Lisa R. Merlin

Synaptic modifications accompanying epileptogenesis in vitro: long-term depression of GABA-mediated inhibition

We used an in vitro model similar to kindling to examine the processes underlying epileptogenesis. A 60 Hz train was applied every 5-10 min to the Schaffer collateral pathways in guinea pig hippocampal slices until epileptiform bursting was elicited in the CA3 region. The resultant alterations in both spontaneous and evoked activities were studied using intracellular recordings from CA3 pyramidal cells. An attempt was made to elucidate the synaptic modifications responsible for the conversion to this state of enhanced excitability. Analyses revealed that the emergence of epileptiform discharge was accompanied by a long-term depression of evoked inhibitory conductances. This tetanus-induced reduction of inhibition involved both the early and late phases of the evoked hyperpolarization, suggesting modification of both the GABAA and GABAB receptor-mediated events. Previous studies have suggested that NMDA receptor activation plays an important role in the induction of epileptiform activity in this model. Our data, showing that depression of inhibition can be induced in the presence of CNQX, is consistent with this hypothesis. The parallel development of long-term depression of inhibition and epileptiform bursting following tetanic stimulation suggests that plasticity of the inhibitory transmission process is a potential source of vulnerability contributing to epileptogenesis.

A competency-based longitudinal core curriculum in medical neuroscience.

Current medical educational theory encourages the development of competency-based curricula. The Accreditation Council for Graduate Medical Educations 6 core competencies for resident education (medical knowledge, patient care, professionalism, interpersonal and communication skills, practice-based learning, and systems-based practice) have been embraced by medical schools as the building blocks necessary for becoming a competent licensed physician. Many medical schools are therefore changing their educational approach to an integrated model in which students demonstrate incremental acquisition and mastery of all competencies as they progress through medical school. Challenges to medical schools include integration of preclinical and clinical studies as well as development of learning objectives and assessment measures for each competency. The Undergraduate Education Subcommittee (UES) of the American Academy of Neurology (AAN) assembled a group of neuroscience educators to outline a longitudinal competency-based curriculum in medical neuroscience encompassing both preclinical and clinical coursework. In development of this curriculum, the committee reviewed United States Medical Licensing Examination content outlines, Liaison Committee on Medical Education requirements, prior AAN-mandated core curricula for basic neuroscience and clinical neurology, and survey responses from educators in US medical schools. The newly recommended curriculum provides an outline of learning objectives for each of the 6 competencies, listing each learning objective in active terms. Documentation of experiences is emphasized, and assessment measures are suggested to demonstrate adequate achievement in each competency. These guidelines, widely vetted and approved by the UES membership, aspire to be both useful as a stand-alone curriculum and also provide a framework for neuroscience educators who wish to develop a more detailed focus in certain areas of study.

Impact of protein kinase C activation on epileptiform activity in the hippocampal slice

There is evidence suggesting that protein kinase C (PKC) activation can prevent the enhanced network excitability associated with status epilepticus and group I metabotropic glutamate receptor (mGluR)-induced epileptogenesis. However, we observed no suppression of mGluR-induced burst prolongation in the guinea pig hippocampal slice when applied in the presence of the PKC activator phorbol-12,13-dibutyrate (PDBu). Furthermore, PDBu alone converted picrotoxin-induced interictal bursts into ictal-length discharges ranging from 2 to 6s in length. This effect could not be elicited by the inactive analog 4-alpha-PDBu and was suppressed with the PKC inhibitor chelerythrine, indicating PKC dependence. PKC activation can enhance neurotransmitter release, and both glutamate and acetylcholine are capable of eliciting similar prolonged synchronized discharges. However, neither mGluR1 nor NMDA receptor antagonist suppressed PDBu-driven burst prolongation, suggesting that increased glutamate release alone is unlikely to account for the PKC-induced expression of ictaform discharges. Similarly, atropine, a broad-spectrum muscarinic receptor antagonist, had no effect on PKC-induced burst prolongation. By contrast, AMPA/kainate receptor antagonist abolished PKC-induced burst prolongation, and mGluR5 antagonist significantly blunted the maximum burst length induced by PKC. These data suggest that PKC-induced prolongation of epileptiform bursts is dependent on changes specific to mGluR5 and AMPA/kainate receptors and not mediated simply by a generalized increase in transmitter release.

Contribution of GABAB receptor-mediated inhibition to the expression and termination of group I mGluR-induced ictaform bursts

In guinea pig hippocampal slices, the GABA(B) receptor antagonist CGP 35348 increased the length of picrotoxin-induced interictal bursts by only 22%, yet it increased the length of group I mGluR-induced ictaform bursts by 85%. These data suggest that (1) suppression of GABAergic inhibition is insufficient to account for group I mGluR agonist-induced ictaform bursts, and (2) although GABA(B) plays a minor role in terminating interictal bursts, it is a major contributor to the termination of mGluR-induced ictaform bursts.

Distinctions between persistent and reversible group I mGluR‐induced epileptiform burst prolongation

We have previously shown that selective activation of group I metabotropic glutamate receptors (mGluRs) results in long‐lasting enhancement of synchronized network activity in the hippocampal slice. Data herein suggest that activation of group I mGluRs need not result in this potentially epileptogenic effect. (1S,3R)‐1‐Aminocyclopentane‐1,3‐dicarboxylic acid (ACPD), a nonselective mGluR agonist, elicits ictaform bursts identical in appearance to those induced by selective agonists, but ACPD‐induced bursts do not persist following removal of the agent. Like the bursts induced by selective agonist, the ACPD bursts are blocked with group I mGluR antagonists and are not dependent on activation of either N‐methyl‐d‐aspartate (NMDA) receptors or protein kinase C. However, they differ from the persistent bursts in that they do not require active protein synthesis and they are not suppressed with L‐cysteine sulfinic acid, an agonist at a phospholipase D‐coupled metabotropic receptor. These novel findings provide evidence that group I mGluR‐induced epileptogenesis may be preventable.

Chloride's Exciting Role in Neonatal Seizures Suggests Novel Therapeutic Approach

Commentary Inhibition in the central nervous system is dependent on chloride conductance through GABAA receptor-associated channels. When chloride channels open, the membrane potential is driven toward chloride’s reversal potential, and because chloride’s reversal potential is negative to the neuron’s threshold for firing action potentials, it normally has an inhibitory effect. GABAA receptors have binding sites for both barbiturates and benzodiazepines, allowing them to enhance the inhibitory effect of GABA at this receptor; it is this mechanism of action that is responsible for the anticonvulsant activity of these agents. Prolonged seizure activity reduces the efficacy of GABAergic antiepileptic drugs (AEDs) such as benzodiazepines and barbiturates. If postsynaptic GABAA receptor responsiveness was reduced, one would expect GABAergic agents to lose efficacy as well. Indeed, a gradual suppression of GABAA receptor responses occurs during in vitro kindling (1) and GABA receptor internalization during ongoing seizures and status epilepticus has been documented (2), accounting for pharmacoresistance to GABAergic agents in these settings. One might also expect reduced inhibitory efficacy to result from intracellular buildup of chloride: prolonged neuronal activity results in elevated extracellular potassium concentrations, and this potassium is cleared in part by neuronal transporters that bring potassium back into the cells. NKCC1 is one such transporter, and when this transporter brings potassium back into the cells, it imports chloride with it. The resultant elevation of intracellular chloride will reduce efficacy of GABAA receptor-mediated synaptic inhibition by making the chloride reversal potential less negative. However, in the mature nervous system, the KCC2 transporter will do its best to counteract this chloride buildup by extruding chloride and restoring normal homeostasis. In the immature nervous system, however, the KCC2 transporter is not fully expressed. This makes the neonate more vulnerable to intracellular chloride buildup mediated by the NKCC1 transporter. Indeed, immature neurons have elevated baseline concentrations of intracellular chloride; as a result, neonatal GABAA receptor responses are not only less inhibitory, they may actually be excitatory (3, 4). The excitatory action of GABA-mediated chloride conductance is believed to contribute to the increased incidence of seizures in human neonates (5). There is a progressive developmental increase in expression of the chloride-extruding transporter KCC2 in the brain that eventually allows for the full expression of hyperpolarizing GABAA receptor responses; in the rat hippocampus, this occurs by the end of the second postnatal week (6). Numerous experiments have been done examining the efficacy of barbiturates in neonatal seizure models in vitro and in vivo, with conflicting results (7, 8). The current paper by Dzhala and colleagues attempts to make sense of this controversial area of investigation. The authors propose that the differences in response to GABAergic AEDs may correlate with Progressive NKCC1-Dependent Neuronal Chloride Accumulation During Neonatal Seizures.

Making Generalizations about Seizure Propagation

The Source of Afterdischarge Activity in Neocortical Tonic–Clonic Epilepsy. Trevelyan AJ, Baldeweg T, van Drongelen W, Yuste R, Whittington M. J Neurosci 2007;27(49):13513–13519. Tonic–clonic seizures represent a common pattern of epileptic discharges, yet the relationship between the various phases of the seizure remains obscure. Here we contrast propagation of the ictal wavefront with the propagation of individual discharges in the clonic phase of the event. In an in vitro model of tonic–clonic epilepsy, the after discharges (clonic phase) propagate with relative uniform speed and are independent of the speed of the ictal wavefront (tonic phase). For slowly propagating ictal wave fronts, the source of the afterdischarges, relative to a given recording electrode, switched as the wavefront passed by, indicating that afterdischarges are seeded from wavefront itself. In tissue that has experienced repeated ictal events, the wavefront generalizes rapidly, and the afterdischarges in this case show a different “flip-flop” pattern, with frequent switches in their direction of propagation. This same flip-flop pattern is also seen in subdural EEG recordings in patients suffering intractable focal seizures caused by cortical dysplasias. Thus, in both slowly and rapidly generalizing ictal events, there is not a single source of afterdischarge activity: rather, the source is continuously changing. Our data suggest a complex view of seizures in which the ictal event and its constituent discharges originate from distinct locations.

The Fragile X Mental Retardation Protein: A Valuable Partner in the Battle against Epileptogenesis

Correction of Fragile X Syndrome in Mice. Dölen G, Osterweil E, Rao BSS, Smith GB, Auerbach BD, Chattarji S, Bear MF. Neuron 2007;56:955–962. Fragile X syndrome (FXS) is the most common form of heritable mental retardation and the leading identified cause of autism. FXS is caused by transcriptional silencing of the FMR1 gene that encodes the fragile X mental retardation protein (FMRP), but the pathogenesis of the disease is unknown. According to one proposal, many psychiatric and neurological symptoms of FXS result from unchecked activation of mGluR5, a metabotropic glutamate receptor. To test this idea we generated Fmr1 mutant mice with a 50% reduction in mGluR5 expression and studied a range of phenotypes with relevance to the human disorder. Our results demonstrate that mGluR5 contributes significantly to the pathogenesis of the disease, a finding that has significant therapeutic implications for fragile X and related developmental disorders. Limbic Epileptogenesis in a Mouse Model of Fragile X Syndrome. Qiu LF, Lu TJ, Hu XL, Yi YH, Liao WP, Xiong ZQ. Cereb Cortex 2009 in press. (doi:10.1093/cercor/bhn163) Fragile X syndrome (FXS), caused by silencing of the Fmr1 gene, is the most common form of inherited mental retardation. Epilepsy is reported to occur in 20–25% of individuals with FXS. However, no overall increased excitability has been reported in Fmr1 knockout (KO) mice, except for increased sensitivity to auditory stimulation. Here, we report that kindling increased the expressions of Fmr1 mRNA and protein in the forebrain of wild-type (WT) mice. Kindling development was dramatically accelerated in Fmr1 KO mice, and Fmr1 KO mice also displayed prolonged electrographic seizures during kindling and more severe mossy fiber sprouting after kindling. The accelerated rate of kindling was partially repressed by inhibiting N-methyl-D-aspartic acid receptor (NMDAR) with MK-801 or mGluR5 receptor with 2-methyl-6-(phenylethynyl)-pyridine (MPEP). The rate of kindling development in WT was not effected by MPEP, however, suggesting that FMRP normally suppresses epileptogenic signaling downstream of metabotropic glutamate receptors. Our findings reveal that FMRP plays a critical role in suppressing limbic epileptogenesis and predict that the enhanced susceptibility of patients with FXS to epilepsy is a direct consequence of the loss of an important homeostatic factor that mitigates vulnerability to excessive neuronal excitation.

The Ups and Downs of Hippocampal Metabotropic Glutamate Receptors: Ramifications for Epileptogenesis and Cognitive Impairment Following Status Epilepticus

Loss of Metabotropic Glutamate Receptor-Dependent Long-Term Depression via Downregulation of mGluR5 after Status Epilepticus. Kirschstein T, Bauer M, Müller L, Rüschenschmidt C, Reitze M, Becker AJ, Schoch S, Beck H. J Neurosci 2007;27(29):7696–7704. Synaptic plasticity is thought to be a key mechanism of information storage in the CNS. Different forms of synaptic long-term potentiation have been shown to be impaired in neurological disorders. Here, we show that metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD), but not NMDA receptor-dependent LTD at Schaffer collateral–CA1 synapses, is profoundly impaired after status epilepticus. Brief application of the group I mGluR agonist (R,S)-3,5-dihydroxyphenylglycine (100 μM; 5 min) induced mGluR LTD in control, but not in pilocarpine-treated rats. Experiments in the presence of selective inhibitors of either mGluR5 [2-methyl-6-(phenylethynyl)-pyridine] or mGluR1 [7-(hydroxyimino)cyclopropachromen-carboxylate ethyl ester and (S)-(+)–amino-4-carboxy-2-methylbenzeneacetic acid] demonstrate that loss of mGluR LTD is most likely attributable to a loss of mGluR5 function. Quantitative real-time reverse transcription PCR revealed a specific downregulation of mGluR5 mRNA, but not of mGluR1 mRNA in the CA1 region. Furthermore, we detected a strong reduction in mGluR5 protein expression by immunofluorescence and quantitative immunoblotting. Additionally, the scaffolding protein Homer that mediates coupling of mGluR5 to downstream signaling cascades was downregulated. Thus, we conclude that the reduction of mGluR LTD after pilocarpine-induced status epilepticus is the result of the subtype-specific downregulation of mGluR5 and associated downstream signaling components. Functional Role of mGluR1 and mGluR4 in Pilocarpine-Induced Temporal Lobe Epilepsy. Pitsch J, Schoch S, Gueler N, Flor PJ, van der Putten H, Becker AJ. Neurobiol Dis 2007;26(3):623–633. Altered expression and distribution of neurotransmitter receptors, including metabotropic glutamate receptors (mGluRs), constitute key aspects in epileptogenesis, impaired hippocampal excitability and neuronal degeneration. mGluR1 mediates predominantly excitatory effects, whereas mGluR4 acts as inhibitory presynaptic receptor. Increased hippocampal expression of mGluR1 and mGluR4 has been observed in human temporal lobe epilepsy (TLE). In this study, we address whether genetic mGluR1 upregulation and mGluR4 knock-down influence seizure susceptibility and/or vulnerability of hippocampal neurons by analyzing transgenic animals in the pilocarpine TLE model. Therefore, we generated transgenic mice expressing mGluR1-enhanced green fluorescent protein (EGFP) fusion protein under control of the human cytomegalovirus (CMV) immediate early promoter. Status epilepticus (SE) was induced in 1) mice overexpressing mGluR1-EGFP and 2) mice deficient for mGluR4 (mGluR4 KO) as well as littermate controls. In the acute epileptic stage after pilocarpine application, mGluR4 KO mice showed a significant increase of severe seizure activity, in contrast to mGluR1 transgenics. Analysis of both transgenic mouse lines in the chronic epileptic phase, using a telemetric EEG-/video-monitoring system, revealed a significant increase in seizure frequency only in mGluR1-EGFP mice. In contrast, enhanced neuronal cell loss was only present in the hippocampus of epileptic mGluR4 KO mice. Our results suggest a role for mGluR1 in promoting seizure susceptibility as well as for mGluR4 to counteract excitatory activity and seizure-associated vulnerability of hippocampal neurons. Therefore, our data strongly recommend both mGluRs as potential drug targets to interfere with the development of hippocampal damage and seizure activity in TLE.

Metabotropic Glutamate Receptors in the Plasticity of Excitatory Responses in the Hippocampus

Excitatory connections in the hippocampal network have been shown to exhibit plasticity when subjected to specific modes of activation. These include short term and long term potentiation (STP and LTP, respectively) as well as long term depression (LTD). Ionotropic glutamate receptor (iGluR) activation, especially that mediated via NMDA receptors, is key in regulating many of these processes and is discussed in more detail in other chapters in this text. This chapter will demonstrate the participation of metabotropic glutamate receptors (mGluRs) in the plastic processes of the hippocampal circuit and will emphasize the possible clinical relevance of these processes. I will first provide a review of the receptor classification and the actions of the various mGluR subgroups. I will briefly summarize the work to date demonstrating the different ways in which each of these subgroups participates in synaptic plasticity. I will discuss ways in which mGluR-mediated activities may influence clinical conditions such as epilepsy. And finally, I will present the results of my studies on the role of these receptors in the production of epilep-tiform activities in the in vitro hippocampus, focusing on their unique capacity for autopotentiation, providing a potential mechanism for epileptogenesis.