Meinrad Drexel

Progressive loss of phasic, but not tonic, GABAA receptor-mediated inhibition in dentate granule cells in a model of post-traumatic epilepsy in rats.

Traumatic brain injury (TBI) is a risk factor for the development of epilepsy, which can occur months to years after the insult. The hippocampus is particularly vulnerable to the pathophysiological effects of TBI. Here, we determined whether there are long-term changes in inhibition in the dentate gyrus that could contribute to the progressive susceptibility to seizures after TBI. We used severe lateral-fluid percussion brain injury to induce TBI in rats. In this model, spontaneous seizure activity, which involves the hippocampus, appears after a long latent period, resembling the human condition. We demonstrate that synaptic GABA(A) receptor-mediated inhibition is profoundly reduced in ipsilateral dentate granule cells 1 month after TBI. Moreover, synaptic inhibition decreases over time, and by 6 months after TBI, it is also significantly decreased contralaterally. Progressive loss of synaptic inhibition is paralleled by a decline in the number of parvalbumin-positive interneurons, but, in contrast to status epilepticus models, GABA(A) receptor subunit expression is largely unaltered. At both time points, the magnitude of tonic GABA(A) receptor-mediated currents after TBI is maintained, indicating a preservation of the inhibitory constraint of granule cells through tonic inhibition. Our results extend the time window during which strategies to target epileptogenesis may be effective.

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).

Sequel of spontaneous seizures after kainic acid-induced status epilepticus and associated neuropathological changes in the subiculum and entorhinal cortex

Injection of the seaweed toxin kainic acid (KA) in rats induces a severe status epilepticus initiating complex neuropathological changes in limbic brain areas and subsequently spontaneous recurrent seizures. Although neuropathological changes have been intensively investigated in the hippocampus proper and the dentate gyrus in various seizure models, much less is known about changes in parahippocampal areas. We now established telemetric EEG recordings combined with continuous video monitoring to characterize the development of spontaneous seizures after KA-induced status epilepticus, and investigated associated neurodegenerative changes, astrocyte and microglia proliferation in the subiculum and other parahippocampal brain areas. The onset of spontaneous seizures was heterogeneous, with an average latency of 15 ± 1.4 days (range 3–36 days) to the initial status epilepticus. The frequency of late spontaneous seizures was higher in rats in which the initial status epilepticus was recurrent after its interruption with diazepam compared to rats in which this treatment was more efficient. Seizure-induced neuropathological changes were assessed in the subiculum by losses in NeuN-positive neurons and by Fluoro-Jade C staining of degenerating neurons. Neuronal loss was already prominent 24 h after KA injection and only modestly progressed at the later intervals. It was most severe in the proximal subiculum and in layer III of the medial entorhinal cortex and distinct Fluoro-Jade C labeling was observed there in 75% of rats even after 3 months. Glutamatergic neurons, labeled by in situ hybridization for the vesicular glutamate transporter 1 followed a similar pattern of cell losses, except for the medial entorhinal cortex and the proximal subiculum that appeared more vulnerable. Glutamate decarboxylase65 (GAD65) mRNA expressing neurons were generally less vulnerable than glutamate neurons. Reactive astrocytes and microglia were present after 24 h, however, became prominent only after 8 days and remained high after 30 days. In the proximal subiculum, parasubiculum and entorhinal cortex the number of microglia cells was highest after 30 days. Although numbers of reactive astrocytes and microglia were reduced again after 3 months, they were still present in most rats. The time course of astrocyte and microglia proliferation parallels that of epileptogenesis.

Parvalbumin interneurons and calretinin fibers arising from the thalamic nucleus reuniens degenerate in the subiculum after kainic acid-induced seizures.

The subiculum is the major output area of the hippocampus. It is closely interconnected with the entorhinal cortex and other parahippocampal areas. In animal models of temporal lobe epilepsy (TLE) and in TLE patients it exerts increased network excitability and may crucially contribute to the propagation of limbic seizures. Using immunohistochemistry and in situ-hybridization we now investigated neuropathological changes affecting parvalbumin and calretinin containing neurons in the subiculum and other parahippocampal areas after kainic acid-induced status epilepticus. We observed prominent losses in parvalbumin containing interneurons in the subiculum and entorhinal cortex, and in the principal cell layers of the pre- and parasubiculum. Degeneration of parvalbumin-positive neurons was associated with significant precipitation of parvalbumin-immunoreactive debris 24 h after kainic acid injection. In the subiculum the superficial portion of the pyramidal cell layer was more severely affected than its deep part. In the entorhinal cortex, the deep layers were more severely affected than the superficial ones. The decrease in number of parvalbumin-positive neurons in the subiculum and entorhinal cortex correlated with the number of spontaneous seizures subsequently experienced by the rats. The loss of parvalbumin neurons thus may contribute to the development of spontaneous seizures. On the other hand, surviving parvalbumin neurons revealed markedly increased expression of parvalbumin mRNA notably in the pyramidal cell layer of the subiculum and in all layers of the entorhinal cortex. This indicates increased activity of these neurons aiming to compensate for the partial loss of this functionally important neuron population. Furthermore, calretinin-positive fibers terminating in the molecular layer of the subiculum, in sector CA1 of the hippocampus proper and in the entorhinal cortex degenerated together with their presumed perikarya in the thalamic nucleus reuniens. In addition, a significant loss of calretinin containing interneurons was observed in the subiculum. Notably, the loss in parvalbumin positive neurons in the subiculum equaled that in human TLE. It may result in marked impairment of feed-forward inhibition of the temporo-ammonic pathway and may significantly contribute to epileptogenesis. Similarly, the loss of calretinin-positive fiber tracts originating from the nucleus reuniens thalami significantly contributes to the rearrangement of neuronal circuitries in the subiculum and entorhinal cortex during epileptogenesis.

GAL3 receptor KO mice exhibit an anxiety-like phenotype

Significance In the modern world, stress-related diseases, including depression and anxiety disorders, are rapidly increasing. Neuropeptides are important modulators of these diseases. The neuropeptide galanin (GAL) has already been implicated in anxiety- and depression-related behaviors, but the relevant receptor subtypes remain to be elucidated. In the present work, we are the first, to our knowledge, to examine the role of the GAL3 receptor in anxiety- and depression-related behaviors in GAL3 receptor-deficient mice. We provide evidence that this receptor subtype is involved in stress-related diseases, and we propose this receptor as a target for alternative treatment strategies for mood disorders. The neuropeptide galanin (GAL) is widely distributed in the central and peripheral nervous systems. It is a modulator of various physiological and pathological processes, and it mediates its effects via three G protein-coupled receptors (GAL1–3 receptors). A role for GAL as a modulator of mood and anxiety was suggested, because GAL and its receptors are highly expressed in limbic brain structures of rodents. In recent years, numerous studies of animal models have suggested an involvement of GAL and GAL1 and GAL2 receptors in anxiety- and depression-related behavior. However, to date, there is sparse literature implicating GAL3 receptors in behavioral functions. Therefore, we studied the behavior of GAL3 receptor-deficient (GAL3-KO) mice to elucidate whether GAL3 receptors are involved in mediating behavior-associated actions of GAL. The GAL3-KO mouse line exhibited normal breeding and physical development. In addition to behavioral tests, phenotypic characterization included analysis of hematology, amino acid profiles, metabolism, and sudomotor function. In contrast to WT littermates, male GAL3-KO mice exhibited an anxiety-like phenotype in the elevated plus maze, open field, and light/dark box tests, and they were less socially affiliated than WT animals to a stranger mouse in a social interaction test. In conclusion, our data suggest involvement of GAL3 receptors in GAL-mediated effects on mood, anxiety, and behavior, making it a possible target for alternative treatment strategies for mood disorders.

Expression of GABA receptor subunits in the hippocampus and thalamus after experimental traumatic brain injury

Traumatic brain injury is a major cause of death and disability worldwide and often associated with post-traumatic epilepsy. We recently demonstrated that TBI induces acquired GABAA receptors channelopathy that associates with hyperexcitability in granule cell layer (GCL). We now assessed the expression of GABAA and GABAB receptor subunit mRNAs between 6 h and 6 months post-TBI in the hippocampus and thalamus. The expression of major GABAA receptor subunit mRNAs (α1, α2, α5, β2, β3, γ2 and δ) was, often bilaterally, down-regulated in the GCL and in the CA3 pyramidal cells. Instead, expression of α4 (GCL, CA3, CA1), α5 (CA1) and γ2 (GCL, CA3, CA1) mRNA was up-regulated after 10 d and/or 4 months. Many of these changes were reversible. In the thalamus, we found decreases in α1, α4, β2, γ2 and δ mRNAs in the laterodorsal thalamus and in the area combining the posterior thalamic nuclear group, ventroposterolateral and ventroposteromedial complex at 6 h to 4 months post-TBI. Unlike in the hippocampus, thalamic subunit down-regulations were irreversible and limited to the ipsilateral side. However, contralaterally there was up-regulation of the subunits δ and α4 6 h and 4 months after TBI, respectively. PCR array analysis suggested a mild long-lasting GABAA receptor channelopathy in the GCL and thalamus after TBI. Whereas TBI induces transient changes in the expression of GABAA receptor subunits in the hippocampus (presumably representing compensatory mechanisms), alterations of GABAA receptor subunit mRNAs in the thalamus are long-lasting and related to degeneration of receptor-containing neurons in thalamo-cortical relay nuclei. This article is part of the Special Issue entitled ‘GABAergic Signaling in Health and Disease’.

Changes in the expression of GABAA receptor subunit mRNAs in parahippocampal areas after kainic acid induced seizures

The parahippocampal areas including the subiculum, pre- and parasubiculum, and notably the entorhinal cortex (EC) are intimately involved in the generation of limbic seizures in temporal lobe epilepsy. We investigated changes in the expression of 10 major GABAA receptor subunit mRNAs in subfields of the ventral hippocampus, ventral subiculum, EC, and perirhinal cortex (PRC) at different intervals (1, 8, 30, and 90 days) after kainic acid (KA)-induced status epilepticus priming epileptogenesis in the rat. The most pronounced and ubiquitous changes were a transient (24 h after KA only) down-regulation of γ2 mRNA and lasting decreases in subunit α5, β3, and δ mRNAs that were prominent in all hippocampal and parahippocampal areas. In the subiculum similarly as in sectors CA1 and CA3, levels of subunit α1, α2, α4, and γ2 mRNAs decreased transiently (1 day after KA-induced status epilepticus). They were followed by increased expression of subunit α1 and α3 mRNAs in the dentate gyrus (DG) and sectors CA1 and CA3, and subunit α1 also in the EC layer II (30 and 90 days after KA). We also observed sustained overexpression of subunits α4 and γ2 in the subiculum and in the Ammon’s horn. Subunit γ2 mRNA was also increased in sector CA1 at the late intervals after KA. Taken together, our results suggest distinct regulation of mRNA expression for individual GABAA receptor subunits. Especially striking was the wide-spread down-regulation of the often peri- or extrasynaptically located subunits α5 and δ. These subunits are often associated with tonic inhibition. Their decrease could be related to decreased tonic inhibition or may merely reflect compensatory changes. In contrast, expression of subunit α4 that may also mediate tonic inhibition when associated with the δ-subunit was significantly upregulated in the DG and in the proximal subiculum at late intervals. Thus, concomitant up-regulation of subunit γ2, α1 and α4 mRNAs (and loss in δ-subunits) ultimately indicates significant rearrangement of GABAA receptor composition after KA-induced seizures.

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.

Somatostatin and Neuropeptide Y Neurons Undergo Different Plasticity in Parahippocampal Regions in Kainic Acid–Induced Epilepsy

Abstract Parahippocampal brain areas including the subiculum, presubiculum and parasubiculum, and entorhinal cortex give rise to major input and output neurons of the hippocampus and exert increased excitability in animal models and human temporal lobe epilepsy. Using immunohistochemistry and in situ hybridization for somatostatin and neuropeptide Y, we investigated plastic morphologic and neurochemical changes in parahippocampal neurons in the kainic acid (KA) model of temporal lobe epilepsy. Although constitutively contained in similar subclasses of &ggr;-aminobutyric acid (GABA)-ergic neurons, both neuropeptide systems undergo distinctly different changes in their expression. Somatostatin messenger RNA (mRNA) is rapidly but transiently expressed de novo in pyramidal neurons of the subiculum and entorhinal cortex 24 hours after KA. Surviving somatostatin interneurons display increased mRNA levels at late intervals (3 months) after KA and increased labeling of their terminals in the outer molecular layer of the subiculum; the labeling correlates with the number of spontaneous seizures, suggesting that the seizures may trigger somatostatin expression. In contrast, neuropeptide Y mRNA is consistently expressed in principal neurons of the proximal subiculum and the lateral entorhinal cortex and labelingfor the peptide persistently increased in virtually all major excitatory pathways of the hippocampal formation. The pronounced plastic changes differentially involving both neuropeptide systems indicate marked rearrangement of parahippocampal areas, presumably aiming at endogenous seizure protection. Their receptors may be targets for anticonvulsive drug therapy.

Calcium‐binding proteins in focal cortical dysplasia

Alterations in γ‐aminobutyric acid (GABA)‐ergic cortical neurons have been reported in focal cortical dysplasia (FCD)Ia/IIIa, a malformation of cortical development associated with drug‐resistant epilepsy. We compared numbers of neurons containing calcium‐binding proteins parvalbumin (PV), calbindin (CB), and calretinin (CR) and densities of respective fibers in lateral temporal lobe surgical specimens of 17 patients with FCD with 19 patients who underwent anterior temporal lobe resection due to nonlesional temporal lobe epilepsy (non‐FCD) as well as with 7 postmortem controls.