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Epileptogenesis

Previous accomplishments and main current research interests of the Neuropharmacology Group:
1) Molecular and Morphological adaptation in Temporal Lobe Epilepsy (TLE)

Using neurochemical methods, changes in various neurotransmitter systems were characterized during and subsequent to kainic acid induced status epilepticus in rats [1, 2]. This kainic acid model closely reflects neuropathological changes seen in human temporal lobe epilepsy and allows investigating neuropathological changes induced by a status epilepticus and taking place during development of epilepsy (epileptogenesis: 10 to 30 days after the acute status epilepticus). As can be expected many neurotransmitter systems are strongly affected by acute seizures. Although GABA-ergic neurons degenerate to some extent, early studies of our group indicated pronounced adaptation of GABA-ergic systems, including over-expression of the GABA-synthesizing enzyme glutamate decarboxylase [2, 3]. In the last two decades our research group was engaged in the characterization of adaptive changes in the GABA-ergic system and in neuropeptide systems in the course of epileptogenesis. Our studies involved animal models of temporal lobe epilepsy as well as studies in tissue from patient with drug resistant temporal lobe epilepsy.

Neuropeptides in animal models and human Temoral Lobe Epilepsy (TLE). (Reviews: [1, 4, 5])
Neuropeptides act as cotransmitters of classical neurotransmitters. Like classical transmitters they become released during epileptic seizures. Interesting acute seizures induce over-expression of some of these peptides (e.g. neurokinin B, somatostatin, cholecystokinin, dynorphin, neuropeptide Y) in brain areas involved in limbic seizures [3, 6-13]. Hippocampal granule cells with their axonal extensions, the mossy fibers, are especially “plastic” in TLE. Like other peptides (neurokinin B, dynorphin), neuropeptide Y (NPY) becomes even ectopically expressed in granule cells of epileptic rats [14-16]. At the same time, also NPY-Y2 receptors (located at the mossy fiber terminals) become up-regulated in these neurons, whereas Y1 receptors (targeted to the dendritic extensions of granule cells) are downregulated [17-20]. We postulated that the over-expressed NPY may be released during epileptic seizures and may mediate an inhibition of glutamate release through Y2 receptors and thus exert an endogenous anticonvulsive mechanism [16, 21, 22]. This postulate was substantiated in the course of an international collaborative project supported by the Human Frontier Science Program (together with Drs. Beck-Sickinger, Leipzig; W. Colmers, Edmonton, Canada; H. Herzog, Sydney; H. Scharfman, New York; A. Vezzani, Milan). Behavioral pharmacological experiments using specific ligands to different NPY receptors, electrophysiological and histochemical studies using receptor knock out mice [23], rats over-expressing NPY [24] and over-expression of NPY after injecting a viral vector carrying the NPY gene [25]substantiated the proposal of an crucial role of NPY in epileptic seizures. 
Similar adaptive mechanism seem to occur also in the hippocampus of patients with drug resistant TLE: Y2 receptors (presumably mediating NPY’s anticonvulsant properties) become up-regulated, whereas Y1 receptors (mediating a modest pro-convulsant action of NPY) effects are down regulated [26]. In contrast to epileptic rats, NPY is not over-expressed in granule cells of the human epileptic hippocampus. Instead, NPY/GABA containing interneurons undergo pronounced sprouting of their terminals presumably “supplying” over-expressed Y2 receptors with NPY, and indicating a similar endogenous protective role for NPY as shown for the rat [26].

The GABA system in animal models and human TLE. (review: [27])
Presynaptic functions of GABA neurons in epilepsy: Also GABA neurons undergo critical changes in epilepsy. Depending on the severity of the status epilepticus many GABA neurons degenerate notably in the hilus of the dentate gyrus. On the other hand, surviving GABA neurons appear to be highly active as judged by increased activity of the GABA synthesizing enzyme glutamate decarboxylase (GAD) and the elevated levels of its mRNA [2, 3]. A striking finding was, however, that GAD and GABA are expressed in the glutamatergic mossy fibers of rats and become upregulated in epileptic animals [28]. This finding implied that mossy fibers (at least in epileptic rats) not only may contain the excitatory transmitter glutamate but also the inhibitory transmitter GABA. A striking finding in this context was that expression of GAD67 was preceded by expression of splice variant of GAD67, otherwise only founding the embryonic brain [29]. The mechanisms mossy fibers release GABA during epileptic seizures are still under debate. We are proposing that reversed transport of GABA by the GABA transporter GAT-1 may be one possible mechanism. This idea is supported by the observation that the cytoplasmatic membrane transporter GAT-1 but not the vesicular GABA transporter (VGAT) seem to be expressed in these neurons [30]. High intracellular sodium concentration (occurring after strong depolarization of the neuron) and high intracellular concentrations of GABA (caused by expression of GAD, possible lack of transport into vesicles and down-regulation of GABA-transaminase) can be triggers for a reversal of GABA transport by GAT-1 [30]. Recent finding indicate a similar strong expression of GAD also in granule cells the human epileptic hippocampus.
GABA receptors in epilepsy. Also on the level of GABA receptors marked changes occur. Both GABA-A and GABA-B receptor reveal marked rearrangement of their subunit composition in animal models of TLE [31-34] and in the epileptic human hippocampus [35, 36].

2) Morphological and molecular plasticity in TLE. (Review: [37, 38])

In collaboration with the University Clinics for Neurology (Prof. Dr. C. Baumgartner) and Neurosurgery (Dr. T. Czech) of the Medical University Vienna this cooperative research project is undertaken. Hippocampal tissue obtained at routine epilepsy surgery from patients with drug resistant TLE is collected after obtaining informed consent. Using histochemical (immunocytochemistry, in situ-hybridization, receptor autoradiography) and neurochemical (Western blots, RT-PCR) methods changes in patients with TLE and Ammon’s horn sclerosis and patients with non-lesional TLE are investigated. Morphological rearrangement of the dentate gyrus (sprouting of mossy fibers, rearrangement of innervation of the dentate molecular layer by GABA-ergic interneurons are investigated my means of different neurotransmitter markers [26, 39]. A main focus is directed towards investigation of the subiculum, the major output area of the hippocampus.

3) Role of NPY in anxiety

In collaboration with the research group of Prof. Dr. N. Singewald (Dept. Pharmacy, Innsbruck), we observed a pronounced anxiolytic phenotype in Y2 receptor knock out mice. This finding is now pursued using conditional Y2 receptor knock out mice in which the receptors are deleted in individual brain areas by local injection of a AAV-based cre-recombinase vector and by using strategies of NPY over-expression.

4) Chemical neuroanatomy

In collaboration with Prof. Werner Sieghart from the Brain Research Institute in Vienna the immunocytochemical distribution of 13 different GABA-A receptor subunits in the rat brain has been undertaken. Major focus was directed towards the characterization of the hippocampus and the basal ganglia. The studies revealed a highly heterogeneous distribution of GABA-A receptor subunits, e. g. indicating preferential expression of a1, b2 and g2 subunits in interneurons [32, 37, 40, 41]. This project is presently continued by studies on co-expression of subunits using immunocytochemistry and affinity purification of different species of the GABA-A receptor complex in subregions of the rat hippocampus.
A current histochemical study involves the characterization of peptidergic and GABA-ergic neurons in the rat and human subicular complex. See also a former study on neurokinin B in the rat brain [42].

References:

1. Sperk, G., Kainic acid seizures in the rat, Prog Neurobiol, 42 (1994) 1-32.
2. Sperk, G., Lassmann, H., Baran, H., Kish, S.J., Seitelberger, F. and Hornykiewicz, O., Kainic acid induced seizures: neurochemical and histopathological changes, Neuroscience, 10 (1983) 1301-1315.
3. Marksteiner, J. and Sperk, G., Concomitant increase of somatostatin, neuropeptide Y and glutamate decarboxylase in the frontal cortex of rats with decreased seizure threshold, Neuroscience, 26 (1988) 379-385.
4. Vezzani, A. and Sperk, G., Overexpression of NPY and Y2 receptor in epileptic brain tissue: an endogenous neuroprotective mechanism in temporal lobe epilepsy, Neuropeptides (2004) in press.
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6. Meyer, D.K., Widmann, R. and Sperk, G., Increased brain levels of cholecystokinin octapeptide after kainic acid-induced seizures in the rat, Neurosci Lett, 69 (1986) 208-211.
7. Sperk, G., Wieser, R., Widmann, R. and Singer, E.A., Kainic acid induced seizures: changes in somatostatin, substance P and neurotensin, Neuroscience, 17 (1986) 1117-1126.
8. Marksteiner, J., Sperk, G. and Maas, D., Differential increases in brain levels of neuropeptide Y and vasoactive intestinal polypeptide after kainic acid-induced seizures in the rat, Naunyn Schmiedebergs Arch Pharmacol, 339 (1989) 173-177.
9. Sperk, G., Marksteiner, J., Saria, A. and Humpel, C., Differential changes in tachykinins after kainic acid-induced seizures in the rat, Neuroscience, 34 (1990) 219-224.
10. Bellmann, R., Humpel, C., Krause, J.E., Marksteiner, J., Saria, A. and Sperk, G., Differential changes in mRNAs encoding for preprotachykinin A and B after kainic acid-induced seizures in the rat, Synapse, 8 (1991) 71-73.
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13. 13 Gruber, B., Greber, S. and Sperk, G., Kainic acid seizures cause enhanced expression of cholecystokinin-octapeptide in the cortex and hippocampus of the rat, Synapse, 15 (1993) 221-228.<
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15. 15 Sperk, G., Marksteiner, J., Gruber, B., Bellmann, R., Mahata, M. and Ortler, M., Functional changes in neuropeptide Y- and somatostatin-containing neurons induced by limbic seizures in the rat, Neuroscience, 50 (1992) 831-846.
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17. 17 Kofler, N., Kirchmair, E., Schwarzer, C. and Sperk, G., Altered expression of NPY-Y1 receptors in kainic acid induced epilepsy in rats, Neurosci Lett, 230 (1997) 129-132.
18. 18 Roder, C., Schwarzer, C., Vezzani, A., Gobbi, M., Mennini, T. and Sperk, G., Autoradiographic analysis of neuropeptide Y receptor binding sites in the rat hippocampus after kainic acid-induced limbic seizures, Neuroscience, 70 (1996) 47-55.
19. 19 Schwarzer, C., Kofler, N. and Sperk, G., Up-regulation of neuropeptide Y-Y2 receptors in an animal model of temporal lobe epilepsy, Mol Pharmacol, 53 (1998) 6-13.
20. 20 Gobbi, M., Gariboldi, M., Piwko, C., Hoyer, D., Sperk, G. and Vezzani, A., Distinct changes in peptide YY binding to, and mRNA levels of, Y1 and Y2 receptors in the rat hippocampus associated with kindling epileptogenesis, J Neurochem, 70 (1998) 1615-1622.
21. 21 Greber, S., Schwarzer, C. and Sperk, G., Neuropeptide Y inhibits potassium-stimulated glutamate release through Y2 receptors in rat hippocampal slices in vitro, Br J Pharmacol, 113 (1994) 737-740.
22. 22 Gruber, B., Greber, S., Rupp, E. and Sperk, G., Differential NPY mRNA expression in granule cells and interneurons of the rat dentate gyrus after kainic acid injection, Hippocampus, 4 (1994) 474-482.
23. 23 El Bahh, B., Balosso, S., Hamilton, T., Herzog, H., Beck-Sickinger, A., Sperk, G., Vezzani, A. and Colmers, W.F., Anticonvulsant response to NPY is mediated by the Y2 receptor, (2004), submitted.
24. 24 Vezzani, A., Michalkiewicz, M., Michalkiewicz, T., Moneta, D., Ravizza, T., Richichi, C., Aliprandi, M., Mule, F., Pirona, L., Gobbi, M., Schwarzer, C. and Sperk, G., Seizure susceptibility and epileptogenesis are decreased in transgenic rats overexpressing neuropeptide Y, Neuroscience, 110 (2002) 237-243.
25. 25 Richichi, C., Lin, E.J., Stefanin, D., Colella, D., Ravizza, T., Grignaschi, G., Veglianese, P., Sperk, G., During, M.J. and Vezzani, A., Anticonvulsant and antiepileptogenic effects mediated by adeno-associated virus vector neuropeptide Y expression in the rat hippocampus, J Neurosci, 24 (2004) 3051-309.
26. 26 Furtinger, S., Pirker, S., Czech, T., Baumgartner, C., Ransmayr, G. and Sperk, G., Plasticity of Y1 and Y2 receptors and neuropeptide Y fibers in patients with temporal lobe epilepsy, J Neurosci, 21 (2001) 5804-12.
27. 27 Sperk, G., Furtinger, S., Schwarzer, C. and Pirker, S., GABA and its receptors in epilepsy. In Recent Advances in Epilepsy, H. E. Scharfman and K. Binder (Ed.), 2003. Eurekah.com and Kluwer Academic/Plenum Publishers, New York.
28. 28 Schwarzer, C. and Sperk, G., Hippocampal granule cells express glutamic acid decarboxylase-67 after limbic seizures in the rat, Neuroscience, 69 (1995) 705-709.
29. 29 Szabo, G., Kartarova, Z., Hoertnagl, B., Somogyi, R. and Sperk, G., Differential regulation of adult and embryonic glutamate decarboxylases in rat dentate granule cells after kainate-induced limbic seizures, Neuroscience, 100 (2000) 287-295.
30. 30 Sperk, G., Schwarzer, C., Heilman, J., Furtinger, S., Reimer, R.J., Edwards, R.H. and Nelson, N., Expression of plasma membrane GABA transporters but not of the vesicular GABA transporter in dentate granule cells after kainic acid seizures, Hippocampus, 13 (2003) 806-815.
31. 31 Tsunashima, K., Schwarzer, C., Kirchmair, E., Sieghart, W. and Sperk, G., GABAA receptor subunits in the rat hippocampus III: altered messenger RNA expression in kainic acid-induced epilepsy, Neuroscience, 80 (1997) 1019-1032.
32. 32 Sperk, G., Schwarzer, C., Tsunashima, K., Fuchs, K. and Sieghart, W., GABAA receptor subunits in the rat hippocampus I: immunocytochemical distribution of 13 subunits, Neuroscience, 80 (1997) 987-1000.
33. 33 Kish, S.J., Sperk, G. and Hornykiewicz, O., Alterations in benzodiazepine and GABA receptor binding in rat brain following systemic injection of kainic acid, Neuropharmacology, 22 (1983) 1303-1309.
34. 34 Furtinger, S., Bettler, B. and Sperk, G., Altered expression of GABAB receptors in the hippocampus after kainic-acid-induced seizures in rats, Brain Res Mol Brain Res, 113 (2003) 107-115.
35. 35 Pirker, S., Schwarzer, C., Czech, T., Baumgartner, C., Pockberger, H., Maier, H., Hauer, B., Sieghart, W., Furtinger, S. and Sperk, G., Increased expression of GABAA receptor beta-subunits in the hippocampus of patients with temporal lobe epilepsy, J Neuropathol Exp Neurol, 62 (2003) 820-834.
36. 36 Furtinger, S., Pirker, S., Czech, T., Baumgartner, C. and Sperk, G., Increased expression of gamma-aminobutyric acid type B receptors in the hippocampus of patients with temporal lobe epilepsy, Neurosci Lett, 352 (2003) 141-145.
37. 37 Pirker, S., Schwarzer, C., Wieselthaler, A., Sieghart, W. and Sperk, G., GABAA receptors: immunocytochemical distribution of 13 subunits in the adult rat brain, Neuroscience, 101 (2000) 815-850.
38. 38 Sieghart, W. and Sperk, G., Subunit composition, distribution and function of GABA(A) receptor subtypes, Curr Top Med Chem, 2 (2002) 795-816.
39. 39 Pirker, S., Czech, T., Baumgartner, C., Maier, H., Novak, K., Furtinger, S., Fischer-Colbrie, R. and Sperk, G., Chromogranins as markers of altered hippocampal circuitry in temporal lobe epilepsy, Ann Neurol, 50 (2001) 216-226.
40. 40 Schwarzer, C., Berresheim, U., Pirker, S., Wieselthaler, A., Fuchs, K., Sieghart, W. and Sperk, G., Distribution of the major gamma-aminobutyric acidA receptor subunits in the basal ganglia and associated limbic brain areas of the adult rat, J Comp Neurol, 433 (2001) 526-549.
41. 41 Sperk, G., Schwarzer, C., Tsunashima, K. and Kandlhofer, S., Expression of GABAA receptor subunits in the hippocampus of the rat after kainic acid-induced seizures, Epilepsy Res, 32 (1998) 129-139.
42. 42 Marksteiner, J., Sperk, G. and Krause, J.E., Distribution of neurons expressing neurokinin B in the rat brain: immunohistochemistry and in situ hybridization, J Comp Neurol, 317 (1992) 341-256.