Synaptic responses in superficial layers of medial entorhinal cortex from rats with kainate-induced epilepsy

Abstract

Mesial temporal lobe epilepsy patients often display shrinkage of the entorhinal cortex, which has been attributed to neuronal loss in medial entorhinal cortex layer III (MEC-III). MEC-III neuronal loss is reproduced in chronic epileptic rats after kainate-induced (KA) status epilepticus. Here we examined, in vitro, functional changes in superficial entorhinal cortex layers. Alterations in superficial layer circuitry were suggested by showing that presubiculum, parasubiculum and deep MEC stimulation evoked 100–300 Hz field potential transients and prolonged EPSPs (superimposed on IPSPs) in superficial MEC which were partially blocked by APV (in contrast to control) and fully blocked by CNQX. Contrary to controls, bicuculline (5 and 30 μM) had minor effects on evoked field potentials in KA rats. GAD65/67 in situ hybridization revealed preserved interneurons in MEC-III. In conclusion, hyperexcitability in superficial MEC neurons is not due to loss of GABAergic interneurons and probably results from alterations in synaptic connectivity within superficial MEC.

Introduction

The entorhinal cortex (EC) provides a major afferent input to the hippocampus (Witter and Amaral, 2004) and has been suggested to be functionally involved in the development and possible maintenance of epileptiform activity in the temporal lobe. In patients with mesial temporal lobe epilepsy (MTLE), seizures often originate in the EC (Spencer and Spencer, 1994, Bartolomei et al., 2004, Bartolomei et al., 2005). Furthermore, several animal studies both in vivo and in vitro showed that the EC is possibly more seizure prone than the hippocampus (Walther et al., 1986, Stanton et al., 1987, Pare et al., 1992, Heinemann et al., 1993, Gloveli et al., 1998, Avoli et al., 2002) and hyperexcitable after induction of epilepsy (Bear et al., 1996, Fountain et al., 1998, Scharfman et al., 1998, Bragin et al., 2002, Kobayashi et al., 2003, Shah et al., 2004, Kumar and Buckmaster, 2006). In MTLE patients shrinkage of the EC is observed, mostly in combination with hippocampal damage (Salmenpera et al., 2000, Bernasconi et al., 2003a, Bernasconi et al., 2003b). The EC atrophy has been attributed to a clear pathology in the EC, with cell loss particularly noticeable in layer III of the medial EC (MEC-III; Du et al., 1993). Although the extent and localization of EC neuronal loss appear to vary among patients (Yilmazer-Hanke et al., 2000) and might not be a consistent neuropathological feature for all MTLE patients (Salmenpera et al., 2000, Dawodu and Thom, 2005), selective MEC-III cell loss has been reproduced in several rat models of MTLE after induction of a status epilepticus (SE; Du et al., 1995, Kobayashi et al., 2003, van Vliet et al., 2004, Kumar and Buckmaster, 2006). This makes these models extremely suitable to study the effect of selective EC atrophy on the excitability of the EC in chronic epilepsy. The neuronal loss in MEC-III from chronic epileptic rats seems to affect mainly principal neurons (Du et al., 1995, Eid et al., 1999, Kobayashi et al., 2003), although interneurons can be lost as well (Du et al., 1995, van Vliet et al., 2004, Kumar and Buckmaster, 2006). We observed in an in vivo study in chronic epileptic rats of the kainate model that presubiculum (prS) or subiculum stimulation revealed hyperexcitability of superficial EC layers (Tolner et al., 2005a, Tolner et al., 2005b). The prS provides a direct anatomical input to MEC-III in rats (Kohler, 1984, Kohler, 1985, Caballero-Bleda and Witter, 1993, Eid et al., 1996, van Haeften et al., 1997, Honda and Ishizuka, 2004) and, unlike MEC-III, does not show neurodegeneration after chronic epilepsy in either humans (Mathern et al., 1996) or rats (Pitkanen et al., 1995, Tolner et al., 2005b, van Vliet et al., 2004). Functional information on cellular and network alterations in superficial MEC after chronic epilepsy is scarce, and has so far come from in vitro studies in chronic epileptic rats of the self-sustained status epilepticus (SSLSE) model (Bear et al., 1996), pilocarpine model (Kobayashi et al., 2003, Kumar and Buckmaster, 2006) and a rat excitotoxicity model using the indirect excitotoxic substance amino-oxyacetic acid (AOAA; Scharfman et al., 1998). It is unclear whether after chronic epilepsy selective changes have occurred in the functional connectivity of superficial MEC with its main input areas prS, parasubiculum and deep MEC layers. Using in vitro entorhinal–hippocampal slices prepared from chronic epileptic rats of the kainate model, in the present study, we aim to (1) examine how does hyperexcitability in the MEC arise, and whether it is related to selective alterations in synaptic inputs from prS, parasubiculum or deep MEC; (2) get better insight regarding the presence and functionality of interneurons in MEC; (3) determine whether alterations in anatomical connectivity occur within MEC in relation to the selective neurodegeneration in MEC-III. We employed field and intracellular recordings to examine in detail the pathophysiology of the superficial layers of MEC. NeuN immunostaining and GAD65/67 in situ hybridization were used to discriminate between loss of principal neurons and interneurons in MEC. Anatomical tracings were used to examine connectivity within MEC.