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Integration of NG2-glia (synantocytes) into the neuroglial network rebekah wigley and arthur m. butt NG2-glia are a distinct class of CNS glial cells that are generally classed as oligodendrocyte progenitor cells. However, in the adult CNS a large fraction of NG2 cells does not appear to divide or generate oligodendrocytes. The functions of these adult NG2-glia, which we have termed synantocytes, are unknown. NG2-glia (synantocytes) form interactive domains with astro- cytes and neurons. Within their domains, NG2-glia and astrocytes contact the same neurons, form multiple heterologous con- tacts with each other, and contact pericytes which regulate cerebral blood flow. NG2-glia receive presynaptic input from neurons and respond to neurotransmitters released at synapses. In addition, NG2-glia are intimately associated with astroglia and respond to astroglial signals, a hitherto neglected aspect of NG2-glial cell physiology. The non-overlapping domain organ- isation of astrocytes is believed to be important in isolating and integrating activity at the synapses and blood vessels within their domains. The domains of NG2-glia overlap with astrocytes, suggesting they could play a role in integrating non- overlapping astrocyte domains. Keywords: NG2, astrocyte, synantocyte, neuron, synapse, pericyte, cerebral blood vessel INTRODUCTION NG2-glia are a distinct class of CNS glial cells that are identified by their specific expression of the NG2 chondroitin sulphate proteoglycan (CSPG) and alpha receptors for platelet-derived growth factor (PDGFaR). NG2-glia are phenotypically distinct from astrocytes and oligodendrocytes (Butt et al., 2002), and generally classed as oligodendrocyte progenitor cells (OPCs) (Nishiyama et al., 1996). It has been shown by genetic fate mapping in transgenic mice that NG2/PDGFaR-expressing cells generate oligodendrocytes during development (Zhu et al., 2008) and in the adult (Rivers et al., 2008). In addition, the same authors showed that in the forebrain at least, NG2/ PDGFaR-expressing cells generate astrocytes during develop- ment (Zhu et al., 2008), but not apparently in the adult (Rivers et al., 2008), and that NG2-glia generate a restricted population of neurons throughout life (Rivers et al., 2008). However, there are regional differences indicated in the literature. The astrogenic potential of NG2-glia is indicated in the pons, where NG2-glia express the astroglial reporter gene enhanced green fluorescent protein (EGFP) driven by glial fibrillary acidic protein (GFAP) in transgenic mice (Grass et al., 2004). We have confirmed this is the case in the pons and provide evidence that NG2-glia ‘turn on’ the GFAP reporter in cerebellar slices ex vivo (Leoni et al., 2009). The neurogenic potential of NG2-glia has been indi- cated in the hippocampus (Belachew et al., 2003; Aguirre et al., 2004), olfactory bulb (Aguirre et al., 2004) and cortex (Dayer et al., 2005). Nonetheless, NG2-glia are equally numerous outside areas of neurogenesis and in areas where oligodendro- cytes are not found, such as the molecular layer of the cerebellum (Butt et al., 2005). Moreover, NG2-glia are highly populous throughout the adult CNS, making up 5–7% of cells, and 50% of these cells and do not appear to be normally involved in generating oligodendrocytes, astrocytes or neurons (Rivers et al., 2008; Psachoulia et al., 2009). It is ambiguous to consider NG2-glia as OPCs, because this implies the fate of all NG2-glia ultimately is to turn into oligodendrocytes. The functions of non- proliferating NG2-glia in the adult CNS are unknown and we have asked the question whether they represent a new class of glia, which we called synantocytes (Butt et al., 2002, 2005). These novel glial cells form exquisite associations with neurons at synapses (Bergles et al., 2000; Lin and Bergles, 2004; Lin et al., 2005; Ge et al., 2009), with unmyelinated axons at ‘synapse- like’ sites (Ziskin et al., 2007), and with myelinated axons at nodes of Ranvier (Butt et al., 1999, 2005; Hamilton et al., 2009). In addition, NG2-glia express a range of ion channels and neurotransmitter receptors, which enable them to respond to neuronal and glial signals (Bergles et al., 2000; Lin and Bergles, 2004; Lin et al., 2005; Karadottir et al., 2005, 2008; Ge et al., 2006, 2009; Hamilton et al., 2009; Bakiri et al., 2009). In this review, we examine how NG2-glia are integrated with neurons, astrocytes and the vasculature of the CNS. NG2-GLIA AND ASTROCYTES ARE SEPARATE POPULATIONS WITH OVERLAPPING DOMAINS We have examined NG2-glia and astrocytes in the brain using immunohistochemistry in transgenic mice in which the expression of EGFP is driven by the human promoter for GFAP (Nolte et al., 2001). We have previously reported that outside the pons we do not normally find co-expression of NG2 immunolabelling and the GFAP-EGFP reporter (Wigley et al., 2007; Leoni et al., 2009). EGFP identifies the true morphological complexity of astrocytes, but it is impor- tant to recognise that not all astrocytes express the reporter (Nolte et al., 2001; Wigley et al., 2007). NG2-glia and astro- cytes have a similar distribution throughout grey matter, but differ in their antigenic and morphological phenotype Corresponding author: Arthur M. Butt Email: [email protected] 21 Neuron Glia Biology, 2009, 5(1/2), 21–28. # Cambridge University Press, 2009 doi:10.1017/S1740925X09990329 https://doi.org/10.1017/S1740925X09990329 Downloaded from https:/www.cambridge.org/core. Open University Library, on 04 Feb 2017 at 14:42:54, subject to the Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms.

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Integration of NG2-glia (synantocytes) intothe neuroglial network

rebekah wigley and arthur m butt

NG2-glia are a distinct class of CNS glial cells that are generally classed as oligodendrocyte progenitor cells However in theadult CNS a large fraction of NG2 cells does not appear to divide or generate oligodendrocytes The functions of these adultNG2-glia which we have termed synantocytes are unknown NG2-glia (synantocytes) form interactive domains with astro-cytes and neurons Within their domains NG2-glia and astrocytes contact the same neurons form multiple heterologous con-tacts with each other and contact pericytes which regulate cerebral blood flow NG2-glia receive presynaptic input fromneurons and respond to neurotransmitters released at synapses In addition NG2-glia are intimately associated with astrogliaand respond to astroglial signals a hitherto neglected aspect of NG2-glial cell physiology The non-overlapping domain organ-isation of astrocytes is believed to be important in isolating and integrating activity at the synapses and blood vessels withintheir domains The domains of NG2-glia overlap with astrocytes suggesting they could play a role in integrating non-overlapping astrocyte domains

Keywords NG2 astrocyte synantocyte neuron synapse pericyte cerebral blood vessel

I N T R O D U C T I O N

NG2-glia are a distinct class of CNS glial cells that are identifiedby their specific expression of the NG2 chondroitin sulphateproteoglycan (CSPG) and alpha receptors for platelet-derivedgrowth factor (PDGFaR) NG2-glia are phenotypically distinctfrom astrocytes and oligodendrocytes (Butt et al 2002) andgenerally classed as oligodendrocyte progenitor cells (OPCs)(Nishiyama et al 1996) It has been shown by genetic fatemapping in transgenic mice that NG2PDGFaR-expressingcells generate oligodendrocytes during development (Zhuet al 2008) and in the adult (Rivers et al 2008) In additionthe same authors showed that in the forebrain at least NG2PDGFaR-expressing cells generate astrocytes during develop-ment (Zhu et al 2008) but not apparently in the adult (Riverset al 2008) and that NG2-glia generate a restricted populationof neurons throughout life (Rivers et al 2008) However thereare regional differences indicated in the literature The astrogenicpotential of NG2-glia is indicated in the pons where NG2-gliaexpress the astroglial reporter gene enhanced green fluorescentprotein (EGFP) driven by glial fibrillary acidic protein (GFAP)in transgenic mice (Grass et al 2004) We have confirmed thisis the case in the pons and provide evidence that NG2-glialsquoturn onrsquo the GFAP reporter in cerebellar slices ex vivo (Leoniet al 2009) The neurogenic potential of NG2-glia has been indi-cated in the hippocampus (Belachew et al 2003 Aguirre et al2004) olfactory bulb (Aguirre et al 2004) and cortex (Dayeret al 2005) Nonetheless NG2-glia are equally numerousoutside areas of neurogenesis and in areas where oligodendro-cytes are not found such as the molecular layer of the cerebellum(Butt et al 2005) Moreover NG2-glia are highly populousthroughout the adult CNS making up 5ndash7 of cells and 50of these cells and do not appear to be normally involved in

generating oligodendrocytes astrocytes or neurons (Riverset al 2008 Psachoulia et al 2009) It is ambiguous to considerNG2-glia as OPCs because this implies the fate of all NG2-gliaultimately is to turn into oligodendrocytes The functions of non-proliferating NG2-glia in the adult CNS are unknown and wehave asked the question whether they represent a new class ofglia which we called synantocytes (Butt et al 2002 2005)These novel glial cells form exquisite associations with neuronsat synapses (Bergles et al 2000 Lin and Bergles 2004 Linet al 2005 Ge et al 2009) with unmyelinated axons at lsquosynapse-likersquo sites (Ziskin et al 2007) and with myelinated axons atnodes of Ranvier (Butt et al 1999 2005 Hamilton et al2009) In addition NG2-glia express a range of ion channelsand neurotransmitter receptors which enable them to respondto neuronal and glial signals (Bergles et al 2000 Lin andBergles 2004 Lin et al 2005 Karadottir et al 2005 2008 Geet al 2006 2009 Hamilton et al 2009 Bakiri et al 2009) Inthis review we examine how NG2-glia are integrated withneurons astrocytes and the vasculature of the CNS

N G 2 - G L I A A N D A S T R O C Y T E S A R ES E P A R A T E P O P U L A T I O N S W I T HO V E R L A P P I N G D O M A I N S

We have examined NG2-glia and astrocytes in the brain usingimmunohistochemistry in transgenic mice in which theexpression of EGFP is driven by the human promoter forGFAP (Nolte et al 2001) We have previously reported thatoutside the pons we do not normally find co-expression ofNG2 immunolabelling and the GFAP-EGFP reporter(Wigley et al 2007 Leoni et al 2009) EGFP identifies thetrue morphological complexity of astrocytes but it is impor-tant to recognise that not all astrocytes express the reporter(Nolte et al 2001 Wigley et al 2007) NG2-glia and astro-cytes have a similar distribution throughout grey matter butdiffer in their antigenic and morphological phenotype

Corresponding authorArthur M ButtEmail Arthurbuttportacuk

21

Neuron Glia Biology 2009 5(12) 21ndash28 Cambridge University Press 2009doi101017S1740925X09990329

httpsdoiorg101017S1740925X09990329Downloaded from httpswwwcambridgeorgcore Open University Library on 04 Feb 2017 at 144254 subject to the Cambridge Core terms of use available at httpswwwcambridgeorgcoreterms

(Fig 1) NG2-glia have a characteristic stellate morphologywith a central cell body and fine processes that extend out-wards radially to distances of up to 50 mm as illustrated inthe hippocampus (Fig 1AndashE) and the cerebellar granule celllayer (Fig 1F) and molecular layer (Fig 1G) Astrocyteshave five or more thick primary processes from which

emanate a multitude of fine processes giving the cell alsquofurryrsquo appearance (Fig 1AndashF) NG2-glia and astrocytesdefine overlapping three-dimensional domains in which thedomains of the same cell type overlap only slightly at theedges but those of different cell types overlap considerably(Fig 1AndashF) NG2-glial domains are significantly larger than

Fig 1 Domain structure of NG2-glia and astroglia Immunolabelling for NG2 (red) in brain sections from GFAP-EGFP mice Confocal images are shown astwo-dimensional snapshots (ABDFG) single z-section (C) and with isosurface rendering (B inset EG inset) NG2-glia and astrocytes are arranged inoverlapping radial domains mice in the molecular layer of the hippocampus (ACD) the dentate gyrus (B) the granule cell layer of the cerebellum (F) andthe molecular layer of the cerebellum (G) NG2-glial cell and astrocyte processes ensheath each other at numerous points other along their length (B inset)(AndashE) Domains of individual astrocytes interact with those of three or more NG2-glia (C) whereas individual NG2-glia interact with six or more astroglia(FG) (DE) NG2-glial cell and astrocyte somata are often directly apposed and have completely overlapping domains with processes that interdigitateextensively NG2-glia and astrocytes extend end feet to blood vessels but those of astrocytes expand to form perivascular end feet (DE arrows) (FG)NG2-glia traverse the different layers of the cerebellum to associate with multiple velate astrocytes and Bergmann glia the process of NG2-glia and primaryprocess of Bergmann glia are extensively apposed along their length (G inset) Scale bars frac14 20 mm in (AFG) and 10 mm in (BCDE) and 15 mm (G inset)

22 rebekah wigley and arthur m butt

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those of astrocytes (P 005 unpaired t-test) with an averagediameter in the hippocampus of 99 + 1 mm (n frac14 17) inNG2-glia and 78 + 2 mm (n frac14 13) in astrocytes Thedomains of individual astrocytes overlap with those of threeor more NG2-glia (Fig 1AndashC) whereas those of NG2-gliaoverlap with the domains of more than six astrocytes(Fig 1F) bearing in mind that this number is likely to begreater because not all astrocytes express GFAP or the repor-ter gene Within overlapping domains the processes ofNG2-glia and astrocytes interdigitate and display bipartiteensheathment whereby short segments of NG2-glial andastroglial processes ensheath each other (Fig 1B inset) Thesomata of some NG2-glia and astrocytes are directlyapposed to one another with the appearance of sister cells(Fig 1D) In these cases the domains of the NG2-glial cellsand astrocyte are almost identical and there is a largesurface area of contact between the somata and their pro-cesses which interdigitate extensively within the overlappingprocess fields of the two adjoining cells (Fig 1E) BothNG2-glia and astrocytes extend processes to blood vesselsbut those of astrocytes expand to form perivascular end-feet(Fig 1DE arrows) In the cerebellum astrocytes display func-tional and morphological specialisation with velate astrocytes

in the granule cell layer (Fig 1F) and Bergmann glia with cellsomata in the Purkinje cell layer and primary processesextending through the molecular layer to the pia (Fig 1G)In contrast NG2-glia do not display morphological hetero-geneity and individual cells extend processes to traverse thedifferent layers to form overlapping domains with velateastrocytes (Fig 1F) and Bergmann glia (Fig 1G) NG2-glialcell processes are directly apposed to the primary radial pro-cesses of Bergmann glia and they ensheath each other regu-larly along their lengths (Fig 1G inset) The domainorganisation of astrocytes is considered to be important forintegrated activity at the synapses in their domain (Bushonget al 2002 Nedergaard et al 2003 Oberheim et al 2006)and presumably this is also true for NG2-glia

I N T E G R A T I O N O F N G 2 - G L I A I N T OT H E N E U R O G L I A L N E T W O R K

We have examined the interrelationships between NG2-gliaastrocytes and neurons in the cortex and cerebellum Doubleimmunolabelling for NG2 and calretinin or calbindin forneurons in the cortex of GFAP-EGFP mice illustrates the

Fig 2 NG2-glia and astrocytes contact the same neurons in the cortex Immunolabelling for NG2 (red) and calretinin (A blue) or calbindin (B blue) in 100 mmmid-sagital brain sections from the cortex of GFAP-EGFP mice Confocal images are shown in three channels (AiBi) and two channels (AiiAiiiBiiBiii) and withisosurface rendering (AivBiv) (A) NG2-glia and astrocytes are associated with 10 or more neurons within their overlapping domains (B) NG2-glia and astroglialprocesses almost circumnavigate the same neuronal somata Scale bars frac14 10 mm in (AindashAiii) and (BindashBiii) and 5 mm in (Aiv) and (Biv)

integration of ng2-glia (synantocytes) into the neuroglial network 23

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Fig 3 Interactions of NG2-glia with astrocytes and neurons in the granule cell layer of the cerebellum Immunolabelling for NG2 (red) and calretinin (blue) inthe cerebellum of GFAP-EGFP mice Confocal images are shown in three channels (Ai) and two channels (AiiAiii) and with isosurface rendering (BC) Astrocytesand NG2-glia contact 20 or more neurons within their overlapping domains Velate astrocyte processes encompass neuronal somata (Aii) whereas NG2-glial cellprocesses from en passant associations (Aiii) The processes of Ng2-glia and astrocytes are directly apposed at the same contact sites with neuronal somatasuggesting they subserve the same synapses (BC) Scale bars frac14 10 mm in (A) 5 mm in (B) and 2 mm in (C)

Fig 4 Integration of NG2-glia with Purkinje neurons and Bergmann glia in the molecular layer of the cerebellum Immunolabelling for NG2 (red) andcalbindin (blue) in the cerebellum of P15 GFAP-EGFP mice Confocal images are shown in three channels (AiBi) and two channels (AiiAiii) and withisosurface rendering (BC) NG2-glial cell somata lie along the primary processes of Bergmann glia and the Purkinje neuron dendrites and extend processesparallel and perpendicular to the primary processes of Bergmann glia The processes of NG2-glia and Bergmann glia are intimately associated with Purkinjeneuron dendrites (CD) Scale bars frac14 20 mm in (A) and 2 mm in (BC)

24 rebekah wigley and arthur m butt

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integration of the three elements (Fig 2) The processes ofNG2-glia like those of astrocytes are directly apposed tomost if not all neuronal somata within their domains(Fig 2A) It is important to note that our results demonstratethe close associations that NG2-glia form with other elementsin the CNS but it is not possible at the confocal microscopiclevel to distinguish whether these contacts are actually func-tional Nonetheless using a combination of electronmicroscopy and patch-clamp electrophysiology it has beenestimated that only a few of the neuronal contacts formed byNG2-glia are electrophysiologically active lsquosynapsesrsquo (Linet al 2005 Ge et al 2009) In contrast astrocytes form

many hundreds of active contacts with neuronal synapses(Araque et al 1999 Grosche et al 1999 Bushong et al2002) NG2-glia and astrocytes form intimate contacts withthe same neurons extending processes that almost completelycircumnavigate neuronal somata (Fig 2B) and perisynapticprocesses from both cells types form a latticework (reticulation)on the cell body (Fig 2Biv) The close apposition of NG2-gliaand astrocytes to the same neurons suggests they may contactthe same synapses but they subserve neurons in differentways Electron microscopy studies demonstrate the differentassociations made by NG2-glia and astrocytes with neuronsNG2-glia form specific points of synaptic like contacts withneurons (Bergles et al 2000) whereas astrocytes ensheathpre- and post-synaptic boutons (Nedergaard et al 2003)This close relationship between NG2-glia astrocytes andneurons is also reflected in the cerebellum (Fig 3) In thegranule cell layer individual neurons are contacted by astro-cytes and NG2-glial cell processes (Fig 3A) However whilstvelate astrocyte processes circumnavigate neuronal cellsomata the processes of NG2-glia extend radially to form enpassant associations with both neurons and astrocytes as theyweave around the somata (Fig 3BC) as noted above it is notcurrently known whether these contacts have any function incommunication In the molecular layer NG2-glia are closelyintegrated with Bergmann glia and Purkinje cells (Fig 4)NG2-glial cell somata often lie along the primary process ofBergmann glial cells and Purkinje cell primary dendrites(Fig 4A) NG2-glia extend processes both parallel to and per-pendicular to Purkinje cell dendritic trees (Fig 4A) and theirprocesses interdigitate with those of Bergmann glia andPurkinje cell dendrites (Fig 4BC) A single NG2-glial cell sub-serves multiple Bergmann glia and Purkinje cells and manyNG2-glia extend processes into both the molecular andgranule cell layers to form additional contacts with velate astro-cytes and granule neurons NG2-glia contact synapses betweenclimbing fibres and Purkinje neurons but they appear to formfunctional contacts with only a small number of synapseswithin their domain (Lin et al 2005) whereas Bergmann gliaform many hundreds of microdomains that interact with thesynapses they ensheath (Grosche et al 1999 2002) Similarlya single hippocampal astrocyte can contact in excess of100000 synapses (Bushong et al 2002) and cortical astrocyteswrap on average four neuronal cell bodies and hundreds of den-drites (Halassa et al 2007b) In contrast NG2-glia do not havethe profuse fine processes of protoplasmic astrocytes and onaverage may form electrophysiologically active contacts withless than 20 synapses (Bergles et al 2000 Lin and Bergles2004 Ge et al 2009) However the intimate associationsbetween NG2-glia and astrocytes independent of neuronsboth on their somata and along their processes are sites of neu-rotransmitter mediated glia-to-glia calcium signalling(Hamilton et al 2009) The overlapping domain organisationof NG2-glia and astrocytes may therefore be important for inte-grating glial and neuronal activity

C O M M U N I C A T I O N B E T W E E NN G 2 - G L I A A N D T H E N E U R O G L I A LN E T W O R K

Electrophysiological studies show that NG2-glia express gluta-mate and GABA receptors and respond to neuronally releasedneurotransmitters (Bergles et al 2000 Lin and Bergles 2004

Fig 5 Synaptophysin is localised to sites of close apposition betweenNG2-glia and astrocytes Immunolabelling for NG2 (red) andsynaptophysin (blue) in the hippocampus of GFAP-EGFP mice Confocalimages are shown in three channels (A) and with isosurface rendering (BC)The processes of NG2-glia and astrocytes are associated with a large densityof synapses (AB) There is immediate apposition of the NG2-glial cell andastrocyte cell bodies and processes and synaptophysin is localised to thesesites of contact (arrows) indicating sites of synaptic communication ontoboth NG2-glia and astrocytes In addition synaptophysin was localisedwithin the soma of the NG2-glia cell confirming our recent report(Hamilton et al 2009) and suggesting NG2-glia are capable of bidirectionalcommunication with astrocytes and neurons Scale bar frac14 20 mm (A) 5 mm(B) and 1 mm (C)

integration of ng2-glia (synantocytes) into the neuroglial network 25

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Lin et al 2005) Calcium imaging and immunocytochemistryshow that NG2-glia express functional AMPA-type glutamatereceptors and metabotropic P2Y and ionotropic P2X recep-tors including the P2X7 subtype (Ge et al 2006 Hamiltonet al 2009 Wang et al 2009) Cerebellar NG2-glia candisplay both GABAergic and glutamatergic inputs (Lin andBergles 2004 Karadottir et al 2005) consistent with ourobservations that individual NG2-glia traverse the cell layersof the cerebellum to contact glutamatergic and GABAergicelements In addition we have used immunolabelling forsynaptophysin to examine the relations of NG2-glia withsynapses (Fig 5) The density of synaptophysin immunolabel-ling in the grey matter precludes the quantification of specificsynapses contacted by NG2-glia (Fig 5A) Nevertheless ourresults provide evidence that NG2-glia and astrocytescontact the same neurons (Figs 3ndash5) and the immediate

apposition of synaptophysin to points of contact betweenNG2-glia and astrocytes suggests they may be integrated atthe same synapses (Fig 5) Moreover synaptophysin is local-ised to sites of immediate apposition between astrocytesand NG2-glia and a region of particular interest is theexpression of synaptophysin between the NG2-glial cellbody and an astrocyte process (Fig 5B) Isoforming at theintegral communication site between the NG2-glial cell andastrocyte process provides further evidence of the localisationof synaptophysin to the NG2-glial cell body and to the inter-face between NG2-glia and astrocytes (Fig 5C) The resultspresented above show that the cell somata of astrocytes andNG2-glia are often directly apposed and that their processesexhibit regions where they tightly ensheath each other (Figs1B inset 1G inset 3BC and 4BC) The localisation of synap-tophysin to these areas indicates these are potential sits of

Fig 6 NG2-glia are in direct contact with pericytes NG2-glia are marked by an asterisk in each image Images show immunolabelling for NG2 in optic nerveof non-transgenic mice (AB) and NG2-glia and pericytes in sections of NG2-dsRed mouse brain (CndashE) NG2-glia contact multiple pericytes and in many casestheir cell somata appear to be in direct contact (CD) and this is confirmed in cross sectional images (E) Scale frac14 10 mm (A) 12 mm (B) 5 mm (C) 8 mm (D) and10 mm (E)

26 rebekah wigley and arthur m butt

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vesicular communication between the two cell typesAstrocytes have mechanisms to release neurotransmitters viavesicles (Parpura et al 1994 Araque et al 1999) We haveprovided evidence that astrocytes also signal onto NG2-gliavia glutamate and ATP (Hamilton et al 2008 2009)Moreover the evidence that NG2-glia express synaptophysinindicates they may be capable of neurotransmitter release andbidirectional communication with astrocytes and neurons

R E L A T I O N S B E T W E E N N G 2 - G L I AA N D T H E C E R E B R A LV A S C U L A T U R E

Astrocytes are able to lsquosensersquo changes in neuronal activity andtransmit signals to the vasculature to regulate local blood flowand provide metabolic support to neurons (Halassa et al2007a) Astrocytes may use glutamate signalling mechanismsto communicate with pericytes on blood vessels that are ableto regulate cerebral blood flow (Peppiatt et al 2006)NG2-glia also contact pericytes on cerebral blood vessels(Fig 6) and they contact astrocytes and neurons withintheir domains (Figs 1ndash5) and express synaptophysin(Hamilton et al 2009) and may therefore play a role in reg-ulating blood flow NG2-glia and pericytes both express theNG2 CSPG (Ozerdem et al 2002) and the two cell typesare distinguished by their morphology NG2-glia as stellateprocess-bearing cells and pericytes as perivascular cells withtwo or more primary processes extending along bloodvessels According to these distinguishing features it can beseen that NG2-glia and pericytes lie in very close proximityto each other (Fig 6AB) The processes of NG2-glia contactpericyte processes on the blood vessel (Fig 6A) In somecases it appears that the pericyte cell soma extends outfrom the capillary to almost contact that of a neighbouringNG2-glial cell (Fig 6B) and in many cases NG2-glial cellsomata are directly apposed to blood vessels identified byimmunolabelling (Fig 6C) and in the NG2-DsRed transgenicmouse (Fig 6DE) Pericytes have a neurogenic potential invitro (Dore-Duffy et al 2006) and proliferate in response toinjury (Gerhardt and Betsholtz 2003) and the intimateassociations between NG2-glia and pericytes is suggestive ofa lineage relationship between the two cell types (Wigleyand Butt unpublished observations) It remains to be seenwhether NG2-glia communicate with pericytes and help regu-late blood flow but this is a possibility since they expresssynaptophysin and contact synapses astrocytes and pericytes

C O N C L U S I O N S

NG2-glia and astrocytes form overlapping neuronndashglialndashvascular domains Within these domains NG2-glia and astro-cytes contact the same neurons and most likely the samesynapses but they perform different functions Astrocyte pro-cesses envelop synapses and one of their main functions is theremoval of excess neurotransmitters NG2-glia form fine con-nections with synapses and do not express transporters andtheir function at synapses is unknown The large domains ofNG2-glia enable them to lsquosensersquo neuronal activity frommany neurons over a large area that overlaps with thedomains of multiple astrocytes Their relation with pericytesindicates that NG2-glia and astrocytes form parallel

communications pathways between neurons and bloodvessels which may be important in the regulation of bloodflow The evidence that NG2-glia also receive input fromastrocytes and the possibility that NG2-glia express mechan-isms for vesicular release of gliotransmitters raises the possi-bility that NG2-glia may also have an output and be able tofeed back to neurons or astrocytes The fact that NG2-gliaand astrocytes form parallel communications pathwayssuggests they have different functions since otherwise thislevel of redundancy is difficult to explain Astrocyte domainorganisation segregates intercellular communication withinneuronal circuits (Houades et al 2008) and astrocytes arecoupled by gap junctions that provide an activity-dependentintercellular pathway for transport between blood vesselsand neurons (Rouach et al 2008) NG2-glia are not coupledby gap junctions but by forming lsquosynapsesrsquo with neuronsastrocytes and pericytes they connect otherwise segregatedastroglial domains An important question for future studiesis to determine how these multiple inputs are integrated inindividual NG2-glia and whether they have a physiologicaloutput

A C K N O W L E D G E M E N T S

Supported by the BBSRC and INTERRIG

Statement of interestNone

R E F E R E N C E S

Aguirre AA Chittajallu R Belachew S and Gallo V (2004)NG2-expressing cells in the subventricular zone are type C-like cellsand contribute to interneuron generation in the postnatal hippo-campus Journal of Cell Biology 165 575ndash589

Araque A Parpura V Sanzgiri RP and Haydon PG (1999) Tripartitesynapses glia the unacknowledged partner Trends in Neuroscience 22208ndash215

Bakiri Y Attwell D and Karadottir R (2009) Electrical signalling prop-erties of oligodendrocyte precursor cells Neuron Glia Biology101017S1740925X09990202

Belachew S Chittajallu R Aguirre AA Yuan X Kirby M AndersonS et al (2003) Postnatal NG2 proteoglycan-expressing progenitorcells are intrinsically multipotent and generate functional neuronsJournal of Cell Biology 161 169ndash186

Bergles DE Roberts JD Somogyi P and Jahr CE (2000)Glutamatergic synapses on oligodendrocyte precursor cells in thehippocampus Nature 405 187ndash191

Bushong EA Martone ME Jones YZ and Ellisman MH (2002)Protoplasmic astrocytes in CA1 stratum radiatum occupy separateanatomical domains Journal of Neuroscience 22 183ndash192

Butt AM Duncan A Hornby MF Kirvell SL Hunter A LevineJM et al (1999) Cells expressing the NG2 antigen contact nodes ofRanvier in adult CNS white matter Glia 26 84ndash91

Butt AM Hamilton N Hubbard P Pugh M and Ibrahim M (2005)Synantocytes the fifth element Journal of Anatomy 207 695ndash706

Butt AM Kiff J Hubbard P and Berry M (2002) Synantocytes newfunctions for novel NG2 expressing glia Journal of Neurocytology31 551ndash565

integration of ng2-glia (synantocytes) into the neuroglial network 27

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Dayer AG Cleaver KM Abouantoun T and Cameron HA (2005)New GABAergic interneurons in the adult neocortex and striatumare generated from different precursors Journal of Cell Biology 168415ndash427

Dore-Duffy P Katychev A Wang X and Van Buren E (2006) CNSmicrovascular pericytes exhibit multipotential stem cell activityJournal of Cerebral Blood Flow and Metabolism 26 613ndash624

Ge WP Yang XJ Zhang Z Wang HK Shen W Deng QD et al(2006) Long-term potentiation of neuron-glia synapses mediated byCa2thorn-permeable AMPA receptors Science 312 1533ndash1537

Ge WP Zhou W Luo Q Jan LY and Jan YN (2009) Dividing glialcells maintain differentiated properties including complex mor-phology and functional synapses Proceedings of the NationalAcademy of Sciences of the USA 106 328ndash333

Gerhardt H and Betsholtz C (2003) Endothelial-pericyte interactions inangiogenesis Cell Tissue Research 314 15ndash23

Grass D Pawlowski PG Hirrlinger J Papadopoulos N RichterDW Kirchhoff F et al (2004) Diversity of functional astroglialproperties in the respiratory network Journal of Neuroscience 241358ndash1365

Grosche J Kettenmann H and Reichenbach A (2002) Bergmann glialcells form distinct morphological structures to interact with cerebellarneurons Journal of Neuroscience Research 68 138ndash149

Grosche J Matyash V Moller T Verkhratsky A Reichenbach A andKettenmann H (1999) Microdomains for neuron-glia interactionparallel fiber signaling to Bergmann glial cells Nature Neuroscience2 139ndash143

Halassa MM Fellin T and Haydon PG (2007a) The tripartite synapseroles for gliotransmission in health and disease Trends in MolecularMedicine 13 54ndash63

Halassa MM Fellin T Takano H Dong JH and Haydon PG(2007b) Synaptic islands defined by the territory of a single astrocyteJournal of Neuroscience 27 6473ndash6477

Hamilton N Vayro S Kirchhoff F Verkhratsky A Robbins J GoreckiD et al (2008) Mechanisms of ATP- and glutamate-mediated calciumsignaling in white matter astrocytes Glia 56 734ndash749

Hamilton N Vayro S Wigley R Robbins J and Butt AM (2009)Axons and astrocytes release glutamate and ATP to evoke calciumsignals in NG2-glia Glia in press

Houades V Koulakoff A Ezan P Seif I and Giaume C (2008) Gapjunction-mediated astrocytic networks in the mouse barrel cortexJournal of Neuroscience 28 5207ndash5217

Karadottir R Cavelier P Bergersen LH and Attwell D (2005) NMDAreceptors are expressed in oligodendrocytes and activated in ischae-mia Nature 438 1162ndash1166

Karadottir R Hamilton NB Bakiri Y and Attwell D (2008) Spikingand nonspiking classes of oligodendrocyte precursor glia in CNS whitematter Nature Neuroscience 11 450ndash456

Leoni G Rattray M and Butt AM (2009) NG2 cells differentiate intoastrocytes in cerebellar slices Molecular and Cellular Neurosciences42 208ndash218

Lin SC and Bergles DE (2004) Synaptic signaling between GABAergicinterneurons and oligodendrocyte precursor cells in the hippocampusNature Neuroscience 7 24ndash32

Lin SC Huck JH Roberts JD Macklin WB Somogyi P andBergles DE (2005) Climbing fiber innervation of NG2-expressingglia in the mammalian cerebellum Neuron 46 773ndash785

Nedergaard M Ransom B and Goldman SA (2003) New roles forastrocytes redefining the functional architecture of the brain Trendsin Neuroscience 26 523ndash530

Nishiyama A Lin X Giese N Heldin C and Stallcup W (1996)Co-localization of NG2 proteoglycan and PDGF alpha-receptor onO2A progenitor cells in the developing rat brain Journal ofNeuroscience Research 43 299ndash314

Nolte C Matyash M Pivneva T Schipke CG Ohlemeyer C HanischUK et al (2001) GFAP promoter-controlled EGFP-expressing trans-genic mice a tool to visualize astrocytes and astrogliosis in living braintissue Glia 33 72ndash86

Oberheim NA Wang X Goldman S and Nedergaard M (2006)Astrocytic complexity distinguishes the human brain Trends inNeuroscience 29 547ndash553

Ozerdem U Monosov E and Stallcup WB (2002) NG2 proteoglycanexpression by pericytes in pathological microvasculatureMicrovascular Research 63 129ndash134

Parpura V Basarsky TA Liu F Jeftinija K Jeftinija S and HaydonPG (1994) Glutamate-mediated astrocyte-neuron signalling Nature369 744ndash747

Peppiatt CM Howarth C Mobbs P and Attwell D (2006)Bidirectional control of CNS capillary diameter by pericytes Nature443 700ndash704

Psachoulia K Young KM and Richardson WD (2009) Separatepopulations of dividing and non-dividing NG2 cells in the postnatalmouse brain Neuron Glia Biology in press

Rivers LE Young KM Rizzi M Jamen F Psachoulia K Wade Aet al (2008) PDGFRANG2 glia generate myelinating oligodendro-cytes and piriform projection neurons in adult mice NatureNeuroscience 11 1392ndash1401

Rouach N Koulakoff A Abudara V Willecke K and Giaume C(2008) Astroglial metabolic networks sustain hippocampal synaptictransmission Science 322 1551ndash1555

Wang LY Cai WQ Chen PH Deng QY and Zhao CM (2009)Downregulation of P2X7 receptor expression in rat oligodendrocyteprecursor cells after hypoxia ischemia Glia 57 307ndash319

Wigley R Hamilton N Nishiyama A Kirchhoff F and Butt AM(2007) Morphological and physiological interactions of NG2-gliawith astrocytes and neurons Journal of Anatomy 210 661ndash670

Zhu X Bergles DE and Nishiyama A (2008) NG2 cells generate botholigodendrocytes and gray matter astrocytes Development 135145ndash157

Ziskin JL Nishiyama A Rubio M Fukaya M and Bergles DE (2007)Vesicular release of glutamate from unmyelinated axons in whitematter Nature Neuroscience 10 321ndash330

A U T H O R S rsquo A D D R E S S

Institute for Biomedical and Biomolecular SciencesSchool of Pharmacy and Biomedical SciencesUniversity of PortsmouthUK

Correspondence should be addressed toArthur M ButtInstitute for Biomedical and Biomolecular SciencesSchool of Pharmacy and Biomedical SciencesUniversity of PortsmouthUKemail Arthurbuttportacuk

28 rebekah wigley and arthur m butt

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(Fig 1) NG2-glia have a characteristic stellate morphologywith a central cell body and fine processes that extend out-wards radially to distances of up to 50 mm as illustrated inthe hippocampus (Fig 1AndashE) and the cerebellar granule celllayer (Fig 1F) and molecular layer (Fig 1G) Astrocyteshave five or more thick primary processes from which

emanate a multitude of fine processes giving the cell alsquofurryrsquo appearance (Fig 1AndashF) NG2-glia and astrocytesdefine overlapping three-dimensional domains in which thedomains of the same cell type overlap only slightly at theedges but those of different cell types overlap considerably(Fig 1AndashF) NG2-glial domains are significantly larger than

Fig 1 Domain structure of NG2-glia and astroglia Immunolabelling for NG2 (red) in brain sections from GFAP-EGFP mice Confocal images are shown astwo-dimensional snapshots (ABDFG) single z-section (C) and with isosurface rendering (B inset EG inset) NG2-glia and astrocytes are arranged inoverlapping radial domains mice in the molecular layer of the hippocampus (ACD) the dentate gyrus (B) the granule cell layer of the cerebellum (F) andthe molecular layer of the cerebellum (G) NG2-glial cell and astrocyte processes ensheath each other at numerous points other along their length (B inset)(AndashE) Domains of individual astrocytes interact with those of three or more NG2-glia (C) whereas individual NG2-glia interact with six or more astroglia(FG) (DE) NG2-glial cell and astrocyte somata are often directly apposed and have completely overlapping domains with processes that interdigitateextensively NG2-glia and astrocytes extend end feet to blood vessels but those of astrocytes expand to form perivascular end feet (DE arrows) (FG)NG2-glia traverse the different layers of the cerebellum to associate with multiple velate astrocytes and Bergmann glia the process of NG2-glia and primaryprocess of Bergmann glia are extensively apposed along their length (G inset) Scale bars frac14 20 mm in (AFG) and 10 mm in (BCDE) and 15 mm (G inset)

22 rebekah wigley and arthur m butt

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those of astrocytes (P 005 unpaired t-test) with an averagediameter in the hippocampus of 99 + 1 mm (n frac14 17) inNG2-glia and 78 + 2 mm (n frac14 13) in astrocytes Thedomains of individual astrocytes overlap with those of threeor more NG2-glia (Fig 1AndashC) whereas those of NG2-gliaoverlap with the domains of more than six astrocytes(Fig 1F) bearing in mind that this number is likely to begreater because not all astrocytes express GFAP or the repor-ter gene Within overlapping domains the processes ofNG2-glia and astrocytes interdigitate and display bipartiteensheathment whereby short segments of NG2-glial andastroglial processes ensheath each other (Fig 1B inset) Thesomata of some NG2-glia and astrocytes are directlyapposed to one another with the appearance of sister cells(Fig 1D) In these cases the domains of the NG2-glial cellsand astrocyte are almost identical and there is a largesurface area of contact between the somata and their pro-cesses which interdigitate extensively within the overlappingprocess fields of the two adjoining cells (Fig 1E) BothNG2-glia and astrocytes extend processes to blood vesselsbut those of astrocytes expand to form perivascular end-feet(Fig 1DE arrows) In the cerebellum astrocytes display func-tional and morphological specialisation with velate astrocytes

in the granule cell layer (Fig 1F) and Bergmann glia with cellsomata in the Purkinje cell layer and primary processesextending through the molecular layer to the pia (Fig 1G)In contrast NG2-glia do not display morphological hetero-geneity and individual cells extend processes to traverse thedifferent layers to form overlapping domains with velateastrocytes (Fig 1F) and Bergmann glia (Fig 1G) NG2-glialcell processes are directly apposed to the primary radial pro-cesses of Bergmann glia and they ensheath each other regu-larly along their lengths (Fig 1G inset) The domainorganisation of astrocytes is considered to be important forintegrated activity at the synapses in their domain (Bushonget al 2002 Nedergaard et al 2003 Oberheim et al 2006)and presumably this is also true for NG2-glia

I N T E G R A T I O N O F N G 2 - G L I A I N T OT H E N E U R O G L I A L N E T W O R K

We have examined the interrelationships between NG2-gliaastrocytes and neurons in the cortex and cerebellum Doubleimmunolabelling for NG2 and calretinin or calbindin forneurons in the cortex of GFAP-EGFP mice illustrates the

Fig 2 NG2-glia and astrocytes contact the same neurons in the cortex Immunolabelling for NG2 (red) and calretinin (A blue) or calbindin (B blue) in 100 mmmid-sagital brain sections from the cortex of GFAP-EGFP mice Confocal images are shown in three channels (AiBi) and two channels (AiiAiiiBiiBiii) and withisosurface rendering (AivBiv) (A) NG2-glia and astrocytes are associated with 10 or more neurons within their overlapping domains (B) NG2-glia and astroglialprocesses almost circumnavigate the same neuronal somata Scale bars frac14 10 mm in (AindashAiii) and (BindashBiii) and 5 mm in (Aiv) and (Biv)

integration of ng2-glia (synantocytes) into the neuroglial network 23

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Fig 3 Interactions of NG2-glia with astrocytes and neurons in the granule cell layer of the cerebellum Immunolabelling for NG2 (red) and calretinin (blue) inthe cerebellum of GFAP-EGFP mice Confocal images are shown in three channels (Ai) and two channels (AiiAiii) and with isosurface rendering (BC) Astrocytesand NG2-glia contact 20 or more neurons within their overlapping domains Velate astrocyte processes encompass neuronal somata (Aii) whereas NG2-glial cellprocesses from en passant associations (Aiii) The processes of Ng2-glia and astrocytes are directly apposed at the same contact sites with neuronal somatasuggesting they subserve the same synapses (BC) Scale bars frac14 10 mm in (A) 5 mm in (B) and 2 mm in (C)

Fig 4 Integration of NG2-glia with Purkinje neurons and Bergmann glia in the molecular layer of the cerebellum Immunolabelling for NG2 (red) andcalbindin (blue) in the cerebellum of P15 GFAP-EGFP mice Confocal images are shown in three channels (AiBi) and two channels (AiiAiii) and withisosurface rendering (BC) NG2-glial cell somata lie along the primary processes of Bergmann glia and the Purkinje neuron dendrites and extend processesparallel and perpendicular to the primary processes of Bergmann glia The processes of NG2-glia and Bergmann glia are intimately associated with Purkinjeneuron dendrites (CD) Scale bars frac14 20 mm in (A) and 2 mm in (BC)

24 rebekah wigley and arthur m butt

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integration of the three elements (Fig 2) The processes ofNG2-glia like those of astrocytes are directly apposed tomost if not all neuronal somata within their domains(Fig 2A) It is important to note that our results demonstratethe close associations that NG2-glia form with other elementsin the CNS but it is not possible at the confocal microscopiclevel to distinguish whether these contacts are actually func-tional Nonetheless using a combination of electronmicroscopy and patch-clamp electrophysiology it has beenestimated that only a few of the neuronal contacts formed byNG2-glia are electrophysiologically active lsquosynapsesrsquo (Linet al 2005 Ge et al 2009) In contrast astrocytes form

many hundreds of active contacts with neuronal synapses(Araque et al 1999 Grosche et al 1999 Bushong et al2002) NG2-glia and astrocytes form intimate contacts withthe same neurons extending processes that almost completelycircumnavigate neuronal somata (Fig 2B) and perisynapticprocesses from both cells types form a latticework (reticulation)on the cell body (Fig 2Biv) The close apposition of NG2-gliaand astrocytes to the same neurons suggests they may contactthe same synapses but they subserve neurons in differentways Electron microscopy studies demonstrate the differentassociations made by NG2-glia and astrocytes with neuronsNG2-glia form specific points of synaptic like contacts withneurons (Bergles et al 2000) whereas astrocytes ensheathpre- and post-synaptic boutons (Nedergaard et al 2003)This close relationship between NG2-glia astrocytes andneurons is also reflected in the cerebellum (Fig 3) In thegranule cell layer individual neurons are contacted by astro-cytes and NG2-glial cell processes (Fig 3A) However whilstvelate astrocyte processes circumnavigate neuronal cellsomata the processes of NG2-glia extend radially to form enpassant associations with both neurons and astrocytes as theyweave around the somata (Fig 3BC) as noted above it is notcurrently known whether these contacts have any function incommunication In the molecular layer NG2-glia are closelyintegrated with Bergmann glia and Purkinje cells (Fig 4)NG2-glial cell somata often lie along the primary process ofBergmann glial cells and Purkinje cell primary dendrites(Fig 4A) NG2-glia extend processes both parallel to and per-pendicular to Purkinje cell dendritic trees (Fig 4A) and theirprocesses interdigitate with those of Bergmann glia andPurkinje cell dendrites (Fig 4BC) A single NG2-glial cell sub-serves multiple Bergmann glia and Purkinje cells and manyNG2-glia extend processes into both the molecular andgranule cell layers to form additional contacts with velate astro-cytes and granule neurons NG2-glia contact synapses betweenclimbing fibres and Purkinje neurons but they appear to formfunctional contacts with only a small number of synapseswithin their domain (Lin et al 2005) whereas Bergmann gliaform many hundreds of microdomains that interact with thesynapses they ensheath (Grosche et al 1999 2002) Similarlya single hippocampal astrocyte can contact in excess of100000 synapses (Bushong et al 2002) and cortical astrocyteswrap on average four neuronal cell bodies and hundreds of den-drites (Halassa et al 2007b) In contrast NG2-glia do not havethe profuse fine processes of protoplasmic astrocytes and onaverage may form electrophysiologically active contacts withless than 20 synapses (Bergles et al 2000 Lin and Bergles2004 Ge et al 2009) However the intimate associationsbetween NG2-glia and astrocytes independent of neuronsboth on their somata and along their processes are sites of neu-rotransmitter mediated glia-to-glia calcium signalling(Hamilton et al 2009) The overlapping domain organisationof NG2-glia and astrocytes may therefore be important for inte-grating glial and neuronal activity

C O M M U N I C A T I O N B E T W E E NN G 2 - G L I A A N D T H E N E U R O G L I A LN E T W O R K

Electrophysiological studies show that NG2-glia express gluta-mate and GABA receptors and respond to neuronally releasedneurotransmitters (Bergles et al 2000 Lin and Bergles 2004

Fig 5 Synaptophysin is localised to sites of close apposition betweenNG2-glia and astrocytes Immunolabelling for NG2 (red) andsynaptophysin (blue) in the hippocampus of GFAP-EGFP mice Confocalimages are shown in three channels (A) and with isosurface rendering (BC)The processes of NG2-glia and astrocytes are associated with a large densityof synapses (AB) There is immediate apposition of the NG2-glial cell andastrocyte cell bodies and processes and synaptophysin is localised to thesesites of contact (arrows) indicating sites of synaptic communication ontoboth NG2-glia and astrocytes In addition synaptophysin was localisedwithin the soma of the NG2-glia cell confirming our recent report(Hamilton et al 2009) and suggesting NG2-glia are capable of bidirectionalcommunication with astrocytes and neurons Scale bar frac14 20 mm (A) 5 mm(B) and 1 mm (C)

integration of ng2-glia (synantocytes) into the neuroglial network 25

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Lin et al 2005) Calcium imaging and immunocytochemistryshow that NG2-glia express functional AMPA-type glutamatereceptors and metabotropic P2Y and ionotropic P2X recep-tors including the P2X7 subtype (Ge et al 2006 Hamiltonet al 2009 Wang et al 2009) Cerebellar NG2-glia candisplay both GABAergic and glutamatergic inputs (Lin andBergles 2004 Karadottir et al 2005) consistent with ourobservations that individual NG2-glia traverse the cell layersof the cerebellum to contact glutamatergic and GABAergicelements In addition we have used immunolabelling forsynaptophysin to examine the relations of NG2-glia withsynapses (Fig 5) The density of synaptophysin immunolabel-ling in the grey matter precludes the quantification of specificsynapses contacted by NG2-glia (Fig 5A) Nevertheless ourresults provide evidence that NG2-glia and astrocytescontact the same neurons (Figs 3ndash5) and the immediate

apposition of synaptophysin to points of contact betweenNG2-glia and astrocytes suggests they may be integrated atthe same synapses (Fig 5) Moreover synaptophysin is local-ised to sites of immediate apposition between astrocytesand NG2-glia and a region of particular interest is theexpression of synaptophysin between the NG2-glial cellbody and an astrocyte process (Fig 5B) Isoforming at theintegral communication site between the NG2-glial cell andastrocyte process provides further evidence of the localisationof synaptophysin to the NG2-glial cell body and to the inter-face between NG2-glia and astrocytes (Fig 5C) The resultspresented above show that the cell somata of astrocytes andNG2-glia are often directly apposed and that their processesexhibit regions where they tightly ensheath each other (Figs1B inset 1G inset 3BC and 4BC) The localisation of synap-tophysin to these areas indicates these are potential sits of

Fig 6 NG2-glia are in direct contact with pericytes NG2-glia are marked by an asterisk in each image Images show immunolabelling for NG2 in optic nerveof non-transgenic mice (AB) and NG2-glia and pericytes in sections of NG2-dsRed mouse brain (CndashE) NG2-glia contact multiple pericytes and in many casestheir cell somata appear to be in direct contact (CD) and this is confirmed in cross sectional images (E) Scale frac14 10 mm (A) 12 mm (B) 5 mm (C) 8 mm (D) and10 mm (E)

26 rebekah wigley and arthur m butt

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vesicular communication between the two cell typesAstrocytes have mechanisms to release neurotransmitters viavesicles (Parpura et al 1994 Araque et al 1999) We haveprovided evidence that astrocytes also signal onto NG2-gliavia glutamate and ATP (Hamilton et al 2008 2009)Moreover the evidence that NG2-glia express synaptophysinindicates they may be capable of neurotransmitter release andbidirectional communication with astrocytes and neurons

R E L A T I O N S B E T W E E N N G 2 - G L I AA N D T H E C E R E B R A LV A S C U L A T U R E

Astrocytes are able to lsquosensersquo changes in neuronal activity andtransmit signals to the vasculature to regulate local blood flowand provide metabolic support to neurons (Halassa et al2007a) Astrocytes may use glutamate signalling mechanismsto communicate with pericytes on blood vessels that are ableto regulate cerebral blood flow (Peppiatt et al 2006)NG2-glia also contact pericytes on cerebral blood vessels(Fig 6) and they contact astrocytes and neurons withintheir domains (Figs 1ndash5) and express synaptophysin(Hamilton et al 2009) and may therefore play a role in reg-ulating blood flow NG2-glia and pericytes both express theNG2 CSPG (Ozerdem et al 2002) and the two cell typesare distinguished by their morphology NG2-glia as stellateprocess-bearing cells and pericytes as perivascular cells withtwo or more primary processes extending along bloodvessels According to these distinguishing features it can beseen that NG2-glia and pericytes lie in very close proximityto each other (Fig 6AB) The processes of NG2-glia contactpericyte processes on the blood vessel (Fig 6A) In somecases it appears that the pericyte cell soma extends outfrom the capillary to almost contact that of a neighbouringNG2-glial cell (Fig 6B) and in many cases NG2-glial cellsomata are directly apposed to blood vessels identified byimmunolabelling (Fig 6C) and in the NG2-DsRed transgenicmouse (Fig 6DE) Pericytes have a neurogenic potential invitro (Dore-Duffy et al 2006) and proliferate in response toinjury (Gerhardt and Betsholtz 2003) and the intimateassociations between NG2-glia and pericytes is suggestive ofa lineage relationship between the two cell types (Wigleyand Butt unpublished observations) It remains to be seenwhether NG2-glia communicate with pericytes and help regu-late blood flow but this is a possibility since they expresssynaptophysin and contact synapses astrocytes and pericytes

C O N C L U S I O N S

NG2-glia and astrocytes form overlapping neuronndashglialndashvascular domains Within these domains NG2-glia and astro-cytes contact the same neurons and most likely the samesynapses but they perform different functions Astrocyte pro-cesses envelop synapses and one of their main functions is theremoval of excess neurotransmitters NG2-glia form fine con-nections with synapses and do not express transporters andtheir function at synapses is unknown The large domains ofNG2-glia enable them to lsquosensersquo neuronal activity frommany neurons over a large area that overlaps with thedomains of multiple astrocytes Their relation with pericytesindicates that NG2-glia and astrocytes form parallel

communications pathways between neurons and bloodvessels which may be important in the regulation of bloodflow The evidence that NG2-glia also receive input fromastrocytes and the possibility that NG2-glia express mechan-isms for vesicular release of gliotransmitters raises the possi-bility that NG2-glia may also have an output and be able tofeed back to neurons or astrocytes The fact that NG2-gliaand astrocytes form parallel communications pathwayssuggests they have different functions since otherwise thislevel of redundancy is difficult to explain Astrocyte domainorganisation segregates intercellular communication withinneuronal circuits (Houades et al 2008) and astrocytes arecoupled by gap junctions that provide an activity-dependentintercellular pathway for transport between blood vesselsand neurons (Rouach et al 2008) NG2-glia are not coupledby gap junctions but by forming lsquosynapsesrsquo with neuronsastrocytes and pericytes they connect otherwise segregatedastroglial domains An important question for future studiesis to determine how these multiple inputs are integrated inindividual NG2-glia and whether they have a physiologicaloutput

A C K N O W L E D G E M E N T S

Supported by the BBSRC and INTERRIG

Statement of interestNone

R E F E R E N C E S

Aguirre AA Chittajallu R Belachew S and Gallo V (2004)NG2-expressing cells in the subventricular zone are type C-like cellsand contribute to interneuron generation in the postnatal hippo-campus Journal of Cell Biology 165 575ndash589

Araque A Parpura V Sanzgiri RP and Haydon PG (1999) Tripartitesynapses glia the unacknowledged partner Trends in Neuroscience 22208ndash215

Bakiri Y Attwell D and Karadottir R (2009) Electrical signalling prop-erties of oligodendrocyte precursor cells Neuron Glia Biology101017S1740925X09990202

Belachew S Chittajallu R Aguirre AA Yuan X Kirby M AndersonS et al (2003) Postnatal NG2 proteoglycan-expressing progenitorcells are intrinsically multipotent and generate functional neuronsJournal of Cell Biology 161 169ndash186

Bergles DE Roberts JD Somogyi P and Jahr CE (2000)Glutamatergic synapses on oligodendrocyte precursor cells in thehippocampus Nature 405 187ndash191

Bushong EA Martone ME Jones YZ and Ellisman MH (2002)Protoplasmic astrocytes in CA1 stratum radiatum occupy separateanatomical domains Journal of Neuroscience 22 183ndash192

Butt AM Duncan A Hornby MF Kirvell SL Hunter A LevineJM et al (1999) Cells expressing the NG2 antigen contact nodes ofRanvier in adult CNS white matter Glia 26 84ndash91

Butt AM Hamilton N Hubbard P Pugh M and Ibrahim M (2005)Synantocytes the fifth element Journal of Anatomy 207 695ndash706

Butt AM Kiff J Hubbard P and Berry M (2002) Synantocytes newfunctions for novel NG2 expressing glia Journal of Neurocytology31 551ndash565

integration of ng2-glia (synantocytes) into the neuroglial network 27

httpsdoiorg101017S1740925X09990329Downloaded from httpswwwcambridgeorgcore Open University Library on 04 Feb 2017 at 144254 subject to the Cambridge Core terms of use available at httpswwwcambridgeorgcoreterms

Dayer AG Cleaver KM Abouantoun T and Cameron HA (2005)New GABAergic interneurons in the adult neocortex and striatumare generated from different precursors Journal of Cell Biology 168415ndash427

Dore-Duffy P Katychev A Wang X and Van Buren E (2006) CNSmicrovascular pericytes exhibit multipotential stem cell activityJournal of Cerebral Blood Flow and Metabolism 26 613ndash624

Ge WP Yang XJ Zhang Z Wang HK Shen W Deng QD et al(2006) Long-term potentiation of neuron-glia synapses mediated byCa2thorn-permeable AMPA receptors Science 312 1533ndash1537

Ge WP Zhou W Luo Q Jan LY and Jan YN (2009) Dividing glialcells maintain differentiated properties including complex mor-phology and functional synapses Proceedings of the NationalAcademy of Sciences of the USA 106 328ndash333

Gerhardt H and Betsholtz C (2003) Endothelial-pericyte interactions inangiogenesis Cell Tissue Research 314 15ndash23

Grass D Pawlowski PG Hirrlinger J Papadopoulos N RichterDW Kirchhoff F et al (2004) Diversity of functional astroglialproperties in the respiratory network Journal of Neuroscience 241358ndash1365

Grosche J Kettenmann H and Reichenbach A (2002) Bergmann glialcells form distinct morphological structures to interact with cerebellarneurons Journal of Neuroscience Research 68 138ndash149

Grosche J Matyash V Moller T Verkhratsky A Reichenbach A andKettenmann H (1999) Microdomains for neuron-glia interactionparallel fiber signaling to Bergmann glial cells Nature Neuroscience2 139ndash143

Halassa MM Fellin T and Haydon PG (2007a) The tripartite synapseroles for gliotransmission in health and disease Trends in MolecularMedicine 13 54ndash63

Halassa MM Fellin T Takano H Dong JH and Haydon PG(2007b) Synaptic islands defined by the territory of a single astrocyteJournal of Neuroscience 27 6473ndash6477

Hamilton N Vayro S Kirchhoff F Verkhratsky A Robbins J GoreckiD et al (2008) Mechanisms of ATP- and glutamate-mediated calciumsignaling in white matter astrocytes Glia 56 734ndash749

Hamilton N Vayro S Wigley R Robbins J and Butt AM (2009)Axons and astrocytes release glutamate and ATP to evoke calciumsignals in NG2-glia Glia in press

Houades V Koulakoff A Ezan P Seif I and Giaume C (2008) Gapjunction-mediated astrocytic networks in the mouse barrel cortexJournal of Neuroscience 28 5207ndash5217

Karadottir R Cavelier P Bergersen LH and Attwell D (2005) NMDAreceptors are expressed in oligodendrocytes and activated in ischae-mia Nature 438 1162ndash1166

Karadottir R Hamilton NB Bakiri Y and Attwell D (2008) Spikingand nonspiking classes of oligodendrocyte precursor glia in CNS whitematter Nature Neuroscience 11 450ndash456

Leoni G Rattray M and Butt AM (2009) NG2 cells differentiate intoastrocytes in cerebellar slices Molecular and Cellular Neurosciences42 208ndash218

Lin SC and Bergles DE (2004) Synaptic signaling between GABAergicinterneurons and oligodendrocyte precursor cells in the hippocampusNature Neuroscience 7 24ndash32

Lin SC Huck JH Roberts JD Macklin WB Somogyi P andBergles DE (2005) Climbing fiber innervation of NG2-expressingglia in the mammalian cerebellum Neuron 46 773ndash785

Nedergaard M Ransom B and Goldman SA (2003) New roles forastrocytes redefining the functional architecture of the brain Trendsin Neuroscience 26 523ndash530

Nishiyama A Lin X Giese N Heldin C and Stallcup W (1996)Co-localization of NG2 proteoglycan and PDGF alpha-receptor onO2A progenitor cells in the developing rat brain Journal ofNeuroscience Research 43 299ndash314

Nolte C Matyash M Pivneva T Schipke CG Ohlemeyer C HanischUK et al (2001) GFAP promoter-controlled EGFP-expressing trans-genic mice a tool to visualize astrocytes and astrogliosis in living braintissue Glia 33 72ndash86

Oberheim NA Wang X Goldman S and Nedergaard M (2006)Astrocytic complexity distinguishes the human brain Trends inNeuroscience 29 547ndash553

Ozerdem U Monosov E and Stallcup WB (2002) NG2 proteoglycanexpression by pericytes in pathological microvasculatureMicrovascular Research 63 129ndash134

Parpura V Basarsky TA Liu F Jeftinija K Jeftinija S and HaydonPG (1994) Glutamate-mediated astrocyte-neuron signalling Nature369 744ndash747

Peppiatt CM Howarth C Mobbs P and Attwell D (2006)Bidirectional control of CNS capillary diameter by pericytes Nature443 700ndash704

Psachoulia K Young KM and Richardson WD (2009) Separatepopulations of dividing and non-dividing NG2 cells in the postnatalmouse brain Neuron Glia Biology in press

Rivers LE Young KM Rizzi M Jamen F Psachoulia K Wade Aet al (2008) PDGFRANG2 glia generate myelinating oligodendro-cytes and piriform projection neurons in adult mice NatureNeuroscience 11 1392ndash1401

Rouach N Koulakoff A Abudara V Willecke K and Giaume C(2008) Astroglial metabolic networks sustain hippocampal synaptictransmission Science 322 1551ndash1555

Wang LY Cai WQ Chen PH Deng QY and Zhao CM (2009)Downregulation of P2X7 receptor expression in rat oligodendrocyteprecursor cells after hypoxia ischemia Glia 57 307ndash319

Wigley R Hamilton N Nishiyama A Kirchhoff F and Butt AM(2007) Morphological and physiological interactions of NG2-gliawith astrocytes and neurons Journal of Anatomy 210 661ndash670

Zhu X Bergles DE and Nishiyama A (2008) NG2 cells generate botholigodendrocytes and gray matter astrocytes Development 135145ndash157

Ziskin JL Nishiyama A Rubio M Fukaya M and Bergles DE (2007)Vesicular release of glutamate from unmyelinated axons in whitematter Nature Neuroscience 10 321ndash330

A U T H O R S rsquo A D D R E S S

Institute for Biomedical and Biomolecular SciencesSchool of Pharmacy and Biomedical SciencesUniversity of PortsmouthUK

Correspondence should be addressed toArthur M ButtInstitute for Biomedical and Biomolecular SciencesSchool of Pharmacy and Biomedical SciencesUniversity of PortsmouthUKemail Arthurbuttportacuk

28 rebekah wigley and arthur m butt

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those of astrocytes (P 005 unpaired t-test) with an averagediameter in the hippocampus of 99 + 1 mm (n frac14 17) inNG2-glia and 78 + 2 mm (n frac14 13) in astrocytes Thedomains of individual astrocytes overlap with those of threeor more NG2-glia (Fig 1AndashC) whereas those of NG2-gliaoverlap with the domains of more than six astrocytes(Fig 1F) bearing in mind that this number is likely to begreater because not all astrocytes express GFAP or the repor-ter gene Within overlapping domains the processes ofNG2-glia and astrocytes interdigitate and display bipartiteensheathment whereby short segments of NG2-glial andastroglial processes ensheath each other (Fig 1B inset) Thesomata of some NG2-glia and astrocytes are directlyapposed to one another with the appearance of sister cells(Fig 1D) In these cases the domains of the NG2-glial cellsand astrocyte are almost identical and there is a largesurface area of contact between the somata and their pro-cesses which interdigitate extensively within the overlappingprocess fields of the two adjoining cells (Fig 1E) BothNG2-glia and astrocytes extend processes to blood vesselsbut those of astrocytes expand to form perivascular end-feet(Fig 1DE arrows) In the cerebellum astrocytes display func-tional and morphological specialisation with velate astrocytes

in the granule cell layer (Fig 1F) and Bergmann glia with cellsomata in the Purkinje cell layer and primary processesextending through the molecular layer to the pia (Fig 1G)In contrast NG2-glia do not display morphological hetero-geneity and individual cells extend processes to traverse thedifferent layers to form overlapping domains with velateastrocytes (Fig 1F) and Bergmann glia (Fig 1G) NG2-glialcell processes are directly apposed to the primary radial pro-cesses of Bergmann glia and they ensheath each other regu-larly along their lengths (Fig 1G inset) The domainorganisation of astrocytes is considered to be important forintegrated activity at the synapses in their domain (Bushonget al 2002 Nedergaard et al 2003 Oberheim et al 2006)and presumably this is also true for NG2-glia

I N T E G R A T I O N O F N G 2 - G L I A I N T OT H E N E U R O G L I A L N E T W O R K

We have examined the interrelationships between NG2-gliaastrocytes and neurons in the cortex and cerebellum Doubleimmunolabelling for NG2 and calretinin or calbindin forneurons in the cortex of GFAP-EGFP mice illustrates the

Fig 2 NG2-glia and astrocytes contact the same neurons in the cortex Immunolabelling for NG2 (red) and calretinin (A blue) or calbindin (B blue) in 100 mmmid-sagital brain sections from the cortex of GFAP-EGFP mice Confocal images are shown in three channels (AiBi) and two channels (AiiAiiiBiiBiii) and withisosurface rendering (AivBiv) (A) NG2-glia and astrocytes are associated with 10 or more neurons within their overlapping domains (B) NG2-glia and astroglialprocesses almost circumnavigate the same neuronal somata Scale bars frac14 10 mm in (AindashAiii) and (BindashBiii) and 5 mm in (Aiv) and (Biv)

integration of ng2-glia (synantocytes) into the neuroglial network 23

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Fig 3 Interactions of NG2-glia with astrocytes and neurons in the granule cell layer of the cerebellum Immunolabelling for NG2 (red) and calretinin (blue) inthe cerebellum of GFAP-EGFP mice Confocal images are shown in three channels (Ai) and two channels (AiiAiii) and with isosurface rendering (BC) Astrocytesand NG2-glia contact 20 or more neurons within their overlapping domains Velate astrocyte processes encompass neuronal somata (Aii) whereas NG2-glial cellprocesses from en passant associations (Aiii) The processes of Ng2-glia and astrocytes are directly apposed at the same contact sites with neuronal somatasuggesting they subserve the same synapses (BC) Scale bars frac14 10 mm in (A) 5 mm in (B) and 2 mm in (C)

Fig 4 Integration of NG2-glia with Purkinje neurons and Bergmann glia in the molecular layer of the cerebellum Immunolabelling for NG2 (red) andcalbindin (blue) in the cerebellum of P15 GFAP-EGFP mice Confocal images are shown in three channels (AiBi) and two channels (AiiAiii) and withisosurface rendering (BC) NG2-glial cell somata lie along the primary processes of Bergmann glia and the Purkinje neuron dendrites and extend processesparallel and perpendicular to the primary processes of Bergmann glia The processes of NG2-glia and Bergmann glia are intimately associated with Purkinjeneuron dendrites (CD) Scale bars frac14 20 mm in (A) and 2 mm in (BC)

24 rebekah wigley and arthur m butt

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integration of the three elements (Fig 2) The processes ofNG2-glia like those of astrocytes are directly apposed tomost if not all neuronal somata within their domains(Fig 2A) It is important to note that our results demonstratethe close associations that NG2-glia form with other elementsin the CNS but it is not possible at the confocal microscopiclevel to distinguish whether these contacts are actually func-tional Nonetheless using a combination of electronmicroscopy and patch-clamp electrophysiology it has beenestimated that only a few of the neuronal contacts formed byNG2-glia are electrophysiologically active lsquosynapsesrsquo (Linet al 2005 Ge et al 2009) In contrast astrocytes form

many hundreds of active contacts with neuronal synapses(Araque et al 1999 Grosche et al 1999 Bushong et al2002) NG2-glia and astrocytes form intimate contacts withthe same neurons extending processes that almost completelycircumnavigate neuronal somata (Fig 2B) and perisynapticprocesses from both cells types form a latticework (reticulation)on the cell body (Fig 2Biv) The close apposition of NG2-gliaand astrocytes to the same neurons suggests they may contactthe same synapses but they subserve neurons in differentways Electron microscopy studies demonstrate the differentassociations made by NG2-glia and astrocytes with neuronsNG2-glia form specific points of synaptic like contacts withneurons (Bergles et al 2000) whereas astrocytes ensheathpre- and post-synaptic boutons (Nedergaard et al 2003)This close relationship between NG2-glia astrocytes andneurons is also reflected in the cerebellum (Fig 3) In thegranule cell layer individual neurons are contacted by astro-cytes and NG2-glial cell processes (Fig 3A) However whilstvelate astrocyte processes circumnavigate neuronal cellsomata the processes of NG2-glia extend radially to form enpassant associations with both neurons and astrocytes as theyweave around the somata (Fig 3BC) as noted above it is notcurrently known whether these contacts have any function incommunication In the molecular layer NG2-glia are closelyintegrated with Bergmann glia and Purkinje cells (Fig 4)NG2-glial cell somata often lie along the primary process ofBergmann glial cells and Purkinje cell primary dendrites(Fig 4A) NG2-glia extend processes both parallel to and per-pendicular to Purkinje cell dendritic trees (Fig 4A) and theirprocesses interdigitate with those of Bergmann glia andPurkinje cell dendrites (Fig 4BC) A single NG2-glial cell sub-serves multiple Bergmann glia and Purkinje cells and manyNG2-glia extend processes into both the molecular andgranule cell layers to form additional contacts with velate astro-cytes and granule neurons NG2-glia contact synapses betweenclimbing fibres and Purkinje neurons but they appear to formfunctional contacts with only a small number of synapseswithin their domain (Lin et al 2005) whereas Bergmann gliaform many hundreds of microdomains that interact with thesynapses they ensheath (Grosche et al 1999 2002) Similarlya single hippocampal astrocyte can contact in excess of100000 synapses (Bushong et al 2002) and cortical astrocyteswrap on average four neuronal cell bodies and hundreds of den-drites (Halassa et al 2007b) In contrast NG2-glia do not havethe profuse fine processes of protoplasmic astrocytes and onaverage may form electrophysiologically active contacts withless than 20 synapses (Bergles et al 2000 Lin and Bergles2004 Ge et al 2009) However the intimate associationsbetween NG2-glia and astrocytes independent of neuronsboth on their somata and along their processes are sites of neu-rotransmitter mediated glia-to-glia calcium signalling(Hamilton et al 2009) The overlapping domain organisationof NG2-glia and astrocytes may therefore be important for inte-grating glial and neuronal activity

C O M M U N I C A T I O N B E T W E E NN G 2 - G L I A A N D T H E N E U R O G L I A LN E T W O R K

Electrophysiological studies show that NG2-glia express gluta-mate and GABA receptors and respond to neuronally releasedneurotransmitters (Bergles et al 2000 Lin and Bergles 2004

Fig 5 Synaptophysin is localised to sites of close apposition betweenNG2-glia and astrocytes Immunolabelling for NG2 (red) andsynaptophysin (blue) in the hippocampus of GFAP-EGFP mice Confocalimages are shown in three channels (A) and with isosurface rendering (BC)The processes of NG2-glia and astrocytes are associated with a large densityof synapses (AB) There is immediate apposition of the NG2-glial cell andastrocyte cell bodies and processes and synaptophysin is localised to thesesites of contact (arrows) indicating sites of synaptic communication ontoboth NG2-glia and astrocytes In addition synaptophysin was localisedwithin the soma of the NG2-glia cell confirming our recent report(Hamilton et al 2009) and suggesting NG2-glia are capable of bidirectionalcommunication with astrocytes and neurons Scale bar frac14 20 mm (A) 5 mm(B) and 1 mm (C)

integration of ng2-glia (synantocytes) into the neuroglial network 25

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Lin et al 2005) Calcium imaging and immunocytochemistryshow that NG2-glia express functional AMPA-type glutamatereceptors and metabotropic P2Y and ionotropic P2X recep-tors including the P2X7 subtype (Ge et al 2006 Hamiltonet al 2009 Wang et al 2009) Cerebellar NG2-glia candisplay both GABAergic and glutamatergic inputs (Lin andBergles 2004 Karadottir et al 2005) consistent with ourobservations that individual NG2-glia traverse the cell layersof the cerebellum to contact glutamatergic and GABAergicelements In addition we have used immunolabelling forsynaptophysin to examine the relations of NG2-glia withsynapses (Fig 5) The density of synaptophysin immunolabel-ling in the grey matter precludes the quantification of specificsynapses contacted by NG2-glia (Fig 5A) Nevertheless ourresults provide evidence that NG2-glia and astrocytescontact the same neurons (Figs 3ndash5) and the immediate

apposition of synaptophysin to points of contact betweenNG2-glia and astrocytes suggests they may be integrated atthe same synapses (Fig 5) Moreover synaptophysin is local-ised to sites of immediate apposition between astrocytesand NG2-glia and a region of particular interest is theexpression of synaptophysin between the NG2-glial cellbody and an astrocyte process (Fig 5B) Isoforming at theintegral communication site between the NG2-glial cell andastrocyte process provides further evidence of the localisationof synaptophysin to the NG2-glial cell body and to the inter-face between NG2-glia and astrocytes (Fig 5C) The resultspresented above show that the cell somata of astrocytes andNG2-glia are often directly apposed and that their processesexhibit regions where they tightly ensheath each other (Figs1B inset 1G inset 3BC and 4BC) The localisation of synap-tophysin to these areas indicates these are potential sits of

Fig 6 NG2-glia are in direct contact with pericytes NG2-glia are marked by an asterisk in each image Images show immunolabelling for NG2 in optic nerveof non-transgenic mice (AB) and NG2-glia and pericytes in sections of NG2-dsRed mouse brain (CndashE) NG2-glia contact multiple pericytes and in many casestheir cell somata appear to be in direct contact (CD) and this is confirmed in cross sectional images (E) Scale frac14 10 mm (A) 12 mm (B) 5 mm (C) 8 mm (D) and10 mm (E)

26 rebekah wigley and arthur m butt

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vesicular communication between the two cell typesAstrocytes have mechanisms to release neurotransmitters viavesicles (Parpura et al 1994 Araque et al 1999) We haveprovided evidence that astrocytes also signal onto NG2-gliavia glutamate and ATP (Hamilton et al 2008 2009)Moreover the evidence that NG2-glia express synaptophysinindicates they may be capable of neurotransmitter release andbidirectional communication with astrocytes and neurons

R E L A T I O N S B E T W E E N N G 2 - G L I AA N D T H E C E R E B R A LV A S C U L A T U R E

Astrocytes are able to lsquosensersquo changes in neuronal activity andtransmit signals to the vasculature to regulate local blood flowand provide metabolic support to neurons (Halassa et al2007a) Astrocytes may use glutamate signalling mechanismsto communicate with pericytes on blood vessels that are ableto regulate cerebral blood flow (Peppiatt et al 2006)NG2-glia also contact pericytes on cerebral blood vessels(Fig 6) and they contact astrocytes and neurons withintheir domains (Figs 1ndash5) and express synaptophysin(Hamilton et al 2009) and may therefore play a role in reg-ulating blood flow NG2-glia and pericytes both express theNG2 CSPG (Ozerdem et al 2002) and the two cell typesare distinguished by their morphology NG2-glia as stellateprocess-bearing cells and pericytes as perivascular cells withtwo or more primary processes extending along bloodvessels According to these distinguishing features it can beseen that NG2-glia and pericytes lie in very close proximityto each other (Fig 6AB) The processes of NG2-glia contactpericyte processes on the blood vessel (Fig 6A) In somecases it appears that the pericyte cell soma extends outfrom the capillary to almost contact that of a neighbouringNG2-glial cell (Fig 6B) and in many cases NG2-glial cellsomata are directly apposed to blood vessels identified byimmunolabelling (Fig 6C) and in the NG2-DsRed transgenicmouse (Fig 6DE) Pericytes have a neurogenic potential invitro (Dore-Duffy et al 2006) and proliferate in response toinjury (Gerhardt and Betsholtz 2003) and the intimateassociations between NG2-glia and pericytes is suggestive ofa lineage relationship between the two cell types (Wigleyand Butt unpublished observations) It remains to be seenwhether NG2-glia communicate with pericytes and help regu-late blood flow but this is a possibility since they expresssynaptophysin and contact synapses astrocytes and pericytes

C O N C L U S I O N S

NG2-glia and astrocytes form overlapping neuronndashglialndashvascular domains Within these domains NG2-glia and astro-cytes contact the same neurons and most likely the samesynapses but they perform different functions Astrocyte pro-cesses envelop synapses and one of their main functions is theremoval of excess neurotransmitters NG2-glia form fine con-nections with synapses and do not express transporters andtheir function at synapses is unknown The large domains ofNG2-glia enable them to lsquosensersquo neuronal activity frommany neurons over a large area that overlaps with thedomains of multiple astrocytes Their relation with pericytesindicates that NG2-glia and astrocytes form parallel

communications pathways between neurons and bloodvessels which may be important in the regulation of bloodflow The evidence that NG2-glia also receive input fromastrocytes and the possibility that NG2-glia express mechan-isms for vesicular release of gliotransmitters raises the possi-bility that NG2-glia may also have an output and be able tofeed back to neurons or astrocytes The fact that NG2-gliaand astrocytes form parallel communications pathwayssuggests they have different functions since otherwise thislevel of redundancy is difficult to explain Astrocyte domainorganisation segregates intercellular communication withinneuronal circuits (Houades et al 2008) and astrocytes arecoupled by gap junctions that provide an activity-dependentintercellular pathway for transport between blood vesselsand neurons (Rouach et al 2008) NG2-glia are not coupledby gap junctions but by forming lsquosynapsesrsquo with neuronsastrocytes and pericytes they connect otherwise segregatedastroglial domains An important question for future studiesis to determine how these multiple inputs are integrated inindividual NG2-glia and whether they have a physiologicaloutput

A C K N O W L E D G E M E N T S

Supported by the BBSRC and INTERRIG

Statement of interestNone

R E F E R E N C E S

Aguirre AA Chittajallu R Belachew S and Gallo V (2004)NG2-expressing cells in the subventricular zone are type C-like cellsand contribute to interneuron generation in the postnatal hippo-campus Journal of Cell Biology 165 575ndash589

Araque A Parpura V Sanzgiri RP and Haydon PG (1999) Tripartitesynapses glia the unacknowledged partner Trends in Neuroscience 22208ndash215

Bakiri Y Attwell D and Karadottir R (2009) Electrical signalling prop-erties of oligodendrocyte precursor cells Neuron Glia Biology101017S1740925X09990202

Belachew S Chittajallu R Aguirre AA Yuan X Kirby M AndersonS et al (2003) Postnatal NG2 proteoglycan-expressing progenitorcells are intrinsically multipotent and generate functional neuronsJournal of Cell Biology 161 169ndash186

Bergles DE Roberts JD Somogyi P and Jahr CE (2000)Glutamatergic synapses on oligodendrocyte precursor cells in thehippocampus Nature 405 187ndash191

Bushong EA Martone ME Jones YZ and Ellisman MH (2002)Protoplasmic astrocytes in CA1 stratum radiatum occupy separateanatomical domains Journal of Neuroscience 22 183ndash192

Butt AM Duncan A Hornby MF Kirvell SL Hunter A LevineJM et al (1999) Cells expressing the NG2 antigen contact nodes ofRanvier in adult CNS white matter Glia 26 84ndash91

Butt AM Hamilton N Hubbard P Pugh M and Ibrahim M (2005)Synantocytes the fifth element Journal of Anatomy 207 695ndash706

Butt AM Kiff J Hubbard P and Berry M (2002) Synantocytes newfunctions for novel NG2 expressing glia Journal of Neurocytology31 551ndash565

integration of ng2-glia (synantocytes) into the neuroglial network 27

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Dayer AG Cleaver KM Abouantoun T and Cameron HA (2005)New GABAergic interneurons in the adult neocortex and striatumare generated from different precursors Journal of Cell Biology 168415ndash427

Dore-Duffy P Katychev A Wang X and Van Buren E (2006) CNSmicrovascular pericytes exhibit multipotential stem cell activityJournal of Cerebral Blood Flow and Metabolism 26 613ndash624

Ge WP Yang XJ Zhang Z Wang HK Shen W Deng QD et al(2006) Long-term potentiation of neuron-glia synapses mediated byCa2thorn-permeable AMPA receptors Science 312 1533ndash1537

Ge WP Zhou W Luo Q Jan LY and Jan YN (2009) Dividing glialcells maintain differentiated properties including complex mor-phology and functional synapses Proceedings of the NationalAcademy of Sciences of the USA 106 328ndash333

Gerhardt H and Betsholtz C (2003) Endothelial-pericyte interactions inangiogenesis Cell Tissue Research 314 15ndash23

Grass D Pawlowski PG Hirrlinger J Papadopoulos N RichterDW Kirchhoff F et al (2004) Diversity of functional astroglialproperties in the respiratory network Journal of Neuroscience 241358ndash1365

Grosche J Kettenmann H and Reichenbach A (2002) Bergmann glialcells form distinct morphological structures to interact with cerebellarneurons Journal of Neuroscience Research 68 138ndash149

Grosche J Matyash V Moller T Verkhratsky A Reichenbach A andKettenmann H (1999) Microdomains for neuron-glia interactionparallel fiber signaling to Bergmann glial cells Nature Neuroscience2 139ndash143

Halassa MM Fellin T and Haydon PG (2007a) The tripartite synapseroles for gliotransmission in health and disease Trends in MolecularMedicine 13 54ndash63

Halassa MM Fellin T Takano H Dong JH and Haydon PG(2007b) Synaptic islands defined by the territory of a single astrocyteJournal of Neuroscience 27 6473ndash6477

Hamilton N Vayro S Kirchhoff F Verkhratsky A Robbins J GoreckiD et al (2008) Mechanisms of ATP- and glutamate-mediated calciumsignaling in white matter astrocytes Glia 56 734ndash749

Hamilton N Vayro S Wigley R Robbins J and Butt AM (2009)Axons and astrocytes release glutamate and ATP to evoke calciumsignals in NG2-glia Glia in press

Houades V Koulakoff A Ezan P Seif I and Giaume C (2008) Gapjunction-mediated astrocytic networks in the mouse barrel cortexJournal of Neuroscience 28 5207ndash5217

Karadottir R Cavelier P Bergersen LH and Attwell D (2005) NMDAreceptors are expressed in oligodendrocytes and activated in ischae-mia Nature 438 1162ndash1166

Karadottir R Hamilton NB Bakiri Y and Attwell D (2008) Spikingand nonspiking classes of oligodendrocyte precursor glia in CNS whitematter Nature Neuroscience 11 450ndash456

Leoni G Rattray M and Butt AM (2009) NG2 cells differentiate intoastrocytes in cerebellar slices Molecular and Cellular Neurosciences42 208ndash218

Lin SC and Bergles DE (2004) Synaptic signaling between GABAergicinterneurons and oligodendrocyte precursor cells in the hippocampusNature Neuroscience 7 24ndash32

Lin SC Huck JH Roberts JD Macklin WB Somogyi P andBergles DE (2005) Climbing fiber innervation of NG2-expressingglia in the mammalian cerebellum Neuron 46 773ndash785

Nedergaard M Ransom B and Goldman SA (2003) New roles forastrocytes redefining the functional architecture of the brain Trendsin Neuroscience 26 523ndash530

Nishiyama A Lin X Giese N Heldin C and Stallcup W (1996)Co-localization of NG2 proteoglycan and PDGF alpha-receptor onO2A progenitor cells in the developing rat brain Journal ofNeuroscience Research 43 299ndash314

Nolte C Matyash M Pivneva T Schipke CG Ohlemeyer C HanischUK et al (2001) GFAP promoter-controlled EGFP-expressing trans-genic mice a tool to visualize astrocytes and astrogliosis in living braintissue Glia 33 72ndash86

Oberheim NA Wang X Goldman S and Nedergaard M (2006)Astrocytic complexity distinguishes the human brain Trends inNeuroscience 29 547ndash553

Ozerdem U Monosov E and Stallcup WB (2002) NG2 proteoglycanexpression by pericytes in pathological microvasculatureMicrovascular Research 63 129ndash134

Parpura V Basarsky TA Liu F Jeftinija K Jeftinija S and HaydonPG (1994) Glutamate-mediated astrocyte-neuron signalling Nature369 744ndash747

Peppiatt CM Howarth C Mobbs P and Attwell D (2006)Bidirectional control of CNS capillary diameter by pericytes Nature443 700ndash704

Psachoulia K Young KM and Richardson WD (2009) Separatepopulations of dividing and non-dividing NG2 cells in the postnatalmouse brain Neuron Glia Biology in press

Rivers LE Young KM Rizzi M Jamen F Psachoulia K Wade Aet al (2008) PDGFRANG2 glia generate myelinating oligodendro-cytes and piriform projection neurons in adult mice NatureNeuroscience 11 1392ndash1401

Rouach N Koulakoff A Abudara V Willecke K and Giaume C(2008) Astroglial metabolic networks sustain hippocampal synaptictransmission Science 322 1551ndash1555

Wang LY Cai WQ Chen PH Deng QY and Zhao CM (2009)Downregulation of P2X7 receptor expression in rat oligodendrocyteprecursor cells after hypoxia ischemia Glia 57 307ndash319

Wigley R Hamilton N Nishiyama A Kirchhoff F and Butt AM(2007) Morphological and physiological interactions of NG2-gliawith astrocytes and neurons Journal of Anatomy 210 661ndash670

Zhu X Bergles DE and Nishiyama A (2008) NG2 cells generate botholigodendrocytes and gray matter astrocytes Development 135145ndash157

Ziskin JL Nishiyama A Rubio M Fukaya M and Bergles DE (2007)Vesicular release of glutamate from unmyelinated axons in whitematter Nature Neuroscience 10 321ndash330

A U T H O R S rsquo A D D R E S S

Institute for Biomedical and Biomolecular SciencesSchool of Pharmacy and Biomedical SciencesUniversity of PortsmouthUK

Correspondence should be addressed toArthur M ButtInstitute for Biomedical and Biomolecular SciencesSchool of Pharmacy and Biomedical SciencesUniversity of PortsmouthUKemail Arthurbuttportacuk

28 rebekah wigley and arthur m butt

httpsdoiorg101017S1740925X09990329Downloaded from httpswwwcambridgeorgcore Open University Library on 04 Feb 2017 at 144254 subject to the Cambridge Core terms of use available at httpswwwcambridgeorgcoreterms

Fig 3 Interactions of NG2-glia with astrocytes and neurons in the granule cell layer of the cerebellum Immunolabelling for NG2 (red) and calretinin (blue) inthe cerebellum of GFAP-EGFP mice Confocal images are shown in three channels (Ai) and two channels (AiiAiii) and with isosurface rendering (BC) Astrocytesand NG2-glia contact 20 or more neurons within their overlapping domains Velate astrocyte processes encompass neuronal somata (Aii) whereas NG2-glial cellprocesses from en passant associations (Aiii) The processes of Ng2-glia and astrocytes are directly apposed at the same contact sites with neuronal somatasuggesting they subserve the same synapses (BC) Scale bars frac14 10 mm in (A) 5 mm in (B) and 2 mm in (C)

Fig 4 Integration of NG2-glia with Purkinje neurons and Bergmann glia in the molecular layer of the cerebellum Immunolabelling for NG2 (red) andcalbindin (blue) in the cerebellum of P15 GFAP-EGFP mice Confocal images are shown in three channels (AiBi) and two channels (AiiAiii) and withisosurface rendering (BC) NG2-glial cell somata lie along the primary processes of Bergmann glia and the Purkinje neuron dendrites and extend processesparallel and perpendicular to the primary processes of Bergmann glia The processes of NG2-glia and Bergmann glia are intimately associated with Purkinjeneuron dendrites (CD) Scale bars frac14 20 mm in (A) and 2 mm in (BC)

24 rebekah wigley and arthur m butt

httpsdoiorg101017S1740925X09990329Downloaded from httpswwwcambridgeorgcore Open University Library on 04 Feb 2017 at 144254 subject to the Cambridge Core terms of use available at httpswwwcambridgeorgcoreterms

integration of the three elements (Fig 2) The processes ofNG2-glia like those of astrocytes are directly apposed tomost if not all neuronal somata within their domains(Fig 2A) It is important to note that our results demonstratethe close associations that NG2-glia form with other elementsin the CNS but it is not possible at the confocal microscopiclevel to distinguish whether these contacts are actually func-tional Nonetheless using a combination of electronmicroscopy and patch-clamp electrophysiology it has beenestimated that only a few of the neuronal contacts formed byNG2-glia are electrophysiologically active lsquosynapsesrsquo (Linet al 2005 Ge et al 2009) In contrast astrocytes form

many hundreds of active contacts with neuronal synapses(Araque et al 1999 Grosche et al 1999 Bushong et al2002) NG2-glia and astrocytes form intimate contacts withthe same neurons extending processes that almost completelycircumnavigate neuronal somata (Fig 2B) and perisynapticprocesses from both cells types form a latticework (reticulation)on the cell body (Fig 2Biv) The close apposition of NG2-gliaand astrocytes to the same neurons suggests they may contactthe same synapses but they subserve neurons in differentways Electron microscopy studies demonstrate the differentassociations made by NG2-glia and astrocytes with neuronsNG2-glia form specific points of synaptic like contacts withneurons (Bergles et al 2000) whereas astrocytes ensheathpre- and post-synaptic boutons (Nedergaard et al 2003)This close relationship between NG2-glia astrocytes andneurons is also reflected in the cerebellum (Fig 3) In thegranule cell layer individual neurons are contacted by astro-cytes and NG2-glial cell processes (Fig 3A) However whilstvelate astrocyte processes circumnavigate neuronal cellsomata the processes of NG2-glia extend radially to form enpassant associations with both neurons and astrocytes as theyweave around the somata (Fig 3BC) as noted above it is notcurrently known whether these contacts have any function incommunication In the molecular layer NG2-glia are closelyintegrated with Bergmann glia and Purkinje cells (Fig 4)NG2-glial cell somata often lie along the primary process ofBergmann glial cells and Purkinje cell primary dendrites(Fig 4A) NG2-glia extend processes both parallel to and per-pendicular to Purkinje cell dendritic trees (Fig 4A) and theirprocesses interdigitate with those of Bergmann glia andPurkinje cell dendrites (Fig 4BC) A single NG2-glial cell sub-serves multiple Bergmann glia and Purkinje cells and manyNG2-glia extend processes into both the molecular andgranule cell layers to form additional contacts with velate astro-cytes and granule neurons NG2-glia contact synapses betweenclimbing fibres and Purkinje neurons but they appear to formfunctional contacts with only a small number of synapseswithin their domain (Lin et al 2005) whereas Bergmann gliaform many hundreds of microdomains that interact with thesynapses they ensheath (Grosche et al 1999 2002) Similarlya single hippocampal astrocyte can contact in excess of100000 synapses (Bushong et al 2002) and cortical astrocyteswrap on average four neuronal cell bodies and hundreds of den-drites (Halassa et al 2007b) In contrast NG2-glia do not havethe profuse fine processes of protoplasmic astrocytes and onaverage may form electrophysiologically active contacts withless than 20 synapses (Bergles et al 2000 Lin and Bergles2004 Ge et al 2009) However the intimate associationsbetween NG2-glia and astrocytes independent of neuronsboth on their somata and along their processes are sites of neu-rotransmitter mediated glia-to-glia calcium signalling(Hamilton et al 2009) The overlapping domain organisationof NG2-glia and astrocytes may therefore be important for inte-grating glial and neuronal activity

C O M M U N I C A T I O N B E T W E E NN G 2 - G L I A A N D T H E N E U R O G L I A LN E T W O R K

Electrophysiological studies show that NG2-glia express gluta-mate and GABA receptors and respond to neuronally releasedneurotransmitters (Bergles et al 2000 Lin and Bergles 2004

Fig 5 Synaptophysin is localised to sites of close apposition betweenNG2-glia and astrocytes Immunolabelling for NG2 (red) andsynaptophysin (blue) in the hippocampus of GFAP-EGFP mice Confocalimages are shown in three channels (A) and with isosurface rendering (BC)The processes of NG2-glia and astrocytes are associated with a large densityof synapses (AB) There is immediate apposition of the NG2-glial cell andastrocyte cell bodies and processes and synaptophysin is localised to thesesites of contact (arrows) indicating sites of synaptic communication ontoboth NG2-glia and astrocytes In addition synaptophysin was localisedwithin the soma of the NG2-glia cell confirming our recent report(Hamilton et al 2009) and suggesting NG2-glia are capable of bidirectionalcommunication with astrocytes and neurons Scale bar frac14 20 mm (A) 5 mm(B) and 1 mm (C)

integration of ng2-glia (synantocytes) into the neuroglial network 25

httpsdoiorg101017S1740925X09990329Downloaded from httpswwwcambridgeorgcore Open University Library on 04 Feb 2017 at 144254 subject to the Cambridge Core terms of use available at httpswwwcambridgeorgcoreterms

Lin et al 2005) Calcium imaging and immunocytochemistryshow that NG2-glia express functional AMPA-type glutamatereceptors and metabotropic P2Y and ionotropic P2X recep-tors including the P2X7 subtype (Ge et al 2006 Hamiltonet al 2009 Wang et al 2009) Cerebellar NG2-glia candisplay both GABAergic and glutamatergic inputs (Lin andBergles 2004 Karadottir et al 2005) consistent with ourobservations that individual NG2-glia traverse the cell layersof the cerebellum to contact glutamatergic and GABAergicelements In addition we have used immunolabelling forsynaptophysin to examine the relations of NG2-glia withsynapses (Fig 5) The density of synaptophysin immunolabel-ling in the grey matter precludes the quantification of specificsynapses contacted by NG2-glia (Fig 5A) Nevertheless ourresults provide evidence that NG2-glia and astrocytescontact the same neurons (Figs 3ndash5) and the immediate

apposition of synaptophysin to points of contact betweenNG2-glia and astrocytes suggests they may be integrated atthe same synapses (Fig 5) Moreover synaptophysin is local-ised to sites of immediate apposition between astrocytesand NG2-glia and a region of particular interest is theexpression of synaptophysin between the NG2-glial cellbody and an astrocyte process (Fig 5B) Isoforming at theintegral communication site between the NG2-glial cell andastrocyte process provides further evidence of the localisationof synaptophysin to the NG2-glial cell body and to the inter-face between NG2-glia and astrocytes (Fig 5C) The resultspresented above show that the cell somata of astrocytes andNG2-glia are often directly apposed and that their processesexhibit regions where they tightly ensheath each other (Figs1B inset 1G inset 3BC and 4BC) The localisation of synap-tophysin to these areas indicates these are potential sits of

Fig 6 NG2-glia are in direct contact with pericytes NG2-glia are marked by an asterisk in each image Images show immunolabelling for NG2 in optic nerveof non-transgenic mice (AB) and NG2-glia and pericytes in sections of NG2-dsRed mouse brain (CndashE) NG2-glia contact multiple pericytes and in many casestheir cell somata appear to be in direct contact (CD) and this is confirmed in cross sectional images (E) Scale frac14 10 mm (A) 12 mm (B) 5 mm (C) 8 mm (D) and10 mm (E)

26 rebekah wigley and arthur m butt

httpsdoiorg101017S1740925X09990329Downloaded from httpswwwcambridgeorgcore Open University Library on 04 Feb 2017 at 144254 subject to the Cambridge Core terms of use available at httpswwwcambridgeorgcoreterms

vesicular communication between the two cell typesAstrocytes have mechanisms to release neurotransmitters viavesicles (Parpura et al 1994 Araque et al 1999) We haveprovided evidence that astrocytes also signal onto NG2-gliavia glutamate and ATP (Hamilton et al 2008 2009)Moreover the evidence that NG2-glia express synaptophysinindicates they may be capable of neurotransmitter release andbidirectional communication with astrocytes and neurons

R E L A T I O N S B E T W E E N N G 2 - G L I AA N D T H E C E R E B R A LV A S C U L A T U R E

Astrocytes are able to lsquosensersquo changes in neuronal activity andtransmit signals to the vasculature to regulate local blood flowand provide metabolic support to neurons (Halassa et al2007a) Astrocytes may use glutamate signalling mechanismsto communicate with pericytes on blood vessels that are ableto regulate cerebral blood flow (Peppiatt et al 2006)NG2-glia also contact pericytes on cerebral blood vessels(Fig 6) and they contact astrocytes and neurons withintheir domains (Figs 1ndash5) and express synaptophysin(Hamilton et al 2009) and may therefore play a role in reg-ulating blood flow NG2-glia and pericytes both express theNG2 CSPG (Ozerdem et al 2002) and the two cell typesare distinguished by their morphology NG2-glia as stellateprocess-bearing cells and pericytes as perivascular cells withtwo or more primary processes extending along bloodvessels According to these distinguishing features it can beseen that NG2-glia and pericytes lie in very close proximityto each other (Fig 6AB) The processes of NG2-glia contactpericyte processes on the blood vessel (Fig 6A) In somecases it appears that the pericyte cell soma extends outfrom the capillary to almost contact that of a neighbouringNG2-glial cell (Fig 6B) and in many cases NG2-glial cellsomata are directly apposed to blood vessels identified byimmunolabelling (Fig 6C) and in the NG2-DsRed transgenicmouse (Fig 6DE) Pericytes have a neurogenic potential invitro (Dore-Duffy et al 2006) and proliferate in response toinjury (Gerhardt and Betsholtz 2003) and the intimateassociations between NG2-glia and pericytes is suggestive ofa lineage relationship between the two cell types (Wigleyand Butt unpublished observations) It remains to be seenwhether NG2-glia communicate with pericytes and help regu-late blood flow but this is a possibility since they expresssynaptophysin and contact synapses astrocytes and pericytes

C O N C L U S I O N S

NG2-glia and astrocytes form overlapping neuronndashglialndashvascular domains Within these domains NG2-glia and astro-cytes contact the same neurons and most likely the samesynapses but they perform different functions Astrocyte pro-cesses envelop synapses and one of their main functions is theremoval of excess neurotransmitters NG2-glia form fine con-nections with synapses and do not express transporters andtheir function at synapses is unknown The large domains ofNG2-glia enable them to lsquosensersquo neuronal activity frommany neurons over a large area that overlaps with thedomains of multiple astrocytes Their relation with pericytesindicates that NG2-glia and astrocytes form parallel

communications pathways between neurons and bloodvessels which may be important in the regulation of bloodflow The evidence that NG2-glia also receive input fromastrocytes and the possibility that NG2-glia express mechan-isms for vesicular release of gliotransmitters raises the possi-bility that NG2-glia may also have an output and be able tofeed back to neurons or astrocytes The fact that NG2-gliaand astrocytes form parallel communications pathwayssuggests they have different functions since otherwise thislevel of redundancy is difficult to explain Astrocyte domainorganisation segregates intercellular communication withinneuronal circuits (Houades et al 2008) and astrocytes arecoupled by gap junctions that provide an activity-dependentintercellular pathway for transport between blood vesselsand neurons (Rouach et al 2008) NG2-glia are not coupledby gap junctions but by forming lsquosynapsesrsquo with neuronsastrocytes and pericytes they connect otherwise segregatedastroglial domains An important question for future studiesis to determine how these multiple inputs are integrated inindividual NG2-glia and whether they have a physiologicaloutput

A C K N O W L E D G E M E N T S

Supported by the BBSRC and INTERRIG

Statement of interestNone

R E F E R E N C E S

Aguirre AA Chittajallu R Belachew S and Gallo V (2004)NG2-expressing cells in the subventricular zone are type C-like cellsand contribute to interneuron generation in the postnatal hippo-campus Journal of Cell Biology 165 575ndash589

Araque A Parpura V Sanzgiri RP and Haydon PG (1999) Tripartitesynapses glia the unacknowledged partner Trends in Neuroscience 22208ndash215

Bakiri Y Attwell D and Karadottir R (2009) Electrical signalling prop-erties of oligodendrocyte precursor cells Neuron Glia Biology101017S1740925X09990202

Belachew S Chittajallu R Aguirre AA Yuan X Kirby M AndersonS et al (2003) Postnatal NG2 proteoglycan-expressing progenitorcells are intrinsically multipotent and generate functional neuronsJournal of Cell Biology 161 169ndash186

Bergles DE Roberts JD Somogyi P and Jahr CE (2000)Glutamatergic synapses on oligodendrocyte precursor cells in thehippocampus Nature 405 187ndash191

Bushong EA Martone ME Jones YZ and Ellisman MH (2002)Protoplasmic astrocytes in CA1 stratum radiatum occupy separateanatomical domains Journal of Neuroscience 22 183ndash192

Butt AM Duncan A Hornby MF Kirvell SL Hunter A LevineJM et al (1999) Cells expressing the NG2 antigen contact nodes ofRanvier in adult CNS white matter Glia 26 84ndash91

Butt AM Hamilton N Hubbard P Pugh M and Ibrahim M (2005)Synantocytes the fifth element Journal of Anatomy 207 695ndash706

Butt AM Kiff J Hubbard P and Berry M (2002) Synantocytes newfunctions for novel NG2 expressing glia Journal of Neurocytology31 551ndash565

integration of ng2-glia (synantocytes) into the neuroglial network 27

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Dayer AG Cleaver KM Abouantoun T and Cameron HA (2005)New GABAergic interneurons in the adult neocortex and striatumare generated from different precursors Journal of Cell Biology 168415ndash427

Dore-Duffy P Katychev A Wang X and Van Buren E (2006) CNSmicrovascular pericytes exhibit multipotential stem cell activityJournal of Cerebral Blood Flow and Metabolism 26 613ndash624

Ge WP Yang XJ Zhang Z Wang HK Shen W Deng QD et al(2006) Long-term potentiation of neuron-glia synapses mediated byCa2thorn-permeable AMPA receptors Science 312 1533ndash1537

Ge WP Zhou W Luo Q Jan LY and Jan YN (2009) Dividing glialcells maintain differentiated properties including complex mor-phology and functional synapses Proceedings of the NationalAcademy of Sciences of the USA 106 328ndash333

Gerhardt H and Betsholtz C (2003) Endothelial-pericyte interactions inangiogenesis Cell Tissue Research 314 15ndash23

Grass D Pawlowski PG Hirrlinger J Papadopoulos N RichterDW Kirchhoff F et al (2004) Diversity of functional astroglialproperties in the respiratory network Journal of Neuroscience 241358ndash1365

Grosche J Kettenmann H and Reichenbach A (2002) Bergmann glialcells form distinct morphological structures to interact with cerebellarneurons Journal of Neuroscience Research 68 138ndash149

Grosche J Matyash V Moller T Verkhratsky A Reichenbach A andKettenmann H (1999) Microdomains for neuron-glia interactionparallel fiber signaling to Bergmann glial cells Nature Neuroscience2 139ndash143

Halassa MM Fellin T and Haydon PG (2007a) The tripartite synapseroles for gliotransmission in health and disease Trends in MolecularMedicine 13 54ndash63

Halassa MM Fellin T Takano H Dong JH and Haydon PG(2007b) Synaptic islands defined by the territory of a single astrocyteJournal of Neuroscience 27 6473ndash6477

Hamilton N Vayro S Kirchhoff F Verkhratsky A Robbins J GoreckiD et al (2008) Mechanisms of ATP- and glutamate-mediated calciumsignaling in white matter astrocytes Glia 56 734ndash749

Hamilton N Vayro S Wigley R Robbins J and Butt AM (2009)Axons and astrocytes release glutamate and ATP to evoke calciumsignals in NG2-glia Glia in press

Houades V Koulakoff A Ezan P Seif I and Giaume C (2008) Gapjunction-mediated astrocytic networks in the mouse barrel cortexJournal of Neuroscience 28 5207ndash5217

Karadottir R Cavelier P Bergersen LH and Attwell D (2005) NMDAreceptors are expressed in oligodendrocytes and activated in ischae-mia Nature 438 1162ndash1166

Karadottir R Hamilton NB Bakiri Y and Attwell D (2008) Spikingand nonspiking classes of oligodendrocyte precursor glia in CNS whitematter Nature Neuroscience 11 450ndash456

Leoni G Rattray M and Butt AM (2009) NG2 cells differentiate intoastrocytes in cerebellar slices Molecular and Cellular Neurosciences42 208ndash218

Lin SC and Bergles DE (2004) Synaptic signaling between GABAergicinterneurons and oligodendrocyte precursor cells in the hippocampusNature Neuroscience 7 24ndash32

Lin SC Huck JH Roberts JD Macklin WB Somogyi P andBergles DE (2005) Climbing fiber innervation of NG2-expressingglia in the mammalian cerebellum Neuron 46 773ndash785

Nedergaard M Ransom B and Goldman SA (2003) New roles forastrocytes redefining the functional architecture of the brain Trendsin Neuroscience 26 523ndash530

Nishiyama A Lin X Giese N Heldin C and Stallcup W (1996)Co-localization of NG2 proteoglycan and PDGF alpha-receptor onO2A progenitor cells in the developing rat brain Journal ofNeuroscience Research 43 299ndash314

Nolte C Matyash M Pivneva T Schipke CG Ohlemeyer C HanischUK et al (2001) GFAP promoter-controlled EGFP-expressing trans-genic mice a tool to visualize astrocytes and astrogliosis in living braintissue Glia 33 72ndash86

Oberheim NA Wang X Goldman S and Nedergaard M (2006)Astrocytic complexity distinguishes the human brain Trends inNeuroscience 29 547ndash553

Ozerdem U Monosov E and Stallcup WB (2002) NG2 proteoglycanexpression by pericytes in pathological microvasculatureMicrovascular Research 63 129ndash134

Parpura V Basarsky TA Liu F Jeftinija K Jeftinija S and HaydonPG (1994) Glutamate-mediated astrocyte-neuron signalling Nature369 744ndash747

Peppiatt CM Howarth C Mobbs P and Attwell D (2006)Bidirectional control of CNS capillary diameter by pericytes Nature443 700ndash704

Psachoulia K Young KM and Richardson WD (2009) Separatepopulations of dividing and non-dividing NG2 cells in the postnatalmouse brain Neuron Glia Biology in press

Rivers LE Young KM Rizzi M Jamen F Psachoulia K Wade Aet al (2008) PDGFRANG2 glia generate myelinating oligodendro-cytes and piriform projection neurons in adult mice NatureNeuroscience 11 1392ndash1401

Rouach N Koulakoff A Abudara V Willecke K and Giaume C(2008) Astroglial metabolic networks sustain hippocampal synaptictransmission Science 322 1551ndash1555

Wang LY Cai WQ Chen PH Deng QY and Zhao CM (2009)Downregulation of P2X7 receptor expression in rat oligodendrocyteprecursor cells after hypoxia ischemia Glia 57 307ndash319

Wigley R Hamilton N Nishiyama A Kirchhoff F and Butt AM(2007) Morphological and physiological interactions of NG2-gliawith astrocytes and neurons Journal of Anatomy 210 661ndash670

Zhu X Bergles DE and Nishiyama A (2008) NG2 cells generate botholigodendrocytes and gray matter astrocytes Development 135145ndash157

Ziskin JL Nishiyama A Rubio M Fukaya M and Bergles DE (2007)Vesicular release of glutamate from unmyelinated axons in whitematter Nature Neuroscience 10 321ndash330

A U T H O R S rsquo A D D R E S S

Institute for Biomedical and Biomolecular SciencesSchool of Pharmacy and Biomedical SciencesUniversity of PortsmouthUK

Correspondence should be addressed toArthur M ButtInstitute for Biomedical and Biomolecular SciencesSchool of Pharmacy and Biomedical SciencesUniversity of PortsmouthUKemail Arthurbuttportacuk

28 rebekah wigley and arthur m butt

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integration of the three elements (Fig 2) The processes ofNG2-glia like those of astrocytes are directly apposed tomost if not all neuronal somata within their domains(Fig 2A) It is important to note that our results demonstratethe close associations that NG2-glia form with other elementsin the CNS but it is not possible at the confocal microscopiclevel to distinguish whether these contacts are actually func-tional Nonetheless using a combination of electronmicroscopy and patch-clamp electrophysiology it has beenestimated that only a few of the neuronal contacts formed byNG2-glia are electrophysiologically active lsquosynapsesrsquo (Linet al 2005 Ge et al 2009) In contrast astrocytes form

many hundreds of active contacts with neuronal synapses(Araque et al 1999 Grosche et al 1999 Bushong et al2002) NG2-glia and astrocytes form intimate contacts withthe same neurons extending processes that almost completelycircumnavigate neuronal somata (Fig 2B) and perisynapticprocesses from both cells types form a latticework (reticulation)on the cell body (Fig 2Biv) The close apposition of NG2-gliaand astrocytes to the same neurons suggests they may contactthe same synapses but they subserve neurons in differentways Electron microscopy studies demonstrate the differentassociations made by NG2-glia and astrocytes with neuronsNG2-glia form specific points of synaptic like contacts withneurons (Bergles et al 2000) whereas astrocytes ensheathpre- and post-synaptic boutons (Nedergaard et al 2003)This close relationship between NG2-glia astrocytes andneurons is also reflected in the cerebellum (Fig 3) In thegranule cell layer individual neurons are contacted by astro-cytes and NG2-glial cell processes (Fig 3A) However whilstvelate astrocyte processes circumnavigate neuronal cellsomata the processes of NG2-glia extend radially to form enpassant associations with both neurons and astrocytes as theyweave around the somata (Fig 3BC) as noted above it is notcurrently known whether these contacts have any function incommunication In the molecular layer NG2-glia are closelyintegrated with Bergmann glia and Purkinje cells (Fig 4)NG2-glial cell somata often lie along the primary process ofBergmann glial cells and Purkinje cell primary dendrites(Fig 4A) NG2-glia extend processes both parallel to and per-pendicular to Purkinje cell dendritic trees (Fig 4A) and theirprocesses interdigitate with those of Bergmann glia andPurkinje cell dendrites (Fig 4BC) A single NG2-glial cell sub-serves multiple Bergmann glia and Purkinje cells and manyNG2-glia extend processes into both the molecular andgranule cell layers to form additional contacts with velate astro-cytes and granule neurons NG2-glia contact synapses betweenclimbing fibres and Purkinje neurons but they appear to formfunctional contacts with only a small number of synapseswithin their domain (Lin et al 2005) whereas Bergmann gliaform many hundreds of microdomains that interact with thesynapses they ensheath (Grosche et al 1999 2002) Similarlya single hippocampal astrocyte can contact in excess of100000 synapses (Bushong et al 2002) and cortical astrocyteswrap on average four neuronal cell bodies and hundreds of den-drites (Halassa et al 2007b) In contrast NG2-glia do not havethe profuse fine processes of protoplasmic astrocytes and onaverage may form electrophysiologically active contacts withless than 20 synapses (Bergles et al 2000 Lin and Bergles2004 Ge et al 2009) However the intimate associationsbetween NG2-glia and astrocytes independent of neuronsboth on their somata and along their processes are sites of neu-rotransmitter mediated glia-to-glia calcium signalling(Hamilton et al 2009) The overlapping domain organisationof NG2-glia and astrocytes may therefore be important for inte-grating glial and neuronal activity

C O M M U N I C A T I O N B E T W E E NN G 2 - G L I A A N D T H E N E U R O G L I A LN E T W O R K

Electrophysiological studies show that NG2-glia express gluta-mate and GABA receptors and respond to neuronally releasedneurotransmitters (Bergles et al 2000 Lin and Bergles 2004

Fig 5 Synaptophysin is localised to sites of close apposition betweenNG2-glia and astrocytes Immunolabelling for NG2 (red) andsynaptophysin (blue) in the hippocampus of GFAP-EGFP mice Confocalimages are shown in three channels (A) and with isosurface rendering (BC)The processes of NG2-glia and astrocytes are associated with a large densityof synapses (AB) There is immediate apposition of the NG2-glial cell andastrocyte cell bodies and processes and synaptophysin is localised to thesesites of contact (arrows) indicating sites of synaptic communication ontoboth NG2-glia and astrocytes In addition synaptophysin was localisedwithin the soma of the NG2-glia cell confirming our recent report(Hamilton et al 2009) and suggesting NG2-glia are capable of bidirectionalcommunication with astrocytes and neurons Scale bar frac14 20 mm (A) 5 mm(B) and 1 mm (C)

integration of ng2-glia (synantocytes) into the neuroglial network 25

httpsdoiorg101017S1740925X09990329Downloaded from httpswwwcambridgeorgcore Open University Library on 04 Feb 2017 at 144254 subject to the Cambridge Core terms of use available at httpswwwcambridgeorgcoreterms

Lin et al 2005) Calcium imaging and immunocytochemistryshow that NG2-glia express functional AMPA-type glutamatereceptors and metabotropic P2Y and ionotropic P2X recep-tors including the P2X7 subtype (Ge et al 2006 Hamiltonet al 2009 Wang et al 2009) Cerebellar NG2-glia candisplay both GABAergic and glutamatergic inputs (Lin andBergles 2004 Karadottir et al 2005) consistent with ourobservations that individual NG2-glia traverse the cell layersof the cerebellum to contact glutamatergic and GABAergicelements In addition we have used immunolabelling forsynaptophysin to examine the relations of NG2-glia withsynapses (Fig 5) The density of synaptophysin immunolabel-ling in the grey matter precludes the quantification of specificsynapses contacted by NG2-glia (Fig 5A) Nevertheless ourresults provide evidence that NG2-glia and astrocytescontact the same neurons (Figs 3ndash5) and the immediate

apposition of synaptophysin to points of contact betweenNG2-glia and astrocytes suggests they may be integrated atthe same synapses (Fig 5) Moreover synaptophysin is local-ised to sites of immediate apposition between astrocytesand NG2-glia and a region of particular interest is theexpression of synaptophysin between the NG2-glial cellbody and an astrocyte process (Fig 5B) Isoforming at theintegral communication site between the NG2-glial cell andastrocyte process provides further evidence of the localisationof synaptophysin to the NG2-glial cell body and to the inter-face between NG2-glia and astrocytes (Fig 5C) The resultspresented above show that the cell somata of astrocytes andNG2-glia are often directly apposed and that their processesexhibit regions where they tightly ensheath each other (Figs1B inset 1G inset 3BC and 4BC) The localisation of synap-tophysin to these areas indicates these are potential sits of

Fig 6 NG2-glia are in direct contact with pericytes NG2-glia are marked by an asterisk in each image Images show immunolabelling for NG2 in optic nerveof non-transgenic mice (AB) and NG2-glia and pericytes in sections of NG2-dsRed mouse brain (CndashE) NG2-glia contact multiple pericytes and in many casestheir cell somata appear to be in direct contact (CD) and this is confirmed in cross sectional images (E) Scale frac14 10 mm (A) 12 mm (B) 5 mm (C) 8 mm (D) and10 mm (E)

26 rebekah wigley and arthur m butt

httpsdoiorg101017S1740925X09990329Downloaded from httpswwwcambridgeorgcore Open University Library on 04 Feb 2017 at 144254 subject to the Cambridge Core terms of use available at httpswwwcambridgeorgcoreterms

vesicular communication between the two cell typesAstrocytes have mechanisms to release neurotransmitters viavesicles (Parpura et al 1994 Araque et al 1999) We haveprovided evidence that astrocytes also signal onto NG2-gliavia glutamate and ATP (Hamilton et al 2008 2009)Moreover the evidence that NG2-glia express synaptophysinindicates they may be capable of neurotransmitter release andbidirectional communication with astrocytes and neurons

R E L A T I O N S B E T W E E N N G 2 - G L I AA N D T H E C E R E B R A LV A S C U L A T U R E

Astrocytes are able to lsquosensersquo changes in neuronal activity andtransmit signals to the vasculature to regulate local blood flowand provide metabolic support to neurons (Halassa et al2007a) Astrocytes may use glutamate signalling mechanismsto communicate with pericytes on blood vessels that are ableto regulate cerebral blood flow (Peppiatt et al 2006)NG2-glia also contact pericytes on cerebral blood vessels(Fig 6) and they contact astrocytes and neurons withintheir domains (Figs 1ndash5) and express synaptophysin(Hamilton et al 2009) and may therefore play a role in reg-ulating blood flow NG2-glia and pericytes both express theNG2 CSPG (Ozerdem et al 2002) and the two cell typesare distinguished by their morphology NG2-glia as stellateprocess-bearing cells and pericytes as perivascular cells withtwo or more primary processes extending along bloodvessels According to these distinguishing features it can beseen that NG2-glia and pericytes lie in very close proximityto each other (Fig 6AB) The processes of NG2-glia contactpericyte processes on the blood vessel (Fig 6A) In somecases it appears that the pericyte cell soma extends outfrom the capillary to almost contact that of a neighbouringNG2-glial cell (Fig 6B) and in many cases NG2-glial cellsomata are directly apposed to blood vessels identified byimmunolabelling (Fig 6C) and in the NG2-DsRed transgenicmouse (Fig 6DE) Pericytes have a neurogenic potential invitro (Dore-Duffy et al 2006) and proliferate in response toinjury (Gerhardt and Betsholtz 2003) and the intimateassociations between NG2-glia and pericytes is suggestive ofa lineage relationship between the two cell types (Wigleyand Butt unpublished observations) It remains to be seenwhether NG2-glia communicate with pericytes and help regu-late blood flow but this is a possibility since they expresssynaptophysin and contact synapses astrocytes and pericytes

C O N C L U S I O N S

NG2-glia and astrocytes form overlapping neuronndashglialndashvascular domains Within these domains NG2-glia and astro-cytes contact the same neurons and most likely the samesynapses but they perform different functions Astrocyte pro-cesses envelop synapses and one of their main functions is theremoval of excess neurotransmitters NG2-glia form fine con-nections with synapses and do not express transporters andtheir function at synapses is unknown The large domains ofNG2-glia enable them to lsquosensersquo neuronal activity frommany neurons over a large area that overlaps with thedomains of multiple astrocytes Their relation with pericytesindicates that NG2-glia and astrocytes form parallel

communications pathways between neurons and bloodvessels which may be important in the regulation of bloodflow The evidence that NG2-glia also receive input fromastrocytes and the possibility that NG2-glia express mechan-isms for vesicular release of gliotransmitters raises the possi-bility that NG2-glia may also have an output and be able tofeed back to neurons or astrocytes The fact that NG2-gliaand astrocytes form parallel communications pathwayssuggests they have different functions since otherwise thislevel of redundancy is difficult to explain Astrocyte domainorganisation segregates intercellular communication withinneuronal circuits (Houades et al 2008) and astrocytes arecoupled by gap junctions that provide an activity-dependentintercellular pathway for transport between blood vesselsand neurons (Rouach et al 2008) NG2-glia are not coupledby gap junctions but by forming lsquosynapsesrsquo with neuronsastrocytes and pericytes they connect otherwise segregatedastroglial domains An important question for future studiesis to determine how these multiple inputs are integrated inindividual NG2-glia and whether they have a physiologicaloutput

A C K N O W L E D G E M E N T S

Supported by the BBSRC and INTERRIG

Statement of interestNone

R E F E R E N C E S

Aguirre AA Chittajallu R Belachew S and Gallo V (2004)NG2-expressing cells in the subventricular zone are type C-like cellsand contribute to interneuron generation in the postnatal hippo-campus Journal of Cell Biology 165 575ndash589

Araque A Parpura V Sanzgiri RP and Haydon PG (1999) Tripartitesynapses glia the unacknowledged partner Trends in Neuroscience 22208ndash215

Bakiri Y Attwell D and Karadottir R (2009) Electrical signalling prop-erties of oligodendrocyte precursor cells Neuron Glia Biology101017S1740925X09990202

Belachew S Chittajallu R Aguirre AA Yuan X Kirby M AndersonS et al (2003) Postnatal NG2 proteoglycan-expressing progenitorcells are intrinsically multipotent and generate functional neuronsJournal of Cell Biology 161 169ndash186

Bergles DE Roberts JD Somogyi P and Jahr CE (2000)Glutamatergic synapses on oligodendrocyte precursor cells in thehippocampus Nature 405 187ndash191

Bushong EA Martone ME Jones YZ and Ellisman MH (2002)Protoplasmic astrocytes in CA1 stratum radiatum occupy separateanatomical domains Journal of Neuroscience 22 183ndash192

Butt AM Duncan A Hornby MF Kirvell SL Hunter A LevineJM et al (1999) Cells expressing the NG2 antigen contact nodes ofRanvier in adult CNS white matter Glia 26 84ndash91

Butt AM Hamilton N Hubbard P Pugh M and Ibrahim M (2005)Synantocytes the fifth element Journal of Anatomy 207 695ndash706

Butt AM Kiff J Hubbard P and Berry M (2002) Synantocytes newfunctions for novel NG2 expressing glia Journal of Neurocytology31 551ndash565

integration of ng2-glia (synantocytes) into the neuroglial network 27

httpsdoiorg101017S1740925X09990329Downloaded from httpswwwcambridgeorgcore Open University Library on 04 Feb 2017 at 144254 subject to the Cambridge Core terms of use available at httpswwwcambridgeorgcoreterms

Dayer AG Cleaver KM Abouantoun T and Cameron HA (2005)New GABAergic interneurons in the adult neocortex and striatumare generated from different precursors Journal of Cell Biology 168415ndash427

Dore-Duffy P Katychev A Wang X and Van Buren E (2006) CNSmicrovascular pericytes exhibit multipotential stem cell activityJournal of Cerebral Blood Flow and Metabolism 26 613ndash624

Ge WP Yang XJ Zhang Z Wang HK Shen W Deng QD et al(2006) Long-term potentiation of neuron-glia synapses mediated byCa2thorn-permeable AMPA receptors Science 312 1533ndash1537

Ge WP Zhou W Luo Q Jan LY and Jan YN (2009) Dividing glialcells maintain differentiated properties including complex mor-phology and functional synapses Proceedings of the NationalAcademy of Sciences of the USA 106 328ndash333

Gerhardt H and Betsholtz C (2003) Endothelial-pericyte interactions inangiogenesis Cell Tissue Research 314 15ndash23

Grass D Pawlowski PG Hirrlinger J Papadopoulos N RichterDW Kirchhoff F et al (2004) Diversity of functional astroglialproperties in the respiratory network Journal of Neuroscience 241358ndash1365

Grosche J Kettenmann H and Reichenbach A (2002) Bergmann glialcells form distinct morphological structures to interact with cerebellarneurons Journal of Neuroscience Research 68 138ndash149

Grosche J Matyash V Moller T Verkhratsky A Reichenbach A andKettenmann H (1999) Microdomains for neuron-glia interactionparallel fiber signaling to Bergmann glial cells Nature Neuroscience2 139ndash143

Halassa MM Fellin T and Haydon PG (2007a) The tripartite synapseroles for gliotransmission in health and disease Trends in MolecularMedicine 13 54ndash63

Halassa MM Fellin T Takano H Dong JH and Haydon PG(2007b) Synaptic islands defined by the territory of a single astrocyteJournal of Neuroscience 27 6473ndash6477

Hamilton N Vayro S Kirchhoff F Verkhratsky A Robbins J GoreckiD et al (2008) Mechanisms of ATP- and glutamate-mediated calciumsignaling in white matter astrocytes Glia 56 734ndash749

Hamilton N Vayro S Wigley R Robbins J and Butt AM (2009)Axons and astrocytes release glutamate and ATP to evoke calciumsignals in NG2-glia Glia in press

Houades V Koulakoff A Ezan P Seif I and Giaume C (2008) Gapjunction-mediated astrocytic networks in the mouse barrel cortexJournal of Neuroscience 28 5207ndash5217

Karadottir R Cavelier P Bergersen LH and Attwell D (2005) NMDAreceptors are expressed in oligodendrocytes and activated in ischae-mia Nature 438 1162ndash1166

Karadottir R Hamilton NB Bakiri Y and Attwell D (2008) Spikingand nonspiking classes of oligodendrocyte precursor glia in CNS whitematter Nature Neuroscience 11 450ndash456

Leoni G Rattray M and Butt AM (2009) NG2 cells differentiate intoastrocytes in cerebellar slices Molecular and Cellular Neurosciences42 208ndash218

Lin SC and Bergles DE (2004) Synaptic signaling between GABAergicinterneurons and oligodendrocyte precursor cells in the hippocampusNature Neuroscience 7 24ndash32

Lin SC Huck JH Roberts JD Macklin WB Somogyi P andBergles DE (2005) Climbing fiber innervation of NG2-expressingglia in the mammalian cerebellum Neuron 46 773ndash785

Nedergaard M Ransom B and Goldman SA (2003) New roles forastrocytes redefining the functional architecture of the brain Trendsin Neuroscience 26 523ndash530

Nishiyama A Lin X Giese N Heldin C and Stallcup W (1996)Co-localization of NG2 proteoglycan and PDGF alpha-receptor onO2A progenitor cells in the developing rat brain Journal ofNeuroscience Research 43 299ndash314

Nolte C Matyash M Pivneva T Schipke CG Ohlemeyer C HanischUK et al (2001) GFAP promoter-controlled EGFP-expressing trans-genic mice a tool to visualize astrocytes and astrogliosis in living braintissue Glia 33 72ndash86

Oberheim NA Wang X Goldman S and Nedergaard M (2006)Astrocytic complexity distinguishes the human brain Trends inNeuroscience 29 547ndash553

Ozerdem U Monosov E and Stallcup WB (2002) NG2 proteoglycanexpression by pericytes in pathological microvasculatureMicrovascular Research 63 129ndash134

Parpura V Basarsky TA Liu F Jeftinija K Jeftinija S and HaydonPG (1994) Glutamate-mediated astrocyte-neuron signalling Nature369 744ndash747

Peppiatt CM Howarth C Mobbs P and Attwell D (2006)Bidirectional control of CNS capillary diameter by pericytes Nature443 700ndash704

Psachoulia K Young KM and Richardson WD (2009) Separatepopulations of dividing and non-dividing NG2 cells in the postnatalmouse brain Neuron Glia Biology in press

Rivers LE Young KM Rizzi M Jamen F Psachoulia K Wade Aet al (2008) PDGFRANG2 glia generate myelinating oligodendro-cytes and piriform projection neurons in adult mice NatureNeuroscience 11 1392ndash1401

Rouach N Koulakoff A Abudara V Willecke K and Giaume C(2008) Astroglial metabolic networks sustain hippocampal synaptictransmission Science 322 1551ndash1555

Wang LY Cai WQ Chen PH Deng QY and Zhao CM (2009)Downregulation of P2X7 receptor expression in rat oligodendrocyteprecursor cells after hypoxia ischemia Glia 57 307ndash319

Wigley R Hamilton N Nishiyama A Kirchhoff F and Butt AM(2007) Morphological and physiological interactions of NG2-gliawith astrocytes and neurons Journal of Anatomy 210 661ndash670

Zhu X Bergles DE and Nishiyama A (2008) NG2 cells generate botholigodendrocytes and gray matter astrocytes Development 135145ndash157

Ziskin JL Nishiyama A Rubio M Fukaya M and Bergles DE (2007)Vesicular release of glutamate from unmyelinated axons in whitematter Nature Neuroscience 10 321ndash330

A U T H O R S rsquo A D D R E S S

Institute for Biomedical and Biomolecular SciencesSchool of Pharmacy and Biomedical SciencesUniversity of PortsmouthUK

Correspondence should be addressed toArthur M ButtInstitute for Biomedical and Biomolecular SciencesSchool of Pharmacy and Biomedical SciencesUniversity of PortsmouthUKemail Arthurbuttportacuk

28 rebekah wigley and arthur m butt

httpsdoiorg101017S1740925X09990329Downloaded from httpswwwcambridgeorgcore Open University Library on 04 Feb 2017 at 144254 subject to the Cambridge Core terms of use available at httpswwwcambridgeorgcoreterms

Lin et al 2005) Calcium imaging and immunocytochemistryshow that NG2-glia express functional AMPA-type glutamatereceptors and metabotropic P2Y and ionotropic P2X recep-tors including the P2X7 subtype (Ge et al 2006 Hamiltonet al 2009 Wang et al 2009) Cerebellar NG2-glia candisplay both GABAergic and glutamatergic inputs (Lin andBergles 2004 Karadottir et al 2005) consistent with ourobservations that individual NG2-glia traverse the cell layersof the cerebellum to contact glutamatergic and GABAergicelements In addition we have used immunolabelling forsynaptophysin to examine the relations of NG2-glia withsynapses (Fig 5) The density of synaptophysin immunolabel-ling in the grey matter precludes the quantification of specificsynapses contacted by NG2-glia (Fig 5A) Nevertheless ourresults provide evidence that NG2-glia and astrocytescontact the same neurons (Figs 3ndash5) and the immediate

apposition of synaptophysin to points of contact betweenNG2-glia and astrocytes suggests they may be integrated atthe same synapses (Fig 5) Moreover synaptophysin is local-ised to sites of immediate apposition between astrocytesand NG2-glia and a region of particular interest is theexpression of synaptophysin between the NG2-glial cellbody and an astrocyte process (Fig 5B) Isoforming at theintegral communication site between the NG2-glial cell andastrocyte process provides further evidence of the localisationof synaptophysin to the NG2-glial cell body and to the inter-face between NG2-glia and astrocytes (Fig 5C) The resultspresented above show that the cell somata of astrocytes andNG2-glia are often directly apposed and that their processesexhibit regions where they tightly ensheath each other (Figs1B inset 1G inset 3BC and 4BC) The localisation of synap-tophysin to these areas indicates these are potential sits of

Fig 6 NG2-glia are in direct contact with pericytes NG2-glia are marked by an asterisk in each image Images show immunolabelling for NG2 in optic nerveof non-transgenic mice (AB) and NG2-glia and pericytes in sections of NG2-dsRed mouse brain (CndashE) NG2-glia contact multiple pericytes and in many casestheir cell somata appear to be in direct contact (CD) and this is confirmed in cross sectional images (E) Scale frac14 10 mm (A) 12 mm (B) 5 mm (C) 8 mm (D) and10 mm (E)

26 rebekah wigley and arthur m butt

httpsdoiorg101017S1740925X09990329Downloaded from httpswwwcambridgeorgcore Open University Library on 04 Feb 2017 at 144254 subject to the Cambridge Core terms of use available at httpswwwcambridgeorgcoreterms

vesicular communication between the two cell typesAstrocytes have mechanisms to release neurotransmitters viavesicles (Parpura et al 1994 Araque et al 1999) We haveprovided evidence that astrocytes also signal onto NG2-gliavia glutamate and ATP (Hamilton et al 2008 2009)Moreover the evidence that NG2-glia express synaptophysinindicates they may be capable of neurotransmitter release andbidirectional communication with astrocytes and neurons

R E L A T I O N S B E T W E E N N G 2 - G L I AA N D T H E C E R E B R A LV A S C U L A T U R E

Astrocytes are able to lsquosensersquo changes in neuronal activity andtransmit signals to the vasculature to regulate local blood flowand provide metabolic support to neurons (Halassa et al2007a) Astrocytes may use glutamate signalling mechanismsto communicate with pericytes on blood vessels that are ableto regulate cerebral blood flow (Peppiatt et al 2006)NG2-glia also contact pericytes on cerebral blood vessels(Fig 6) and they contact astrocytes and neurons withintheir domains (Figs 1ndash5) and express synaptophysin(Hamilton et al 2009) and may therefore play a role in reg-ulating blood flow NG2-glia and pericytes both express theNG2 CSPG (Ozerdem et al 2002) and the two cell typesare distinguished by their morphology NG2-glia as stellateprocess-bearing cells and pericytes as perivascular cells withtwo or more primary processes extending along bloodvessels According to these distinguishing features it can beseen that NG2-glia and pericytes lie in very close proximityto each other (Fig 6AB) The processes of NG2-glia contactpericyte processes on the blood vessel (Fig 6A) In somecases it appears that the pericyte cell soma extends outfrom the capillary to almost contact that of a neighbouringNG2-glial cell (Fig 6B) and in many cases NG2-glial cellsomata are directly apposed to blood vessels identified byimmunolabelling (Fig 6C) and in the NG2-DsRed transgenicmouse (Fig 6DE) Pericytes have a neurogenic potential invitro (Dore-Duffy et al 2006) and proliferate in response toinjury (Gerhardt and Betsholtz 2003) and the intimateassociations between NG2-glia and pericytes is suggestive ofa lineage relationship between the two cell types (Wigleyand Butt unpublished observations) It remains to be seenwhether NG2-glia communicate with pericytes and help regu-late blood flow but this is a possibility since they expresssynaptophysin and contact synapses astrocytes and pericytes

C O N C L U S I O N S

NG2-glia and astrocytes form overlapping neuronndashglialndashvascular domains Within these domains NG2-glia and astro-cytes contact the same neurons and most likely the samesynapses but they perform different functions Astrocyte pro-cesses envelop synapses and one of their main functions is theremoval of excess neurotransmitters NG2-glia form fine con-nections with synapses and do not express transporters andtheir function at synapses is unknown The large domains ofNG2-glia enable them to lsquosensersquo neuronal activity frommany neurons over a large area that overlaps with thedomains of multiple astrocytes Their relation with pericytesindicates that NG2-glia and astrocytes form parallel

communications pathways between neurons and bloodvessels which may be important in the regulation of bloodflow The evidence that NG2-glia also receive input fromastrocytes and the possibility that NG2-glia express mechan-isms for vesicular release of gliotransmitters raises the possi-bility that NG2-glia may also have an output and be able tofeed back to neurons or astrocytes The fact that NG2-gliaand astrocytes form parallel communications pathwayssuggests they have different functions since otherwise thislevel of redundancy is difficult to explain Astrocyte domainorganisation segregates intercellular communication withinneuronal circuits (Houades et al 2008) and astrocytes arecoupled by gap junctions that provide an activity-dependentintercellular pathway for transport between blood vesselsand neurons (Rouach et al 2008) NG2-glia are not coupledby gap junctions but by forming lsquosynapsesrsquo with neuronsastrocytes and pericytes they connect otherwise segregatedastroglial domains An important question for future studiesis to determine how these multiple inputs are integrated inindividual NG2-glia and whether they have a physiologicaloutput

A C K N O W L E D G E M E N T S

Supported by the BBSRC and INTERRIG

Statement of interestNone

R E F E R E N C E S

Aguirre AA Chittajallu R Belachew S and Gallo V (2004)NG2-expressing cells in the subventricular zone are type C-like cellsand contribute to interneuron generation in the postnatal hippo-campus Journal of Cell Biology 165 575ndash589

Araque A Parpura V Sanzgiri RP and Haydon PG (1999) Tripartitesynapses glia the unacknowledged partner Trends in Neuroscience 22208ndash215

Bakiri Y Attwell D and Karadottir R (2009) Electrical signalling prop-erties of oligodendrocyte precursor cells Neuron Glia Biology101017S1740925X09990202

Belachew S Chittajallu R Aguirre AA Yuan X Kirby M AndersonS et al (2003) Postnatal NG2 proteoglycan-expressing progenitorcells are intrinsically multipotent and generate functional neuronsJournal of Cell Biology 161 169ndash186

Bergles DE Roberts JD Somogyi P and Jahr CE (2000)Glutamatergic synapses on oligodendrocyte precursor cells in thehippocampus Nature 405 187ndash191

Bushong EA Martone ME Jones YZ and Ellisman MH (2002)Protoplasmic astrocytes in CA1 stratum radiatum occupy separateanatomical domains Journal of Neuroscience 22 183ndash192

Butt AM Duncan A Hornby MF Kirvell SL Hunter A LevineJM et al (1999) Cells expressing the NG2 antigen contact nodes ofRanvier in adult CNS white matter Glia 26 84ndash91

Butt AM Hamilton N Hubbard P Pugh M and Ibrahim M (2005)Synantocytes the fifth element Journal of Anatomy 207 695ndash706

Butt AM Kiff J Hubbard P and Berry M (2002) Synantocytes newfunctions for novel NG2 expressing glia Journal of Neurocytology31 551ndash565

integration of ng2-glia (synantocytes) into the neuroglial network 27

httpsdoiorg101017S1740925X09990329Downloaded from httpswwwcambridgeorgcore Open University Library on 04 Feb 2017 at 144254 subject to the Cambridge Core terms of use available at httpswwwcambridgeorgcoreterms

Dayer AG Cleaver KM Abouantoun T and Cameron HA (2005)New GABAergic interneurons in the adult neocortex and striatumare generated from different precursors Journal of Cell Biology 168415ndash427

Dore-Duffy P Katychev A Wang X and Van Buren E (2006) CNSmicrovascular pericytes exhibit multipotential stem cell activityJournal of Cerebral Blood Flow and Metabolism 26 613ndash624

Ge WP Yang XJ Zhang Z Wang HK Shen W Deng QD et al(2006) Long-term potentiation of neuron-glia synapses mediated byCa2thorn-permeable AMPA receptors Science 312 1533ndash1537

Ge WP Zhou W Luo Q Jan LY and Jan YN (2009) Dividing glialcells maintain differentiated properties including complex mor-phology and functional synapses Proceedings of the NationalAcademy of Sciences of the USA 106 328ndash333

Gerhardt H and Betsholtz C (2003) Endothelial-pericyte interactions inangiogenesis Cell Tissue Research 314 15ndash23

Grass D Pawlowski PG Hirrlinger J Papadopoulos N RichterDW Kirchhoff F et al (2004) Diversity of functional astroglialproperties in the respiratory network Journal of Neuroscience 241358ndash1365

Grosche J Kettenmann H and Reichenbach A (2002) Bergmann glialcells form distinct morphological structures to interact with cerebellarneurons Journal of Neuroscience Research 68 138ndash149

Grosche J Matyash V Moller T Verkhratsky A Reichenbach A andKettenmann H (1999) Microdomains for neuron-glia interactionparallel fiber signaling to Bergmann glial cells Nature Neuroscience2 139ndash143

Halassa MM Fellin T and Haydon PG (2007a) The tripartite synapseroles for gliotransmission in health and disease Trends in MolecularMedicine 13 54ndash63

Halassa MM Fellin T Takano H Dong JH and Haydon PG(2007b) Synaptic islands defined by the territory of a single astrocyteJournal of Neuroscience 27 6473ndash6477

Hamilton N Vayro S Kirchhoff F Verkhratsky A Robbins J GoreckiD et al (2008) Mechanisms of ATP- and glutamate-mediated calciumsignaling in white matter astrocytes Glia 56 734ndash749

Hamilton N Vayro S Wigley R Robbins J and Butt AM (2009)Axons and astrocytes release glutamate and ATP to evoke calciumsignals in NG2-glia Glia in press

Houades V Koulakoff A Ezan P Seif I and Giaume C (2008) Gapjunction-mediated astrocytic networks in the mouse barrel cortexJournal of Neuroscience 28 5207ndash5217

Karadottir R Cavelier P Bergersen LH and Attwell D (2005) NMDAreceptors are expressed in oligodendrocytes and activated in ischae-mia Nature 438 1162ndash1166

Karadottir R Hamilton NB Bakiri Y and Attwell D (2008) Spikingand nonspiking classes of oligodendrocyte precursor glia in CNS whitematter Nature Neuroscience 11 450ndash456

Leoni G Rattray M and Butt AM (2009) NG2 cells differentiate intoastrocytes in cerebellar slices Molecular and Cellular Neurosciences42 208ndash218

Lin SC and Bergles DE (2004) Synaptic signaling between GABAergicinterneurons and oligodendrocyte precursor cells in the hippocampusNature Neuroscience 7 24ndash32

Lin SC Huck JH Roberts JD Macklin WB Somogyi P andBergles DE (2005) Climbing fiber innervation of NG2-expressingglia in the mammalian cerebellum Neuron 46 773ndash785

Nedergaard M Ransom B and Goldman SA (2003) New roles forastrocytes redefining the functional architecture of the brain Trendsin Neuroscience 26 523ndash530

Nishiyama A Lin X Giese N Heldin C and Stallcup W (1996)Co-localization of NG2 proteoglycan and PDGF alpha-receptor onO2A progenitor cells in the developing rat brain Journal ofNeuroscience Research 43 299ndash314

Nolte C Matyash M Pivneva T Schipke CG Ohlemeyer C HanischUK et al (2001) GFAP promoter-controlled EGFP-expressing trans-genic mice a tool to visualize astrocytes and astrogliosis in living braintissue Glia 33 72ndash86

Oberheim NA Wang X Goldman S and Nedergaard M (2006)Astrocytic complexity distinguishes the human brain Trends inNeuroscience 29 547ndash553

Ozerdem U Monosov E and Stallcup WB (2002) NG2 proteoglycanexpression by pericytes in pathological microvasculatureMicrovascular Research 63 129ndash134

Parpura V Basarsky TA Liu F Jeftinija K Jeftinija S and HaydonPG (1994) Glutamate-mediated astrocyte-neuron signalling Nature369 744ndash747

Peppiatt CM Howarth C Mobbs P and Attwell D (2006)Bidirectional control of CNS capillary diameter by pericytes Nature443 700ndash704

Psachoulia K Young KM and Richardson WD (2009) Separatepopulations of dividing and non-dividing NG2 cells in the postnatalmouse brain Neuron Glia Biology in press

Rivers LE Young KM Rizzi M Jamen F Psachoulia K Wade Aet al (2008) PDGFRANG2 glia generate myelinating oligodendro-cytes and piriform projection neurons in adult mice NatureNeuroscience 11 1392ndash1401

Rouach N Koulakoff A Abudara V Willecke K and Giaume C(2008) Astroglial metabolic networks sustain hippocampal synaptictransmission Science 322 1551ndash1555

Wang LY Cai WQ Chen PH Deng QY and Zhao CM (2009)Downregulation of P2X7 receptor expression in rat oligodendrocyteprecursor cells after hypoxia ischemia Glia 57 307ndash319

Wigley R Hamilton N Nishiyama A Kirchhoff F and Butt AM(2007) Morphological and physiological interactions of NG2-gliawith astrocytes and neurons Journal of Anatomy 210 661ndash670

Zhu X Bergles DE and Nishiyama A (2008) NG2 cells generate botholigodendrocytes and gray matter astrocytes Development 135145ndash157

Ziskin JL Nishiyama A Rubio M Fukaya M and Bergles DE (2007)Vesicular release of glutamate from unmyelinated axons in whitematter Nature Neuroscience 10 321ndash330

A U T H O R S rsquo A D D R E S S

Institute for Biomedical and Biomolecular SciencesSchool of Pharmacy and Biomedical SciencesUniversity of PortsmouthUK

Correspondence should be addressed toArthur M ButtInstitute for Biomedical and Biomolecular SciencesSchool of Pharmacy and Biomedical SciencesUniversity of PortsmouthUKemail Arthurbuttportacuk

28 rebekah wigley and arthur m butt

httpsdoiorg101017S1740925X09990329Downloaded from httpswwwcambridgeorgcore Open University Library on 04 Feb 2017 at 144254 subject to the Cambridge Core terms of use available at httpswwwcambridgeorgcoreterms

vesicular communication between the two cell typesAstrocytes have mechanisms to release neurotransmitters viavesicles (Parpura et al 1994 Araque et al 1999) We haveprovided evidence that astrocytes also signal onto NG2-gliavia glutamate and ATP (Hamilton et al 2008 2009)Moreover the evidence that NG2-glia express synaptophysinindicates they may be capable of neurotransmitter release andbidirectional communication with astrocytes and neurons

R E L A T I O N S B E T W E E N N G 2 - G L I AA N D T H E C E R E B R A LV A S C U L A T U R E

Astrocytes are able to lsquosensersquo changes in neuronal activity andtransmit signals to the vasculature to regulate local blood flowand provide metabolic support to neurons (Halassa et al2007a) Astrocytes may use glutamate signalling mechanismsto communicate with pericytes on blood vessels that are ableto regulate cerebral blood flow (Peppiatt et al 2006)NG2-glia also contact pericytes on cerebral blood vessels(Fig 6) and they contact astrocytes and neurons withintheir domains (Figs 1ndash5) and express synaptophysin(Hamilton et al 2009) and may therefore play a role in reg-ulating blood flow NG2-glia and pericytes both express theNG2 CSPG (Ozerdem et al 2002) and the two cell typesare distinguished by their morphology NG2-glia as stellateprocess-bearing cells and pericytes as perivascular cells withtwo or more primary processes extending along bloodvessels According to these distinguishing features it can beseen that NG2-glia and pericytes lie in very close proximityto each other (Fig 6AB) The processes of NG2-glia contactpericyte processes on the blood vessel (Fig 6A) In somecases it appears that the pericyte cell soma extends outfrom the capillary to almost contact that of a neighbouringNG2-glial cell (Fig 6B) and in many cases NG2-glial cellsomata are directly apposed to blood vessels identified byimmunolabelling (Fig 6C) and in the NG2-DsRed transgenicmouse (Fig 6DE) Pericytes have a neurogenic potential invitro (Dore-Duffy et al 2006) and proliferate in response toinjury (Gerhardt and Betsholtz 2003) and the intimateassociations between NG2-glia and pericytes is suggestive ofa lineage relationship between the two cell types (Wigleyand Butt unpublished observations) It remains to be seenwhether NG2-glia communicate with pericytes and help regu-late blood flow but this is a possibility since they expresssynaptophysin and contact synapses astrocytes and pericytes

C O N C L U S I O N S

NG2-glia and astrocytes form overlapping neuronndashglialndashvascular domains Within these domains NG2-glia and astro-cytes contact the same neurons and most likely the samesynapses but they perform different functions Astrocyte pro-cesses envelop synapses and one of their main functions is theremoval of excess neurotransmitters NG2-glia form fine con-nections with synapses and do not express transporters andtheir function at synapses is unknown The large domains ofNG2-glia enable them to lsquosensersquo neuronal activity frommany neurons over a large area that overlaps with thedomains of multiple astrocytes Their relation with pericytesindicates that NG2-glia and astrocytes form parallel

communications pathways between neurons and bloodvessels which may be important in the regulation of bloodflow The evidence that NG2-glia also receive input fromastrocytes and the possibility that NG2-glia express mechan-isms for vesicular release of gliotransmitters raises the possi-bility that NG2-glia may also have an output and be able tofeed back to neurons or astrocytes The fact that NG2-gliaand astrocytes form parallel communications pathwayssuggests they have different functions since otherwise thislevel of redundancy is difficult to explain Astrocyte domainorganisation segregates intercellular communication withinneuronal circuits (Houades et al 2008) and astrocytes arecoupled by gap junctions that provide an activity-dependentintercellular pathway for transport between blood vesselsand neurons (Rouach et al 2008) NG2-glia are not coupledby gap junctions but by forming lsquosynapsesrsquo with neuronsastrocytes and pericytes they connect otherwise segregatedastroglial domains An important question for future studiesis to determine how these multiple inputs are integrated inindividual NG2-glia and whether they have a physiologicaloutput

A C K N O W L E D G E M E N T S

Supported by the BBSRC and INTERRIG

Statement of interestNone

R E F E R E N C E S

Aguirre AA Chittajallu R Belachew S and Gallo V (2004)NG2-expressing cells in the subventricular zone are type C-like cellsand contribute to interneuron generation in the postnatal hippo-campus Journal of Cell Biology 165 575ndash589

Araque A Parpura V Sanzgiri RP and Haydon PG (1999) Tripartitesynapses glia the unacknowledged partner Trends in Neuroscience 22208ndash215

Bakiri Y Attwell D and Karadottir R (2009) Electrical signalling prop-erties of oligodendrocyte precursor cells Neuron Glia Biology101017S1740925X09990202

Belachew S Chittajallu R Aguirre AA Yuan X Kirby M AndersonS et al (2003) Postnatal NG2 proteoglycan-expressing progenitorcells are intrinsically multipotent and generate functional neuronsJournal of Cell Biology 161 169ndash186

Bergles DE Roberts JD Somogyi P and Jahr CE (2000)Glutamatergic synapses on oligodendrocyte precursor cells in thehippocampus Nature 405 187ndash191

Bushong EA Martone ME Jones YZ and Ellisman MH (2002)Protoplasmic astrocytes in CA1 stratum radiatum occupy separateanatomical domains Journal of Neuroscience 22 183ndash192

Butt AM Duncan A Hornby MF Kirvell SL Hunter A LevineJM et al (1999) Cells expressing the NG2 antigen contact nodes ofRanvier in adult CNS white matter Glia 26 84ndash91

Butt AM Hamilton N Hubbard P Pugh M and Ibrahim M (2005)Synantocytes the fifth element Journal of Anatomy 207 695ndash706

Butt AM Kiff J Hubbard P and Berry M (2002) Synantocytes newfunctions for novel NG2 expressing glia Journal of Neurocytology31 551ndash565

integration of ng2-glia (synantocytes) into the neuroglial network 27

httpsdoiorg101017S1740925X09990329Downloaded from httpswwwcambridgeorgcore Open University Library on 04 Feb 2017 at 144254 subject to the Cambridge Core terms of use available at httpswwwcambridgeorgcoreterms

Dayer AG Cleaver KM Abouantoun T and Cameron HA (2005)New GABAergic interneurons in the adult neocortex and striatumare generated from different precursors Journal of Cell Biology 168415ndash427

Dore-Duffy P Katychev A Wang X and Van Buren E (2006) CNSmicrovascular pericytes exhibit multipotential stem cell activityJournal of Cerebral Blood Flow and Metabolism 26 613ndash624

Ge WP Yang XJ Zhang Z Wang HK Shen W Deng QD et al(2006) Long-term potentiation of neuron-glia synapses mediated byCa2thorn-permeable AMPA receptors Science 312 1533ndash1537

Ge WP Zhou W Luo Q Jan LY and Jan YN (2009) Dividing glialcells maintain differentiated properties including complex mor-phology and functional synapses Proceedings of the NationalAcademy of Sciences of the USA 106 328ndash333

Gerhardt H and Betsholtz C (2003) Endothelial-pericyte interactions inangiogenesis Cell Tissue Research 314 15ndash23

Grass D Pawlowski PG Hirrlinger J Papadopoulos N RichterDW Kirchhoff F et al (2004) Diversity of functional astroglialproperties in the respiratory network Journal of Neuroscience 241358ndash1365

Grosche J Kettenmann H and Reichenbach A (2002) Bergmann glialcells form distinct morphological structures to interact with cerebellarneurons Journal of Neuroscience Research 68 138ndash149

Grosche J Matyash V Moller T Verkhratsky A Reichenbach A andKettenmann H (1999) Microdomains for neuron-glia interactionparallel fiber signaling to Bergmann glial cells Nature Neuroscience2 139ndash143

Halassa MM Fellin T and Haydon PG (2007a) The tripartite synapseroles for gliotransmission in health and disease Trends in MolecularMedicine 13 54ndash63

Halassa MM Fellin T Takano H Dong JH and Haydon PG(2007b) Synaptic islands defined by the territory of a single astrocyteJournal of Neuroscience 27 6473ndash6477

Hamilton N Vayro S Kirchhoff F Verkhratsky A Robbins J GoreckiD et al (2008) Mechanisms of ATP- and glutamate-mediated calciumsignaling in white matter astrocytes Glia 56 734ndash749

Hamilton N Vayro S Wigley R Robbins J and Butt AM (2009)Axons and astrocytes release glutamate and ATP to evoke calciumsignals in NG2-glia Glia in press

Houades V Koulakoff A Ezan P Seif I and Giaume C (2008) Gapjunction-mediated astrocytic networks in the mouse barrel cortexJournal of Neuroscience 28 5207ndash5217

Karadottir R Cavelier P Bergersen LH and Attwell D (2005) NMDAreceptors are expressed in oligodendrocytes and activated in ischae-mia Nature 438 1162ndash1166

Karadottir R Hamilton NB Bakiri Y and Attwell D (2008) Spikingand nonspiking classes of oligodendrocyte precursor glia in CNS whitematter Nature Neuroscience 11 450ndash456

Leoni G Rattray M and Butt AM (2009) NG2 cells differentiate intoastrocytes in cerebellar slices Molecular and Cellular Neurosciences42 208ndash218

Lin SC and Bergles DE (2004) Synaptic signaling between GABAergicinterneurons and oligodendrocyte precursor cells in the hippocampusNature Neuroscience 7 24ndash32

Lin SC Huck JH Roberts JD Macklin WB Somogyi P andBergles DE (2005) Climbing fiber innervation of NG2-expressingglia in the mammalian cerebellum Neuron 46 773ndash785

Nedergaard M Ransom B and Goldman SA (2003) New roles forastrocytes redefining the functional architecture of the brain Trendsin Neuroscience 26 523ndash530

Nishiyama A Lin X Giese N Heldin C and Stallcup W (1996)Co-localization of NG2 proteoglycan and PDGF alpha-receptor onO2A progenitor cells in the developing rat brain Journal ofNeuroscience Research 43 299ndash314

Nolte C Matyash M Pivneva T Schipke CG Ohlemeyer C HanischUK et al (2001) GFAP promoter-controlled EGFP-expressing trans-genic mice a tool to visualize astrocytes and astrogliosis in living braintissue Glia 33 72ndash86

Oberheim NA Wang X Goldman S and Nedergaard M (2006)Astrocytic complexity distinguishes the human brain Trends inNeuroscience 29 547ndash553

Ozerdem U Monosov E and Stallcup WB (2002) NG2 proteoglycanexpression by pericytes in pathological microvasculatureMicrovascular Research 63 129ndash134

Parpura V Basarsky TA Liu F Jeftinija K Jeftinija S and HaydonPG (1994) Glutamate-mediated astrocyte-neuron signalling Nature369 744ndash747

Peppiatt CM Howarth C Mobbs P and Attwell D (2006)Bidirectional control of CNS capillary diameter by pericytes Nature443 700ndash704

Psachoulia K Young KM and Richardson WD (2009) Separatepopulations of dividing and non-dividing NG2 cells in the postnatalmouse brain Neuron Glia Biology in press

Rivers LE Young KM Rizzi M Jamen F Psachoulia K Wade Aet al (2008) PDGFRANG2 glia generate myelinating oligodendro-cytes and piriform projection neurons in adult mice NatureNeuroscience 11 1392ndash1401

Rouach N Koulakoff A Abudara V Willecke K and Giaume C(2008) Astroglial metabolic networks sustain hippocampal synaptictransmission Science 322 1551ndash1555

Wang LY Cai WQ Chen PH Deng QY and Zhao CM (2009)Downregulation of P2X7 receptor expression in rat oligodendrocyteprecursor cells after hypoxia ischemia Glia 57 307ndash319

Wigley R Hamilton N Nishiyama A Kirchhoff F and Butt AM(2007) Morphological and physiological interactions of NG2-gliawith astrocytes and neurons Journal of Anatomy 210 661ndash670

Zhu X Bergles DE and Nishiyama A (2008) NG2 cells generate botholigodendrocytes and gray matter astrocytes Development 135145ndash157

Ziskin JL Nishiyama A Rubio M Fukaya M and Bergles DE (2007)Vesicular release of glutamate from unmyelinated axons in whitematter Nature Neuroscience 10 321ndash330

A U T H O R S rsquo A D D R E S S

Institute for Biomedical and Biomolecular SciencesSchool of Pharmacy and Biomedical SciencesUniversity of PortsmouthUK

Correspondence should be addressed toArthur M ButtInstitute for Biomedical and Biomolecular SciencesSchool of Pharmacy and Biomedical SciencesUniversity of PortsmouthUKemail Arthurbuttportacuk

28 rebekah wigley and arthur m butt

httpsdoiorg101017S1740925X09990329Downloaded from httpswwwcambridgeorgcore Open University Library on 04 Feb 2017 at 144254 subject to the Cambridge Core terms of use available at httpswwwcambridgeorgcoreterms

Dayer AG Cleaver KM Abouantoun T and Cameron HA (2005)New GABAergic interneurons in the adult neocortex and striatumare generated from different precursors Journal of Cell Biology 168415ndash427

Dore-Duffy P Katychev A Wang X and Van Buren E (2006) CNSmicrovascular pericytes exhibit multipotential stem cell activityJournal of Cerebral Blood Flow and Metabolism 26 613ndash624

Ge WP Yang XJ Zhang Z Wang HK Shen W Deng QD et al(2006) Long-term potentiation of neuron-glia synapses mediated byCa2thorn-permeable AMPA receptors Science 312 1533ndash1537

Ge WP Zhou W Luo Q Jan LY and Jan YN (2009) Dividing glialcells maintain differentiated properties including complex mor-phology and functional synapses Proceedings of the NationalAcademy of Sciences of the USA 106 328ndash333

Gerhardt H and Betsholtz C (2003) Endothelial-pericyte interactions inangiogenesis Cell Tissue Research 314 15ndash23

Grass D Pawlowski PG Hirrlinger J Papadopoulos N RichterDW Kirchhoff F et al (2004) Diversity of functional astroglialproperties in the respiratory network Journal of Neuroscience 241358ndash1365

Grosche J Kettenmann H and Reichenbach A (2002) Bergmann glialcells form distinct morphological structures to interact with cerebellarneurons Journal of Neuroscience Research 68 138ndash149

Grosche J Matyash V Moller T Verkhratsky A Reichenbach A andKettenmann H (1999) Microdomains for neuron-glia interactionparallel fiber signaling to Bergmann glial cells Nature Neuroscience2 139ndash143

Halassa MM Fellin T and Haydon PG (2007a) The tripartite synapseroles for gliotransmission in health and disease Trends in MolecularMedicine 13 54ndash63

Halassa MM Fellin T Takano H Dong JH and Haydon PG(2007b) Synaptic islands defined by the territory of a single astrocyteJournal of Neuroscience 27 6473ndash6477

Hamilton N Vayro S Kirchhoff F Verkhratsky A Robbins J GoreckiD et al (2008) Mechanisms of ATP- and glutamate-mediated calciumsignaling in white matter astrocytes Glia 56 734ndash749

Hamilton N Vayro S Wigley R Robbins J and Butt AM (2009)Axons and astrocytes release glutamate and ATP to evoke calciumsignals in NG2-glia Glia in press

Houades V Koulakoff A Ezan P Seif I and Giaume C (2008) Gapjunction-mediated astrocytic networks in the mouse barrel cortexJournal of Neuroscience 28 5207ndash5217

Karadottir R Cavelier P Bergersen LH and Attwell D (2005) NMDAreceptors are expressed in oligodendrocytes and activated in ischae-mia Nature 438 1162ndash1166

Karadottir R Hamilton NB Bakiri Y and Attwell D (2008) Spikingand nonspiking classes of oligodendrocyte precursor glia in CNS whitematter Nature Neuroscience 11 450ndash456

Leoni G Rattray M and Butt AM (2009) NG2 cells differentiate intoastrocytes in cerebellar slices Molecular and Cellular Neurosciences42 208ndash218

Lin SC and Bergles DE (2004) Synaptic signaling between GABAergicinterneurons and oligodendrocyte precursor cells in the hippocampusNature Neuroscience 7 24ndash32

Lin SC Huck JH Roberts JD Macklin WB Somogyi P andBergles DE (2005) Climbing fiber innervation of NG2-expressingglia in the mammalian cerebellum Neuron 46 773ndash785

Nedergaard M Ransom B and Goldman SA (2003) New roles forastrocytes redefining the functional architecture of the brain Trendsin Neuroscience 26 523ndash530

Nishiyama A Lin X Giese N Heldin C and Stallcup W (1996)Co-localization of NG2 proteoglycan and PDGF alpha-receptor onO2A progenitor cells in the developing rat brain Journal ofNeuroscience Research 43 299ndash314

Nolte C Matyash M Pivneva T Schipke CG Ohlemeyer C HanischUK et al (2001) GFAP promoter-controlled EGFP-expressing trans-genic mice a tool to visualize astrocytes and astrogliosis in living braintissue Glia 33 72ndash86

Oberheim NA Wang X Goldman S and Nedergaard M (2006)Astrocytic complexity distinguishes the human brain Trends inNeuroscience 29 547ndash553

Ozerdem U Monosov E and Stallcup WB (2002) NG2 proteoglycanexpression by pericytes in pathological microvasculatureMicrovascular Research 63 129ndash134

Parpura V Basarsky TA Liu F Jeftinija K Jeftinija S and HaydonPG (1994) Glutamate-mediated astrocyte-neuron signalling Nature369 744ndash747

Peppiatt CM Howarth C Mobbs P and Attwell D (2006)Bidirectional control of CNS capillary diameter by pericytes Nature443 700ndash704

Psachoulia K Young KM and Richardson WD (2009) Separatepopulations of dividing and non-dividing NG2 cells in the postnatalmouse brain Neuron Glia Biology in press

Rivers LE Young KM Rizzi M Jamen F Psachoulia K Wade Aet al (2008) PDGFRANG2 glia generate myelinating oligodendro-cytes and piriform projection neurons in adult mice NatureNeuroscience 11 1392ndash1401

Rouach N Koulakoff A Abudara V Willecke K and Giaume C(2008) Astroglial metabolic networks sustain hippocampal synaptictransmission Science 322 1551ndash1555

Wang LY Cai WQ Chen PH Deng QY and Zhao CM (2009)Downregulation of P2X7 receptor expression in rat oligodendrocyteprecursor cells after hypoxia ischemia Glia 57 307ndash319

Wigley R Hamilton N Nishiyama A Kirchhoff F and Butt AM(2007) Morphological and physiological interactions of NG2-gliawith astrocytes and neurons Journal of Anatomy 210 661ndash670

Zhu X Bergles DE and Nishiyama A (2008) NG2 cells generate botholigodendrocytes and gray matter astrocytes Development 135145ndash157

Ziskin JL Nishiyama A Rubio M Fukaya M and Bergles DE (2007)Vesicular release of glutamate from unmyelinated axons in whitematter Nature Neuroscience 10 321ndash330

A U T H O R S rsquo A D D R E S S

Institute for Biomedical and Biomolecular SciencesSchool of Pharmacy and Biomedical SciencesUniversity of PortsmouthUK

Correspondence should be addressed toArthur M ButtInstitute for Biomedical and Biomolecular SciencesSchool of Pharmacy and Biomedical SciencesUniversity of PortsmouthUKemail Arthurbuttportacuk

28 rebekah wigley and arthur m butt

httpsdoiorg101017S1740925X09990329Downloaded from httpswwwcambridgeorgcore Open University Library on 04 Feb 2017 at 144254 subject to the Cambridge Core terms of use available at httpswwwcambridgeorgcoreterms