Susanne Pirker

GABA and Its Receptors in Epilepsy

Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the mammalian brain. It acts through 2 classes of receptors, GABAA receptors that are ligand-operated ion channels and the G-protein-coupled metabotropic GABAB receptors. Impairment of GABAergic transmission by genetic mutations or application of GABA receptor antagonists induces epileptic seizures, whereas drugs augmenting GABAergic transmission are used for antiepileptic therapy. In animal epilepsy models and in tissue from patients with temporal lobe epilepsy, loss in subsets of hippocampal GABA neurons is observed. On the other hand, electrophysiological and neurochemical studies indicate a compensatory increase in GABAergic transmission at certain synapses. Also, at the level of the GABAA receptor, neurodegeneration-induced loss in receptors is accompanied by markedly altered expression of receptor subunits in the dentate gyrus and other parts of the hippocampal formation, indicating altered physiology and pharmacology of GABAA receptors. Such mechanisms may be highly relevant for seizure induction, augmentation of endogenous protective mechanisms, and resistance to antiepileptic drug therapy. Other studies suggest a role of GABAB receptors in absence seizures. Presynaptic GABAB receptors suppress neurotransmitter release. Depending on whether this action is exerted in GABAergic or glutamatergic neurons, there may be anticonvulsant or proconvulsant actions.

A Pilot Study on Brain-to-Plasma Partition of 10,11-Dyhydro-10-hydroxy-5H-dibenzo(b,f)azepine-5-carboxamide and MDR1 Brain Expression in Epilepsy Patients Not Responding to Oxcarbazepine

Summary:  Purpose: We measured the brain‐to‐plasma partition of 10,11‐dihydro‐10‐hydroxy‐5H‐dibenzo(b,f)azepine‐5‐carboxamide (10‐OHCBZ) in epilepsy patients undergoing surgery to alleviate drug‐resistant seizures and administered with different oral doses of oxcarbazepine (OXC). We addressed the possible contribution of the multidrug transporter P‐glycoprotein (P‐gp or MDR1) in determining 10‐OHCBZ brain levels by measuring whether this active metabolite is a substrate of P‐gp and the relation between the level of expression of MDR1 and the drug concentration in the same brain tissue specimens.

Neuronal plasticity in animal models and the epileptic human hippocampus

Prolonged status epilepticus in humans as in experimental animals can initiate the development of temporal lobe epilepsy (TLE) (Kapur, 1999). Therefore, application of potent convulsant substances such as kainic acid or pilocarpine in rats induces acute status epilepticus that, after a silent period of 1–2 weeks, is followed by spontaneous convulsions. The status epilepticus is characterized by severe limbic seizures and sequelae of neuropathologic signs including opening of the blood–brain barrier, local brain edema, bleeding into the brain, and activation of microglia and astrocytes followed by neurodegeneration in the hippocampus, amygdala, entorhinal cortex, and other brain areas (Sperk et al., 1983; Du et al., 1993; Rizzi et al., 2003). Induced by the seizure activity, neurotransmitters such as γ-aminobutyric acid (GABA), glutamate, or amine transmitters are released from their stores and mechanisms of their resynthesis are strongly activated (Sperk et al., 1983). In addition, pronounced changes in the expression of multiple functionally important proteins have been found in brains of experimental animals and humans (Herdegen et al., 1993; Sperk, 1994; McNamara, 1999; Morimoto et al., 2004).

Increased expression of Nogo-A in hippocampal neurons of patients with temporal lobe epilepsy

Mesial temporal lobe epilepsy (TLE) is associated with pronounced anatomical and biochemical changes in the hippocampal formation including extensive neurodegeneration, reorganization of mossy fibres and sprouting of interneurons. Although the anatomical features and some of the physiological consequences of hippocampal remodeling have been well documented, the molecular mechanisms underlying the profound and orientated outgrowth of hippocampal neurons in TLE are not yet understood. The reticulon protein Nogo‐A has been associated with an inhibitory action on axon growth and plasticity. Using immunohistochemistry and in situ hybridization, we investigated the expression of Nogo‐A in specimens obtained at surgery from patients with TLE compared with those obtained from autopsy controls. In control specimens, Nogo‐A immunoreactivity and mRNA were mainly confined to oligodendrocytes. Only ≈ 40% of the specimens revealed low expression of Nogo‐A mRNA in neurons. In contrast, in TLE patients with and without Ammons horn sclerosis, Nogo‐A mRNA and immunoreactivity were markedly up‐regulated in most neurons (3.6‐ and 4.4‐fold increases in Nogo‐A mRNA in granule cells of sclerotic and nonsclerotic specimens) and their processes throughout the hippocampal formation. Similar elevations in Nogo‐A mRNA and protein levels were determined by quantitative RT‐PCR and Western blotting. Since Nogo‐A expression was also up‐regulated in specimens without hippocampal sclerosis, it may be induced by seizures prior to progressing neurodegeneration.

Increased expression of γ-aminobutyric acid type B receptors in the hippocampus of patients with temporal lobe epilepsy

Abstract Malfunctioning of the GABA-ergic system has been postulated as a possible cause of epilepsy. We investigated changes in the mRNA expression of the GABA B receptor subtypes GABA B -R1 and GABA B -R2 and of GABA B receptor binding in the hippocampus of patients with temporal lobe epilepsy (TLE) compared with post-mortem controls. In patients with Ammons horn sclerosis, significant decreases in [ 3 H]CG54626A binding were observed in subfields CA1 and CA3 of the hippocampus proper and the dentate hilus. On the other hand, both GABA B receptor mRNAs and receptor binding were enhanced after correction for neuronal loss in dentate granule cells and in the molecular layer, respectively, and the subiculum of patients with and without hippocampal sclerosis. These increases were even more pronounced when correcting the values for cell losses in the respective areas and indicated also increased expression of GABA B -R in the dentate hilus. Increased expression of both subtypes of GABA B receptors indicates augmented presynaptic inhibition of glutamate release as a possible protective mechanism in TLE.

Chromogranins as markers of altered hippocampal circuitry in temporal lobe epilepsy

Chromogranins are polypeptides which are widely expressed in the central nervous system. They are stored in dense core vesicles of nerve terminals, from where they are released upon stimulation. Using immunocytochemistry, we investigated the distribution of chromogranin A, chromogranin B, secretoneurin, and, for comparison, dynorphin in hippocampal specimens removed at routine surgery from patients with drug‐resistant mesial temporal lobe epilepsy and in autopsy tissues from nonneurologically deceased subjects. In post mortem controls (n = 21), immunoreactivity for all four peptides (most prominently for chromogranin B and dynorphin) was observed in the terminal field of mossy fibers. For chromogranins, staining was observed also in sectors CA1 to CA3 and in the subiculum. Chromogranin B immunoreactivity was found in the inner molecular layer of the dentate gyrus, the area of terminating associational‐commissural fibers. Secretoneurin and dynorphin immunoreactivity labeled the outer molecular layer and the stratum lacunosum moleculare of sectors CA1 to CA3, where projections from the entorhinal cortex terminate. In specimens with Ammons horn sclerosis (n = 25), staining for all three chromogranins and for dynorphin was reduced in the hilus of the dentate gyrus. Instead, intense staining was observed in the inner molecular layer, presumably delineating terminals of sprouted mossy fibers. Specimens obtained from temporal lobe epilepsy patients without Ammons horn sclerosis (n = 4) lacked this pronounced rearrangement of mossy fibers. In the stratum lacunosum moleculare of sector CA1, secretoneurin and dynorphin immunoreactivity was reduced in sclerotic, but not in nonsclerotic, specimens, paralleling the partial loss of fibers arising from the entorhinal cortex. Instead, presumably sprouted secretoneurin‐immunoreactive fibers were found in the outer dentate molecular layer in sclerotic specimens. These changes in staining patterns for chromogranins and dynorphin mark profound plastic and functional rearrangement of hippocampal circuitry in temporal lobe epilepsy.

Glutamate Decarboxylase67 is Expressed in Hippocampal Mossy Fibers of Temporal Lobe Epilepsy Patients

Recently, expression of glutamate decarboxylase‐67 (GAD67), a key enzyme of GABA synthesis, was detected in the otherwise glutamatergic mossy fibers of the rat hippocampus. Synthesis of the enzyme was markedly enhanced after experimentally induced status epilepticus. Here, we investigated the expression of GAD67 protein and mRNA in 44 hippocampal specimens from patients with mesial temporal lobe epilepsy (TLE) using double immunofluorescence histochemistry, immunoblotting, and in situ hybridization. Both in specimens with (n = 37) and without (n = 7) hippocampal sclerosis, GAD67 was highly coexpressed with dynorphin in terminal areas of mossy fibers, including the dentate hilus and the stratum lucidum of sector CA3. In the cases with Ammons horn sclerosis, also the inner molecular layer of the dentate gyrus contained strong staining for GAD67 immunoreactivity, indicating labeling of mossy fiber terminals that specifically sprout into this area. Double immunofluorescence revealed the colocalization of GAD67 immunoreactivity with the mossy fiber marker dynorphin. The extent of colabeling correlated with the number of seizures experienced by the patients. Furthermore, GAD67 mRNA was found in granule cells of the dentate gyrus. Levels, both of GAD67 mRNA and of GAD67 immunoreactivity were similar in sclerotic and nonsclerotic specimens and appeared to be increased compared to post mortem controls. Provided that the strong expression of GAD67 results in synthesis of GABA in hippocampal mossy fibers this may represent a self‐protecting mechanism in TLE. In addition GAD67 expression also may result in conversion of excessive intracellular glutamate to nontoxic GABA within mossy fiber terminals.

In vitro responsiveness of human-drug-resistant tissue to antiepileptic drugs: Insights into the mechanisms of pharmacoresistance

Pharmacoresistance in epileptic patients may be ascribed to at least two, not mutually exclusive, mechanisms: a pharmacokinetic mechanism and a decreased sensitivity or availability of targets to antiepileptic drugs (AEDs; i.e., carbamazepine and phenytoin (CBZ, PHT)). Brain:plasma drug concentration ratios were determined intraoperatively during lobectomies performed to alleviate drug-resistant seizures. The brain:plasma ratio of CBZ was 1.48 when therapeutic serum levels (15-34 microM) were achieved. When concentrations of CBZ found in multiple-drug-resistant brain were directly applied to human cortical slices from drug-resistant patients made hyperexcitable and hypersynchronous by Mg(2+)-free media, bursting frequency was not significantly affected and overall excitability was reduced by 40%. Similar results were obtained for PHT. At higher AED concentrations (60-200 microM), a dose-dependent decrease of bursting frequency and amplitude was observed. Slices from drug-resistant epileptic patients made hypersynchronous/hyperexcitable by elevated potassium or inhibition of GABA-A receptors behaved similarly. Of note is the response of slices from human multiple-drug-resistant brain, which was greater than in rodent cortex from naive animals. Taken together, our results support the hypothesis that multiple drug resistance to AEDs involves cerebrovascular changes that impede the achievement of appropriate drug levels in the central nervous system.

Dynamic up-regulation of prodynorphin transcription in temporal lobe epilepsy

Dynorphin neuropeptides are believed to act as endogenous anticonvulsants, though direct evidence for such a role in humans is sparse. We now report pronounced increases of prodynorphin mRNA expression in the dentate gyrus of patients with temporal lobe epilepsy in comparison to controls. We detected a conspicuously right skewed, bimodal distribution of mRNA levels among patients, suggestive of a dynamic up‐regulation of prodynorphin expression in epilepsy. Highest transcript levels were seen postictally. Our data argue for an essential role of dynorphin in the termination of seizures.

Altered Expression of Neuropeptide Y, ‐Y1, and ‐Y2 Receptors in the Hippocampus of Patients with Mesial Temporal Lobe Epilepsy

It is well established that neuropeptide Y (NPY) and its Y2 receptors become upregulated in interneurons and granule cells of the dentate gyrus in various animal models of epilepsy (1). In these rats, NPY may exert an endogenous anticonvulsive action amelioriating seizure activity. We now investigated NPY immunoreactivity and Y1and Y2-receptor binding in specimens obtained at routine surgery from patients with temporal lobe epilepsy (TLE) and in post mortem controls. Specimens from TLE patients had either Ammon’s horn sclerosis (n 31) or were without major signs of sclerosis (n 5), as assessed by high-resolution magnetic resonance imaging and neuropathologic examination. In control specimens, modest NPY-IR was observed, mainly in interneurons of the dentate hilus and in various other subfields of the hippocampus, in distinct fibers and, more diffusely, in the terminal field of mossy fibers. Similar to that in the rat, Y1-receptor binding was mainly localized in the dentate molecular layer and dense Y2receptor binding in the terminal field of mossy fibers. In specimens with Ammon’s horn sclerosis (and to a considerably lesser extent in the nonsclerotic specimens), a marked increase in the number of NPY-positive fibers was observed in all parts of the hippocampus, especially in the outer molecular layer of the dentate gyrus. At the same time, marked increases in NPY mRNA concentrations were observed in hilar interneurons of both TLE groups, indicating [in accordance with data by (3) and (2)], sprouting of NPY fibers. A significant increase in Y2-receptor binding (by 43– 48%) in the terminal field of mossy fibers in spite of a reduction to 54% in the number of granule cells in the specimens with Ammon’s horn sclerosis. Y1-receptor binding was reduced by 62% in sclerotic specimens, indicating a reduction of binding sites in the range of granule cell loss. NPY is thought to mediate an inhibition of glutamate release from mossy fibers through presynaptic Y2 receptors. Sprouting NPY-containing neurons and upregulation of Y2 receptors in mossy fibers in human TLE specimens thus may represent a possible endogenous protective mechanism.