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.

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.

Selective hippocampal cell loss and altered neurotransmitter receptor expression have been proposed as pathogenic mechanisms in the development of chronic mesial temporal lobe epilepsy (TLE). Studies in animal models point to metabotropic glutamate receptors (mGluRs) as modulators of hippocampal epileptogenesis. In addition, mGluRs may constitute specific targets for the development of novel anticonvulsive drugs. As mGluR4 represents an inhibitory class III mGluR associated with the reduction of intracellular cyclic AMP levels and calcium influx, we have analyzed the regional and cellular expression of mGluR4 in surgical hippocampal specimens obtained from patients with TLE by using immunohistochemistry and in situ hybridization. Although the hippocampi of control specimens (n = 11) were almost devoid of mGluR4 immunolabeling, all TLE specimens (n = 35) showed a striking up-regulation of mGluR4 immunoreactivity, in particular within the dentate gyrus. Immunoelectron microscopy localized the receptor protein to the periphery of presynaptic and postsynaptic membranes. In situ hybridization revealed increased transcript levels of mGluR4 in dentate granule cells and residual CA4 neurons of TLE specimens compared with controls. Our results suggest a potential role of mGluR4 in counteracting excitatory hippocampal activity and in modulating seizure-associated vulnerability of hippocampal neurons. These data may also provide a basis for pharmacological studies of mGluR4 agonists as potential novel drugs in the treatment of TLE.

In the present study, we have investigated the developmental expression of the transmitter-synthesizing enzymes choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) in rat medial septal neurons by using in situ hybridization histochemistry. In addition, we have employed immunostaining for ChAT and the calcium-binding protein parvalbumin, known to be contained in septohippocampal GABAergic neurons. A large number of GAD67 mRNA-expressing neurons were already observed in the septal complex on embryonic day (E) 17, the earliest time point studied. During later developmental stages, there was mainly an increase in the intensity of labeling. Neurons expressing ChAT mRNA were first recognized at E 20, and their number slowly increased during postnatal development of the septal region. The adult pattern of ChAT mRNA-expressing neurons was observed around postnatal day (P) 16. By using a monoclonal ChAT antibody, the first immunoreactive cells were not seen before P 8. Similarly, the first weakly parvalbumin-immunoreactive neurons were seen in the septal complex by the end of the 1st postnatal week. These results indicate that in situ hybridization histochemistry may be an adequate method to monitor the different development of transmitter biosynthesis in cholinergic and GABAergic septal neurons. Moreover, the late onset of ChAT mRNA expression would be compatible with a role of target-derived factors for the differentiation of the cholinergic phenotype.