Semaphorins: contributors to structural stability of hippocampal networks?

M.A. Hofman, G.J. Boer, A.J.G.D. Holtmaat, E.J.W. Van Someren, Progress in Brain Research, Vol. 138 0 2002 Elsevier Science B.V. All rights reserved

J. Verhaagen and D.F. Swaab (Eds.)

CHARTER 2

Semaphorins: contributors to structural stability of hippocampal networks? Anthony J.G.D. Holtmaat ‘v*, Fred De Winter ‘, Joris De Wit ‘, Jan A. Gorter 2, Fernando H. Lopes da Silva 2 and Joost Verhaagen’ 2 Swammerdam

I Netherlands Institute

Institute for Brain Research, Meibergdreef for Life Sciences, University of Amsterdam,

Introduction During development of the nervous system axons and dendrites are guided towards their targets with great precision. The molecular cues that govern the guidance of growing axons consist of surrounding molecules that are either released from a distance or attached to glial or neuronal membranes (TessierLavigne and Goodman, 1996). The molecules encode attractive and repulsive information that is interpreted by receptors present on the growth cones. It appears that the signal transduction pathway for many axon guidance signals ends at the actin cytoskeleton, which is the ‘motor’ for axon and dendrite motility. So far four main families of guidance molecules with their receptors have been described: the semaphorins with neuropilins and plexins, the ephrins with Eph-receptors (or vice versa), the netrins with DCCs, and slits with Robos. Many of these ligands and receptors are found in the mature nervous system. This is remarkable, since it has long been believed that neurons in the adult central nervous system do not extend neurites over long distances,

* Correspondence to: A.J.G.D. Holtmaat, Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands. E-mail: [email protected]

33, 1105 AZ Amsterdam, The Netherlands Kruislaan 320, 1098 SM Amsterdam, The Netherlands

as they do during development. It is now known that there is some long distance outgrowth of axons in particular parts of the mature nervous system, but this does not compare to the extensive presence of guidance factors at all sorts of locations in the CNS during adulthood. Thus, what would be the function of axon guidance factors in the mature nervous system? Are they needed to govern subtle changes in the morphology of mature neurons or are they needed to stabilize existing connections? Or, as a third option, are they involved in neuronal plasticity through interactions with regulators of synaptic transmission? For some members of the ephrin family, convincing evidence is emerging that they are involved in structural and synaptic plasticity. However, for most of the guidance factors their function in the adult nervous system is still a mystery. In this review we will focus on semaphorins. It is not our intention to provide a complete survey of the current knowledge on semaphorin signaling or their biological function, neither to give a detailed description of their role in nervous system development in general. As a matter of introduction we will summarize the molecular interactions and signaling of semaphorins and subsequently focus on the putative role that semaphorins and their receptors play in hippocampal development. Subsequently, we will discuss the implications of their presence in the adult hippocampus for structural plasticity.

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Semaphorin signaling The family of semaphorins comprises secreted and membrane-attached proteins characterized by one common extracellular domain, the semaphorin domain (Kolodkin et al., 1993; Luo et al., 1993, 1995; Puschel et al., 1995). They are categorized in eight subclasses of which the first two represent the invertebrate forms and the eighth class comprises the viral variants. The other classes are found in vertebrates. Class 3 comprises the secreted molecules while classes 4-7 represent semaphorins that are anchored in or attached to the cell membrane (Semaphorin-nomenclature- and committee, 1999). Generally, semaphorins are thought to exert their function via receptors named plexins, although some transmembrane semaphorins have been found to have intracellular binding partners and thereby could function as bi-directional signaling molecules as well (Eckhardt et al., 1997; Winberg et al., 1998; Takahashi et al., 1999; Tamagnone et al., 1999; Wang et al., 1999; Klostermann et al., 2000; Nakamura et al., 2000; Rohm et al., 2000; Inagaki et al., 2001; Leighton et al., 2001; Ohoka et al., 2001; Schultze et al., 2001). The vertebrate plexin family consists of nine members, which are divided into four subfamilies (plexin-Al-A4, plexin-Bl-B3, plexin-Cl and plexin-Dl). All Plexins contain an extracellular semaphorin domain and a conserved intracellular sex-plexin domain (Tamagnone et al., 1999). Although catalytic functions of this intracellular domain have not yet been defined it is believed that these regions somehow constitute the starting point for the intracellular signaling cascade. Only a few functional interactions between semaphorins and plexins have been described. Plexin-Bl appears to function as a receptor for Sema4D, and plexin-Cl interacts with Sema7A and the viral semaphorins (Tamagnone et al., 1999). The plexin-A subfamily acts as mediators for signaling of secreted semaphorins, however, they do not bind secreted semaphorins on their own (Takahashi et al., 1999; Tamagnone et al., 1999). For proper functioning, the secreted semaphorins need to bind a complex of plexins and neuropilins (He and Tessier-Lavigne, 1997; Kolodkin et al., 1997; Takahashi et al., 1999). Two types of neuropilins have been described

(neuropilin-1 and -2), that both need to be present as homo/heterodimers in the complex with plexins and semaphorins. Neuropilins lack a functional signaling domain at their carboxy terminus and therefore do not directly transmit semaphorin signals. Instead, it is thought that dimers of neuropilins serve as the ligand binding components in the receptor complex while the dimers of plexin molecules, via their cytoplasmic region, transduce the signal into the cytoplasm (Takahashi et al., 1999; Tamagnone et al., 1999; Rohm et al., 2000). In addition, there is evidence, at least for Sema3A signaling, that the cell adhesion molecule L 1 takes part in the receptor complex as well (Castellani et al., 2000). It is not known whether Ll acts as a mediator for the formation of the receptor complex or whether it is a modulator of intracellular components that are needed for semaphorin signaling (He, 2000). The interactions of secreted semaphorins with plexins and neuropilins are very divers. First, neuropilin-1 binds to all secreted semaphorins, but is unlikely to mediate the biological function of Sema3F. Second, neuropilin-2 binds all secreted semaphotins except for Sema3A. Third, Sema3A uses homodimers of neuropilin-1 while Sema3F uses homodimers of neuropilin-2 to exert biological function (Chen et al., 1998; Giger et al., 1998~). Fourth, Sema3C probably functions via binding to heterodimers of neuropilin-1 and -2 (Chen et al., 1998). The other class 3 semaphotins bind either to homodimers or heterodimers of neuropilins, but the exact composition that is needed for proper functioning is not known (Feiner et al., 1997; Nakamura et al., 2000). To illustrate the complexity of secreted semaphorin signaling, it is interesting to note that semaphorin 3B and 3C can act as agonists of neuropilin-2, but act as antagonists of neuropilin-1 receptors (Takahashi et al., 1998). Furthermore, COS cell contraction assays (an in vitro assay based on the contraction of the cytoskeleton of the non neuronal COS-7 tumor cells; developed by Takahashi et al., 1999) have revealed that Sema3A and Sema3F induce contraction via plexin-Al or plexin-A2 (when present in a complex with neuropilin-1 and neuropilin-2, respectively). Sema3C has remained functionless in these assays as well as plexin-A3 (Takahashi and Strittmatter, 2001). However, recently it has been found that particular neurons from

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plexin-A3 knockout mice show a strong to minor reduction of responsiveness to Sema3F and Sema3A, respectively, indicating that plexin-A3 does play a major role in the signal transduction of Sema3F and in part regulates responsiveness to Sema3A (Cheng et al., 2001). It is not known how exactly plexins exert their activity, but the signaling cascade seems to involve a wide range of intracellular proteins, including collapsin response mediator proteins (CRMPs), Rho family GTPases, cGMP, LIM-kinase and cofilin (for reviews, see Nakamura et al., 2000; Tamagnone and Comoglio, 2000; Liu and Strittmatter, 2001). Most likely all responses are directly or indirectly modifying the actin filamentous network thereby influencing the morphology of neurite endings (Fritsche et al., 1999; Aizawa et al., 2001). For example, upon addition of purified Sema3A to dorsal root ganglion (DRG) cell cultures, the axons respond by a collapse of their growth cone and subsequent growth arrest, an activity of Sema3A reflected in the name that was first given to its chicken analogue, collapsin-1 (Luo et al., 1993). It is believed that low levels of secreted semaphorins (for example, as present in gradients from point sources) elicit a partial growth cone collapse and thereby allow the growth cone to turn away from the semaphorin source, resulting in the typical bending and avoidance of particular environments by growing axons, as seen in the nervous system in vivo (Fan and Raper, 1995; Giger et al., 1996; Song et al., 1998). The latter aspect of growth cone guidance is probably best illustrated in vitro by repulsion assays, stripe assays or turning assays in which growing axons either avoid or preferentially enter an unidirectional gradient of chemorepellents or attractants, respectively (Lumsden and Davies, 1986; Walter et al., 1987; Fitzgerald et al., 1993; Song et al., 1998). A relevant function of Sema3A was first described for dorsal root ganglion (DRG) projections in the developing spinal cord. From repulsion assays it could be concluded that during initial stages of DRG axon growth into the spinal cord (around El35 in mouse) all DRG axons are repelled by Sema3A (Puschel et al., 1996; Shepherd et al., 1997). Since Sema3A is present in all aspects of the spinal cord around that time the axons could be prevented to prematurely enter the spinal cord. Later on, Sema3A expression becomes restricted to the ventral part of the

spinal cord, which thereby would permit DRG-fibers to enter the dorsal region of the spinal cord. Concomitantly NT-3-dependent DRG neurons become insensitive to Sema3A while axons of the NGFdependent population of neurons remain repelled by Sema3A (Messersmith et al., 1995; Puschel et al., 1996). Since the NT-3-dependent fibers are not sensitive to the ventral Sema3A source they are allowed to enter the ventral region to form the proprioceptive and mechanoreceptive connection, while the NGFdependent fibers are forced to terminate in the dorsal part of the spinal cord to form the nociceptive projection (Messersmith et al., 1995; Puschel et al., 1996; Shepherd et al., 1997; Reza et al., 1999). This was confirmed by experiments with organotypic explants of the spinal cord, in which the entrance of nociceptive sensory DRG afferents was blocked by viral vector mediated ectopic expression of Sema3A in the dorsal aspect of the spinal cord (Pasterkamp et al., 2000). A role of Sema3A and other secreted semaphorins in the development of several cranial and peripheral nerves has now become evident as well (Kitsukawa et al., 1997; Kobayashi et al., 1997; Taniguchi et al., 1997; Varela-Echavarria et al., 1997; Giger et al., 1998b, 2000; Ulupinar et al., 1999; Chen et al., 2000; Nakamura et al., 2000; Raper, 2000; Renzi et al., 2000; Rochlin et al., 2000). In vitro studies have indicated that secreted semaphorins can affect neurites from the central nervous system (e.g. Bagnard et al., 1998; Chedotal et al., 1998; de Castro et al., 1999; Rabacchi et al., 1999; Steup et al., 1999). However, there is not much in vivo evidence for an important role of secreted semaphorins in central nerve projections since two different Sema3A knockout mouse lines only show very mild phenotypic changes in central neuronal trajectories (Behar et al., 1996; Taniguchi et al., 1997; Catalan0 et al., 1998) and obvious defects in the central nervous system of Sema3C knockout mice have not yet been reported (Feiner et al., 2001). It is thought that a lack of phenotypes in the central nervous system of these mice is caused by compensatory mechanisms or redundancy within the function of different secreted semaphorin family members. One can envision that this is likely to occur given the fact that an extreme diversity of possible semaphorin-neuropilin-plexin interactions exists.

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This idea is corroborated by the notion that when necessary components of semaphorin receptors (neuropilin-1 and -2) are knocked out, mice do show severe abnormalities in the central nervous system. This indicates that secreted semaphorins indeed play important roles in central nervous system development (Kitsukawa et al., 1997; Chen et al., 2000; Giger et al., 2000). Another surprise provided by Sema3A, Sema3C and neuropilin-1 knockout mice was the role that several semaphorins play in the morphogenesis of non-neuronal tissue, such as the cardiovascular system, bones and cartilage (Behar et al., 1996; Kitsukawa et al., 1997; Taniguchi et al., 1997; Chen et al., 2000; Giger et al., 2000; Cheng et al., 2001; Feiner et al., 2001). It is beyond the scope of this review to go into detail on all these aspects of the biological function of semaphorins, but it is important to note that more and more evidence is emerging that the function of semaphorins is not confined to the nervous system. In addition to a chemotropic action on neurites, semaphorins appear to exert a range of other cellular responses (e.g. apoptosis, cell migration, cell adhesion, tumor formation; Sekido et al., 1996; Yamada et al., 1997; Gagliardini and Fankhauser, 1999; Shirvan et al., 1999; Brambilla et al., 2000; Bagnard et al., 2001). Likewise, it has become clear that secreted semaphorins are not always acting as repellents. One single semaphorin can be a repellent for a particular population of neurons while it is an attractant for another. Sema3C, for example, has been shown to act as an attractant for El6 cortical axons, while it is a repellent for neurons from the embryonic septum that are projecting to the hippocampus (Bagnard et al., 1998; Steup et al., 2000). Furthermore, it has become apparent that Sema3A (as well as slit-2, an axon guidance molecule from a different family) can at the same time act as a repellent and as an attractant for different processes of a single cortical neuron (Polleux et al., 1998, 2000; Whitford et al., 2002). The levels of the intracellular messengers CAMP and cGMP are critical determinants of the final action resulting from the exposure to extracellular guidance signals (Song et al., 1998; Castellani et al., 2000). Song et al. (1998) showed that Sema3A becomes an attractant for growing axons when cytoplasmic levels of cGMP are artificially enhanced. Polleux et al. (2000) subsequently showed

that in a single developing cortical neuron the level of cGMP is high in the presumptive dendrite while it is low in the axonal segment, which probably explains why dendrites and axons from the same neuron are attracted and repelled, respectively, by an apical source of Sema3A in the developing cortex. Semaphorins play a role in hippocampal development The development of the hippocampus is a fascinating process since projections within this structure become laminated during maturation and mainly have a unidirectional character (Fig. 1). The most illustrative examples of lamination in the hippocampal formation are the segregated termination of two main afferents of granule cells in the dentate gyrus and of pyramidal cells in the hippocampus proper. The entorhinal cortex layer ll and III project axons to the outer and medial dendritic segments of granule cells (i.e. the middle and outer molecular layer) and to the most distal portion of pyramidal cell dendrites in the stratum lacunosum moleculare, while most of the commissural/associational fibers terminate on the most proximal parts of the granule cell dendrites in the inner molecular layer of the dentate gyrus and of the pyramidal cell dendrites in the stratum oriens and stratum radiatum (Andersen et al., 1969; Witter, 1993; Super and Soriano, 1994; Frotscher and Heimrich, 1995; Frotscher et al., 1997; Sanes and Yamagata, 1999). The other striking feature of the hippocampal network, the unidirectional connectivity, is most pronounced in the projection from the entorhinal cortex to the dentate gyrus (the perforant path), the projection of granule cell axons to CA3 (the mossy fiber path) and the projection of CA3 pyramidal cell axons to CA1 (the Schaffer collateral path; for an excellent review of hippocampal anatomy, see Amaral and Witter, 1995). The notion that the hippocampus develops to such a remarkable neuronal network prompts the questions what are the cellular and molecular cues that guide the afferents to their exact location and what could be the determinants of its unidirectional organization? These questions have formed the basis of many in vitro studies on development of the hippocampal formation, which have revealed that it is mainly the intrinsic properties of the hippocampus

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Fig. 1. Formation of main hippocampal projections. Neurons in the entorhinal cortex project through the angular bundle towards the hippocampus and terminate in the outer molecular layer of the dentate gyms and stratum lacunosum moleculare of CAl-CA3. Here they finally contact the distal segments of granule and pyramidal cell dendrites. Mossy cells in the polymorphic layer of the dentate gyms mainly project to the inner molecular layer to contact the proximal parts of granule cell dendrites. The granule cells send out axons (mossy fibers) into the stratum lucidum of CA3 and contact the proximal parts of pyramidal cell dendrites. Pyramidal cells in CA3 project to CA1 @chaffer collaterals), both in the stratum radiatum and stratum oriens. Septal fibers initially project through the fimbria into the stratum oriens and later also terminate in several other layers in the dentate gyms and CA regions. Note that only some of the extrinsic and intrinsic connections have been drawn. For sake of clarity, not all final terminations have been indicated and commissural projections have been omitted. EC II + III, entorhinal cortex layer II and III; DG, dentate gyrus; ml, molecular layer of the dentate gyms; po, polymorphic layer of the dentate gyrus; gel, granule cell layer; sl, stratum lucidum; slm, stratum lacunosum mole&are; f, fimbria; sp, stratum pyramidale, sr, stratum radiatum; so, stratum oriens; Su, subiculum.

that determine the pattern of projection of its afferents. Recently it has become clear that both, transient pioneer cells and several axon guidance molecules putatively constitute these intrinsic properties. Role of pioneer cells in the lamination of entorhinal-hippocampal and commissural/associationalJiber projections Initially studies have focused on the temporal aspects of afferent termination, since it was believed that in general ‘early-arriving’ axons project to distal parts of dendrites and ‘late-arriving’ axons prefer to terminate on proximal portions (Bayer and Altman, 1987). However, from studies by Soriano and oth-

ers it could be concluded that the temporal aspects of axonal ingrowth are not the single determinants of lamination. It turned out that during late embryonic development Cajal Retzius cells occupy the hippocampal outer marginal zone (the presumptive stratum lacunosum moleculare), at the time that entorhinal axons enter this region and pyramidal cell dendrites are still short. Thereby they provide a transient target for entorhinal synapses that are later transferred to the distal portions of the pyramidal cell dendrites. The inner marginal zone (the presumptive stratum radiatum), however, is occupied by GABA-ergic pioneer neurons that provide synaptic substrates for the commissural/associational projection that stays confined to the proximal dendritic

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segments (Super and Soriano, 1994; Del Rio et al., 1997; Super et al., 1998). It would be interesting to know what are the molecular cues used by entorhinal and commissural/associational fibers to find and project to these temporary targets and what are the mechanisms involved in the transfer of the synapses to their final targets. Furthermore, what are the cues that serve the maintenance of the laminar organization of the molecular layer of the dentate gyms and hippocampus after these pioneer neurons have (partly) disappeared or when structural changes within the hippocampus are evoked (for review, see Deller and Frotscher, 1997)? It would be likely that in addition to these pioneer cells the spatial organization of molecular cues derived from the final target dendrites themselves and/or differences in sensitivity of afferents to molecular cues determine which afferents project to which region of the dendrites. Like in the spinal cord, for example, the laminated fashion of termination of hippocampal afferents could be regulated by secreted semaphorins that are present as gradients in forbidden territories or by membrane bound semaphorins that are present on inappropriate targets. In addition, a particular afferent could compete for synaptic space by expressing a chemorepellent semaphorin to which the competitor projection is sensitive. Especially the secreted semaphorins are potentially interesting in this respect since they would provide a means to influence the competitor afferent from a distance and thereby prevent its entrance in a territory that is already claimed. An important role of semaphorin mediated chemorepulsion in the development of the hippocampal formation has emerged from knockout studies and from organotypic co-culture studies and repulsion assays using different parts of the developing hippocampus (Chedotal et al., 1998; Steup et al., 1999, 2000; Chen et al., 2000; Giger et al., 2000; Cheng et al., 2001; Pozas et al., 2001). These studies have indicated that semaphorins are major contributors to the laminar and unidirectional projection of intrahippocampal connections and afferents. Suffice it to say that semaphorins are not the single molecular players and that various other repulsive, attractive and adhesive cues (e.g. netrins, ephrins and NCAM) have been shown to contribute to the targeting of these projections (Stein et al., 1999; Seki and

Rutishauser, 1998; Steup et al., 2000; for review, see Skutella and Nitsch, 2001). A putative role of semaphorins 3A and/or 3F in development and lamination of the entorhino-hippocampal projection Sema3A and Sema3F are expressed in the embryonic entorhinal and hippocampal formation (Giger et al., 1996; Chedotal et al., 1998). Sema3A seems to be expressed exclusively in the hippocampal and neocortical area at E15, while Sema3F expression is more widespread. Later on, at perinatal stages, Sema3A expression becomes restricted to layer II and III neurons of the entorhinal cortex and becomes moderately expressed in dentate granule cells (Giger et al., 1996; Chedotal et al., 1998). Repulsion assays have elucidated that Sema3A, but not Sema3F repels embryonic entorhinal cortex-derived axons. At perinatal stages, both Sema3A and 3F are able to repel entorhinal axons, although the time frame of Sema3F mediated repulsion is very small and only lasts until postnatal day 2 (Pozas et al., 2001). Based on these observations it was hypothesized that early and high expression of Sema3A in the embryonic (e.g. at E15) entorhinal cortex pushes developing axons out of the entorhinal cortex, thereby forcing them to grow into the hippocampal area, and that this process is being enforced and fine tuned by Sema3F, confining the entorhino-hippocampal projection to the stratum lacunosum moleculare (Pozas et al., 2001). This hypothesis also holds for involvement of Sema3A and Sema3F in regulating the projection of entorhinal axons into the molecular layer of the dentate gyrus: expression of Sema3A and Sema3F by granule cells at perinatal ages might prevent entorhinal axons from overshooting their presumptive targets, the distal parts of granule cell dendrites. It is interesting to note in this respect that, despite the fact that Sema3A knockout mice display a high degree of phenotypical compensations, they do exhibit ectopic projections of entorhinal axons in the stratum radiatum and dentate hilus, indicating that Sema3A plays a potent role in regulating entorhinohippocampal projections (Pozas et al., 2001). The plexin-A3 knockout mouse does not display faults in entorhino-hippocampal projections, and there have not been any reports of obvious defects in these

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projections in neuropilin-2 mutant mice. This indicates that probably Sema3F-neuropilin-2/plexin-A3 signaling is not the most important factor to guide the initial outgrowth of this hippocampal afferent (Chen et al., 2000; Giger et al., 2000; Cheng et al., 2001). The laminar organization of mossy fibers and commissural/associational projections could be regulated by Sema3A and/or Sema3F

Commissural/associational projections arrive somewhat later in their target area as compared to entorhinal axons. Since the entorhinal neurons that project to the hippocampus display high expression of Sema3A one could envision that local secretion of Sema3A in the stratum lacunosum moleculare and outer molecular layer of the dentate gyrus produces a gradient of a chemorepellent towards the pyramidal and granule cell layer, and thereby prevents the termination of commissural/associational fibers in the outer layers of the hippocampus and dentate gyms (Pozas et al., 2001). Likewise, the expression of Sema3F in the hippocampal plate could form a repulsive boundary locally in the stratum pyramidale. Chedotal et al. (1998) argued that secreted semaphorins do not necessarily diffuse over long distances since they exhibit a highly charged, and thus sticky carboxy terminus. Indeed, in a followup study Pozas et al. (2001) showed, using a stripe assay, that Sema3A and Sema3F can lay out spatial cues for hippocampal axons to grow in a tight bundle towards their targets. Thus, Sema3A from the stratum lacunosum moleculare and Sema3F from the stratum pyramidale could constitute a trap, in the form of repulsive stripes, for mossy fibers and commissural/associational fibers and thereby confine their projection to the stratum lucidum and stratum radiatum or stratum oriens, respectively. Baranes et al. (1996) have shown that, when cultured in vitro, the connections of mossy fibers to CA3 pyramidal cells are not restricted to the proximal parts of the dendrites, indicating that the correct localization of mossy fiber synapses is likely to be extrinsically regulated. Neuropilin-2 and plexin-A3 knockouts display typical mossy fiber termination defects that corroborate this idea (Chen et al., 2000; Giger et al., 2000; Cheng et al., 2001). In these mice,

the mossy fiber projection respects the boundary in between the stratum lucidum and stratum radiatum, however, they do not respect the boundary laid out at the stratum pyramidale. This results in aberrant projections of mossy fibers into the pyramidal cell layer and beyond, the infra-pyramidal blade of the dentate gyms and stratum oriens. This indicates that Sema3F-neuropilin-2/plexin-A3 signaling is a main determinant for confining mossy fibers to the stratum lucidum. Does Sema3C regulate development of the difSuse projection of the septohippocampal pathway?

In rats, Sema3C is highly expressed in the developing hippocampus, at Pl and P19 but not at El5 (Steup et al., 2000). The co-receptors for Sema3C, neuropilin-1 and -2 are both expressed in the septal area. Neuropilin-1 is expressed in the septum and mammillary body nucleus from El55 on (Kawakami et al., 1996) and neuropilin-2 is at least expressed in the septal region from El55 on (Chen et al., 1997). The septohippocampal projection in rat reaches the hippocampus as early as El9 (it is present in the fimbrial pole at E18; in mice the first fibers are detected in the hippocampus at E17) and the adult projection pattern is reached at PlO (Linke and Frotscher, 1993; Super and Soriano, 1994). The fact that Sema3C is able to repel medial septal axons and is expressed in the hippocampus at around the time that septohippocampal fibers grow into the hippocampus indicates that Sema3C could be a stop signal for septal fibers in vivo, and function to prevent septal fibers from entering the pyramidal cell layer, directing them to the stratum oriens (Steup et al., 2000). However, to date, there is no definitive proof for such a role of Sema3C in the development of the septohippocampal system. Conclusions on the biological function of Sema3C are especially difficult to draw since its interaction with neuropilins could be enormously complex. Sema3C can exert its effects either via heterodimers of neuropilin- 1/neuropilin-2 or to a lesser extent via homodimers of neuropilin-2 (Chen et al., 1998; Takahashi et al., 1998) and Sema3C can also function as an antagonist for neuropilin-1 homodimer receptors (for example, to inhibit the action of Sema3A on neuropilin-1 containing receptor com-

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plexes; Takahashi et al., 1998). In addition, Sema3C has been shown to exhibit attractive actions on developing cortical axons (Bagnard et al., 1998). Direct effects of Sema3C on septal fibers at postnatal stages have not yet been investigated, which leaves the possibility open that Sema3C starts to attract these afferents to individual parts of the hippocampus at later stages of development. Furthermore, septal fibers are insensitive to Sema3A, while they do express neuropilin-1 (Steup et al., 2000). This could indicate that additional mechanisms are modulating the responses of septal fibers to hippocampus-derived semaphorins. For example, plexin-Al and plexin-A3 are expressed in the embryonic septum, but plexinA2 is not (Murakami et al., 2001). Plexin-A3 knockouts do not display gross misrouting of septal fibers, which leaves only plexin-Al as a functional receptor for semaphorin-mediated guidance of septal fibers. Furthermore, expression of Sema3C by the septum itself, which, to our knowledge, has not been investigated as yet, and in the fimbria, which is one of the major routes that septal fibers follow towards the hippocampus could render particular fibers insensitive to Sema3A and thereby contribute to the more diffuse pattern of projections of septal fibers into the hippocampus compared to other afferents (Chedotal et al., 1998). For example, the (supra)mammillary nucleus expresses plexin-A l-A3 and neuropilin- 1, and thereby most likely equips these hypothalamic neurons with receptor complexes that render them sensitive to several semaphorins (Kawakami et al., 1996; Murakami et al., 2001). Interestingly, despite the fact that the supramammillary-hippocampal projection grossly follows the same trajectory as the septohippocampal pathway, its termination is much more confined to a narrow supra- and infragranular zone in the dentate gyrus and the hippocampal CA2 region as compared to the septohippocampal projection which is diffuse over the whole hippocampus (Haglund et al., 1984; Vertes, 1992; Amaral and Witter, 1995). This indicates that, if at all secreted semaphorins form a main directive cue for the targeting of septum- and hypothalamus-derived afferents, differences in lamination between the septohippocampal and supramammillary-hippocampal projections could be explained by (temporal) variations in the expression profile of semaphorin receptor complexes.

Class 3 semaphorins putatively regulate the one way direction of entorhino-hippocampal and intrahippocampal projections

As discussed earlier, entorhino-hippocampal cocultures and repulsion assays have indicated that Sema3A in the entire entorhinal cortex and Sema3F in the cortical plate (for a short time frame) could direct entorhinal axons out of the entorhinal cortex, into the hippocampus (Chedotal et al., 1998; Steup et al., 1999). Chedotal et al. (1998) have shown that the neocortex exerts only a mild repulsive activity to entorhinal explants, suggesting that in addition to perforant pathway formation, expression of Sema3A in the neocortex and prolonged expression of Sema3A in the outer region of the entorhinal cortex could contribute to sparse recurrent projections of subtypes of entorhinal fibers to other subcortical areas (Chedotal et al., 1998). The fact that entorhinal cortex efferents project to other cortical areas (e.g. the perirhinal cortex) from which it also receives inputs underscores the notion that the cortical projections of the entorhinal cortex are not included in the unidirectional path of the hippocampal formation (Annual and Witter, 1995). The projection of the entorhinal cortex to hippocampus, however, is unidirectional since the entorhinal cortex does not receive recurrent projections from the dentate or pyramidal cells directly. In co-cultures the entorhinal cortex repels dentate gyrus and CA-region axons, an effect that can be mimicked by COS cell aggregates that secrete Sema3A or Sema3F (Chedotal et al., 1998; Steup et al., 1999; Pozas et al., 2001). This suggests that, during development, Sema3A and/or Sema3F from the entorhinal cortex repels granule and pyramidal cell axons, directing their growth away from the cortex. Thereby, these fibers would be forced to project into the hippocampus proper and are prevented to form a recurrent projection from the dentate or CA3 region to the entorhinal cortex (Chedotal et al., 1998; for review, see Skutella and Nitsch, 2001). Also the connection of the dentate gyrus with CA3 in the hippocampus proper is a unidirectional projection. The dentate granule cell axons (mossy fibers) terminate on dendrites of CA3 pyramidal cells, but these pyramidal cells do not project back to granule cells. In turn CA3 fibers project to CAl, but do not receive projections back from CAl. As

25 mentioned above, CA3 and CA1 fibers are repelled by pieces of embryonic entorhinal cortex, an effect that could be blocked by neuropilin- 1 antibodies, but not by neuropilin-2 antibodies. This indicates that Sema3A from the entorhinal cortex could also be involved in directing the Schaffer collaterals away from the dentate towards CA1 and prohibits CA1 fibers to project back to CA3. Pozas et al. (2001) showed that at postnatal stages the inner layers of the entorhinal cortex start to attract CA1 axons, suggesting that this allows or even promotes the formation of CA1 projections towards the subiculum and entorhinal cortex. It is interesting to note that the dentate gyrus itself does not seem to have repulsive effects on CA3 or CA1 axons (Chedotal et al., 1998). Sema3A is expressed in low levels in the dentate gyrus at embryonic and perinatal ages and thus, in principle, the dentate would have to be repulsive for CA3 axons. The lack of an effect in co-culture assays could be explained by the fact that the expression levels might be too low or, like discussed earlier, display a too limited diffusion to account for a significant repulsive gradient in the dish. In vivo, the low levels are probably condensed in the granule cell layer, which could locally produce very pronounced effects. An alternative explanation might be that Sema3A is released specifically by the axons of entorhinal cortex and granule cells as soon as they have reached their targets. Since these axons would be chopped off by culturing, the explants could thereby lose their repulsive activity. In vivo, secretion by the axon would produce a local gradient of Sema3A in the molecular layer of the dentate gyrus (by entorhinal axons) and in the CA3 (by granule cell axons), in the vicinity of their neuronal targets and in turn push their axons away towards CA3 and CAl, respectively. It is not known what local cues in CA3, CA1 and subiculum could be responsible for the exact termination of mossy fibers in CA3 and of Schaffer collaterals in CAl. Differential expression of other cues, such as the transmembrane semaphorins 5A and 5B (5A expression is very pronounced in DG while 5B expression is most pronounced in CA3; Mark et al., 1997) could provide spatially specific stop signals in these regions. Gene expression profiling of different regions of the hippocampus would provide a powerful method to determine whether particular proteins are

operating specifically at boundary regions between hippocampal subregions (Zhao et al., 2001). Do semaphorins play a role in structural plasticity of adult hippocampal circuits? The first indication (and the only direct evidence to date) that semaphorins can act as morphogenic molecules for neuronal structures during adulthood came from a study by Tanelian et al. (1997). In this study, Sema3A was ectopically expressed in the rabbit cornea using gene gun delivery of Sema3A expression plasmids. Expression of Sema3A appeared to repel established sensory projections within the cornea and to inhibit reinnervation of the wounded cornea by regenerating trigeminal fibers. All the other indications that semaphorins could be involved in structural changes in the mature nervous system are based on the anatomical distribution of semaphorins and their receptors in highly plastic brain regions, such as the hippocampus and the olfactory system, and on spatiotemporal changes of semaphorin expression following seizure-induced sprouting, and following lesions leading to regenerative success or regenerative failure (for reviews, see Gavazzi, 2001; Pasterkamp and Verhaagen, 2001). Here, we will discuss the potential relevance of particular semaphorins for structural plasticity in the hippocampus, based on the anatomy of expression and the changes in expression during seizureassociated plasticity processes in the hippocampal formation. Semaphorins and their receptors are expressed in the adult hippocampus Studies on the anatomical distribution of the different members of the semaphorin gene family revealed that many semaphorins are expressed in the adult hippocampal formation in very specific patterns (Fig. 2). In Table 1, the expression patterns of all semaphorins, neuropilins and plexins that are known so far, in the adult hippocampal formation of rats or mice are summarized. Sema3A is persistently and prominently expressed in the adult entorhinal cortex (Giger et al., 1998a). Retrograde tracing experiments revealed that many of the Sema3A-expressing cells are stellate

26

Sema4B

Sema5A

Fig. 2. Semaphorin expression in the adult hippocampus. mRNA in situ hybridizations of several semaphorins (A-D) and neuropilins (E,F) in the adult hippocampal formation of a rat. Note the high expression of some membrane-attached semaphorins in the dentate gyrus and pyramidal cell layer (C,D). Neuropilin-2 is exquisitely present in cells in the polymorphic layer of the dentate gyms.

21 TABLE 1 Semaphorin expression in the late postnatal or adult murhre hippocampal formation Semaphorin subtype

Area of expression

Reference

Sema3A

high in EC; none to very low in grDG, none in poDG, pyCA; moderate in Su low throughout H (high in human H, areas not specified) b

Giger et al., 1998a

Sema3B Sema3C Sema3D Sema3E Sema3F Sema4A Sema4B

very low in EC; low in grDG and poDG, low to moderate in pyCA3-CA 1 not known low in grDG, moderate to high in poDG and pyCA3-CAl low to moderate in grDG and poDG; moderate in pyCA3-CAl

F.D.W., unpublished observation; Sekido et al., 1996 Mark et al., 1997; Steup et al., 2000; Fig. 2

a

Miyazaki et al., 1999 Sekido et al., 1996; Hirsch et al., 1999; R.J. Giger, personal communications

low in grDG and poDG, low to moderate in pyCA3CAl and Su none in EC; high in grDG; moderate to high in poDG, CA3-CAl; low in Su low in H, areas not specified b low throughout H not known detected in H, not specified in which areas b not known

Mark et al., 1997 Fig. 2

Mark et al., 1997; Fig. 2

Sema5B

none in EC; very high in grDG, low in poDG, high in pyCA3; low to moderate in CA1 and Su high in grDG, poDG and pyCA3; moderate in CA1 and Su

Sema6A

low thoughout H (detected in E18.5 H)

Sema6B Sema6C

very high thoughout H, including interneurons in CA3-CAl low in grDG and poDG, high in pyCAlXA3

F.D.W., unpublished observations; Zhou et al., 1997 F.D.W., unpublished observations Kikuchi et al., 1999

Sema7A

very high throughout H and EC, prominent in pyCA2

Xu et al., 1998; Fig. 2

Neuropilin- 1

moderate to high in EC; very high in grDG, poDG and pyCA3-CAl; moderate to high in Su none to low in EC; high in grDG; very high in poDG and pyCA3; low to moderate in pyCA1 and Su

Giger et al., 1998b; Fig. 2

Sema4C Sema4D Sema4E Sema4F Sema4G Sema5A

Neuropilin-2 Plexin-Al c Plexin-A2 c Plexin-A3 c Plexin-A4 ’ Other plexins ’

low to moderate in grDG and poDG, moderate to high in pyCA3-CAl and Su moderate to high in all aspects of H moderate to high in all aspects of H moderate in grDG; low to moderate in poDG, pyCA3-CAl; su low to moderate in all aspects of H

Wang et al., 1999 F.D.W., unpublished observations Encinas et al., 1999

Mark et al., 1997; F.D.W., unpublished data

Chen et al., 2000; Giger et al., 2000; Fig. 2 Cheng et al., 2001; Murakami et al., 2001

high in

Cheng et al., 2001; Murakami et al., 2001 Cheng et al., 2001; Murakami et al., 2001 Cheng et al., 2001 Cheng et al., 2001

EC, entorhinal cortex; H, hippocampus; grDG, granule cell layer in the dentate gyms; poDG, polymorphic cell layer in the dentate gyms; pyCA, pyramidal cell layer in the CA areas; Su, subiculum. aPartly based on data obtained from human brain tissue. b Based on Northern blots. ’ Data are based on expression in young (Pl-P3) mice.

cells in entorhinal cortex layer II that project to the molecular layer of the dentate gyrus. In order to find support for the idea that the stellate cells secrete

Sema3A into the dentate gyrus, we have raised an antibody directed against the carboxy terminus of Sema3A and have started Sema3A immunostainings

28

Fig. 3. Sema3A against Sema3A secrete Sema3A molecular layer;

antibody staining in the molecular layer of the dentate gyms. A crude rabbit serum containing antibodies di rected labels the outer molecular layer of the dentate gyms (indicated by two arrows). This suggests that entorhinal fibers into the molecular layer where it could form a gradient towards the granul e cell layer. gel, granule cell layer; iml, inner oml, outer molecular layer.

on adult hippocampus using crude preparations of antiSema3A sera from rabbits. This revealed a dotted pattern of Sema3A immunoreactivity in the outer layers of the DG, which indeed suggests that Sema3A is transported to the DG by entorhinal fibers (Fig. 3). Furthermore, Sema3A receptors (neuropilin-1 and the plexin-A subfamily) are expressed in adult hippocampal granule, hilar and pyramidal cells. Neuropilin- 1 in situ hybridization reveals very high expression in granule cells, hilus cells and pyramidal cells in CA3-CA1 (Fig. 2). Interestingly, we find massive binding of Sema3A-AP in the stratum lucidum, which probably reflects the projection of mossy fibers to the basal portion of the CA3 pyramidal cells (Fig. 4). Moderate alkaline phosphatase staining was detected in the hilus, which could also represent granule cell axon collaterals. Other hippocampal areas in which we detect very weak binding are the inner molecular layer of

the dentate gyrus, the stratum radiatum and stratum oriens. The expression of Sema3A receptors and the binding of Sema3A in adult hippocampal structures suggest that the potential for Sema3A-signaling continuous to be present in the hippocampal formation after the major connections have been formed (Giger et al., 1998a; Cheng et al., 2001; Murakami et al., 2001). Taken together, these findings suggest that entorhinal neurons may secrete Sema3A into the molecular layer of the dentate gyms and stratum lacunosum moleculare of the hippocampus where it forms a chemorepulsive gradient that gradually declines towards the granule cell layer, and putatively signals through neuropilin- 1 and plexin-A receptors, present on neurons in the dentate gyrus and hippocampus proper. Many neurons in the medial septum and vertical diagonal band express neuropilin-2, but do not express neuropilin-1 (Fig. 5). In contrast, the lateral

Fig. 4. Binding of Sema3A and SemaSF to their receptors in the adult hippocampus. Alkaline phosphatase @)-tagged Sema3A and Sema3F were used to perform an in situ labeling of hippocampal fibers that express their receptors. The staining was revealed by an alkaline phosphatase color reaction. Note the strong labeling of mossy fibers by AP-Sema3A (arrow in B) and of the inner molecular layer by AP-Sema3F (arrow in C).

septum expresses both, neuropilin- 1 and neuropilin2. Interestingly, the neurons from the medial septumvertical diagonal band maintain projections to the hippocampus, mainly in the stratum oriens, stratum radiatum, the polymorphic layer and molecular layer of the dentate gyms. The lack of neuropilin-1 expression in the septohippocampal projection suggests that these neurons are insensitive to Sema3A in the adult hippocampus, while expression of neuropilin-2 would render them sensitive to Sema3F. The exact pattern of Sema3F expression in the adult murine hippocampal formation has as yet not been described. However, we and other groups have found indications that low to moderate levels of Sema3F are expressed in the dentate gyrus and pyramidal cells of CA3-CA1 (R.J. Giger, personal communications; F.D.W. unpublished data; Hirsch et al., 1999). Neuropilin-2 is highly expressed in dentate granule cells, and very high in the hilus and pyrami-

da1 cells of CA3 (Fig. 2). Neuropilin-2 is exquisitely present in particular large cells in the hilus, which could represent mossy cells. Pyramidal cells in CA1 exhibit low to moderate expression of neuropilin2. The in situ hybridization pattern of neuropilin-2 mRNA is confirmed by in situ binding of Sema3F. AP-Sema3F binds mainly in the inner molecular layer of the dentate gyms and stratum lucidum, and moderately in the stratum radiatum and stratum oriens (Fig. 4) The binding of AP-Sema3F in the inner molecular layer of the dentate gyms probably reflects the very high expression of neuropilin-2 in mossy cells of the hilus, which precisely project to this layer. Sema3C is expressed in low to moderate levels in granule cells of the dentate gyms and pyramidal cells of CA3-CA1 (Fig. 2). It is hardly detected in dentate hilus cells.

Fig. 5. Neuropilin expression in the septum. mRNA in situ hybridization of neuropilin-1 and neuropilin-2 in the septal area of an adult rat (A,B). The boxes (a-d) represent the magnifications at the right, of the lateral septal nucleus intermediate part (LSI; a,c) and the vertical limb of the diagonal band (VDB; b,d). Note that neuropilin-2 is expressed in the medial septum (B,d), while neuropilin-1 is not (A,b). This indicates that the septohippocampal projection, which mainly originates from the medial part of the septum, does not contain neuropilin-l-expressing fibers during adulthood.

31 The potential role of secreted semaphorins

The observation that Sema3A is expressed in the adult entorhino-hippocampal projection prompts the question what would be the function of Sema3A in this established system? One could envision a dual function: (1) to serve maintenance of the one-way projection of mossy fibers; (2) to serve maintenance of the lamination of the molecular layer in the dentate gyrus and stratum lacunosum moleculare in the hippocampus. It is known for quite some time that granule cells in the hippocampal dentate gyrus are continuously generated throughout life (Altman and Das, 1965). More recently it has become clear that these newly formed neurons also develop axons and produce functional connections to hilar and pyramidal cells within the adult hippocampus (Stanfield and Trite, 1988; Markakis and Gage, 1999), suggesting that even in the adult hippocampus steering cues are needed to guide growing axons towards their target. In line with its presumptive role during development, Sema3A produced by entorhinal cortex afferents could function to direct newly generated granule cell axons away from the molecular layer, towards the hilar and CA3 region in the adult hippocampus. Secretion of Sema3A in the stratum lacunosum moleculare could prevent the entrance of mossy fibers in this region and thereby confine their termination to the proximal parts of the CA3 dendrites. In addition, Sema3A could, similar to its function in the developing cortex, attract dendrites of newly generated granule cells towards the outer molecular layer of the dentate gyms (Polleux et al., 2000).

In addition to promoting a laminar projection of mossy fibers in the stratum lucidum, Sema3A could also contribute to the persistent layer specificity of particular projections in the molecular layer of the dentate gyrus. For example, commissural/associational axons that project to the inner molecular layer are able to sprout and occupy vacant synaptic space in the molecular layer throughout life (Deller and Frotscher, 1997). However, despite the fact that unilateral lesions of the entorhinalhippocampal pathway induce growth of these axons towards the outer molecular layer, the majority does not overlap with the few remaining fibers from the

contralateral entorhinal cortex and thus respect the laminar boundary even during robust reactive sprouting (Deller and Frotscher, 1997). Thus, Sema3A, secreted by contralateral entorhinal fibers could repel many of the sprouts of commissural/associational fibers following unilateral lesions and thereby serve to maintain the lamination following induction of structural rearrangements. What could be the function of Sema3F and/or Sema3C signaling in the hippocampus? It is difficult to assess the possible function of Sema3F and Sema3C in the adult hippocampus on basis of expression profiles. Their expression is moderate in the hippocampus proper and neither is found to be prominent in structures that are directly associated with the hippocampus. However, since neuropilin- 1 and neuropilin-2, and to a lower extent the plexin-A subfamily, are highly expressed in adult hippocampal neurons (see Fig. 2), one could argue that their axons are very sensitive to secreted semaphorins and therefore low levels of Sema3F and 3C can still exert chemotropic actions on these neurons. In analogy with a role of Sema3F in layer-specific termination of mossy fibers during development, its role in the adult CA3 could be to guide newly formed axons into the stratum lucidum and prevent them from overshooting proximal pyramidal cell dendrites towards the stratum oriens. Indeed, as mentioned earlier, neuropilin-2 and plexin-A3 mutant mice display aberrant terminations of mossy fibers in the infrapyramidal blade and stratum oriens (Chen et al., 2000; Giger et al., 2000). In young postnatal mice AP-Sema3F strongly binds to the stratum lucidum, which supports the finding that Sema3F/neuropilin2 interactions regulate the targeting of mossy fibers (Giger et al., 2000). However, at later ages, the binding of AP-Sema3F appears to decrease, so it is highly speculative to reason that this function of Sema3F is extended into adulthood (R.J. Giger, personal communication). It is interesting to note here that prolonged expression of GAP-43, an intraneuronal axon growth promoting protein, in adult dentate granule cells induces sprouting of mossy fibers into the stratum oriens, indicating that this boundary can readily be overcome by mossy fibers when the balance between growth-promoting molecules and repulsive cues is disturbed (Aigner et al., 1995). This would argue

32

for the fact that low levels of Sema3F or Sema3C do not provide a strong repulsive signal in the stratum pyramidale during adulthood. Remarkably, the binding of AP-Sema3F to mossy fibers is not as strong as compared to the binding of AP-Sema3A to mossy fibers (see Fig. 4). This is in line with the fact that neuropilin-2 is not as highly expressed in granule cells as compared to neuropilin-1 and suggests that secreted semaphorin cues other than Sema3A might be overcome relatively easy. Noteworthy is the strong binding of AP-Sema3F to the inner molecular layer, which is likely to be the result of binding to neuropilin-2 present on mossy cell axons. This indicates that Sema3C potentially affects these neurons as well. Two hippocampus-associated regions in the adult brain that are moderately to highly expressing Sema3C are entorhinal cortex layer II and the locus coeruleus (Fig. 2; F.D.W., unpublished data). As reasoned for the role of Sema3A, Sema3C from the entorhino-hippocampal projection could also provide an anterograde repulsive gradient towards the inner molecular layer. The locus coeruleus mainly projects, among others, to the polymorphic layer of the dentate gyrus and thus could, together with the granule cells, deliver a retrograde gradient of Sema3C from the hilus towards the molecular layer (Haring and Davis, 1983). These two gradients together could form a cue to confine mossy cell axons to the inner molecular layer. Additionally, sparse cells in the medial septum-vertical diagonal band produce Sema3C and thus could deliver Sema3C to an infragranular band in the dentate gyms (F.D.W., unpublished data). However, neuropilin-2 mutants do not display changes in the projection of mossy cell axons to the inner molecular layer, so one could doubt whether Sema3F/Sema3C-neuropilin-2 interactions has any relationship with gross structural stability in the molecular layer of the dentate gyrus (Chen et al., 2000). What could be the role of transmembrane semaphorins? As presented in Fig. 2 and Table 1, many membraneassociated semaphorins are highly expressed in the hippocampal formation, but as yet there have been no studies performed that point to an obvious role of these molecules in either hippocampal development

or structural stability. Some biochemical aspects of particular transmembrane semaphorins and their receptors suggest that they are potentially interesting with respect to plasticity processes. Sema4C and Sema4F interact with PSD-95, a molecule that is important for synapse maturation (El-Husseini et al., 2000; Inagaki et al., 2001; Schultze et al., 2001). Sema6B binds to c-Src, which is implicated in NMDA receptor function (Eckhardt et al., 1997; Lu et al., 1999). Plexin-Bl, which is the receptor for Sema4D interacts with active Rat, a Rho GTPase that is related to both axon guidance and synaptic stability (Luo, 2000; Vikis et al., 2000; Driessens et al., 2001). Thus, the presence of particular membrane-attached semaphorins and their receptors in the hippocampus together with signaling molecules that regulate plasticity processes suggests that these semaphorins can contribute to the modulation hippocampal synaptic plasticity. Sema3A is downregulated during mossy fiber sprouting in a TLE model In order to get an idea about the importance of Sema3A expression in the entorhinal cortex for the maintenance of the unidirectional fashion of the mossy fiber projection in the adult hippocampus we started to analyze its expression profile during experimentally induced temporal lobe epilepsy (Holtmaat et al., 1998). Temporal lobe epilepsy is characterized by a robust sprouting of mossy fibers, resulting in the formation of recurrent mossy fiber projections in the molecular layer (Sutula et al., 1988; Buckmaster and Dudek, 1997). The generation of synaptic contacts between mossy fiber collaterals and granule cell dendrites could contribute to the formation of a recurrent excitatory circuit which potentially facilities the spontaneous recurrence of focal seizures and propagation of the epileptic state (Babb et al., 1992; for review, see McNamara, 1999). If Sema3A, secreted by axons from the entorhinal cortex serves to prevent extending axon collaterals to enter the molecular layer, we hypothesized that its expression might be temporarily downregulated in this model of temporal lobe epilepsy. Indeed, shortly after induction of status epilepticus we observed an almost complete disappearance of Sema3A messenger RNA in the stellate cells of the entorhinal cortex (Fig. 6).

33

Fig. 6. Downregulation of Sema3A in the entorbinal cortex during status epilepticus. mRNA in situ hybridizations of Sema3A in the entorhinal cortex of a control rat (A) and an epileptic rat (B), 1 day after induction of status epilepticus. Note that the stellate cells in layer II of the entorhinal cortex (ECII) almost completely lose expression of Sema3A after induction of seizures.

The levels were lowest at 24 h after induction of the first seizures and returned back to normal during the day thereafter. The downregulation of Sema3A appeared to happen concomitantly with an upregulation of GAP-43 in the granule cells. The latter is known to stimulate mossy fiber sprouting and has been observed in many models for epilepsy (Aigner et al., 1995; for review, see McNamara, 1999). Thus, in this model for temporal lobe epilepsy, the chemorepellent Sema3A is likely to disappear in the molecular layer, in the same time frame during which granule cell axons are prompted to form sprouts, as induced e.g. by GAP-43. When the electrical stimulation of the angular bundle, used to induce seizures, did not result in a complete status epilepticus the downregulation of Sema3A did not occur, but the upregulation of GAP-43 did. Interestingly, incomplete induction of status epilepticus does not normally result in robust recurrent projections of mossy fibers, indicating that mossy fiber sprouts do not massively enter the molecular layer. This suggests that the downregulation of Sema3A in the molecular layer of the dentate gyms is a molecular event that is required to allow recurrent mossy fiber sprouting. Barnes et al. (1999) used kainic-acid injections to

induce status epilepticus and observed that the Sema3C content of CA1 was downregulated within the first week of epileptogenesis. This suggests that an inactivation of the Sema3C signaling plays a role in the formation of recurrent excitatory synapses in CAl, which does occur in the kainic acid model. It will be interesting to determine whether ectopic or overexpression of secreted semaphorins in rat or mouse status epilepticus models could block sprouting events in the hippocampal formation. This would provide a way to elucidate whether the formation of aberrant connections plays a pivotal role in the spontaneous recurrence of epileptic seizures in temporal lobe epilepsy. Conclusion Many members of the semaphorin family of axon guidance molecules are prominently and differentially expressed in the developing and adult hippocampus and/or in hippocampus-associated regions. It has become evident that the secreted members of the semaphorin family play an important role in guiding axonal projections towards, from and within the hippocampus, probably by acting at a dis-

34

tance during early development and locally during postnatal development. Thereby these molecules are likely to be important to determine the layer-specific and laminar organization of certain projections, and for the formation of unidirectional pathways within the hippocampus. Many of the semaphorins that are expressed during development continue to be expressed during adulthood, which suggests that they are also involved in morphogenic processes after establishment of hippocampal structures. It is as yet unclear how exactly we should interpret their presence in the adult hippocampus. The expression patterns of secreted semaphorins in relation to layer-specific projections and the downregulation of Sema3C and Sema3A during axonal sprouting in epilepsy models point towards a function of secreted semaphorins in maintaining the structural properties of hippocampal pathways. However, in order to formulate conclusive statements we will have to await results of targeted manipulation of semaphorins in vivo. Although null mutant mice have revealed the importance of Sema3A, neuropilin-2 and plexin-A3 in certain aspects of hippocampal pathway formation, one has not been able to ascertain their function in adult structural plasticity due to the fact that in these mice, defects already appear during development. Conditional mutant or transgenic mice will be necessary to address this question specifically in the adult hippocampus. Furthermore, sensitive readout methods, such as ‘single neuron labeling’ (Feng et al., 2000; Luo and Zong, 2001), ‘vital labeling of synapses’ (Gan et al., 1999; Matus, 2000) and ‘time lapse two-photon imaging’ (Denk and Svoboda, 1997) will be needed to monitor only subtle changes that are likely to occur after manipulations of these morphogenic molecules in established neuronal circuits such as the hippocampus. Acknowledgements We would like to thank Alex L. Kolodkin and Roman J. Giger for AP-Sema3A/3F expression vectors and neuropilin cDNAs, Andreas W. Piischel for semaphorin cDNAs that were used for in situ mapping of semaphorin mRNAs, and Chris W. Pool and Joke Wortel for help with Sema3A antibody production.

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