hyaluronan-associated molecules play a crucial role in the segregation of fiber projections to the hippocampus. MATERIALS AND METHODS

In the hippocampus ingrowing afferents terminate in specific layers. Lamina-specific fiber termination also develops in organotypic slice cultures suggesting the preservation of specific laminar recognition cues for distinct fiber systems under these in vitro conditions (Yamamoto et al., 1992; Yamamoto et al., 1999; Frotscher and Heimrich, 1993; Li et al., 1993; Li et al., 1994; Li et al., 1995; Li et al., 1996; Del Río et al., 1997). In recent years, a variety of experimental strategies have been used to identify these layer-specific recognition cues for growing axons in the hippocampus. After ablation of hippocampal Cajal-Retzius (CR) cells in hippocampal slice cultures, entorhinal fibers fail to innervate their appropriate target layers, indicating that CR cells play a key role in guiding entorhinal fibers to distinct hippocampal laminae (Del Río et al., 1996; Del Río et al., 1997; Frotscher, 1998; Ceranik et al., 1999, Ceranik et al., 2000). CR cells synthesize the extracellular matrix (ECM) glycoprotein reelin (d‘Arcangelo et al., 1995; d’Arcangelo et al., 1997), but reelin is unlikely to be the molecule governing the correct laminaspecific termination of entorhinal fibers. In the hippocampus of reeler mice lacking reelin, entorhinal fibers terminate with correct laminar specificity (Del Río et al., 1997; Borrell et al., 1999; Deller et al., 1999). Membrane preparations of hippocampal cells have been found to guide entorhinal axons in vitro, indicating that membrane-associated molecules play a role in target layer recognition by entorhinal fibers (Skutella et al., 1999). Pathfinding of entorhinal fibers may also involve diffusible guidance cues (Chédotal et al., 1998; Steup et al., 1999; Stein et al., 1999; Barallobre et al., 2000). We have recently shown that dissociated entorhinal neurons and microspheres coated with entorhinal membranes adhere with laminar specificity on living slices of hippocampus (Förster et al., 1998) suggesting a role of these adhesive cues in the positioning of entorhinal fibers to the hippocampus. 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 laminaspecific 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.