Astrocyte and oligodendrocyte connexins of the glial syncytium in relation to astrocyte anatomical domains and spatial buffering

Astroctyes express a set of three connexins (Cx26, Cx30, and Cx43) that are contained in astrocyte-to-astrocyte (A/A) gap junctions; oligodendrocytes express a different set of three connexins (Cx29, Cx32, and Cx47) that are contained in the oligodendrocyte side of necessarily heterotypic astrocyte-to-oligodendrocyte (A/O) gap junctions, and there is little ultrastructural evidence for gap junction formation between individual oligodendrocytes. In addition, primarily Cx29 and Cx32 are contained deeper in myelin sheaths, where they form autologous gap junctions at sites of uncompacted myelin. The presence of six connexins in macroglial cell populations has revealed unprecedented complexity of potential connexin coupling partners, and with restricted deployment of gap junctional intercellular communication (GJIC) within the "pan-glial" syncytium. New implications for the organization and regulation of spatial buffering mediated by glial GJIC are derived from recent observations of the existence of separate astrocyte anatomical domains, with only narrow regions of overlap between astrocyte processes at domain borders. Thus, widespread spatial buffering in the CNS may occur not successively through a multitude of processes arising from different astrocytes, but rather in a more orderly fashion from one astrocyte domain to another via intercellular coupling that occurs only at restricted regions of overlap between astrocyte domains, augmented by autocellular coupling that occurs within each domain.     

Substance P Receptors on O-2A Progenitor Cells and Type-2 Astrocytes In Vitro

Abstract: Bradykinin- and substance P (SP)-stimulated second messenger studies in isolated subsets of neuroglia showed bradykinin-stimulated synthesis of phospho- inositides (PI) in type-1 astrocytes and oligodendrocytes. SP-stimulated PI accumulation was restricted to oligoden- drocyte/type-2 astrocyte progenitor cells and type-2 astrocytes. These data were confirmed by analysis of calcium transients in single cells. In a regional study, SP-stimulated PI accumulation in primary astrocyte cultures was restricted to white matter. We conclude that regional heterogeneity in the expression of peptide receptors in cultures of primary astrocytes arises from a restricted distribution on subsets of macroglia. SP receptors restricted on cells of the oligodendrocyte/type-2 astrocyte type-2 lineage in vitro, coupled with in vivo observations by others, suggests that SP receptor expression is conserved on subsets of macroglia in vitro and possibly reactive astrocytes in vivo.     

Astrocyte and Oligodendrocyte Connexins of the Glial Syncytium in Relation to Astrocyte Anatomical Domains and Spatial Buffering

Astroctyes express a set of three connexins (Cx26, Cx30, and Cx43) that are contained in astrocyte-to-astrocyte (A/A) gap junctions; oligodendrocytes express a different set of three connexins (Cx29, Cx32, and Cx47) that are contained in the oligodendrocyte side of necessarily heterotypic astrocyte-to-oligodendrocyte (A/O) gap junctions, and there is little ultrastructural evidence for gap junction formation between individual oligodendrocytes. In addition, primarily Cx29 and Cx32 are contained deeper in myelin sheaths, where they form autologous gap junctions at sites of uncompacted myelin. The presence of six connexins in macroglial cell populations has revealed unprecedented complexity of potential connexin coupling partners, and with restricted deployment of gap junctional intercellular communication (GJIC) within the “pan-glial” syncytium. New implications for the organization and regulation of spatial buffering mediated by glial GJIC are derived from recent observations of the existence of separate astrocyte anatomical domains, with only narrow regions of overlap between astrocyte processes at domain borders. Thus, widespread spatial buffering in the CNS may occur not successively through a multitude of processes arising from different astrocytes, but rather in a more orderly fashion from one astrocyte domain to another via intercellular coupling that occurs only at restricted regions of overlap between astrocyte domains, augmented by autocellular coupling that occurs within each domain.     

Identification of cells expressing Cx43, Cx30, Cx26, Cx32 and Cx36 in gap junctions of rat brain and spinal cord

We have identified cells expressing Cx26, Cx30, Cx32, Cx36 and Cx43 in gap junctions of rat central nervous system (CNS) using confocal light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling (FRIL). Confocal microscopy was used to assess general distributions of connexins, whereas the 100-fold higher resolution of FRIL allowed co-localization of several different connexins within individual ultrastructurally-defined gap junction plaques in ultrastructurally and immunologically identified cell types. In >4000 labeled gap junctions found in >370 FRIL replicas of gray matter in adult rats, Cx26, Cx30 and Cx43 were found only in astrocyte gap junctions; Cx32 was only in oligodendrocytes, and Cx36 was only in neurons. Moreover, Cx26, Cx30 and Cx43 were co-localized in most astrocyte gap junctions. Oligodendrocytes shared intercellular gap junctions only with astrocytes, and these heterologous junctions had Cx32 on the oligodendrocyte side and Cx26, Cx30 and Cx43 on the astrocyte side. In 4 and 18 day postnatal rat spinal cord, neuronal gap junctions contained Cx36, whereas Cx26 was present in leptomenigeal gap junctions. Thus, in adult rat CNS, neurons and glia express different connexins, with "permissive" connexin pairing combinations apparently defining separate pathways for neuronal vs. glial gap junctional communication.     

Identification of Cells Expressing Cx43, Cx30, Cx26, Cx32 and Cx36 in Gap Junctions of Rat Brain and Spinal Cord

We have identified cells expressing Cx26, Cx30, Cx32, Cx36 and Cx43 in gap junctions of rat central nervous system (CNS) using confocal light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling (FRIL). Confocal microscopy was used to assess general distributions of connexins, whereas the 100-fold higher resolution of FRIL allowed co-localization of several different connexins within individual ultrastructurally-defined gap junction plaques in ultrastructurally and immunologically identified cell types. In >4000 labeled gap junctions found in >370 FRIL replicas of gray matter in adult rats, Cx26, Cx30 and Cx43 were found only in astrocyte gap junctions; Cx32 was only in oligodendrocytes, and Cx36 was only in neurons. Moreover, Cx26, Cx30 and Cx43 were co-localized in most astrocyte gap junctions. Oligodendrocytes shared intercellular gap junctions only with astrocytes, and these heterologous junctions had Cx32 on the oligodendrocyte side and Cx26, Cx30 and Cx43 on the astrocyte side. In 4 and 18 day postnatal rat spinal cord, neuronal gap junctions contained Cx36, whereas Cx26 was present in leptomenigeal gap junctions. Thus, in adult rat CNS, neurons and glia express different connexins, with “permissive” connexin pairing combinations apparently defining separate pathways for neuronal vs. glial gap junctional communication.     

Glutamate pretreatment affects Ca2+ signaling in processes of astrocyte pairs

Simultaneous somatic patch-pipette recording of a single astrocyte to evoke voltage-gated calcium currents, and Ca(2+) imaging, were used to study the spatial and temporal profiles of depolarization-induced changes in intracellular Ca(2+) ([Ca(2+)](i)) in the processes of cultured rat cortical astrocytes existing as pairs. Transient Ca(2+) changes locked to depolarization were observed as microdomains in the processes of the astrocyte pairs, and the responses were more pronounced in the adjoining astrocyte. Considering the functional significance of higher concentrations of glutamate observed in certain pathological conditions, Ca(2+) transients were recorded following pretreatment of cells with glutamate (500 microM for 20 min). This showed distance-dependent incremental scaling and attenuation in the presence of the metabotropic glutamate receptor (mGluR) antagonist, alpha-methyl(4-carboxy-phenyl) glycine (MCPG). Estimation of local Ca(2+) diffusion coefficients in the astrocytic processes indicated higher values in the adjoining astrocyte of the glutamate pretreated group. Intracellular heparin introduced into the depolarized astrocyte did not affect the Ca(2+) transients in the heparin-loaded astrocyte but attenuated the [Ca(2+)](i) responses in the adjoining astrocyte, suggesting that inositol 1,4,5 triphosphate (IP(3)) may be the transfer signal. The uncoupling agent, 1-octanol, attenuated the [Ca(2+)](i) responses in both the control and glutamate pretreated astrocytes, indicating the role of gap junctional communication. Our studies indicate that individual astrocytes have distinct functional domains, and that the glutamate-induced alterations in Ca(2+) signaling involve a sequence of intra- and intercellular steps in which phospholipase C (PLC), IP(3), internal Ca(2+) stores, VGCC and gap junction channels appear to play an important role.     

Macroglial cell development in embryonic rat brain: studies using monoclonal antibodies, fluorescence activated cell sorting, and cell culture

Astrocytes, ependymal cells, and oligodendrocytes have been shown to develop on the same schedule in dissociated cell cultures of early embryonic rat brain as in vivo. Subsequent studies showed that there are two major types of astrocyte (type-1 and type-2), which, in cultures of perinatal optic nerve, develop as two distinct lineages. In such cultures, type-2 astrocytes and oligodendrocytes develop from the same, bipotential, (O-2A) progenitor cells, which differentiate into type-2 astrocytes in 10% fetal calf serum (FCS) and into oligodendrocytes in less than or equal to 0.5% FCS. In light of these findings, we now have extended our studies on macroglial cell development in rat brain and show the following: (i) The first astrocytes to develop have a type-1 phenotype, while astrocytes with a type-2 phenotype do not develop until almost 2 weeks later, just as in the optic nerve. (ii) Most importantly, type-2 astrocytes, like the other macroglial cells, develop on the same schedule in cultures of early embryonic (less than or equal to E15) brain as they do in vivo. (iii) By contrast, both oligodendrocytes and type-2 astrocytes develop prematurely in cultures of E17 brain, and FCS influences this development in the same way it does in perinatal optic nerve cultures. (iv) Type-2 astrocyte precursors are labeled by the A2B5 monoclonal antibody, as shown previously for oligodendrocyte precursors in brain and for O-2A progenitor cells in optic nerve. Taken together with our previous findings, these results suggest that oligodendrocytes and type-2 astrocytes in brain develop from bipotential O-2A progenitor cells, whose choice of developmental pathway and timing of differentiation depend on mechanisms that operate independently of brain morphogenesis.     

Selective stimulation of astrocyte calcium in situ does not affect neuronal excitatory synaptic activity

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

A quantitative model of purinergic junctional transmission of calcium waves in astrocyte networks

A principal means of transmitting intracellular calcium (Ca2+) waves at junctions between astrocytes involves the release of the chemical transmitter adenosine triphosphate (ATP). A model of this process is presented in which activation of purinergic P2Y receptors by ATP triggers the release of ATP, in an autocrine manner, as well as concomitantly increasing intracellular Ca2+. The dependence of the temporal characteristics of the Ca2+ wave are shown to critically depend on the dissociation constant (K(R)) for ATP binding to the P2Y receptor type. Incorporating this model astrocyte into networks of these cells successfully accounts for many of the properties of propagating Ca2+ waves, such as the dependence of velocity on the type of P2Y receptor and the time-lag of the Ca2+ wave behind the ATP wave. In addition, the conditions under which Ca2+ waves may jump from one set of astrocytes across an astrocyte-free lane to another set of astrocytes are quantitatively accounted for by the model. The properties of purinergic transmission at astrocyte junctions may determine many of the characteristics of Ca2+ propagation in networks of these cells.     

Immunocytochemical and RT-PCR analysis of connexin36 in cultures of mammalian glial cells

The expression of connexin36 (Cx36) was studied in primary cultures of rat brain glial cells: mature astrocytes, ameboid and ramified microglia and immature oligodendrocytes (at middle period of myelinogenesis). The data from these cells were compared with those obtained from cultures of neocortical and hypothalamic neurons. mRNA encoding Cx36 was investigated by RT-PCR, the Cx36 protein by immunocytochemistry using a polyclonal antibody against Cx36 in cells characterized by antibodies specific for the single cell types. The Cx36 was found in oligodendrocytes, both ameboid and ramified microglial cells and in neurons. Astrocytes showed no detectable expression of the Cx36. The expression of Cx36 in oligodendrocytes and microglial cells suggests an involvement of the direct cell-cell communication channels formed by Cx36 in myelin formation and in brain development, damage and repair processes.     

Purified astrocytes promote the in vitro division of a bipotential glial progenitor cell.

Optic nerves of neonatal rats contain a bipotential glial progenitor cell which can be induced by tissue culture conditions to differentiate into either an oligodendrocyte (the myelin-forming cell of the CNS) or a type 2 astrocyte (an astrocyte population found only in the myelinated tracts of the CNS). In our previous studies most oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells differentiated within 3 days in vitro with relatively little division of the progenitors or their differentiated progeny. We have now found that the O-2A progenitors are stimulated to divide in culture by purified populations of type 1 astrocytes, another glial cell-type found in the rat optic nerve. This cell-cell interaction appears to be mediated by a soluble factor(s) and results in the production of large numbers of both progenitor cells and oligodendrocytes. As type 1 astrocytes are the major glial cell-type in the optic nerve when oligodendrocytes first begin to be produced in large numbers in vivo, our results suggest that this astrocyte subpopulation may play an important role in expanding the oligodendrocyte population during normal development.     

The potential to induce glial differentiation is conserved between Drosophila and mammalian glial cells missing genes

Drosophila glial cells missing (gcm) is a key gene that determines the fate of stem cells within the nervous system. Two mouse gcm homologs have been identified, but their function in the nervous system remains to be elucidated. To investigate their function, we constructed retroviral vectors harboring Drosophila gcm and two mouse Gcm genes. Expression of these genes appeared to influence fibroblast features. In particular, mouse Gcm1 induced the expression of astrocyte-specific Ca(2+)-binding protein, S100beta, in those cells. Introduction of the mouse Gcm1 gene in cultured cells from embryonic brains resulted in the induction of an astrocyte lineage. This effect was also observed by in utero injection of retrovirus harboring mouse Gcm1 into the embryonic brain. However, cultures from mouse Gcm1-deficient mouse brains did not exhibit significant reductions in the number of astrocytes. Furthermore, in situ hybridization analysis of mouse Gcm1 mRNA revealed distinct patterns of expression in comparison with other well-known glial markers. The mammalian homolog of Drosophila gcm, mouse Gcm1, exhibits the potential to induce gliogenesis, but may function in the generation of a minor subpopulation of glial cells.     

Role of capacitative calcium entry on glutamate-induced calcium influx in type-I rat cortical astrocytes

Capacitative calcium entry (CCE) has been described in a variety of cell types. To date, little is known about its role in the CNS, and in particular in the cross-talk between glia and neurons. We have first analyzed the properties of CCE of astrocytes in culture, in comparison with that of the rat basophilic leukemia cell line (RBL-2H3), a model where calcium release-activated Ca2+ (CRAC) channels have been unambiguously correlated with CCE. We here show that (i) in astrocytes CCE activated by store depletion and Ca2+ influx induced by glutamate share the same pharmacological profile of CCE in RBL-2H3 cells and (ii) glutamate-induced Ca2+ influx in astrocytes plays a primary role in glutamate-dependent intracellular Ca2+ concentration ([Ca2+]i) oscillations, being these latter reduced in frequency and amplitude by micromolar concentrations of La3+. Finally, we compared the expression of various mammalian transient receptor potential genes (TRP) in astrocytes and RBL-2H3 cells. Despite the similar pharmacological properties of CCE in these cells, the pattern of TRP expression is very different. The involvement of CCE and TRPs in glutamate dependent activation of astrocytes is discussed.     

Type-2 astrocyte development in rat brain cultures is initiated by a CNTF-like protein produced by type-1 astrocytes

O-2A progenitor cells are bipotential glial precursors that give rise to both oligodendrocytes and type-2 astrocytes on a precise schedule in the rat CNS. Studies in culture suggest that oligodendrocyte differentiation occurs constitutively, while type-2 astrocyte differentiation requires an exogenous inducer such as fetal calf serum. Here we describe a rat brain cell culture system in which type-2 astrocytes develop on schedule in the absence of exogenous inducers. Coincident with type-2-astrocyte development, the cultures produce an approximately 20 kd type-2-astrocyte-inducing factor(s). Purified cultures of type-1 astrocytes can produce a similar factor(s). Under conditions where they produce type-2-astrocyte-inducing factor(s), both brain and type-1 astrocyte cultures produce a factor(s) with ciliary neurotrophic (CNTF)-like activity. Purified CNTF, like the inducers from brain and type-1 astrocyte cultures, prematurely induces type-2 astrocyte differentiation in brain cultures. These findings suggest that type-2 astrocyte development is initiated by a CNTF-like protein produced by type-1 astrocytes.     

Deregulation of dorsoventral patterning by FGF confers trilineage differentiation capacity on CNS stem cells in vitro

The CNS is thought to develop from self-renewing stem cells that generate neurons, astrocytes, and oligodendrocytes. Other data, however, have suggested that astrocytes and oligodendrocytes are generated from separate progenitor populations. To reconcile these observations, we have prospectively isolated progenitors that do or do not express Olig2, an oligodendrocyte bHLH determination factor. Both Olig2(-) and Olig2(+) progenitors can behave as tripotential CNS stem cells (CNS-SCs) in vitro. Growth in FGF-2 causes induction of Olig2 in the former population, permitting oligodendrocyte differentiation; extinction of Olig2 in the latter cells permits astrocyte differentiation. The induction of Olig2 by FGF-2 is mediated, in part, via endogenous Sonic Hedgehog. These data indicate that clonogenic competence to generate neurons, astrocytes, and oligodendrocytes reflects a deregulation of dorsoventral patterning during expansion in vitro, raising the question of whether such trifatent cells actually exist in vivo.