Elsevier

Neurobiology of Disease

Volume 19, Issue 3, August 2005, Pages 451-460
Neurobiology of Disease

Entorhinal cortex entrains epileptiform activity in CA1 in pilocarpine-treated rats

https://doi.org/10.1016/j.nbd.2005.01.016Get rights and content

Abstract

Layer III neurons of the medial entorhinal cortex (mEC) project to CA1 via the temporoammonic pathway and exert a powerful feed-forward inhibition of CA1 pyramidal neurons. The present study evaluates the hypothesis that disrupted inhibition of CA1 pyramidal neurons causes an eased propagation of entorhinal seizures to the hippocampus via the temporoammonic pathway. Using a method to induce a confined epileptic focus in brain slices, we investigated the spread of epileptiform activity from the disinhibited mEC to CA1 in control and pilocarpine-treated rats that had displayed status epilepticus and spontaneous recurrent seizures. In pilocarpine-treated rats, the mEC showed a moderate layer III cell loss and an enhanced susceptibility to epileptiform discharges compared to control animals. Entorhinal discharges propagated to CA1 in pilocarpine-treated rats but not in controls. Disconnecting CA3 from CA1 did not affect the spread of epileptiform activity to CA1 excluding its propagation via the trisynaptic hippocampal loop. Mimicking the invasion of epileptiform discharges by repetitive stimulation of the temporoammonic pathway caused a facilitation of field potentials in CA1 that were contaminated by population spikes and afterdischarges in pilocarpine-treated but not control rats. Single cell recordings of CA1 pyramidal neurons revealed a dramatic loss of feed-forward inhibition and the occurrence of strong postsynaptic excitatory potentials in pilocarpine-treated rats. Excitatory responses in CA1 were characterized by multiple NMDA receptor-mediated afterdischarges and a strong paired-pulse facilitation in response to activation of the temporoammonic pathway. Our results suggest that, irrespective of the enhanced seizure-susceptibility of the mEC in epileptic rats, the loss of feed-forward inhibition and the enhanced NMDA receptor-mediated excitability CA1 pyramidal cells ease the spread of epileptiform activity from the mEC to CA1 via the temporoammonic pathway bypassing the classical trisynaptic hippocampal loop.

Introduction

The EC seems to be critically involved in temporal lobe epilepsy (TLE). In patients suffering from TLE and in several in vivo and in vitro models of TLE, the entorhinal cortex (EC) shows an enhanced susceptibility to seizures and epileptiform discharges, respectively (Collins et al., 1983, Dasheiff and McNamara, 1982, Rutecki et al., 1989, Spencer and Spencer, 1994). The increased excitability is associated with a pronounced neurodegeneration in the medial entorhinal cortex (mEC) layer III in patients and models of temporal lobe epilepsy. Previous studies showed that status epilepticus causes a preferential loss of glutamatergic neurons sparing GABAergic neurons in layer III of the mEC (Du et al., 1995, Eid et al., 1999, Kobayashi et al., 2003). Interestingly, in aminooxyacetic acid-treated animals, layer III cell loss correlates with hyperexcitability in this cell layer (Scharfman et al., 1998).

The EC mediates the majority of reciprocal connections between the hippocampus and the neocortex. The entorhinal input to the hippocampus originates in the superficial layers of the EC and consists of two branches. While cells in layer II of the EC send their axons predominantly to the dentate gyrus and CA3/CA2 of the hippocampus, the projection that originates from cells in layer III, the so called temporoammonic pathway, terminates exclusively in CA1 and the subiculum (Amaral and Witter, 1989, Witter et al., 1989). In addition, anatomical data demonstrated that deep layer (IV–VI) neurons of the EC also project to the dentate gyrus and the subiculum (Deller et al., 1996, Dugladze et al., 2001).

Numerous in vitro studies investigated the propagation of epileptiform activity from the EC to the dentate gyrus which has traditionally been considered as the main gate to the hippocampus (Barbarosie and Avoli, 1997, Barbarosie et al., 2000, Dreier and Heinemann, 1991, Jones and Lambert, 1990, Walther et al., 1986). While in control animals the dentate gyrus was shown to function as a filter preventing the spread of epileptiform activity from the EC to the hippocampus (Heinemann et al., 1992, Lothman et al., 1992), in chronic epileptic tissue, this gating mechanism breaks down facilitating the spread of epileptiform activity (Behr et al., 1996, Behr et al., 1998). The role of the temporoammonic pathway to CA1 has achieved less attention in previous studies on seizure propagation. The entorhinal input to CA1 is characterized by a strong feed-forward inhibition providing substrate for projected inhibition and a control of excitatory Schaffer collateral input originating in CA3 (Colbert and Levy, 1992, Empson and Heinemann, 1995). In kainic acid-treated rats, an enhancement of the temporoammonic input to CA1 wash shown in vivo (Wu and Leung, 2003). The authors inferred a loss of inhibition in CA1. Denslow and colleagues showed that selective loss of layer III neurons induced by focal injection of the excitotoxin aminooxyacetic acid also disrupts inhibitory function in CA1. Interestingly, this alteration was independent of the lesion in the EC but rather a consequence of the AOAA-induced seizure. This finding indicates a loss of inhibitory mechanisms within CA1 itself (Denslow et al., 2001). Consistently, Cossart et al. demonstrated a selective loss of interneurons in stratum oriens of CA1 in pilocarpine-treated rats suggesting a shift in the synaptic input from inhibition in controls to excitation in chronic epileptic animals (Cossart et al., 2001).

In the present study, we evaluate the hypothesis that a disrupted inhibition of CA1 pyramidal cells causes an eased propagation of entorhinal seizures to the hippocampal area CA1 via the temporoammonic pathway. Using a local application system to induce focal epileptiform activity, we demonstrate an enhanced propensity of the mEC to epileptiform activity in pilocarpine-treated rats. This activity propagates to CA1 via the temporoammonic pathway bypassing the classical trisynaptic hippocampal loop. Our data indicate that irrespective of the enhanced susceptibility of the mEC to epileptiform discharges, the disruption of feed-forward inhibition and the enhanced NMDA receptor-mediated excitability in CA1 ease the spread of epileptiform activity from the mEC to CA1 via the temporoammonic pathway.

Section snippets

Pilocarpine-treated animals

All experimental procedures concerning treatment and animal care were approved by the Regional Berlin Animal Ethics Committee. Young adult Wistar rats (150–180 g) were treated with pilocarpine according to established procedures (Gabriel et al., 1998). Thirty minutes after pretreatment with scopolamine hydrobromide (1 mg/kg s.c.), a generalized convulsive status epilepticus was induced by injection of pilocarpine hydrochloride (340 mg/kg i.p.). Duration of the status was limited to 2 h by

Nissl staining

Like in previous studies (Du et al., 1995, Kobayashi et al., 2003), there was a loss of layer III cells in pilocarpine-treated rats. Representative slices from control and epileptic rats are shown in Fig. 1. We determined the density of layer III neurons in 4 sections from 4 control and in 4 sections from 4 pilocarpine-treated rats. All of the rats that had experienced status epilepticus and spontaneous recurrent seizures demonstrated reduced density of Nissl-stained neuron profiles in layer

Discussion

Using a method to induce a confined epileptic focus in brain slices, we investigated the propagation of epileptiform activity from the disinhibited mEC to CA1 by way of the temporoammonic pathway. In pilocarpine-treated rats, the mEC revealed an enhanced susceptibility to epileptiform discharges compared to control animals. Entorhinal discharges showed a propagation to CA1 in epileptic rats but not in controls. This spread of epileptiform activity was unaffected by microsurgical cuts separating

Acknowledgments

We are grateful to A. Piechotta, S. Walden and Drs. H.-J. Gabriel and H. Siegmund for excellent technical help. This work was supported by a DFG grant to J. Behr and U. Heinemann (SFB-TR3).

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