Entorhinal Axons Research Articles

Developmental Hypothyroidism Influences the Development of the Entorhinal-Dentate Gyrus Pathway of Rat Offspring.

BackgroundDevelopmental hypothyroidism impairs learning and memory in offspring, which depend on extensive neuronal circuits in the entorhinal cortex, together with the hippocampus and neocortex. The entorhinal-dentate gyrus pathway is the main entrance of memory circuits. We investigated whether developmental hypothyroidism impaired the morphological development of the entorhinal-dentate gyrus pathway.MethodsWe examined the structure and function of the entorhinal-dentate gyrus pathway in response to developmental hypothyroidism induced using 2-mercapto-1-methylimidazole.Results1,1´-Dioctadecyl-3,3,3´,3´-tetramethylindocarbocyanine perchlorate tract tracing indicated that entorhinal axons showed delayed growth in reaching the outer molecular layer of the dentate gyrus at postnatal days 2 and 4 in hypothyroid conditions. The proportion of fibers in the outer molecular layer was significantly smaller in the hypothyroid group than in the euthyroid group at postnatal day 4. At postnatal day 10, the pathway showed a layer-specific distribution in the outer molecular layer, similar to the euthyroid group. However, the projected area of entorhinal axons was smaller in the hypothyroid group than in the euthyroid group. An electrophysiological examination showed that hypothyroidism impaired the long-term potentiation of the perforant and the cornu ammonis 3–cornu ammonis 1 pathways. Many repulsive axon guidance molecules were involved in the formation of the entorhinaldentate gyrus pathway. The hypothyroid group had higher levels of erythropoietin-producing hepatocyte ligand A3 and semaphorin 3A than the euthyroid group.ConclusionWe demonstrated that developmental hypothyroidism might influence the development of the entorhinal-dentate gyrus pathway, contributing to impaired long-term potentiation. These findings improve our understanding of neural mechanisms for memory function.

Crossed Entorhino-Dentate Projections Form and Terminate With Correct Layer-Specificity in Organotypic Slice Cultures of the Mouse Hippocampus.

The entorhino-dentate projection, i.e., the perforant pathway, terminates in a highly ordered and laminated fashion in the rodent dentate gyrus (DG): fibers arising from the medial entorhinal cortex (MEC) terminate in the middle molecular layer, whereas fibers arising from the lateral entorhinal cortex (LEC) terminate in the outer molecular layer of the DG. In rats and rabbits, a crossed entorhino-dentate projection exists, which originates from the entorhinal cortex (EC) and terminates in the contralateral DG. In contrast, in mice, such a crossed projection is reportedly absent. Using single and double mouse organotypic entorhino-hippocampal slice cultures, we studied the ipsi- and crossed entorhino-dentate projections. Viral tracing revealed that entorhino-dentate projections terminate with a high degree of lamina-specificity in single as well as in double cultures. Furthermore, in double cultures, entorhinal axons arising from one slice freely intermingled with entorhinal axons originating from the other slice. In single as well as in double cultures, entorhinal axons exhibited a correct topographical projection to the DG: medial entorhinal axons terminated in the middle and lateral entorhinal axons terminated in the outer molecular layer. Finally, entorhinal neurons were virally transduced with Channelrhodopsin2-YFP and stimulated with light, revealing functional connections between the EC and dentate granule cells. We conclude from our findings that entorhino-dentate projections form bilaterally in the mouse hippocampus in vitro and that the mouse DG provides a permissive environment for crossed entorhinal fibers.

Preferential Targeting of Lateral Entorhinal Inputs onto Newly Integrated Granule Cells.

Mature dentate granule cells in the hippocampus receive input from the entorhinal cortex via the perforant path in precisely arranged lamina, with medial entorhinal axons innervating the middle molecular layer and lateral entorhinal cortex axons innervating the outer molecular layer. Although vastly outnumbered by mature granule cells, adult-generated newborn granule cells play a unique role in hippocampal function, which has largely been attributed to their enhanced excitability and plasticity (Schmidt-Hieber et al., 2004; Ge et al., 2007). Inputs from the medial and lateral entorhinal cortex carry different informational content. Thus, the distribution of inputs onto newly integrated granule cells will affect their function in the circuit. Using retroviral labeling in combination with selective optogenetic activation of medial or lateral entorhinal inputs, we examined the functional innervation and synaptic maturation of newly generated dentate granule cells in the mouse hippocampus. Our results indicate that lateral entorhinal inputs provide the majority of functional innervation of newly integrated granule cells at 21 d postmitosis. Despite preferential functional targeting, the dendritic spine density of immature granule cells was similar in the outer and middle molecular layers, which we speculate could reflect an unequal distribution of shaft synapses. However, chronic blockade of neurotransmitter release of medial entorhinal axons with tetanus toxin disrupted normal synapse development of both medial and lateral entorhinal inputs. Our results support a role for preferential lateral perforant path input onto newly generated neurons in mediating pattern separation, but also indicate that medial perforant path input is necessary for normal synaptic development.SIGNIFICANCE STATEMENT The formation of episodic memories involves the integration of contextual and spatial information. Newly integrated neurons in the dentate gyrus of the hippocampus play a critical role in this process, despite constituting only a minor fraction of the total number of granule cells. Here we demonstrate that these neurons preferentially receive information thought to convey the context of an experience. Each newly integrated granule cell plays this unique role for ∼1 month before reaching maturity.

New functions of Semaphorin 3E and its receptor PlexinD1 during developing and adult hippocampal formation

The development and maturation of cortical circuits relies on the coordinated actions of long and short range axonal guidance cues. In this regard, the class 3 semaphorins and their receptors have been seen to be involved in the development and maturation of the hippocampal connections. However, although the role of most of their family members have been described, very few data about the participation of Semaphorin 3E (Sema3E) and its receptor PlexinD1 during the development and maturation of the entorhino-hippocampal (EH) connection are available. In the present study, we focused on determining their roles both during development and in adulthood. We determined a relevant role for Sema3E/PlexinD1 in the layer-specific development of the EH connection. Indeed, mice lacking Sema3E/PlexinD1 signalling showed aberrant layering of entorhinal axons in the hippocampus during embryonic and perinatal stages. In addition, absence of Sema3E/PlexinD1 signalling results in further changes in postnatal and adult hippocampal formation, such as numerous misrouted ectopic mossy fibers. More relevantly, we describe how subgranular cells express PlexinD1 and how the absence of Sema3E induces a dysregulation of the proliferation of dentate gyrus progenitors leading to the presence of ectopic cells in the molecular layer. Lastly, Sema3E mutant mice displayed increased network excitability both in the dentate gyrus and the hippocampus proper.

Viral interference with neuronal integrity: what can we learn from the Borna disease virus?

The neurotropic Borna disease virus (BDV) is unusual in that it can persistently infect neurons of the central nervous system (CNS) without causing general cell death, reflecting its favourable adaptation to the brain. The activity-dependent enhancement of neuronal network activity is however disturbed after BDV infection, possibly by its effect on the protein kinase C signalling pathway. The best model for studying BDV, which has a non-cytolytic replication strategy in primary neurons, is the rat. Infection of adult rats results in a fatal immune-mediated disease, whereas BDV establishes persistent infection of the brain in newborn rats resulting in progressive neuronal cell loss in defined regions of the CNS. Our recently developed system of BDV-infected hippocampal slice cultures has clearly shown that the onset of granule cell loss begins after the formation of the mossy fibre projection. Quantitative analysis has revealed a significant increase in synaptic density on identified remaining granule cell dendrites at 6weeks after infection, followed by a decline. Granule cells are the major target of entorhinal afferents. However, despite an almost complete loss of dentate granule cells during BDV infection, entorhinal axons persist in their correct layer, both in vivo and in slice cultures, possibly exploiting rewiring capabilities and thereby allowing new synapse formation with available targets. These morphological observations, together with electrophysiological and biochemical data, indicate that BDV is a suitable model virus for studying virus-induced morphological and functional changes of neurons and connectivity patterns.

Borna disease virus infection alters synaptic input of neurons in rat dentate gyrus

Granule cells are major targets of entorhinal afferents terminating in a laminar fashion in the outer molecular layer of the dentate gyrus. Since Borna disease virus (BDV) infection of newborn rats causes a progressive loss of granule cells in the dentate gyrus, entorhinal fibres become disjoined from their main targets. We have investigated the extent to which entorhinal axons react to this loss of granule cells. Unexpectedly, anterograde DiI tracing has shown a prominent layered termination of the entorhinal projection, despite an almost complete loss of granule cells at 9 weeks after infection. Combined light- and electron-microscopic analysis of dendrites at the outer molecular layer of the dentate gyrus at 6 and 9 weeks post-infection has revealed a transient increase in the synaptic density of calbindin-positive granule cells and parvalbuminergic neurons after 6 weeks. In contrast, synaptic density reaches values similar to those of uninfected controls 9 weeks post-infection. These findings indicate that, after BDV infection, synaptic reorganization processes occur at peripheral dendrites of the remaining granule cells and parvalbuminergic neurons, including the unexpected persistence of entorhinal axons in the absence of their main targets.

Regulation of Axonal Elongation and Pathfinding from the Entorhinal Cortex to the Dentate Gyrus in the Hippocampus by the Chemokine Stromal Cell-Derived Factor 1α

During the early developmental stage, a neural circuit is established between the entorhinal cortex (EC) and the hippocampal dentate gyrus (DG) via the perforant pathway. However, the manner in which the perforant fibers are navigated has mostly remained a mystery. Here, we analyzed the functional role of a chemokine, namely, stromal cell-derived factor 1alpha (SDF-1alpha), in the navigation of the perforant fibers. SDF-1alpha was observed to promote neurite growth, which is dependent on mDia1, in cultured entorhinal cortical neurons obtained from rats at postnatal day 0. We then used entorhino-hippocampal cocultures comprising green fluorescence-labeled EC and DG slices to assess the projection of the perforant fibers from the EC. Although the specific laminar termination of the entorhinal axons was observed with this system, the number of appropriately terminating entorhinal axons decreased significantly when the SDF-1alpha signaling pathway was blocked by a neutralizing antibody against SDF-1alpha or by the specific SDF-1alpha receptor antagonist AMD3100 (1,1'-[1,4-phenylenebis(methylene)]bis-1,4,8,11-tetra-azacyclotetradecane octahydrochloride). Furthermore, inhibition of the SDF-1alpha signaling pathway resulted in a decrease in the immunoreactivity for PSD-95 (postsynaptic density protein-95) in the DG, possibly because of a reduction in the number of projecting perforant fibers. These results demonstrate that SDF-1alpha plays a critical role in promoting the growth of perforant fibers from the EC to the DG.

Modulation of synaptic transmission and plasticity by cell adhesion and repulsion molecules

Adhesive and repellent molecular cues guide migrating cells and growing neurites during development. They also contribute to synaptic function, learning and memory in adulthood. Here, we review the roles of cell adhesion molecules of the immunoglobulin superfamily (Ig-CAMs) and semaphorins (some of which also contain Ig-like domains) in regulation of synaptic transmission and plasticity. Interestingly, among the seven studied Ig-CAMs, the neuronal cell adhesion molecule proved to be important for all tested forms of hippocampal plasticity, while its associated unusual glycan polysialic acid is necessary and sufficient part for synaptic plasticity only at CA3-CA1 synapses. In contrast, Thy-1 and L1 specifically regulate long-term potentiation (LTP) at synapses formed by entorhinal axons in the dentate gyrus and cornu ammonis, respectively. Contactin-1 is important for long-term depression but not for LTP at CA3-CA1 synapses. Analysis of CHL1-deficient mice illustrates that at intermediate stages of development a deficit in a cell adhesion molecule is compensated but appears as impaired LTP during early and late postnatal development. The emerging mechanisms by which adhesive Ig-CAMs contribute to synaptic plasticity involve regulation of activities of NMDA receptors and L-type Ca2+ channels, signaling via mitogen-activated protein kinase p38, changes in GABAergic inhibition and motility of synaptic elements. Regarding repellent molecules, available data for semaphorins demonstrate their activity-dependent regulation in normal and pathological conditions, synaptic localization of their receptors and their potential to elevate or inhibit synaptic transmission either directly or indirectly.

Regeneration of lesioned entorhino‐hippocampal axons in vitro by combined degradation of inhibitory proteoglycans and blockade of Nogo‐66/NgR signaling

Damaged axons do not regenerate after axotomy in the adult mammalian central nervous system (CNS). This may be due to local inhibitory factors at the site of injury, such as overexpression of chondroitin sulfate (CS) proteoglycans (CSPG), and the presence of myelin-associated inhibitors (MAI). To overcome CSPG- or myelin-induced inhibition, strategies based on extrinsic and intrinsic treatments have been developed. For example, NEP1-40 is a synthetic peptide that promotes axonal regeneration by blocking Nogo-66/NgR interaction and chondroitinase ABC (ChABC), which degrades CS, thereby also promoting axon regrowth. Here, we examined whether the combination of these complementary strategies facilitates regeneration of the lesioned entorhino-hippocampal pathway (EHP) in slice cultures. In this model, overexpressed CSPG and MAI impaired axon regrowth, which mimics regeneration failure in vivo. Both CS cleavage with ChABC and NEP1-40 strongly facilitated the regrowth of entorhinal axons after axotomy, permitting the re-establishment of synaptic contacts with target cells. However, the combined treatment did not improve the regeneration induced by ChABC alone, and the delayed treatment of ChABC, but not NEP1-40, had a less pronounced effect on axonal regrowth compared with acute treatment. These results provide insight into the development of new assays and strategies to enhance axon regeneration in injured cortical connections.

The repulsive guidance molecule RGMa is involved in the formation of afferent connections in the dentate gyrus.

In the developing dentate gyrus, afferent fiber projections terminate in distinct laminas. This relies on an accurately regulated spatiotemporal network of guidance molecules. Here, we have analyzed the functional role of the glycosylphosphatidylinositol (GPI)-anchored repulsive guidance molecule RGMa. In situ hybridization in embryonic and postnatal brain showed expression of RGMa in the cornu ammonis and hilus of the hippocampus. In the dentate gyrus, RGM immunostaining was confined to the inner molecular layer, whereas the outer molecular layers targeted by entorhinal fibers remained free. To test the repulsive capacity of RGMa, different setups were used: the stripe and explant outgrowth assays with recombinant RGMa, and entorhino-hippocampal cocultures incubated either with a neutralizing RGMa antibody (Ab) or with the GPI anchor-digesting drug phosphatidylinositol-specific phospholipase C. Entorhinal axons were clearly repelled by RGMa in the stripe and outgrowth assays. After disrupting the RGMa function, the specific laminar termination pattern in entorhino-hippocampal cocultures was lost, and entorhinal axons entered inappropriate hippocampal areas. Our data indicate an important role of RGMa for the layer-specific termination of the perforant pathway as a repulsive signal that compels entorhinal fibers to stay in their correct target zone.

Myelination in the hippocampus during development and following lesion.

Myelin is crucial for the stabilization of axonal projections in the developing and adult mammalian brain. However, myelin components also act as a non-permissive and repellent substrate for outgrowing axons. Therefore, one major factor which accounts for the lack of axonal regeneration in the mature brain is myelin. Here we report on the appearance of mature, fully myelinated axons during hippocampal development and following entorhinal lesion with the myelin-specific marker Black Gold. Although entorhinal axons enter the hippocampal formation at embryonic day 17, light and ultrastructural analysis revealed that mature myelinated fibers in the hippocampus occur in the second postnatal week. During postnatal development, increasing numbers of myelinated fibers appear and the distribution of myelinated fibers at postnatal day 25 was similar to that found in the adult. After entorhinal cortex lesion, a specific anterograde denervation in the hippocampus takes place, accompanied by a long-lasting loss of myelin. Quantitative analysis of myelin and myelin breakdown products at different time points after lesion revealed a temporally close correlation to the degeneration and reorganization pha-ses in the hippocampus. In contrast, electroconvulsive seizures resulted in brief demyelination and a faster recovery time course. In conclusion, we could show that the appearance of mature axons in the hippocampus is temporally regulated during development. In the adult hippocampus, demyelination was found after anterograde degeneration and also following seizures, suggesting that independent types of insult lead to demyelination. Reappearing mature axons were found in the hippocampus following axonal sprouting. Therefore, our quantitative analysis of mature axons and myelination effectively reflects the readjusted axonal density and possible electrophysiological balance following lesion.

Involvement of Cajal-Retzius cells in robust and layer-specific regeneration of the entorhino-hippocampal pathways.

Severed adult CNS axons can extend over long distances when a permissive 'milieu', such as grafted Schwann cells or ensheathing cells, is provided. Moreover, functional blocking of endogenous inhibitory factors, such as Nogo-A or proteoglycans, enhances the regeneration of axotomized neurons. Here we examine whether guidance cues available during the development of axonal pathways could also potentiate the regeneration of lesioned adult circuits. The Cajal-Retzius cells in the hippocampus are transient pioneer neurons that guide entorhino-hippocampal afferents to their target layers. By using an in vitro model of axotomy of the entorhino-hippocampal pathway we show that Cajal-Retzius cells triggered the regeneration of the axotomized entorhino-hippocampal pathway. Furthermore, the regrowth induced by Cajal-Retzius cells was robust and its pattern was indistinguishable from that of the unlesioned entorhino-hippocampal pathway. Thus, regenerating axons regrew in a layer-specific fashion towards the appropriate target layers, making synaptic contacts with target pyramidal neurons. Interestingly, the ability of lesioned entorhinal axons to regrow was maintained for at least 9 days after axotomy. These results show that the growth-promoting cells controlling the development of neural circuits will be a relevant approach to promoting the regeneration of lesioned adult CNS pathways.

Entorhinal axons exhibit sprouting in CA1 subfield of the adult hippocampus in a rat model of temporal lobe epilepsy

Intracerebroventricular kainic acid administration in rat, a model of temporal lobe epilepsy, results in CA3 pyramidal neuron degeneration leading to deafferentation of CA1 pyramidal neurons. Denervation in CA1 shows a near-complete recovery of synaptic density over 2-3 months, but the source of axons participating in the reinnervation is not clear. This study investigated the contribution of the entorhinal cortex in this reinnervation by comparing the distribution of the entorhinal axons in the CA1 subfield between the intact hippocampus and the CA3-lesioned hippocampus at 3 months after administration of kainic acid. Entorhinal axons were visualized by anterograde tracing using injections of the biotinylated dextran amine into the entorhinal cortex. In the CA1 subfield of the intact hippocampus, entorhinal axons were conspicuous in the alveus and the stratum lacunosum moleculare. The distribution in the strata oriens, pyramidale, and radiatum was sparse and was characterized by isolated entorhinal fibers of the alvear pathway crossing these strata to the stratum lacunosum moleculare. However, after kainic acid-induced CA3 lesion, the density of entorhinal axons increased significantly in the CA1 stratum radiatum (375% of the intact hippocampus), as a large number of axons emanating from the entorhinal fiber plexus in the stratum lacunosum moleculare invaded the stratum radiatum. The stratum radiatum also exhibited wavy entorhinal axons filled with boutons and oriented parallel to the stratum pyramidale, suggesting collateral sprouting from entorhinal axons traversing the stratum radiatum. Thus, a significant aberrant sprouting of entorhinal axons occurs into the CA1 stratum radiatum after CA3 lesion. The sprouted fibers appear to come from both entorhinal fiber plexus in the stratum lacunosum moleculare (translaminar sprouting) and entorhinal axons traversing the stratum radiatum (intralaminar sprouting). However, the major contribution appears to be from the entorhinal plexus in the stratum lacunosum moleculare. This aberrant sprouting may lead to altered afferent excitatory connectivity in the CA1 subfield and contribute to the persistent CA1 hyperexcitability that occurs after the CA3 lesion.

Age-Dependent Effects of Secreted Semaphorins 3A, 3F, and 3E on Developing Hippocampal Axons: In Vitro Effects and Phenotype of Semaphorin 3A (−/−) Mice

We studied the role of Semaphorins in the formation of hippocampal connections at embryonic and early postnatal stages. We show that the embryonic entorhinal cortex has a repulsive effect on embryonic hippocampal axons that disappears gradually at postnatal stages. Such chemorepulsion is blocked by Neuropilin-1 and -2 blocking antibodies. However, at perinatal stages, the inner layers of the entorhinal cortex attract CA1 axons. At these stages, Sema3A and Sema3F bind commissural and entorhinal axons. Sema3A and Sema3F repel hippocampal axons at E14-P2, but not at E13. A similar spatiotemporal pattern of chemorepulsion is observed for Sema3A on entorhinal axons, in contrast to Sema3F, which repels these axons only at postnatal ages. Sema3E also repels hippocampal axons but exclusively at E14. We show that Sema3A and Sema3F can induce the collapse of hippocampal growth cones and that membrane-bound Sema3A and Sema3F can guide hippocampal axons in the stripe assay. In sema3A (−/−) mice, the entorhinohippocampal projection is largely normal although single axons innervate aberrantly the stratum radiatum and the hilus. Thus, the chemorepulsion evoked by Sema3A, Sema3E, and Sema3F is dynamically regulated in the developing hippocampal formation.

Regeneration of Entorhinal Fibers in Mouse Slice Cultures Is Age Dependent and Can Be Stimulated by NT-4, GDNF, and Modulators of G-Proteins and Protein Kinase C

Axonal regeneration after lesions is normally not possible in the mature central nervous system, but occurs in the embryonic and neonatal nervous system. Slice cultures offer a convenient experimental system to study the decline of axonal regeneration with increasing maturation of central nervous system tissue. We have used mouse entorhinohippocampal slice cultures to assess regeneration of entorhinal fibers after mechanical lesions in vitro. We found that entorhinal axons regenerate well in cultures derived from postnatal days 5–7 mouse pups when the lesion is made at the second and fourth days in vitro (DIV 2 and DIV 4). Only little regenerative outgrowth is seen after lesions made at DIV 6 and DIV 10. This indicates that a maturation of the cultures occurs within a short time period in vitro resulting in a loss of the regenerative potential. We have used this system to screen for neurotrophic factors and pharmacological compounds that may promote axonal regeneration. Treatments were added to the cultures 1 day before the lesion was made. We found that most added factors did not promote regeneration. Only treatment with the neurotrophic factors NT-4 and GDNF stimulated regeneration in cultures where normally little regeneration is found. A similar improvement of regeneration was found after treatment with pertussis toxin, an inhibitor of Gi-proteins, and with GF109203X, an inhibitor of protein kinase C. These substances may promote regeneration by interfering with intracellular signaling pathways activated by outgrowth inhibitors. Our findings indicate that the application of neurotrophic factors and the modulation of intracellular signal transduction pathways could be useful strategies to enhance axonal regeneration in a complex microenvironment.

Aberrant trajectory of entorhino-dentate axons in the mutant Shaking Rat Kawasaki: a Dil-labelling study.

The Shaking Rat Kawasaki (SRK) is a neurological mutant that exhibits abnormalities of cell migration and lamination, with many similarities to the mouse reeler mutant. We recently used lamina-specific antibody staining to show that despite severe aberrations in the laminar organization of the SRK dentate gyrus, the entorhinal terminal field in the outer dentate molecular layer appeared relatively normal (Woodhams & Terashima, 1999, J. Comp. Neurol. 409 p57). However, neurofilament immunostaining suggested that entorhino-dentate afferents take an abnormal trajectory in reaching their appropriate targets, the granule cells dendrites. In the present study, anterograde tracing with the carbocyanine dye 1, 1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) has been used to delineate directly the path that entorhinal axons take to the dentate gyrus, confirming that in SRK entorhinal axons do indeed reach their appropriate terminal fields in the molecular layer, with laminar segregation between projections from the lateral and medial entorhinal cortices. However, these fibres fail to cross the hippocampal fissure between the subiculum and the dentate gyrus, coursing instead parallel to it until they curve round the deepest point of the fissure in field CA3. Similar findings were seen in the murine reeler mutant. Insertion of DiI crystals into the entorhinal cortex of neonatal rats also retrogradely labelled the developmentally transient Cajal-Retzius cells at the hippocampal fissure; these survive for longer in SRK than in normal littermates. The presence of a marked astrogliosis at the SRK hippocampal fissure may play a part in determining the abnormal trajectory taken by entorhino-dentate afferents in this mutant.

Development of the entorhino-hippocampal projection: guidance by Cajal-Retzius cell axons.

The entorhinal cortex gives rise to a massive projection to the hippocampus and fascia dentata. In the rat, this projection forms early in development with first entorhinal axons reaching the hippocampus around embryonic day (E) 17. From the very beginning, the entorhinal axons recognize their appropriate termination zones in the hippocampus proper and fascia dentata, i.e., stratum lacunosum-moleculare and the outer molecular layer of the dentate. This is remarkable, because at the time of entorhinal fiber ingrowth, the definitive target cells of entorhinal axons, pyramidal cells and granule cells, are not yet fully developed, and the majority of their distal dendritic tips have not yet reached these layers. This raises the question as to the cellular and molecular signals guiding the entorhinal axons to and keeping them in their target layers. Here we hypothesize that early generated Cajal-Retzius (CR) cells located in stratum lacunosum-moleculare and the outer molecular layer of the dentate, and in particular their axons projecting to the entorhinal cortex, provide a template that is used by the entorhinal axons to find their target layers in the hippocampus.

Outgrowth-promoting molecules in the adult hippocampus after perforant path lesion.

Lesion-induced neuronal plasticity in the adult central nervous system of higher vertebrates appears to be controlled by region- and layer-specific molecules. In this study we demonstrate that membrane-bound hippocampal outgrowth-promoting molecules, as present during the development of the entorhino-hippocampal system and absent or masked in the adult hippocampus, appear 10 days after transection of the perforant pathway. We used an outgrowth preference assay to analyse the outgrowth preference of axons from postnatal entorhinal explants on alternating membrane lanes obtained from hippocampus deafferented from its entorhinal input taken 4, 10, 20, 30 and 80 days post-lesion and from adult control hippocampus. Neurites from the entorhinal cortex preferred to extend axons on hippocampal membranes disconnected from their entorhinal input for 10 days in comparison with membranes obtained from unlesioned adult animals. Membranes obtained from hippocampi disconnected from their entorhinal input for 10 days were equally as attractive for growing entorhinal cortex (EC) axons as membranes from early postnatal hippocampi. Further analysis of membrane properties in an outgrowth length assay showed that entorhinal axons extended significantly longer on stripes of lesioned hippocampal membranes in comparison with unlesioned hippocampal membranes. This effect was most prominent 10 days after lesion, a time point at which axonal sprouting and reactive synaptogenesis are at their peak. Phospholipase treatment of membranes obtained from unlesioned hippocampi of adult animals strongly promoted the outgrowth length of entorhinal axons on these membranes but did not affect their outgrowth preference for deafferented hippocampal membranes. Our results indicate that membrane-bound outgrowth-promoting molecules are reactivated in the adult hippocampus following transection of the perforant pathway, and that neonatal entorhinal axons are able to respond to these molecules. These findings support the hypothesis of a temporal accessibility of membrane-bound factors governing the layer-specific sprouting of remaining axons following perforant path lesion in vivo.

Hippocampal Cajal-Retzius cells project to the entorhinal cortex: retrograde tracing and intracellular labelling studies.

Cajal-Retzius (CR) cells are characteristic horizontally orientated, early-generated transient neurons in the marginal zones of the neocortex and hippocampus that synthesize the extracellular matrix protein reelin. They have been implicated in the pathfinding of entorhino-hippocampal axons, but their role in this process remained unclear. Here we have studied the axonal projection of hippocampal CR cells. Following injection of the carbocyanine dye DiI into the entorhinal cortex of aldehyde-fixed rat embryos and young postnatal rats, neurons in the outer molecular layer of the dentate gyrus and stratum lacunosum-moleculare of the hippocampus proper with morphological characteristics of CR cells were retrogradely labelled. In a time course analysis, the first retrogradely labelled CR cells were observed on embryonic day 17. This projection of hippocampal CR cells to the entorhinal cortex was confirmed by retrograde tracing with Fast Blue in new-born rats and by intracellular biocytin filling of CR cells in acute slices from young postnatal rat hippocampus/entorhinal cortex and in entorhino-hippocampal slice cocultures using infrared videomicroscopy in combination with the patch-clamp technique. In double-labelling experiments CR cells were identified by their immunocytochemical staining for reelin or calretinin, and their interaction with entorhino-hippocampal axons labelled by anterograde tracers was analysed. Future studies need to investigate whether this early transient projection of hippocampal CR cells to the entorhinal cortex is used as a template by the entorhinal axons growing to their target layers in the hippocampus.

A Role for the Eph Ligand Ephrin-A3 in Entorhino-Hippocampal Axon Targeting

Neurons of layers II and III of the entorhinal cortex constitute the major afferent connection of the hippocampus. The molecular mechanisms that target the entorhinal axons to specific layers in the hippocampus are not known. EphA5, a member of the Eph receptor family, which has been shown to play critical roles in axon guidance, is expressed in the entorhinal cortex, the origin of the perforant pathway. In addition, ligands that interact with EphA5 are expressed in distinct hippocampal regions during development of the entorhino-hippocampal projection. Of these ligands, ephrin-A3 mRNA is localized both in the granular cell layer of the dentate gyrus and in the pyramidal cell layer of the cornu ammonis, whereas ephrin-A5 mRNA is only expressed in the pyramidal cell layer of the cornu ammonis. In the dentate gyrus, the ligand protein is not present in the termination zone of the entorhinal efferents (the outer molecular layer of the dentate gyrus) but is concentrated in the inner molecular layer into which entorhinal efferents do not grow. We used outgrowth and stripe assays to test the effects of ephrin-A3 and ephrin-A5 on the outgrowth behavior of entorhinal axons. This functional analysis revealed that entorhinal neurites were repelled by ephrin-A3 but not by ephrin-A5. These observations suggest that ephrin-A3 plays an important role in the layer-specific termination of the perforant pathway and that this ligand may interact with the EphA5 receptor to restrict entorhinal axon terminals in the outer molecular layer of the dentate gyrus.