Memory impairment in temporal lobe epilepsy: the role of entorhinal lesions

Memory impairment in temporal lobe epilepsy: the role of entorhinal lesions

Epilepsy Research 50 (2002) 161– 177 www.elsevier.com/locate/epilepsyres Memory impairment in temporal lobe epilepsy: the role of entorhinal lesions ...

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Epilepsy Research 50 (2002) 161– 177 www.elsevier.com/locate/epilepsyres

Memory impairment in temporal lobe epilepsy: the role of entorhinal lesions Robert Schwarcz a,*, Menno P. Witter b a

Maryland Psychiatric Research Center, Uni6ersity of Maryland School of Medicine, P.O. Box 21247, Baltimore, MD 21228, USA b Research Institute Neurosciences, Department of Anatomy, Vrije Uni6ersiteit Medical Center, Amsterdam, The Netherlands

Abstract Temporal lobe epilepsy (TLE) patients are frequently afflicted with deficits in spatial and other forms of declarative memory. This impairment is likely associated with the medial temporal lobe, which suffers widespread damage in the disease. Physiological and lesion studies, as well as examinations of the complex connectivity of the medial temporal lobe in animals and humans, have identified the entorhinal cortex (EC) as a key structure in the function and dysfunction of this brain region. Lesions in EC layer III, which normally provides monosynaptic input to area CA1 of the hippocampus, frequently occur in TLE and may be causally related to the memory impairments seen in the disease. Lesions that are initially largely restricted to EC layer III can be produced in rats by focal intra-entorhinal injections of ‘indirect excitotoxins’ such as aminooxyacetic acid or g-acetylenic GABA. These animals eventually show more extensive neurodegeneration in temporal lobe structures and, after a latent period, exhibit spontaneously recurring seizure activity. These progressive features, which may mimic events that occur in TLE, provide new opportunities to explore the role of the EC in memory deficits associated with TLE. These animals will also be useful for evaluating new treatment strategies that focus on the prevention of pathological events in the EC. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Cognition; Excitotoxicity; Medial temporal lobe dysfunction; Parahippocampal region

1. Introduction Patients suffering from temporal lobe epilepsy (TLE) often experience cognitive problems, most frequently impaired memory function. This deficit, which has been the subject of a large number of clinical and pre-clinical studies (see * Corresponding author. Tel.: +1-410-402-7635; fax: + 1410-747-2434 E-mail address: [email protected] (R. Schwarcz).

Abrahams et al., 1999; Ploner et al., 2000; Guerreiro et al., 2001, for recent discussions), is not only of obvious medical relevance but may provide clues regarding the mechanisms that underlie the acquisition and retrieval of memory under physiological conditions. Thus, detailed knowledge of the amnesia observed in TLE, which in most patients manifests itself in a relatively mild form, can be used to dissect various components of the memory system. Although the influence of excessive neuronal excitation, i.e. seizure activity, on the formation and storage of

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memory constitutes a limiting factor of this approach, the evaluation of TLE patients offers a unique opportunity for research. Studies of memory impairment in TLE are distinguished by the ability to work with a relatively homogeneous human population, by considerable knowledge of the neural substrates that account for the impairment, and because it is possible to examine the effects of seizure reduction as a consequence of anticonvulsant medication or surgical intervention. Moreover, it is possible to use electrophysiological information, which is obtained during pre-surgical assessment, for the generation and testing of hypotheses related to cognitive processes. Analysis of the amnestic characteristics of TLE patients has, therefore, provided invaluable information for the field of cognitive neuroscience. Notably, all these studies are greatly aided by the availability of animal models, which duplicate most of the defining features of TLE, including memory deficits (Leite et al., 1990; Stafstrom et al., 1993; Letty et al., 1995; Nissinen et al., 2000; Yang et al., 2000). The nature of the memory impairment in TLE has been investigated in numerous studies. It is now generally agreed that patients present with deficits in declarative memory, i.e. in their ability to acquire facts and events related to their personal past, and show particularly pronounced deficiencies in the performance of spatial memory tasks. Since these memory processes require the integrity of the same medial temporal lobe structures that are preferentially damaged in TLE, several investigators have proposed a causal relationship between the selective brain lesions seen in the disease and the observed cognitive abnormalities (Abrahams et al., 1999; Ploner et al., 2000; Guerreiro et al., 2001). Recent evidence from studies in humans and experimental animals suggests that the entorhinal cortex (EC), the gatekeeper region controlling the bi-directional information flow to and from the hippocampus, plays a particularly important role in the declarative memory deficits of TLE patients. These results imply that the EC could be targeted in attempts to treat or prevent the cognitive impairments that accompany the disease.

The following review synthesizes currently available data favoring the concept that (damage to) the EC is critically involved in the pathophysiology of TLE. Using examples from neuroanatomical, neuropathological and behavioral investigations, we will place special emphasis on the function of the EC as an integral relay station in the temporal lobe and explain how entorhinal dysfunction may account for memory deficits in TLE.

2. Neuronal degeneration in TLE: the hippocampus and beyond The involvement of the hippocampus and other temporal lobe structures in epilepsy was first recognized in the 19th century through visual inspection and microscopic analysis of brains post-mortem. Notably, these early studies, in particular the landmark publication of Sommer in 1880, not only called attention to the preferential degeneration of a band of pyramidal cells in the hippocampus–now known as the CA1 or Sommer sector— but also remarked on the probable pathophysiological significance of extrahippocampal damage (Sommer, 1880). The existence of uncal (i.e. parahippocampal) lesions in ‘psychomotor epilepsy’, an old term that alluded to the cognitive disturbances seen in TLE, was repeatedly confirmed in the first half of the 20th century, and its importance was emphasized by the neurosurgeons who dominated epilepsy research during that era (Zimmerman, 1938; Gastaut, 1956). However, though the involvement of both hippocampal and extrahippocampal structures was appreciated by all leading researchers in the field, emphasis soon began to shift to the hippocampus at the expense of extrahippocampal structures. This was primarily due to the fact that the surgical removal of the hippocampus, which shows characteristic neuropathological features (‘Ammon’s horn sclerosis’) in approximately 70% of TLE patients (Babb and Brown, 1987), rendered a majority of medication–refractory TLE patients seizure-free. The centrality of the hippocampus in TLE was supported by the advent of credible hypotheses, which suggested that

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intrahippocampal events are solely responsible for the occurrence of spontaneously recurring seizures, the defining clinical hallmark of the disease. Indeed, increasingly sophisticated neurophysiological, neuropathological and imaging studies revealed cellular and molecular abnormalities in the epileptic hippocampus, which were compatible with the facilitation of focally generated, repetitive ictal events (Sloviter, 1987, 1994; Buckmaster and Dudek, 1997). Behavioral studies in experimental animals, demonstrating deficits in spatial learning and memory following hippocampal lesions (Stubley-Weatherly et al., 1996), and the discovery of hippocampal place cells that code spatial information (Moser and Paulsen, 2001), further substantiated the concept that hippocampal damage is the key to understanding the cognitive deficits seen in TLE. Hippocampal atrophy in TLE, which is frequently unilateral, is most striking in the CA1 field of the hippocampus proper and in the bordering, proximal part of the subiculum. In contrast, dentate granule cells, while abnormally dispersed (Houser, 1990), are relatively spared by the disease process. Neuron loss is also evident, but not prominent, in the CA2 and CA3 sectors of the pyramidal band (Babb and Brown, 1987; Sloviter, 1994; Proper et al., 2000) and is frequently seen in the hilar region of the dentate gyrus (DG). In the hilus, excitatory mossy cells and inhibitory somatostatin-containing cells degenerate preferentially both in TLE and in a variety of relevant animal models, whereas the parvalbumin-positive GABAergic basket cells survive (Babb et al., 1989; Sloviter, 1987; but see Gorter et al., 2000). Another interesting and possibly pathophysiologically important feature seen in the hippocampus of TLE patients as well as in animal models is the strong and consistent sprouting of mossy fibers into the inner molecular layer of the DG (Tauck and Nadler, 1985; Sutula et al., 1989), where they establish synaptic contacts with granule cell dendrites (Zhang and Houser, 1999). Although details of the neuropathological characteristics of the epileptic hippocampus and their neurophysiological consequences are still somewhat controversial, there is agreement that the overall net effect onto granule cells is an increase in excitation.

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Renewed emphasis on the pathology and possible etiological role of extrahippocampal structures in TLE was prompted by advances in several disciplines and involved both human and animal studies (Schwarcz et al., 2002). Most importantly, the development of modern neuroanatomical techniques allowed new insights into the circuitry of the medial temporal lobe, revealing an intricate network of fibers connecting the hippocampus with other structures (see Fig. 1 and below for details). Electrophysiological studies in several instances demonstrated the excitatory, glutamatergic nature of these pathways, providing a construct for the existence of a hyperexcitable, reverberating ‘epileptic circuit’ (Lopes da Silva et al., 1990; Bertram, 1997). This concept made it less reasonable to view the hippocampus in isolation and sparked interest in the nature of the reciprocal interactions between elements of the extrahippocampal system in the course of TLE. Important evidence for the participation of extrahippocampal brain areas in the evolution of TLE was provided by using improved imaging methodologies, which permit the detection of both structural and functional abnormalities in the human brain with high anatomical resolution. For example, recent studies showed abnormalities of the amygdala and midline thalamic nuclei in line with an involvement in both epileptogenesis and chronic epilepsy (Pitka¨ nen et al., 1998; Juhasz et al., 1999), thus supporting the suggestions of the early pioneers in epilepsy research, who had made these conclusions without the benefit of modern technology. Notably, though these brain areas have so far not undergone careful neuropathological evaluation in TLE patients, they reliably present with distinct patterns of neuronal loss and metabolic impairment in various animal models of the disease, in agreement with the concept of an interdependent role of these extrahippocampal regions in the pathophysiology of TLE (cf. Bertram, 1997). Of all temporal lobe structures outside the hippocampus, the highly epileptogenic parahippocampal region, comprised of pre- and parasubiculum, EC and the perirhinal and parahippocampal cortices (Scharfman et al., 2000), appears to play a particularly important

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Fig. 1. (A) Horizontal section through the hippocampal formation and parahippocampal region of the rat, taken at mid-dorsoventral level and stained with cresyl violet. Indicated are the borders (black arrowheads) between the different subfields of the hippocampal formation (dentate gyrus: DG; note that the border between the hilus of DG and CA3 is indicated by the white dashed line; fields of the Ammon’s horn: CA3 and CA1; subiculum: Sub) and of the parahippocampal region (presubiculum: PrS; parasubiculum: PaS; medial and lateral entorhinal areas: MEA and LEA, respectively; perirhinal cortex: PER). The perforant pathway components originating from layers II or III, terminating in the molecular layer of DG and CA3 in stratum lacunosummoleculare of CA1 and Sub, respectively, are indicated with white solid arrows. Solid black arrows indicate the intrinsic hippocampal connectivity, whereas dashed black arrows indicate the cortical inputs from association cortex (Assoc.Ctx), reaching the superficial layers of EC through a synapse in the PER, the inputs from PrS, as well as the inputs originating in deep entorhinal layers V and VI. Grey arrows indicate the projections from CA3, CA1 and Sub by way of the fornix. (B) Schematic diagram summarizing the main features of the entorhinal – hippocampal network in relation to major cortical and subcortical connections (see text for further details).

role in the pathogenesis of TLE. Within this region, the EC, by providing monosynaptic input to both hippocampal dentate granule cells and CA1 pyramidal cells, normally controls the information flow to the hippocampus. Detailed electrophysiological and anatomical studies have recently elaborated the complex connectivities within the parahippocampal region and, furthermore, between the six layers of the EC (Ko¨ hler, 1986, 1988; Caballero-Bleda and Witter, 1993; Suzuki and Amaral, 1994a,b; Van Haeften et al., 1997; Burwell and Amaral, 1998a,b; Bilkey and Heinemann, 1999; Dhillon and Jones, 2000; Dickson et al., 2000; see Fig. 1 and below). Taken together, these data provide a reference system that can begin to explain the consequences of entorhinal damage on the structure’s gateway function. Not unexpectedly, abnormalities in the EC acutely trigger discrete changes in the hippocampus (Heinemann et al., 2000). More prolonged or permanent entorhinal malfunction or damage causes both structural and functional alterations in the hippocampus, which often mimic the effects of direct hippocampal injury. Thus, lasting impairment of either EC or hippocampus can induce hyperexcitability and cognitive deficits, making it difficult to delineate cause–effect relationships between the two brain areas (see below). Imaging and microscopic analyses in TLE revealed that marked changes take place in the anterior portion of the EC. In particular, there is pronounced neuronal loss in the outer layers of the structure, preferentially in layer III (Du et al., 1993; Bernasconi et al., 1999; Fig. 2). A qualitatively very similar, striking decrease in the number of neurons is observed in layer III of the medial EC, most extensively in its ventro–medial portion, in animal models of TLE (Du et al., 1995; Fig. 3). In all instances where this neuronal degeneration has been assessed in detail, principal pyramidal cells were found to be most susceptible to damage. In contrast, interneurons, at least certain specific populations, seem to be rather well spared. These neuropathological characteristics and the connectivity of these vulnerable EC neurons within the temporal lobe may provide important clues concerning the pathophysiology of TLE.

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parahippocampal region, especially to the EC (Amaral and Insausti, 1990; Witter et al., 2000b). Details of these and related connectivities are summarized schematically in Fig. 1B and are described in more detail below.

Fig. 2. EC pathology in TLE. (A) Coronal section through the anterior part of the EC of a control individual. Indicated are the two rostral entorhinal fields ECo (olfactory division) and ECr (rostral division; terminology according to Amaral and Insausti, 1990). (B) Coronal section comparable to that in A, taken from an EC specimen from a TLE patient. Note overall cortical shrinkage caused by neuronal degeneration in layer III. (C) High power magnification, indicated in A of the control EC (box). (D) High power magnification (from ECr box in B) of the EC in TLE. Scale bar in A equals 1 mm (also for B). Scale bar in C equals 200 mm (also for D). Reproduced from Du et al. (1993), with permission.

3. Circuit connectivity in the medial temporal lobe

3.1.1. General principles The anatomical organization of the neuronal circuitry relevant to TLE is illustrated in Fig. 1A. The first link of the circuit, the perforant pathway, arises in the EC and terminates in the DG, fields CA3 – CA1 and the subiculum. The second link originates in the granule cells of DG and terminates onto CA3 pyramidal cells. The third link is between CA3 pyramidal cells and neurons in area CA1. Finally, CA1 gives rise to a strong projection to the subiculum. At that point, both CA1 and the subiculum distribute hippocampallyprocessed information to a variety of cortical and subcortical structures, mainly by way of two different output routes: the fornix, which projects to ventral striatum, lateral septum, the hypothalamic region and the prefrontal cortex; and a non-fornical output, which is mainly directed to the

3.1.2. The perforant pathway The perforant pathway comprises different components, characterized by their origin in different laminae or separate subdivisions of the EC. Thus, neurons in layer II project primarily to DG and fields CA3/CA2, whereas the projections to CA1 and the subiculum originate almost exclusively in EC layer III (Steward and Scoville, 1976; Witter and Amaral, 1991; Witter et al., 2000b). The EC is divided into the lateral and medial entorhinal area (LEA and MEA), giving rise to the lateral and medial perforant pathways, respectively. These projections show different terminal distributions in the hippocampus, with cells in layer II of LEA projecting to the transverse extent of the outer one-third of the molecular layer/stratum lacunosum-moleculare of DG and CA3. In contrast, the projection originating in layer II of MEA terminates throughout the transverse extent of the middle one-third of these hippocampal fields. Unlike the lateral and medial components of EC layer II, which appear to influence the same cells in DG and CA3 (McNaughton and Barnes, 1977), layer III fibers of the lateral pathway target neurons in CA1 and subiculum that are different from those receiving input from the medial pathway. Thus, projections from layer III of LEA terminate specifically in the distal part of CA1 and the proximal part of the subiculum (i.e. around the border of CA1/subiculum). In contrast, fibers from MEA terminate in the proximal part of CA1 and the distal part of the subiculum. In the monkey, and most likely in the human, the radial differentiation between the lateral and medial components of the layer II projection is not as clearcut, whereas the organization of the layer III projection system is strikingly similar to that in the rat (Witter et al., 1989, 2000a; Witter and Amaral, 1991). Interestingly, the origin and terminal distributions of the projections from CA1 and the subicu-

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lum back to the EC are in register with the perforant pathway. The proximal part of the subiculum and the adjacent distal part of CA1 project preferentially to LEA, whereas the projections from the distal part of the subiculum and the proximal part of CA1 mainly reach MEA (Tamamaki and Nojyo, 1995). The entorhinal– hippocampal system, therefore, comprises functional re-entrant loops, which may serve oscillatory activity between the EC and the hippocampus (Iijima et al., 1996; Naber et al., 2000). This concept is further supported by the fact that cells in EC layer V give rise to rather restricted axonal cylinders targeting neurons in EC layers III and II (Lorente de No, 1933).

3.1.3. Entorhinal extra-hippocampal connections The most prominent input/output relations of the EC are with the adjacent perirhinal and postrhinal/parahippocampal cortices (Suzuki and Amaral, 1994a,b; Burwell and Amaral, 1998a,b). Additional, but weaker reciprocal connections exist with other higher order polymodal association cortices such as parts of the prefrontal cortex and anterior and posterior cingular cortex (Suzuki and Amaral, 1994a; Insausti et al., 1997; Burwell and Amaral, 1998b). Finally, rather substantial inputs have been described originating in the pre- and parasubiculum (Ko¨ hler, 1985; Van Haeften et al., 1997). In all species studied so far, inputs from the perirhinal and postrhinal/parahippocampal cortex, as well as projections arising in the pre- and parasubiculum, distribute predominantly to the superficial layers I– III of the EC. In addition to these cortical afferents, a major input from the amygdaloid complex, mainly originating in the basolateral nucleus, projects to EC (mainly to layer III; Pitka¨ nen et al., 2000). Finally, nuclei in the midline of the thalamus, in particular the nucleus reuniens, are the source of rather prominent fibers terminating in layers I, III and V of the EC (Wouterlood et al., 1990). In view of the organizational differences described above for the lateral and medial perforant pathway, it is of interest that the major inputs to the EC preferentially target either LEA or MEA. For example, perirhinal and amygdaloid afferents mainly project to parts of LEA, whereas fibers originating in the postrhinal cortex or the pre-

subiculum preferentially or exclusively terminate in MEA. The parasubiculum and the midline thalamus distribute their projections to both LEA and MEA.

4. Connectivity changes in TLE Specifics of the connectivity of layer III of the MEA are of particular interest in view of the striking preferential vulnerability of these neurons in TLE. Using anatomical tracing methods, layer III of MEA was found to receive a unique input, originating in the superficial layers of the presubiculum and targeting both principal and interneurons (Eid et al., 1996; Van Haeften et al., 1997). Ipsilateral and contralateral components of this bilateral presubicular projection are mirror images of each other both in terms of density and topographical distribution (Shipley, 1975; Ko¨ hler, 1985; Caballero-Bleda and Witter, 1993). Dorsal presubicular efferents project selectively to dorso– caudal parts of MEA, whereas ventral portions of the presubiculum target more ventral portions of MEA (Caballero-Bleda and Witter, 1993). Ultrastructural evidence confirmed that presubicular fibers target both principal neurons and interneurons (Van Haeften et al., 1997). The dorsally originating projection is partially GABAergic. These fibers project strictly ipsilaterally and preferentially terminate on interneurons in layer III, thus most likely leading to local entorhinal disinhibition when the presubiculum is active. In contrast, the ventral projection is not GABAergic. This difference possibly accounts for the preferential vulnerability of the ventral portion of MEA in TLE. In EC layer III, presubicular efferents also make synaptic contacts with apical dendrites originating from EC layer V pyramidal cells (Van Haeften et al., 2000). These presubicular fibers survive in epileptic rats (Tolner et al., 2001), supporting the idea that excitotoxic, ‘axon-sparing’ mechanisms are responsible for seizure-induced death of EC layer III neurons (Du et al., 1995). Following the degeneration of its postsynaptic elements, this presubicular axonal plexus conceivably undergoes reorganization, resulting in the innervation of new

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cellular targets or a strengthening of the innervation of parvalbumin-positive GABAergic neurons in layer III (Eid et al., 1999) and layer V cells. Such local changes in the EC may eventually result in a hyperexcitable network and contribute to the development of chronic epilepsy. Similarly, the dramatic neuronal death in area CA1 of the hippocampus in TLE will certainly lead to marked changes in the local circuitry of CA1 and will also affect both the monosynaptic afferents from area CA3 (and possibly surviving fibers originating in EC layer III) and the efferent connections to the subiculum and the EC. Jointly, these changes are likely to have pronounced, deleterious effects on hippocampal and parahippocampal function. Several scenarios can be predicted from knowledge of the normal connectivity of area CA1, and may have significance in the pathophysiology of TLE. For example, afferent connections from area CA3 and the EC will either deteriorate or reorganize such that new postsynaptic contacts will be established onto surviving neurons. Likewise, massive local reorganizations in synaptic connectivity are expected at the level of the subiculum, where CA1 neurons normally establish a dense innervation of interneurons and along dendrites and soma of principal cells. At the level of EC layer V, the most likely fiber system to participate in a presumed reorganization in TLE is the remaining subiculoentorhinal connection (cf. Fig. 1B). The specific functional roles of area CA1, either locally or through its intricate interconnections with other components of the parahippocampal– hippocampal network, have only recently begun to undergo experimental scrutiny. Thus, the relevance of the CA3-to-CA1 projection was studied by disconnecting CA1 from CA3 so that the majority of pyramidal cells in CA1 had excitatory connections only with the EC (Brun et al., 2002). The results of the electrophysiological and behavioral analyses indicated that the direct projection from EC and the local circuitry of CA1 are sufficient for converting spatially modulated signals from EC to accurate place representations in CA1. However, efficient storage of spatial memory requires the additional participation of CA3. Although this finding does not reveal the overall

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functional effects of major neuron death in area CA1, as seen in TLE, these data suggest that such pathology will lead to dramatic effects on learning and memory capacities of the system (see below). Comparable experiments are clearly needed to assess the effect of disconnecting CA1 from the subiculum and the EC. Complementary information will also come from new animal models of epilepsy that are caused by initial damage to layer III pyramidal cells in the EC (see below). These animals provide an opportunity to assess the excitatory CA3-to-CA1 connection in vivo following the functional isolation of CA1 pyramidal cells from their excitatory entorhinal input.

5. EC lesions and memory impairment Physiological studies in humans and non-human primates strongly indicate a role of the EC in memory function. Supportive data come from experiments determining neural activity (see Suzuki, 1999, for review), cerebral blood flow (Klingberg et al., 1994), and chemical activation patterns (Sybirska et al., 2000) in various learning paradigms. Thus, the EC constitutes a relay station, mediating the reciprocal interactions between perirhinal and parahippocampal cortices on one hand, and the hippocampus on the other. Together with other regions of the medial temporal lobe, the EC-and possibly certain ‘modules’ within the structure (Solodkin and Van Hoesen, 1996) - appears to be of particular importance for establishing long-term memory for facts and events (declarative memory) (Squire and ZolaMorgan, 1991). More specifically, the extrahippocampal structures most likely mediate some form of ‘semantic information’, whereas the hippocampus is responsible for the recollection of particular episodes. Parahippocampally generated associations between distinct stimuli can thus be used through interactive circuits of and with the hippocampus as a basis for conscious recollection (Eichenbaum, 2002). The role of the EC and associated brain areas in memory is frequently examined in humans and animals with medial temporal lobe damage. This

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includes Alzheimer’s disease patients, whose early cognitive impairments can be traced to lesions in the EC (Van Hoesen et al., 2000) and TLE patients, who present with lesions that preferentially affect layer III of the EC (see above). Experimental lesion studies in animals ranging from rodents to non-human primates have essentially confirmed the centrality of the medial temporal lobe, and especially the EC, in declarative memory functions. In common to most of these studies is the conclusion that qualitatively distinct impairments occur after focal damage to the hippocampus and parahippocampal structures, respectively. However, species differences in the anatomy of the medial temporal lobe, the use of diverse memory tasks, experimental designs and lesioning tools, and variability in the location and extent of the tissue destruction, have greatly complicated data interpretation and comparisons between laboratories (Jarrard, 2001). One of the major uncertainties, of relevance to the cognitive dysfunction seen in TLE, concerns the question whether processes involved in spatial learning and memory can be localized to the EC. A review of 25 articles in the primary literature, published since 1977, suggests a substantial correlation but does not resolve the issue unequivocally (Table 1). The majority of these studies were performed in rats, and many compared the effects of lesions placed within the EC with lesions placed in the EC and a surrounding area (such as the subiculum or the perirhinal cortex) or with focal hippocampal damage. Furthermore, efforts were made in some studies to distinguish between the effects of lesions in the medial and lateral EC. Conclusions were generally based on the animals’ performance in a complex radial maze, an elevated T-maze or a water maze, assessing either retention of pre-surgery training (retrograde memory) or acquisition following surgery (anterograde memory). These hippocampal and retrohippocampal lesions were placed either unilaterally or bilaterally and were originally often performed by a brief, focal application of electric current (electrolytical lesion), microscopically guided tissue resection, or physical separation of known anatomical pathways by knife cuts. Without exception, all these procedures, as well as the ‘axon-

sparing’ lesions caused by stereotaxic, focal microinjections of excitotoxins such as N-methylD-aspartate (NMDA), ibotenate or quinolinate, resulted in some impairment of spatial memory. However, this deficit did not appear to depend on entorhinal involvement in 20–25% of the studiesprobably a reflection of differences in experimental design and methodologies (Table 1). Notably, the importance of the role of the EC in spatial memory is further supported by the outcome of studies in which entorhinal afferents, for example from the presubiculum (Liu et al., 2001) or from the perirhinal cortex (Liu and Bilkey, 1998), are selectively removed. These experiments, though also dependent on lesion placement and the nature of the memory task (Glenn and Mumby, 1998), suggest that the EC may in fact serve a more critical function, especially for the acquisition of declarative memory, than assumed until recently. Thus, deafferentation appears to influence entorhinal activity and cognitive processes in a similar fashion as focal entorhinal neurodegeneration or tissue ablation. The involvement of retrohippocampal structures in spatial memory was recently reviewed by Aggleton et al. (2000), who suggested that information reaches the hippocampus through a number of routes, each of which may support different types of spatial processes. A recurring issue in the literature deals with the question whether the medial temporal lobe structures are dedicated to spatial tasks or whether they mediate memory processes in general (Eichenbaum et al., 1999). Indeed, there is ample evidence favoring a role of the EC in non-spatial, declarative memory. Test paradigms generally involve contextual learning and object–stimulus discrimination, and contextual fear conditioning has been used extensively to assess the role of the medial temporal lobe in configural or relational memory (cf. Maren et al., 1997; Bucci et al., 2000). The results of these studies are remarkably consistent, indicating a preferential role for the perirhinal cortex, and possibly the EC, but not necessarily the hippocampus itself. For example, hippocampal lesions in rats result in only mild impairments in delayed non-match to sample

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Table 1 Experimental EC/retrohippocampal lesions and spatial memory impairment Species/test

Location/nature of lesion

EC involvement

Reference

Rat/T/R Rat/M/R Rat/W/A Rat/M/A Rat/W,T/A Rat/W/A Mouse/M/A Rat/M/A Monkey/O/R Rat/W/A Rat/W/A Rat/M/R Rat/W/A Rat/M/A Rat/M/A Rat/T/A Rat/M/R Rat/W/A Monkey/O/A Rat/W/A Rat/W/A Rat/W/A Rat/W,T/A Dog/O/R Rat/W/A

EC/resection EC/electrolytic EC, preS, paraS, S/radiofrequency EC, EC+S, H/excitotoxic EC+PreS+ParaS+S+H/resection EC+S/excitotoxic EC/excitotoxic EC/excitotoxic H, H+EC/resection EC+PR/resection EC/electrolytic EC/electrolytic EC/electrolytic EC/excitotoxic EC+PR/resection EC, MS/resection EC, PreS, ParaS/electrolytic EC+H/knife cut PHG, H/resection, excitotoxic EC/excitotoxic EC/electrolytic, excitotoxic EC+S/excitotoxic EC, FF/resection, excitotoxic EC+PR, H, EC+PR+H/resection EC/resection

Yes No Yes No Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes No No Yes Yes Yes Yes Yes No/yes No Yes

Loesche and Steward, 1977 Ramirez and Stein, 1984 Schenk and Morris, 1985 Bouffard and Jarrard, 1988 Goodlett et al., 1989 Hagan et al., 1992 Cho and Jaffard, 1994 Holscher and Schmidt, 1994 Zola-Morgan et al., 1994 Nagahara et al., 1995 Glasier et al., 1995 Cho and Kesner, 1996 Fugger et al., 1997 Zajaczkowski and Danysz, 1997 Otto et al., 1997 Marighetto et al., 1998 Kesner and Giles, 1998 Kirkby and Higgins, 1998 Murray et al., 1998 Pouzet et al., 1999 Eijkenboom et al., 2000 Oswald and Good, 2000 Bannerman et al., 2001 Kowalska et al., 2001 Davis et al., 2001

Memory tasks: radial maze (M), elevated T-maze (T), water maze (W) or other (O). R: retrograde memory; A: anterograde memory. ‘Yes/No’ indicates the authors’ interpretation. Consult individual publications for additional details. FF, fornix/fimbria; H, hippocampus; MS, medial septum; ParaS, parasubiculum; PHG, parahippocampal gyrus; PR, perirhinal cortex; PreS, presubiculum.

paradigms (Mumby et al., 1992), whereas combined perirhinal/entorhinal lesions produce marked deficits (Otto and Eichenbaum, 1992; Mumby and Pinel, 1994 cf. Eichenbaum, 2002, for further details). The reported damage of thalamic midline nuclei, more specifically the nucleus reuniens, too, may contribute to cognitive dysfunctions in TLE. Thus, stimulation of the nucleus reuniens excites neurons in area CA1 of the hippocampus and in the subiculum, and in addition leads to feed-forward inhibition through local inhibitory neurons (Dolleman-van der Weel and Witter, 1995; Dolleman-van der Weel et al., 1997; Bertram and Zhang, 1999; Bertram et al., 2001). It can be assumed from the scheme illustrated in Fig. 1B that reuniens activity will affect EC activity in a similar fashion as area CA1, and lesions of this

thalamic structure indeed indicate a role in memory processes (Datiche et al., 1995; Dolleman-Van der Weel and Witter, 2002 for a recent review, see Van der Werf et al., 2002).

6. TLE-like EC lesions through focal injection of indirect excitotoxins Neither the en bloc resection of various retrohippocampal structures, knife cuts or focal, intraentorhinal injections of classic excitotoxic agents such as NMDA, ibotenate or quinolinate result in the preferential EC layer III damage seen in TLE patients. Animals lesioned by these procedures, which are bound to affect a multitude of complex anatomical circuits in the medial temporal lobe and beyond, are therefore inappropriate for

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studying the role of the EC in the memory deficits of TLE. The neurobiological features of TLE, including the topographic distribution of nerve cell loss, are far better duplicated in TLE models that are induced by the systemic administration of chemoconvulsants such as kainate (Schwob et al., 1980) or pilocarpine (Cavalheiro et al., 1991) or by prolonged electrical stimulation of various regions in the temporal lobe (Lothman et al., 1989; Nissinen et al., 2000). All these animals present with the degeneration of EC layer III that is characteristic for seizure-related injury, including the relative resistance of GABAergic, parvalbumin-positive interneurons (Du et al., 1995; Fig. 3C and D). Afferent fibers originating in the presubiculum are critical for the manifestation of entorhinal cell loss in these models since presubicular deafferentation is neuroprotective (Eid et al., 2001). Notably, lesioned animals display cognitive deficits (Leite et al., 1990; Stafstrom et al., 1993; Letty et al., 1995; Nissinen et al., 2000; Yang et al., 2000), but widespread neurodegeneration

Fig. 3. Low (A, C, E) and high power (B, D, F, from boxes in A, C and E, respectively) micrographs taken from horizontal sections through the hippocampal formation and parahippocampal region of a control rat (A, B), and of animals that received a systemic injection of kainate (C, D; 10 mg/kg, i.p.) or a local injection of GAG (E, F; 4 mg/1 ml) in the EC (cannula track in layer VI/white matter indicated by arrow) 4 days earlier. Note the striking absence of neurons in layer III of the medial EC in C –F. Scale bar in A equals 500 m, (also for C and E). Scale bar in B equals 200 mm (also for D). Scale bar in F equals 100 m. A –D reproduced with permission from Du et al. (1995). E and F are taken from Wu and Schwarcz (1998), with permission.

(Schwob et al., 1980; Cavalheiro et al., 1991; Nissinen et al., 2000) makes it impossible to isolate specific contributions of the EC damage. The discovery that TLE-like injury in the EC can be caused in rats by focal intra-entorhinal injections of either of two ‘indirect’ excitotoxins, aminooxyacetic acid (AOAA) or g-acetylenic GABA (GAG), provides a new opportunity to study the role of EC layer III lesions in memory dysfunction. Intra-entorhinal AOAA and GAG application results in acute seizure activity that is readily observed electrographically but only rarely manifests itself behaviorally. Notably, these compounds do not activate excitatory amino acid receptors directly but appear to increase neuronal vulnerability to normally innocuous concentrations of endogenous excitotoxins such as glutamate or quinolinate (Du and Schwarcz, 1992; Wu and Schwarcz, 1998). The precise mechanisms underlying this cell death are not fully understood but may involve abnormal cellular energy metabolism leading to a local increase of NMDA receptor function (Scharfman, 1996). Pyramidal neurons in layer III of the MEA are most vulnerable to the toxic effects of either AOAA or GAG, whereas the LEA and other layers in the EC are less susceptible (Du and Schwarcz, 1992; Du et al., 1998; Wu and Schwarcz, 1998; Fig. 3E and F). Interestingly, and accentuating the resemblance of these lesions with those observed in TLE and several animal models, GABAergic neurons frequently survive where pyramidal cells are greatly reduced in number (Eid et al., 1999). Most important for the present discussion, no substantial extra-entorhinal damage is seen during the first days after a unilateral or bilateral, focal application of AOAA or GAG (Du et al., 1998; Wu and Schwarcz, 1998).

7. Progressive neurodegeneration after EC layer III damage Animals with restricted, seizure-related lesions in EC layer III can be used to address several questions related to the pathophysiology of TLE. Of particular interest, AOAA- and GAG-lesioned rats have been assessed by electrophysiological and microscopic means to examine long-term con-

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sequences of limited entorhinal cell loss. So far, these studies have revealed chronic hyperexcitability of the deafferentated area CA1 in the hippocampus (Scharfman et al., 1998; Denslow et al., 2001), in line with an increased activity of postsynaptic elements following the degeneration of the excitatory projection from EC layer III (Witter et al., 1992; Desmond et al., 1994; Yeckel and Berger, 1995; Canning and Leung, 1997). They have also shown synaptic rearrangements involving surviving GABAergic interneurons in the lesioned EC layer III itself, leading to the suggestion that these newly wired cells might participate in a reverberating epileptic circuit that eventually results in spontaneously recurring seizure activity (Eid et al., 1999). In fact, the eventual appearance of such chronic seizures, which has so far been best documented in rats receiving bilateral entorhinal GAG injections (Wu et al., 2000), indicates that restricted entorhinal lesions caused by relatively minor seizure activity can indeed progress to create chronic epilepsy. Histological analyses demonstrate that critical events take place between 3 and 14 days following intra-entorhinal GAG injections. Thus, area CA1 of the hippocampus shows no apparent cell damage after 3 days, but is dramatically affected after 2 weeks when the pattern of cell loss resembles Ammon’s horn sclerosis (Fig. 4). Similarly, hilar cell loss is marginal at 3 days but pronounced after 2 weeks (Wu and Schwarcz, unpublished data). These data suggest a sequence of events originating in the EC (where the toxins are applied), leading to restricted neurodegeneration in EC layer III and, subsequently, hippocampal neurodegeneration. Notably, no spontaneous seizures are seen until this secondary loss of hippocampal neurons occurs (Wu et al., 2000). Eventually, however, synaptic rearrangements and neurochemical adaptations in various components of the temporal lobe result in chronic seizure activity.

8. EC lesions and memory deficits in TLE: conceptual and therapeutic implications Although the scenario described here may con-

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stitute merely one of several ways to generate an epileptic circuit in TLE, animals with AOAA- or GAG-induced EC lesions create several interesting, novel opportunities for epilepsy research. For example, it is now possible, using a variety of in vivo and ex vivo approaches, to study the molecular and structural changes that produce secondary neurodegeneration by 14 days following the initial insult. Dissection of the mechanisms underlying the progression from small, restricted EC lesions to more widespread neuronal damage, is of particular interest since the recruitment of extra-entorhinal structures does not seem to be mediated by intermittent seizure activity or profoundly abnormal electrophysiological changes (Wu et al., 2000). In addition, this model allows the assessment of the temporal sequence of events that result in spontaneously recurring seizures after damage is brought on in several medial temporal lobe areas. All these studies are of obvious rele-

Fig. 4. Low (A, B) and high (C – F) power micrographs of Nissl-stained coronal sections through the dorsal hippocampus of rats 3 days (A, C, E) or 14 days (B, D, F) after bilateral intra-entorhinal injections of 4 mg GAG. Boxes indicate location of the high power scenes depicted in C – F, respectively. Note that an extensive lesion in area CA1 and the hilus is only observed in the animal that survived 14 days after the GAG treatment. Scale bars, 500 mm (A, B); 100 mm (C – F).

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vance to the pathophysiology of TLE and will generate new hypotheses regarding the link between nerve cell loss and chronic epilepsy that can subsequently be tested in both animals and TLE patients. Cognitive processes have so far not been examined in animals with EC lesions caused by AOAA or GAG, but these studies can be expected to be quite informative. One of the particularly interesting questions, given the duplication of several cardinal features of TLE in the model (i.e. neuropathological characteristics, latent period prior to the emergence of chronic disease), is whether and when memory deficits occur in these animals. Currently available data, summarized above and in Table 1, suggest that abnormalities, maybe limited to declarative memory, will occur. The nature, selectivity, onset and duration of these anticipated impairments are difficult to predict, however, especially in view of the anatomical complexities of entorhinal connections (Fig. 1). In such studies, which should soon be extended to AOAA- or GAG-treated non-human primates to examine higher order cognitive functions, it will be particularly important to use a multidisciplinary approach to detect correlations and, ideally, establish cause– effect relationships. Such studies could include, for example, the performance of memory tests in conjunction with brain imaging or in vivo microdialysis. Thus, the emergence and characteristics of cognitive deficiencies could be studied longitudinally, allowing comparisons to the clinical situation. This approach will also make it feasible to evaluate an important but still controversial issue in TLE research, namely the effects of therapeutic surgical interventions on memory processes (Gleissner et al., 2002; Lee et al., 2002). Assuming that EC lesions play an integral and possibly primary role in the pathophysiology of TLE, animals receiving intra-entorhinal injections of AOAA or GAG also constitute useful models to test various drug intervention strategies. Taking advantage of the temporal cascade of pathological events that are initiated by seizure-induced focal EC layer III lesions, both anti-epileptogenic and anticonvulsant medications could be evaluated. These studies, like those designed to exam-

ine the cellular and molecular mechanisms underlying TLE, will need to use electrophysiological, microscopic and biochemical methods to monitor the effects of pharmacological treatment. Assessments of memory and other cognitive processes in these animals will permit investigators to elucidate several issues that are pertinent to the area of cognitive neuroscience and are particularly important for epilepsy research. Thus, timed interventions should allow differentiation between the cognitive consequences of focal entorhinal versus more extensive temporal lobe lesions, and of acute versus chronic seizure activity. It will also be possible to address the therapeutically relevant question whether entorhinal neuroprotection is desirable or even necessary to prevent or arrest memory impairment in TLE patients. As an offshoot, these studies may shed light on the nature and cause of cognitive disturbances that have occasionally been reported as a side effect of anti-epileptic medication (Seidel and Mitchell, 1999; Thompson et al., 2000; Martin et al., 2001).

9. Summary and conclusions Many TLE patients suffer from declarative memory impairments, but it is not clear whether these deficits are causally related to seizures or to the seizure-related neurodegeneration in the medial temporal lobe that frequently occurs in the disease. Data reviewed here suggest that the EC, the major relay center orchestrating the information flow to and from the hippocampus, plays an important role in this cognitive dysfunction. Thus, a large number of studies in animals and humans have identified the EC and associated structures of the parahippocampal region as critical for spatial and other forms of declarative memory. In addition to damage in several other parts of the medial temporal lobe, TLE patients often present with lesions in EC layer III, which normally provides a direct excitatory input to area CA1 of the hippocampus. The fact that remarkably similar EC lesions, as well as cognitive impairments, are seen in relevant animal models of chronic epilepsy, led to the idea that the degeneration of EC layer III may be an early event in the patho-

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physiology of TLE. A recently introduced TLE model, created by the intra-entorhinal injection of indirect excitotoxins such as AOAA or GAG in rats, has provided evidence in support of this concept and should prove valuable for the study of memory deficits in the disease. These animals will also allow investigators to explore new antiepileptogenic and anticonvulsant intervention strategies that focus on the prevention of pathological events in the EC.

Acknowledgements The work described here was in part supported by USPHS grant NS 16102 (to R. Schwarcz) and grants 903-47-008 and 903-47-051 from the Netherlands Organization for Scientific Research (NWO) and by a grant (QLG3-CT-1999-00192) from the fifth Framework RTD Programme of the European Commission (to M.P. Witter). We greatly appreciate the assistance of Dr Hui-Qiu Wu, who provided the unpublished material illustrated in Fig. 4.

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