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Review

Where the thoughts dwell: The physiology of neuronal–glial“diffuse neural net”

Alexei Verkhratskya,b,d,⁎, Vladimir Parpurac, José J. Rodríguezb,d,e

aFaculty of Life Sciences, The University of Manchester, Manchester, UKbInstitute of Experimental Medicine, ASCR, Prague, Czech RepubliccDepartment of Neurobiology, Center for Glial Biology in Medicine, Civitan International Research Center,Atomic Force Microscopy and Nanotechnology Laboratories, and Evelyn F. McKnight Brain Institute, University of Alabama, Birmingham, USAdIKERBASQUE, Basque Foundation for Science, 48011, Bilbao, SpaineDepartment of Neurosciences, University of the Basque Country UPV/EHU, 48940, Leioa, Spain

A R T I C L E I N F O

⁎ Corresponding author. The University of MaE-mail address: alex.verkhratsky@manch

0165-0173/$ – see front matter © 2010 Elsevidoi:10.1016/j.brainresrev.2010.05.002

A B S T R A C T

Article history:Accepted 17 May 2010Available online 9 June 2010

The mechanisms underlying the production of thoughts by exceedingly complex cellularnetworks that construct the human brain constitute the most challenging problem ofnatural sciences. Our understanding of the brain function is very much shaped by theneuronal doctrine that assumes that neuronal networks represent the only substrate forcognition. These neuronal networks however are embedded into much larger and probablymore complex network formed by neuroglia. The latter, although being electrically silent,employ many different mechanisms for intercellular signalling. It appears that astrocytescan control synaptic networks and in such a capacity they may represent an integralcomponent of the computational power of the brain rather than being just brain “connectivetissue”. The fundamental question of whether neuroglia is involved in cognition andinformation processing remains, however, open. Indeed, a remarkable increase in thenumber of glial cells that distinguishes the human brain can be simply a result ofexceedingly high specialisation of the neuronal networks, which delegated all matters ofsurvival and maintenance to the neuroglia. At the same time potential power of analogueprocessing offered by internally connected glial networks may represent the alternativemechanism involved in cognition.

© 2010 Elsevier B.V. All rights reserved.

Keywords:Human brainGliaNeuroneHistoryEvolution of gliaCognitionNeuronal–glial networkHomeostasisNeuropathology

Contents

1. The neural cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1342. Neuroglia organises and maintains the nervous system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

2.1. Evolutionary aspect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1382.2. Developmental aspect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1402.3. Anatomical aspect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1402.4. Homeostatic aspect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

nchester, Oxford Road, Manchester, M13 9PT, UK.ester.ac.uk (A. Verkhratsky).

er B.V. All rights reserved.

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2.4.1. Ion and water homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1402.4.2. Neurotransmitter homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1412.4.3. Control of local blood flow and metabolic support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

2.5. Physiological aspect: neuroglia and information processing in the brain . . . . . . . . . . . . . . . . . . . . . 1412.5.1. Glial receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1412.5.2. Synaptic transmission onto glia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1422.5.3. Signalling in glial syncytia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1422.5.4. Gliotransmission and exocytotic release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1432.5.5. Neuroglia controls connectivity in neural networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

2.6. Pathophysiological aspect. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1443. Conclusion: the neuronal–glial networks form the substrate of brain function. . . . . . . . . . . . . . . . . . . . . . 145Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

1. The neural cells

“Omnis cellula e cellula” this aphorism initially coined byFrançois-Vincent Raspail and subsequently popularised byRudolf Virchow is an epitome of the biological revolution of19th century, which begun from the identification of cellularnature of life and ended in theoretical understanding ofevolution and genetic code.

The general idea about elementary units of life, fromwhichall tissues and organisms are formed, began to circulate in theearly 17th century, being promulgated, for example, by PierreGassendi and Robert Boyle. The origins of cellular theory arerooted in the discoveries of the first microscopists (e.g. RobertHooke, Marcello Malpighi and Nehemiah Grew) made in the17th century. It all began with themicroscopic observations ofthe plants, and it was Robert Hooke, who, when observing thefine structures of cork, visualised the regular structures thatreminded him of the monk's cells in the monasterialdormitories — and the term “cell” was born (Hooke, 1665).The first animal cells were discovered, to all likelihood, byAntonius van Leeuwenhoek, who in his many letters to theRoyal Society described bacteria (and named them animalculesor little animals) and erythrocytes, observed single musclefibres, followed the movements of live spermatozoids, andwas the first to see the regular structure (representing singleaxons) in the sagittal slice of the peripheral nerve (Fig. 1,(Bentivoglio, 1996; Leeuwenhoek, 1673–1696; Leeuwenhoek,1798)). Leeuwenhoek reflected on the latter observation madein 1717: “Often and not without pleasure, I have observed thestructure of the nerves to be composed of very slender vesselsof an indescribable fineness, running lengthwise to form thenerve” (cited fromBentivoglio, 1996). The fine cylindrical nervefibreswere also described by Felice Gaspar Ferdinand Fontana,who mechanically dissected the nerve and observed hispreparations under 700× magnification (Bentivoglio, 1996).More cell types were discovered at the beginning of 19thcentury, and around 1830 Robert Brown found the nucleus(Ford, 1992). The cellular theory was then formalised byTheodor Schwann and Matthias Jakob Schleiden (Schleiden,1838; Schwann, 1839; Schwann and Schleiden, 1847) and wasrapidly embraced by the scientific community.

The first cells of the nervous system were visualised in1830-ies. Probably the first observations were done by Chris-

tian Gottfried Ehrenberg who was investigating the nervoussystem of the leech (Ehrenberg, 1836) and by Johann Evange-lista Purkinje (Figs. 1B andC)whowas studying cerebellumanddescribed the cell named after him (Purkinje, 1837). Purkinje'spupil, Gustav Gabriel Valentin came with the first publisheddrawing of the neurone (which he called Kugeln — Fig. 1D)where the nucleus and other intracellular structures werevisible (Valentin, 1836). Incidentally, Valentin alsowas the firstto suggest that the nervous system contains both active andpassive elements.

The passive elements of the brain were soon conceptua-lised by Rudolf Virchow who introduced connective tissue tothe nervous system under the name of neuroglia ((Virchow,1856; Virchow, 1858), for the more detailed account on thehistory of glia see Kettenmann and Ransom (2005), Ketten-mann and Verkhratsky (2008), Verkhratsky (2006b) andVerkhratsky (2009)). Virchow did not recognise neuroglia as aspecific class of neural cells; the drawings of some roundstructures, which he reproduced in the Die Cellularpathologie,show, if anything, activated microglia. This did not matter,because for Virchow the neuroglia was a connective tissue ofmesodermal origin; the misconception, which was embracedby many histologists (e.g. by Andriezen, Robertson andWeigart) and which was still believed by Cajal as late as in1920 (Ramón y Cajal, 1920).

In the meantime discoveries of the neural cells continued.In 1838 Robert Remakmade the description of nerve fibres andvisualised the covering sheath around them (Remak, 1838). In1851HeinrichMüller (1851) produced the first images of retinalradial glia (the cells which were subsequently named Müllercells by Rudolf Albert von Kölliker (1852b). Von Kölliker byhimself also described nerve elements in the acoustic nerve ofthe ox (Kölliker, 1852a). In 1858 Max Schulze made a detailedinvestigation of the Müller cells and produced probably thebest possible drawings of them in the pre-staining era ofhistology. Simultaneously Karl Bergmann (1857) had identifiedradial glial cells in the cerebellum (the Bergmann glial cells). In1862 the neuro-muscular junction was described by WilhelmFriedrich Kühne, who named it the “endplate” (Kühne, 1862).Slightly later the very detailed images of both neurones andstellate glial cells (that to all probability were astrocytes —Figs. 1E, F and G) weremade by young (who untimely deceasedat 29) Otto Deiters (1865), and some years later Jacob Henle and

Fig. 1 – First images of neural cells. A. Antonius van Leeuwenhoek (1632–1723) and his drawing of a “small Nerve (BCDEF),”composed by many “vessels” in which “the lines or strokes denote the cavities or orifices of those vessels. This Nerve issurrounded, in part, by five other Nerves (GGGGG)” in which only “external coats” are represented. The image was kindlyprovided by Prof. Marina Bentivoglio. B, C. Johann Evangelista Purkinje (1787–1869) and the first drawings of the Purkinjeneurone made by him for the Congress of Physicians and Scientists Conference in Prague, in 1837. Reproduced fromLopez-Munoz et al. (2006). D. The first published drawing of neurone made by Gustav Gabriel Valentin (1810–1883) (Valentin,1836). E, F, G. Otto Friedrich Karl Deiters (1834–1863) and his drawings (Deiters, 1865) of motoneurones and “connective tissuecells” (astrocytes). Reproduced with permission from Bentivoglio (1996) and Kettenmann and Ransom (2005). H. Section of thespinal cord of the ox demonstrating the network of stellate cells. Cells were stained by carmine after chromic acid preparation(Henle and Merkel, 1869). Reproduced with permission from Kettenmann and Ransom (2005).

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Friedrich Merkel visualised the glial network in the greymatter (Fig. 1H — (Henle and Merkel, 1869)).

It has to be stressed that all these early cellular imageswere done mostly on unstained preparations followingpainstaking cells isolation by microsurgery. The histologicalrevolution occurred in 1873 when Camillo Golgi developed thesilver-chromate staining technique (the famous “reazione

nera” or black staining — (Golgi, 1873; Golgi, 1903)), which,for the first time, allowed neurohistologists to obtain imagesof neural cells in their entity (Fig. 2; for a comprehensive andvividly written account of the dissemination of Golgi stainingtechnique through the world see Mazzarello (2010)).

As early as 1871 Golgi recognised glial cells and he was thefirst to demonstrate that the glia represent cellular population

Fig. 2 – Astroglial cells stained by the silver-chromate technique. A: Protoplasmic astroglial cells in the grey matter stained anddrawn by Camillo Golgi; the astrocytes form numerous contacts (the endfeet) with brain capillaries (Golgi, 1883). The imagewaskindly provided by Prof. Paolo Mozzarello. B. Morphological heterogeneity of human astrocytes. The astrocytes in the brainslices of human foetuses were stained by silver-chromate technique (Retzius, 1894).

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distinct fromnervous cells, and that the glial cells (whichwereto all probability the protoplasmic astrocytes) send theprocesses to the blood vessels where they establish theendfoot structures (Fig. 2A); he was also the first to proposethat the glial cells are instrumental in transferring themetabolic substances from the blood to the brain parenchyma(Golgi, 1875; Golgi, 1894), this concept subsequently gained theuniversal acceptance. The application of Golgi staining led to afurther in depth characterisation of glial cells of the greymatter, and in 1891 the term “astrocyte” was introduced byMichael von Lenhossek (Lenhossek, 1891; Lenhossek, 1893);slightly later the astroglia was further subdivided intoprotoplasmic (Andriezen, 1893) and fibrous (Kölliker, 1893)astrocytes. At the same years Wilhelm His made a funda-mental discovery, when, in 1889, he found the neuronal originof neuroglia and directly demonstrated that both nerve cellsand neuroglia derive from the neuroectoderm (His, 1889a; His,1889b).

The end of 1880-ies marked the arrival of the neurone andof Santiago Ramón y Cajal, who was to champion and developthe neuronal doctrine over the next decades. The first papersof Cajal dedicated to the fine structure of the nervous systembegun to appear in 1888, about one year after he learned theblack staining technique. With characteristic determinationand originality Cajal made a special journal for his papers, theRevista Truimestral de Histologia Normal y Patologica, of which henaturally became the Editor-in-Chief, and the first issues ofthis journal were almost entirely occupied by his papers(Mazzarello, 2010). In the first paper published in the first issueof his journal Cajal made the seminal statement that “eachnerve cell is a totally autonomous physiological canton”(Ramón y Cajal, 1888). One year later, in October of 1889,Cajal attended the German Anatomical congress where hedemonstrated his numerous microscopic preparations (Fig. 3)to the delegates; this instantly made his internationalreputation. In 1891 Heinrich Wilhelm von Waldeyer, a greatadmirer and follower of Cajal, coined the term “neurone”(Waldeyer von, 1891) and neuronal doctrine begun to conquerthe minds of neuroscientists. The neurone was endowed withdendrites (the term was introduced by Wilhelm His in 1889)and very soon it acquired the axon (which was named so byAlfred Kölliker in 1896).

The previous (and the first) grand theory about the brainfunctional organisation was formalised by Josef von Gerlach(1871), who proposed the diffuse network of neurofilamentsand neural cell processes, that are internally connected andact in concert. This “reticular” theory dominated neurosci-ence for good 20 years and recruited many supportersincluding Golgi, who was very much convinced in theexistence of “diffuse neural net”. The history of the neuron-ism–reticularism conflict was widely popularised, and thecurios reader may find all the dramatic details in severalcomprehensive treatises (Jacobson, 2005; Lopez-Munoz et al.,2006; Mazzarello, 2007; Mazzarello, 2010; Ramón y Cajal,1933; Shepherd, 1991).

Here we shall continue with a brief outline of furtherdiscoveries of glial cells (Fig. 3), which, curiously enough, arevery much connected with Cajal's school and with hispupils, Nicolás Achúcarro and Pío del Río-Hortega. First,Cajal developed gold chloride-sublimate staining method

that was specific for both protoplasmic and fibrous astro-cytes (Garcia-Marin et al., 2007). We now know that thisstain targeted intermediate filaments consisting mainly ofglial fibrillary acidic protein (GFAP), a protein used today asan astrocytic marker (reviewed in Kimelberg, 2004). Usingthis technique Cajal confirmed earlier ideas of the origin ofastrocytes from radial glia, and also demonstrated thatastrocytes can divide in the adult brain, thus laying thebasis for much later discoveries of the stem properties ofastroglia (Ramón y Cajal, 1913a; Ramón y Cajal, 1913b;Ramón y Cajal, 1916). Subsequently Pío del Río-Hortegaidentified two other principal classes of glial cells, initiallyconsidered by Cajal as the “third element” (a group of“adendritic” cells) and what represented, in fact, theoligodendrocytes and the microglia.

Oligodendrocytes were initially observed and described byWilliam Ford Robertson who developed for this purpose aspecific platinum stain technique; Robertson however did notrealise the myelinating capacity and role of these cells andthought themexternal invaders to the brain, hence identifyingthem as mesoglia (Robertson, 1899; Robertson, 1900). Almost20 years later Río-Hortega rediscovered these cells and gavethem a name of oligodendrocytes (Del Río-Hortega, 1921;Gibson, 1994); he also demonstrated that oligodendrocyteswere myelinating cells in the CNS being thus analogous to theSchwann cells in the periphery. It was also Río-Hortega whodescribed the microglial cells, the only glia members of non-neuronal origin (for staining of both oligodendrocytes andmicroglia Río-Hortega deployed specifically developed ammo-niacal silver carbonate method (Del Río-Hortega, 1917)).Initially Río-Hortega called these cells “garbage collectors”; inthe literature they also were known as “cells of Hortega”(Gibson, 1993; Gibson, 1994). Del Río-Hortega gave microglia avery detailed and comprehensive characterisation. He foundthat microglial precursors invade the brain shortly after birth,disseminate over the brain parenchyma and develop a distinctphenotype. He also found that these cells reacted to braindamage by morphological and functional modification knownas microglial activation (Del Río-Hortega, 1919a; Del Río-Hortega, 1919b; Del Río-Hortega, 1920; Del Río-Hortega, 1927;Del Río-Hortega, 1932).

Thus by the 1920-ies the overall understanding of mor-phological organisation of the brain and the spinal cord hasbeen generally completed, and the following developmentswere mostly dedicated to uncovering the functional mechan-isms by which neural cells communicate and by which theymay perform higher brain functions. By then the neuronaldoctrine became absolutely dominating, and the synapse, thename of which was introduced by Charles Scott Sherrington(Foster and Sherrington, 1897; Sherrington, 1906) has receiveda particular attention as the principal place of integration inthe nervous system. Rapidly developing electrophysiologicalexperimental techniques allowedmonitoring of the functionalactivity of neurones; and the image of neuronal networkwhere binary signals (action potentials) swiftly convey theinformation though neuronal membranes and activate syn-aptic release of neurotransmitters that subsequently triggerchanges in postsynaptic potential, which, in turn, provide forthe electronic summation thus integrating the incomingsignals, was universally accepted.

Fig. 3 – Glial cells by the eyes of Santiago Ramón y Cajal and Pío del Río-Hortega. A: Cajal's drawing of Golgi impregnated gliashowing human cortical neuroglial cells of the plexiform layer (A–D), cells of the second and third layers (E–H and K, R) andperivascular glia (I, J). B, C: Astrocytes in the stratum lucidum of the human CA1 area of the hippocampus with particularemphasis on the anatomy of perivascular astrocytes in the CA1 stratum radiatum. D, E: Drawings of Pío del Río-Hortegashowing the different morphological types of microglial cells in the rabbit Ammon's horn and cortical perivascular neuroglia.

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2. Neuroglia organises and maintains thenervous system

“It may seem strange that, since I have always beenopposed to the neurone theory – although acknowledgingthat its starting-point is to be found in my own work – Ihave chosen this question of the neurone as the subject ofmy lecture, and that it comes at a time when this doctrineis generally recognized to be going out of favour.”(CamilloGolgi starting the Nobel lecture (Golgi, 1906))

Thenature of informationprocessing in thehumanbrain andthe role of different cell types and their temporal and spatialrelations, the role of fast and slow processes and the nature of

signalling events that occur between andwithin the cells as wellas comprehension of the relevance of all these immenselycomplex processes in the generations of thoughts, emotions andcreative ideas remain very far fromunderstanding. Theneuronaldoctrine regards neuronal networks as a sole substance forinformation processing; the knowledge accumulated in the last2 decades indicates that theoverall picture canbemore complex.Here we shall briefly overview the cell biology and physiology ofneuroglia from the perspective of the integrated neuronal–glialnetworks as a substrate of brain function.

2.1. Evolutionary aspect

The primordial glial cells appeared early in evolution (Fig. 4),probably already at the stage of diffuse nervous system when

Fig. 4 – Evolution of the neuroglia; the tree of life is taken from Haeckel (1879).

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they assisted ancestral neurones in their functions (Bacaj etal., 2008; Reichenbach and Pannicke, 2008). Development ofneuronal masses organised first in ganglia and then in thefunctionally segregated centralised nervous system, stimulat-ed the evolution of supporting cells, the proto-astrocytes,which controlled the extracellular homeostasis, providedmetabolic support, regulated development and assisted neu-rones in fulfilling their primary functions. In Caenorhabditiselegansworm the artificial elimination of glia (some of the glialcells are associated with the nematode sensory organ) doesnot result in wide-spread neuronal death, but dramaticallyalters function and sensitivity of sensory neurones (Bacaj etal., 2008). Incidentally a number of signalling and enzymaticcascades present in glial cells are exceptionally highlyconserved in evolution, being similar in the worms, insectsandmammals (Barres, 2008). At the higher evolutionary stagesastroglial cells remain an important part of both sensoryorgans and the CNS, where they assume multiple roles, andmuch higher significance — elimination of astroglia from the

CNS results in neuronal demise. The high degree of special-isation acquired by neurones in the centralised nervoussystem renders them helpless in the absence of the glialsupport; this support (which contributes at many levels ofbrain homeostasis) is the ancestral function of astrocytes thatremains throughout the evolution. Another important func-tion that appeared very early in the evolutionary ladder wasthe isolation of the nervous tissue from the rest of the body bythe blood–brain barrier (BBB), which is made solely byastrocytes in crustacean, insects and cephalopods. Interest-ingly that astroglial blood–brain barrier is also present in thebrain of sharks; some of the capillaries are completelysurrounded by astroglial process being thus endocellularvessels (Long et al., 1968). At the later stages of phylogenesisthe BBB function is shifted to endothelial cells, the latternonetheless remain under astrocytic control (Abbott, 2005;Abbott and Pichon, 1987).

An increase in the size of living organisms naturallypresented an additional survivalist task — an increase of the

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speed of communication between the periphery and thecentral neural organs. Demand for an increased velocity ofelectrical propagation through the axons led to an appearanceof myelin isolators provided by oligodendrocytes andSchwann cells. The myelin isolation appeared sometimes inOrdovican period (Fields, 2008a; Hartline, 2008; Roots, 2008) inthe ancestral invertebrates (e.g. in oligochaetes (earthworms)),in malacostraca (shrimps and prawns), in copepods (smallcrustaceans) and in gnathostomes (the jawed fishes; althoughthe class includes not only sharks and rays but alsotetrapodes).

Finally, the isolated position of the nervous tissue requiredself-defence systemand the immune cells begun to invade theganglia in bivalve, crustacean and insects thus forming theancestral microglia, which is present in brain parenchyma atall subsequent evolutionary stages (Liu et al., 1996; Magazineet al., 1996; Stefano et al., 1996).

Neuroglia attained maximal development in mammals,and moreover an increased complexity of the brain, togetherwith an increased intellectual power was accompanied withremarkable increase in both numbers and complexity of glia(Oberheim et al., 2006; Reichenbach, 1989; Verkhratsky, 2009;Verkhratsky and Butt, 2007). Astrocytes became the mostnumerous cells in the human brain, outnumbering neurones.For example, in layers I and IV of the rat cerebral cortex,neurones/glial cells (astrocytes and oligodendrocytes) ratio is∼2.5 to 1 (Bass et al., 1971). This is inversed in the humans: theglia to neurones ratio in human cortex is ∼1.65:1 (Nedergaardet al., 2003; Sherwood et al., 2006). Furthermore, the morphol-ogy of human astrocytes is unique when compared to all othermammals (Oberheim et al., 2009); the size of human proto-plasmic astrocyte is about 2.5–3 times and fibrous astrocytes∼2.2 times larger than in rodents. The human protoplasmicastrocytes have ∼10 times more primary processes, andcorrespondingly much more complex processes arborisationthan rodent astroglia. As a result, human protoplasmicastrocyte contacts and integrates ∼2 millions of synapsesresiding in its territorial domain, whereas rodent astrocytescover ∼20,000–120,000 synaptic contacts.

2.2. Developmental aspect

Neurones and neuroglia both originate from the neuroepithe-lial cells (Alvarez-Buylla et al., 2001), which phylogeneticallycorrespond to the ancestral neuroepithelium. The lattercomprised secretory epithelial cells, which were connectedby gap junctions and probably expressed some sets of voltage-gated channels. All in all, the very primordial neural elementswere, most likely, physiologically closer to neuroglia, than tothe neurones judging on their excitability and syncytia-drivensignal propagation.

Be it all as it may, the neuroepithelial cells transform intothe radial glia, which act as a universal neural precursor; theasymmetrical division of radial glia produce neuronal pre-cursors that migrate to their destinations using the processesof their progenitor as a scaffolding guide-line. The radial gliaalso act as progenitors (through several transitional forms) forboth astrocytes and oligodendrocytes. Some of the astrocytes,dwelling in the germinal centres of the adult brain, retain thestem cell properties throughout the life span and underlie the

adult neuro- and glio-genesis (see Alvarez-Buylla et al. (2001),Doetsch et al. (1999), Gotz and Huttner (2005) and Mori et al.(2005) for a comprehensive review). In addition neuroglial cellsare instrumental in promoting neuronal survival at differentdevelopmental stages through the release of numerousneurotrophic factors (e.g. epidermal growth factor, EGF, Glialcell-derived neurotrophic factor, GDNF, etc); and by theactivation of astrocytic transcriptome-associated genes suchas ApoE, ApoJ, MFGE8 and cystatin C (Banker, 1980; Barres,2008; Wagner et al., 2006).

2.3. Anatomical aspect

In mammalian brain the astroglial cells define the micro-architecture of the parenchyma by dividing the grey matter(through the process known as “tiling” (Bushong et al., 2004))into relatively independent structural units. The protoplasmicastrocytes occupy their own territory and create the micro-anatomical domains within the limits of their processes(Bushong et al., 2002; Halassa et al., 2007b; Nedergaard et al.,2003; Oberheim et al., 2009; Ogata and Kosaka, 2002;Wilhelms-son et al., 2006). Within the confines of these anatomicaldomains the membrane of the astrocyte covers synapses andneuronal membranes as well as sends the process to plasterthe wall of the neighbouring blood vessel with the endfoot.

The individual astroglial domains are integrated into thesuperstructure of astroglial syncytia through gap junctions(Giaume and Venance, 1998) localised on the peripheralprocesses of astroglial cells. These astroglial syncytia arealso anatomically segregated being formed within definedanatomical structures, for example in individual barrels of thesomatosensory cortex (Giaume et al., 2009).

The second main class of neuroglia, the oligodendroglia, iscentral in moulding and maintaining the white matter. In thehuman brain the white matter is much larger as compared toother species; it makes more that 50% of the whole brain,which most likely reflects a high demand for interneuronalconnections (see Fields (2008b) for comprehensive review).The oligodendrocytes demonstrate an exceptionally highdegree of plasticity andmyelination can be rapidly modulatedin response to various learning paradigms (Fields, 2008b). Theoligodendrocytes are actively communicating with neurones,and this reciprocal signalling, details of which only begin to beuncovered, profoundly contributes to overall brain plasticityand cognitive processes.

2.4. Homeostatic aspect

Astroglia represents the main cellular element of extracellularhomeostasis in the brain. Indeed the control over the concen-trations of ions, neurotransmitters, neuromodulators, metabo-lites and other active substances is of a paramount importancefor normal operation of neural networks, because even rela-tively small changes in a number of molecules may result insubstantial shifts of their respective concentrations in tiny andoften diffusionally isolated extracellular compartments.

2.4.1. Ion and water homeostasisAstroglia assume the leading role in regulation of extracellularK+. The concentration of the latter may rise up to 10–12 mM

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following frequent action potential firing that may substan-tially affect the excitability of neuronal networks. Astrocytesremove the excess of extracellular K+ via local K+ uptake(accomplished by inward rectifier K+ channels) and K+ spatialbuffering (Coles and Orkand, 1983; Kofuji and Newman, 2004;Newman, 1995). The latter mechanism provides for theredistribution of K+ from the areas with elevated [K+]o to theregions with low [K+]o and may occur either in the single glialcells (K+ siphoning in retinalMüller cells (Newman et al., 1984))or in the glial syncytia. The glial syncytia and aquaporinechannels expressed in astrocytes also play the most criticalrole in regulation of brain water homeostasis (Simard andNedergaard, 2004).

2.4.2. Neurotransmitter homeostasisThe second important homeostatic task accomplished byastroglial cells is the control over extracellular concentrationof neurotransmitters, and most importantly, glutamate. Thelatter, when released in excess or for a long-time, acts as apowerful neurotoxin that triggers neuronal cell death in amultitude of acute and chronic brain lesions (Caudle andZhang, 2009; Obrenovitch et al., 2000; Schousboe et al., 1997;Westbrook, 1993). Interestingly, the glial function to “chemi-cally split or take up” transmitters was speculated upon by anItalian psychiatrist Ernesto Lugaro in 1907 (Lugaro, 1907).Astrocytes are responsible for the bulk of glutamate removalfrom the extracellular space: they accumulate 80% of theglutamate released, whereas the remaining 20% goes toneurones (Swanson, 2005; Verkhratsky and Kirchhoff, 2007a).Astrocytic uptake of glutamate from extracellular space is alsoan important modulator of the time-course of synapticneurotransmission (Mennerick and Zorumski, 1994).

There are five types of glutamate transporters operative inthe human brain (Gadea and Lopez-Colome, 2001); these areknown as EAAT1 to EAAT5 (where EAAT stands for excitatoryamino acid transporter). Astroglial cells express almostexclusively the EAAT1 and EAAT2 (known in rodent brain asglutamate/aspartate transporter, GLAST, and glutamate trans-porter-1, GLT-1) that accomplish the bulk of glutamate uptake(Danbolt, 2001); some expression of GLT-1a has been recentlyreported at neuronal pre-synaptic terminals (Furness et al.,2008; Melone et al., 2009). The glutamate transporters are co-transporters which utilise the energy saved in the form oftransmembrane Na+ gradient so that the transport of a singleglutamate molecule requires an influx of 3 Na+ ions and 1 H+

ion coupled with the efflux of 1 K+ ion (Kirischuk et al., 2007).The substantial sodium accumulation accompanying gluta-mate accumulation is counterbalanced by Na+ efflux throughNa+/Ca2+ exchanger working in the reverse mode (Kirischuk etal., 1997; Kirischuk et al., 2007); both the Na+/Ca2+ exchangerand glutamate transporters are co-localised in perisynapticastroglial processes (Minelli et al., 2007).

Astroglial glutamate transport is crucial for neuronalglutamatergic transmission by operating the glutamate–glutamine shuttle (Verkhratsky and Butt, 2007). Glutamate,accumulated by astrocytes is enzymatically converted intoglutamine by the astrocytic-specific glutamine synthetase(Martinez-Hernandez et al., 1977). Glutamine can be safelytransported to pre-synaptic terminals through the extracellu-lar space; after entering the neuronal compartment glutamine

is transformed into glutamate. It is also of importance thatastrocytes possess the enzyme pyruvate carboxylase, andthus act as a main source for de novo glutamate synthesis(Hertz et al., 1999).

2.4.3. Control of local blood flow and metabolic supportThe astroglial cells are the central elements of the neurovas-cular units that integrate neural circuitry with local blood flowand metabolic support. The astrocyte, which determines theconfines of the neurovascular unit, bridges the brain paren-chyma and local vasculature by virtue of perivascular processand the endfeet. Increased activity of neurones integrated intothe neurovascular unit triggers Ca2+ signals in the astrocyte,which, in turn, stimulate release of vasocoactive agents thatregulate the local blood flow (Metea and Newman, 2006;Mulligan and MacVicar, 2004; Zonta et al., 2003). Similarly,astrocytes are also responsible for local metabolic support ofactive neurones though the postulated glucose–lactate shut-tle; the latter being under the control of cytosolic Na+

concentration (Magistretti, 2006; Magistretti, 2009).

2.5. Physiological aspect: neuroglia and informationprocessing in the brain

The glia is electrically non-excitable, and therefore is unable touse the plasmalemmal ion conductances to rapidly convey theinformation. Nonetheless, glial cells are capable of expressingthe excitable molecules: the voltage-gated ion channels, theneurotransmitter receptors, and the intracellular signallingchains. Furthermore, the neuroglia is capable of developingpropagating signals either through the gap junctions orthrough the release of gliotransmitters. Conceptually, glialcells can perform all physiological processes as neurones do(recognise the incoming information, generate propagatingsignals, and secrete the transmitters that form the informa-tional output), albeit the neuroglia executes these physiolog-ical processes in a specific and distinct way.

2.5.1. Glial receptorsNeuroglial cells are capable to express virtually every receptorto neurotransmitters, neurohormones and neuromodulators.This fundamentally important property was discovered in themultitude of early experiments on cultured glial cells thatdemonstrated the sensitivity of glia to a variety of stimulatingagents (Condorelli et al., 1999; Cornell Bell et al., 1990; Dave etal., 1991; Fraser et al., 1995; Gallo and Ghiani, 2000; Verkh-ratsky et al., 1998; Verkhratsky and Steinhauser, 2000). Thispromiscuity of cultured glia towards neuroactive ligands isgenerally regarded as an obstacle, and indeed the culturedneuroglial cells are very poor models of the glia in vivo, bothmorphologically and functionally. Nonetheless, these earlyexperiments were specifically important because theyshowed, beyond any doubt, that the neuroglia is endowedwith molecular machinery to participate in chemical trans-mission in neural networks.

The receptor patterns expressed by glial cells in situ are verydifferent, as the complement of receptors is quite restrictivedepending on the brain region, and is, most likely, controlledby the local neurotransmitter environment. Usually themodality of neurotransmitter receptors expressed by astroglia

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is similar to that expressed in their neuronal neighbours(Verkhratsky and Kettenmann, 1996; Verkhratsky et al., 1998).The most abundant neurotransmitter receptors expressed byneuroglia are glutamate receptors and purinoceptors, al-though the expression of particular subtypes varies betweenbrain regions. Most of astrocytes and oligodendrocytesexpress metabotropic glutamate receptors of mGluR3 andmGluR5 variety; the latter receptors are instrumental forgeneration of glial Ca2+ signals (Hamilton et al., 2008; Kirischuket al., 1999; Tamaru et al., 2001; Verkhratsky and Kirchhoff,2007a). Astro- and oligodendroglia in hippocampus, corpuscallosum, optic nerve and cerebellum show relatively highlevels of expression of ionotropic AMPA receptors, quite oftenof high-Ca2+-permeable form (Garcia-Barcina and Matute,1998; Matute and Miledi, 1993; Muller et al., 1992; Seifert andSteinhauser, 2001; Steinhäuser and Gallo, 1996; Verkhratskyand Kirchhoff, 2007a). Cortical astrocytes and oligodendro-cytes in the corpus callosum, cerebellum and the optic nerveexpress “glial” NMDA receptors characterised by weak Mg2+

block (Karadottir et al., 2005; Lalo et al., 2006; Micu et al., 2006;Salter and Fern, 2005; Verkhratsky and Kirchhoff, 2007b).

All glial cells express a broad spectrum of purinoceptors.The purinergic neurotransmission (Abbracchio et al., 2009;North and Verkhratsky, 2006), operative in many regions ofthe brain, is particularly important for neuronal–glial, glial–glial and glial–neuronal signalling. The purinergic signallingsystem employing ATP, adenosine and adenine nucleotidesas mediators represents the ancient system for intercellularcommunications, and as such it is exceptionally broadlydistributed in different types of cells and tissues (Burnstock etal., 2010; Burnstock and Verkhratsky, 2009). The glial cells,arguably, inherited the sensitivity to purines from theirepithelial ancestors; be it as it may, all glial cells havesensitivity to purines mediated through several types of P2(ATP/nucleotide) and/or P1 (adenosine) receptors. Activationof purinoceptors controls many vital functions of glial cellsbeing instrumental for glial signalling, release of gliotrans-mitters, initiation of astrogliosis and activation of microglia(Verkhratsky et al., 2009). The actual mapping of purinocep-tors in various types of glia has not been accomplished;nonetheless majority of astrocytes and oligodendrocytespossess metabotropic A1,2A,2B,3 adenosine receptors andP2Y1,2,4,6 nucleotide receptors coupled to the InsP3-signallingchain and Ca2+ signalling (Abbracchio and Burnstock, 1998;Boison et al., in press; Kirischuk et al., 1995a; Kirischuk et al.,1995b; Verkhratsky et al., 2009). Glial expression of ionotropicP2X receptors is much less clear; the P2X1/5 mediated currentswere found in cortical astroglia (Lalo et al., 2008), yet P2Xcurrents were detected neither in hippocampal astrocytes(Jabs et al., 2007) nor in Bergmann glial cells (Kirischuk et al.,1995a). There is some evidence of functional expression ofP2X7 receptors in oligodendrocytes, although they mayoperate mostly in pathological conditions (Domercq et al.,2010; Matute, 2008). Finally, multiple P2Y and P2X4,7 receptorsare abundantly expressed in microglia; these receptorscontrol activation, motility, release of cytokines and exertnumerous trophic effects upon microglia (Farber and Ketten-mann, 2006b; Haas et al., 1996; Hanisch and Kettenmann,2007; Inoue, 2008; Inoue and Tsuda, 2009; Inoue et al., 2005;Moller et al., 2000).

The two main receptor systems, glutamatergic and pur-inergic, expressed in glial cells are complemented by almostlimitless combinations of other receptors that allow glia toaccurately receive all types of chemical transmission thatoccurs in the brain. In addition, the morphological proximityof astroglialmembranes and synaptic structures (which in factmake the central synapses tripartite — i.e. comprising pre-and postsynaptic compartments as well as astroglial perisy-naptic process (Araque, 2008; Araque et al., 1999; Halassa et al.,2007a)) almost certainly indicates the continuous involvementof glia in the ongoing synaptic transmission.

2.5.2. Synaptic transmission onto gliaAstrocytes are able to receive inputs from synaptic transmis-sion during the spill-over of a neurotransmitter for which theyexpress appropriate receptors. Dani et al. (1992), usingcultured organotypic hippocampal slices, have demonstratedthat neuronal activity at mossy fibre—CA3 synapses cantrigger [Ca2+]i increases and intercellular waves in an astro-cytic network mediated by glutamate released from synapticterminals. Similarly, astrocytes can respond to neurotrans-mitter release in acute slices. Porter and McCarthy (1996),using acutely isolated hippocampal slices, have observed thatelectrical stimulation of Schaffer collaterals caused increasesin [Ca2+]i in astrocytes located in the stratum radiatum of theCA1 region. At the lower level of synaptic activity this effectwas mediated by mGluRs, while the higher levels of neuronalactivity recruited both mGluRs and AMPA/kainate ionotropicGluRs on astrocytes. Glutamate is not the onlymediator of thisneurone–astrocyte signalling via spill-over. For examplesynaptically released acetylcholine also evokes [Ca2+]i eleva-tions in astrocytes in hippocampal slices through the activa-tion of metabotropic acetylcholine receptors (Araque et al.,2002).

Besides glial cells sensing spill-over of synaptically re-leased neurotransmitters, they can receive direct synapse-like(synaptoid) or even classical synaptic connections. Stimula-tion of pituitary stalk can depolarize stellate astrocyte-likeglia, pituicytes, in situ, an effect mediated by γ-aminobutyricacid (GABA) and dopamine receptors on this glial cell type(Mudrick-Donnon et al., 1993) and occurring via direct inputsfrom neurones through synaptoid contacts, where axonalprojections end on pituicytes (Buijs et al., 1987; van Leeuwen etal., 1983; Wittkowski and Brinkmann, 1974). Similarly, norepi-nephrine terminals make synaptoid contacts onto septohip-pocampal astrocytes (Milner et al., 1995). Direct neuronalglutamatergic and GABAergic synapses made onto oligoden-drocyte precursor cells were observed in the hippocampus(Bergles et al., 2000; Lin and Bergles, 2004).

2.5.3. Signalling in glial syncytiaThe extended complement of plasmalemmal receptors,expressed by glial cells is coupled to intracellular signallingcascades, which provide glia with specific excitabilitymechanisms. The glial excitability, as we can perceive ittoday, is based on the excitability of the endomembrane(which forms the endoplasmic reticulum, the ER) endowedwith Ca2+ release channels, represented by InsP3 receptors andryanodine receptors (Berridge et al., 2003; Kostyuk andVerkhratsky, 1994; Verkhratsky, 2005). Stimulation of the

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majority of metabotropic receptors expressed in neurogliainduces formation of InsP3, which in turn triggers Ca2+ releasefrom the ER producing thus Ca2+ signals; these Ca2+ signals aregenerally associated with glial activation and act as asubstrate for glial excitability (Deitmer et al., 1998; Farberand Kettenmann, 2006a; Verkhratsky et al., 1998). The Ca2+

release channels are regulated by both cytosolic and intra-ERCa2+ concentrations (Burdakov et al., 2005); importantly thisCa2+ sensitivity (together with the internal continuity of the ERCa2+ store (Jones et al., 2009; Petersen and Verkhratsky, 2007;Solovyova and Verkhratsky, 2003)) is instrumental in gener-ating intracellular propagating Ca2+ waves. These propagatingwaves of endomembrane excitation cross cell-to-cell bound-aries in the astroglial syncytia thus providing the means forlong-range signalling (Cornell Bell et al., 1990; Haas et al., 2006;Scemes and Giaume, 2006; Verkhratsky and Toescu, 2006). Theactual mechanisms of generation and maintenance of inter-cellular Ca2+ waves are complex and involve InsP3 diffusionthrough gap junctions as well as gliotransmitter (most notablyATP) release (Anderson et al., 2004; Arcuino et al., 2002;Guthrie et al., 1999; Haas et al., 2006; Scemes and Giaume,2006; Suadicani et al., 2004; Verkhratsky, 2006a).

The gap junctional route can also form the pathways foralternative signalling in the astroglial syncytia, which mayinvolve various second messengers, metabolic substrates(Rouach et al., 2008), and maybe some other molecules. Weknow very little about these alternative pathways, and yetthese can be important for the plasticity and informationprocessing by neuroglia. Furthermore not only Ca2+ but alsoother ionsmay serve as glial signallers. This, for example,maybe an important function of Na+ ions. The concentration of Na+

is often increased in glia following activation of transportersystems and ionotropic receptors (Deitmer and Rose, 2010;Kirischuk et al., 2007; Langer and Rose, 2009). Sodium ions arecapable of activating enzymatic systems. An increase inastroglial [Na+]i activates Na+–K+ ATPase, which, in turn,stimulates phosphoglycerate kinase thus initiating produc-tion of lactate and lactate shuttling to neurones.

The glial networks established by the plethora of gapjunctions formed between glial cells can also connect them toneuronal networks. Although gap junctions between glial cellsand neurones are not common, their existence has beendemonstrated in crayfish (Peracchia, 1981). Additionally,direct heterocellular coupling of mammalian neurones andglia has been demonstrated in neuronal-glial co-cultures(Nedergaard, 1994; Froes et al., 1999), in acute slices from thelocus coeruleus (Alvarez-Maubecin et al., 2000) and in thecerebellum (Pakhotin and Verkhratsky, 2005).

2.5.4. Gliotransmission and exocytotic releaseAstrocytes and other glial cells can release a variety oftransmitters into the extracellular space. The criteria for achemical released fromglia to be classified as a gliotransmitterhave been defined (Do et al., 1997; Martin et al., 2007; VolterraandMeldolesi, 2005). Gliotransmitters are classified based on aworking set of criteria: (i) synthesis by and/or storage in glia; (ii)regulated release triggered by physiological and/or patholog-ical stimuli; (iii) activation of rapid (milliseconds to seconds)responses in neighbouring cells; and (iv) a role in (patho)physiological processes (Parpura and Zorec, 2010).

Since the first demonstration of the release of GABA fromglial cells in superior cervical ganglia (Bowery et al., 1976), aquest for an understanding the mechanisms and conditionsthat underlie transmitter release is underway. Several differ-ent mechanisms have been implicated in release of agliotransmitter: (i) through channels like anion channelopening, induced by cell swelling (Pasantes Morales andSchousboe, 1988), release through functional unpaired “hemi-channels” (connexins/pannexins) located at the plasmamem-brane (Cotrina et al., 1998; Iglesias et al., 2009) and P2X7

ionotropic purinergic receptors (Duan et al., 2003); (ii) throughtransporters, e.g. by reversal of uptake by plasma membraneexcitatory amino acid transporters (Szatkowski et al., 1990),exchange via the cystine–glutamate antiporter (Warr et al.,1999) or organic anion transporters (Rosenberg et al., 1994);and (iii) through Ca2+-dependent exocytosis (Parpura et al.,1994). Gliotransmitters can be divided into three generalgroups: (i) amino acids and their derivatives, such asglutamate and D-serine; (ii) nucleotides, like ATP, and (iii)peptides (recently reviewed in Parpura and Zorec, 2010). Atpresent, Ca2+-dependent vesicular release of glutamate andATP from astrocytes can readily occur under physiologicalconditions, while there are questions raised as to whetherother mechanisms might solely operate during pathophysio-logical circumstances.

The first documented description of general secretion fromglial cells came from the work of a French neuroanatomist-physician Jean Nageotte who proposed that these cells mayrelease substances into the blood, acting like an endocrinegland (Nageotte, 1910). He observed various secretory granulesin glial cells of grey matter (astrocytes) using the Altmannmethod of fucsin labelling (Fig. 5).

He reported: “The facts that I have just observed seemsto me shed new light on the physiology of the neuroglialcells, not only of cells that are associated with neuronalcells, which have been named satellite cells, but also, andparticularly, of cells that are in connection with thevasculature walls.

Indeed, I was able to present evidence of robust and activesecretion phenomenon in the protoplasm of these cells inrabbit and guinea pig. This observation was visible especiallywithin the protoplasmic expansions which cross the emptyspace created by the retraction of tissues around the vascularwalls, on which the neuroglial cells attach using an enlargedfoot.

In a previous note, I have described the mitochondria thatexist in these protoplasmic expansions, and I have shown thatmany, and maybe all of the granulations located in the greysubstance outside the protoplasm of neuronal cells, in realitybelong to the neuroglia. Today, I am poised to follow theevolution that occurs within these granulations and to showtheir progressive transformation into secretion grains. Thesephenomena are exactly similar as those described by Altmannin the glandular cells; the granulations observed are of threetypes: 1° round grains excessively small that, by the Altmannmethod, colour intensively in red; 2° more voluminous grains,with clear centres; 3° grains that do not colourwith fucsin. Thelast ones are slightly smaller than the more voluminous redgrains. All intermediates exist between the three types, whichrepresent the successive phases of the transformation of

Fig. 5 –Neuroglial cells of grey matter in rabbit medulla, mostlikely astrocytes, contain various secretory granules. Scalebar, 5 μm. Reproduced from Nageotte (1910).

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mitochondria in to secretion grains.” (Translated from French;Nageotte, 1910).

Only recently, however, has there been accumulatingevidence of biochemical and morphological correlates of theexocytotic process in astrocytes (reviewed in Montana et al.2006) as briefed below.

Astrocytes express proteins of the core SNARE complex:synaptobrevin 2, syntaxin 1, synaptosome-associated proteinof 23 kDa, as well as proteins important for sequesteringglutamate/ATP into vesicles: the vacuolar type of protonATPase (V-ATPase), which drives protons into the vesicularlumen creating the proton concentration gradient necessaryfor glutamate/ATP transport intovesicles, and the threeknownisoforms of vesicular glutamate transporters (VGLUTs) 1, 2 and3 and vesicular nucleotide transporter (VNUT) (reviewed inParpura and Zorec, 2010). Immunoelectronmicroscopy studiesdemonstrated that VGLUTs 1 or 2 in astrocytes in situ associatewith small clear vesicles with a mean diameter of ∼30 nm(Bezzi et al., 2004). Astrocytes also display large dense coregranules with diameters of ∼115 nm, containing the secretorypeptide secretogranin II (Calegari et al., 1999) and ATP (Coco etal., 2003). These two populations of vesicles appear to bebiochemically and morphologically distinct.

The consequences of Ca2+-dependent exocytotic glutamaterelease from astrocytes can be seen in neurones as: (i) anelevation of neuronal [Ca2+]i (Parpura et al., 1994); (ii) a slowinward current (Araque et al., 1998a; Araque et al., 1998b);(iii) an increase of excitability (Hassinger et al., 1995);(iv) modulation of spontaneous and action potential evokedsynaptic transmission (Araque et al., 1998a; Araque et al.,1998b); (v) synchronization of synaptic events (Fellin et al.,2004), induction of long-term potentiation, LTP (Perea andAraque, 2007) or various combinations of the above (reviewedin Ni et al., 2007). Such effects of the glutamate-mediated

astrocyte–neurone signalling pathway have been observed inneurones of different brain regions, including visual cortex,hippocampus, thalamus and nucleus accumbens.

Besides glutamate there are several other vesicularly re-leasedmediators of astrocyte–neurone signalling, most notablyD-serine andATP. Astrocytes can exocytotically release D-serine(Mothet et al., 2000) an endogenous NMDA receptor agonist(Schell et al., 1995). Indeed, release of D-serine from glia cancontribute to the physiological activation of NMDA receptorsexpressed on retinal ganglion cells (Stevens et al., 2003), as wellas to the hippocampal LTP (Yang et al., 2003). Similarly, ATP canbe released from astrocytes and it serves as the major humoralfactor in support of astroglial Ca2+ wave propagation (Arcuinoet al., 2002; Cotrina et al., 2000; Guthrie et al., 1999; Stout et al.,2002). ATP can also contribute to astrocyte–microglia (VerderioandMatteoli, 2001), astrocyte–endothelial cell (Braet et al., 2001),andastrocyte–neurone signalling (Newman, 2003). For example,in the case of astrocyte–neurone signalling, stimulated astro-cytes in the whole mount retina can release ATP intoextracellular space, where it is rapidly converted by ectoen-zymes to adenosine. Subsequent activation of adenosinereceptors located on retinal ganglion cells induces hyperpolar-ization of these cells thus reducing spontaneous firing rate ofneurones exhibiting spontaneous spike activity. Similarly, ATPreleased from astrocytes can cause tonic and activity-depen-dent suppression of excitatory synaptic transmission in acuteslices (Zhang et al., 2003). Taken together, glia–neuronesignalling pathway might be a wide-spread phenomenonthroughout the brain with glia/astrocytes having the means(by releasing various gliotransmitters) to participate in manyinformation processing functions of the CNS.

2.5.5. Neuroglia controls connectivity in neural networksThe operational connectivity between the elements of thenervous system are of a paramount importance for itsfunction; by and by the maintenance and plastic remodellingof the connectivity status is the raison d'etre of the nervoustissue. The connections in the brain are represented by manystructures that include chemical and electrical synapses, theneuronal–glial and glial–glial contacts. The synapse appear-ance, maintenance survival and death — these all arecontrolled by the neuroglia. Indeed, astrocytes secrete numer-ous factors indispensable for synaptogenesis (Liauw et al.,2008; Nieweg et al., 2009), and without astrocytes formation ofsynapses is greatly depressed (Pfrieger, 2009; Pfrieger andBarres, 1996). The astroglia in the tripartite synapse (which isthe most common in the CNS) maintains the efficacy ofsynaptic transmission through homeostatic control over thesynaptic cleft contents and through metabolic support.Removal of astroglial coverage results in synaptic shutdownand synapse elimination (Nagele et al., 2004). The second levelof CNS connectivity accomplished by the white matter isalmost exclusively maintained by the oligodendrocytes, andoligodendroglial failure severely alters the brain function.

2.6. Pathophysiological aspect

The pathological potential of neuroglia was recognised veryearly, and several prominent neurologists of 19th–beginningof 20th centuries have stressed the role of glial cells in the

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progression of various neurological diseases (Alzheimer, 1910;Del Río-Hortega, 1919a; Frommann, 1878; Nissl, 1899). Ourknowledge of the pathological importance of neurogliaremains fragmentary, because of a long-lasting prevalence ofneurocentric views in neurology and neuropathology.

Despite substantial gaps in our knowledge we may safelyconclude that neuroglia takes the leading role in initiation,progression and outcome of neurological diseases; further-more we can regard neurological diseases primarily as thegliopathologies (for the state of the art reviews and compre-hensive literature see Fields (2008b), Giaume et al. (2007),Halassa et al. (2007a), Heneka et al. (2010), Matute (2010),Nedergaard and Dirnagl (2005), Nedergaard et al. (2010),Rodriguez et al. (2009), Rossi and Volterra (2009) and Seifertet al. (2010)). Indeed, the ultimate specialisation of neurones,which underlie the sophistication of neuronal network in thehuman brain, robbed the former from the house-holdingabilities. Indeed in the higher mammals, the neuroglia is fullyresponsible for brain homeostasis, for the brain metabolicsupport and for the brain defence, and every type of lesion tothe nervous system tests the ability of neuroglia to withstandand repair the damage.

The neuroglial cells are involved in all types of brainpathology from acute lesions (trauma or stroke) to chronicneurodegenerative processes (such as Alzheimer's disease,Parkinson's disease, multiple sclerosis and many others) andpsychiatric diseases. The pathologically relevant neuroglialprocesses are many, and they include various programmes ofactivation, which are essential for limiting the areas of damage,producing neuro-immune responses and for the post-insultremodelling and recovery of neural function (Hanisch andKettenmann, 2007; Pekny and Nilsson, 2005; Rolls et al., 2009).Recent studies also emphasised the role of astroglial degener-ation and atrophy in the early stages of various neurodegener-ative disorders, which may be important for cognitiveimpairments (see Heneka et al., 2010; Olabarria et al., 2010;Rodriguez et al., 2009; Rossi and Volterra, 2009). Similarly,microglia being the innate CNS defence system is intimatelyinvolved in all types of brain pathology including the neurode-generative diseases (Heneka et al., 2010; Rodríguez et al., 2010).

3. Conclusion: the neuronal–glial networksform the substrate of brain function

The human brain is unique in its cognitive power that resultsfrom many levels of integration within complex cellularnetworks. The brain is organised by combining the clearlydelineated functional modules (responsible for sensory inputsand executive outputs) with the diffuse information proces-sing and analysis that involves many different structures. Inthat, when performing the complex functions related to self-consciousness, generation of thoughts and creativity the brainvery much acts like a “diffuse neural net” contemplated byCamillo Golgi.

Our understanding of the brain function is very muchshaped by neuronal doctrine that assumes that the neuronalnetworks represent the sole substrate for cognition. Theseneuronal networks are, however, embedded into muchlarger and probably more complex networks made by

neuroglia. The latter, although being electrically silent,employ many different mechanisms for inter- and intracel-lular signalling. It appears that astrocytes can controlsynaptic networks and in such a capacity they mayrepresent an integral component of the computationalpower of the brain rather than being just a brain “connectivetissue”. Consequently, it is not surprising that in anascending phylogeny, the glia (astrocytes):neurones ratioincreases and peaks within the human brain, which has bothrelatively and absolutely the greatest number of glia.Interestingly, Albert Einstein's brain contained a significant-ly higher ratio of glia:neurons in his left Brodmann's area 39(Diamond et al., 1985). This makes us wonder.

The fundamental question of whether neuroglia is in-volved in cognition and information processing remains,however, open. Indeed, the “glial explosion” that distinguishesthe human brain can be simply a result of exceedingly highspecialisation of the neuronal networks that delegated allmatters of survival andmaintenance to the neuroglia; to do sothe latter obviously is in need of sensing the neuronal activityand communicating with fellow glia. At the same timepotential power of analogue processing offered by internallyconnected glial networks may represent the alternativemechanism of cognitive amplification. Where is the truth?This is the future, which holds the answer.

Acknowledgments

We thank Drs. Vedrana Montana and Mathieu Lesort for theirtranslation of Nageotte's publication. Authors' research wassupported by the Alzheimer's Research Trust (UK) Programmegrant (ART/PG2004A/1) to AV and JJR; by the National Instituteof Health (NIH) grant to VP (R01 MH 069791); by the NationalScience Foundation (CBET 0943343) grant to VP, and by theGrant Agency of the Czech Republic (GACR 309/09/1696) to JJRand (GACR 305/08/1381, GACR 305/08/1384) to AV.

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