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Comment on "On the Origin of Interictal Activity in Human Temporal Lobe Epilepsy in Vitro"
For more than a decade, there has been extensive discussionabout whether hippocampal sclerosis causes enhanced neuronalexcitability as a prerequisite for seizure generation. The disorderknown as Ammon's horn sclerosis (AHS) is characterized by pronouncedcell loss and gliosis in various regions of the hippocampalformation, leaving the subiculum generally intact (1, 2). Giventhe hypothesis that epileptic activity is generated within thehippocampal formation when the CA3 and CA1 regions are damagedor even absent, it is feasible that the adjacent subiculum isuniquely responsible for the generation of limbic seizures.Using multielectrode recordings in hippocampal brain slicesof patients with temporal lobe epilepsy (TLE) and hippocampalsclerosis, Cohen et al. (3) detected spontaneous, rhythmic spikesin the subiculum— but rarely in the CA3 or CA1 regions.This activity closely resembled the discharges seen on the electroencephalograms(EEGs) of these patients. Cohen et al. (3) therefore concludedthat in patients with AHS, deafferentation of the subiculuminitiates an epileptogenic plasticity that includes changesin glutamatergic or -aminobutyric acid (GABA)-ergic signaling.
We find that even in nonsclerotic hippocampal tissue, as gradedby Wyler (4), the subiculum shows cellular and synaptic changeswhich suffice to generate an epileptic focus. To elucidate thisissue further, we investigated the contribution of subicularcells to interictal activity recorded in EEGs of TLE patientswith (AHS, Wyler score W3, W4; n = 7) and without hippocampalsclerosis (non-AHS, W0-W2; n = 6). In AHS tissue, the majority(75%) of subicular cells were regular firing cells [n = 18 (5)],whereas only six cells were bursting cells (Fig. 1A). More thanhalf of the recorded cells (both cell types) displayed spontaneous,rhythmic activity of 1.45 ± 0.31 Hz that correlated withthe occurrence and frequency (0.75 to 2 Hz) of interictal dischargesrecorded in the EEGs of the corresponding patients (Fig. 1B;n = 5 patients). Intracellular recordings showed spontaneousexcitatory postsynaptic potentials that were suppressed by 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), but not by D,L-2-amino-5-phosphovalericacid (APV), and inhibitory postsynaptic potentials that weresuppressed by bicuculline; together, these results indicatedan AMPA/kainate- and GABAA receptor–mediated synapticcircuit (Fig. 1C, a and b). We never found APV-/NBQX-insensitiveexcitatory events that would suggest spontaneous depolarizingGABAergic responses. In contrast to (3), two patients with severesclerosis in area CA1 (W4) showed no rhythmic activity in theEEGs or in the subicular slice preparations (n = 7 cells).
Fig. 1. Relation of hippocampal sclerosis, interictal activity, and membrane properties in human TLE. (A) Membrane properties and discharge patterns of a human subicular bursting cell (left trace) and regular firing cell (right trace) upon negative and positive current steps [range: ± 0.9 nA; duration:300 ms; representative traces recorded in AHS tissue (5, 7)]. In AHS and non-AHS tissue, the majority of subicular cells were regular firing cells (75% and 79%, respectively). Arrow indicates spontaneous activity recorded in AHS tissue. (B) 56% (AHS tissue) and 28% (non-AHS tissue) of subicular principle cells displayed spontaneous, rhythmic activity (in vitro), which was always correlated with interictal spike and wave events recorded with sphenoidal electrodes (in vivo) in the same patient. (C) The synchronized spontaneous activity consisted of excitatory and inhibitory postsynaptic potentials that were suppressed by the AMPA/kainate receptor-antagonist NBQX (but not by the NMDA receptor-antagonist APV) (a) and the GABAA receptor-antagonist bicuculline (b). (D) fAHP and sAHP following a train of depolarization-induced action potentials (1 nA, 1 s) were significantly decreased in cells showing spontaneous rhythmic activity compared to cells without activity (9). Arrow indicates spontaneous activity in the course of the recording.
[View Larger Version of this Image (27K GIF file)]
As in AHS tissue, the majority (79%) of cells in non-AHS tissuewere regular firing cells (n = 15) and only four cells werebursting cells (6). Notably, nearly one-third of the cells showedspontaneous activity reminiscent of the activity recorded insclerotic tissue (0.65 ± 0.15 Hz). As with AHS patients,this activity corresponded to the interictal spike and waveevents of the EEG records (Fig. 1B; n = 3 patients).
In addition to synaptic alterations, persistent changes in neuronalmembrane properties contribute to epileptogenesis. We foundno differences in resting membrane potential, input resistance,and discharge properties of subicular neurons between AHS andnon-AHS tissue (5, 6). Surprisingly, the ratio between burstingcells and regular firing cells in both tissues was virtuallyinverse compared to the ratio observed in the rat subiculum(7). These results challenge theories on seizure generationthat support a crucial role for bursting cells in seizure activity(8). However, the fast afterhyperpolarization (fAHP) and slowafterhyperpolarization (sAHP) recorded following a train ofaction potentials were significantly decreased in cells showingspontaneous activity compared with "silent" cells [Fig. 1D;(9)].
Our data demonstrate that histologically identified sclerosisof the CA1 region does not necessarily promote spontaneous rhythmicactivity in the adjacent subiculum in vivo or in vitro. Furthermore,spontaneous activity driven by excitatory and inhibitory circuitsalso occurs in the absence of AHS. Thus, the presence of hippocampalsclerosis is not mandatory for the development of an epilepticfocus in the major output pathway of the hippocampus, as proposedby Cohen et al. (3). However, we do concede that according tothe Wyler grading system, a moderate neuronal dropout may occurin a non-AHS specimen (4). Therefore, even in nonsclerotic tissue,a loss of afferents from CA1 to the subiculum is conceivable.Even though AHS in the resected hippocampus has important prognosticimplications for freedom from seizures postoperatively, ourdata show that functional alterations on both the synaptic andthe cellular level enhance seizure susceptibility in cases wheredeafferentation of the subiculum is absent or not as severeas the one observed in classical AHS (10).
Christian Wozny Anatol Kivi
Neuroscience Research Center at the Charité Humboldt University of Berlin Schumannstrasse 20/21 10117 Berlin, Germany
Thomas-Nicolas Lehmann
Department of Neurosurgery, Charité Humboldt University of Berlin Augustenburgerplatz 1 13353 Berlin, Germany
Christoph Dehnicke
Epilepsy Center Berlin-Brandenburg Herzbergstrasse 79 10365 Berlin, Germany
Uwe Heinemann
Johannes-Müller-Institute of Physiology, Charité Humboldt University of Berlin Tucholskystrasse 2 10117 Berlin, Germany
Joachim Behr
Neuroscience Research Center at the Charité Humboldt University of Berlin E-mail: joachim.behr{at}charite.de
References and Notes
1. G. W. Mathern et al., in Epilepsy: A Comprehensive Textbook, J. Engel Jr., T. A. Pedley, Eds. (Lippincott-Raven, Philadelphia, PA, 1997).
3. I. Cohen, V. Navarro, S. Clemenceau, M. Baulac, R. Miles, Science298, 1418 (2002).[Abstract/Free Full Text]
4. A. R. Wyler, F. C. Dohan Jr., J. B. Schweitzer, A. D. Berry III, J. Epilepsy5, 220 (1992). [CrossRef] [ISI]
5. In AHS tissue, regular firing cells had an average resting membrane potential of – 68.4 ± 1.1 mV and an input resistance of 35.6 ± 3.4 M (n = 18). Bursting cells that are characterized by a burst of 2 to 5 action potentials upon a depolarizing current pulse injection displayed an average resting membrane potential of – 68.7 ± 2.6 mV and an input resistance of 29.6 ± 3.6 M (n = 6; P > 0.5).
6. In non-AHS tissue, regular firing cells had an average resting membrane potential of – 69.3 ± 1.0 mV and an input resistance of 34.8 ± 3.3 M (n = 15). The resting membrane potential of bursting cells was not significantly different from regular firing cells (– 64.0 ± 3.6 mV; n = 4; P = 0.23). Bursting cells had an input resistance of 23.8 ± 4.2 M (n = 4; P = 0.08). All parameters were not significantly different from those recorded in AHS-tissue.
7. S. M. O'Mara, S. Commins, M. Anderson, J. Gigg, Prog. Neurobiol.64, 129 (2001). [CrossRef] [ISI] [Medline]
8. Y. Yaari, H. Beck, Brain Pathol.12, 234 (2002). [ISI] [Medline]
9. fAHP:Spontaneously active cells 3.9 ± 0.2 mV (n = 9) versus nonactive cells 4.9 ± 0.2 mV (n = 12; P < 0.01). sAHP:Spontaneously active cells 0.7 ± 0.1 mV (n = 9) versus 2.8 ± 0.1 (n = 12; P < 0.01).
10. EEGs with sphenoidal electrodes were correlated with spontaneous activity recorded in subicular neurons of the resected epileptic tissue. The study was approved by the local ethics committee and each patient gave informed consent to the studies on the removed tissue. Patients were between 20 and 67 years old in the AHS group (34 ± 6) and had suffered from TLE for 24 ± 5 years (n = 7). In the non-AHS group, patients ranged from 15 to 46 years (28 ± 5; P > 0.4) with a seizure history of 13 ± 5 years (n = 6; P > 0.1). Hippocampal specimens were obtained in the operating room and immediately incubated at 4°C in carbogenated transport solution containing 3 mM KCl, 1.25 mM NaH2PO4, 10 mM glucose, 2 mM MgSO4, 2mM MgCl2, 1.6 mM CaCl2, 21 mM NaHCO3, 200 mM sucrose, and 0.1 mM -tocopherol; pH 7.4, osmolarity 300 mosm/L. Sodium chloride was reduced to prevent hypoxia-induced sodium influx into neurons, and -tocopherol was added as a scavenger of free radicals. Transport from the operating room to the lab lasted about 30 min. Tissue was coronally dissected into slices 500 µm thick using a vibratome (Campden Instruments, Leicester, UK). Slices were immediately transferred into an interface chamber, perfused at a rate of 1.5 to 2.0 ml/min with pre-warmed (32° to 35°C) carbogenated artificial cerebrospinal fluid (ACSF) containing 129 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 10 mM glucose, 2 mM MgCl2, 1.6 mM CaCl2, and 21 mM NaHCO3; pH 7.4. Recording started 4 to 5 hours after preparation to permit recovery of the slices. Intracellular recordings were performed with 2.5 M potassium acetate containing sharp microelectrodes (resistance 40 to 100 M). Recordings were made in bridge mode technique using a SEL 05L or SEL 10 amplifier (npi instruments, Tamm, Germany). Signals were filtered at 3 kHz and sampled at 10 kHz using a TIDA interface card. All data were analyzed offline using TIDA-software (HEKA, Lambrecht/Pfalz, Germany). Data were expressed as means ± SEM and statistical comparison was done by applying student's t test (Excel, Microsoft). Significance niveau was set to P < 0.05. The following drugs were bath-applied:5 µM bicuculline methiodide (BCM, Sigma, Deisenhofen, Germany), 10 µM NBQX (a gift from Novo Nordisk), and 60 µM APV (Tocris, UK).
11. This work was supported by a DFG-Grant to J. B. (2011/3-1 and SFB-TR 3) and by the Graduate School 238 to C. W. We thank A. von Deimling and his colleagues for the support and contribution in scoring the human tissue according the classification by Wyler. We thank S. Gabriel for helpful discussions during the experiments and S. Walden for her excellent technical assistance.
Received for publication 7 March 2003. Accepted for publication 2 June 2003.
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Ivan Cohen, Vincent Navarro, Gilles Huberfeld, Stéphane Clemenceau, Michel Baulac, and Richard Miles (25 July 2003) Science301 (5632), 463d.
[DOI: 10.1126/science.1085812] |Full Text »|PDF »
REPORTS
Ivan Cohen, Vincent Navarro, Stéphane Clemenceau, Michel Baulac, and Richard Miles (15 November 2002) Science298 (5597), 1418.
[DOI: 10.1126/science.1076510] |Abstract »|Full Text »|PDF »
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