Hippocampal AMPA and NMDA mRNA levels and subunit immunoreactivity in human temporal lobe epilepsy patients and a rodent model of chronic mesial limbic epilepsy
Introduction
In the vertebrate central nervous system, most fast excitatory neurotransmission involves ionotrophic glutamate receptors (Monaghan et al., 1989). Based on their electrophysiologic and pharmacologic characteristics, ionotrophic glutamate receptors are usually classified into three subtypes: N-methyl-d-aspartate (NMDA), kainate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA). The molecular cloning of the glutamate receptors has identified several genomic subunits, and this discovery has greatly enhanced our understanding of the neurobiology and channel properties of these excitatory receptors (Hollmann and Heinemann, 1994). For example, the AMPA receptor is composed of four possible subunits, termed GluR1–4, and the NMDA receptor is made up of the NMDAR1 subunit in combination with NMDAR2a–d subunits (Sommer and Seeburg, 1992). Native AMPA and NMDA ionotrophic receptors are mostly heteromeric compositions of several subunits. Furthermore, based on recombinant molecular studies, subtle alterations in the composition of glutamate receptor subunits can alter the receptor's pharmacology and channel characteristics (Monaghan and Wenthold, 1997). Hence, it has been hypothesized that small changes in glutamate receptor subunit composition and/or concentrations could lead to alterations in excitatory neurotransmission, and possibly contribute to seizure generation and propagation (McNamara, 1994, Mathern et al., 1997c).
For temporal lobe epilepsy patients, an important clinical research aim is to comprehend the neurobiology of this disease so as to develop rationally based treatment plans. This understanding requires the elucidation of the neuropathologic substrates that underlie the human seizure disorders. Clinical-pathologic studies indicate that two neuropathologic sub-categories account for nearly all symptomatic temporal lobe seizures, and removal of these substrates is associated with successful post-surgery seizure control (Babb and Brown, 1987, Mathern et al., 1997b). The most frequent pathology is focal and severe hippocampal neuron loss, especially in the hilus and CA1 regions, in a pattern termed hippocampal sclerosis (HS) (Babb and Brown, 1987, Mathern et al., 1997b). HS cases also show aberrant molecular layer mossy fiber sprouting as demonstrated by neo-Timm's stains (Babb et al., 1991, Mathern et al., 1997b). The second most common pathologic substrate are temporal lobe mass lesions such as tumors or areas of cortical dysplasia (non-HS) (Babb and Brown, 1987, Mathern et al., 1995a, Mathern et al., 1997b). Non-HS hippocampi generally do not show as much neuronal loss or synaptic reorganization as HS patients. However, in both pathology groups the electrophysiologic focus, as determined by ictal intracerebral electrode recordings, most often appears first in the hippocampus and other mesial limbic structures (Babb et al., 1984, Wieser et al., 1993, Mathern et al., 1995b). Based on these electroclinical-pathologic finding, it has been hypothesized that in HS patients, the damaged hippocampus is a necessary factor in generating mesial limbic seizures. By comparison, in non-HS cases the hippocampus may be the passive recipient of seizures, or may amplify and propagate seizures originating from nearby cortical lesions. It is unclear, however, if non-HS hippocampi can generate mesial limbic seizures independent of the associated neocortical lesions.
In non-HS hippocampi, the absence of severe hippocampal neuron loss and axon sprouting suggests that other pathophysiologic mechanisms may be involved in producing neuronal hyperexcitability. These mechanisms may include an increase in glutamate-mediated synaptic excitation and/or a decrease in GABAergic inhibition (Sloviter, 1991, Babb and Pretorius, 1993, McNamara, 1994, Mathern et al., 1997a, Mathern et al., 1997b). The goals of our current clinical-pathologic research studies have been to determine in non-HS and HS hippocampi if there are changes in ionotrophic glutamate receptor concentrations and/or subunit compositions that could explain the neuronal hyperexcitability and seizure susceptibility noted in both temporal lobe pathology groups.
Previous human studies suggest that temporal lobe seizures may be associated with increased hippocampal AMPA and NMDA receptors, but the findings have not been consistent. For example, pharmacologic and electrophysiologic studies support the notion that glutamate receptors, especially of the NMDA family, may be increased or show greater sensitivity to glutamate stimulation in patients or animals with seizures (Avoli and Olivier, 1987, Isokawa and Lévesque, 1991, Köhr and Mody, 1994, Kraus et al., 1994). However, human anatomic studies involving hippocampal AMPA and NMDA receptor autoradiography have been confusing; various studies have reported increased, decreased, or unchanged binding compared to autopsies (Geddes et al., 1990, Hosford et al., 1991, McDonald et al., 1991). This apparent variability may be related to the research technique, or the common practice of combining HS and non-HS patients (i.e. with different amounts of hippocampal neuron loss) in the same study. Therefore, in mesial temporal lobe epilepsy patients, it remains uncertain if hippocampi show anatomic evidence that AMPA and NMDA ionotrophic receptor subunits are altered compared to autopsies.
An equally relevant basic science question is whether animal models of chronic mesial limbic seizures show changes in ionotrophic glutamate receptors that parallel the human disease. Several animal preparations have been proposed as possible models of human mesial temporal lobe epilepsy, and many of them show clinical and/or hippocampal pathologic characteristics that potentially mimic either HS or non-HS cases. For example, studies of the rat self sustained limbic status epilepticus (SSLSE) model show that acute limbic status for many hours generates hippocampal hilar and CA1 neuron loss and mossy fiber sprouting similar to human HS patients (Bertram et al., 1990, Lothman et al., 1990, Bertram and Cornett, 1993, Bertram and Lothman, 1993, Bertram and Cornett, 1994). Several weeks following the status episode, spontaneous limbic seizures develop, a pattern that resembles the clinical characteristics of human HS cases (Mathern et al., 1997b). In contrast, the rat kindling model shows less hippocampal neuron loss and fascia dentata mossy fiber sprouting than SSLSE rats (Cavazos et al., 1991, Cavazos et al., 1994, Mathern et al., 1997a), and kindled animals rarely demonstrate spontaneous limbic seizures. Kindled hippocampi, therefore, may be a model for human non-HS hippocampi. Hence, another research question is whether SSLSE and/or kindled animals show changes in hippocampal AMPA and NMDA receptor subunits that parallel human HS cases and/or non-HS hippocampi.
This communication is a synopsis of recent studies looking at AMPA and NMDA receptor subunit mRNA levels and immunoreactivity (IR) in patients with temporal lobe epilepsy and in rodent models of limbic seizures. In humans we determined if mesial limbic seizures were associated with differential increases in AMPA and NMDA receptor subunit mRNA levels or subunit IR. In rodent models, we examined in the fascia dentata from SSLSE and/or kindled rats for changes in AMPA and NMDA receptor subunit IR, especially as such changes might parallel findings in human HS and non-HS cases.
Section snippets
Patient evaluation and tissue collection
Patients with intractable temporal lobe seizures were evaluated using a protocol approved by the institution's Human Subject Protection Committee, and written permission was obtained to use any clinical-pathologic information for research purposes (Engel et al., 1991, Mathern et al., 1995a). Evaluation included detailed history and neurological examinations, interictal and ictal scalp EEG, a neuropsychological test battery, and intracarotid amobarbital injections (Wada test) for memory and
Examples of human AMPA and NMDA in situ hybridization
Fig. 1 shows examples of the human hippocampus from an autopsy (top row), non-HS (middle row), and HS patient (bottom row). They are presented by showing the Nissl stain (panels A, B, and C) followed by darkfields illustrating mRNA hybridization densities for AMPA GluR1 (panels D, E, and F), GluR2 (panels G, H, and I), GluR3 (panels J, K, and L), NMDAR1 (panels M, N, and O), and NMDAR2 (panels P, Q, and R).
In the autopsy case, hybridization densities were greater than background levels over the
Discussion
By comparing non-HS and HS patients to autopsies, and SSLSE rats to kindled animals and controls, these studies found that chronic spontaneous mesial limbic seizures, but not kindled events, were associated with increased fascia dentata AMPA and NMDA receptor subunit IR and mRNA levels per granule cell. These findings support the hypothesis that, at least in the fascia dentata, chronic mesial temporal lobe seizures are associated with alterations in hippocampal glutamate-mediated excitatory
Acknowledgements
This work was supported at UCLA from NIH Grants K08 NS 01603 and NS 02808. The work at Ribeirão Preto was supported by FAPESP 95/09248-5, CNPq, and FAEPA. EHB was supported by NIH Grant NS 21671, NS 25605, and the University of Virginia. Surgical material was obtained from: UCLA (I. Fried, W.J. Peacock, G.W. Mathern); Ribeirão Preto, University of São Paulo; (J.A. Assirati); and the University of Pittsburgh (P.D. Adelson). Leila Chimelli at Ribeirão Preto and Harry V. Vinters at UCLA generously
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