CHAPTER 2

Neuron–glial cell cooperation

There are roughly twice as many glial cells as there Glial cells have morphological as well as functional are in the central . They occupy and metabolic characteristics that distinguish them the space between neurons and neuronal processes and from neurons: separate neurons from blood vessels. As a result, the extracellular space between the plasma membranes of • They do not generate or conduct action potentials. different cells is narrow, of the order of 15–20 nm. Thus, although they extend processes, these are only Virchow (1846) was the first to propose the existence of one type and are neither nor . of non-neuronal tissue in the . • They do not establish chemical between He named it ‘nevroglie’ ( glue), because it themselves, with neurons, or any other cell type. appeared to stick the neurons together. Following this, • Unlike most neurons in humans, glial cells are capa- Deiters (1865) and Golgi (1885) identified glial cells as ble of division for at least several years postnatally. making up the nevroglie and distinguished them from neurons. is made compact by glial cells and for There are several categories of glial cells. Depend- this reason they are often ascribed the role of support- ing on their anatomical position they are classed as ing tissue. However, as we will see in this chapter, they follows: have additional functions. We will explain in this chapter the roles of , and Schwann • Central are found in the central nervous system, cells, only. and comprise four cell types: astrocytes, oligoden- drocytes, (these three types are also known as interstitial glia, because they are found in interneuronal spaces) and ependymal cells which 2.1 ASTROCYTES FORM A VAST form the epithelial surface covering the walls of the CELLULAR NETWORK OR cerebral ventricles and of the central canal of the SYNCYTIUM BETWEEN NEURONS, spinal cord. BLOOD VESSELS AND THE • Peripheral glia comprise a single type: Schwann cells. SURFACE OF THE BRAIN These cells ensheath the axons and encapsulate the cell bodies of neurons. In the latter case, they are 2.1.1 Astrocytes are star-shaped cells also called satellite cells. characterized by the presence of glial filaments in their cytoplasm Glial cells, excluding microglia, have an ectodermal ori- gin. Those of the central nervous system derive from Astrocytes are small star-shaped cells with numerous the germinal neural epithelium (neural tube), while fine, tortuous, ramified processes covered with vari- peripheral glia (Schwann cells) are derived from the cosities (Figure 2.1). The cell body is typically 9–10 µm neural crest. Microglia, in contrast, have a mesodermal in diameter and the processes extend radially over origin. 40–50 µm. These often have enlarged terminals in

21 22 2. –GLIAL CELL COOPERATION

Soma of fibrillary

Terminal end foot

Blood vessel

FIGURE 2.1 Fibrillary astrocyte. Micrograph of a fibrillary astrocyte stained with a Golgi stain observed through an optical microscope. The processes of this astrocyte make contact with a blood vessel: these are the terminal end feet. Photograph by Olivier Robain. contact with neurons or non-neuronal tissue (like the . This characteristic has been exploited walls of blood vessels). as a method of identifying astrocytes. By using an anti- Two kinds of astrocytes are recognized. Some astro- serum to glial fibrillary acidic protein (anti-GFAP) cytes contain in their cytoplasm numerous glial fila- linked to fluorescein, one can stain astrocytes, in situ or ments: these are fibrillary astrocytes, principally located in culture, without marking either neurons or other in the (Figure 2.1). They have numerous, types of glial cells. radial processes which are infrequently branched and Astrocytes, like all glial cells, do not form chemical covered with ‘expansions en brindilles’. Other astro- synapses. They do, however, mutually form junctional cytes contain few, if any, glial filaments: these are proto- complexes. Two types of junctions have been demon- plasmic astrocytes, found normally in the . strated: communicating junctions (or gap junctions) and They have more delicate processes, some of which are desmosomes (puncta adhaerentia). Coupled to each velate (veil-like). Both types of astrocytes send out other by numerous junctional complexes, astrocytes processes that end on the walls of blood vessels or therefore constitute a vast cellular network, or syn- beneath the pial surface of the brain and spinal cord. cytium, extending from neurons to blood vessels and The principal ultrastructural characteristics of astro- the external surface of the brain. cytes are the glial filaments and glycogen granules present in the cytoplasm of their somata and processes. 2.1.2 Astrocytes maintain the blood–brain The filaments are ‘intermediate filaments’ with an barrier in the adult brain average diameter of 8–10 µm. They are composed of a protein specific to astrocytes, glial fibrillary acidic pro- The essential characteristic of astrocytic processes is tein (GFAP), consisting of a single type of subunit with their termination on the walls of blood vessels in astro- a molecular weight of 50 kD, different from that of cytic end feet (Figure 2.2). Here the end feet are joined 2.1 ASTROCYTES FORM A VAST CELLULAR NETWORK OR SYNCYTIUM BETWEEN NEURONS, BLOOD VESSELS AND THE SURFACE 23

Astrocyte terminal barrier, and that astrocytes thus contribute to regulation end feet of the brain extracellular fluid. However, astrocytes have other important roles in controlling the composi- tion of the extracellular fluid. We shall consider as an Capillary lumen example the regulation of the extracellular potassium concentration. The extracellular potassium concentration needs to be tightly regulated: if potassium increased it would depolarize neurons. This would first increase neuronal excitability and then inactivate propa- gation. Regulation of the extracellular potassium con- centration must occur in the face of large fluxes of potassium ions into the extracellular space during Blood vessel in the CNS neuronal activity, when potassium ions leave neurons FIGURE 2.2 Diagram of the covering formed by astrocyte end through voltage-activated potassium channels (see feet around a capillary in the central nervous system (CNS). Sections 4.3 and 5.3). Astrocytes are thought to regulate From Goldstein G and Betz L (1986) La barrière qui protège le extracellular potassium by the mechanism of ‘spatial cerveau. Pour la Science, November, 84–94, with permission. buffering’. This means that astrocytes take up potas- sium ions in regions where the concentration rises and by gap junctions and desmosomes, forming a ‘palisade’ eventually release through their end feet an equivalent between neurons and vascular endothelial cells. The amount of potassium ions into the vicinity of blood space between the layer of astrocyte end feet and the vessels or across the glia limitans externa. The details of endothelial cells is about 40–100 nm and is occupied by the process are complicated, but potassium ions are a basal lamina. Astrocytes also send processes to the thought to enter astrocytes via channels or the sodium external surface of the central nervous system where pump and to exit at the end feet through channels. This the astrocyte end feet, together with the basal lamina potassium buffering role of astrocytes is likely to be of that they produce, form the ‘glia limitans externa’, particular importance at the nodes of Ranvier, where which separates the pia mater from the nervous tissue. marked accumulation of potassium ions in the Astrocytes therefore constitute a barrier between neu- restricted extracellular space can occur, due to the con- rons and the external medium (blood), preventing duction of action potentials. access of substances foreign to the central nervous sys- tem. They thus protect neurons. This barrier is not, how- 2.1.4 Astrocytes take part in the ever, totally impermeable and astrocytes are involved in neurotransmitter cycle selective exchange processes. Astrocyte end feet are not the blood–brain barrier. After neurotransmitters are released during synaptic This is formed, in most regions of the central nervous transmission, they need to be removed from the extra- system, by vascular endothelial cells joined together cellular space to prevent the extracellular neurotransmit- by tight junctions. Even though the astrocyte end feet ter concentration from rising. Steady high concentrations do not form the blood–brain barrier, they have an of transmitter would interfere with synaptic transmis- important role in its development and maintenance. sion, and long-lasting activation of receptors (particu- Thus, if the layer of astrocyte end feet in the adult is larly glutamate receptors) can damage neurons. Most destroyed, by a tumour or by allergic illnesses, for transmitters are removed from the extracellular space example, the capillary endothelial cells immediately by reuptake into cells (but acetylcholine is hydrolyzed; take on the characteristics normally observed in capil- see Figure 6.12). Transmitters are taken up by special- laries outside the central nervous system: they are no ized carrier molecules in the . Although longer bound by tight junctions and become ‘fenes- both neurons and glia express such carrier proteins, it trated’. In such capillaries the blood–brain barrier no seems that uptake into astrocytes is of particular longer exists. importance. This is especially clear for the case of glu- tamate: astrocytes have an enormous capacity to take 2.1.3 Astrocytes regulate the ionic up this transmitter, presumably reflecting the abun- composition of the extracellular fluid dance of this transmitter and the toxicity to neurons of high glutamate concentrations. We have seen that astrocyte end feet are involved in Besides their role in transmitter clearance from the the formation and maintenance of the blood–brain synaptic cleft (by recapture), astrocytes play a role in the 24 2. NEURON–GLIAL CELL COOPERATION synthesis of transmitters and particularly glutamate and GABA. For example, thanks to the presence of glutamine synthetase in astrocytes (see Figure 10.13), glutamine is formed from glutamate. Glutamine is then uptaken by Myelinated neurons and transformed back in glutamate.

2.2 OLIGODENDROCYTES FORM Myelinated THE SHEATHS OF AXONS axon Myelinated IN THE CENTRAL NERVOUS SYSTEM axon AND ALLOW THE CLUSTERING OF NAϩ CHANNELS AT NODES process OF RANVIER Cell body of Oligodendrocyte Two types of oligodendrocyte are recognized: inter- fascicular or myelinizing oligodendrocytes, found in the white matter where they make the sheaths of myelinated axons; and satellite oligodendrocytes which surround neuronal somata in the grey matter. We will deal with the former type in detail. Their major role is, by forming the myelin sheath, to electrically isolate segments of axons, induce the formation of ϩ clusters of Na channels at nodes of Ranvier and there- FIGURE 2.3 Myelinating oligodendrocyte. fore to allow the fast propagation of Naϩ action poten- Electron micrograph of an oligodendrocyte. The cell body and one of tials (see Section 4.4). its processes enwrapping several axons can be seen. Section taken through the spinal cord. Photograph by Olivier Robain. 2.2.1 Processes of interfascicular oligodendrocytes electrically isolate segments of central axons by forming The myelin sheath is a compact roll of the plasmalemma the lipid-rich myelin sheath of an oligodendrocyte process: this glial membrane is rich in lipids The cell bodies of interfascicular oligodendrocytes are situated between bundles of axons Myelinated axons are surrounded by a succession of myelin segments, each about 1 mm long. The covered Interfascicular, or myelinizing, oligodendrocytes have regions of axons alternate with short exposed lengths small spherical or polyhedral cell bodies of diameter where the axonal membrane () is not covered. 6–8 µm and few processes. They are called interfascicu- These unmyelinated regions (of the order of a micron) lar because their cell bodies are aligned between bun- are called nodes of Ranvier (Figures 2.4 and 2.5a). dles (fascicles) of axons. They are distinguished from Amyelinated segment comprises the length of axon astrocytes by the sites of termination of their processes: covered by an oligodendrocyte. One oligodendrocyte oligodendrocyte processes enwrap axons and make no can form 20–70 myelin segments around different axons contact with blood vessels. (Figure 2.4). Thus the degeneration or dysfunction of Observed by electron microscopy, the nucleus and a single oligodendrocyte leads to the disappearance perikaryon of oligodendrocytes appear dark (Figure 2.3), of myelin segments on several different axons. there are no glial filaments, and there are many micro- tubules in the somatic and dendritic cytoplasm. Because Formation and ultrastructure of a myelin segment of this, oligodendrocyte processes may be confused with fine dendrites, and it is by the absence of chemical Myelinization represents a crucial stage in the onto- synapses that the glial processes are identified. genesis of the nervous system. In the human at birth, Oligodendrocytes can be identified by immunohisto- myelinization is only just beginning, and in some chemistry. This is done using an antigalactoceramide regions is not complete even by the end of the second immune serum (anti-gal-C), galactoceramide being year of life. The first step in the process is migration of a glycolipid found exclusively in the membrane of oligodendrocytes into the bundles of axons, then the processes of myelinizing oligodendrocytes. myelinization of some, but not all, axons. Once contact 2.2 OLIGODENDROCYTES FORM THE MYELIN SHEATHS OF AXONS IN THE CENTRAL NERVOUS SYSTEM 25

Node of also disappears, and the external leaflets also stick to Ranvier each other. This apposition is, however, less close and a small space remains between the external leaflets. The apposed external leaflets form the minor, or inter- period, dense line (Figure 2.5b). Thus, a cross-section of a myelinated axon observed by electron microscopy shows alternating dark and light lines forming a spiral around the axon. The major Cell body dense line terminates where the internal leaflets sepa- rate to enclose the cytoplasm within the external loop. External loop The interperiod dense line disappears at the surface of the sheath at the end of the spiral (Figure 2.5b). In the central nervous system there is no basal lam- ina around myelin segments, so myelin segments of adjacent axons may adhere to each other forming an interperiod dense line.

Myelin Myelin consists of a compact spiral (without intra- cellular or extracellular space) of glial plasma mem- Internal loop Axon brane of a very particular composition. Lipids make up about 70% of the dry weight of myelin and proteins FIGURE 2.4 Diagram of a myelinating oligodendrocyte and its only 30%. Compared with the membranes of other numerous processes. Each form a segment of myelin around a different axon in the central cells, this represents an inversion of the lipid:protein nervous system. Two myelin segments are represented, one partially ratio (Figure 2.6). unrolled, the other completely unrolled. Drawing by Tom Prentiss. This lipid-rich membrane is highly enriched in In Morell P and Norton W (1980) La myéline et la sclérose en glycosphingolipids and cholesterol. The major glyco- plaques. Pour la Science 33, with permission. in myelin are galactosylceramide and its sulfated derivative (20% of lipid dry weight). has been made between the oligodendrocyte and axon, There is also an unusually high proportion of ethano- the initial turn of myelin around the axon is rapidly lamine phosphoglycerides in the plasmalogen form, formed. Myelin is then slowly deposited over a period which accounts for one-third of the . which in humans can reach several months. Myelin- In myelin, a number of structural classes of proteins ization is responsible for a large part of the increase in are present. Some proteins are extremely hydrophobic weight of the central nervous system following the end membrane-embedded polypeptides, some integral of neurogenesis. membrane proteins have a single transmembrane In order to form the compact spiral of myelin mem- domain and clearly define extra- and intracellular brane, the oligodendrocyte process must roll itself domains, and some of the myelin proteins are cytoso- around the axon many times (up to 40 turns) (Figure lic; however, they are often intimately associated with 2.5). It is the terminal portion of the process, called the the myelin membrane. inner loop, situated at the interior of the roll, which pro- Myelin basic protein (MBP) and the proteolipid pro- gressively spirals around the axon. This movement teins (PLP/DM20) are the two major myelin proteins necessitates the sliding of myelin sheets which are not in the CNS. Myelin basic proteins are found on the firmly attached. During this period, the oligodendro- cytoplasmic side and play a role in the adhesion of the cyte synthesizes several times its own weight of myelin internal leaflets of the specialized oligodendroglial membrane each day. plasma membrane. Proteolipid proteins are integral Within the spiral the cytoplasm disappears entirely membrane proteins. Though they are in high abun- (except at the internal and external loops). The internal dance (they represent around 50% of the total myelin leaflets of the plasma membranes can thus adhere to protein in the central nervous system) their exact each other. This adhesion is so intimate that the inter- biological role has not yet been elucidated. nal leaflets virtually fuse, forming the period, or major, We have seen that myelin has an inverted lipid:pro- dense line of thickness of 3 nm (Figure 2.5b). The extra- tein ratio, while the cell body membrane of the oligo- cellular space between the different turns of membrane dendrocyte has a ratio comparable to that of other cell 26 2. NEURON–GLIAL CELL COOPERATION

(a) Oligodendrocyte (b) (cell body) Glial process Glial process

External loop Major dense line

Area enlarged in (b) Interperiod line

CNS axon Myelin sheath

CNS axon Node of Ranvier

Paranodal region

FIGURE 2.5 Myelin sheath of central axons. (a) Three-dimensional diagram of the myelin sheath of an axon in the central nervous system (CNS). The sheath is formed by a succession of compact rolls of glial processes from different oligodendrocytes. (b) Cross-section through a myelin sheath. The dark lines, or major dense lines, and clear bands (in the middle of which are found the interperiod lines) visible with electron microscopy are accounted for by the manner in which the myelin membrane surrounds the axon, and by the composition of the membrane. The dark lines represent the adhesion of the internal leaflets of the myelin membrane while the interperiod lines represent the adhesion of the external leaflets. The lines are formed by membrane proteins while the clear bands are formed by the . Drawing (a) from Bunge MB, Bunge RP, Ris H (1961) Ultrastructural study of in an experimental lesion in adult cat spinal cord. J. Biophys. Biochem. Cytol. 10, 67–94, with permission of Rockerfeller University Press. Drawing (b) by Tom Prentiss. In Morell P and Norton W (1980) La myéline et la sclérose en plaques. Pour la Science 33, with permission.

100 100 Nodes of Ranvier Protein Phospholipids In the central nervous system the nodes of Ranvier, 75 75 Protein Phospholipids regions between myelin segments, are relatively long (several microns) compared with those in the periph- 50 50 Glycolipids eral nervous system. Here the axolemma is exposed

Percentage Percentage Lipid and an accumulation of dense material is seen on the Glycolipids 25 25 cytoplasmic side. The myelin sheath does not termi- Lipid Cholesterol Cholesterol nate abruptly. Successive layers of myelin membrane 0 0 terminate at regularly spaced intervals along the axon, Hepatic cell membrane Myelin the internal layers (close to the axon) terminating first. FIGURE 2.6 Comparison of the lipid content of plasma mem- This staggered termination of the different layers of brane and myelin. myelin constitutes the paranodal region (Figure 2.5a). The protein:lipid ratio is inverted between the two membranes. The proportions of the three groups of lipids are also different. 2.2.2 Myelination enables rapid conduction membranes. As the myelin of the oligodendrocyte of action potentials for two reasons process is in continuity with the plasma membrane of Isolation of internode axonal segments the cell body, it is necessary to postulate gradients in the composition of lipids and proteins (in opposite The high lipid content and compact structure of the directions to each other) between the cell body and the myelin sheath help make it impermeable to hydrophilic various processes. During the active phase of myelina- substances such as ions. It prevents transmembrane ion tion, each oligodendrocyte must produce as much as fluxes and acts as a good electrical insulator between 5–50,000 µm2 of myelin membrane surface area per day. the intracellular (i.e. intra-axonal) and extracellular 2.2 OLIGODENDROCYTES FORM THE MYELIN SHEATHS OF AXONS IN THE CENTRAL NERVOUS SYSTEM 27 media. Between the nodes of Ranvier the axon therefore oligodendrocyte contact with axon influences Naϩ behaves as an insulated cable. This permits rapid, channel distribution, nodes of Ranvier in the brain of action potentials along the axon of hypomyelinating mouse Shiverer are examined. (see Section 5.4). Shiverer mice have oligodendrocytes that ensheath axons but do not form compact myelin and axoglial junctions. In these mutant mice, there are far fewer Formation of Ranvier nodes with a high density of ϩ Naϩ channels Na channel clusters than in control littermates and aberrant locations of Naϩ channels are observed. If Naϩ channels are clustered in very high density within Naϩ channel clustering depends only on the presence the nodal gap whereas voltage-dependent Kϩ channels of oligodendrocytes and is independent of myelin and are segregated in juxta-paranodal regions, beneath oligodendroglial contact, one would expect to find overlying myelin (see Figure 2.5a). To test whether normal Naϩ channel distribution along axons.

Internal loop (a) Major dense line Minor dense line External loop

Nucleus Axon body Myelin sheath Internal loop External loop

Periaxonal space (extracellular) Node of Ranvier

Paranodal region

(b) 1 2 Schwann cell

Schwann Internal loop PNS axon External loop

Periaxonal space 3

Myelin sheath

FIGURE 2.7 Myelin sheath of a peripheral axon. (a) Three-dimensional diagram of the myelin sheath of an axon of the peripheral nervous system (PNS). The sheath is formed by successive rolled Schwann cells. (b) Process of myelinization. The internal loop wraps around the axon several times. During this process the axon grows and the myelin becomes compact. Contact between the Schwann cell and axon occurs only at the paranodal and nodal regions. Elsewhere an extracellu- lar, or periaxonal, space always remains. Drawing (a) adapted from Maillet M (1977) Le Tissu Nerveux, Paris: Vigot, with permission. Drawing (b) by Tom Prentiss. In Morell P and Norton W (1980) La myéline et la sclérose en plaques. Pour la Science 33, with permission. 28 2. NEURON–GLIAL CELL COOPERATION

2.3 SCHWANN CELLS ARE THE non-myelinating Schwann cell surrounds several axons GLIAL CELLS OF THE PERIPHERAL (about 5–20) for a distance of 200–500 µm in man. NERVOUS SYSTEM; THEY FORM THE In addition, spinal and cranial ganglia contain a MYELIN SHEATH OF AXONS OR large number of Schwann cells that do not produce ENCAPSULATE NEURONS myelin. These Schwann cells cover the somata of the ganglionic cells, leaving an extracellular space of about 20 nm between themselves and the surface of the There are three types of Schwann cell: covered neuron. • those forming the myelin sheath of peripheral The lipid and protein composition of the plasma myelinated axons (myelinating Schwann cells); membrane of non-myelinating Schwann cells is the • those encapsulating non-myelinated peripheral same as that of other eukaryotic cells (30% lipid, 70% axons (non-myelinating Schwann cells); those that protein). encapsulate the bodies of cells (non- Apart from their role in the saltatory conduction of myelinating Schwann cells or satellite cells). action potentials (myelinating Schwann cells), Schwann cells also play a role in the regeneration of peripheral 2.3.1 Myelinating Schwann cells make the nerve cells. It has long been known that cut peripheral myelin sheath of peripheral axons can, within certain limits, regrow and reinner- vate deafferented regions while central axons are not Along an axon, several Schwann cells form succes- capable of this. This property of regeneration is due in sive segments of the myelin sheath. In contrast to large part to an enabling effect of Schwann cells on oligodendrocytes, it is not a process that enwraps the axon regrowth. peripheral axon to form the segment of myelin, but the whole Schwann cell (Figure 2.7). Each Schwann cell therefore forms only one myelin segment. FURTHER READING The composition of peripheral myelin differs from that of central myelin only in the proteins it contains. The principal protein constituents of peripheral myelin Dupree JL, Mason JL, Marcus JR, et al. (2005) Oligodendrocytes assist in the maintenance of sodium channel clusters independent of the are: peripheral myelin protein 2 (P2), protein zero (P0) myelin sheath. Neuron Glia Biol. 1, 1–14. and myelin basic proteins (MBPs). The first two proteins Farber K and Kettenmann H (2005) Physiology of microglial cells. are specific to peripheral myelin. MBP comprises a Brain Res Brain Res Rev. 48, 133–143. major part of the cytosolic protein of myelin and is pres- Freeman MR and Doherty J (2006) Glial cell biology in Drosophila 29 ent both in the CNS and PNS. Protein zero is a glyco- and vertebrates. Trends Neurosci. , 82–90. Greer JM and Lees MB (2002) Myelin proteolipid protein – the first protein that has adhesive properties and is located in the 50 years. Int J Biochem & Cell Biology 34, 211–215. interperiod line. It functions, in part, as a homotypic Montague P, McCallion AS, Davies RW, Griffiths IR (2006) Myelin- adhesion molecule throughout the full thickness of the associated oligodendrocytic basic protein: a family of abundant myelin sheath. It is a good marker for myelinating CNS myelin proteins in search of a function. Dev Neurosci. 28, Schwann cells as it represents over 50% of total PNS 479–487. Peles E and Salzer JL (2000) Molecular domains of myelinated axons. myelin protein. Curr Opin Neurobiol. 10, 558–565. Pfeiffer SE, Warrington AE, Bansal R (1993) The oligodendrocyte and its many cellular processes. Trends Cell Biol. 3, 191–197. 2.3.2 Non-myelinating Schwann cells Popko B (2000) Myelin galactolipids: mediators of axon–glial inter- encapsulate the axons and cell bodies actions? Glia 29, 149–153. of peripheral neurons Shapiro L, Doyle JP, Hensley P, Colman DR, Hendrickson WA (1996) Crystal structure of the extracellular domain from P0, the major Non-myelinated axons are not uncovered in the structural protein of peripheral nerve myelin. Neuron 17, 435–449. peripheral nervous system as they are in the central Streit WJ (2000) Microglial response to brain injury: a brief synopsis. nervous system; they are encapsulated. A single Toxicol. Pathol. 28, 28–30.