Primary cultures as a model for studying ependymal functions: glycogen metabolism in ependymal cells

Brain Research 920 (2001) 74–83 www.elsevier.com / locate / bres

Research report

Primary cultures as a model for studying ependymal functions: glycogen metabolism in ependymal cells ¨ Christian Prothmann a , John Wellard a , Jurgen Berger b , Bernd Hamprecht a , a, Stephan Verleysdonk * a

¨ , Hoppe-Seyler-Str. 4, D-72076 Tubingen ¨ , Germany Physiologisch-chemisches Institut der Universitat b ¨ Entwicklungsbiologie, Spemannstr. 35, D-72076 Tubingen ¨ Max-Planck-Institut f ur , Germany Accepted 17 August 2001

Abstract Ependymal cells form a single-layered, ciliated epithelium at the interface between the cerebrospinal fluid and the brain parenchyma. Although their morphology has been studied in detail, ependymal functions remain largely speculative. We have established and characterized a previously described cell culture model to investigate ependymal glycogen metabolism. During growth in minimal medium lacking many non-essential amino acids including L-glutamate, but containing glucose at physiological concentration, the cells contained negligible amounts of glycogen (763 nmol glucosyl residues / mg protein) despite the presence of insulin. However, during a period of 24 h, the cells accumulated glycogen to very high levels after transferal to a medium containing insulin, glucose at a 5-fold higher concentration, and all proteinogenic amino acids except L-asparagine and L-serine (9906112 nmol glucosyl residues / mg protein). Omission of insulin resulted in a 50% reduction in glycogen accumulation. Upon glucose deprivation, glycogen was degraded with a half-life of 21 min. The ependymal primary cultures contained 8065 mU glycogen phosphorylase (Pho) / mg protein and stained positively with antibodies raised against this enzyme. Astroglial cultures built up less glycogen and had less Pho activity under identical conditions. Ependymal glycogen was mobilized by noradrenaline and serotonin. Our results indicate that ependymal cells maintain glycogen as a functional energy store, subject to rapid turnover dependent on the availability of energy substrates and the presence of appropriate signal molecules. Thus ependymocytes appear to be active players in the multitude of processes resulting in normal brain function, and ependymal primary cultures are suggested as a suitable model for studying the role of ependymal cells in these processes.  2001 Elsevier Science B.V. All rights reserved. Theme: Other systems of the CNS Topic: Brain metabolism and blood flow Keywords: Ependymal cell; Cell culture; Glial metabolism; Glycogen; Glycogen phosphorylase; Insulin

1. Introduction Ependymal cells form a single-layered, mostly ciliated epithelium separating the cerebrospinal fluid-filled spaces of the central nervous system (CNS) from its parenchyma. Ependymal cells do not constitute a homogeneous cell population but fall into different subtypes based on differences in morphology. The cuboidal to squamous, ciliabearing ventricular epithelial cells make up the most *Corresponding author. Tel.: 149-707-1297-3331; fax: 149-7071295-360. E-mail address: [email protected] (S. Verleysdonk).

prominent variation and are traditionally viewed as ‘normal’ ependymal cells [14,39]. Ependymal subtypes include tanycytes [13,43] and sparsely ciliated choroid plexus cells [25]. The most prominent feature of the ventricular epithelial cells is their bundle of cilia, which are thought to cause a streaming of the CSF and a dispersion of materials in this fluid [34]. The severe symptoms associated with the human immotile cilia syndromes are generally not linked to the CNS [1]. In contrast to ependymal cells from the ventricles of lower vertebrates those from higher vertebrates lack regenerative capacity [32]. These facts have contributed to the notion that the ependyma are vestigial in these species. However, there are strong indications that

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mammalian ependymal cells are metabolically active. They possess a wide array of membrane transporters, are equipped with enzymes involved in hormone metabolism, express receptors for a plethora of signal molecules and might have secretory activity (for reviews, see [2,5]). The ventricular epithelial cells probably contain the highest amount of glycogen of all cell types in the CNS [3]. Furthermore, these cells possess the highest levels of glycogen phosphorylase activity in the brain [3]. As glycogen is an energy store that can be mobilized during periods of increased cellular energy requirement, the presence of both high levels of glycogen and its associated degradative enzyme suggests that these cells have the capacity to rapidly mobilize large amounts of glucosyl residues. In times of increased neuronal activity astrocytes break down their glycogen reserves and furnish the lactate formed therefrom as fuel for the generation of energy to the neighbouring neurons [10,40]. The anatomical location of ependymal cells and their mass ratio to the other cell types of the CNS render it unlikely that ependymal cells contribute significantly to the supply of the brain parenchyma with energy substrates. Rather, the ependymal glycogen stores are suggested to serve as an internal energy supply under conditions of increased ependymocyte activity. The modulation of ependymal glycogen levels by particular stimuli, such as insulin, neurotransmitters and related agonists, or changes in levels of extracellular glucose or amino acids, would provide some insight into the functional status of these glycogen stores. Such experiments are very difficult to carry out in situ, but ependymal cell culture models have been reported [15,42]. Whilst the morphology of such cell cultures has been described in detail, they have yet to be utilised in biochemical studies. In this study we describe the results of using a modified version of a previously reported ependymal cell culture system [42]. Biochemical manipulation of this system has provided a first insight into factors controlling ependymal glycogen metabolism and thus provides the basis for further biochemical studies of both ependymal cell metabolism and function.

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tamycin and D-fructose (Frc) were purchased from Fluka (Sigma, Deisenhofen, Germany). Amyloglucosidase, ATP, glucose-6-phosphate dehydrogenase, glutamate:pyruvate aminotransferase, hexokinase, lactate dehydrogenase and phosphoglucomutase were from Roche (Mannheim, Germany). Human thrombin was a gift from Centeon, now Aventis Behring (Marburg, Germany). Fibronectin was purified from bovine plasma according to the method described in [20]. A monoclonal anti-tubulin antibody (hybridoma supernatant) was kindly provided by Dr. M. Schliwa (University of Munich, Germany). Cy3-conjugated anti-mouse IgG was purchased from Dianova (Hamburg, Germany). Streptavidin Texas Red conjugated and biotinylated (Fab) 2 -fragments of sheep anti-mouse IgG were obtained from Amersham (Braunschweig, Germany). Rabbit anti-GFAP serum was from Dakopatts (Hamburg, Germany). The monoclonal antibody directed against glycogen phosphorylase was described in [27]. ‘Falcon’ cell culture dishes 35 mm in diameter were from Becton Dickinson (Schubert Medizinprodukte, Wackersdorf, Germany).

2.2. Cell culture

2. Materials and methods

Ependymal primary cultures were prepared from the brains of newborn rats by dissociating whole brains as described in [17]. The cells were resuspended in MEM c (MEM supplemented with 0.5 g / l fatty acid-free BSA, 5 mg / l insulin and 10 mg / l transferrin) and seeded on fibronectin-coated dishes as described in [42]. After 48 h, the culture medium was changed to MEM c T (MEM c supplemented with 500 U / l thrombin) and renewed on every third day. Rat astroglia-rich primary cultures were prepared as described in [17]. For maximal glycogen accumulation, cells were transferred to WM c T (WM supplemented with 0.5 g / l fatty acid-free BSA, 5 mg l insulin, 10 mg / l transferrin and 500 U / l thrombin). In other cases, WM supplemented with 0.5 g / l BSA and 10 mg / ml transferrin (WM NC ) was used for glycogen accumulation studies and enzyme assays. For immunocytochemistry, cells were grown on fibronectin-coated coverslips that had been attached to the surface of a culture dish with sterile silicon grease.

2.1. Materials

2.3. Immunocytochemistry

Dulbecco’s modified Eagles medium (DMEM, [11,22]), Minimal Essential Medium (MEM, [12]), Waymouth Medium 705 / 1 (WM, [41]) and fetal calf serum (FCS) were purchased from Life Technologies (Eggenstein, Germany). DMEM without glucose, fatty-acid free bovine serum albumin (BSA), D-galactose (Gal), D-mannose (Man), bovine insulin, transferrin and fluorescein conjugated anti-rabbit IgG antibodies were obtained from Sigma (Deisenhofen, Germany). Penicillin G and streptomycin sulfate were from Serva (Heidelberg, Germany). Gen-

After removal of the culture media, the coverslips were washed three times with ice-cold phosphate-buffered saline (PBS), and the cells growing on the coverslips were fixed by immersion in 3.5% paraformaldehyde / PBS for 10 min. After washing twice with PBS, the remaining paraformaldehyde was inactivated by incubation with 0.1% glycine in PBS for 10 min. To enhance antibody penetration, the cells were incubated in PBS / 0.3% Triton X-100 for 10 min. The coverslips were subsequently covered with the appropriately diluted (GFAP, 1:100, glycogen phosphorylase,

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1:30) primary antibodies in PBS / 0.1% Triton X-100 / 10% normal goat serum and incubated for 2 h. After washing three times with PBS / 0.1% Triton X-100, the cells were incubated in the dark with the appropriately diluted secondary antibodies in PBS / 0.1% Triton X-100 / 10% normal goat serum for 1 h. The dilutions were: FITC conjugated anti-rabbit IgG, 1:100, biotinylated sheep antimouse (Fab) 2 -fragments, 1:100, streptavidin Texas Red, 1:100, Cy3-conjugated anti-mouse IgG, 1:800. The cells were washed three times with PBS and in the case of the biotinylated secondary antibody, incubated in the dark with streptavidin-Texas Red conjugate for 30 min. After a final washing step with PBS / 0.1% Triton X-100, the coverslips were mounted cells down on slides in 70% glycerol / PBS and inspected through a Zeiss IM 35 fluorescence microscope equipped with interference filters. For negative controls (photomicrographs not shown), primary antibodies were replaced by normal rabbit serum.

2.4. Electron microscopy For scanning electron microscopy, ependymal cells growing on coverslips for 15 d were washed three times with PBS and fixed with 2.5% glutardialdehyde in PBS at room temperature for 10 min. Postfixing was carried out in 1% osmium tetroxide / PBS at 08C for 1 h. After dehydration in a graded ethanol series and critical-point drying in CO 2 , the specimens were sputter-coated with 8 nm gold palladium and inspected in a Hitachi S-800 scanning electron microscope.

2.5. Histochemical glycogen staining The culture dishes were washed three times with icecold PBS and fixed in methanol at 2208C for 5 min. The methanol was then replaced by 1% (w / v) periodic acid in 70% ethanol, and the incubation was continued for 30 min. The cells were subsequently washed twice with water and stained with Schiff’s reagent [4] for 1 h. After rinsing the culture dishes with water for at least 30 min, the cells were inspected under a Zeiss IM 35 phase contrast microscope.

2.6. Glycogen assay The glycogen content of the cultures was assayed according to the procedure described in [7]. Briefly, the cells were washed once in ice-cold PBS and lysed by the addition of 400 ml 0.1 M NaOH. The lysates were incubated at 808C for 1 h, and the glycogen was precipitated by the addition of 2.5 volumes of ethanol. After centrifugation, the pellet was dried in a vacuum concentrator and resuspended in 50 mM sodium acetate buffer, pH 4.8. The glycogen was digested with amyloglucosidase for 90 min at 378C and the resulting glucose was assayed in a microtiter plate using a reaction mixture containing 113 mM triethanolamine / KOH, pH 7.6, 1.5 mM MgSO 4 ,

1 mM ATP, 0.76 mM NADP, 0.35 U glucose-6-phosphate dehydrogenase and 0.56 U hexokinase.

2.7. Glycogen phosphorylase assay The culture medium was aspirated, and the cells were washed three times with ice-cold PBS. They were then scraped off the bottom of the culture dish in 200 ml 10 mM Tris / HCl / 10% glycerol buffer, pH 7.4 containing 2 mM EDTA and 5 mM mercaptoethanol. The lysed cells were homogenized in a Potter-Elvehjem homogenizer and centrifuged (10 min; 13 000 g). Glycogen phosphorylase activities were determined in aliquot parts of the resulting supernatant. The experiments were carried out in a total volume of 1 ml, consisting of 975 ml of a solution of 50 mM potassium phosphate buffer (pH 7.5), 5 mM EDTA, 10 mM MgCl 2 , 0.67 mM NADP, 0.5 mg glycogen, 10 ml of a solution of 100 mM AMP, 5 ml of a solution containing 1.2 U phosphoglucomutase, 1 U glucose-6phosphate dehydrogenase, and 10 ml sample.

3. Results The method for culturing ependymal cells described in [42] was simplified by using whole brains of newborn rats as starting material. The resulting ependymal primary cultures were characterized by immunocytochemistry and scanning electron microscopy. To date, no definitive ependyma-specific immunological markers have been identified, and ependymocyte-specific antibodies have only been obtained by chance in the form of minor components in unrelated antisera [18,29]. Therefore the presence of cilia and morphological criteria were used as identifiers for ependymal cells. Cilia were often hard to discern in the phase-contrast view (Fig. 1A). However, staining with an antibody directed against a-tubulin facilitated the reliable detection of cilitated cells, even if only one cilium was present (Fig. 1B). Additionally, the concomitant staining of the cytoskeleton allowed the discrimination of ependymal subtypes based on their morphology (see below). Although ependymal cells have been reported to express GFAP [19,42], staining with anti-GFAP antibodies did not permit the easy recognition of ependymal cells by their epithelial morphology (Fig. 1C). In accordance with previous reports [42], the weak GFAP staining was concentrated around the nuclei. Occasionally, the processes of tanycyte-like cells were stained positively for GFAP (not shown). The process-bearing cells possessed only one cilium as described for tanycytes [13]. The percentage of ciliated ependymal cells in the cultures was estimated by comparing counts of ciliated cells visualized by antitubulin staining, with total cell numbers per area determined by staining of the nuclei with bisbenzimide (Fig. 1D). Of 900 counted cells from two independent ependymal cultures, 70% were ciliated. The scanning electron

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Fig. 1. Primary ependymal cultures were inspected by light and scanning electron microscopy (S.E.M.). The phase-contrast view of a fixed 15-day-old ependymal culture does not show details of the ependymal epithelium (A), but staining with an anti-tubulin antibody clearly defines cell borders and cilia (B). Counterstaining with an anti-GFAP antibody reveals weak expression of the intermediary filament protein in some but not all ependymal cells (C). Staining of the nuclei facilitated the counting of total cells per field of view (D). The S.E.M. micrographs of a 15-day-old ependymal culture show a lawn of epithelial cells with variation in cell size and number of cilia (E, G, H) and number of microvilli (F, H). F shows a cell with a centrally located bundle of cilia. G exhibits cells lacking cilia. H displays cells with only one cilium. Only a few non-epithelial cells are visible. The bars in A–D, F and H correspond to 10 mm, in E and G to 50 mm.

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micrographs of such cultures (Fig. 1E–H) displayed a confluent lawn of epithelial cells covering most parts of the bottom of the culture dish. The cells differed in both cell size and number of cilia (Fig. 1E, G, H). The predominant subtype exhibited a central bundle of approximately 40 cilia on the apical surface (Fig. 1F). Cells with similar shape, but possessing no cilia (Fig. 1G) or only one cilium (Fig. 1H) were also present. Most cells possessed varying numbers of microvilli. The appearance of the strongly ciliated cells was similar to electron micrographs obtained from intact ventricular epithelium [6]. When ependymal cultures grown in MEM c T were stained using the periodic acid Schiff base (PAS) reaction, almost no glycogen staining was observed. However, after 24 h of incubation in WM c T, a very high glycogen content was evident (Fig. 2). The staining pattern was heterogeneous with very intensely stained areas and some patches of only faintly stained cells, possibly representing a different ependymal subtype. Similar results were obtained by immunocytochemical staining of the epithelial layer with an antibody directed against the glycogen degrading enzyme, glycogen phosphorylase (Fig. 3). Presumably, only cells with high glycogen content expressed high levels of this enzyme. Table 1 shows the overall glycogen content of ependymal cultures incubated with different media for 24 h. During growth in MEM c T, only 763 nmol glycosyl residues were present per mg cellular protein, which was in agreement with the absence of a PAS reaction. A switch of the culture medium to WM c T resulted in a substantial accumulation of glycogen equivalent to 9906112 nmol / mg protein after 24 h. If insulin was omitted from the WM c T (WM c T-I), the accumulation of glycogen was only 50% of that in the presence of insulin. Thus, insulin increased the glycogen content of ependymal cells in culture two-fold. Differences in cell culture media had a profound influence on ependymal glycogen content. Cultivation in DMEM / FCS resulted in glycogen levels as high as those observed after incubation with WM c T-I, whereas omission of fetal calf serum dropped the levels to approximately one third of these values. If the glucose in DMEM was replaced by other hexoses of the same concentration (25 mM), glycogen accumulation was further reduced. A switch to mannose resulted in 70% of the glycogen obtained with DMEM, while fructose and galactose yielded 17% and 8% of the glycogen content after exposure to DMEM, respectively. As shown in Table 2, cultured ependymal cells were found to possess on average more than 200% of the glycogen present in astroglia-rich primary cultures after 24 h of incubation in WM c T. Total glycogen phosphorylase activity in ependymal cell cultures was found to be 150% of the activity in astroglia-rich primary cultures under these conditions. Furthermore, glycogen phosphorylase activities varied by a factor of 2 dependent on the incubation media. Similar to what has been described for astroglial primary

cultures [7], ependymal cells in primary culture rapidly decreased their glycogen content when deprived of glucose (Fig. 4). This process appeared to follow first-order kinetics, and a half-life of 21 min could be calculated from the semi-logarithmic plot of glycogen content versus time. The initial rate of glycogen degradation was 26 nmol / min. This was 32% of the maximal possible rate based on the glycogen phosphorylase activity present in the culture. The glycogen content of ependymal primary cultures was not only changed by glucose deprivation, but also by hormones in the presence of glucose (Table 3). Incubation of the ependymal primary cultures in the presence of 1 mM noradrenaline or serotonin resulted in significantly reduced glycogen contents, the levels having fallen by 25–40% within the hour of exposure. In contrast, the presence of 1 mM insulin led to a doubling of stored glycogen over the course of 24 h.

4. Discussion The simplified protocol for the generation of ependymal cell cultures according to [42] did not negatively affect the quality of the resulting cultures. These consisted primarily of polygonal epithelial cells with a variable number of cilia and microvilli. The morphologies of such cells closely resembled the morphologies of fully differentiated ependymal cells found in various regions of the cerebral ventricles in vivo. The expression of GFAP by a number of the cultured ependymal cells, however, suggests that some or all of them may have been in a stage of differentiation that occurs during fetal development [31]. In adult rats, most of the GFAP immunoreactivity associated with the ventricular walls is found in the subependymal layer [19]. The observed differences between individual cultured cells with respect to the number of cilia and microvilli mirrors the heterogeneity of the ventricular epithelium. In the fourth ventricle of monkey brain, densely ciliated cells cover the ventricular floor, with the degree of ciliation decreasing towards the median sulcus. Part of the sulcus is formed by cells devoid of cilia, but possessing large amounts of microvilli [36]. The regional morphology of the ventricular epithelium seems to be governed by the underlying tissue. Cells from the rabbit lateral ventricle are either cuboidal, heavily ciliated and rich in microvilli when covering the caudate nucleus, or they are squamous with less cilia when covering periventricular white matter [24]. The ependymal primary cultures consist of ependymal cells exhibiting the same variation in morphology that has been described in vivo. In contrast to the situation in the ventricle, however, the different morphological subtypes are found in a random fashion throughout the epithelial layer rather than in a regionally organized manner. This emphasizes the influence of the underlying tissue on ependymal morphology. The non-ciliated epithelial cells, interspersed with the ciliated cells in the cultures are most

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Fig. 2. Ependymal cultures (15-day-old) were stained for glycogen with the PAS technique 24 h after a change of the culture medium to either MEM C T (A) or WM C T (B). No glycogen is visible in the cells treated with the minimal medium MEM C T, while many of the cells exposed to WM C T show intensive red staining for glycogen.

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Fig. 3. Staining of a 15-day-old ependymal primary culture (A) with an antibody against glycogen phosphorylase (Pho) revealed a nonhomogeneous distribution of the enzyme (B). Counterstaining with an anti-GFAP antibody pointed to a stronger expression of the intermediary filament by the Pho-positive rather than the Pho-negative cells (C). Staining of the nuclei by bisbenzimide facilitated the counting of total cells per visual field (D). The bars in A–D correspond to 10 mm.

likely also ependymal cells. Therefore the proportion of ependymal cells in the culture is well above the percentage of 70% for the ciliated cells. Thus purity of the ependymal primary cultures was within the previously reported range [42]. The anti-tubulin staining in conjunction with the staining of the nuclei by bisbenzimide proved to be a reliable method for assessing the enrichment of ciliated ependymal cells. Depending on the cell culture medium, the ependymal primary cultures contained variable amounts of glycogen.

Despite the presence of insulin, glycogen content was very low in MEM c T. This medium contains 5.6 mM glucose, substantially less than present in DMEM (25 mM) or WM c T (27.8 mM). It is also devoid of the amino acids L-aspartate and L-glutamate, which are known to increase cellular glycogen in astroglial cells [16,38]. Furthermore, it lacks L-alanine, L-serine, L-proline and glycine, which must consequently be synthesized from glucose. The combination of low initial glucose content and the requirement for biosynthesis of almost all of the non-essential amino acids

Table 1 Glycogen content of 11-day-old ependymal primary cultures after incubation in different media for 24 h

Table 2 Glycogen phosphorylase activities and glycogen contents in ependymal (EPK) as well as astroglial primary cultures (APK) after 24 h of incubation in different media

Media

Glycogen content (nmol glucosyl residues / mg protein)

WM c T WM c T-insulin DMEM / FCS DMEM DMEM–Glc / Gal DMEM–Glc / Frc DMEM–Glc / Man MEM c T

9906112 501652 5356107 197611 1564 3464 138624 763

The cells were cultivated in MEM c T prior to the change to the indicated medium. Each value is the mean6S.D. of five individual determinations. Abbreviations: Glc, glucose; Gal, galactose; Frc, fructose; Man, mannose.

Media

DMEM / FCS MEM c T WM NC WM c T

EPK

APK

Glycogen (nmol / mg)

Pho activity (mU / mg)

Glycogen (nmol / mg)

Pho activity (mU / mg)

n.d. n.d. n.d. 8776124

n.d 4463 4962 8065

n.d. n.d. n.d. 416647

3763 4361 5065 5966

Glycogen content is expressed as nmol glucosyl residues per mg cellular protein, and Pho activity is given in mU per mg protein. Each value corresponds to the mean6S.D. of three individual assays performed on two different cultures. The cells were 9-days-old in both cases. n.d., not determined.

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Fig. 4. Glycogen was degraded with a half-life of 21 min in 10-day-old primary ependymal cultures after onset of glucose deprivation. The cells had been preincubated in WM c T for 24 h before they were exposed to glucose-free DMEM. The curved line was obtained by fitting a one-phase exponential decay function to the data weighed by 1 / S.D.2 . The inset shows a semi-logarithmic representation of the data. The straight line depicted is the result of a linear regression analysis.

might be one explanation for the inability of insulin to promote glycogen accumulation. Notably, the activity of glycogen synthase in adult rat hepatocytes increases by a factor of 19 after elevation of the glucose concentration from 8.3 to 30 mM [44]. If ependymal cells responded in a similar way, a large increase in intracellular glycogen should be expected after transferal of the cells to a high glucose medium. Indeed, ependymal culture glycogen levels rose substantially upon change from MEM c T to DMEM. This was likely due to the presence of a number of non-essential amino acids and the increased glucose concentration. The addition of FCS led to further elevated glycogen levels, explainable by the hormone and amino acid content of the serum. WM c T devoid of insulin resulted in higher glycogen levels than were obtained in DMEM, probably owing to the presence of all proteinogenic amino acids except L-asparagine and L-serine, as well as to a slightly higher glucose concentration. Upon addition of insulin, the glycogen content was further increased by a factor of two. Thus the qualitative ependymal Table 3 Modulation of ependymal glycogen content by exposure to different hormones (1 mM) Hormone

Glycogen content (% of control6S.D.)

NA 5HT Insulin

6166 7564 20469

For glycogen accumulation, dishes from a 9-day-old culture were washed once with WM NC and then kept in WM NC for 24 h. In the case of insulin, the agonist was present during the 24 h incubation period. In the case of NA and 5HT, the agonist was added after 24 h of incubation in WM NC , and glycogen was assayed after a further 1 h incubation period. Abbreviations: NA, noradrenaline; 5HT, serotonin.

response to L-aspartate, L-glutamate and insulin was similar to what has been described for astroglial cells [7,20]. The extent of glycogen accumulation after transferal to WM c T, however, differed by a factor of two in favor of the ependymal cells (Table 2). This corresponds to the in vivo observation that ependymal cells show the highest glycogen content of all brain cell types [3]. Similar to the situation in astroglial cultures, ependymal cells were able to utilize mannose [9] more efficiently than fructose or galactose [8] as a substrate for glycogen accumulation. Therefore, the enzyme, mannose-6-phosphate isomerase, is apparently present in ependymal cells. Its physiological purpose might be involvement in the metabolism of mannose residues stemming from glycoprotein degradation. A source of such glycoproteins could be the CSF. The failure of mannose-fed ependymal cells to reach the glycogen levels of glucose-fed controls indicates that the activity of mannose-6-phosphate isomerase might be lower in ependymal than in astroglial cells. The glycogen phosphorylase activity in ependymal as well as in astroglial primary cultures was dependent on the culture medium. Expression of the glycogen phosphorylase gene has been shown to be under regulation by insulin and cAMP in rat skeletal muscle [28]. In the liver, enzyme levels are regulated by the insulin / glucagon ratio [30]. Studies on cultured astrocytes have revealed a protein biosynthesis-dependent effect of VIP and noradrenaline on glycogen metabolism [37]. These findings make it appear likely that insulin and other hormonally active components occurring in FCS influence the expression levels of glycogen phosphorylase also in culture. Additionally, other media ingredients may modulate the activity of the enzyme either directly or indirectly. The heterogeneous distribution of glycogen phosphorylase in the cultures as evident from

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immunocytochemical staining (Fig. 3) emphasizes the biochemical differences between the morphologically distinguished ependymal subpopulations. These subpopulations might consist of either generically distinct cells or different stages of differentiation of one cell type, or a combination of both. A number of anatomical studies have indicated an innervation of the ventricular wall and the ependymocytes by serotoninergic nerve fibers originating from the Raphe nuclei [21,35]. The expression of noradrenaline transporters by ependymal cells [33] warrants the assumption that also noradrenaline released from endings of nerve fibers originating in the nucleus coeruleus could be involved in the regulation of ependymal functions. Both serotonin and noradrenaline triggered glycogen degradation in ependymal cultures. Therefore the metabolic activity of ependymocytes seems to be under the control of serotoninergic and adrenergic innervations of the ventricular area. While the final result of ependymal activation remains unknown, mobilization of cellular glycogen stores shown here on cultured ependymal cells and changes in beat frequency of cilia of ependymal cells in brain slices [23], both indicate a regulation of ependymal functions by neuronal centers including the Raphe nuclei. These ependymal functions could involve substrate exchange across the ventricular boundary, accelerated by the increase in ciliary beat frequency. The increased energy demand accompanying ependymal activation could be met by glycogen phosphorylase mediated mobilization of ependymal glycogen. The simultaneous presence in situ of the two indispensible components of glycogen metabolism, glycogen [3] and glycogen phosphorylase [3,26] makes it highly likely that glycogen and the regulation of its metabolism play important physiological roles in ependymal cells in brain. The ependymal cell culture system used in the present study appears to be a useful model for analyzing the details of ependymal metabolism and might hold the key to unraveling the precise functions of this enigmatic cell type of the CNS.

Acknowledgements The authors wish to thank Dr. Mirna Rapp, Aventis Behring GmbH, Marburg, Germany, for a generous gift of human thrombin, Dr. Manfred Schliwa, University of Munich, Germany, for a gift of a monoclonal anti-tubulin ¨ antibody and Dr. Heinz Schwarz, Max-Planck Institut fur ¨ Entwicklungsbiologie, Tubingen, Germany, for help with the scanning electron microscopy.

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