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Ependymal Cells
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Ependymal Cells H Wolburg, K Wolburg-Buchholz, and A F Mack, University of Tu¨bingen, Tu¨bingen, Germany A Reichenbach, University of Leipzig, Leipzig, Germany ã 2009 Elsevier Ltd. All rights reserved.
Introduction As early as 1837 Valentin and Purkinje visualized the lining of the inner cavities of the brain, the brain ventricle system, by the observation of striking cilia on its surface. This lining was called ‘ependyma’ (Greek meaning ‘upon garment’), and its constituent cells are termed ependymal cells. The ependymal cells belong to the family of neuroglial cells (glia) and are thus also called ependymoglial cells or ependymal glia. After its early discovery, it required many decades of research to elucidate this cellular layer(s) as an extremely important component of the developing and mature brain. During ontogenetic development, the ventricular lining is composed of proliferating cells termed the ‘matrix zone,’ which generates all types of neuroectodermal brain cells, including neurons and their ‘local successors,’ the ependymoglial cells. In the adult central nervous system (CNS), the ependymoglial cells contribute to the secretion of the cerebrospinal fluid (CSF) and are an essential component of an important biological barrier system segregating different compartments such as blood, CSF, and CNS interstitial space (blood–brain and blood–CSF barriers; Figure 1). Originally, Paul Ehrlich found that a blood-infused dye failed to stain the brain tissue, and his pupil, Ernst Goldmann, observed complementarily that the very same dye, if applied into the CSF, did stain the brain tissue. This led to the concept of a biological barrier between blood and brain. Due to the free access of the dye from brain ventricle to brain tissue, they concluded that there is no CSF–brain barrier. However, the staining of circumventricular organs and the choroid plexus in Goldmann’s experiment applying the dye into the general circulation (Goldmann-I experiment) and the avoidance of staining of these organs in the experiment applying the dye into the CSF (Goldmann-II experiment) suggested the existence of a barrier between the CSF and the blood (Figure 1). The cellular basis of these barriers has been located within the endothelium (endothelial blood–brain barrier in most vertebrates; only in elasmobranchs (sharks and rays) is the blood–brain barrier located in astrocytes), in the epithelial choroid plexus cells, and in
the tanycytes of the circumventricular organs (glial blood–CSF barrier).
Definition of Ependymoglial Cells Macroglial cells, including oligodendrocytes, astrocytes, and ependymoglial cells, may be classified according to the shapes and contacts of their cell processes. Distinctively, only ependymoglial cells establish contact with the ventricular surface (or, in the case of Mu¨ller glial cells, with the subretinal space, a remnant of the embryonic ventricle system). Astroglial cells are consistently characterized by the fact that at least one of their processes bears endfeet contacting a basal lamina around blood vessels (glia limitans perivascularis), the pia mater (glia limitans superficialis), or both. Ependymoglial cells share this property; opposite to their ventricular processes, these bipolar cells establish endfeet at a basal lamina (the majority of the subependymal blood vessels become obliterated in later stages of embryogenesis and most of the ependymocytes contact the remaining ‘basement membrane labyrinths’ rather than true blood vessels or the pia). Generally, we have to distinguish between these ‘normal’ cilia-bearing ependymocytes lining the major part of the ventricular surface and the choroid plexus epithelial cells and the tanycytes (Figure 1). Both of the latter cell types form the blood–CSF barrier to avoid free diffusion between blood and CSF. This is essentially due to the absence of an endothelial barrier: in the choroid plexus, the endothelium has to be permeable in order to allow the formation of the CSF from the blood by the plexus epithelial cells (Figures 1–3). In the circumventricular organs and in the hypothalamus–hypophyseal system, the vessels are permeabilized in order to receive neurosecretory input from the neurons and to deliver signal compounds from the blood to the neurons (Figures 2 and 3).
Morphology of Ependymoglial Cell Types The morphology of ependymoglia is diverse (Figure 4). Much of this diversity is related to structural and functional interactions of a given cell with its microenvironment, which includes blood vessels, the pia mater, and/or the ventricular space. Where macroglial cells form a ‘border sheath’ against the ventricular space, pia, or blood vessels, they form epitheloid aggregates. This is observed in ependymocytes, choroid plexus cells, and retinal pigment epithelial cells which also belong to ependymoglial cells. The term ‘radial glia’ should be restricted to bipolar ependymoglial
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= Gap junctions and/or zonulae adherentes
BL
Pia mater Glia limitans
Tight junctions (tight) Tight junctions (leaky) BL BL Circumventricular organs
BL
Perivascular glia Brain parenchyma Ependymal cells
BL
Choroid plexus
Cerebrospinal fluid Figure 1 Schematic drawing of the topology of the compartments within the central nervous system (CNS). Within the CNS, there is the neural compartment containing neurons and the neuropil, glial cells, and the vasculature consisting of endothelial cells surrounded by a basal lamina, pericytes, and astroglial endfeet. At the outer surface of the CNS, superficial astroglial endfeet interconnected by gap junctions form the glial limiting membrane, a border toward the meningeal cells which, however, is no physiological barrier. The blood vessels within the pia mater have blood–brain barrier properties. At the inner surface of the CNS, ependymal cells line the ventricular space. Here as well, they have no barrier properties but are interconnected by gap junctions and poorly developed, leaky tight junctions. Due to the fact that the cerebrospinal fluid (CSF) is produced by the plexus epithelium from the blood, there is no barrier between blood and choroid plexus epithelium; however, between blood and cerebrospinal fluid, there is the so-called blood–cerebrospinal fluid barrier. This barrier is formed by tight junctions between choroid plexus epithelial cells and tanycytes in the circumventricular organs. The endothelial cells in these organs and in the choroid plexus are highly permeable and fenestrated.
CSF
Epithelium
Blood
HCO−3 CI−Na+ H2O CI− Na+
2K+ 3 Na+ Cl− HCO3−
Na+ H+
H+ HCO3−
HCO3− CO2
Cl−
H2CO3 BL
Figure 2 Highly schematic view of the equipment of a choroid plexus epithelial cell with transporter molecules and the Naþ, Kþ-ATPase. Prerequisite for cerebrospinal fluid (CSF) production is the fenestration of the choroid plexus blood vessels.
cells that extend long processes throughout the thickness of the tissue. Early in embryonic development, the immature brain (i.e., the wall of the ventricular system) is spanned by a scaffold of many fetal radial glial cells. In the mature CNS, the descendants of these cells are further differentiated into multipolar astrocytes, or they are maintained as tanycytes in the circumventricular organs or as Mu¨ller cells in the retina (Figure 4).
Tanycytes
Tanycytes are a common type of macroglia in the CNS of lower vertebrates (and even deuterostomic invertebrates). In adult mammals, they are restricted to certain brain regions where the tissue is thin, such as some circumventricular organs (e.g., the subcommissural organ), the stalk of the pituitary, and the velum medullare, and to the raphe region of the spinal cord. In the circumventricular organs of all vertebrates except sharks, the capillaries are fenestrated; in these regions, the tanycytes (as well as the choroid plexus cells) constitute the blood–CSF barrier by expressing an extensive apical tight junctional network (Figure 3(f)). Some of these tanycytes are specialized for the secretion of signaling molecules and the material constituting the Reissner’s fiber. Ependymocytes, Choroid Plexus Cells, and Retinal Pigment Epithelial Cells
Ependymocytes, choroid plexus cells, and retinal pigment epithelial cells are specialized glial cells lining the ventricle (or the subretinal space, respectively; Figure 4). At their basal pole, most mature ependymocytes contact remnants of embryonic blood vessels (so-called basement membrane labyrinths; Figure 4)
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Figure 3 Electron micrographs of the ependyma and choroid plexus. (a) Ultrathin section through a mitotic ependymal cell of a prenatal mouse brain showing the proliferative property of this periventricular matrix zone. To the left of this mitotic cell, a differentiated ependymal cell already has developed a cilium. The framed box is shown in b, showing the cilium with the basal apparatus and the tight and adhesion junctions interconnecting this cell with its neighbors. (c) Ultrathin section of choroid plexus with epithelial cells (top) and a blood vessel (bottom). The framed box of the apical region of the epithelial cell is magnified in d, showing the microvilli and the junctional domain between two cells. (e) Ultrathin section through the basal labyrinth of a choroid plexus epithelial cell underlined by a basal lamina. Below this basal lamina, the fenestrated endothelium of a blood vessel can clearly be recognized. Arrows indicate fenestrations. (f) Freezefracture replica of the apical tight junction of a choroid plexus epithelial cell constituting the blood–cerebrospinal fluid barrier and consisting of parallel strands mainly associated with the inner (protoplasmic) leaflet of the membrane (P-face (PF)). EF, E-face, the outer (external) leaflet of the membrane.
rather than intact blood vessels. At their other pole (i.e., the ventricular pole), they possess, in addition to microvilli, kinocilia to support the movement of CSF (Figures 3(a), 4, and 5(c)). The latter is mainly secreted by the choroid plexus cells, characterized by a high density of Naþ, Kþ-ATPase molecules at their microvillous membrane and a high concentration of the water channel protein aquaporin-1 (Figures 2 and 5(d)). This secretion requires a high permeability of the fenestrated plexus endothelial cells (Figure 3(e)), which is now known to be induced by the delivery of the vascular endothelial growth factor (VEGF). VEGF is recognized by the endothelial VEGF receptor followed by a signal cascade leading to the formation of fenestrations. To prevent the diffusion of blood-borne substances into the ventricle, the plexus epithelium is equipped with a densely meshed tight junctional apparatus (Figure 3(f)) constituted by the zonula occludens protein 1 (ZO-1), occludin, and claudin family members 1, 2, and 11 (Figure 5) and also probably by unknown molecules. Retinal pigment epithelial (RPE) cells reveal features which are related to those of choroid plexus
epithelial cells. They line the subretinal space opposite to the neuroretina. Their apical surface forms two types of microvilli: (1) long (5–7 mm) thin microvilli maximizing the membrane area available for transepithelial transport and (2) specially arranged shorter microvilli termed photoreceptor sheaths. The basal surface of RPE cells contains numerous invaginations to increase the surface area. Like the choroid plexus cells, RPE cells are in close apposition to many blood vessels, secrete VEGF in order to induce fenestrations in the choroid vessels, are specialized for transmembrane transport, and form the blood–CSF (or, in this case, –subretinal) fluid barrier by their tight junctions. However, there is an important difference between RPE and plexus epithelial cells (and all other ependymoglial cells): RPE cells do not secrete fluid across their apical microvillous membrane but, rather, perform a net fluid uptake from the subretinal space. This water resorption is mediated via Naþ/bicarbonate exchange and prevents (together with the intraocular pressure and with cellular adhesion molecules) a detachment of the neuroretina from the pigment epithelium.
1136 Ependymal Cells Pia
‘Neuropil’ Basal lamina Tanycyte
Tanycyte
Blood vessels
Blood vessels
Basement membrane labyrinth(s)
Ependyma
Microvilli Choroid plexus epithelial cell
Cilium
Ventricle
Ependymocyte
Figure 4 Semischematic survey of the main types of ependymoglial cells and their localization in different regions of the central nervous system. The situation shown at left is also true for the pigment epithelial cells of the eye. The left tanycyte can also be called radial glial cell due to contact to both the pial surface of the brain and the brain ventricle. Another example of this type of tanycyte is the retinal Mu¨ller (glial) cell: in this case, the basal lamina corresponds to the inner limiting membrane of the retina. The microvilli-bearing lower part is oriented toward the pigment epithelial microvilli within the subretinal space which is equivalent to the ventricle of the early eye cup. The right tanycyte is the vascular tanycyte, which is the site of the blood–cerebrospinal fluid barrier in the circumventricular organs. The classical ependymocytes line the ventricle and bear cilia on their surface which provide for the steady movement of the cerebrospinal fluid.
Marker Molecules of Ependymoglial Cells
Ependymoglial cells can be visualized by immunocytochemical labeling of certain antigens that are, at least within the CNS, restricted to these cells. The expression of these molecules by certain cell types may change with differentiation or during pathological processes (Table 1). Furthermore, not all members of a given cell population (otherwise considered homogeneous) must express the same antigen (at detectable levels). Table 1 provides an overview of antigens commonly identified by immunolabeling procedures on specific types of astro- and ependymoglial cells.
Ultrastructural Features of Ependymoglial Cells
The somata of some types of ependymoglial cells contain conspicuous melanin pigment granula (RPE cells and choroid epithelial cells). In most of these epitheloid glial cells (including the ependymocytes), the lateral membranes of the somata are interconnected by zonulae adherentes and tight junctions, thus forming the blood–brain (or –retina) barrier (Figures 3 and 5). In ependymoglial cell nuclei, the nucleoplasm is rather evenly distributed compared to that in oligodendrocytes and microglial cells. In some ependymoglial cells, such as in many tanycytes, the cell nuclei are very irregularly shaped and may display deep incisions. The nuclei (and somata) of Mu¨ller cells seem to be ‘indented’ by neighboring neurons; atomic force microscopy has shown that Mu¨ller cell somata are ‘more soft’ (i.e., possess a lower module of elasticity) than the somata of the neighboring bipolar neurons. Stem processes are those cellular processes which directly arise from the soma. Typically, they contain bundles of intermediate filaments. Particularly high densities of intermediate filaments are found in the basal (i.e., endfoot-bearing) processes of tanycytes and Mu¨ller cells. The stem processes usually contain numerous mitochondria. An interesting exception is Mu¨ller cell processes in species with avascular retinae which contain mitochondria only at their apical pole (i.e., close to the choroid, which is the only source of oxygen supply), whereas their stem processes are devoid of these organelles. It has been argued that due to their dominant glycolytic energy metabolism, Mu¨ller cells are free to move and place their mitochondria toward sites of high pO2 rather than toward sites of high energy demand as observed in the neurons with their dominant aerobic metabolism. The stem processes of tanycytes and Mu¨ller cells do not show the regular dichotomic branching pattern characteristic of neuronal dendrites but, rather, are the origins of specialized endings or side branches described in the following sections. Ependymoglial and astrocytic endfeet cover almost all basal laminae within the CNS (along the blood vessels, the pia mater, and the vitreous body in the eye). In the case of tanycytes and Mu¨ller cells, the endfeet are densely filled with smooth endoplasmic reticulum. Bundles of intermediate filaments extend into the endfeet but fail to occupy the cytoplasm close to the basal lamina-contacting endfoot membrane. These filaments consist primarily of vimentin when the endfoot is in contact with CSF or vitreous humor and of glial fibrillary acidic protein (GFAP) when a blood vessel is contacted; however, this rule may be modified in some cases. With the exception of Mu¨ller
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Figure 5 Confocal laser scanning images of the ependyma and the choroid plexus. (a) Occludin was the first known tight junctional component within the membrane. It is very well expressed in the epithelial cells of the choroid plexus. (b) Transitional zone between ependyma and choroid plexus. GFAP (red) is present in astrocytes and ependymal cells but not in choroid plexus epithelial cells, whereas the tight junctional protein occludin (green) is expressed in choroid plexus epithelial cells only and not in astrocytes. However, as shown in (c), occludin is expressed in both ependymal cells (as recognized by cilia) and plexus epithelial cells (top). (d) The choroid plexus epithelium specifically expresses the water channel protein aquaporin-1 (AQP1), which is not found in ependymal cells. (e) Ependymal cells, together with astrocytes, synthesize AQP4, which in turn is not found in the choroid plexus (top). (f) Glucose transporter-1 isoform (GLUT-1) immunostaining. Where the paracellular route is occluded by highly resistant tight junctions as in plexus epithelial cells, glucose has to be transported by specific transporter molecules such as the sodium-independent GLUT-1. Because the ependymal cells have only leaky (occludin-positive) tight junctions (c), the glucose transporter is not needed in ependymal cells (not shown). (g and h) Within the choroid plexus, the endothelial tight junction molecule claudin-5 is restricted to blood vessels, whereas the tight junctions of epithelial cells contain, besides claudin-2 and -11 (not shown), claudin-3 (h). (i) The epithelial and endothelial basal laminae of the choroid plexus can be recognized by antibodies against fibronectin. The epithelial tight junctions can be visualized not only by classical tight junctional components such as occludin and claudins but also by the coxsackievirus–adenovirus receptor (CAR). Scale bar ¼ 20 mm.
cells in species with completely avascular retinae (i.e., missing both intra- and supraretinal blood vessels), the endfeet are rich in mitochondria. The occurrence of caveolae, coated pits, and vesicles concerned with endo-, exo-, or pinocytosis indicates active material exchange with the compartment on the other side of the basal lamina (i.e., blood plasma, vitreous body, or subarachnoidal fluid). A secretory function has been ascribed to the tanycytes of some circumventricular organs. Enigmatic membrane structures of ependymoglial cells are the so-called orthogonal arrays of intramembranous particles (OAPs), which have been most
extensively described in astrocytes. There, they are concentrated in membrane domains that directly contact a basal lamina, particularly the perivascular basal lamina. In periventricular ependymocytes which do not possess a basal lamina, OAPs occur across the whole cellular surface with the preference of the basolateral membrane. Interestingly, the existence of OAPs and tight junctions is inversely correlated: the ependymoglial cells have OAPs but no or less developed tight junctions, whereas the choroid plexus epithelial cells have well-developed tight junctions but no OAPs. It is known that OAPs are mainly composed of the water channel protein aquaporin-4
1138 Ependymal Cells Table 1 ‘Marker antigens’ suitable to visualize and/or to identify the various types of ependymoglial cells during ontogenetic development, in the normal mature CNS, and during reactive changes in cases of pathology Cell type
Antigen
Developing
Adult
Ependymoglia
GFAP Vimentin Cytokeratin RAN-2 Occludin AQP4 Vimentin CRALBP R-cadherinC Occludin GLUT-1 Cytokeratin GFAP Vimentin Neurofilament* Occludin Claudin-2,-3,-11 CAR AQP1 GLUT-1
(+)
++ ++ +++ ++ ++ ++ ++ +++ +++ +++ ++ ++ ++ +++ +++ +++ +++ +++
RPE
Choroid plexus epithelium
++ +
Reactive
++
Whereas the table basically reflects the situation in mammals, many of the antigens can also be found in the corresponding cell types of other vertebrates. In cases in which an antigen is only found in nonmammalian cells, it is labeled by an exposed letter: C, chicken. An asterisk indicates that the listed antigen (or antibody, respectively) does not label selectively glial cells but (in other regions of the CNS) also labels neurons. Antigens expressed in the cytoplasmic membranes, such as ion channels, receptors, or adhesion molecules, are excluded since immunocytochemistry for these antigens usually results in ‘diffuse’ labeling of the neuropil at the light microscopical level. However, tight junctional molecules are included (Figure 4). AQP1/4, water channel proteins aquaporin-1 and -4; BLBP, brain lipid-binding protein; CAR coxsackievirus–adenovirus receptor; CRALBP, cellular retinaldehyde-binding protein; GFAP, glial fibrillary acidic protein; GLUT-1, sodium-independent glucose transporter isoform 1; RAN-2, rat neural antigen-2.
(AQP4). In astrocytes, the role of OAP/AQP4 for water homeostasis of the brain and for maintenance of the blood–brain barrier has been widely discussed; in periventricular cells, the role of OAP/AQP4 may be connected to osmotic regulation as well. The development of the OAP/AQP4-related polarity of astrocytes seems to correlate with the time of agrin expression. Agrin is a heparan sulfate proteoglycan of the extracellular matrix present on ependymoglial endfeet. It has been shown to be essential for the clustering of acetylcholine receptors in the postsynaptic membrane of the motor endplate. Agrin also binds to a-dystroglycan, a member of the dystrophin– dystroglycan complex which localizes at glial endfoot membranes in a similar manner as OAP/AQP4. In mice with an induced deletion of the gene encoding Dp71 (i.e., the Mu¨ller cell-specific member of the dystrophins), a redistribution of AQP4 and Kir4.1 in the Mu¨ller cell membrane was observed. Kir4.1 is a weakly inward rectifying potassium channel involved in the glial buffering capacity of extracellular Kþ ions. These and other data support the hypothesis that agrin and dystrophins cooperate in modulating the
OAP/AQP4- (and Kir4.1-) related polarity of astrocytes and ependymoglial cells, such as retinal Mu¨ller cells and tanycytes. When, under pathological or experimental conditions, apicolateral membranes of RPE cells, Mu¨ller cells, or ependymocytes are confronted with mesenchyma, they lose their original features and develop an endfoot-like structure. This response suggests that mesenchymal contact (i.e., collagen/laminin/agrin and/or other molecules) stimulates the insertion of Kir4.1 Kþ channels and the production of a basal lamina by adjacent mesenchymal cells. Ventricular Contacts
Cell processes contacting the ventricular (or subretinal) space occur in ependymoglial cells such as tanycytes and Mu¨ller cells (but not in astrocytes). They always display an enlarged surface area by extending microvilli into the fluid volume; furthermore, the apical pole contains abundant mitochondria. Both features are indicative of a high metabolic activity that is presumably related to active exchange of substances with the luminal fluid.
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Neighboring glial ventricular contact processes (and adjacent neuronal cell processes, if present) are connected by various types of apicolateral junctions. These (particularly desmosomes) are general ‘markers’ of virtually all epithelial cells and occur very early in development. However, their nature varies considerably depending on the local microenvironment. In regions where no endothelial blood–brain barrier exists (e.g., in most circumventricular organs and in the RPE), ependymoglial cells form a CSF–brain barrier by expressing tight junctions. Apicolateral gap junctions normally occur between retinal Mu¨ller cells in frogs but not in mammals. However, when rabbit Mu¨ller cells form homogeneous cultures in vitro, gap and even tight junctions can be observed, suggesting that the retinal microenvironment in situ inhibits the establishment of such intercellular contacts. RPE cells proliferating in areas of retinal detachment may lose their basal lamina contact (Bruch’s membrane) and face the subretinal fluid around their entire surface. In these cases, they lose their membrane polarity. The apical surface of typical ependymocytes is characterized by the presence of 12–60 kinocilia, which vary in number according to the species. The cilia are 10–20 mm long and are of the 9 þ 2 type. This term means that the cilia contain an axoneme structure consisting of two centrally arranged single microtubules and nine circularly arranged doublets of microtubules. These cilia beat rhythmically at a frequency of approximately 200 beats per minute, and they appear to assist the rostrocaudad flow of CSF.
Choroid Plexus and Tanycytes as the Site of the Blood–Cerebrospinal Fluid Barrier and Immunological Aspects of It As mentioned previously, the choroid plexus epithelium and the tanycytes of the circumventricular organs form a physiologically important barrier between the CSF and the blood. This is necessarily a consequence of the facts that (1) the CSF has free access to the brain parenchyma through the leaky lining of ependymocytes and (2) the neurosecretory cells of the circumventricular organs and the tuberohypophyseal system need the direct contact with leaky blood vessels in order to release their hormones into the vasculature. Likewise, plexus epithelial cells need the direct contact with leaky blood vessels to produce the CSF from the blood. The intense interplay between neurons, ependymoglial cells, and blood vessels may be demonstrated by the observation that hormonal or osmotic stimulation of neurons in the tuberohypophyseal system leads to changes in the covering of blood vessels by tanycytic endfeet, the ultrastructure of which changes with the physiological state.
These changes modify the interface area available for neurohumoral secretion. In rhesus monkeys, the structure of hypothalamic tanycytes differs between males and females; moreover, the apical protrusions of tanycytes in females change their size and number depending on the estrous cycle. Much of the morphological diversity of astrocytes and ependymoglial cells results from the different local microenvironments into which a given cell is born (or migrating). Inevitably, mesenchymal contact will induce the formation of endfeet with OAP-rich membranes, whereas contact with CSF will induce the outgrowth of microvilli and the formation of stabilizing cell–cell junctions. Where neuronal elements are contacted, the glial cells form delicate side branches that end in lamellar sheaths or fingerlike branchlets. The number, size, and shape of these glial ‘end structures’ are precisely adjusted to the morphological and functional features of the adjacent neuronal elements. This adjustment continues after ontogeny as a lifelong process of plasticity. A role of the choroid plexus in the pathogenesis of multiple sclerosis (MS) or its animal model experimental allergic encephalomyelitis (EAE) – that is, as an alternative entry site for circulating lymphocytes directly into CSF – has not been seriously considered. The morphological correlate of the blood–CSF barrier is the tight junctions of choroid plexus epithelium. Interestingly, in EAE the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are upregulated on the choroid plexus epithelium in parallel with their upregulation on blood–brain barrier endothelium. Ultrastructural studies have revealed a polar localization of ICAM-1, VCAM-1, and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on the apical surface of choroid plexus epithelial cells and their complete absence on the fenestrated endothelial cells within the choroid plexus parenchyma. Furthermore, ICAM-1, VCAM-1, and MAdCAM-1, expressed in choroid plexus epithelium, mediate the binding of lymphocytes via their known ligands. Massive ultrastructural changes can be observed during EAE within the choroid plexus; however, at the ultrastructural level the tight junctions seem to remain intact. Choroid plexus epithelial tight junctions are morphologically unique (resembling only those of the central myelin sheaths) because they are characterized by their parallel and poorly anastomosing strands (Figure 3(f)). This specific type of tight junctions of CNS myelin sheaths was found to be exclusively constituted by oligodendrocyte-specific protein (OSP), which has been identified as a member of the claudin family (claudin-11). A possible role for OSP/claudin-11 in the development of EAE was
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demonstrated by the induction of the disease in susceptible mice after immunization with OSP/claudin11 and by the demonstration of antibodies directed against OSP/claudin-11 in the CSF of patients with MS. Although the presence of anti-claudin-11 antibodies in the CSF of MS patients is still taken as a measure for myelin degradation, it might well reflect a modulation of the blood–CSF barrier localized at the choroid plexus epithelium. In addition, because choroid plexus epithelial cells have been found to express major histocompatibility complex class I and II molecules on their surface, it will be interesting to determine the contribution of this barrier to autoimmune inflammation within the CNS.
Developmental Aspects of the Ependymoglial Cells Since the nineteenth century, it has been recognized that the periventricular zone is a matrix zone of growth and differentiation. There was a long-standing debate on whether this matrix contains segregated pools of glial and neuronal progenitors or common progenitors for macroglial cells and neurons. Data suggest that even in adult mammals neural stem cells in the matrix zone generate precursor cells of both the neuronal and the glial lineage, and that the determination of the cleavage plane during mitosis may also determine the fate of the stem cells. Considerable effort is being spent to understand the extremely complex molecular mechanisms that control the differentiation processes. Apparently, there is a complex balance between neurogenic and gliogenic factors and their receptors, such as neurogenin, notch receptor, bone morphogenetic proteins, or leukemia inhibitory factor receptor, which decide together (and/or under the control of the transcription factor Pax6) which one eventually determines the cell fate for a given postmitotic cell. Similarly, the radial glial (Mu¨ller) cells in the retina are considered as a potential source of neural regeneration after damage of retinal neurons, at least in the chicken. Again, Pax6 and other transcription factors play an important role in this glio-neuronal transdifferentiation process.
See also: Bergmann Glial Cells; Glial Growth Factors; Macroglial Lineages; Oligodendrocyte Morphology; Retinal Pharmacology: Inner Retinal Layers; Retinal Glia; Retinal Development: Cell Type Specification.
Further Reading Campbell K and Go¨tz M (2002) Radial glia: Multi-purpose cells for vertebrate brain development. Trends in Neuroscience 25: 235–238. Doetsch F (2003) The glial identity of neural stem cells. Nature Neuroscience 6: 1127–1134. Engelhardt B (ed.) (2001) The choroid plexus in health and disease. Microscopy Research and Technique 521. Fischer AJ and Reh TA (2001) Mu¨ller glia are a potential source of neural regeneration in the postnatal chicken retina. Nature Neuroscience 4: 247–252. Go¨tz M and Huttner WB (2005) The cell biology of neurogenesis. Nature Reviews Molecular Cell Biology 6: 777–788. Hatton GI (1997) Function-related plasticity in hypothalamus. Annual Review of Neuroscience 20: 375–397. Kro¨ger S (1997) Differential distribution of agrin isoforms in the developing and adult avian retina. Molecular and Cellular Neuroscience 10: 149–161. Leonhardt H (1980) Ependym und circumventricula¨re Organe. In: Oksche A (ed.) Neuroglia I, vol. 4, part 10 of Handbuch der mikroskopischen Anatomie des Menschen (Oksche A and Vollrath L, series eds.), pp. 177–666. Berlin: Springer. Reichenbach A (1989) Attempt to classify glial cells by means of their process specialization using the rabbit retinal Mu¨ller cell as an example of cytotopographic specialization of glial cells. Glia 2: 250–259. Reichenbach A and Robinson SR (1995) Phylogenetic constraints on retinal organization and development: An Haeckelian perspective. Progress in Retinal Research 15: 139–171. Reichenbach A and Wolburg H (2005) Astrocytes and ependymal glia. In: Ransom BR and Kettenmann H (eds.) Neuroglial Cells, pp. 19–35. Oxford University Press: Oxford. Sarthy V and Ripps H (2001) The Retinal Mu¨ller Cell. Structure and Function. New York: Kluwer Academic/Plenum. Saunders NR and Dziegielewska KM (eds.) (2000) Barriers in the brain. Cellular and Molecular Neurobiology 20: 1–2. Venero JL, Vizuete ML, Machado A, and Cano J (2001) Aquaporins in the central nervous system. Progress in Neurobiology 63: 321–336. Wolburg H (1995) Orthogonal arrays of intramembranous particles. A review with special reference to astrocytes. Journal of Brain Research 36: 239–258. Wolburg H and Lippoldt A (2002) Tight junctions of the blood– brain barrier: Development, composition and regulation. Vascular Pharmacology 20: 1–15.
Ependymal Cells H Wolburg, K Wolburg-Buchholz, and A F Mack, University of Tu¨bingen, Tu¨bingen, Germany A Reichenbach, University of Leipzig, Leipzig, Germany ã 2009 Elsevier Ltd. All rights reserved.
Introduction As early as 1837 Valentin and Purkinje visualized the lining of the inner cavities of the brain, the brain ventricle system, by the observation of striking cilia on its surface. This lining was called ‘ependyma’ (Greek meaning ‘upon garment’), and its constituent cells are termed ependymal cells. The ependymal cells belong to the family of neuroglial cells (glia) and are thus also called ependymoglial cells or ependymal glia. After its early discovery, it required many decades of research to elucidate this cellular layer(s) as an extremely important component of the developing and mature brain. During ontogenetic development, the ventricular lining is composed of proliferating cells termed the ‘matrix zone,’ which generates all types of neuroectodermal brain cells, including neurons and their ‘local successors,’ the ependymoglial cells. In the adult central nervous system (CNS), the ependymoglial cells contribute to the secretion of the cerebrospinal fluid (CSF) and are an essential component of an important biological barrier system segregating different compartments such as blood, CSF, and CNS interstitial space (blood–brain and blood–CSF barriers; Figure 1). Originally, Paul Ehrlich found that a blood-infused dye failed to stain the brain tissue, and his pupil, Ernst Goldmann, observed complementarily that the very same dye, if applied into the CSF, did stain the brain tissue. This led to the concept of a biological barrier between blood and brain. Due to the free access of the dye from brain ventricle to brain tissue, they concluded that there is no CSF–brain barrier. However, the staining of circumventricular organs and the choroid plexus in Goldmann’s experiment applying the dye into the general circulation (Goldmann-I experiment) and the avoidance of staining of these organs in the experiment applying the dye into the CSF (Goldmann-II experiment) suggested the existence of a barrier between the CSF and the blood (Figure 1). The cellular basis of these barriers has been located within the endothelium (endothelial blood–brain barrier in most vertebrates; only in elasmobranchs (sharks and rays) is the blood–brain barrier located in astrocytes), in the epithelial choroid plexus cells, and in
the tanycytes of the circumventricular organs (glial blood–CSF barrier).
Definition of Ependymoglial Cells Macroglial cells, including oligodendrocytes, astrocytes, and ependymoglial cells, may be classified according to the shapes and contacts of their cell processes. Distinctively, only ependymoglial cells establish contact with the ventricular surface (or, in the case of Mu¨ller glial cells, with the subretinal space, a remnant of the embryonic ventricle system). Astroglial cells are consistently characterized by the fact that at least one of their processes bears endfeet contacting a basal lamina around blood vessels (glia limitans perivascularis), the pia mater (glia limitans superficialis), or both. Ependymoglial cells share this property; opposite to their ventricular processes, these bipolar cells establish endfeet at a basal lamina (the majority of the subependymal blood vessels become obliterated in later stages of embryogenesis and most of the ependymocytes contact the remaining ‘basement membrane labyrinths’ rather than true blood vessels or the pia). Generally, we have to distinguish between these ‘normal’ cilia-bearing ependymocytes lining the major part of the ventricular surface and the choroid plexus epithelial cells and the tanycytes (Figure 1). Both of the latter cell types form the blood–CSF barrier to avoid free diffusion between blood and CSF. This is essentially due to the absence of an endothelial barrier: in the choroid plexus, the endothelium has to be permeable in order to allow the formation of the CSF from the blood by the plexus epithelial cells (Figures 1–3). In the circumventricular organs and in the hypothalamus–hypophyseal system, the vessels are permeabilized in order to receive neurosecretory input from the neurons and to deliver signal compounds from the blood to the neurons (Figures 2 and 3).
Morphology of Ependymoglial Cell Types The morphology of ependymoglia is diverse (Figure 4). Much of this diversity is related to structural and functional interactions of a given cell with its microenvironment, which includes blood vessels, the pia mater, and/or the ventricular space. Where macroglial cells form a ‘border sheath’ against the ventricular space, pia, or blood vessels, they form epitheloid aggregates. This is observed in ependymocytes, choroid plexus cells, and retinal pigment epithelial cells which also belong to ependymoglial cells. The term ‘radial glia’ should be restricted to bipolar ependymoglial
1134 Ependymal Cells
= Gap junctions and/or zonulae adherentes
BL
Pia mater Glia limitans
Tight junctions (tight) Tight junctions (leaky) BL BL Circumventricular organs
BL
Perivascular glia Brain parenchyma Ependymal cells
BL
Choroid plexus
Cerebrospinal fluid Figure 1 Schematic drawing of the topology of the compartments within the central nervous system (CNS). Within the CNS, there is the neural compartment containing neurons and the neuropil, glial cells, and the vasculature consisting of endothelial cells surrounded by a basal lamina, pericytes, and astroglial endfeet. At the outer surface of the CNS, superficial astroglial endfeet interconnected by gap junctions form the glial limiting membrane, a border toward the meningeal cells which, however, is no physiological barrier. The blood vessels within the pia mater have blood–brain barrier properties. At the inner surface of the CNS, ependymal cells line the ventricular space. Here as well, they have no barrier properties but are interconnected by gap junctions and poorly developed, leaky tight junctions. Due to the fact that the cerebrospinal fluid (CSF) is produced by the plexus epithelium from the blood, there is no barrier between blood and choroid plexus epithelium; however, between blood and cerebrospinal fluid, there is the so-called blood–cerebrospinal fluid barrier. This barrier is formed by tight junctions between choroid plexus epithelial cells and tanycytes in the circumventricular organs. The endothelial cells in these organs and in the choroid plexus are highly permeable and fenestrated.
CSF
Epithelium
Blood
HCO−3 CI−Na+ H2O CI− Na+
2K+ 3 Na+ Cl− HCO3−
Na+ H+
H+ HCO3−
HCO3− CO2
Cl−
H2CO3 BL
Figure 2 Highly schematic view of the equipment of a choroid plexus epithelial cell with transporter molecules and the Naþ, Kþ-ATPase. Prerequisite for cerebrospinal fluid (CSF) production is the fenestration of the choroid plexus blood vessels.
cells that extend long processes throughout the thickness of the tissue. Early in embryonic development, the immature brain (i.e., the wall of the ventricular system) is spanned by a scaffold of many fetal radial glial cells. In the mature CNS, the descendants of these cells are further differentiated into multipolar astrocytes, or they are maintained as tanycytes in the circumventricular organs or as Mu¨ller cells in the retina (Figure 4).
Tanycytes
Tanycytes are a common type of macroglia in the CNS of lower vertebrates (and even deuterostomic invertebrates). In adult mammals, they are restricted to certain brain regions where the tissue is thin, such as some circumventricular organs (e.g., the subcommissural organ), the stalk of the pituitary, and the velum medullare, and to the raphe region of the spinal cord. In the circumventricular organs of all vertebrates except sharks, the capillaries are fenestrated; in these regions, the tanycytes (as well as the choroid plexus cells) constitute the blood–CSF barrier by expressing an extensive apical tight junctional network (Figure 3(f)). Some of these tanycytes are specialized for the secretion of signaling molecules and the material constituting the Reissner’s fiber. Ependymocytes, Choroid Plexus Cells, and Retinal Pigment Epithelial Cells
Ependymocytes, choroid plexus cells, and retinal pigment epithelial cells are specialized glial cells lining the ventricle (or the subretinal space, respectively; Figure 4). At their basal pole, most mature ependymocytes contact remnants of embryonic blood vessels (so-called basement membrane labyrinths; Figure 4)
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Figure 3 Electron micrographs of the ependyma and choroid plexus. (a) Ultrathin section through a mitotic ependymal cell of a prenatal mouse brain showing the proliferative property of this periventricular matrix zone. To the left of this mitotic cell, a differentiated ependymal cell already has developed a cilium. The framed box is shown in b, showing the cilium with the basal apparatus and the tight and adhesion junctions interconnecting this cell with its neighbors. (c) Ultrathin section of choroid plexus with epithelial cells (top) and a blood vessel (bottom). The framed box of the apical region of the epithelial cell is magnified in d, showing the microvilli and the junctional domain between two cells. (e) Ultrathin section through the basal labyrinth of a choroid plexus epithelial cell underlined by a basal lamina. Below this basal lamina, the fenestrated endothelium of a blood vessel can clearly be recognized. Arrows indicate fenestrations. (f) Freezefracture replica of the apical tight junction of a choroid plexus epithelial cell constituting the blood–cerebrospinal fluid barrier and consisting of parallel strands mainly associated with the inner (protoplasmic) leaflet of the membrane (P-face (PF)). EF, E-face, the outer (external) leaflet of the membrane.
rather than intact blood vessels. At their other pole (i.e., the ventricular pole), they possess, in addition to microvilli, kinocilia to support the movement of CSF (Figures 3(a), 4, and 5(c)). The latter is mainly secreted by the choroid plexus cells, characterized by a high density of Naþ, Kþ-ATPase molecules at their microvillous membrane and a high concentration of the water channel protein aquaporin-1 (Figures 2 and 5(d)). This secretion requires a high permeability of the fenestrated plexus endothelial cells (Figure 3(e)), which is now known to be induced by the delivery of the vascular endothelial growth factor (VEGF). VEGF is recognized by the endothelial VEGF receptor followed by a signal cascade leading to the formation of fenestrations. To prevent the diffusion of blood-borne substances into the ventricle, the plexus epithelium is equipped with a densely meshed tight junctional apparatus (Figure 3(f)) constituted by the zonula occludens protein 1 (ZO-1), occludin, and claudin family members 1, 2, and 11 (Figure 5) and also probably by unknown molecules. Retinal pigment epithelial (RPE) cells reveal features which are related to those of choroid plexus
epithelial cells. They line the subretinal space opposite to the neuroretina. Their apical surface forms two types of microvilli: (1) long (5–7 mm) thin microvilli maximizing the membrane area available for transepithelial transport and (2) specially arranged shorter microvilli termed photoreceptor sheaths. The basal surface of RPE cells contains numerous invaginations to increase the surface area. Like the choroid plexus cells, RPE cells are in close apposition to many blood vessels, secrete VEGF in order to induce fenestrations in the choroid vessels, are specialized for transmembrane transport, and form the blood–CSF (or, in this case, –subretinal) fluid barrier by their tight junctions. However, there is an important difference between RPE and plexus epithelial cells (and all other ependymoglial cells): RPE cells do not secrete fluid across their apical microvillous membrane but, rather, perform a net fluid uptake from the subretinal space. This water resorption is mediated via Naþ/bicarbonate exchange and prevents (together with the intraocular pressure and with cellular adhesion molecules) a detachment of the neuroretina from the pigment epithelium.
1136 Ependymal Cells Pia
‘Neuropil’ Basal lamina Tanycyte
Tanycyte
Blood vessels
Blood vessels
Basement membrane labyrinth(s)
Ependyma
Microvilli Choroid plexus epithelial cell
Cilium
Ventricle
Ependymocyte
Figure 4 Semischematic survey of the main types of ependymoglial cells and their localization in different regions of the central nervous system. The situation shown at left is also true for the pigment epithelial cells of the eye. The left tanycyte can also be called radial glial cell due to contact to both the pial surface of the brain and the brain ventricle. Another example of this type of tanycyte is the retinal Mu¨ller (glial) cell: in this case, the basal lamina corresponds to the inner limiting membrane of the retina. The microvilli-bearing lower part is oriented toward the pigment epithelial microvilli within the subretinal space which is equivalent to the ventricle of the early eye cup. The right tanycyte is the vascular tanycyte, which is the site of the blood–cerebrospinal fluid barrier in the circumventricular organs. The classical ependymocytes line the ventricle and bear cilia on their surface which provide for the steady movement of the cerebrospinal fluid.
Marker Molecules of Ependymoglial Cells
Ependymoglial cells can be visualized by immunocytochemical labeling of certain antigens that are, at least within the CNS, restricted to these cells. The expression of these molecules by certain cell types may change with differentiation or during pathological processes (Table 1). Furthermore, not all members of a given cell population (otherwise considered homogeneous) must express the same antigen (at detectable levels). Table 1 provides an overview of antigens commonly identified by immunolabeling procedures on specific types of astro- and ependymoglial cells.
Ultrastructural Features of Ependymoglial Cells
The somata of some types of ependymoglial cells contain conspicuous melanin pigment granula (RPE cells and choroid epithelial cells). In most of these epitheloid glial cells (including the ependymocytes), the lateral membranes of the somata are interconnected by zonulae adherentes and tight junctions, thus forming the blood–brain (or –retina) barrier (Figures 3 and 5). In ependymoglial cell nuclei, the nucleoplasm is rather evenly distributed compared to that in oligodendrocytes and microglial cells. In some ependymoglial cells, such as in many tanycytes, the cell nuclei are very irregularly shaped and may display deep incisions. The nuclei (and somata) of Mu¨ller cells seem to be ‘indented’ by neighboring neurons; atomic force microscopy has shown that Mu¨ller cell somata are ‘more soft’ (i.e., possess a lower module of elasticity) than the somata of the neighboring bipolar neurons. Stem processes are those cellular processes which directly arise from the soma. Typically, they contain bundles of intermediate filaments. Particularly high densities of intermediate filaments are found in the basal (i.e., endfoot-bearing) processes of tanycytes and Mu¨ller cells. The stem processes usually contain numerous mitochondria. An interesting exception is Mu¨ller cell processes in species with avascular retinae which contain mitochondria only at their apical pole (i.e., close to the choroid, which is the only source of oxygen supply), whereas their stem processes are devoid of these organelles. It has been argued that due to their dominant glycolytic energy metabolism, Mu¨ller cells are free to move and place their mitochondria toward sites of high pO2 rather than toward sites of high energy demand as observed in the neurons with their dominant aerobic metabolism. The stem processes of tanycytes and Mu¨ller cells do not show the regular dichotomic branching pattern characteristic of neuronal dendrites but, rather, are the origins of specialized endings or side branches described in the following sections. Ependymoglial and astrocytic endfeet cover almost all basal laminae within the CNS (along the blood vessels, the pia mater, and the vitreous body in the eye). In the case of tanycytes and Mu¨ller cells, the endfeet are densely filled with smooth endoplasmic reticulum. Bundles of intermediate filaments extend into the endfeet but fail to occupy the cytoplasm close to the basal lamina-contacting endfoot membrane. These filaments consist primarily of vimentin when the endfoot is in contact with CSF or vitreous humor and of glial fibrillary acidic protein (GFAP) when a blood vessel is contacted; however, this rule may be modified in some cases. With the exception of Mu¨ller
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Figure 5 Confocal laser scanning images of the ependyma and the choroid plexus. (a) Occludin was the first known tight junctional component within the membrane. It is very well expressed in the epithelial cells of the choroid plexus. (b) Transitional zone between ependyma and choroid plexus. GFAP (red) is present in astrocytes and ependymal cells but not in choroid plexus epithelial cells, whereas the tight junctional protein occludin (green) is expressed in choroid plexus epithelial cells only and not in astrocytes. However, as shown in (c), occludin is expressed in both ependymal cells (as recognized by cilia) and plexus epithelial cells (top). (d) The choroid plexus epithelium specifically expresses the water channel protein aquaporin-1 (AQP1), which is not found in ependymal cells. (e) Ependymal cells, together with astrocytes, synthesize AQP4, which in turn is not found in the choroid plexus (top). (f) Glucose transporter-1 isoform (GLUT-1) immunostaining. Where the paracellular route is occluded by highly resistant tight junctions as in plexus epithelial cells, glucose has to be transported by specific transporter molecules such as the sodium-independent GLUT-1. Because the ependymal cells have only leaky (occludin-positive) tight junctions (c), the glucose transporter is not needed in ependymal cells (not shown). (g and h) Within the choroid plexus, the endothelial tight junction molecule claudin-5 is restricted to blood vessels, whereas the tight junctions of epithelial cells contain, besides claudin-2 and -11 (not shown), claudin-3 (h). (i) The epithelial and endothelial basal laminae of the choroid plexus can be recognized by antibodies against fibronectin. The epithelial tight junctions can be visualized not only by classical tight junctional components such as occludin and claudins but also by the coxsackievirus–adenovirus receptor (CAR). Scale bar ¼ 20 mm.
cells in species with completely avascular retinae (i.e., missing both intra- and supraretinal blood vessels), the endfeet are rich in mitochondria. The occurrence of caveolae, coated pits, and vesicles concerned with endo-, exo-, or pinocytosis indicates active material exchange with the compartment on the other side of the basal lamina (i.e., blood plasma, vitreous body, or subarachnoidal fluid). A secretory function has been ascribed to the tanycytes of some circumventricular organs. Enigmatic membrane structures of ependymoglial cells are the so-called orthogonal arrays of intramembranous particles (OAPs), which have been most
extensively described in astrocytes. There, they are concentrated in membrane domains that directly contact a basal lamina, particularly the perivascular basal lamina. In periventricular ependymocytes which do not possess a basal lamina, OAPs occur across the whole cellular surface with the preference of the basolateral membrane. Interestingly, the existence of OAPs and tight junctions is inversely correlated: the ependymoglial cells have OAPs but no or less developed tight junctions, whereas the choroid plexus epithelial cells have well-developed tight junctions but no OAPs. It is known that OAPs are mainly composed of the water channel protein aquaporin-4
1138 Ependymal Cells Table 1 ‘Marker antigens’ suitable to visualize and/or to identify the various types of ependymoglial cells during ontogenetic development, in the normal mature CNS, and during reactive changes in cases of pathology Cell type
Antigen
Developing
Adult
Ependymoglia
GFAP Vimentin Cytokeratin RAN-2 Occludin AQP4 Vimentin CRALBP R-cadherinC Occludin GLUT-1 Cytokeratin GFAP Vimentin Neurofilament* Occludin Claudin-2,-3,-11 CAR AQP1 GLUT-1
(+)
++ ++ +++ ++ ++ ++ ++ +++ +++ +++ ++ ++ ++ +++ +++ +++ +++ +++
RPE
Choroid plexus epithelium
++ +
Reactive
++
Whereas the table basically reflects the situation in mammals, many of the antigens can also be found in the corresponding cell types of other vertebrates. In cases in which an antigen is only found in nonmammalian cells, it is labeled by an exposed letter: C, chicken. An asterisk indicates that the listed antigen (or antibody, respectively) does not label selectively glial cells but (in other regions of the CNS) also labels neurons. Antigens expressed in the cytoplasmic membranes, such as ion channels, receptors, or adhesion molecules, are excluded since immunocytochemistry for these antigens usually results in ‘diffuse’ labeling of the neuropil at the light microscopical level. However, tight junctional molecules are included (Figure 4). AQP1/4, water channel proteins aquaporin-1 and -4; BLBP, brain lipid-binding protein; CAR coxsackievirus–adenovirus receptor; CRALBP, cellular retinaldehyde-binding protein; GFAP, glial fibrillary acidic protein; GLUT-1, sodium-independent glucose transporter isoform 1; RAN-2, rat neural antigen-2.
(AQP4). In astrocytes, the role of OAP/AQP4 for water homeostasis of the brain and for maintenance of the blood–brain barrier has been widely discussed; in periventricular cells, the role of OAP/AQP4 may be connected to osmotic regulation as well. The development of the OAP/AQP4-related polarity of astrocytes seems to correlate with the time of agrin expression. Agrin is a heparan sulfate proteoglycan of the extracellular matrix present on ependymoglial endfeet. It has been shown to be essential for the clustering of acetylcholine receptors in the postsynaptic membrane of the motor endplate. Agrin also binds to a-dystroglycan, a member of the dystrophin– dystroglycan complex which localizes at glial endfoot membranes in a similar manner as OAP/AQP4. In mice with an induced deletion of the gene encoding Dp71 (i.e., the Mu¨ller cell-specific member of the dystrophins), a redistribution of AQP4 and Kir4.1 in the Mu¨ller cell membrane was observed. Kir4.1 is a weakly inward rectifying potassium channel involved in the glial buffering capacity of extracellular Kþ ions. These and other data support the hypothesis that agrin and dystrophins cooperate in modulating the
OAP/AQP4- (and Kir4.1-) related polarity of astrocytes and ependymoglial cells, such as retinal Mu¨ller cells and tanycytes. When, under pathological or experimental conditions, apicolateral membranes of RPE cells, Mu¨ller cells, or ependymocytes are confronted with mesenchyma, they lose their original features and develop an endfoot-like structure. This response suggests that mesenchymal contact (i.e., collagen/laminin/agrin and/or other molecules) stimulates the insertion of Kir4.1 Kþ channels and the production of a basal lamina by adjacent mesenchymal cells. Ventricular Contacts
Cell processes contacting the ventricular (or subretinal) space occur in ependymoglial cells such as tanycytes and Mu¨ller cells (but not in astrocytes). They always display an enlarged surface area by extending microvilli into the fluid volume; furthermore, the apical pole contains abundant mitochondria. Both features are indicative of a high metabolic activity that is presumably related to active exchange of substances with the luminal fluid.
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Neighboring glial ventricular contact processes (and adjacent neuronal cell processes, if present) are connected by various types of apicolateral junctions. These (particularly desmosomes) are general ‘markers’ of virtually all epithelial cells and occur very early in development. However, their nature varies considerably depending on the local microenvironment. In regions where no endothelial blood–brain barrier exists (e.g., in most circumventricular organs and in the RPE), ependymoglial cells form a CSF–brain barrier by expressing tight junctions. Apicolateral gap junctions normally occur between retinal Mu¨ller cells in frogs but not in mammals. However, when rabbit Mu¨ller cells form homogeneous cultures in vitro, gap and even tight junctions can be observed, suggesting that the retinal microenvironment in situ inhibits the establishment of such intercellular contacts. RPE cells proliferating in areas of retinal detachment may lose their basal lamina contact (Bruch’s membrane) and face the subretinal fluid around their entire surface. In these cases, they lose their membrane polarity. The apical surface of typical ependymocytes is characterized by the presence of 12–60 kinocilia, which vary in number according to the species. The cilia are 10–20 mm long and are of the 9 þ 2 type. This term means that the cilia contain an axoneme structure consisting of two centrally arranged single microtubules and nine circularly arranged doublets of microtubules. These cilia beat rhythmically at a frequency of approximately 200 beats per minute, and they appear to assist the rostrocaudad flow of CSF.
Choroid Plexus and Tanycytes as the Site of the Blood–Cerebrospinal Fluid Barrier and Immunological Aspects of It As mentioned previously, the choroid plexus epithelium and the tanycytes of the circumventricular organs form a physiologically important barrier between the CSF and the blood. This is necessarily a consequence of the facts that (1) the CSF has free access to the brain parenchyma through the leaky lining of ependymocytes and (2) the neurosecretory cells of the circumventricular organs and the tuberohypophyseal system need the direct contact with leaky blood vessels in order to release their hormones into the vasculature. Likewise, plexus epithelial cells need the direct contact with leaky blood vessels to produce the CSF from the blood. The intense interplay between neurons, ependymoglial cells, and blood vessels may be demonstrated by the observation that hormonal or osmotic stimulation of neurons in the tuberohypophyseal system leads to changes in the covering of blood vessels by tanycytic endfeet, the ultrastructure of which changes with the physiological state.
These changes modify the interface area available for neurohumoral secretion. In rhesus monkeys, the structure of hypothalamic tanycytes differs between males and females; moreover, the apical protrusions of tanycytes in females change their size and number depending on the estrous cycle. Much of the morphological diversity of astrocytes and ependymoglial cells results from the different local microenvironments into which a given cell is born (or migrating). Inevitably, mesenchymal contact will induce the formation of endfeet with OAP-rich membranes, whereas contact with CSF will induce the outgrowth of microvilli and the formation of stabilizing cell–cell junctions. Where neuronal elements are contacted, the glial cells form delicate side branches that end in lamellar sheaths or fingerlike branchlets. The number, size, and shape of these glial ‘end structures’ are precisely adjusted to the morphological and functional features of the adjacent neuronal elements. This adjustment continues after ontogeny as a lifelong process of plasticity. A role of the choroid plexus in the pathogenesis of multiple sclerosis (MS) or its animal model experimental allergic encephalomyelitis (EAE) – that is, as an alternative entry site for circulating lymphocytes directly into CSF – has not been seriously considered. The morphological correlate of the blood–CSF barrier is the tight junctions of choroid plexus epithelium. Interestingly, in EAE the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are upregulated on the choroid plexus epithelium in parallel with their upregulation on blood–brain barrier endothelium. Ultrastructural studies have revealed a polar localization of ICAM-1, VCAM-1, and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on the apical surface of choroid plexus epithelial cells and their complete absence on the fenestrated endothelial cells within the choroid plexus parenchyma. Furthermore, ICAM-1, VCAM-1, and MAdCAM-1, expressed in choroid plexus epithelium, mediate the binding of lymphocytes via their known ligands. Massive ultrastructural changes can be observed during EAE within the choroid plexus; however, at the ultrastructural level the tight junctions seem to remain intact. Choroid plexus epithelial tight junctions are morphologically unique (resembling only those of the central myelin sheaths) because they are characterized by their parallel and poorly anastomosing strands (Figure 3(f)). This specific type of tight junctions of CNS myelin sheaths was found to be exclusively constituted by oligodendrocyte-specific protein (OSP), which has been identified as a member of the claudin family (claudin-11). A possible role for OSP/claudin-11 in the development of EAE was
1140 Ependymal Cells
demonstrated by the induction of the disease in susceptible mice after immunization with OSP/claudin11 and by the demonstration of antibodies directed against OSP/claudin-11 in the CSF of patients with MS. Although the presence of anti-claudin-11 antibodies in the CSF of MS patients is still taken as a measure for myelin degradation, it might well reflect a modulation of the blood–CSF barrier localized at the choroid plexus epithelium. In addition, because choroid plexus epithelial cells have been found to express major histocompatibility complex class I and II molecules on their surface, it will be interesting to determine the contribution of this barrier to autoimmune inflammation within the CNS.
Developmental Aspects of the Ependymoglial Cells Since the nineteenth century, it has been recognized that the periventricular zone is a matrix zone of growth and differentiation. There was a long-standing debate on whether this matrix contains segregated pools of glial and neuronal progenitors or common progenitors for macroglial cells and neurons. Data suggest that even in adult mammals neural stem cells in the matrix zone generate precursor cells of both the neuronal and the glial lineage, and that the determination of the cleavage plane during mitosis may also determine the fate of the stem cells. Considerable effort is being spent to understand the extremely complex molecular mechanisms that control the differentiation processes. Apparently, there is a complex balance between neurogenic and gliogenic factors and their receptors, such as neurogenin, notch receptor, bone morphogenetic proteins, or leukemia inhibitory factor receptor, which decide together (and/or under the control of the transcription factor Pax6) which one eventually determines the cell fate for a given postmitotic cell. Similarly, the radial glial (Mu¨ller) cells in the retina are considered as a potential source of neural regeneration after damage of retinal neurons, at least in the chicken. Again, Pax6 and other transcription factors play an important role in this glio-neuronal transdifferentiation process.
See also: Bergmann Glial Cells; Glial Growth Factors; Macroglial Lineages; Oligodendrocyte Morphology; Retinal Pharmacology: Inner Retinal Layers; Retinal Glia; Retinal Development: Cell Type Specification.
Further Reading Campbell K and Go¨tz M (2002) Radial glia: Multi-purpose cells for vertebrate brain development. Trends in Neuroscience 25: 235–238. Doetsch F (2003) The glial identity of neural stem cells. Nature Neuroscience 6: 1127–1134. Engelhardt B (ed.) (2001) The choroid plexus in health and disease. Microscopy Research and Technique 521. Fischer AJ and Reh TA (2001) Mu¨ller glia are a potential source of neural regeneration in the postnatal chicken retina. Nature Neuroscience 4: 247–252. Go¨tz M and Huttner WB (2005) The cell biology of neurogenesis. Nature Reviews Molecular Cell Biology 6: 777–788. Hatton GI (1997) Function-related plasticity in hypothalamus. Annual Review of Neuroscience 20: 375–397. Kro¨ger S (1997) Differential distribution of agrin isoforms in the developing and adult avian retina. Molecular and Cellular Neuroscience 10: 149–161. Leonhardt H (1980) Ependym und circumventricula¨re Organe. In: Oksche A (ed.) Neuroglia I, vol. 4, part 10 of Handbuch der mikroskopischen Anatomie des Menschen (Oksche A and Vollrath L, series eds.), pp. 177–666. Berlin: Springer. Reichenbach A (1989) Attempt to classify glial cells by means of their process specialization using the rabbit retinal Mu¨ller cell as an example of cytotopographic specialization of glial cells. Glia 2: 250–259. Reichenbach A and Robinson SR (1995) Phylogenetic constraints on retinal organization and development: An Haeckelian perspective. Progress in Retinal Research 15: 139–171. Reichenbach A and Wolburg H (2005) Astrocytes and ependymal glia. In: Ransom BR and Kettenmann H (eds.) Neuroglial Cells, pp. 19–35. Oxford University Press: Oxford. Sarthy V and Ripps H (2001) The Retinal Mu¨ller Cell. Structure and Function. New York: Kluwer Academic/Plenum. Saunders NR and Dziegielewska KM (eds.) (2000) Barriers in the brain. Cellular and Molecular Neurobiology 20: 1–2. Venero JL, Vizuete ML, Machado A, and Cano J (2001) Aquaporins in the central nervous system. Progress in Neurobiology 63: 321–336. Wolburg H (1995) Orthogonal arrays of intramembranous particles. A review with special reference to astrocytes. Journal of Brain Research 36: 239–258. Wolburg H and Lippoldt A (2002) Tight junctions of the blood– brain barrier: Development, composition and regulation. Vascular Pharmacology 20: 1–15.