Mossy cells in epilepsy: rigor mortis or vigor mortis?

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1 140 Opinion Mossy cells in epilepsy: rigor mortis or vigor mortis? Anna d. H. Ratzliff, Vijayalakshmi Santhakumar, Allyson Howard and Ivan Soltesz Mossy cells are bi-directionally connected through a positive feedback loop to granule cells, the principal cells of the dentate gyrus. This recurrent circuit is strategically placed between the entorhinal cortex and the hippocampal region. In spite of their potentially pro-convulsive arrangement with granule cells, have not been seriously considered to promote seizures, because, allegedly one of the most vulnerable cell types in the entire mammalian brain, have long been known to die en masse in epilepsy. However, new data suggest that rumors of the rapid demise of the might have been greatly exaggerated. Anna d. H. Ratzliff Vijayalakshmi Santhakumar Allyson Howard Ivan Soltesz* Dept of Anatomy and Neurobiology, University of California, Irvine, CA , USA. * isoltesz@uci.edu A characteristic histological sign of temporal lobe epilepsy in hippocampal specimens from both humans [1 4] and experimental animals [5 14] is the loss of hilar cells in the dentate gyrus of the hippocampal formation. Hilar neurons have been reported to die after traumatic brain injury [5,6], ischemia [7,8] and seizures produced by electrical stimulation [9] or by injection of excitotoxins [10 14]. Among hilar cells, it is the glutamatergic excitatory [15] that are the most populous, constituting about half of all hilar cells, while the other half is composed of several groups of inhibitory, GABAergic interneurons [13,16]. Mossy cells, albeit subject to synaptic inhibition [17 20], are highly excitable [21]. Because of their numerical dominance and excitability, it has been generally assumed that bear the brunt of hilar cell loss that occurs after various insults. The perception that simply and conveniently die off in epilepsy has also been sustained because, until recently, there was no mossy cell-specific marker that could assess directly the actual degree of mossy cell loss in various models of epilepsy. The confluence of these factors has contributed to the general belief that because are lost, they did not have to be taken into account in the design of experiments and the development of models aimed at understanding the mechanisms that underlie limbic seizure generation. However, new results suggest that mossy cells are not destined to be drop-outs, and that their survival might be just as important as their loss in generating seizures [22]. Mossy cells: a case of neurons living dangerously? A caricature of is that they are so vulnerable they behave like fuses, and that their rapid death breaks the circuit after a dangerous surge in electrical activity in the network [21]. What are the mossy cell properties that could enable these cells to blow so easily and serve as self-sacrificing neuronal fuses? First and foremost, give and receive excitatory synapses to and from granule cells, an arrangement that provides a positive feedback circuit [23 26]. Granule cells, being the principal cells of the dentate gyrus, outnumber by about 30 to one [27]. The axons of project for millimeters even in the relatively small rat brain [18,28], connecting perhaps thousands of granule cells into functional groups along the longitudinal axis of the hippocampus [27] (Fig. 1a,b). Physiologically, show a characteristically high rate of spontaneous excitatory postsynaptic potentials (sepsps) [29,30] (Fig. 1c). In fact, the frequency of sepsps in in vitro is an order of magnitude higher than the rate of spontaneous inhibitory events [31], which is unique in cortical cells. In addition, the sepsps have been shown to undergo an unusual form of potentiation after a period of depolarization in [29]. mossy cell survival might be just as important as mossy cell loss in generating seizures. Several of these normal mossy cell properties could appear rather risky in terms of epilepsy: mossy cells receive massive glutamatergic inputs from mossy fibers (the axons of granule cells) (Fig. 1b), their EPSPs can self-potentiate after a short period of depolarization and cause more depolarization (Fig. 1c), and project back to the cells that provided them with powerful excitatory inputs in the first place (Fig. 1a,b). Thus, even though the anatomical and physiological properties of must serve some strong advantage in terms of normal hippocampal functions [18,21,32], it appears that the very same features (especially the strong glutamatergic inputs) might also predispose these cells to excitotoxic cell death [11]. But does apparent fragility mean certain untimely death for all mossy cells in epilepsy? Mossy cells lost and found Owing to the lack of mossy cell-specific neuronal markers, most of the data upon which the ideas of selective vulnerability and widespread loss of mossy cells rested are based on indirect evidence, such as equating hilar cell loss from Nissl stains with mossy cell loss or studying changes in the number of cells that lack expression for glutamate decarboxylase (GAD65/67), the synthesizing enzyme for the inhibitory neurotransmitter GABA. However, inferences based on such indirect approaches are fraught with serious pitfalls. Indeed, even before the /02/$ see front matter 2002 Elsevier Science Ltd. All rights reserved. PII: S (00)

2 { Opinion 141 (a) Interneuron subtypes { Mossy cells Mossy cell Granule cell CA1 H DG Trauma and seizures (b) Hilar cell loss (c) Loss of all Loss and survival of Survival of all Depolarizing current pulse Fig. 1. Recurrent excitatory feedback circuits involving in the dentate hilus. (a) A single mossy cell sends its axon for millimeters along the longitudinal axis of the hippocampal formation, synaptically coupling spatially distant granule cells. (b) The axons (the mossy fibers) from numerous granule cells form strong excitatory synaptic contacts on a single mossy cell (left), and a single mossy cell contacts many postsynaptic granule cells (right). In both cases, the excitatory synapses are close to the soma, indicating relatively small signal attenuation from the site of origin to the site of action potential initiation. (c) Electrophysiological recordings from reveal frequent spontaneous excitatory postsynaptic potentials (EPSPs) (top trace). After a period of depolarization, reportedly show a period of transient potentiation of both the amplitude and frequency of the spontaneous EPSPs (bottom trace). Such depolarization-induced potentiation of EPSPs, together with the strong excitatory input output functions of, suggest that mossy hilar cells are likely to participate, distribute and amplify seizure-like electrical discharges in the epileptic limbic system. Abbreviations: CA1,, Cornu Ammonis regions of the hippocampus; DG, dentate gyrus; H, hilus. development of mossy cell-specific markers, a decrease in the number of hilar cells labeled with interneuronal markers (e.g. GAD and somatostatin) was repeatedly noted in several models of hyperexcitability [33], indicating that are not the only hilar cells that die. In several experimental animal models of hyperexcitability, hilar cell loss is either nonsignificant (e.g. seizures in infant rats induced by hyperthermia [34] or hypoxia [35]) or smaller than 50% (e.g. lithium pilocarpine-induced seizures in developing rats [36], the kindling model of epilepsy [10] and mild head trauma [6]). As long as the hilar cell loss is less than 50%, some will definitely survive, simply based on the fact that constitute half of the hilar neuronal population. In addition, several good, if not perfect, markers for have been identified recently, including antibodies against GluR2 and/or GluR3 (in humans, Fig. 2. Mossy cell survival and hilar cell loss. The neurons of the hilus are represented by a circle, composed of 50% interneurons (of various subtypes, e.g. parvalbumin-containing basket cells, somatostatincontaining dendritically projecting hilar cells, etc.) and 50%. Evidently, as long as the cell loss is less than 50% (e.g. after experimental febrile seizures, kindling or mild head injury), some will survive. Furthermore, new evidence from both human and animal studies suggests that, contrary to previous assumptions, can frequently survive in the epileptic brain even when the degree of hilar cell loss is around 50% (as shown). Therefore, out of the three hypothetical outcomes of hilar cell loss [exclusive mossy cell loss (left), hilar cell loss involving both and interneurons (middle), and exclusive interneuron loss (right)], the most likely scenario is the one in the middle. Naturally, various insults can result in small differences in the numbers of and subpopulations of interneurons lost. However, the survival of any in the epileptic brain is likely to have pro-convulsive consequences. rats and monkeys [37,38]), CGRP (in rats [39]), calretinin (in mice and gerbils [7,40,41]), and mglur 7b (in humans [3]). These mossy cell-specific markers make it possible, for the first time, to assess mossy cell loss and survival directly. These investigations, together with studies that have relied on various intracellular tracers, have revealed that although many die after seizures, ischemia and trauma in animal models and in humans, many also survive. Even in cases such as moderate head trauma, where the total hilar cell loss can exceed 50%, numerous GluR2- and/or GluR3-immunoreactive cells (presumed ) could be observed weeks and months after the impact [6]. Calretinin-containing also have been found to survive in gerbils after excitotoxic insults [7]. Mossy cells can also survive in humans with epilepsy [3,4]. In individuals with temporal lobe epilepsy with focal lesions that do not involve the hippocampus proper, mossy cell density (determined by mglur 7b -immunoreactivity) reportedly decreases by only 30%, and can even be detected in some individuals with advanced Ammon s horn sclerosis [3]. Naturally, all immunocytochemical cell-type markers have their inherent limitations, and the

3 142 Opinion (a) Cell loss-induced (b) Dormant basket (c) sprouting hypothesis cell hypothesis Loss of Mossy fiber sprouting Loss of Hypoactive inhibition Irritable mossy cell hypothesis Survival of Increased excitability Epilepsy Epilepsy Epilepsy Fig. 3. Three hypotheses concerning the role of the death versus survival of in epilepsy. (a) In this scenario, the loss of is the major trigger for mossy fiber sprouting, a likely proconvulsive modification. (b) According to the dormant basket cell hypothesis, the loss of is a key event because it removes excitation from the surviving interneurons, resulting in hypoactive inhibition and hence seizures. (c) The irritable mossy cell hypothesis emphasizes the survival, and not only the loss, of in the generation of hyperexcitability in the dentate gyrus. The surviving are suggested to distribute and amplify hyperexcitability, as indicated by the stronger connections in the middle right panel. The actual source of hyperexcitability could be mossy fiber sprouting (probably triggered by the loss of both the interneurons and ), potentiation of synapses along the mossy fiber mossy cell granule cell circuits (perhaps involving repetitive selfsustaining depolarization-induced potentiation of excitatory postsynaptic potentials in the mossy cells themselves) or a combination of both modifications. Abbreviation:, Cornu Ammonis region of the hippocampus. mossy cell markers are no exceptions [42]. In addition, the expression of markers can be altered from normal patterns and levels in compromised neuronal circuits [38,43]. Therefore, it is a key finding that intracellular labeling techniques, which do not rely on endogenously expressed markers, have verified the presence of in the hyperexcitable dentate gyrus after head injury [22], as well as after pilocarpine-induced seizures [44]. Numerous mossy cells have also been found after ischemia using a neuronal tracer technique, even in rats that show an almost complete loss of CA1 pyramidal cells [8]. Therefore, irrespective of whether the actual rate of mossy cell loss is higher or lower than that of the interneurons, it is safe to conclude that although mossy cell loss is frequently a part of overall hilar cell loss, often remain in the hilus in epileptic, hyperexcitable and injured neuronal circuits of the dentate gyrus (Fig. 2). Further data obtained using the available mossy cell-specific markers will be necessary to determine the degree of mossy cell loss and survival in various epilepsy models. Can both loss and survival of be proconvulsive? As far as the long-term decrease in threshold for seizures is concerned, is it the lost or the surviving that are truly important? While the answer to this question remains to be determined, it is instructive to compare the predictions of three theories. The first of these is essentially the mossy cell loss causes mossy fiber sprouting hypothesis (Fig. 3a). As mossy fibers (the axons of granule cells) heavily innervate, it has generally been believed that the loss of is a key trigger for mossy fiber sprouting, which is a hallmark of epileptic hippocampi in humans and animal models [33,45]. Normally, mossy fibers do not project to other granule cells, but the sprouted mossy fibers make excitatory synapses between granule cells, essentially short-circuiting the dentate network [46,47]. However, the surprising truth is that there is no clearcut evidence that it is specifically the mossy cell loss, as opposed to hilar interneuronal loss, that triggers mossy fiber sprouting [48]. The alleged causal connection between mossy cell loss and mossy fiber sprouting becomes especially tenuous in light of recent neuroanatomical data showing that mossy fibers actually make more synapses with interneurons than with [49 51]. In addition, there are reports that argue that epilepsy might develop in the presence of protein synthesis inhibitors that block mossy fiber sprouting [52,53]. It is entirely possible that mossy cell loss and mossy cell survival with hyperexcitable response properties represent two sides of the same coin. The second idea, the dormant basket cell hypothesis, suggests that mossy cell loss is important for another reason (Fig. 3b). According to this hypothesis, mossy cell loss removes excitatory synapses from interneurons (e.g. basket cells that inhibit granule cells), making these interneurons hypoactive ( dormant ) [9] and thereby creating a seizure-prone dentate network (for discussions of the evidence for and against this view, see Refs [9,54 57]). The third scheme, called the irritable mossy cell hypothesis, emphasizes not the lost, but the surviving [22]. This third hypothesis proposes that some survive in most cases of dentate hyperexcitability, and that the surviving amplify the hyperexcitable (spontaneous or elicited) activity patterns of granule cells (Fig. 3c). The amplification of dentate hyperexcitability by mossy cells could take place in various ways. For example, could undergo persistent alterations in their intrinsic or synaptic properties that could enhance their input output relationship in response to excitatory inputs. Alternatively, could amplify dentate hyperexcitability simply through their so-called risky properties (already outlined).

4 Opinion 143 Acknowledgements This work was supported by the NIH (grant NS35915 to I.S.). Like the two previous hypotheses, the irritable mossy cell hypothesis, which arose from observations from a model of post-traumatic hyperexcitability, is proposed to be generally applicable to any model of epilepsy or hyperexcitability involving the limbic system. Indeed, electrophysiological recordings from morphologically identified have demonstrated that show significantly enhanced excitability both after head trauma [22] and after pilocarpine-induced seizures [44], indicating that participate in epileptiform discharges taking place in the hyperexcitable dentate gyrus. It is not known which, if any, of these three ideas will be found to represent the true fate and role of in epilepsy. It is entirely possible that mossy cell loss and mossy cell survival with hyperexcitable response properties represent two sides of the same coin. For example, mossy cell loss, together with hilar interneuronal loss, could contribute to mossy fiber sprouting, which could result either directly or indirectly in enhanced mossy cell discharges. The enhanced mossy cell firing could then activate more granule cells, contributing to excitotoxicity in (or other hilar cells) and completing a vicious circle. Although we do not yet have the answer, it is encouraging that experiments can be designed to test these hypotheses. For example, if either interneurons or could be labeled (e.g. with a cell type-specific promoter), these two groups of cells could be selectively lesioned in organotypic slice cultures to determine which one is the major trigger for mossy fiber sprouting. Similarly, cell type-specific deletion of might one day reveal whether mossy cell loss increases (as predicted by the dormant basket cell hypothesis) or decreases (as predicted by the irritable mossy cell hypothesis) granule cell excitability. Is the hilus half full or half empty in epilepsy? Seizures beget seizures [58]; mossy fiber activity triggers mossy cell loss that triggers mossy fiber sprouting [45]; interneurons hyper-synchronize principal cells [59]; interneuronal heterogeneity regulates excitability [60]; increased inhibition [61 69] becomes converted to hyperexcitability [70]: these are just some of the challenging ideas that have arisen from recent results in epilepsy research. These possibilities vividly illustrate the web-like complexity and the frequently unexpected, even paradoxical, nature of the interactions of cellular and synaptic events that eventually result in seizures. This complexity and bewildering inter-relatedness of the various factors is certainly daunting. However, the fact that these paradoxical interactions are now recognized and taken into account indicates that the Möbius strip of the dentate hilus is beginning to unravel. 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