Autoradiography reveals selective changes in serotonin binding in neocortex of patients with temporal lobe epilepsy

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Abstract

The main goal of the present study was to evaluate binding to serotonin in the neocortex surrounding the epileptic focus of patients with mesial temporal lobe epilepsy (MTLE). Binding to 5-HT, 5-HT1A, 5-HT4, 5-HT7 receptors and serotonin transporter (5-HTT) in T1–T2 gyri of 15 patients with MTLE and their correlations with clinical data, neuronal count and volume were determined. Autopsy material acquired from subjects without epilepsy (n = 6) was used as control. The neocortex from MTLE patients demonstrated decreased cell count in layers III–IV (21%). No significant changes were detected on the neuronal volume. Autoradiography experiments showed the following results: reduced 5-HT and 5-HT1A binding in layers I–II (24% and 92%, respectively); enhanced 5-HT4 binding in layers V–VI (32%); no significant changes in 5-HT7 binding; reduced 5-HTT binding in all layers (I–II, 90.3%; III–IV, 90.3%, V–VI, 86.9%). Significant correlations were found between binding to 5-HT4 and 5-HT7 receptors and age of seizure onset, duration of epilepsy and duration of antiepileptic treatment. The present results support an impaired serotoninergic transmission in the neocortex surrounding the epileptic focus of patients with MTLE, a situation that could be involved in the initiation and propagation of seizure activity.

Introduction

Serotonin has been shown to be involved in the mechanisms regulating the seizure activity (Bagdy et al., 2007). The idea of inhibition of seizures by serotonin was initially suggested by Bonnycastle et al. (1957), who described that some anticonvulsants, including phenytoin, augment brain serotonin levels. At present, it is known that serotonin may induce inhibitory and excitatory effects on seizure activity. Low extracellular concentrations of serotonin produce protective and anticonvulsant effects, whereas high levels significantly aggravated seizures (Clinckers et al., 2004). Selective inhibitors of the serotonin uptake protect against seizures (Prendiville and Gale, 1993, Pasini et al., 1992, Wada et al., 1993). Concerning serotonin receptor subtypes, the activation of 5-HT1A receptors plays an inhibitory role on seizure activity (Lopez-Meraz et al., 2005, Wada et al., 1997). Similarly, the serotoninergic neurotransmission mediated by 5-HT2C receptors suppresses neuronal network hyperexcitability and convulsive activity (Tecott et al., 1995, Brennan et al., 1997, Applegate and Tecott, 1998). In contrast, the activation of 5-HT4 receptors enhances the epileptiform activity in the rat hippocampus (Tokarski et al., 2002), and the administration of 5-HT7 receptor antagonists protects from convulsions in experimental models (Bourson et al., 1997, Graf et al., 2004).

In patients with mesial temporal lobe epilepsy (MTLE), one of the most frequent types of human epilepsy associated with neuronal loss and gliosis in the hippocampus (Babb et al., 1984), positron emission tomography (PET) studies demonstrated decreased 5-HT1A receptor binding in both, medial and lateral temporal regions ipsilateral to the epileptic foci (Toczek et al., 2003, Savic et al., 2004), a situation that correlates to the degree of epileptogenicity (Merlet et al., 2004). Studies on resected tissues revealed elevated concentrations of serotonin concentration and of its metabolite 5-hydroxyindolacetic acid (5-HIIA) in the epileptogenic cortex, this increase being greater in regions where frequent interictal spikes are recorded (Pintor et al., 1990, Louw et al., 1989, Naffah-Mazzacoratti et al., 1996). Concerning experimental models of temporal lobe epilepsy in rats, an increase in binding to 5-HT1A receptors has been identified during the late chronic phase, bilaterally in the hippocampus, after kainic acid injection (Van Bogaert et al., 2001) or kindling (Clark et al., 1993, Cagnotto et al., 1998).

It is well known that the neocortex may play an important role in seizure generation and/or propagation (Chagnac-Amitai and Connors, 1989, Chervin et al., 1988, Connors, 1999). Studies indicate that the neocortex surrounding the epileptic focus of patients with MTLE presents a fine disorganization in synaptic circuits which consists of an increase and decrease of excitatory and inhibitory synapses, respectively (Marco and DeFelipe, 1997, De Felipe, 1997, De Felipe et al., 1998), and a loss of inhibitory neurons (De Felipe et al., 1993, Ferrer et al., 1994). These subtle changes in the apparent normal neocortex could explain why some patients that underwent epilepsy surgery eventually present epileptic seizures, whereas other patients remain free of subsequent convulsions (Kilpatrick et al., 1999).

Previous in vitro studies of the actions of serotonin in cortex concentrated on layer V pyramidal neurons (Foehring et al., 2002). However, understanding serotoninergic effects on cortical function associated with epileptic activity requires knowledge of the serotonin transmission on the cortical microorganization. The aim of the present study was to evaluate the binding to the 5-HT transporter (5-HTT) and receptor subtypes positively (5-HT4 and 5-HT7) or negatively (5-HT1A) coupled to adenylate cyclase, in temporal neocortex without morphological alterations detected by magnetic resonance imaging (MRI) and obtained from patients with intractable MTLE. We carried out in vitro autoradiography experiments in order to label different binding to serotonin and avoid a competition of endogenous ligands with the radioligand. Our major finding was that binding to serotonin in the apparent normal neocortex of epileptic patients presents significant changes that depend on the cortical layer and receptor subtype evaluated.

Section snippets

Patient histories and tissue collection

Temporal neocortical tissue was obtained from a group of 15 patients (8 males and 7 females), aged 19–52 years (33.2 ± 9.2 years, mean ± S.D.) with MTLE. Table 1 provides a summary of the relevant clinical data and side of the epileptic focus for each patient. Each MTLE patient underwent an extensive presurgical evaluation, including video electroencephalogram (EEG), MRI and single photon emission computed tomography (SPECT) within the Epilepsy Surgery Program from “Instituto Nacional de Neurologia

Control autopsy neocortex and serotonin binding

Tissue obtained at autopsy from subjects with no evidence of neurological disease was used as controls since previous reports indicate that 5-HT receptors and serotonin, tryptophan and 5-HIIA tissue content are preserved for several hours after death (Craven et al., 2005, Varnas et al., 2004a, Musshoff et al., 2004). Cell density values for control tissue were as follows: layers I–II, 73,279 ± 9220 cells/mm3; layers III–IV, 77,232 ± 17,531 cells/mm3; and layers V–VI, 77,945 ± 25,525 cells/mm3 (Fig. 1

Discussion

Our autoradiography experiments revealed that the temporal neocortex surrounding the epileptic focus of patients with MTLE presents diminished 5-HTT binding in all cortical layers. Furthermore, external cortical layers (I–II) demonstrate decreased 5-HT and 5-HT1A receptor binding, while internal layers (V–VI) show enhanced 5-HT4 receptor binding. In addition, some binding values correlate with clinical data of the epileptic patients. These results support significant changes in the binding to

Acknowledgements

We thank Ms. Leticia Neri Bazán and Mr. Héctor Vázquez Espinosa for their excellent technical assistance. This study was supported by Consejo Nacional de Ciencia y Tecnololgía (Grant 45943-M).

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