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Hyaluronan-associated adhesive cues control fiber segregation in the hippocampus

Institute of Anatomy, University of Freiburg, PO Box 111, D-79001, Freiburg, Germany

View larger version (103K): [in a new window]   Fig. 1. (A) Schematic illustration of hippocampal lamination. So, stratum oriens; sp, pyramidal cell layer (areas CA1 and CA3 are indicated); sr, stratum radiatum; slm, stratum lacunosum-moleculare; ml, dentate molecular layer (oml=outer ml; iml=inner ml); g, granule cell layer; h, hilus; FD, fascia dentata; F, fimbria. (B) Fluorescent microspheres (4 µm diameter) were coated with membrane preparations from cortical cells (P0) and plated on hippocampal slices (P6). After incubation, non-adherent particles were removed. Microspheres adhere only to the stratum oriens, the stratum lacunosum moleculare, the outer molecular layer and the hilar region. Microspheres do not adhere to the pyramidal cell layer, the granule cell layer, and to the stratum radiatum. Bar, 200 µm. (C) Lamina-specific adhesion of membrane-coated fluorescent microspheres to the outer molecular layer of a DAPI-stained hippocampal control slice (P6). Bar, 50 µm. (D) Lamina-specific adhesion of microspheres is abolished on a hippocampal slice (P6) which was treated with hyaluronidase before the adhesion assay. Bar, 50 µm.

View larger version (18K): [in a new window]   Fig. 2. Influence of different enzyme treatments on microsphere adhesion to hippocampal slices. Densities on the different hippocampal laminae, stratum oriens (so), stratum pyramidale (sp), stratum radiatum (sr), stratum lacunosum-moleculare + dentate outer molecular layer (slm/oml), inner molecular layer + granule cell layer (iml/g), hilus (h), were determined for 10 hippocampal slices (individual columns). (A) Densities of adherent microspheres on different hippocampal laminae on untreated control slices. Densities of adherent microspheres differed significantly between adjacent laminae (Wilcoxon Rank Sum Test, two tailed: P<0.05). (B) Hippocampal slices were incubated with 200 TRU/ml hyaluronidase before the adhesion assay. Note the massive reduction of microsphere adhesion in the layers oml/slm when compared to control slices. Densities of adherent microspheres still differed significantly between oml/slm and iml (Wilcoxon Rank Sum Test, two tailed: P<0.05). (C) Hippocampal slices were incubated with neuraminidase before the adhesion assay. Densities of adherent microspheres on different hippocampal laminae were similar to those on untreated control slices (compare with A) and differed significantly between adjacent laminae (Wilcoxon Rank Sum Test, two-tailed: P<0.05). (D) Hippocampal slices were incubated with 0.25 U/ml chondroitinase before the adhesion assay. Membrane-coated microspheres adhere with laminar specificity to the layers oml/slm. However, the number of adherent microspheres in the layers oml/slm is slightly reduced when compared to untreated control slices (A) or neuraminidase-treated slices (B). (Wilcoxon Rank Sum Test, two-tailed: P<0.05).

View larger version (21K): [in a new window]   Fig. 3. Increased aggregation of membrane-coated microspheres in a solution containing hyaluronan and a mixture of CSPGs. (A) Control: Membrane-coated microspheres after 30 minutes incubation in 0.1 M PB. (B) Membrane-coated microspheres after 30 min of incubation in 500 µg/ml hyaluronan in 0.1 M PB. (C) Membrane-coated microspheres after 30 minutes incubation in 0.1 M PB containing 100 µg/ml of a CSPG mixture. (D) Membrane-coated microspheres after 30 minutes incubation in 0.1 M PB containing 500 µg/ml hyaluronan and 100 µg/ml of a CSPG mixture. Note the increased number of aggregated microspheres.

View larger version (93K): [in a new window]   Fig. 4. Effect of hyaluronidase treatment on the lamina-specific growth of entorhinal fibers in entorhino-hippocampal cocultures. (A) Control: Entorhino-hippocampal coculture after 7 days in vitro (DIV) without addition of enzyme. The entorhinal fibers are visualized by tracing with biocytin. Entorhino-hippocampal fibers (arrow) are restricted to the stratum lacunosum moleculare (slm) and dentate outer molecular layer (oml). Note that entorhinal fibers do not invade the inner molecular layer (iml). The tissue section is counterstained with Cresyl Violet. DG, dentate gyrus; g, granule cell layer; EC, entorhinal cortex; hippocampal areas CA1 and CA3 are indicated. Bar: 100 µm. (B) Hyaluronidase-treated entorhino-hippocampal coculture after 7 DIV. As in control cultures, entorhino-hippocampal fibers project to the slm and the molecular layer (arrow). However, entorhinal fibers are not restricted to the oml but also invade the iml. Bar, 100 µm.

View larger version (86K): [in a new window]   Fig. 5. Analysis of the protein content in the slice incubation buffer after hyaluronidase treatment. Silver-stained polyacrylamide gels are shown after electrophoresis of supernatants from hippocampal slices (P6): C, control without addition of enzyme; Hy, after hyaluronidase treatment; ABC, after treatment with chondroitinase ABC; Hy/ABC, after treatment with both hyaluronidase and chondroitinase. (A) Protein bands in the range between 200 kDa and 400 kDa are shown. Note the presence of an additional band (*) of approximately 300 kDa after hyaluronidase (Hy) treatment of the slices. This band is not seen in control supernatants (C) or in supernatants from chondroitinase (ABC) treated slices, suggesting specific release of this protein by hyaluronidase treatment. Absence of this band in supernatant that was treated with both hyaluronidase and chondroitinase (Hy/ABC) suggests that the band represents a protein that is modified by chondroitinsulfate. (B) Protein bands below 140 kDa are shown. Note the presence of an additional band (*) of approx. 130 kDa after hyaluronidase (Hy) treatment of the slices. This band is not seen in control supernatants (C) or in supernatants from chondroitinase (ABC) treated slices, suggesting release of this protein only by hyaluronidase treatment of the slices. However, this band is unaffected by treatment of slices with both hyaluronidase and chondroitinase, suggesting it is not modified by chondroitinsulfate.

View larger version (15K): [in a new window]   Fig. 6. Western blot for neurocan. Proteins in supernatants from hyaluronidase-treated rat hippocampal slices were separated by gel electrophoresis and analyzed with an antiserum which detects neurocan core proteins (NC-1). Lane 1: Protein size standard. Lane 2: Supernatant after hyaluronidase treatment alone; no signal was detected by the neurocan antiserum. Lane 3: The supernatant from lane 2 was subsequently treated with chondroitinase ABC to remove chondroitinsulfate side chains from CSPGs. After this double enzymatic treatment, a band of approx. 130 kDa, which is characteristic for a neurocan core protein, was detected by the antiserum. Thus, neurocan, substituted with CS, is present in hyaluronidase-treated supernatants from hippocampal slices.

View larger version (11K): [in a new window]   Fig. 7. Hypothetical role of hyaluronan and hyaluronan-associated molecules in the lamina-specific growth of entorhinal fibers to the dentate outer molecular layer. (A) Hyaluronan-associated cues direct a growing entorhinal fiber to the dentate outer molecular layer and prevent the growth cone from entering the inner molecular layer. (B) The boundary which is normally formed by hyaluronan-associated cues is disrupted by hyaluronidase treatment. Hyaluronan-associated cues are no longer able to prevent the entorhinal fiber from entering the inner molecular layer.

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