Drug connectivity mapping and functional analysis reveal therapeutic small molecules that differentially modulate myelination.

Disruption or loss of oligodendrocytes (OLs) and myelin has devastating effects on CNS function and integrity, which occur in diverse neurological disorders, including Multiple Sclerosis (MS), Alzheimer's disease and neuropsychiatric disorders. Hence, there is a need to develop new therapies that promote oligodendrocyte regeneration and myelin repair. A promising approach is drug repurposing, but most agents have potentially contrasting biological actions depending on the cellular context and their dose-dependent effects on intracellular pathways. Here, we have used a combined systems biology and neurobiological approach to identify compounds that exert positive and negative effects on oligodendroglia, depending on concentration. Notably, next generation pharmacogenomic analysis identified the PI3K/Akt modulator LY294002 as the most highly ranked small molecule with both pro- and anti-oligodendroglial concentration-dependent effects. We validated these in silico findings using multidisciplinary approaches to reveal a profoundly bipartite effect of LY294002 on the generation of OPCs and their differentiation into myelinating oligodendrocytes in both postnatal and adult contexts. Finally, we employed transcriptional profiling and signalling pathway activity assays to determine cell-specific mechanisms of action of LY294002 on oligodendrocytes and resolve optimal in vivo conditions required to promote myelin repair. These results demonstrate the power of multidisciplinary strategies in determining the therapeutic potential of small molecules in neurodegenerative disorders.

Store-operated calcium entry is essential for glial calcium signalling in CNS white matter.

'Calcium signalling' is the ubiquitous response of glial cells to multiple extracellular stimuli. The primary mechanism of glial calcium signalling is by release of calcium from intracellular stores of the endoplasmic reticulum (ER). Replenishment of ER Ca2+ stores relies on store-operated calcium entry (SOCE). However, despite the importance of calcium signalling in glial cells, little is known about their mechanisms of SOCE. Here, we investigated SOCE in glia of the mouse optic nerve, a typical CNS white matter tract that comprises bundles of myelinated axons and the oligodendrocytes and astrocytes that support them. Using quantitative RT-PCR, we identified Orai1 channels, both Stim1 and Stim2, and the transient receptor potential M3 channel (TRPM3) as the primary channels for SOCE in the optic nerve, and their expression in both astrocytes and oligodendrocytes was demonstrated by immunolabelling of optic nerve sections and cultures. The functional importance of SOCE was demonstrated by fluo-4 calcium imaging on isolated intact optic nerves and optic nerve cultures. Removal of extracellular calcium ([Ca2+]o) resulted in a marked depletion of glial cytosolic calcium ([Ca2+]i), which recovered rapidly on restoration of [Ca2+]o via SOCE. 2-aminoethoxydiphenylborane (2APB) significantly decreased SOCE and severely attenuated ATP-mediated calcium signalling. The results provide evidence that Orai/Stim and TRPM3 are important components of the 'calcium toolkit' that underpins SOCE and the sustainability of calcium signalling in white matter glia.

Expression of Kir4.1 and Kir5.1 inwardly rectifying potassium channels in oligodendrocytes, the myelinating cells of the CNS.

The inwardly rectifying K+ channel subtype Kir5.1 is only functional as a heteromeric channel with Kir4.1. In the CNS, Kir4.1 is localised to astrocytes and is the molecular basis of their strongly negative membrane potential. Oligodendrocytes are the specialised myelinating glia of the CNS and their resting membrane potential provides the driving force for ion and water transport that is essential for myelination. However, little is known about the ion channel profile of mature myelinating oligodendrocytes. Here, we identify for the first time colocalization of Kir5.1 with Kir4.1 in oligodendrocytes in white matter. Immunolocalization with membrane-bound Na+/K+-ATPase and western blot of the plasma membrane fraction of the optic nerve, a typical CNS white matter tract containing axons and the oligodendrocytes that myelinate them, demonstrates that Kir4.1 and Kir5.1 are colocalized on oligodendrocyte cell membranes. Co-immunoprecipitation provides evidence that oligodendrocytes and astrocytes express a combination of homomeric Kir4.1 and heteromeric Kir4.1/Kir5.1 channels. Genetic knock-out and shRNA to ablate Kir4.1 indicates plasmalemmal expression of Kir5.1 in glia is largely dependent on Kir4.1 and the plasmalemmal anchoring protein PSD-95. The results demonstrate that, in addition to astrocytes, oligodendrocytes express both homomeric Kir4.1 and heteromeric Kir4.1/Kir5.1 channels. In astrocytes, these channels are essential to their key functions of K+ uptake and CO2/H+ chemosensation. We propose Kir4.1/Kir5.1 channels have equivalent functions in oligodendrocytes, maintaining myelin integrity in the face of large ionic shifts associated with action potential propagation along myelinated axons.

Opposing actions of fibroblast growth factor-2 on early and late oligodendrocyte lineage cells in vivo.

In vitro studies indicate that fibroblast growth factor 2 (FGF2) has diverse effects on cells of the early and late oligodendrocyte lineage. Here, we have examined this in vivo by comparing the actions of FGF2 on the developing and developed anterior medullary velum (AMV) of postnatal rats. FGF2, or saline vehicle in controls, was administered into the cerebrospinal fluid of anaesthetised rats between postnatal day (P)6 and P9 either for 1 day (1d), 2d, or 3d, and AMV were analysed at P8 or P9. Immunolabelling for NG2 was used to identify oligodendrocyte progenitor cells (OPCs) and Rip for premyelinating and myelin-forming oligodendrocytes. At P6-9, the AMV was clearly demarcated into myelinated caudal and premyelinated rostral areas. The caudal AMV was populated by differentiated myelin-forming oligodendrocytes and 'adult' OPCs, whilst the rostral AMV contained mixed populations of 'perinatal' OPCs, and both premyelinating and myelin-forming oligodendrocytes. Administration of FGF2 resulted in the accumulation of OPCs in both the developing and developed AMV. Notably, FGF2 had a bipartite action on premyelinating oligodendrocytes, at first dramatically expanding their population throughout the premyelinated and myelinated AMV, but subsequently causing the loss of these previously generated cells. In addition, FGF2 induced the loss of existing myelin-forming oligodendrocytes in the developed AMV, and arrested the generation of new myelin-forming cells in the developing AMV. This study provides evidence that FGF2 has opposing positive and negative actions on early and late oligodendrocyte lineage cells in vivo.

P2X and P2Y purinoreceptors mediate ATP-evoked calcium signalling in optic nerve glia in situ.

It is known that ATP acts as an extracellular messenger mediating Ca2+ signalling in glial cells. Here, the mechanisms involved in the ATP-evoked increase in glial [Ca2+]i were studied in situ, in the acutely isolated rat optic nerve. ATP and agonists for P2X (a,b-metATP) and P2Y (2MeSATP) purinoreceptors triggered raised glial [Ca2+]i, and there was no significant difference between cells identified morphologically as astrocytes and oligodendrocytes. Dose-response curves indicated that P2Y receptors were activated at nanomolar concentrations, whereas P2X purinoreceptors were only activated above 10 microM. The rank order of potency for several agonists indicated optic nerve glia expressed heterogeneous purinoreceptors, with P2Y1< or = P2Y2/4< or = P2X. The ATP evoked increase in [Ca2+]i was reversibly blocked by the P2X/Y purinoreceptor antagonist suramin (100 microM) and markedly reduced by thapsigargin (10 microM), which blocks IP3-dependent release of Ca2+ from intracellular stores. Removal of extracellular Ca2+ reduced the ATP evoked increase in [Ca2+]i and completely blocked its recovery, indicating that refilling of intracellular stores was ultimately dependent on Ca2+ influx from the extracellular milieu. The results implicate ATP as an important signal in CNS white matter astrocytes and oligodendrocytes in situ, and indicate that metabotropic P2Y purinoreceptors mobilize intracellular Ca2+ at physiological concentrations of ATP, whereas ionotropic P2X purinoreceptors induce Ca2+ influx across the plasmalemma only at high concentrations of ATP, such as occur following CNS injury.

Morphology of astrocytes and oligodendrocytes during development in the intact rat optic nerve.

The detailed three-dimensional morphology of macroglial cells was determined throughout postnatal development in the intact rat optic nerve, a central nervous system white matter tract. Over 750 cells were analyzed by intracellular injection of horseradish peroxidase or Lucifer Yellow to provide a new perspective of glial differentiation in situ. Retrograde analysis of changes in glial morphology allowed us to identify developmental timetables for three morphological subclasses of astrocytes and oligodendrocytes, and to estimate their time of emergence from undifferentiated glial progenitors. Glial progenitors were recognised throughout postnatal development and persisted in 35-day-old nerves, where we suggest they represent adult progenitor cells. Astrocytes were present at birth, but the majority of these cells developed over the first week as three morphological classes emerged having either transverse, random, or longitudinal process orientation. Several lines of evidence led us to believe that the majority of astrocytes in the rat optic nerve were morphological variations of a single cell type. Young oligodendrocytes were first observed 2 days after birth, indicating that they diverged from progenitors at or near this time. During early development these cells extended a large number of fine processes, which then bifurcated and extended along axons. Later, as myelination proceeded, oligodendrocytes exhibited fewer processes which grew symmetrically and uniformly along the axons, resulting in a highly stereotypic mature oligodendrocyte form. Our analysis of oligodendrocyte growth suggests that these cells did not myelinate axons in a random manner and that axons may influence the myelinating processes of nearby oligodendrocytes.

Morphological changes in oligodendrocytes in the intact mouse optic nerve following intravitreal injection of tumour necrosis factor.

Intracellular dye-injection was used to determine the whole-cell morphology of oligodendrocytes in intact optic nerves of mice following intravitreal injection of tumour necrosis factor-alpha (TNF alpha) or heat-inactivated TNF alpha to act as controls. Oligodendrocytes in control nerves had a stereotypic morphology, and provided an average of 20 axons with single internodal myelin segments of around 200 microns internodal length. Oligodendrocytes with abnormal morphological features were identified 8-14 days following intravitreal injection of TNF alpha. Internodal myelin segments developed swellings along their lengths, became attenuated, and in extreme cases receded completely. This study provides a new insight into the process of demyelination, especially of the early stages which are not amenable with other techniques. Furthermore, it confirms that injection of TNF alpha into the vitreous, a fluid compartment of the CNS, instigates a sequence of events which results in oligodendrocyte disruption and demyelination. The mechanism by which intravitreally injected TNF alpha mediates these changes in optic nerve oligodendrocytes are yet unknown.

Optic neuritis in chronic relapsing experimental allergic encephalomyelitis in Biozzi ABH mice: demyelination and fast axonal transport changes in disease.

The encephalitogenicity of optic nerve tissue was demonstrated in Biozzi ABH (H-2(dq1)) mice. Acute experimental allergic encephalomyelitis (EAE) occurred in 11/14 animals and 4/5 exhibited relapse. The involvement of the optic nerve in spinal cord homogenate induced chronic relapsing EAE (CREAE) was demonstrated by mononuclear cell infiltration and myelin degradation in the optic nerve prior to and during clinical disease. During the relapse phase gross pathological assessment revealed swollen and translucent plaques on the optic nerves. Advanced lesions showed widespread demyelination, astrocytic gliosis and fibrotic changes of the blood vessels. Physiologically, the fast axonal transport of proteins from the retina to the optic nerve and superior colliculus was significantly decreased during relapse. The association of inflammation and demyelination with physiological deficit in the optic nerve highlights the usefulness of this model in the study of multiple sclerosis in which acute monosymptomatic unilateral optic neuritis is a common manifestation. Furthermore, the novel induction of CREAE with optic nerve homogenate suggests that optic neuritis is a common significant role in the pathophysiology and progression of neurological disease in CREAE which may be relevant to studies of optic neuritis in multiple sclerosis.

High potassium selective permeability and extracellular ion regulation in the glial perineurium (blood-brain barrier) of the crayfish.

Selective ion permeability, ion transport properties, and electrical resistance of the perineurial barrier, as they relate to interstitial ion regulation, where studied and characterized electrophysiologically in ion substitution experiments. In high external [K+] a transient spike-like voltage was generated across the perineurial barrier which fell over 1-2 min to a slowly decaying voltage. The glial perineurium had at least a 10 times greater permeability to K+ than Cl-, and was effectively impermeant to Na+. The potential, in high external [K+], was determined by the K+ and Cl- gradients and their relative permeabilities across the sheath. For other cations the selectivity sequence of the perineurial barrier, as determined from electrophysiological measurements, was K+ greater than or equal to Rb+ much greater than NH4+ greater than Cs+ greater than Li+ greater than Na+ corresponding most closely to the Eisenman sequence IV. The perineurium had a resistance of 260 +/- 23 omega cm2 in crayfish physiological solution. In high [K+]0 the resistance fell by over half during the transient spike potential and then recovered towards resting levels as the voltage decayed. In the intact nerve cord interstitial [K+] rose to only 10-20 mM during a 2-min exposure to 100 mM K0+. K influx and efflux were related to the change in barrier permeability and an increased selectivity to K+ which, in these studies, was determined primarily by its electrochemical gradient across the perineurial barrier. The results suggest that the crayfish perineurium is a leaky epithelium capable of a high degree of ion regulation. Trans-perineurial barrier potential and conductance in high external [K+] are primarily functions of passive processes of the perineurial glial cell membranes and of the paracellular conductance channels driven by the electrochemical gradient for the K+. Accordingly, the mass transport of [K+] showed the same quantitative relationship in both directions.

Calcium-dependent regulation of potassium permeability in the glial perineurium (blood-brain barrier) of the crayfish.

The physiological basis of the high selective potassium permeability of the crayfish glial perineurium was studied. The transient spike-like perineurial potential generated in high external [K+] was used as a measure of barrier K+ permeability. The medial giant axon membrane potential was used to monitor interstitial [K+]. Perineurial current-voltage relations of the perineurium were used to measure electrical resistance and to determine changes in K+ conductance of the perineurial barrier. Of a range of cations studied only Rb+, in addition to K+ generated a large transient sheath potential. In some experiments "regenerative" multiple spikes were observed during the continued exposure of the perineurium to high [Rb+]0. This degree of ion selectivity is typical of glial cell membranes and K channels. Barrier conductance increased only very briefly in Rb+; the potential falling rapidly to a steady 5-10 mV. The PCl/PRb and the PCl/PK ratios at the peak transient potential were similar suggesting the permeability site for these cations was the same. The permeability of Rb+ in the plateau phase was significantly lower than K+ suggesting that high [Rb+]0 may act to block K+ channels. The K(+)-selective permeability was reversibly blocked by extracellular Ba2+ at both the peak and the plateau phase, in a concentration-dependent manner. Other K-channel blocking agents, tetraethylammonium ions (10 mM), caesium ions (20 mM), and 3,4-diaminopyridine (0.5 mM) were ineffective. The effect of Ba2+ on the peak potential was similar to the removal of external Ca2+ or exposure to the Ca2(+)-channel blockers, verapamil (10(-4) M) or La3+ (5 mM). The time- and concentration-dependent reversible block of the K+ permeability of the perineurium was consistent with the known action of these agents on voltage-gated Ca2+ channels in nerve and glia. La3+ caused an irreversible decrease in perineurial conductance and K+ influx. Lanthanum titration of the negative charges of glial membranes and mucopolysaccharide matrix of the intercellular space suggest they may be important factors in determining the magnitude of the perineurial leak and paracellular K+ permeability. Electron microscopic examination of La3+ distribution demonstrated a diffusion barrier at the outer layer of perineurial glia. The binding of La3+ at the basolateral membranes of the glial barrier suggested this was the site at which La3+ had its physiological actions. The results suggest that the increase in glial membrane K+ conductance in high [K+]0 was most likely due to voltage-gated Ca2+ and K+ channels and Ca2(+)-activated K+ channels of the membranes of perineurial glia.

The astrocyte response to gamma-aminobutyric acid attenuates with age in the rat optic nerve.

There is increasing evidence that glial cells respond to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), and astrocytes have been shown to possess GABAA receptors both in vivo and in vitro. A recent study by Sakatani et al. (Proc. R. Soc. Lond. B247, 155 (1992)) demonstrated the transient expression of functional GABAA receptors in the developing rat optic nerve, but axonal and glial components of the response were not distinguished. To help address this problem, we have determined the electrophysiological response to GABA in astrocytes of the isolated intact optic nerves from neonatal rats, identified morphologically following intracellular injection of horseradish peroxidase. Astrocytes responded to GABA by a GABAA receptor-mediated depolarization which attenuated gradually during post-natal development; astrocytes in 21-day-old nerves were not observed to respond to GABA. The results indicate the transient presence of functional GABAA receptors in developing rat optic nerve astrocytes in situ, and we speculate upon a role for GABA in glial signalling and the organization of axonglial interrelations during development.

Modulation of a glial blood-brain barrier.

CNS interstitial fluid homeostasis by the glial perineurial blood-brain barrier in the crayfish and cockroach is dependent on glial uptake mechanisms, low paracellular permeability, and the cation-binding properties of the extracellular matrix. Potassium selective permeability of the crayfish perineurium is modulated by a Ca(2+)-dependent mechanism at the basolateral membranes of the glial barrier and is ion and voltage dependent. In addition, extracellular charged sites are significant in perineurial K+ and Ca2+ homeostasis and may be modified by changes in pH. In the cockroach, and probably the crayfish, perineurial K+ transport may also be modulated by receptor-mediated changes in glial membrane permeability. The factors acting at the crayfish and cockroach blood-brain barrier are summarized in FIGURE 8 and would be well suited for providing efficient K+ spatial buffering of the CNS. Analogous processes have been described in vertebrate glial cells and in the endothelial blood-brain barrier, which implies a common primary function. The CNS is protected from large fluctuations in the body fluids by the blood-brain barrier, whereas glial uptake mechanisms control the composition of the brain interstitial fluid, and modulation of both barrier permeability and glial transport by the altered chemical environment following neuronal activity allows precise adjustment of the brain extracellular fluids to the changing needs of the CNS. The insect and crustacean ventral nerve cord and perineurial blood-brain barrier provide an excellent preparation in which the interactions between these factors can be investigated in intact CNS tissue.

Macroglial cell types, lineage, and morphology in the CNS.

The characterization of macroglial antigenic phenotypes has provided considerable information on the classification of glial subtypes and lineage, but the picture in vivo appears to more complex than originally surmised from in vitro systems. Whether type-2 astrocytes and O-2A progenitors play a significant part in vivo remains controversial, but these questions have generated a valuable reassessment of inferences made from in vitro studies. It is not known whether disparate regions of the nervous system have different glial cell types with distinct cell lineages, but results in peripheral nerve and the cortex (see Price and colleagues, and Jessen and colleagues, this volume) suggest that this is likely the case. The heterogeneity of astrocytic form and the implied divergence of function in vivo parallel but have yet to be equated with the wide range of antigenic and electrophysiologic characteristics that can be induced in glia under certain culture conditions in vitro. Whether similar conditions prevail within the developing nervous system is not clear. The continued development of immunocytochemical markers and the use of retroviral vectors, combined with morphologic and electrophysiologic studies in vivo and in vitro, will undoubtedly answer these and other questions.

Effect of inflammatory agents on electrical resistance across the blood-brain barrier in pial microvessels of anaesthetized rats.

The effect of histamine, bradykinin and serotonin on blood-brain barrier permeability was investigated using in situ measurement of transendothelial electrical resistance in pial microvessels of anaesthetized rats. Mean resistance of vessels superfused with artificial cerebrospinal fluid was 1800 omega cm2, indicating a tight barrier with extremely low ion permeability. In paired experiments from continuous measurements in single vessels, addition of 10(-3) M serotonin to the solution bathing the brain had no marked effect on resistance; whereas both histamine and bradykinin, applied at a concentration of 10(-4) M, caused a rapid and reversible decrease in resistance. Mean resistance was 408 and 505 omega cm2 in 10(-4) M histamine and bradykinin, respectively, and approximately 50% of vessels had a resistance less than 250 omega cm2, compared to 12% in controls, indicating a leaky blood-brain barrier that is not capable of normal brain ion homeostasis. Histamine and bradykinin had similar dose-response relations, and a maximal effect was observed between 20 and 50 microM. Thus, histamine and bradykinin act at the abluminal (brain-facing) membranes of the cerebral endothelium to mediate blood-brain barrier opening. These results support a role for histamine and bradykinin in brain oedema formation.

Effect of histamine and antagonists on electrical resistance across the blood-brain barrier in rat brain-surface microvessels.

The effect of histamine on blood-brain barrier permeability was investigated using in situ measurement of transendothelial electrical resistance in brain-surface microvessels of anaesthetized rats. Mean resistance of vessels superfused with artificial cerebrospinal fluid was 1500 omega.cm2, indicating a tight barrier with low ion permeability. The addition of 10(-4) M histamine resulted in a 75% decrease in resistance, in both arterial and venous vessels, indicating a marked increase in barrier permeability. To determine the nature of the response to histamine, rats were given presurgical intraperitoneal injections of promethazine (H1 receptor antagonist), cimetidine (H2 receptor antagonist) or indomethacin (cyclo-oxygenase inhibitor), singularly and in combinations. Cimetidine completely blocked the histamine-mediated increase in barrier permeability whereas promethazine only had a small effect and indomethacin was ineffective. In addition, cimetidine treatment resulted in a 100% increase in basal resistance in both arterial and venous vessels, suggesting endogenous histamine was acting to increase blood-brain barrier permeability. It is concluded that histamine causes an increase in blood-brain barrier permeability which is mediated via endothelial H2 receptors, and that the electrical resistance in cimetidine-treated rats most closely represents the true permeability of the blood-brain barrier.

The NG2 chondroitin sulfate proteoglycan: role in malignant progression of human brain tumours.

The expression and function of NG2, a transmembrane chondroitin sulfate proteoglycan was studied in human gliomas of various histological types in culture using immunocytochemistry and flow cytometry. NG2 was differentially expressed in the neoplasms, with higher expression in high compared to low-grade gliomas. In acutely isolated cells from human biopsies, NG2 +ve and NG2 -ve populations were morphologically distinct from each other, and NG2 +ve cells were more proliferative than NG2 -ve cells. The mitogens platelet derived growth factor (PDGF-AA) and basic fibroblast growth factor (bFGF) added in combination to serum-free medium (SFM) upregulated NG2 expression on glioblastoma multiforme cells in culture but had little effect on NG2 expression on the anaplastic astrocytoma cells. Furthermore, NG2 was colocalised with the platelet derived growth factor alpha receptor (PDGFalphaR) and antibody blockade of the PDGF-alphaR ablated NG2 expression on the glioblastoma multiforme cells, suggesting that increased NG2 expression in the presence of PDGF-AA is mediated via the PDGF-alphaR. Assays of migration and invasion indicate that NG2 +ve glioma cells migrated more efficiently on collagen IV and that NG2 -ve cells were more invasive than their NG2 +ve counterparts. The results indicate that NG2 may be, respectively, positively and negatively related to the proliferative and invasive capacity of glioma cells. Thus, expression of the NG2 proteoglycan may have major implications for malignant progression in glial neoplasms and may prove a useful target for future therapeutic regimens.

Response of astrocytes to gamma-aminobutyric acid in the neonatal rat optic nerve.

The electrophysiological response to gamma-aminobutyric acid (GABA) was determined in astrocytes of the isolated intact optic nerves of rats aged 8 to 12 days old, identified morphologically following intracellular injection of horseradish peroxidase. At this age, astrocytes had a mean (+/- S.E.M.) resting membrane potential of -62.25 +/- 1.9 mV (n = 32), and responded to GABA by a depolarization characterized by an initial peak of mean 9.1 +/- 0.6 mV, which was not sustained and fell to a plateau level. The effect of GABA was mimicked by the GABAA-receptor agonist muscimol, but not by the GABAB-receptor agonist baclofen, and was reduced in the presence of the GABAA-receptor antagonist bicuculline. Astrocytes responded to 10 mM [K+]o by a single phase depolarization of 16 +/- 2 mV (n = 5). It is concluded that GABA acts directly on astrocytes and its effect is not mediated by K+ released by axons. This study indicates the presence of functional GABAA receptors in neonatal rat optic nerve astrocytes in situ. The results suggest immature astrocytes may be the source of the GABA response described in two recent studies on the rat whole optic nerve preparation. The astrocyte response to GABA may be important in axon-glial signalling during development.

Response of oligodendrocytes to glutamate and gamma-aminobutyric acid in the intact mouse optic nerve.

The electrophysiological response to glutamate and gamma-aminobutyric acid (GABA) is determined in oligodendrocytes of the isolated intact mouse optic nerve, identified by their characteristic morphology following iontophoretic injection with horseradish peroxidase (HRP). In this study, mature myelin-forming oligodendrocytes are shown for the first time to respond to glutamate and GABA in situ, by a 2-3 mV depolarization. Morphologically homogeneous oligodendrocytes exhibit a heterogeneous response to glutamate and GABA; some cells respond to both excitatory amino acids, whereas others respond to one but not the other, and some oligodendrocytes do not respond to either. Oligodendrocytes uniformly depolarize in elevated [K+]o and it is concluded that the effect of glutamate and GABA is not mediated by an increase in [K+]o released from axons or astrocytes. The oligodendrocyte response to amino acids may be important in axon-to-oligodendrocyte signalling at the nodes of Ranvier.

Adenosine 5' triphosphate evoked mobilization of intracellular calcium in central nervous system white matter of adult mouse optic nerve.

Although it has been established that immature glial cells express functional purinergic receptors, the responsiveness of mature glial cells in vivo had not been elucidated. This question was addressed using fura-2 ratiometric measurements of [Ca2+]i in the adult mouse optic nerve, a central nervous system (CNS) white matter tract, taking advantage of the facts that (i), the optic nerve contains glial cells but not neurons and (ii), that fura-2 loads primarily astrocytes in isolated intact optic nerves. We show that adenosine 5' triphosphate (ATP) evoked an increase in [Ca2+]i in a concentration-dependent manner with a half-maximal effect at 3 microm ATP, and with a rank order of agonist potency of ATP > ADP > alpha,beta-methyline-ATP > UDP > adenosine. The results indicate mainly P2Y and P2X components, consistent with the in vitro astroglial purinergic receptor profile. The in vivo response of mature glia to ATP may be important in their response to CNS damage.

Changes in P2Y and P2X purinoceptors in reactive glia following axonal degeneration in the rat optic nerve.

Purinoceptors have been shown to be important in mediating Ca(2+) signalling in glial cells and it has been proposed that they may have a role in their response to injury. To investigate this, the glial response to adenosine 5'triphosphate (ATP) was measured in situ, in optic nerves from juvenile rats that were enucleated at postnatal day (P) 1; age-matched normal nerves were used as controls. The optic nerve is a typical central nervous system (CNS) white matter tract containing axons and glial cells, but not neurones or synapses. Following neonatal enucleation, axons degenerate and oligodendrocytes do not develop, so that the optic nerve is populated predominantly by reactive astrocytes, with a minor population of activated microglia. Application of 1 mM ATP evoked a large and rapid increase in glial [Ca(2+)](i) in fura-2 ratiometric whole nerve recordings from normal and gliotic axon-free nerves. Significantly, the response to ATP had a prolonged duration in gliotic axon-free nerves and there was as shift in the agonist rank order of potency from ATP = ADP > UTP >> alpha,beta-metATP to ATP > ADP = UTP = alpha,beta-metATP. The results indicate an in situ role for ATP signalling in reactive astrocytes, via metabotropic P2Y(1) and P2Y(2/4) purinoceptors and ionotropic P2X purinoreceptors. The change in the purinoceptor profile following axon degeneration suggests a special role for P2X purinoceptors in mediating the glial reaction to CNS injury.