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Differential display of hippocampal tissue after entorhinal cortex lesion (ECL) revealed decreases in mRNA encoding the neuronal hyperpolarization-activated, cyclic nucleotide-gated channel HCN1. In situ hybridization confirmed that hippocampal transcripts of HCN1, but not HCN2/3/4, are down-regulated after ECL. Expression recovered at approximately 21 days after lesion (dal). Immunohistochemistry demonstrated a corresponding regulation of HCN1 protein expression in CA1-CA3 dendrites, hilar mossy cells and interneurons, and granule cells. Patch-clamp recordings in the early phase after lesion from mossy cells and hilar interneurons revealed an increase in the fast time constant of current activation and a profound negative shift in voltage activation of Ih. Whereas current activation recovered at 30 dal, the voltage activation remained hyperpolarized in mossy cells and hilar interneurons. Granule cells, however, were devoid of any detectable somatic Ih currents. Hence, denervation of the hippocampus decreases HCN1 and concomitantly the Ih activity in hilar neurons, and the recovery of h-current activation kinetics occurs parallel to postlesion sprouting.

We used the fluorescent dye Fluoro-Jade, capable of selectively staining degenerating neurons and their processes, in order to analyze degenerative effects of transecting the hippocampus from its main input, the entorhinal cortex in vivo and in organotypical hippocampal slice culture. Degenerating fibers stained with Fluoro-Jade were present as early as 1 day postlesion in the outer molecular layer of the dentate gyrus and could be detected up to 30 days postlesion. However, the intensity of the Fluoro-Jade staining in the outer molecular layer faded from postlesional day 20 onward. Punctate staining, various cells and neural processes became visible in this area suggesting that degenerating processes were phagocytosed by microglial cells or astrocytes. We conclude that Fluoro-Jade is an early and sensitive marker for studying degenerating neurites in the hippocampal system.

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.

Myelin is crucial for the stabilization of the entorhinohippocampal projection during late development and is a non-permissive substrate for regrowing axons after lesion in the adult brain. We used two in vitro assays to analyse the impact of myelin on rat entorhinohippocampal projection neurons. A stripe assay was used to study the impact of myelin on the choice behaviour of axons from the entorhinal cortex (EC). Given a choice between alternating hippocampal membrane lanes from developmental stages ranging from early postnatal to adult, EC axons preferred to extend on early postnatal hippocampal membranes. Neither the neutralization of myelin-associated factors by a specific antibody (IN-1) nor the separation of myelin from membranes interfered with the axons' choice behaviour. The entorhinal axons showed no preference in the membrane combination of adult and myelin-free adult hippocampal membranes. These stripe assay experiments demonstrate that support for EC axon choice in the developing hippocampus is maturation-dependent and is not influenced by myelin. The application of IN-1 in the outgrowth assay and the separation of myelin from membranes, enhanced elongation of outgrowing entorhinal axons on adult hippocampal membranes, whereas a control antibody did not. This shows that myelin-associated factors have a strong inhibitory effect on the outgrowth length of entorhinal axons. In conclusion, we suggest that axonal elongation in the entorhinohippocampal system during development is strongly influenced by myelin-associated growth inhibition factors and that specific target finding of entorhinal axons is regulated by a different mechanism.