Institute of Anatomy, University of Freiburg, PO Box 111, D-79001, Freiburg, Germany
*Author for correspondence (e-mail: foerster{at}uni-freiburg.de)
Accepted May 9, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Cell adhesion, Hippocampus, Entorhinal cortex, Axon pathfinding, Hyaluronan, Rat
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present study, we took advantage of our finding that lamina-specific adhesion of dissociated cells is mimicked by fluorescent microspheres coated with isolated membranes from these cells (Förster et al., 1998). We used the microsphere adhesion assay to analyze the role of different extracellular matrix (ECM) components for a potential function in lamina-specific adhesion and entorhinal fiber growth. We show that lamina-specific adhesion on hippocampal slices is specifically abolished by the hyaluronan (HA) degrading enzyme hyaluronidase. This dramatic loss of lamina-specific adhesion was not seen after treatment of the slices with the enzymes neuraminidase or chondroitinase. Furthermore, we provide evidence that not hyaluronan itself, but hyaluronan-associated molecules mediate lamina-specific adhesion. Hyaluronidase treatment of entorhino-hippocampal cocultures also abolished target layer recognition of entorhinal fibers to the hippocampus in vitro but did not interfere with pathfinding. We conclude that hyaluronan-associated molecules play a crucial role in the segregation of fiber projections to the hippocampus.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of membranes and coating of microspheres
Membranes and membrane-coated microspheres were prepared as described (Förster et al., 1998). Briefly, cortices from P0 rats were removed under sterile conditions and transferred to homogenization buffer containing PBS (pH 7.4), urea (4 M), and spermidine (10 mM). Immediately before homogenization, a cocktail of protease inhibitors (Boehringer Mannheim) was added. Cortices were homogenized with 20 strokes of a dounce homogenizer. Cell nuclei and unlyzed cells were pelleted in a swing-out rotor (16000 g, 30 minutes at 4°C). The supernatant was centrifuged again in a fixed-angle rotor (125000 g, 60 minutes at 4°C), and cell membranes were pelleted. The supernatant was discarded and the pellet was washed three times in PBS by resuspension and centrifugation with a fixed-angle rotor (57000 g, 10 minutes) at 4°C in order to completely remove urea. Membranes were resuspended in PBS. Protein concentration was adjusted to 1 mg/ml (Bradford, 1976). Fluorescent microspheres (Molecular Probes, 4 µm diameter) were coated with membrane fractions before use, according to the manufacturers instructions. Membrane-coated microspheres were resuspended in 0.1 M phosphate buffer (PB) and sonicated before use.
Enzyme treatments of hippocampal slices
Enzymes were added to the slice incubation medium in the following concentrations: 200 TRU/ml Streptomyces hyaluronidase, protease free (Calbiochem No. 389561) or 2 units/ml chondroitinase, protease free (Calbiochem No. 230681) or 0.25 units/ml neuraminidase, protease free (Calbiochem No. 480712), and a cocktail of protease inhibitors (Boehringer Mannheim). Hippocampal slices were then incubated individually in a drop of incubation medium for 3 hours at 37°C. To confirm the involvement of proteins in lamina-specific adhesion, slices were incubated for 10 minutes in 0.1 M PB containing 0.01% trypsin before the adhesion assay. After the protease treatment the slices were rinsed in incubation medium and the adhesion assay was performed. As a control, slices were incubated under the same conditions, however, without addition of enzyme. After incubation, the slices were carefully rinsed in 0.1 M PB and then transferred to a large volume of incubation medium. Membrane-coated microspheres were then seeded onto each hippocampal slice, as described previously (Förster et al., 1998; Förster and Kaltschmidt, 1999). Slices with adherent microspheres were carefully transferred to a large volume of incubation medium. To remove non-adherent microspheres, the slices were washed by carefully pipetting them up and down (standardized to 10x up and down) with a 1 ml Eppendorf pipette (pipette tips were cut to a large diameter with a razor blade).
Analysis of microsphere adhesion
Slices were mounted on microscope slides and were analyzed under a Zeiss fluorescence microscope using a 10x, 20x or 40x objective lens. Hippocampal laminae such as the dentate molecular layer, the granule cell layer or the pyramidal cell layer, were discernable under the microscope. Slices were counterstained with the fluorescent dye DAPI (Boehringer Mannheim) to confirm the laminar boundaries under UV fluorescence as described previously (Förster et al., 1998).
Microsphere aggregation assay
To test whether HA has the capacity to mediate adhesion, membrane-coated microspheres were incubated for 30 minutes in 0.1 M PB containing soluble purified HA (Serva) in concentrations ranging from 50 µg/ml to 500 µg/ml. To test the influence of CSPGs on microsphere adhesion, membrane-coated microspheres were incubated in 0.1 M PB containing 500 µg/ml HA + 100 µg/ml of a CSPG mixture (Chemicon CC117; major components of the mixture are neurocan, phosphacan, aggrecan and versican), or in 0.1 M PB containing only 100 µg/ml CSPGs without addition of HA. As a control, membrane-coated microspheres were incubated in 0.1 M PB, without addition of HA. After the incubation, samples of the incubated microspheres were carefully pipetted onto microscope slides and analyzed under the fluorescence microscope.
Preparation and hyaluronidase treatment of entorhino-hippocampal cocultures
Slices containing the hippocampus and the adjacent entorhinal cortex were cultured as static cultures (Stoppini et al., 1991; Ceranik et al., 1999) for 7 days. Briefly, newborn mouse pups (P0) were decapitated and the hippocampus plus entorhinal cortex dissected. 300-µm-thick slices were cut perpendicular to the longitudinal axis of the hippocampus as described previously (Li et al., 1993; Ceranik et al., 1999). Tissue sections were then placed onto millipore membranes and transferred to a six-well plate with 1 ml/well nutrition medium (25% heat-inactivated horse serum, 25% Hanks balanced salt solution, 50% minimal essential medium, 2 mM glutamine, pH 7.3). Slice cultures were incubated in 5% CO2, 37°C. Medium was changed every 2 days. Hyaluronidase (Calbiochem No. 389561; final concentration 70 TRU/ml) was added to the medium at 2 days in vitro (DIV) and at 4 DIV. No enzyme was added to control cultures.
Tracing of entorhino-hippocampal fibers
Tracing was performed as previously described (Frotscher and Heimrich, 1993). Briefly, a crystal of biocytin (Sigma) was placed on the entorhinal cortex after 5 DIV. After 7 DIV, the cultures were fixed with 4% paraformaldehyde, sectioned (50 µm), and incubated with avidin-biotin-peroxidase complex (Vector Laboratories). Sections were developed with diaminobenzidine/nickel (DAB-Ni) and counterstained with Cresyl Violet.
Analysis of proteins released from enzyme-treated hippocampal slices
Hippocampal slices were first carefully rinsed in 0.1 M PB. The buffer was then replaced by fresh PB containing either 200 TRU/ml hyaluronidase or 2 U/ml chondroitinase or, as a control, no enzyme and a cocktail of protease inhibitors. Slices were then incubated for 3 hours at 37°C. After the incubation, the slice supernatants were pipetted into 1.5 ml Eppendorf tubes and insoluble cell debris was removed by centrifugation for 10 minutes (14000 rpm, 4°C, Eppendorf centrifuge 5402). The remaining soluble proteins in the supernatants were separated by electrophoresis on 3-8% polyacrylamide (PAA) gradient gels (Novex) for 3 hours, 75 V in Tris-acetate buffer, pH 7. Protein bands were visualized by subjecting the PAA gels to a silver-staining procedure according to Blum et al. (Blum et al., 1987).
Western blot analysis with antiserum against neurocan
Supernatants from hyaluronidase-treated slices were subjected to PAA gel electrophoresis as described above. An aliquot of the hyaluronidase-treated supernatant was additionally treated with 2 U/ml chondroitinase ABC for 3 hours before electrophoresis, to obtain neurocan core proteins. Blotting of separated proteins on polyvinylidene difluoride membranes (Boehringer Mannheim) was performed with the NUPAGE electrophoresis system (Invitrogen) according to the manufacturers instructions. For the detection of neurocan core proteins, a polyclonal rabbit antiserum against neurocan (NC-1) was used (Haas et al., 1999; the antiserum against neurocan was kindly provided by Drs R. U. Margolis and R. K. Margolis, New York University, New York). Immunoreactive bands were developed with the BM chromogenic western blotting kit (Roche Diagnostics).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Neuraminidase treatment of hippocampal slices does not affect lamina-specific adhesion
To find out whether other glycosidases may similarly abolish lamina-specific adhesion, hippocampal slices (P6; n=30) were incubated with 0.25 units/ml neuraminidase for 3 hours, 37°C before the adhesion assay (see Materials and Methods). Neuraminidase catalyzes the release of sialic acid residues, a frequent posttranslational modification of glycoproteins, such as NCAM (Cremer et al., 1994), or of chondroitinsulfate proteoglycans (CSPGs), such as neurocan (Retzler et al., 1996), both being molecules known to be expressed in the oml/slm (Miller et al., 1993; Haas et al., 1999). After neuraminidase treatment, the microsphere adhesion assay was performed as described above. When looking at the slices under the fluorescence microscope, the characteristic lamina-specific pattern of adherent microspheres was seen, similar to that on the untreated control slices (not shown). Quantification confirmed that microsphere adhesion on neuraminidase-treated slices was not reduced when compared to untreated control slices (Fig. 2C). Thus, laminar adhesive cues are not susceptible to neuraminidase treatment. This finding is in line with previously obtained results by Nakanishi (Nakanishi, 1983), demonstrating that neuraminidase treatment of brain tissue slices does not affect the staining of the ECM glycans HA and chondroitinsulfate.
Lamina-specific adhesion after chondroitinase treatment
Chondroitinsulfate (CS) is a glycosaminoglycan which is covalently linked to the core proteins of CSPGs, a class of ECM proteoglycans associated with HA (Bignami et al., 1993). The enzyme chondroitinase ABC degrades all forms of CS (and dermatansulfate). To examine whether CS may be involved in mediating lamina-specific adhesion, hippocampal slices (P6; n>30) were incubated with 2 units/ml chondroitinase ABC for 3 hours, 37°C. This treatment has been shown to remove most of the CS from embryonic (Nakanishi, 1983; Miller et al., 1995; Emerling and Lander, 1996) and from postnatal cortical slices (Köppe et al., 1997). After chondroitinase treatment, the adhesion assay was performed as described above. When analyzing the slices under the fluorescence microscope, again the characteristic lamina-specific pattern of adherent microspheres could be seen (not shown). However, quantification of adherent microspheres indicated a reduction in the number of adherent microspheres in the oml/slm when compared to untreated control slices (Fig. 2A,D). This minor reduction of microsphere adhesion could be interpreted two ways. (1) It is likely that the reduction of microsphere adhesion is due to partial degradation of HA, since hyaluronidase activity has also been reported for chondroitinase (Yamagata et al., 1968). (2) Alternatively, CS might also be involved in mediating lamina-specific adhesion, suggesting a role for HA-associated CSPGs.
HA fails to aggregate membrane-coated microspheres
To test whether hyaluronan could be the molecule that mediates adhesion, membrane-coated microspheres were incubated for 30 minutes at 37°C in 0.1 M PB containing solubilized HA in concentrations ranging from 50-500 µg/ml. As a control, membrane-coated microspheres were incubated in 0.1 M PB without addition of HA. After the incubation, microspheres were analyzed under a fluorescence microscope (Fig. 3). However, this assay did not give any evidence that HA could directly mediate adhesion of membrane-coated microspheres (Fig. 3A,B). None of the tested HA concentrations increased aggregation of microspheres when compared to controls. These results suggest that HA is not the molecule that mediates lamina-specific microsphere adhesion on hippocampal slices, but molecules that are bound to HA. Aggregates between HA and proteoglycans are formed via non-covalent binding of link proteins to HA (Bignami et al., 1993). To test the influence of proteoglycans on microsphere aggregation, membrane-coated microspheres were incubated in 0.1 M PB containing 100 µg/ml of a CSPG-mixture, or 500 µg/ml HA + 100 µg/ml CSPGs (see Materials and Methods). In the solution containing CSPGs but not HA, only a few aggregated microspheres were detected (Fig. 3C). In contrast, in the solution containing both HA and CSPGs, an increased number of aggregated microsphere was seen (Fig. 3D). Thus, HA-associated CSPGs could also play a role in mediating lamina-specific adhesion.
|
Laminar specificity of the entorhinal-hippocampal projection is abolished by hyaluronidase treatment
To test whether hyaluronidase treatment also affects the lamina-specific growth of entorhinal fibers to the dentate outer molecular layer, entorhino-hippocampal cocultures were prepared from newborn mice (P0; see Frotscher and Heimrich, 1993). Hyaluronidase (70 TRU/ml final concentration) was added to the incubation medium of the cocultures (n=12) after 2 and 4 days in vitro (DIV). No enzyme was added to control cultures (n=12). Thereafter, biocytin crystals were positioned on the entorhinal cocultures to trace the entorhinal fibers. Following 2 further days of incubation the cultures were processed to visualize the labeled entorhinal fibers as previously described (Del Río et al., 1997; see Methods). In control cultures, the characteristic lamina-specific termination of entorhinal fibers in the slm and oml was seen (Fig. 4A), confirming our previous results (Frotscher and Heimrich, 1993; Del Río et al., 1997). Virtually no entorhinal fibers invaded the inner molecular layer, and a sharp boundary between the iml and the oml could be discerned (Fig. 4A). In hyaluronidase-treated cultures, entorhinal fibers still invaded the slm and the oml, suggesting that axonal pathfinding to the hippocampus was not affected by the enzyme treatment. However, entorhinal fibers were no longer restricted to the oml but densely innervated the iml (Fig. 4B), suggesting that the distribution of laminar guidance cues, which normally keep entorhinal fibers restricted to the oml, had been disrupted by the hyaluronidase treatment. Thus, hyaluronan-associated cues play a crucial role in keeping entorhinal fibers restricted to the oml. Biocytin-filled entorhinal neurons in hyaluronidase-treated cultures displayed a normal morphology (not shown), demonstrating that hyaluronidase treatment did not cause gross alterations of these projection neurons.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Role of hyaluronan
Hyaluronic acid is thought to play a major role in the organization of brain extracellular matrix (Bignami et al., 1993). In the present study, we tested the susceptibility of hippocampal laminar adhesive cues to different enzymes that degrade ECM glycans. We found that the laminar adhesive cues in the hippocampus are abolished by the HA degrading enzyme hyaluronidase. What may be the role of HA in mediating this type of adhesion? The possibility has to be considered that HA binding sites were present in the membrane preparations that we used for coating microspheres. The family of hyaluronate receptor proteins (H-CAM or CD44; Underhill and Toole, 1979; Underhill, 1992) are expressed by various cell types including astrocytes (Vogel et al., 1992) and neurons (Sretavan et al., 1994) and might therefore be present in membrane preparations from cortical cells. It is, however, unlikely that HA is the adhesive molecule in our experimental system, since solubilized HA fails to aggregate membrane-coated microspheres. Furthermore, CD44 is only expressed by subsets of cells, which argues against a prominent role of this molecule in our adhesion experiments. Thus, it appears more likely that adhesion is mediated by hippocampal HA-associated molecules. This assumption is strengthend by the observation that laminar adhesive cues are susceptible to mild trypsinization as well as by our finding that a defined set of proteins and proteoglycans are released from tissue sections by hyaluronidase treatment. Aggregates between HA and proteoglycans are formed via noncovalent binding of core proteins containing a HA-binding domain (Bignami et al., 1993; see also Fig. 7A), and release of proteoglycans from tissue sections by hyaluronidase digestion has been reported previously (Asher et al., 1991; see also Fig. 7B).
|
If CSPGs mediate lamina-specific adhesion and fiber growth, it has to be assumed that their core proteins are involved rather than their CS side chains, since we found that chondroitinase treatment only slightly reduced lamina-specific adhesion, probably owing to a weak hyaluronidase activity of chondroitinase (Yamagata et al., 1968). In fact, CSPG core proteins were shown to contain domains similar to those in some adhesive proteins (Zimmermann and Ruoslahti, 1989; Rauch et al., 1992). In line with this, Köppe et al. (Köppe et al., 1997) showed that chondroitinase treatment of brain slices did not alter immunolabelling of CSPG core proteins whereas staining of CS failed, suggesting that some CSPG core proteins remain in the ECM inspite of the removal of their CS side chains. We have to consider, however, that hyaluronan-associated molecules other than CSPGs may play a role in lamina-specific adhesion and fiber growth.
Release of HA-associated cues from the ECM by hyaluronidase treatment
Analysis of the slice incubation buffer after hyaluronidase treatment by polyacrylamide gel electrophoresis suggests that specific HA-associated proteins or proteoglycans are released by the hyaluronidase treatment. Two discrete novel bands could be detected on silver-stained PAA gels after hyaluronidase treatment, a chondroitinase-sensitive 300 kDa band and a chondroitinase-insensitive 130 kDa band. By western blot analysis of hyaluronidase-treated supernatants with an antiserum against neurocan, we could provide evidence that the 300 kDa band is the CSPG neurocan. In the early postnatal period, neurocan is highly expressed in the hippocampus; expression during this period has been shown in the outer molecular layer and the stratum lacunosum moleculare, suggesting a role of neurocan in axon tract formation in the hippocampus (Wilson and Snow, 2000). Furthermore, we could show that at least one additional, yet unknown protein is released into the slice supernatant, specifically after hyaluronidase treatment. It is, of course, likely that additional CSPGs and proteins are released as well by hyaluronidase treatment.
Several studies on the role of laminar hippocampal cues in the growth of entorhinal fibers have concentrated on membrane bound cues (Skutella et al., 1999; Stein et al., 1999). However, the membrane preparations used in these studies (Walter et al., 1987) do not contain CSPGs, CS or tenascin (Tuttle et al., 1995; Skutella et al., 1999). In contrast, our present experiments provide evidence that soluble, HA-associated molecules, such as CSPGs, also may play a prominent role in the lamina-specific growth of entorhinal fibers (Fig. 7).
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Asher, R., Perides, G., Vanderhaegen, J.-J. and Bignami, A. (1991). The extracellular matrix of central nervous system white matter: demonstration of an hyaluronate-protein complex. J. Neurosci. Res. 28, 410-421.[Medline]
Barallobre, M. J., Del Río, J. A., Alcántara, S., Borrell, V., Aguado, F., Ruiz, M., Carmona, M. A., Martín, M., Fabre, M., Yuste, R. et al. (2000). Aberrrant development of hippocampal circuits and altered neural activity in netrin 1-deficient mice. Development 127, 4797-4810.
Bignami, A., Hosley, M. and Dahl, D. (1993). Hyaluronic acid and hyaluronic acid-binding proteins in brain extracellular matrix. Anat. Embryol. 188, 419-433.[Medline]
Blackstad, T. W. (1958). On the termination of some afferents to the hippocampus and fascia dentata: an experimental study in the rat. Acta Anat. (Basel) 35, 202-214.
Blum, H., Beier, H. and Gross, H. J. (1987). Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Anal. Biochem. 119, 115-119.
Borrell, V., Del Río, J. A., Alcántara, S., Derer, M., Martínez, A., DArcangelo, G., Nakajima, K., Mikoshiba K., Derer P., Curran, T. and Soriano, E. (1999). Reelin regulates the development and synaptogenesis of the layer-specific entorhino-hippocampal connections. J. Neurosci. 19, 1345-1358.
Bradford, M. M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.[Medline]
Ceranik, K., Deng, J., Heimrich, B., Lübke, J., Shanting, Z., Förster, E. and Frotscher, M. (1999). Hippocampal Cajal-Retzius cells project to the entorhinal cortex: retrograde tracing and intracellular labelling studies. Eur. J. Neurosci. 11, 4278-4290.[Medline]
Ceranik, K., Zhao, S. and Frotscher, M. (2000). Development of the entorhino-hippocampal projection: Guidance by Cajal-Retzius cell axons. Ann. New York Acad. Sci. 911, 43-54.
Chédotal, A., Del Río, J. A., Ruiz, M., Zhigang, H., Borrell, V., de Castro, F., Ezan, F., Goodman, C. S., Tessier-Lavigne, M., Sotelo, C. and Soriano, E. (1998). Semaphorins III and IV repel hippocampal axons via two distinct receptors. Development 125, 4313-4323.
Cremer, H., Lange, R., Christoph, A., Plomann, M., Vopper, G., Roes, J., Brown, R., Baldwin, S., Kramer, P., Scheff, S. et al. (1994). Inactivation of the N-CAM gene in mice results in size reduction of the olfactory bulb and deficits in spatial learning. Nature 367, 455-459.[Medline]
DArcangelo, G., Miao, G. G., Chen, S. C., Soares, H. D., Morgan, J. I. and Curran, T. (1995). A protein related to extracellular matrix proteins deleted in the mouse mutant reeler. Nature 374, 719-723.[Medline]
DArcangelo, G., Nakajima, K., Miyata, T., Ogawa, M., Mikoshiba, K. and Curran, T. (1997). Reelin is a secreted glycoprotein recognized by the CR-50 monoclonal antibody. J. Neurosci. 17, 23-31.
Del Río, J. A., Heimrich, B., Super, H., Borrell, V., Frotscher M. and Soriano, E. (1996). Differential survival of Cajal-Retzius cells in organotypic cultures of hippocampus and neocortex. J. Neurosci. 16, 6896-6907.
Del Río, J. A., Heimrich, B., Borrell, V., Förster, E., Drakew, A., Alcantara, S., Nakajima, K., Miyata, T., Ogawa, M., Mikoshiba, K. et al. (1997). A role for Cajal-Retzius cells and reelin in the development of hippocampal connections. Nature 385, 70-74.[Medline]
Deller, T., Haas, C. A., Naumann, T., Joester, A., Faissner, A. and Frotscher, M. (1997). Upregulation of astrocyte-derived tenascin-C correlates with neurite outgrowth in the rat dentate gyrus after unilateral entorhinal cortex lesion. Neuroscience 81, 829-846.[Medline]
Deller, T., Drakew, A. and Frotscher, M. (1999). Different primary target cells are important for fiber lamination in the fascia dentata: A lesson from reeler mutant mice. Exp. Neurol. 156, 239-253.[Medline]
Deller T., Haas C. A. and Frotscher, M. (2000). Reorganization of the rat fascia dentata after a unilateral entorhinal cortex lesion. Ann. New York Acad. Sci. 911, 207-220.
Derer P. and Nakanishi, S. (1983). Extracellular matrix distribution during neocortical wall ontogenesis in normal and Reeler mice. J. Hirnforsch. 24, 209-224.[Medline]
Emerling, D. E. and Lander, A. D. (1996). Inhibitors and promotors of thalamic neuron adhesion and outgrowth in embryonic neocortex: Functional association with chondroitin sulfate. Neuron 17, 1089-1100.[Medline]
Faissner, A., Clement, A., Lochter, A., Streit, A., Mandl, C. and Schachner, M. (1994). Isolation of a neural chondroitinsulfate proteoglycan with neurite outgrowth promoting properties. J. Cell Biol. 126, 783-799.[Abstract]
Faissner, A. and Steindler, D. (1995). Boundaries and inhibitory molecules in developing neural tissues. Glia 13, 233-254.[Medline]
Förster, E., Kaltschmidt, C., Deng, J., Cremer, H., Deller, T. and Frotscher, M. (1998). Lamina-specific cell adhesion on living slices of hippocampus. Development 125, 3399-3410.
Förster, E. and Kaltschmidt, C. (1999). Improved protocol for a microsphere adhesion assay on living tissue slices. BioTechniques 26, 466-472.[Medline]
Frotscher, M. and Heimrich, B. (1993). Formation of layer-specific fiber projections to the hippocampus in vitro. Proc. Natl. Acad. Sci. USA 90, 10400-10403.[Abstract]
Frotscher, M. (1998). Cajal-Retzius cells, Reelin, and the formation of layers. Curr. Opin. Neurobiol. 8, 570-575.[Medline]
Grumet, M., Milev, P., Sakurai, T., Kathikeyan, L., Bourdon, M., Margolis, R. K. and Margolis, R. U. (1994). Interactions with tenascin and differential effects on cell adhesion of neurocan and phosphacan, two major chondroitin sulfate proteoglycans of the nervous system. J. Biol. Chem. 269, 12142-12146.
Haas, C. A., Rauch, U., Thon, N., Merten T. and Deller, T. (1999). Entorhinal cortex lesion in adult rats induces the expression of the neural chondroitin sulfate proteoglycan neurocan in reactive astrocytes. J. Neurosci. 19, 9953-9963.
Hernon, M. E. and Lander, A. D. (1990). A diverse set of developmentally regulated proteoglycans is expressed in the rat central nervous system. Neuron 4, 949-961.[Medline]
Köppe, G., Brückner, G., Härtig, W., Delpech, B. and Bigl, V. (1997). Characterization of proteoglycan-containing perineuronal nets by enzymatic treatments of rat brain sections. Histochem. J. 29, 11-20.[Medline]
Li, D., Field, P. M., Starega, U., Li, Y. and Raisman, G. (1993). Entorhinal axons project to dentate gyrus in organotypic slice co-culture. Neuroscience 52, 799-813.[Medline]
Li, D., Field, M., Yoshioka, N. and Raisman, G. (1994). Axons regenerate with correct specificity in horizontal slice culture of the postnatal rat entorhino-hippocampal system. Eur. J. Neurosci. 6, 1026-1037.[Medline]
Li, D., Field, M. and Raisman, G. (1995). Failure of axon regeneration in postnatal rat entorhinal cortex is due to maturation of the axon, not that of the pathway or target. Eur. J. Neurosci. 7, 1164-1171.[Medline]
Li, D., Field, M. and Raisman, G. (1996). Connectional specifications of regenerating entorhinal projection neuron classes cannot be overridden by altered target availability in postnatal organotypic slice co-culture. Exp. Neurol. 142, 151-160.[Medline]
Margolis, R. U. and Margolis, R. K. (1997). Chondroitinsulfate proteoglycans as mediators of axon growth and pathfinding. Cell Tissue Res. 290, 343-348.[Medline]
Meyer-Puttlitz, B., Milev, P., Junker, E., Zimmer, I., Margolis, R. U. and Margolis, R. K. (1995). Chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of nervous tissue: Developmental changes of neurocan and phosphacan. J. Neurochem. 65, 2327-2337.[Medline]
Milev, P., Maurel, P., Chiba, A., Mevissen, M., Popp, S., Yamaguchi,Y., Margolis, R. K. and Margolis, R. U. (1998). Differential regulation of expression of hyaluronan-binding proteoglycans in developing brain: aggrecan, versican, neurocan, and brevican. Biochem. Biophys. Res. Commun. 247, 207-212.[Medline]
Miller, P. D., Chung, W.-W., Lagenaur, C. F. and DeKosky, S. T. (1993). Regional distribution of neural cell adhesion molecule (N-CAM) and L1 in human and rodent hippocampus. J. Comp. Neurol. 327, 341-349.[Medline]
Miller, B., Sheppard, A. M., Bicknese, A. R. and Pearlman, A. L. (1995). Chondroitin sulfate proteoglycans in the developing cerebral cortex: The distribution of neurocan distinguishes forming afferent and efferent axonal pathways. J. Comp. Neurol. 355, 615-628.[Medline]
Nakanishi S. (1983). Extracellular matrix during laminar pattern formation of neocortex in normal and reeler mutant mice. Dev. Biol. 95, 305-316.[Medline]
Pearlman, A. L. and Sheppard, A. M. (1996). Extracellular matrix in early cortical development. Prog. Brain Res. 108, 119-134.
Perides, G., Erickson, H. P., Rahemtulla, F. and Bignami, A. (1993). Colocalization of tenascin with versican, a hyaluronate-binding chondroitin sulfate proteoglycan. Anat. Embryol. 188, 467-479.[Medline]
Rauch, U., Karthikeyan, L., Maurel, P., Margolis, R. U. and Margolis, R. K. (1992). Cloning and primary structure of neurocan, a developmentally regulated, aggregating chondroitin sulphate proteoglycan of brain. J. Biol. Chem. 267, 19536-19547.
Rauch, U. (1997) Modeling an extracellular environment for axonal pathfinding and fasciculation in the central nervous system. Cell Tissue Res. 290, 349-356.[Medline]
Retzler, C., Göhring, W. and Rauch, U. (1996). Analysis of neurocan structures interacting with the neural cell adhesion molecule N-CAM. J. Biol. Chem. 271, 27304-27310.
Sheppard, A. M. and Pearlman, A. L. (1997). Abnormal reorganization of preplate neurons and their associated extracellular matrix: an early manifestation of altered neocortical development in the reeler mutant mouse. J. Comp. Neurol. 378, 173-179.[Medline]
Skutella, T., Savaskan, N. E., Ninnemann, O. and Nitsch, R. (1999). Target- and maturation-specific membrane-associated molecules determine the ingrowth of entorhinal fibers into the hippocampus. Dev. Biol. 211, 277-292.[Medline]
Sretavan, D. W., Feng, L., Pure, E. and Reichardt, L. F. (1994). Embryonic neurons of the developing optic chiasm express L1 and CD44, cell surface molecules with opposing effects on retinal axon growth. Neuron 12, 957-975.[Medline]
Stein, E., Savaskan, N. E., Ninnemann, O., Nitsch, R., Zhou, R. and Skutella, T. (1999). A role for the Eph ligand ephrin-A3 in entorhino-hippocampal axon targeting. J. Neurosci. 15, 8885-8893.
Steup, A., Ninnemann, O., Savaskan, N. E., Nitsch, R., Püschel, A. W. and Skutella, T. (1999). Semaphorin D acts as a repulsive factor for entorhinal and hippocampal neurons. Eur. J. Neurosci. 11, 729-734.[Medline]
Stoppini, L., Buchs, P. A. and Muller, D. (1991). A simple method for organotypic cultures of nervous tissue. J. Neurosci. Methods 37, 173-182.[Medline]
Thon, N., Haas, A. C., Rauch, U., Merten, T., Fässler, R., Frotscher, M. and Deller, T. (2000). The chondroitin sulphate proteoglycan brevican is upregulated by astrocytes after entorhinal cortex lesions in adult rats. Eur. J. Neurosci. 12, 2547-2558.[Medline]
Tuttle, R., Schlaggar, B. L. and O'Leary, D. D. M. (1995). Maturation-dependent upregulation of growth-promoting molecules in developing cortical plate controls thalamic and cortical neurite growth. J. Neurosci. 19, 3039-3052.
Underhill, C. B. and Toole, B. P. (1979). Binding of hyaluronate to the surface of cultured cells. J. Cell Biol. 82, 475-484.[Abstract]
Underhill, C. (1992). CD44 the hyaluronan receptor. J. Cell Sci. 103, 293-298.
Vogel, H., Butcher, E. C. and Picker, L. J. (1992). H-CAM expression in the human nervous system: evidence for a role in diverse glial interactions. J. Neurocytol. 21, 363-373.[Medline]
Walter, J., Kern-Veits, B., Huf, J., Stolze, B. and Bonhoeffer, F. (1987). Recognition of position-specific properties of tectal cell membranes by retinal axons in vitro. Development 101, 685-696.[Abstract]
Wilson, M. T. and Snow, D. M. (2000). Chondroitin sulfate proteoglycan expression in hippocampal development: potential regulation of axon tract formation. J. Comp. Neurol. 424, 532-546.[Medline]
Yamada, H., Fredette, B., Shitara, K., Hagihara, K., Miura, R., Ranscht, B., Stallcup, W. B. and Yamaguchi, Y. (1997). The brain chondroitinsulfate proteoglycan brevican associates with astrocytes ensheating cerebellar glomeruli and inhibits neurite outgrowth from granule neurons. J. Neurosci. 17, 7784-7795.
Yamagata, T., Saito, H., Habuchi, O. and Suzuki, S. (1968). Purification and properties of bacterial chondroitinases and chondrosulfatases. J. Biol. Chem. 243, 1523-1535.
Yamamoto, N., Yamada, K., Kurotani, T. and Toyama, K. (1992). Laminar specificity of extrinsic cortical connections studied in coculture preparations. Neuron 9, 217-228.[Medline]
Yamamoto, N., Matsuyama, Y., Harada, A., Inui, K., Murakami, F., Hanamura, K. (1999). Characterization of factors regulating lamina-specific growth of thalamocortical axons. J. Neurobiol. 42, 56-68.
Zimmermann, D. R. and Ruoslahti, E. (1989). Multiple domains of the large fibroblast proteoglycan, versican. EMBO J. 8, 2975-2981.[Abstract]