrophils and this Ca 2 + influx was inhibited by treating the cells with phorbol ester (Alonso-Torre and Garcia-Sancho, 1997). In cultured human mesangial cells, angiotensin II-induced capacitative Ca 2 + influx was inhibited when treated with phorbol ester in a dose-dependent manner, suggesting that the capacitative Ca 2 + entry was controlled by PKC activity (Mene et al., 1996). Capacitative Ca 2 + influx follows discharge of intracellularly stored Ca 2 + by Ca2+store-depleting agents such as thapsigargin or CPA. Ca 2 + discharged from the internal store results in an initial rise of [Ca2+]i followed by a sustained or plateau phase due to transmembrane Ca influx (Alonso-Torre and Garcia-Sancho, 1997; Mene et al., 1996; 70 Putney, 1997; Wu et al., 1997). Similar results were demonstrated in cultured mouse OLGs. Treating OLGs with 5 uM CPA resulted in 2-fold increase of [Ca2+]i from the basal level and reached a peak at approximately 200 nM over a period of 2 minutes. In the absence of extracellular Ca 2 + , CPA treatment only resulted in a transient increase in [Ca2+]j which increased again to reach a plateau upon reestablishment of extracellular Ca 2 + . The increased [Ca2+]i was then maintained for at least another 20 minutes (see Results). This pattern of capacitative Ca entry resembled the one found in mouse thymocytes (Ross and Cahalan, 1995), in which treating cells with thapsigargin resulted in a steady increase of[Ca2+]ifor 100 seconds which declined slowly after reaching the peak. During the plateau phase of the rise in [Ca2+]i, it was demonstrated that Ca 2 + influx was required to maintain the [Ca2+]j increase in human gastric mucous cells (Schofl et al., 1995) and in thyroid FRTL-5 cells (Tornquist, 1993). Although [Ca2+]j increase due to capacitative Ca 2 + entry was demonstrated in OLGs, it is not yet certain whether PDB-mediated [Ca2+]i increase was due to capacitative Ca 2 + entry. Employing antagonists for capacitative Ca 2 + entry when OLGs are treated with PDB should clarify whether capacitative Ca 2 + entry is indeed the mechanism responsible for [Ca2+]i increase in response to PDB stimulation. Unfortunately, no such specific antagonists for capacitative Ca 2 + influx have been identified yet. The mechanism of how OLGs maintain the [Ca2+], plateau after Ca 2 + influx was induced with phorbol ester treatment is unclear. During the sustained [Ca2+]i plateau phase, increased influx should be compensated for by increased Ca 2 + efflux (either into the extracellular space or into internal stores) to give a net equilibrium between Ca 2 + influx and efflux without any accumulation of cytosolic Ca 2 +. One likely mechanism to enhance Ca 2 + 71 efflux is by PKC increasing Ca 2 + pump activity of ER and refilling the internal store during phorbol ester treatment. It is possible that PKC stimulation induces not only Ca 2 + influx, but also activates the ER Ca 2 + pump to compensate for the resulting [Ca2+]j increase, allowing cells to achieve a steady level of [Ca2+],. I employed CPA, which has been shown to specifically inhibit ER or sarcoplasmic reticulum (SR) Ca 2 + ATPase (Goeger and Riley, 1989; Seidler et al., 1989), to evaluate the functional importance of the ER Ca 2 + pump during the PDB-mediated Ca 2 + influx in OLGs. When pretreated with CPA (5 pM), OLGs exhibited much higher levels of [Ca2+]i in response to the PDB treatment compared to controls. This observation, in turn, suggests that: i) PDB induced-Ca2+ increase was due to transmembrane Ca 2 + influx since OLGs were able to generate a Ca 2 + rise when intracellular Ca 2 + stores were depleted and ii) ER Ca 2 + ATPase pump activity was involved in regulating [Ca2+]j level. After OLGs were exposed to phorbol ester, the activity of the ER Ca 2 + ATPase was disturbed by adding CPA during the sustained phase of the Ca 2 + response. The plateau level of [Ca2+]i seen after phorbol ester treatment was modified and [Ca2+]i increased (from 200 to 350 nM). This result further supports the notion that phorbol ester-mediated PKC activation induces Ca 2 + influx and also enhances Ca 2 + buffering into ER by increasing the ER Ca2+ATPase activity. Reports by other investigators that phorbol ester-mediated PKC activation enhanced ER Ca pump activity in T lymphocytes (Balasubramanyam and Gardner, 1995), further support the notion that phorbol ester-induced PKC activation might also increase ER Ca 2 + ATPase in OLGs. Another mechanism of Ca 2 + handling after a rise in [Ca2+]i might be due the action of calmodulin-dependent kinases. Schulman postulated that a Ca 2 + rise may serve as a cytosolie messenger to activate calmodulin-regulated protein kinases leading to 72 phosphorylation of a specific subset of cellular proteins (Schulman, 1993). This causes an enhanced efflux of Ca 2 + from the cell because of Ca2+-calmodulin-dependent activation of the Ca 2 + pump on the plasma membrane (see review by Alkon and Rasmussen, 1988; Rasmussen et al., 1995). It is possible that in OLGs calmodulin-dependent kinases might enhance Ca pump activity during phorbol ester treatment, compensating for the sustained influx of Ca 2 +. 4.3 Effect of extracellular Ca 2 + on fCa2+l; in OLGs The dependence of [Ca2+]i on extracellular Ca 2 + has been previously demonstrated in a number of different cell types. In rat kidney cells, raising extracellular [Ca2+] from 1.1 to 5.0 mM resulted in a 3-fold increase in [Ca2+]i (Wang et al., 1997). A similar increase in [Ca2+]i when extracellular [Ca2+] was increased, was also observed in mouse keratinocytes (Li et al., 1995). Reducing extracellular [Ca2+] to half the normal level (1.8 mM) caused a significant decrease of [Ca2+]i in mast cells (Amano et al., 1997). Similar observations have been made in cultured rat cortical neurons (Villabla et al., 1994) and in the motoneuron cell line NSC-19 (Hasham et al., 1997). None of these studies have been done on OLGs, and I report here that changing the extracellular [Ca2+] affects [Ca2+]i in OLGs. Decreasing the Ca 2 + content of the buffer solution caused a decline of [Ca2+]i, whereas increasing [Ca2+] above the control Ca 2 + level (1.8 mM Ca2 +) resulted in an increase of [Ca2+] in OLGs. When OLGs were incubated in Ca2+-free buffer, the basal [Ca2+]i level (99 ± 6 nM) decreased by approximately 4 fold (down to 26 ± 4 nM). When extracellular [Ca2+] was 3.6 mM, [Ca2+]i rose by 4 fold (from 92 ± 8 nM to 414 ± 20 nM) and that level was sustained for approximately 10 minutes before returning towards the basal level. Incubating cells in 7.2 mM Ca 2 + resulted in a higher 73 amplitude of [Ca2+]i increase (up to 563 ± 37 nM) with a more transient pattern of [Ca2+]i rise compared to incubation in 3.6 mM Ca 2 +. At 7.2 mM, the [Ca2+]i plateau was sustained only for about 5 min before falling towards the basal level. Even though the changes in [Ca2+]i were measured under exactly the same experimental conditions, the transient pattern of the [Ca2+]i rise with increasing extracellular [Ca2+] seen in OLGs was different from the sustained pattern observed in NSC-19 cells (Hasham et al., 1997). When NSC-19 cells were exposed to 10 mM extracellular Ca 2 + , [Ca2+]; rose to about 380 nM from 90 nM and leveled off afterwards. The amplitude of the [Ca2+]i increase was smaller than when OLGs were exposed to 7.2 mM (560 nM). A transient rise in [Ca2+]i was only evident when NSC-19 cells were exposed to 25 mM [Ca2+] which resulted in [Ca2+]i rise to approximately 600 nM. This level of [Ca2+]i increase was comparable to when OLGs were exposed to 7.2 mM. It is likely that the transient [Ca2+]i response seen in OLGs which were exposed to 7.2 mM [Ca2+] may have been due to activation of a Ca2+-dependent, Ca 2 + efflux-inducing process that was activated by increasing Ca 2 + in OLGs, but inactive in NSC-19 cells until exposed to extracellular [Ca2+] greater than 10 mM (the transient Ca 2 + response was seen in NSC-19 cells at 25 mM and 50 mM). The increased [Ca2+]i has been shown to activate calmodulin which increases the affinity of the Ca2+-ATPase pump by twenty- to thirty-fold (Miller et al., 1991). OLGs exposed to high extracellular [Ca2+] (7.2 mM) might have increased ER Ca 2 + pump activity and the resultant increase of net Ca 2 + efflux into ER might account for the decline of [Ca2+]i after reaching the peak. When OLGs were exposed to 7.2 mM [Ca2+], the amplitude of the [Ca2+]i rise was larger and the plateau phase was shorter compared to OLGs in 3.6 mM. It is probable then when OLGs were treated with 7.2 mM of Ca 2 +, the higher amplitude of Ca 2 + 74 signalling resulted in higher activity of the Ca 2 + filling mechanism and thus a shorter duration of the sustained phase. Although transient, the [Ca2+]i did not return fully to the basal level following incubation in both 3.6 mM and 7.2 mM solutions and were sustained at a slightly higher level than the basal level, suggesting that there could be sustained cellular responses to these stimuli (Alkon and Rasmussen, 1988). The means by which OLGs elevate [Ca2+]i in response to increasing extracellular Ca 2 + was not completely evaluated. In order to evaluate whether the [Ca2+]i increase is due to transmembrane Ca 2 + influx or Ca 2 + mobilization from internal store, OLGs were depleted of intracellular Ca 2 + stores by being treated with CPA and exposed to 7.2 mM extracellular [Ca2+]. OLGs still responded to being exposed to high extracellular [Ca2+] by increasing [Ca2+]j, indicating that the [Ca2+]i increase was due to transmembrane Ca 2 + influx. The amplitude of this [Ca2+]i increase was, however, significantly larger (up to 880 ± 57 nM) than that observed in OLGs not treated with CPA, and the transient pattern was no longer present. These results indicate that Ca 2 + filling into ER through Ca 2 + pump was an important mechanism for OLGs to maintain [Ca2+]j homeostasis and drive the increased [Ca2+]i towards the basal level while exposed to high extracellular [Ca2+]. Since the results indicate that the [Ca2+]i increase in response to high extracellular [Ca2+] is due to Ca 2 + influx, the possibility arises that capacitative Ca 2 + influx might be the mechanism responsible for this [Ca2+]i increase. This extracellular [Ca2+]-induced Ca 2 + influx occurs through PKC-independent pathway, since OLGs preincubated with RO-31-8220 to inhibit PKC activity still responded to exposure to high extracellular [Ca2+] (see Results). High extracellular [Ca2+]-derived capacitative Ca 2 + entry has been previously reported elsewhere. An elevation of extracellular 75 Ca 2 + to 22 mM initiated an inward Ca 2 + current by capacitative Ca 2 + entry that declined slowly in lymphocytes (Zweifach and Lewis, 1995). Considering the observation that capacitative Ca 2 + entry is likely present in OLGs (see Results) and that the [Ca2+]j increase in response to high extracellular [Ca2+] is due to Ca 2 + influx, it is possible, then, that the elevation of extracellular Ca 2 + might have activated capacitative Ca 2 + entry in OLGs. Another possible mechanism for the [Ca2+]; rise in response to extracellular Ca 2 + is the activation of a Ca2+-sensing receptor (CaR). This receptor has been shown to "sense" the extracellular Ca 2 + level and induce Ca 2 + elevation with increasing extracellular Ca 2 + (Brown et al., 1996). Ca 2 + elevation triggered by CaR activation in response to changes in extracellular [Ca2+] has been demonstrated in rat epithelial cells (Wang et al., 1997, Champigneulle et al., 1997). Shorte and Schofield also demonstrated that increasing extracellular [Ca2+] to 20 mM resulted in 64% increase of [Ca2+]j and proposed that this [Ca2+]i elevation was due to the activation of CaR coupled to release of intracellular Ca 2 + store (Shorte and Schofield, 1996). However, this possibility was not systematically explored in this study. 4.4 Effect of extracellular Ca 2 + on OLG process extension Extracellular [Ca2+] has been shown to affect various cellular responses in different cell types. The evidence includes histamine release by mast cells (Amano et al., 1997), the membrane integrity of myocytes and astrocytes (Turman et al., 1995), proliferation of epithelial cells (Buras et al., 1995), epidermal growth factor (EGF)-induced osteoblastic cell proliferation and superoxide production by neutrophils (Brown and Ganey, 1995). The results 76 from this study indicate that in OLGs, increasing extracellular [Ca2+] augmented process outgrowth of OLGs, as evidenced by three indices of assessing OLG process outgrowth. Counting the percentage of OLGs with processes equal to or greater than 3-times the cell body diameter has been previously used by Yong et al. as an index of OLG process extension (Yong et al., 1988, 1991, 1995; Oh et al., 1997). The current study was designed to assess the extent of OLG process formation in various extracellular [Ca2+]. In the lowest [Ca2+] (0.01 mM), approximately 25 ± 3% of OLGs met the criteria of displaying long processes. Assessing OLG process formation was strengthened by other methods as well. Counting the number of primary processes has been a popular method used to assess neurite outgrowth (Hilborn et al., 1997; Xie et al., 1995). I also attempted to measure the degree of OLG process formation by devising an immunoassay method utilizing anti- PLP or anti- GalC antibodies, but the immunoassay method was not sensitive enough to detect very subtle differences of PLP-immunoreactivity under different [Ca2+]. As an alternative approach, we developed a method of measuring the total PLP-positive areas on the coverslips to replicate these assay conditions. Circles were drawn around the extremities of OLG processes including primary, secondary and tertiary processes, so that the circles covered most of PLP-positive sheathes. All the data collected from coverslips containing various extracellular [Ca ] was tested by an analysis of covariance (ANCOVA). This analysis gives the average of the product of deviations of data points from their respective means. Covariance is a measure of the relationship between two sets of data and covariance can be used to determine whether two ranges of data demonstrate a positive correlation. In order to determine whether OLG process formation is associated with extracellular [Ca2+], ANCOVA was used to 77 evaluate any statistically significant effect of extracellular [Ca2+] on OLG process formation. The strength of the test results was expressed in terms of the slope of the logarithmic function between the measure of OLG process formation and extracellular [Ca2+]. The results indicate that OLG process formation is related to the extracellular [Ca ] present in the feeding media. There was a gradual increase of the percentages of OLGs with long processes throughout the various [Ca2+]; at the lowest [Ca2+] (0.01 mM), approximately 25% of cells had long processes, which increased up to near 60% at the highest [Ca2+] (7.2 mM). The lowest extracellular [Ca2+] was provided by adding 0.5% horse serum alone without any additional supplement of Ca 2 +. Horse serum itself contained a known amount of Ca 2 + which was calculated to be 0.01 mM at 0.5%. The number of primary processes increased from 3 processes per cell body at the 0.01 mM to 5 processes per cell body at 7.2 mM extracellular [Ca2+]. In addition, there was an approximately 3-fold increase in the PLP-positive areas (from 0.25 pm2 at 0.01 mM to 0.80 pm2 at 7.2 mM [Ca2+]). The results from ANCOVA indicate that there was statistically significant association of OLG process formation with extracellular [Ca2+]. The regression line between the measurements of OLG process formation and extracellular [Ca2+] had a statistically significant positive slope (p<0.001). OLGs immuno-positive for PLP represent already fully differentiated OLGs (Kalwy and Smith, 1994; McLaurin and Yong, 1995) having primary, secondary and tertiary processes. Among the techniques used to measure OLG process formation, a caution has to be made regarding counting the numbers of primary processes, since extension of secondary and tertiary processes also participates in the formation of OLG processes. On the other hand, 78 approximating the total areas covered by PLP-positive sheathes was a method that addressed the extension of secondary and tertiary processes as well. OLGs were also treated with PDB (100 nM), which has been shown to enhance OLG process extension (Yong et al., 1988; Althaus et al., 1991), in various extracellular [Ca2+]. The regression lines between the measurements of OLG process formation and extracellular [Ca2+] in the presence of PDB also had statistically significant slopes (p<0.001) that were parallel to the slopes in the absence of PDB. The percentage of PDB-treated OLGs with long processes increased from approximately 33 % at 0.01 mM to 61% at 7.2 mM Ca 2 +. The number of primary OLGs increased from 3 processes at 0.01 mM to over 5 processes per cell body at 7.2 mM. The PLP-positive areas also increased from 0.46 pm2 at 0.01 mM to 1.17 pm2 at 7.2 mM. The level of the regression line in the presence of PDB was compared to that obtained in the absence of PDB. The presence of phorbol ester increased the overall number of primary processes the level of regression line between the number of primary processes per cell body and extracellular [Ca2+] in the presence of PDB was higher than that in the absence of PDB. PLP-positive areas also increased when PDB was present in various extracellular [Ca2+] in averages. However, this overall increase was not statistically significant since the level of the regression line between the average PLP-positive area and [Ca2+] was not statistically higher than that in the absence of PDB. Taken together, across the overall range of extracellular [Ca2+], the presence of PDB did not have, except for the number of primary processes, significant effect as revealed by ANCOVA. Comparing OLG process formation between PDB-treated OLGs and untreated OLGs revealed slightly greater process formation in PDB-treated OLGs than non-treated OLGs in 79 low extracellular [Ca2+] (0.03 - 0.45 mM). It is possible that PKC activation and the resultant [Ca2+]i increase in phorbol ester-treated OLGs grown in low extracellular [Ca2+] might have compensated for the lack of external Ca 2 + at these extracellular [Ca2+]. I evaluated whether / 2 + there might be a synergistic effect of PDB treatment and high extracellular [Ca ]. However, in high extracellular [Ca2+] (3.6 mM and 7.2 mM), there was not significant difference in the extent of OLG process formation between PDB-treated and untreated OLGs. One could, then, propose that the [Ca2+], influx alone triggered by incubating cells in high [Ca2+] could provide a sufficient signal to activate OLG process formation. The importance of Ca 2 + influx has been also demonstrated in neurons. Ca 2 + influx through voltage-dependent Ca 2 + channels has been demonstrated to cause neurite outgrowth in PC12 cells (Doherty et al., 1993). In cultured Helisoma neurons, Ca 2 + influx was reported to accelerate the rate and extent of neurite outgrowth (Williams et al, 1995). The Ca 2 + influx-induced neurite outgrowth has been shown to occur through the activation of MAP kinase (Rusanescu et al., 1995), a kinase that has been reported to be important for OLG process extension as well (Stariha et al., 1997). Fibroblast growth factor (FGF), which causes Ca 2 + influx in neurons, has been reported to mediate neurite outgrowth of 3T3 cerebellar neuron cell line (Williams et al., 1995). FGF has been also shown to enhance OLG process extension in human OLGs (Oh et al., 1996). Neurite outgrowth was also enhanced by sustained Ca 2 + wave resultant from Ca 2 + influx (Gu and Spitzer, 1994, 1995). Accumulating evidence suggests that neurite outgrowth and OLG process extension share many aspects of process outgrowth, and the evidence of the importance of Ca 2 + influx in neurite outgrowth may reveal 80 clues to the mechanism by which Ca 2 + influx plays an important role in OLG process extension. Previous studies suggest that cell adhesion is an important factor in OLG process formation. It has been found that OLG adhesion activated PKC followed by phosphorylation of myelin basic protein (MBP) (Vartanian et al., 1986) and an increase of cAMP levels (Vartanian et al., 1988). cAMP has been demonstrated to be an important molecule in myelination and OLG differentiation as evidenced by the increased production of myelin marker including MBP (Raible and McMorris, 1989; Anderson and Miskimins, 1994). Derivatives of cAMP have been shown to play a regulatory role in differentiation of OLGs. Unidentified bipotential glial cells in human brains, which express both oligodendrocytic and astrocytic markers, were found to differentiate into mature OLGs that express GalC when OLGs were treated with derivatives of cAMP in culture (Kim et al., 1985, Kim 1990). Since adhesion factor might be an important parameter during process formation of OLGs, it was also necessary to investigate whether adhering OLGs to Matrigel substrate, a laminin-rich reconstituted basement membrane matrix that was used to coat the coverslips for attaching OLGs for immunocytochemical studies, had any effect on [Ca2+]i. The reason for using Matrigel for immunocytochemical studies stemmed from the fact that mouse OLGs attached better on Matrigel-coated coverslips than on poly-L-lysine-coated Aclar coverslips. Cellular adhesion to Matrigel itself has been demonstrated to elicit a sustained elevation of [Ca2+]i in cultured human keratinocytes (Goberdhan et al., 1997) and in rat Sertoli cells (Ravindranath et al., 1996). The basal levels of [Ca2+]j were compared between OLGs grown on poly-L-lysine and Matrigel in order to see whether there was sustained increase in the level of[Ca2 +] ; 81 due to cellular adhesion to Matrigel. It was found that there was no significant difference in the basal [Ca2+]j levels between OLGs grown on the different substrates (94.4 ±1.6 nM for poly-L-lysine and 96.6 ± 5.8 nM for Matrigel, p=0.80). In addition, OLGs grown on Matrigel substrate also increased [Ca2+]i in response to phorbol ester treatment and exposure to high extracellular [Ca2+]. Taken together, cellular adhesion to Matrigel did not appear to have significant effects on intracellular Ca 2 + signalling in OLGs. In order to investigate how increasing extracellular [Ca2+] affected OLG process formation, OLGs were pretreated with RO-31-8220, a specific PKC inhibitor, before being exposed to high extracellular [Ca2+]. ANOVA was used to see any significantly significant effect of the inhibitor. The results indicated that 1 pM and 0.5 pM RO-31-8220 significantly diminished OLG process formation in both normal (1.8 mM) and high (7.2 mM) extracellular [Ca2+] (p<0.001, see Results 3.5), suggesting that increasing extracellular [Ca2+] could be accompanied by PKC activation. Treating OLGs with 1 pM of RO-31-8220 resulted in an approximately 50% reduction in the percentages of OLGs with long processes (from 55% to 25% in 1.8 mM, and from 67% to 32% in 7.2 mM of Ca2+). Treatment with 0.2 pM RO-31-8220 did not have any significant effects. OLGs, pretreated with BAPTA, an intracellular Ca 2 + chelator, also showed an inhibition of OLG process formation compared to control OLGs in both 1.8 mM and 7.2 mM Ca 2 +. Pretreating OLGs with 10 pM BAPTA reduced the percentage of OLGs with long processes to 42% from 55% in 1.8 mM Ca 2 + (a reduction of 20%) and to 49% from 67% in 7.2 mM Ca 2 + (a reduction of 27%, p<0.5, see Results 3.5). These results suggests that both Ca 2 + signalling and PKC activation might be necessary to elicit the augmentation of OLG process formation. In view of the finding that PKC activity is 82 proportionately related to [Ca2+]i elevation induced by high extracellular [Ca2+] in NSC-19 motoneuron cell line (Hasham et al., 1997) and in cardiocytes (Miyawaki et al., 1996), it is possible that high extracellular [Ca2+] might activate PKC in OLG, leading to the augmented process formation. It should be noted, however, the inhibiting effects of RO-31-8220 and BAPTA on OLG process formation might have been due to non-specific toxic effects of these agents, since I found that higher concentrations of RO-31-8220 (10 uM) and BAPTA (100 pM) were toxic to OLGs. When higher concentrations of these substances were used, there were significant reductions in OLG population and retractions of OLG processes compared to the controls, suggesting that these compounds were toxic to OLGs at these concentrations. An assay to detect cytotoxicity such as measuring the levels of LDH could have been useful to determine whether there were any non-specific toxic effects at the working concentrations of the agents. Other studies have evaluated the importance of Ca 2 + signalling in OLG differentiation. He et al. found that Ca 2 + signalling regulated by axonal contact played an important role in communication between OLGs and neurons by increasing the percentage of OLGs responding to ATP, carbachol, glutamate and histamine (He et al., 1996). Studies of the activation of muscarinic receptors in OLGs have shown that stimulation with carbachol, an acetylcholine analog, resulted in an increase in intracellular Ca 2 + levels, which were dependent both on the mobilization of internal stores and transmembrane influx of extracellular Ca 2 + (Cohen and Almazan, 1994). This muscarinic activation and Ca 2 + increase were accompanied by increased IP3 production and attenuated cAMP levels. They postulated that muscarinic receptor activation might be important in OLG development, since accumulation of cAMP 83 induced OLG differentiation (McMorris, 1990) and phosphorylation of CNP, a major component in myelin (Agrawal et al., 1994). Basic fibroblast growth factor (bFGF), which enhances OLG process formation (Oh et al., 1997), led to phosphorylation of mitogen activated protein kinase (MAPK), an important kinase for OLG process formation (Stariha et al., 1997), and MAPK activity was influenced by [Ca2+]i in OLGs (Pende et al., 1997). It should be noted, however, that [Ca2+]j elevation does not always have enhancing effects on OLG process outgrowth. Complement activation has been shown to mediate transient increases in Ca 2 + in rat optic nerve OLGs (Scolding et al., 1989). T-cell perforin attack on rat optic nerve OLGs also produces an increase in Ca 2 + by the mechanism of transmembrane Ca2" influx through the perforin pore formed after the perforin attack. (Jones et al., 1991). The 2+ [Ca ]i elevation due to perforin attack or complement activation results in rapid OLG death. The Ca 2 + ionophores A23187 and ionomycin mimic both complement and perforin attack, causing OLG lysis at concentrations that did not lyse other glial cells (Scolding et al., 1992). The detrimental effects of a rise in [Ca2+]i have also been reported by Benjamin and her associates. Antibodies to GalC caused a transmembrane influx of Ca 2 + in OLGs (Dyer and Benjamin, 1990). Treatment of OLGs with thapsigargin, which elevated [Ca2+]i, as well as 2+ the Ca lonophore, A23187, resulted in retraction of membrane sheets and cell death in mature mouse OLGs (Benjamin and Nedelkoska, 1996). However, there was a substantial difference in the amplitude of [Ca2+]i signalling between the [Ca2+]i elevations observed in this study and [Ca2+], elevation in the study of Benjamin and Nedelkoska. 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