Karin Borges

Neuronal and glial pathological changes during epileptogenesis in the mouse pilocarpine model.

The rodent pilocarpine model of epilepsy exhibits hippocampal sclerosis and spontaneous seizures and thus resembles human temporal lobe epilepsy. Use of the many available mouse mutants to study this epilepsy model would benefit from a detailed neuropathology study. To identify new features of epileptogenesis, we characterized glial and neuronal pathologies after pilocarpine-induced status epilepticus (SE) in CF1 and C57BL/6 mice focusing on the hippocampus. All CF1 mice showed spontaneous seizures by 17-27 days after SE. By 6 h there was virtually complete loss of hilar neurons, but the extent of pyramidal cell death varied considerably among mice. In the mossy fiber pathway, neuropeptide Y (NPY) was persistently upregulated beginning 1 day after SE; NPY immunoreactivity in the supragranular layer after 31 days indicated mossy fiber sprouting. beta2 microglobulin-positive activated microglia, normally absent in brains without SE, became abundant over 3-31 days in regions of neuronal loss, including the hippocampus and the amygdala. Astrogliosis developed after 10 days in damaged areas. Amyloid precursor protein immunoreactivity in the thalamus at 10 days suggested delayed axonal degeneration. The mortality after pilocarpine injection was very high in C57BL/6 mice from Jackson Laboratories but not those from Charles River, suggesting that mutant mice in the C57BL/6(JAX) strain will be difficult to study in the pilocarpine model, although their neuropathology was similar to CF1 mice. Major neuropathological changes not previously studied in the rodent pilocarpine model include widespread microglial activation, delayed thalamic axonal death, and persistent NPY upregulation in mossy fibers, together revealing extensive and persistent glial as well as neuronal pathology.

AMPA receptors: Molecular and functional diversity

Publisher Summary This chapter summarizes the current knowledge of the pharmacology and molecular biology of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors, with special attention to understanding the link between molecular and functional diversity and the resulting insights for drug discovery. The AMPA subtypes of glutamate receptor are the workhorse excitatory neurotransmitter receptors of the brain. AMPA receptors are postsynaptic ion channels that, when gated open by synaptically released glutamate, mediate the influx of Na + into the neuron and the efflux of K + . Under some conditions, AMPA receptor activation can produce pathophysiological or toxic effects. Neurolathyrism is a crippling neurodegenerative condition caused by the ingestion of a plant, which naturally contains an excitotoxic glutamate analog, β-N-methylamino-L-alanine (BMAA), which activates AMPA receptors. AMPA receptor antagonists are effective neuroprotectant drugs in animal models of global ischemia. Excessive AMPA receptor activation may also play a role in the excitotoxic damage associated with some chronic degenerative disorders such as Huntingtons disease, Parkinsons disease, and amyelotrophic lateral sclerosis.

AMPA/kainate receptor activation in murine oligodendrocyte precursor cells leads to activation of a cation conductance, calcium influx and blockade of delayed rectifying K+ channels

Studies during the last few years have shown that glial cells can express a large repertoire of neurotransmitter receptors. In this study, we have characterized the properties of a glutamate receptor in oligodendrocytes and their precursor cells from cultures of mouse brain, using the patch-clamp technique to measure ligand-activated currents and a fura-2 imaging system to determine changes in free cytosolic Ca2+ concentration ([Ca2+]i). The precursor cells were identified by their characteristic morphology and their voltage-gated currents as described previously [Sontheimer H. et al. (1989) Neuron 2, 1135-1145]. The ligands kainate, domoate and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), as well as L-glutamate but not trans-1-amino-1,3-cyclopentanedicarboxylate elicited inward currents at a holding potential of -70 mV and the antagonist 6-cyano-7-nitroquinoxaline-2,3-dione blocked the glutamate- and kainate-induced response reversibly, indicating the expression of an AMPA/kainate-type glutamate receptor. The response is due to the activation of a cationic conductance as revealed by analysing the reversal potential of the kainate-activated current. Receptor activation is accompanied by two additional responses: (i) an increase in [Ca2+]i mediated by depolarization and a subsequent activation of voltage-gated Ca2+ channels and (ii) a transient blockade of a delayed rectifying K+ current, but not of the A-type K+ current. The blockade of the K+ current was not due to the increase in [Ca2+]i since it was also observed in Ca(2+)-free bathing solution when no increase in [Ca2+]i was detectable after exposure to kainate. In contrast to precursor cells, oligodendrocytes responded weakly or not at all to glutamate or related ligands. We conclude that glutamate activates a complex pattern of physiological events in the glial precursor cells, which may play a role during the differentiation process of these cells.

Anticonvulsant effects of a triheptanoin diet in two mouse chronic seizure models

We hypothesized that in epileptic brains citric acid cycle intermediate levels may be deficient leading to hyperexcitability. Anaplerosis is the metabolic refilling of deficient metabolites. Our goal was to determine the anticonvulsant effects of feeding triheptanoin, the triglyceride of anaplerotic heptanoate. CF1 mice were fed 0-35% calories from triheptanoin. Body weights and dietary intake were similar in mice fed triheptanoin vs. standard diet. Triheptanoin feeding increased blood propionyl-carnitine levels, signifying its metabolism. 35%, but not 20%, triheptanoin delayed development of corneal kindled seizures. After pilocarpine-induced status epilepticus (SE), triheptanoin feeding increased the pentylenetetrazole tonic seizure threshold during the chronically epileptic stage. Mice in the chronically epileptic stage showed various changes in brain metabolite levels, including a reduction in malate. Triheptanoin feeding largely restored a reduction in propionyl-CoA levels and increased methylmalonyl-CoA levels in SE mice. In summary, triheptanoin was anticonvulsant in two chronic mouse models and increased levels of anaplerotic precursor metabolites in epileptic mouse brains. The mechanisms of triheptanoins effects and its efficacy in humans suffering from epilepsy remain to be determined.

Anticonvulsant profile of a balanced ketogenic diet in acute mouse seizure models

Anticonvulsant effects of the ketogenic diet (KD) have been reported in the mouse, although previous studies did not control for intake of vitamins, minerals and antioxidants. The aim of this study was to examine the effects of balanced ketogenic and control diets in acute mouse seizure models. The behavior in four mouse seizure models, plasma d-beta-hydroxybutyrate (d-BHB) and glucose levels were determined after feeding control diet, 4:1 and 6:1 KDs with matched vitamins, minerals and antioxidants. Feeding 4:1 and 6:1 KDs ad lib to 3-week-old (adolescent) mice resulted in 1.2-2.2mM d-BHB in plasma, but did not consistently change glucose levels. The 6:1 KD reproducibly elevated the CC50 (current that initiates seizures in 50% mice tested) in the 6-Hz model after 14 days of feeding to adolescent CD1 mice. Higher plasma d-BHB levels correlated with anticonvulsant effects. Despite ketosis, no consistent anticonvulsant effects of KDs were found in the fluorothyl or pentylenetetrazole CD1 mouse models. The 4:1 KD was neither anticonvulsant nor neuroprotective in hippocampus in the C3H mouse kainate model. Taken together, the KDs anticonvulsant effect was limited to the 6-Hz model, required chronic feeding with 6:1 fat content, and was independent from lowering plasma glucose.

Triheptanoin--a medium chain triglyceride with odd chain fatty acids: a new anaplerotic anticonvulsant treatment?

The triglyceride of heptanoate (C7 fatty acid), triheptanoin, is a tasteless oil used to treat rare metabolic disorders in USA and France. Heptanoate is metabolized by β-oxidation to provide propionyl-CoA, which after carboxylation can produce succinyl-CoA, resulting in anaplerosis - the refilling of the tricarboxylic acid cycle. Heptanoate is also metabolized by the liver to the C5 ketones, β-ketopentanoate and/or β-hydroxypentanoate, which are released into the blood and thought to enter the brain via monocarboxylate transporters. Oral triheptanoin has recently been discovered to be reproducibly anticonvulsant in acute and chronic mouse seizures models. However, current knowledge on alterations of brain metabolism after triheptanoin administration and anaplerosis via propionyl-CoA carboxylation in the brain is limited. This review outlines triheptanoins unique anticonvulsant profile and its clinical potential for the treatment of medically refractory epilepsy. Anaplerosis as a therapeutic approach for the treatment of epilepsy is discussed. More research is needed to elucidate the anticonvulsant mechanism of triheptanoin and to reveal its clinical potential for the treatment of epilepsy and other disorders of the brain.

Quantitative Transcriptional Neuroanatomy of the Rat Hippocampus: Evidence for Wide-Ranging, Pathway-Specific Heterogeneity Among Three Principal Cell Layers

We have used laser‐capture microdissection and microarray hybridization to characterize gene expression in the three principal neuron layers of rat hippocampus. Correlative and clustering analyses revealed all three layers to be easily differentiated from one another based on gene expression profile alone. A greater disparity in gene expression exists between dentate granule and pyramidal cell layers, reflecting phenotypic and ontological differences between those cell populations. Remarkably, the level of more than 45% of expressed transcripts was significantly different among the three neuron populations, with more than a third of those (>1,000 transcripts) being at least twofold different between layers. Even CA1 and CA3 pyramidal cell layers were dramatically different on a transcriptional level with a separate analysis indicating that nearly 20% of transcripts are differentially expressed between them. Only a small number of transcripts were specific for a given hippocampal cell layer, suggesting that functional differences are more likely secondary to wide‐ranging expression differences of modest magnitude rather than very large disparities in a few genes. Categorical analysis of transcript abundance revealed concerted differences in gene expression among the three cell layers referable to specific cellular pathways. For instance, transcripts encoding proteins involved in glucose metabolism are most highly expressed in the CA3 pyramidal layer, which may reflect an underlying greater metabolic rate of these neurons and partially explain their exquisite vulnerability to seizure‐induced damage. Conversely, transcripts related to MAP kinase signaling pathways and transcriptional regulator activity are prominent in the dentate granule cell layer, which could contribute to its resistance to damage following seizure activity by positioning these neurons to respond to external stimuli by altering transcription. Taken together, these data suggest that unique physiological characteristics of major cell layers, such as neuronal activity, neuronal plasticity, and vulnerability to neurodegeneration, are reflected in substantial transcriptional heterogeneity within the hippocampus.

Gene expression changes after seizure preconditioning in the three major hippocampal cell layers

Rodents experience hippocampal damage after status epilepticus (SE) mainly in pyramidal cells while sparing the dentate granule cell layer (DGCL). Hippocampal damage was prevented in rats that had been preconditioned by brief seizures on 2 consecutive days before SE. To identify neuroprotective genes and biochemical pathways changed after preconditioning we compared the effect of preconditioning on gene expression in the CA1 and CA3 pyramidal and DGCLs, harvested by laser capture microscopy. In the DGCL the expression of 632 genes was altered, compared to only 151 and 58 genes in CA1 and CA3 pyramidal cell layers. Most of the differentially expressed genes regulate tissue structure and intra- and extracellular signaling, including neurotransmission. A selective upregulation of energy metabolism transcripts occurred in CA1 pyramidal cells relative to the DGCL. These results reveal a broad transcriptional response of the DGCL to preconditioning, and suggest several mechanisms underlying the neuroprotective effect of preconditioning seizures.

Comparison of two schemes for automatic keyword extraction from MEDLINE for functional gene clustering

One of the key challenges of microarray studies is to derive biological insights from the unprecedented quantities of data on gene-expression patterns. Clustering genes by functional keyword association can provide direct information about the nature of the functional links among genes within the derived clusters. However, the quality of the keyword lists extracted from biomedical literature for each gene significantly affects the clustering results. We extracted keywords from MEDLINE that describe the most prominent functions of the genes, and used the resulting weights of the keywords as feature vectors for gene clustering. By analyzing the resulting cluster quality, we compared two keyword weighting schemes: normalized z-score and term frequency-inverse document frequency (TFIDF). The best combination of background comparison set, stop list and stemming algorithm was selected based on precision and recall metrics. In a test set of four known gene groups, a hierarchical algorithm correctly assigned 25 of 26 genes to the appropriate clusters based on keywords extracted by the TDFIDF weighting scheme, but only 23 of 26 with the z-score method. To evaluate the effectiveness of the weighting schemes for keyword extraction for gene clusters from microarray profiles, 44 yeast genes that are differentially expressed during the cell cycle were used as a second test set. Using established measures of cluster quality, the results produced from TFIDF-weighted keywords had higher purity, lower entropy, and higher mutual information than those produced from normalized z-score weighted keywords. The optimized algorithms should be useful for sorting genes from microarray lists into functionally discrete clusters.

Brain mitochondrial metabolic dysfunction and glutamate level reduction in the pilocarpine model of temporal lobe epilepsy in mice

Although certain metabolic characteristics such as interictal glucose hypometabolism are well established for temporal lobe epilepsy (TLE), its pathogenesis still remains unclear. Here, we performed a comprehensive study of brain metabolism in a mouse model of TLE, induced by pilocarpine-status epilepticus (SE). To investigate glucose metabolism, we injected mice 3.5-4 weeks after SE with [1,2- 13 C]glucose before microwave fixation of the head. Using 1 H and 13 C nuclear magnetic resonance spectroscopy, gas chromatography—mass spectrometry and high-pressure liquid chromatography, we quantified metabolites and 13 C labeling in extracts of cortex and hippocampal formation (HF). Hippocampal levels of glutamate, glutathione and alanine were decreased in pilocarpine-SE mice compared with controls. Moreover, the contents of N-acetyl aspartate, succinate and reduced nicotinamide adenine dinucleotide (phosphate) NAD(P)H were decreased in HF indicating impairment of mitochondrial function. In addition, the reduction in 13 C enrichment of hippocampal citrate and malate suggests decreased tricarboxylic acid (TCA) cycle turnover in this region. In cortex, we found reduced 13 C labeling of glutamate, glutamine and aspartate via the pyruvate carboxylation and pyruvate dehydrogenation pathways, suggesting slower turnover of these amino acids and/or the TCA cycle. In conclusion, mitochondrial metabolic dysfunction and altered amino-acid metabolism is found in both cortex and HF in this epilepsy model.