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SEIZURES | Seizure-Induced Gene Expression
1302 Seizures _ Seizure-Induced Gene Expression Almeida LB (eds.) Spatiotemporal Models in Biological and Artificial Systems, pp. 81–88. Amsterdam: IOS Press. Iasemidis LD, Sackellares JC, Zaveri HP, and Williams WJ (1990) Phase space topography of the electrocorticogram and the Lyapunov exponent in partial seizures. Brain Topography 2: 187–201. Iasemidis LD, Sackellares JC, Zaveri HP, Williams WJ, and Hood TW (1988) Nonlinear dynamics of electrocorticographic data. Journal of Clinical Neurophysiology 5: 339.
Lennox WG (1946) Science and Seizures. New York: Harper. Mormann F, Kreuz T, Andrzejak RG, et al. (2003) Epileptic seizures are preceded by a decrease in synchronization. Epilepsy Research 53: 173–185. Tsakalis K and Iasemidis LD (2006) Control aspects of a theoretical model for epileptic seizures. International Journal of Bifurcation and Chaos 16(7): 2013–2027.
Seizure-Induced Gene Expression K Lukasiuk, The Nencki Institute of Experimental Biology, Warsaw, Poland A Pitka¨nen, University of Kuopio, Kuopio, Finland; Kuopio University Hospital, Kuopio, Finland ã 2009 Elsevier Ltd. All rights reserved.
Introduction Seizures, defined as a pathological increase in neuronal synchronization, are not just the characteristic symptoms of epilepsy, but can also have direct effects on the brain. In particular, prolonged seizures can produce neuronal damage and trigger cascades of biochemical events that can have long-term effects. These seizure events activate second messengers, transcription factors and gene expression, plastic phenomena (such as mossy fiber sprouting), neurogenesis, and altered neurotransmission – all of which may underlie consequent sensory-motor, developmental, and behavioral and cognitive deficits. Seizureinduced gene expression is of particular importance, since production of new proteins (or proteins at altered levels) provides the means for long-lasting alterations in brain functions. Analysis of alterations in gene expression can single out particular molecular mechanisms involved in such long lasting, potentially harmful, effects of seizures.
Background Regulation of Gene Expression In this article we focus on seizure-induced gene expression. While approaching this subject, one should be aware that the regulation of gene expression is a very complex phenomenon. By definition, regulation of gene expression refers to the control of the amount and timing of appearance of functional gene products, that is, RNAs and proteins. The process includes transcription as well as posttranslational modification of protein, which can be modulated at any step via any number of mechanisms.
The first level of regulation of gene expression relies on modification of DNA. Chemical modification refers to methylation of CpG islands by methyltransferase, a common way for silencing a gene. Structural modifications of DNA arise from phosphorylation or methylation of histones; histone acetylation is of special importance for regulation of transcription, since histone deacetyltransferases enable the dissociation of DNA from the histones and therefore allow transcription to proceed. Progression of transcription and the efficiency of RNA synthesis depend on several components of the RNA polymerase machinery, including several regulatory proteins that bind to regulatory sites, usually located near the promoter. The regulatory proteins can be categorized according to their function as: (1) specificity factors that alter specificity of RNA polymerase for given promoter and repressors that bind to noncoding DNA sequences in the vicinity of the promoter and impede RNA polymerase’s progress along the DNA strand; (2) basal factors which position RNA polymerase at the start of transcription, and activators that enhance RNA polymerase interaction at a given promoter either by interaction with RNA polymerase subunits or changing the structure of the DNA; and (3) enhancers that lead to changes in the three-dimensional structure of DNA and bring specific promoters into the vicinity of the initiation complex. When RNA is synthesized, it undergoes further modifications which influence its stability, and therefore determine the yield of translation into protein. Capping protects mRNA from 50 exonuclease, splicing removes introns – noncoding regions – from RNA and allows translation into proper protein, and addition of poly(A) tail increases mRNA’s stability by slowing down 30 exonuclease action.
Seizures _ Seizure-Induced Gene Expression
All these regulatory mechanisms determine which genes are transcribed and at what efficiency. The number of mRNA copies of a given gene determines the translation into the protein and protein content in the cell. Additionally, translation and protein stability and trafficking are tightly regulated. The ultimate outcome is the tight control of the cell’s structure and metabolism at a given moment in time. It is, therefore, reasonable to ask whether gene transcription in the brain is regulated by seizures. Are there any regulatory mechanisms triggered by seizures? Studies have now shown that seizures can influence the transcriptional events on several levels. On the level of DNA modification, seizures regulate histone acetylation and phosphorylation on several promoters. There are also data showing that seizures affect the transcriptional machinery by activation of transcription factors, including transcription factors linked to maintenance of CpG islands like SP-1 or transcription factor complexes that provide means of tight control of transcription AP-1, cAMP response element-binding protein (CREB), the activating transcription factor-4 (ATF4), DREAM (downstream response element antagonistic modulator), NF-kappa-B, Nurr1, and others. The fact that transcriptional events can be regulated by seizures leads us to the next question: What are the target genes which expression is regulated? We address this question in the coming sections, following a brief description of the methods used to study regulation of gene expression.
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gene-specific probe to have access to the cellular compartments and to hybridize with the mRNA present in the section. Radioactively labeled probe is then detected with X-ray film. To obtain cellular details, the slides can be coated with photographic emulsion; following development, the signal will be seen as silver grains (black dots) over the area containing the mRNA of interest. If fluorescently labeled probes are used, the results of hybridization are analyzed using standard fluorescent microscopy techniques. Another powerful technique for quantifying the mRNA content is reverse transcription polymerase chain reaction (RT-PCR). The principle of this method rests on the propagation of mRNA species of interest from the minute quantities found in the starting material. For gene expression analysis, the first step is synthesis of cDNA on the basis of mRNA extracted from the studied tissue, using the enzyme called reverse transcriptase. This cDNA serves then to amplify cDNA representing the gene of interest; the procedure involves the application of a thermosTable DNA polymerase and gene-specific oligonucleotide primers which hybridize to cDNA of interest and allow the enzyme to attach to and synthesize DNA. The PCR reaction is cycled trough a series of temperature changes which provide the conditions for repeated cycles of: separation of DNA strands (e.g., at 94 C), primer annealing (usually at 42–65 C), and synthesis (at 72 C). PCR results in very efficient amplification of DNA and is successfully used for evaluation of the level of mRNA expression, especially if real-time PCR is used, which allows quantification of amplification product in each reaction cycle.
Methods Methods Used for Studies of Activation of Gene Expression Quantitative analysis of the mRNA content of selected genes of interest
One way of approaching the transcriptional activity of genes of interest is simply to evaluate the amount of its mRNA in the tissue. Through the years a number of methods have been mastered to achieve this goal. One of the oldest methods is Northern blot. For this application, RNA is isolated from the sample and then is separated by size, using gel electrophoresis. The RNA is transferred to a membrane which is then exposed to a radiolabeled, gene-specific probe. If the mRNA of interest is in the sample, the probe will specifically hybridize to the mRNA and produce a signal at the predicted size on the autoradiogram. In situ hybridization provides a means of determining the pattern of mRNA expression at the cellular level. For in situ hybridization, fixed tissue sections with preserved cellular anatomy are used. The tissue sections are mounted on slides and prepared in a way that allows the labeled,
Global analysis of gene expression During the last decade, we have witnessed the rapid development of methods for the analysis of the whole transcriptome. Such methods do not require preselection of a gene of interest, and thus provide a means for unbiased analysis of global gene expression – which may result in detection of expression level alterations of previously unknown genes. Historically, one of the first approaches to study a large fraction of the transcriptome was Differential Display. It is a RT-PCR-based method, used to identify differentially expressed genes. In differential display, mRNAs from the specimen are first reverse transcribed and then amplified using nonspecific primers. The array of bands obtained from a series of such amplifications is separated on a sequencing gel, and compared with analogous arrays from different samples. Any bands unique to a particular sample represent differentially expressed genes. The bands can be purified from the gel and sequenced. SAGE (serial analysis of gene expression) and MPSS (massively parallel signature sequencing) are methods that involve creation of large number of short cDNA tags
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(produced on the basis of studied mRNA), their subsequent sequencing, counting, and assigning to corresponding genes using bioinformatics tools. The frequency of a specific tag is related to the abundance of the corresponding mRNA in the cell, and this information allows for a comparison of mRNA expression of both known and novel genes in different samples. No a priori knowledge of the transcript sequence or mRNA abundance is required. DNA microarrays are increasingly popular in studies of gene expression. They enable evaluation of the expression levels of thousands of genes at the same time, and thereby provide global insight into transcriptional events taking place in a studied phenomenon. The principle of DNA microarray technology is the hybridization of labeled cDNA synthesized on the basis of mRNA derived from the tissue of interest, to a microarray consisting of a large number of gene probes placed with high density on a solid surface. The strength of the hybridization signal associated with a particular gene probe reflects the abundance of the respective mRNA in the sample. There are currently two main types of microarrays: spotted microarrays and Affymetrix Gene Chips. In the case of spotted microarrays, the probe is usually spotted on a surface and is hybridized with fluorescently labeled cDNA. It is possible to perform simultaneous hybridization with cDNA from two different conditions (for example control vs. treated), with each condition labeled with a different fluorochrome. Detection of transcript using GeneChips, produced by Affymetrix (Affymetrix, Inc., Santa Clara, CA), relays on perfect match versus mismatch oligonucleotide probe pairs; several probes for each gene increase the specificity and reproducibility of the quantitative results. Microarray experiments require specialized bioinformatic tools for analysis of raw hybridization data as well as for extracting biological meaning from such data. Tools that suggest assignment of experimental data to known cellular and molecular pathways are readily available, and new possibilities for in-depth analyses are constantly emerging.
Promoter activity assays
An alternative method to study regulation of transcription is evaluating the transcriptional activity of the promoter of a gene of interest (instead of quantifying its mRNA level). This approach is usually carried out by introducing into the cells under study a plasmid or viral vector containing the reference gene under control of a promoter of interest. The reference gene usually codes for fluorescent protein (luciferase or green fluorescent protein), so that it can be easily measured by fluorescence or luminescence counter. The activity of the promoter is directly related to the amount of the detected reference protein. In this way the influence of a stimulus (e.g., a seizure) on transcriptional activity of a selected promoter can be assayed.
Animal Models of Seizures The prerequisite for undertaking any experimental study on seizure-induced gene expression is identification of a proper animal model. Although human tissue coming from resections or autopsy is widely used in epilepsy research, it is of limited use in search for seizure-induced events. The molecular state of the tissue is heavily influenced by disease history and medication, which would be indistinguishable from the effects of seizures alone in such tissues. Selection of the animal model depends on the specific questions being asked, and a wide range of characterized models is available (see Pitka¨nen and coworkers, 2006 in Further Reading). In some models, seizures can be evoked in otherwise normal animals by application of proconvulsant. Many such convulsive substances have been tested; the most frequently used are GABAA receptor blockers (pentylenetetrazol, bicuculine, picrotoxin, penicillin), excitatory amino acid receptor activators (kainic acidquisqualic acid, NMDA), acetylocholine-related substances (pilocarpine), strychnine, flurothyl, topical application of metals, and many others. Another way of inducing seizures in normal, nondiseased brain is electrical stimulation of the whole brain (electroshock) or defined brain structures (hippocampus, amygdala). Depending on the specific protocol and method of seizure induction, one can produce short seizures, or status epilepticus (which has much more pronounced consequences for the brain). Seizures can also be studied in experimental animals that generate seizures spontaneously – that is, that are ‘epileptic.’ There are a number of rat (e.g., GAERS and WAG/Rij) and mouse (e.g., EL, tottering, staggerer, weaver) models that are genetically prone to develop seizures. A number of genetically modified mice have also been created that express an epileptic (i.e., spontaneous seizure) phenotype. Epilepsy can also be induced experimentally – by chemically – or by electrically induced status epilepticus, stroke, traumatic brain injury, or extensive kindling. Such animals become epileptic with time and express spontaneous seizures. Some of these models have been successfully used to study seizure-induced gene expression.
Recent Results The expression of a large variety of genes has been studied in response to seizure activity. Most of the data come from studies using status epilepticus as the seizure model, and only a few studies have monitored gene expression after single induced or spontaneous seizures. Selected papers describing changes in gene expression following brief, chemically or electrically induced seizures or kindling are summarized in Table 1. Recently, reviews of status epilepticus-induced gene expression as well as of
Seizures _ Seizure-Induced Gene Expression Table 1
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Selected studies demonstrating seizure induced changes in gene expression
Gene
Model
Author
Transcription factors c-fos
Audiogenic seizures
Shin et al. Brain Res. 1990;518:308–12. Simler et al. Neurosci Lett. 1994;175:58–62. Dragunow et al. Nature. 1987;329:441–2. Clark et al. Mol Brain Res. 1991;11:55–64. Teskey et al. Mol Brain Res. 1991;11:1–10. Simonato et al Mol Brain Res 1991;11:115–24. Morgan et al. Science 1987;237:192–7. Kanter et al. Exp Neurol 1994;129:290–8. Retchkiman et al. Neurobiol Aging 1996;17:41–4. Del Bel Ea et al. Cell Mol Neurobiol 1998;18:339–46. Saffen Proc Natl Acad Sci USA 1988;85:7795–9. Yount et al. Mol Brain Res 1994;21:219–24. Winston et al. J Neurochem 1990;54:1920–5. Marcheselli et al. J Neurosci Res 1994;37:54–61. Ebert et al. Brain Res 1995;671:338–44. Kim et al. Neuroreport 2001;12:3243–6. Kim et al. Neuroreport 2001;12:3243–6. Clark et al. Brain Res 1992;582:101–6. Helton et al. Mol Brain Res 1993;20:285–8. Saffen Proc Natl Acad Sci USA 1988;85:7795–9. Yount et al. Mol Brain Res 1994;21:219–24. Simonato et al. Mol Brain Res 1991;11:115–24. Winston et al. J Neurochem 1990;54:1920–5. Saffen Proc Natl Acad Sci USA 1988 Oct;85(20):7795–9. Saffen Proc Natl Acad Sci USA 1988;85:7795–9. Yount et al. Mol Brain Res. 1994;21:219–24. Simonato et al. Brain Res Mol Brain Res. 1991 Sep;11(2):115–24. Marcheselli et al. J Neurosci Res 1994;37:54–61. Helton et al. Mol Brain Res 1993;20:285–8.
Kindling
Metrazol
ECS
c-jun
Electrical stimulation Febrile seizures Febrile seizures Cocaine-induced seizures Metrazol
jun-B zif/268
Kindling ECS Metrazol Metrazol
ICER, CREM CREB NeuroD-related factor DREAM Zac1 Receptors, channels, transporters GluR1 GluR2 GluR3 KA1 and KA2 NMDAR1 GABAA receptor alpha 1 GABAA receptor alpha 4 GABAA receptor beta 1 GABAA receptor beta 3 GABAA receptor gamma 2 GABA(A)-receptor delta GABAA receptor alpha 1, alpha 4, beta 2, and gamma 2 Kv1.2, and Kv4.2 m1 and m3 muscarinic cholinergic receptor HCN1 and HCN2 voltage-dependent calcium channel alpha1 T-type low-voltage-activated calcium channels alpha1G
Kindling ECS Cocaine-induced seizures ECS Metrazol Metrazol Metrazol Metrazol ECS Hilar lesion Metrazol kindling Hilar lesion Hilar lesion ECS Metrazol Metrazol kindling Kindling Kindling Kindling Kindling Kindling Metrazol g-Hydroxybutyric acid induced seizures Metrazol Kindling ECS Febrile seizures Kindling Absence seizures
Fitzgerald et al. J Neurochem 1996;66:429–32. Pi et al. Brain Res. 2004;127:60–7. Konishi et al. Brain Res. 2001;97:129–36. Matsu-ura et al. Mol Brain Re. 2002;109:198–206. Valente et al. Neuroscience 2004;128:323–36. Wong et al. Epilepsy Res 1993;14:221–7. Gold et al. J Comp Neurol 1996;365:541–55. Lason et al. Brain Res 1998;785:355–8. Gold et al. J Comp Neurol 1996;365:541–55. Gold et al. J Comp Neurol 1996;365:541–55. Porter et al. Brain Res 1996;710:97–102. Jensen et al. Mol Brain Res 1997;44:157–62. Lason et al. Brain Res 1998;785:355–8. Kokaia et al. Mol Brain Res 1994;23:323–32. Clark et al. Mol Brain Res 1994;26:309–19. Clark et al. Mol Brain Res 1994;26:309–19. Kokaia et al. Mol Brain Res 1994;23:323–32. Clark et al. Mol Brain Res 1994;26:309–19. Kokaia et al. Mol Brain Res 1994;23:323–32. Penschuck et al. Mol Brain Res 1997;51:212–9. Benerjee et al. Exp Neurol 1998;154:213–23. Tsaur et al. Neuron 1992;8:1055–67. Mingo et al. Brain Res 1998;810:9–15. Brewster et al. J Neurosci 2002;22:4591–9. Hendriksen et al. Mol Brain Res 1997;50(1–2):257–66. Talley et al. Brain Res Mol Brain Res 2000;75:159–65. Continued
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Table 1
Continued
Gene
Model
Author
GLUT1 and GLUT3 Peptides and receptors Prodynorphin/dynorphin
Metrazol
Nehlig et al. Brain Res 2006;1082:32–42.
Kindling
Xie et al. Brain Res 1989;495:156–60. Moneta et al. Neurosci Lett 1990;110:273–8. Yount et al. Brain Res 1994;21:219–24. Helton et al. Mol Brain Res 1993 Nov;20(3):285–8.
Proenkephalin/enkephalin
dynorphin neuropeptide Y
Metrazol Cocaine-induced seizures ECS Cocaine induce seizures Kindling Cocaine-induced seizures Metrazol Metrazol ECS
Kindling Neuropeptide Y receptor Y1 and Y2 Neuropeptide Y receptor Y1, Y2 and Y5-R somatostatin Oxitocin vasopresin
cholecystokinin Trophic factors and receptors NGF (nerve growth factor)
bFGF (basic fibroblast growth factor) BDNF (brain derived nerve growth factor)
FGF-2 (fibroblast growth factor 2) neurotrophin-3 trkB trkC
Other glucocorticoid and mineralocorticoid receptor thyrotropin-releasing hormone neuronal nitric oxide synthase tPA (tissue plasminogen activator)
ECS Kindling Kindling ECS Metrazol Metrazol Kindling
Kindling
Hilar lesion ECS Kindling ECS Kindling
ECS Febrile seizures Hilar lesion Kindling Kindling Hilar lesion Kindling Kindling Intracerebroventricular injection of bicuculline
Hong et al. Neuropeptides 1985;5:557–60. Przewlocka et al. Neurosci Lett 1994;168:81–4. Moneta et al. Neurosci Lett 1990;110:273–8. Rosen et al. Mol Brain Res 1994;27:71–80. Helton et al. Mol Brain Res 1993;20:285–8. Yount et al. Mol Brain Res 1994;21:219–24. Yount et al. Mol Brain Res 1994;21:219–24. Mikkelsen et al. Mol Brain Res 1994;23:317–22. Madsen et al. Neuroscience 2000;98:33–9. Mikkelsen et al. J Psychiatr Res 2006;40:153–9. Kopp et al. Mol Brain Res 1999;72:17–29. Rosen et al. Mol Brain Res 1994;27:71–80. Madsen et al. Neuroscience 2000;98:33–9. Kopp et al. Mol Brain Res. 1999;72:17–29. Shinoda et al. Mol Brain Res 1989;5:243–6. Mikkelsen et al. J Psychiatr Res 2006;40:153–9. Carter et al. Neurosci Lett 1993;160:135–8. Carter et al. Neurosci Lett 1993;160:135–8. Greenwood et al. Mol Brain Res. 1994;26:286–92. Greenwood et al. Mol Brain Res 1994;24:20–6. Greenwood et al. Neurosci Lett 1997;224:66–70. Zhang et al. Mol Brain Res 1996;35:278–84. Burazom et al. Neurosci Lett 1996;209:65–8. Rocamora et al. Mol Brain Res. 1992;13:27–33. Follesa et al. Exp Neurol 1994;127:37–44. Bengzon et al. Neuroscience 1993;53:433–46. Follesa et al. Exp Neurol 1994;127:37–44. Bengzon et al. Neuroscience 1993;53:433–46. Bregola et al. Epilepsia 2000;41 S6:S122–6. Morimoto et al. Brain Res Bull 1998;45:599–605. Jacobsen et al. Brain Res 2004;1024:183–92. Kim et al. Neuroreport 2001;12:3243–6. Rocamora et al. Mol Brain Res 1992;13:27–33. Bregola et al. Epilepsia. 2000;41 S6:S122–6. Bengzon et al. Neuroscience 1993;53:433–46. Rocamora et al. Mol Brain Res. 1992;13:27–33. Bengzon et al. Neuroscience 1993;53:433–46. Bengzon et al. Neuroscience 1993;53:433–46. Mudo et al. J Mol Neurosci 1995;6:11–22.
Kindling
Clark et al. Neuroendocrinology 1994;59:451–6.
Kindling Kindling Kindling Metrazol
Rosen et al. Mol Brain Res 1994;27:71–80. Elmer et al. Neuroreport 1996;7:1335–9. Qian et al. Nature 1993;361:453–7. Popa-Wagner J Gerontol A Biol Sci Med Sci 2000;55:B242–8. Continued
Seizures _ Seizure-Induced Gene Expression
Table 1
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Continued
Gene
Model
Author
cyclophilin HSP73 (heat shock protein 73) Hsc70 (heat shock cognate protein 70) pannexin1
Hilus lession ECS, kindling ECS
Yount et al. Mol Brain Res 1992;14:139–42. Wong et al. Mol Brain Res. 1992 Mar;13(1–2):19–25. Kaneko et al. Neurosci Lett 1993;157:195–8.
4-aminopyridineinduced seizures Hilus lesion Metrazol
Zappala et al. Neuroscience 2006;141:167–78.
agrin MAP 1B (microtubule-associated protein 1B) synapsin I ferritin heavy chain glycerol phosphate dehydrogenase sialyltransferase isoenzymes ST3Gal IV, ST6GalNAc II, ST3Gal I, ST8Sia IV cyclooxygenase-2 G protein signaling protein subtypes-2,-4,-7,-8 and -10 Claudin 8 orphanin FQ/nociceptin calbindin-D28k GAD (glutamate decarboxylase)
Kindling Absence seizures Metrazol Kindling
O’Connor et al. Mol Brain Res 1995;33:277–87. Popa-Wagner et al. Exp Neurol 1997;148:73–82. Popa-Wagner et al. Neuroscience. 1999;94(2):395–403. Morimoto et al. Seizure 1998;7:229–35. Lakaye et al. Exp Neurol 2000;162:112–20. Link et al. J Neurochem 2000;75:1419–28. Okabe et al. J Neurochem 2001;77:1185–97.
Kindling ECS
Tu et al. Exp Neurol 2003;179:167–75. Gold et al. J Neurochem 2002;82:828–38.
Kindling Kindling Kindling Kindling
Lamas et al. Mol Brain Res 2002;104:250–4. Bregola et al. Epilepsia 2002;43 S5:18–9. Sonnenberg et al. Mol Brain Res 1991;9:179–90. Sonnenberg et al. Mol Brain Res 1991;9:179–90.
global analyses of gene expression have been published (see Further Reading section). In the coming sections, we summarize some of the genes that are most studied. Transcription Factors Seizures not only influence the activity of preexisting transcription factor proteins but also act by inducing their mRNA transcription. A number of seizure-induced transcription factors are immediate early genes. By definition, induction of immediate-early genes (IEGs) mRNA expression is rapid and does not depend on protein synthesis. Kindling, electroconvulsive seizures and chemoconvulsants have all been shown to markedly induce the expression of transcription factor mRNAs in the hippocampus and/or cerebral cortex. Among the best studied are components of AP-1 transcription factor (c-fos, c-jun, c-myc, jun-B, fra-1), krox-20, erg-1, erg-2, erg-3, Nurr 1 or CREM/ICER. Genes coding for transcription factors, following translation, can regulate the expression of other sets of genes. Targets genes for some transcription factors have been identified. For example, ATF4 and CREB regulate the expression of GABABR1a and GABABR1b isoforms of GABAB receptors, thus influencing neuronal excitability. The DREAM transcription factor regulates genes related to neuronal plasticity such as c-fos and preprodynorphin. The AP-1 transcription factor has been shown to regulate the expression of nerve growth factor (NGF), proenkephalin, and prodynorphin. Changes in the expression of such transcription factors can have long-lasting and pronounced consequences.
Neurotransmitter Receptors, Transporters and Ion Channels Alterations in the expression of proteins related to the ligand and voltage-gated ion channels would influence the electrophysiological properties of neurons and glia, and therefore represent obvious candidates for studies of network excitability. Many changes in the expression of mRNAs coding for neurotransmitter receptors and ion channels have been described. For example, changes in mRNA expression for selected GABAA receptor (ligand-gated chloride channels that mediate fast inhibitory synaptic transmission) subunits have been reported following kainic acid, pilocarpine, or electrically induced status epilepticus as well as following kindling-induced seizures. Changes in the expression of mRNAs encoding metabotropic GABAB receptors occur following chemically or electrically induced status epilepticus or kindling. Expression of genes coding for the glutamate receptors, the major excitatory receptors in the brain, has been extensively described. Seizures induce alteration in the expression of several glutamate-gated ion channels, including NMDA, AMPA, and kainate receptor subunits, as well as metabotropic mGLUR5 and mGLUR1 glutamate receptors. Finally, expression of genes coding for sodium channels, calcium channels, hyperpolarization-activated cyclic nucleotide-gated cation channels, and glutamate transporters can be regulated by seizures. All alterations in the gene expression of neurotransmitter receptors and ion channels will lead to changes in the quantity or subunit composition of receptors and channels, at the synapse or
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on other parts of cell membranes, and thus, influence synaptic function. Alteration in neurotransmitter transporter will modify neurotransmitter metabolism and the availability of neurotransmitter for synaptic transmission. Thus, such seizure-induced changes can have pronounced effects on neuronal excitability, and these changes can lead to functional abnormalities.
proteins like chromogranin, secretogranin or clathrin; structural synaptic proteins like dystrophin; or extracellular proteins like tenascin or extracellular metaloproteinases and their inhibitors.
Neuromodulators
Recent technologic developments allow the analysis of gene expression at the level of nearly the whole transcriptome, an approach that provides an unbiased insight into molecular events that occur in the brain following seizures or status epilepticus. Data coming from such experiments have shed new light on molecular phenomena and metabolic pathways involved in determining the effect of seizures on brain function. Studies using animal models (of both seizures and status epilepticus) have confirmed previous results about the effects of seizures on the expression of genes coding for neurotrophic factors, growth factors, ion channels and receptors, a number of plasticity-related genes, and other genes mentioned earlier. In addition, several new groups of genes have been identified, including those involved in angiogenesis, cell cycle and proliferation, protein synthesis, and chemokine signaling. Advanced bioinformatics tools have been used for data mining in order to group these genes into meaningful functional classes, and have been useful for highlighting the most prominent metabolic pathways.
In addition to channels and receptors, many other factors – such as neurotrophins or growth factors – can affect neuronal excitability. Since neurotrophins can influence neuronal network excitability, their expression following seizures has been extensively studied. For example, seizures can induce neuropeptide Y (NPY) expression, which in turn can have a significant role in suppression of seizure activity. Seizures can also regulate the expression of BDNF (increase or decrease), NGF or NT-3, as well as neurotrophin receptors. Seizures have also strong potential for regulating expression of neuropeptides like dynorphin, somatostatin or prohormone convertase. All these changes in expression levels can contribute to seizures-induced plastic changes in the neuronal circuits.
Defense Proteins Upregulation of the expression of genes coding for immune and defense proteins has been described repeatedly, mainly following status epilepticus. The proteins most often identified include interleukin-1 and-6 (IL-1, Il-6), tumor necrosis factor (TNF-a), leukemia inhibiting factor (LIF), and numerous heat shock proteins (Hsp) or heat shock cognate proteins (Hsc). These expression changes may be caused by status epilepticus-induced neurodegeneration, may reflect cell death, or may reflect the cell’s attempt to switch on tissue repair mechanisms. Some of the immune proteins, like IL-6, have been shown to have a direct influence on neuronal excitability. Structural Proteins Seizures can influence the expression of number of genes which, by different means, can influence structural features of neurons, including structure of the synapse. A few of the many such genes include homer-1 (which is induced as an immediate early gene and codes for a scaffold protein that anchors metabotropic glutamate receptors to the cytoskeleton and regulates pyramidal neuron excitability) and immediate early gene Arc (an activity-regulated cytoskeletal-associated gene; Arc mRNA is targeted to recently activated synapses and locally translated into protein). Seizures also regulate cytoskeletal protein like a-tubulin or GFAP; vesicle
Insight from Global Analysis of Gene Expression Studies
Future Goals The work of several laboratories over few past decades has uncovered a large number of genes that are induced in the brain by seizures or status epilepticus. These genes belong to a variety of functional classes, and can have effects on many aspects of cell metabolism/function. Despite this knowledge, we still lack a unifying theory about the role of seizure-induced gene expression in the brain. Especially now, in this ‘‘genomic era,’’ we are faced with many new genes with unknown roles in the brain. Before we can understand the outcome of changed expression of the whole assembly of genes, we have to address several questions for each gene. Given the phenotypic cell diversity in the brain, the expression of a given gene of interest in neuron versus microglia versus astroglia versus endothelium can have quite a different outcome from the point of view of brain function. Further, neurons and glia display great diversity, and can be divided into a large number of subclasses. In addition to cellular localization of the transcription, one should consider localization within the brain (e.g., hippocampus, cortex) or localization within brain structures (e.g., cortical layer, hippocampal division).
Seizures _ Seizure Initiation and Termination
In the case of many novel seizure-induced genes, it is still a challenge to define the consequences of altered expression because of the lack of information about the function of protein products. Even if we know the localization of transcription, in many cases the only other available information is gene sequence. The information available about the function of a gene (or gene product) in other tissues or cell lines cannot always be easily translated to the brain. Therefore, much basic research is needed before we understand the net effect of global alterations of gene expression in the brain, or even attempt to determine if a given alteration in gene expression is beneficial of harmful. See also: Epileptogenesis: Epileptogenesis and Plasticity; Gene Expression in Immature and Mature Hippocampus After Status Epilepticus; Patterns of Gene Expression during Epileptogenesis: Micro-Array Studies in Rats; Surrogate Markers for Epileptogenesis; Plasticity: Proteomic Approaches to the Analysis of Protein Alterations at the Synapse in Kindling; Post-Traumatic Epilepsy: Posttraumatic Models.
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Further Reading Crino PB (2007) Gene expression, genetics, and genomics in epilepsy: Some answers, more questions. Epilepsia 48(supplement 2): 42–50. Lodish H, Berk A, Zipursky SZ, Matsudaira P, Baltimore D, and Darnell JE (2000) Molecular Cell Biology. New York: W. H. Freeman. Lukasiuk K and Pitka¨nen A (2004) Large scale analysis of gene expression in epilepsy research: Is synthesis already possible? Neurochemisty Research 29: 1169–1178. Lukasiuk K, Dabrowski M, Adach A, and Pitkanen A (2006) Epileptogenesis-related genes revisited. Progress in Brain Research 158: 223–241. Lukasiuk K, Dingledine R, Lowenstein DH, and Pitkanen A (2007) Gene expression underling changes in network exccitability. In: Engel J and Pedley TA (eds.) Epilepsy: A Comprehensive Textbook, 2nd edn. Philadelphia, PA: Lippincott Williams & Wilkins. Majores M, Schoch S, Lie A, and Becker AJ (2007) Molecular neuropathology of temporal lobe epilepsy: Complementary approaches in animal models and human disease tissue. Epilepsia 48(supplement 2): 4–12. Sambrook J and Russell DW (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbour Laboratory Press. Pitkanen A, Schwartzkroin PA, and Moshe SL (2006) Models of Seizures and Epilepsy. Oxford: Elsevier Academic Press. Pitkanen A, Kharatishvili I, Karhunen H, et al. (2007) Epileptogenesis in experimental models. Epilepsia 48(supplement 2): 13–20. Zagulska-Szymczak S, Filipkowski RK, and Kaczmarek L (2001) Kainate-induced genes in the hippocampus: Lessons from expression patterns. Neurochemisty International 38: 485–501.
Seizure Initiation and Termination J L Stringer, Baylor College of Medicine, Houston, TX, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction A seizure is defined as an episode of excessive, synchronized neuronal activity associated with disturbance of sensation, loss of consciousness, convulsive movements, or combinations of these clinical signs. The precise combination is thought to reflect the brain regions that are synchronized in the discharge. Ever since electrical activity has been recorded in the brain, there has been reference to ‘spontaneous’ activity, the most marked form of which is seizure activity. Normally, brain activity does not appear to be highly synchronized. However, during a seizure, there is abnormal synchronization of brain circuits. Importantly, however, the occurrence of a seizure does not mean that the brain is abnormal. For example, seizures can be triggered in ‘normal’ brain by metabolic abnormalities such as hyponatremia and hypoglycemia, or by infection or inflammation. Every brain has a capacity to express a seizure.
The brain produces a variety of transient oscillations – synchronized activity in populations of neurons – that do not initiate seizures. This observation can be viewed in two ways. One is that there is a specific type of ‘trigger’ necessary to initiate a seizure. This trigger – such as an electrical shock – may itself produce excessive synchronization or may lower some threshold for seizure initiation. Alternatively, one could view the brain as having mechanisms that control the degree of synchronization; if these limiting factors fail, a seizure may begin. An example of such a process would be loss of inhibitory function in the cortex. Interestingly, if both excitation and inhibition are blocked, neurons tend to fire at a higher rate and in a more rhythmic pattern compared with activity during baseline conditions. This finding supports the idea that there are intrinsic mechanisms that determine neuronal excitability in brain networks, and that these intrinsic mechanisms could limit seizure initiation. Certainly, an active input (a trigger) could interact with loss of an
Lennox WG (1946) Science and Seizures. New York: Harper. Mormann F, Kreuz T, Andrzejak RG, et al. (2003) Epileptic seizures are preceded by a decrease in synchronization. Epilepsy Research 53: 173–185. Tsakalis K and Iasemidis LD (2006) Control aspects of a theoretical model for epileptic seizures. International Journal of Bifurcation and Chaos 16(7): 2013–2027.
Seizure-Induced Gene Expression K Lukasiuk, The Nencki Institute of Experimental Biology, Warsaw, Poland A Pitka¨nen, University of Kuopio, Kuopio, Finland; Kuopio University Hospital, Kuopio, Finland ã 2009 Elsevier Ltd. All rights reserved.
Introduction Seizures, defined as a pathological increase in neuronal synchronization, are not just the characteristic symptoms of epilepsy, but can also have direct effects on the brain. In particular, prolonged seizures can produce neuronal damage and trigger cascades of biochemical events that can have long-term effects. These seizure events activate second messengers, transcription factors and gene expression, plastic phenomena (such as mossy fiber sprouting), neurogenesis, and altered neurotransmission – all of which may underlie consequent sensory-motor, developmental, and behavioral and cognitive deficits. Seizureinduced gene expression is of particular importance, since production of new proteins (or proteins at altered levels) provides the means for long-lasting alterations in brain functions. Analysis of alterations in gene expression can single out particular molecular mechanisms involved in such long lasting, potentially harmful, effects of seizures.
Background Regulation of Gene Expression In this article we focus on seizure-induced gene expression. While approaching this subject, one should be aware that the regulation of gene expression is a very complex phenomenon. By definition, regulation of gene expression refers to the control of the amount and timing of appearance of functional gene products, that is, RNAs and proteins. The process includes transcription as well as posttranslational modification of protein, which can be modulated at any step via any number of mechanisms.
The first level of regulation of gene expression relies on modification of DNA. Chemical modification refers to methylation of CpG islands by methyltransferase, a common way for silencing a gene. Structural modifications of DNA arise from phosphorylation or methylation of histones; histone acetylation is of special importance for regulation of transcription, since histone deacetyltransferases enable the dissociation of DNA from the histones and therefore allow transcription to proceed. Progression of transcription and the efficiency of RNA synthesis depend on several components of the RNA polymerase machinery, including several regulatory proteins that bind to regulatory sites, usually located near the promoter. The regulatory proteins can be categorized according to their function as: (1) specificity factors that alter specificity of RNA polymerase for given promoter and repressors that bind to noncoding DNA sequences in the vicinity of the promoter and impede RNA polymerase’s progress along the DNA strand; (2) basal factors which position RNA polymerase at the start of transcription, and activators that enhance RNA polymerase interaction at a given promoter either by interaction with RNA polymerase subunits or changing the structure of the DNA; and (3) enhancers that lead to changes in the three-dimensional structure of DNA and bring specific promoters into the vicinity of the initiation complex. When RNA is synthesized, it undergoes further modifications which influence its stability, and therefore determine the yield of translation into protein. Capping protects mRNA from 50 exonuclease, splicing removes introns – noncoding regions – from RNA and allows translation into proper protein, and addition of poly(A) tail increases mRNA’s stability by slowing down 30 exonuclease action.
Seizures _ Seizure-Induced Gene Expression
All these regulatory mechanisms determine which genes are transcribed and at what efficiency. The number of mRNA copies of a given gene determines the translation into the protein and protein content in the cell. Additionally, translation and protein stability and trafficking are tightly regulated. The ultimate outcome is the tight control of the cell’s structure and metabolism at a given moment in time. It is, therefore, reasonable to ask whether gene transcription in the brain is regulated by seizures. Are there any regulatory mechanisms triggered by seizures? Studies have now shown that seizures can influence the transcriptional events on several levels. On the level of DNA modification, seizures regulate histone acetylation and phosphorylation on several promoters. There are also data showing that seizures affect the transcriptional machinery by activation of transcription factors, including transcription factors linked to maintenance of CpG islands like SP-1 or transcription factor complexes that provide means of tight control of transcription AP-1, cAMP response element-binding protein (CREB), the activating transcription factor-4 (ATF4), DREAM (downstream response element antagonistic modulator), NF-kappa-B, Nurr1, and others. The fact that transcriptional events can be regulated by seizures leads us to the next question: What are the target genes which expression is regulated? We address this question in the coming sections, following a brief description of the methods used to study regulation of gene expression.
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gene-specific probe to have access to the cellular compartments and to hybridize with the mRNA present in the section. Radioactively labeled probe is then detected with X-ray film. To obtain cellular details, the slides can be coated with photographic emulsion; following development, the signal will be seen as silver grains (black dots) over the area containing the mRNA of interest. If fluorescently labeled probes are used, the results of hybridization are analyzed using standard fluorescent microscopy techniques. Another powerful technique for quantifying the mRNA content is reverse transcription polymerase chain reaction (RT-PCR). The principle of this method rests on the propagation of mRNA species of interest from the minute quantities found in the starting material. For gene expression analysis, the first step is synthesis of cDNA on the basis of mRNA extracted from the studied tissue, using the enzyme called reverse transcriptase. This cDNA serves then to amplify cDNA representing the gene of interest; the procedure involves the application of a thermosTable DNA polymerase and gene-specific oligonucleotide primers which hybridize to cDNA of interest and allow the enzyme to attach to and synthesize DNA. The PCR reaction is cycled trough a series of temperature changes which provide the conditions for repeated cycles of: separation of DNA strands (e.g., at 94 C), primer annealing (usually at 42–65 C), and synthesis (at 72 C). PCR results in very efficient amplification of DNA and is successfully used for evaluation of the level of mRNA expression, especially if real-time PCR is used, which allows quantification of amplification product in each reaction cycle.
Methods Methods Used for Studies of Activation of Gene Expression Quantitative analysis of the mRNA content of selected genes of interest
One way of approaching the transcriptional activity of genes of interest is simply to evaluate the amount of its mRNA in the tissue. Through the years a number of methods have been mastered to achieve this goal. One of the oldest methods is Northern blot. For this application, RNA is isolated from the sample and then is separated by size, using gel electrophoresis. The RNA is transferred to a membrane which is then exposed to a radiolabeled, gene-specific probe. If the mRNA of interest is in the sample, the probe will specifically hybridize to the mRNA and produce a signal at the predicted size on the autoradiogram. In situ hybridization provides a means of determining the pattern of mRNA expression at the cellular level. For in situ hybridization, fixed tissue sections with preserved cellular anatomy are used. The tissue sections are mounted on slides and prepared in a way that allows the labeled,
Global analysis of gene expression During the last decade, we have witnessed the rapid development of methods for the analysis of the whole transcriptome. Such methods do not require preselection of a gene of interest, and thus provide a means for unbiased analysis of global gene expression – which may result in detection of expression level alterations of previously unknown genes. Historically, one of the first approaches to study a large fraction of the transcriptome was Differential Display. It is a RT-PCR-based method, used to identify differentially expressed genes. In differential display, mRNAs from the specimen are first reverse transcribed and then amplified using nonspecific primers. The array of bands obtained from a series of such amplifications is separated on a sequencing gel, and compared with analogous arrays from different samples. Any bands unique to a particular sample represent differentially expressed genes. The bands can be purified from the gel and sequenced. SAGE (serial analysis of gene expression) and MPSS (massively parallel signature sequencing) are methods that involve creation of large number of short cDNA tags
1304 Seizures _ Seizure-Induced Gene Expression
(produced on the basis of studied mRNA), their subsequent sequencing, counting, and assigning to corresponding genes using bioinformatics tools. The frequency of a specific tag is related to the abundance of the corresponding mRNA in the cell, and this information allows for a comparison of mRNA expression of both known and novel genes in different samples. No a priori knowledge of the transcript sequence or mRNA abundance is required. DNA microarrays are increasingly popular in studies of gene expression. They enable evaluation of the expression levels of thousands of genes at the same time, and thereby provide global insight into transcriptional events taking place in a studied phenomenon. The principle of DNA microarray technology is the hybridization of labeled cDNA synthesized on the basis of mRNA derived from the tissue of interest, to a microarray consisting of a large number of gene probes placed with high density on a solid surface. The strength of the hybridization signal associated with a particular gene probe reflects the abundance of the respective mRNA in the sample. There are currently two main types of microarrays: spotted microarrays and Affymetrix Gene Chips. In the case of spotted microarrays, the probe is usually spotted on a surface and is hybridized with fluorescently labeled cDNA. It is possible to perform simultaneous hybridization with cDNA from two different conditions (for example control vs. treated), with each condition labeled with a different fluorochrome. Detection of transcript using GeneChips, produced by Affymetrix (Affymetrix, Inc., Santa Clara, CA), relays on perfect match versus mismatch oligonucleotide probe pairs; several probes for each gene increase the specificity and reproducibility of the quantitative results. Microarray experiments require specialized bioinformatic tools for analysis of raw hybridization data as well as for extracting biological meaning from such data. Tools that suggest assignment of experimental data to known cellular and molecular pathways are readily available, and new possibilities for in-depth analyses are constantly emerging.
Promoter activity assays
An alternative method to study regulation of transcription is evaluating the transcriptional activity of the promoter of a gene of interest (instead of quantifying its mRNA level). This approach is usually carried out by introducing into the cells under study a plasmid or viral vector containing the reference gene under control of a promoter of interest. The reference gene usually codes for fluorescent protein (luciferase or green fluorescent protein), so that it can be easily measured by fluorescence or luminescence counter. The activity of the promoter is directly related to the amount of the detected reference protein. In this way the influence of a stimulus (e.g., a seizure) on transcriptional activity of a selected promoter can be assayed.
Animal Models of Seizures The prerequisite for undertaking any experimental study on seizure-induced gene expression is identification of a proper animal model. Although human tissue coming from resections or autopsy is widely used in epilepsy research, it is of limited use in search for seizure-induced events. The molecular state of the tissue is heavily influenced by disease history and medication, which would be indistinguishable from the effects of seizures alone in such tissues. Selection of the animal model depends on the specific questions being asked, and a wide range of characterized models is available (see Pitka¨nen and coworkers, 2006 in Further Reading). In some models, seizures can be evoked in otherwise normal animals by application of proconvulsant. Many such convulsive substances have been tested; the most frequently used are GABAA receptor blockers (pentylenetetrazol, bicuculine, picrotoxin, penicillin), excitatory amino acid receptor activators (kainic acidquisqualic acid, NMDA), acetylocholine-related substances (pilocarpine), strychnine, flurothyl, topical application of metals, and many others. Another way of inducing seizures in normal, nondiseased brain is electrical stimulation of the whole brain (electroshock) or defined brain structures (hippocampus, amygdala). Depending on the specific protocol and method of seizure induction, one can produce short seizures, or status epilepticus (which has much more pronounced consequences for the brain). Seizures can also be studied in experimental animals that generate seizures spontaneously – that is, that are ‘epileptic.’ There are a number of rat (e.g., GAERS and WAG/Rij) and mouse (e.g., EL, tottering, staggerer, weaver) models that are genetically prone to develop seizures. A number of genetically modified mice have also been created that express an epileptic (i.e., spontaneous seizure) phenotype. Epilepsy can also be induced experimentally – by chemically – or by electrically induced status epilepticus, stroke, traumatic brain injury, or extensive kindling. Such animals become epileptic with time and express spontaneous seizures. Some of these models have been successfully used to study seizure-induced gene expression.
Recent Results The expression of a large variety of genes has been studied in response to seizure activity. Most of the data come from studies using status epilepticus as the seizure model, and only a few studies have monitored gene expression after single induced or spontaneous seizures. Selected papers describing changes in gene expression following brief, chemically or electrically induced seizures or kindling are summarized in Table 1. Recently, reviews of status epilepticus-induced gene expression as well as of
Seizures _ Seizure-Induced Gene Expression Table 1
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Selected studies demonstrating seizure induced changes in gene expression
Gene
Model
Author
Transcription factors c-fos
Audiogenic seizures
Shin et al. Brain Res. 1990;518:308–12. Simler et al. Neurosci Lett. 1994;175:58–62. Dragunow et al. Nature. 1987;329:441–2. Clark et al. Mol Brain Res. 1991;11:55–64. Teskey et al. Mol Brain Res. 1991;11:1–10. Simonato et al Mol Brain Res 1991;11:115–24. Morgan et al. Science 1987;237:192–7. Kanter et al. Exp Neurol 1994;129:290–8. Retchkiman et al. Neurobiol Aging 1996;17:41–4. Del Bel Ea et al. Cell Mol Neurobiol 1998;18:339–46. Saffen Proc Natl Acad Sci USA 1988;85:7795–9. Yount et al. Mol Brain Res 1994;21:219–24. Winston et al. J Neurochem 1990;54:1920–5. Marcheselli et al. J Neurosci Res 1994;37:54–61. Ebert et al. Brain Res 1995;671:338–44. Kim et al. Neuroreport 2001;12:3243–6. Kim et al. Neuroreport 2001;12:3243–6. Clark et al. Brain Res 1992;582:101–6. Helton et al. Mol Brain Res 1993;20:285–8. Saffen Proc Natl Acad Sci USA 1988;85:7795–9. Yount et al. Mol Brain Res 1994;21:219–24. Simonato et al. Mol Brain Res 1991;11:115–24. Winston et al. J Neurochem 1990;54:1920–5. Saffen Proc Natl Acad Sci USA 1988 Oct;85(20):7795–9. Saffen Proc Natl Acad Sci USA 1988;85:7795–9. Yount et al. Mol Brain Res. 1994;21:219–24. Simonato et al. Brain Res Mol Brain Res. 1991 Sep;11(2):115–24. Marcheselli et al. J Neurosci Res 1994;37:54–61. Helton et al. Mol Brain Res 1993;20:285–8.
Kindling
Metrazol
ECS
c-jun
Electrical stimulation Febrile seizures Febrile seizures Cocaine-induced seizures Metrazol
jun-B zif/268
Kindling ECS Metrazol Metrazol
ICER, CREM CREB NeuroD-related factor DREAM Zac1 Receptors, channels, transporters GluR1 GluR2 GluR3 KA1 and KA2 NMDAR1 GABAA receptor alpha 1 GABAA receptor alpha 4 GABAA receptor beta 1 GABAA receptor beta 3 GABAA receptor gamma 2 GABA(A)-receptor delta GABAA receptor alpha 1, alpha 4, beta 2, and gamma 2 Kv1.2, and Kv4.2 m1 and m3 muscarinic cholinergic receptor HCN1 and HCN2 voltage-dependent calcium channel alpha1 T-type low-voltage-activated calcium channels alpha1G
Kindling ECS Cocaine-induced seizures ECS Metrazol Metrazol Metrazol Metrazol ECS Hilar lesion Metrazol kindling Hilar lesion Hilar lesion ECS Metrazol Metrazol kindling Kindling Kindling Kindling Kindling Kindling Metrazol g-Hydroxybutyric acid induced seizures Metrazol Kindling ECS Febrile seizures Kindling Absence seizures
Fitzgerald et al. J Neurochem 1996;66:429–32. Pi et al. Brain Res. 2004;127:60–7. Konishi et al. Brain Res. 2001;97:129–36. Matsu-ura et al. Mol Brain Re. 2002;109:198–206. Valente et al. Neuroscience 2004;128:323–36. Wong et al. Epilepsy Res 1993;14:221–7. Gold et al. J Comp Neurol 1996;365:541–55. Lason et al. Brain Res 1998;785:355–8. Gold et al. J Comp Neurol 1996;365:541–55. Gold et al. J Comp Neurol 1996;365:541–55. Porter et al. Brain Res 1996;710:97–102. Jensen et al. Mol Brain Res 1997;44:157–62. Lason et al. Brain Res 1998;785:355–8. Kokaia et al. Mol Brain Res 1994;23:323–32. Clark et al. Mol Brain Res 1994;26:309–19. Clark et al. Mol Brain Res 1994;26:309–19. Kokaia et al. Mol Brain Res 1994;23:323–32. Clark et al. Mol Brain Res 1994;26:309–19. Kokaia et al. Mol Brain Res 1994;23:323–32. Penschuck et al. Mol Brain Res 1997;51:212–9. Benerjee et al. Exp Neurol 1998;154:213–23. Tsaur et al. Neuron 1992;8:1055–67. Mingo et al. Brain Res 1998;810:9–15. Brewster et al. J Neurosci 2002;22:4591–9. Hendriksen et al. Mol Brain Res 1997;50(1–2):257–66. Talley et al. Brain Res Mol Brain Res 2000;75:159–65. Continued
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Table 1
Continued
Gene
Model
Author
GLUT1 and GLUT3 Peptides and receptors Prodynorphin/dynorphin
Metrazol
Nehlig et al. Brain Res 2006;1082:32–42.
Kindling
Xie et al. Brain Res 1989;495:156–60. Moneta et al. Neurosci Lett 1990;110:273–8. Yount et al. Brain Res 1994;21:219–24. Helton et al. Mol Brain Res 1993 Nov;20(3):285–8.
Proenkephalin/enkephalin
dynorphin neuropeptide Y
Metrazol Cocaine-induced seizures ECS Cocaine induce seizures Kindling Cocaine-induced seizures Metrazol Metrazol ECS
Kindling Neuropeptide Y receptor Y1 and Y2 Neuropeptide Y receptor Y1, Y2 and Y5-R somatostatin Oxitocin vasopresin
cholecystokinin Trophic factors and receptors NGF (nerve growth factor)
bFGF (basic fibroblast growth factor) BDNF (brain derived nerve growth factor)
FGF-2 (fibroblast growth factor 2) neurotrophin-3 trkB trkC
Other glucocorticoid and mineralocorticoid receptor thyrotropin-releasing hormone neuronal nitric oxide synthase tPA (tissue plasminogen activator)
ECS Kindling Kindling ECS Metrazol Metrazol Kindling
Kindling
Hilar lesion ECS Kindling ECS Kindling
ECS Febrile seizures Hilar lesion Kindling Kindling Hilar lesion Kindling Kindling Intracerebroventricular injection of bicuculline
Hong et al. Neuropeptides 1985;5:557–60. Przewlocka et al. Neurosci Lett 1994;168:81–4. Moneta et al. Neurosci Lett 1990;110:273–8. Rosen et al. Mol Brain Res 1994;27:71–80. Helton et al. Mol Brain Res 1993;20:285–8. Yount et al. Mol Brain Res 1994;21:219–24. Yount et al. Mol Brain Res 1994;21:219–24. Mikkelsen et al. Mol Brain Res 1994;23:317–22. Madsen et al. Neuroscience 2000;98:33–9. Mikkelsen et al. J Psychiatr Res 2006;40:153–9. Kopp et al. Mol Brain Res 1999;72:17–29. Rosen et al. Mol Brain Res 1994;27:71–80. Madsen et al. Neuroscience 2000;98:33–9. Kopp et al. Mol Brain Res. 1999;72:17–29. Shinoda et al. Mol Brain Res 1989;5:243–6. Mikkelsen et al. J Psychiatr Res 2006;40:153–9. Carter et al. Neurosci Lett 1993;160:135–8. Carter et al. Neurosci Lett 1993;160:135–8. Greenwood et al. Mol Brain Res. 1994;26:286–92. Greenwood et al. Mol Brain Res 1994;24:20–6. Greenwood et al. Neurosci Lett 1997;224:66–70. Zhang et al. Mol Brain Res 1996;35:278–84. Burazom et al. Neurosci Lett 1996;209:65–8. Rocamora et al. Mol Brain Res. 1992;13:27–33. Follesa et al. Exp Neurol 1994;127:37–44. Bengzon et al. Neuroscience 1993;53:433–46. Follesa et al. Exp Neurol 1994;127:37–44. Bengzon et al. Neuroscience 1993;53:433–46. Bregola et al. Epilepsia 2000;41 S6:S122–6. Morimoto et al. Brain Res Bull 1998;45:599–605. Jacobsen et al. Brain Res 2004;1024:183–92. Kim et al. Neuroreport 2001;12:3243–6. Rocamora et al. Mol Brain Res 1992;13:27–33. Bregola et al. Epilepsia. 2000;41 S6:S122–6. Bengzon et al. Neuroscience 1993;53:433–46. Rocamora et al. Mol Brain Res. 1992;13:27–33. Bengzon et al. Neuroscience 1993;53:433–46. Bengzon et al. Neuroscience 1993;53:433–46. Mudo et al. J Mol Neurosci 1995;6:11–22.
Kindling
Clark et al. Neuroendocrinology 1994;59:451–6.
Kindling Kindling Kindling Metrazol
Rosen et al. Mol Brain Res 1994;27:71–80. Elmer et al. Neuroreport 1996;7:1335–9. Qian et al. Nature 1993;361:453–7. Popa-Wagner J Gerontol A Biol Sci Med Sci 2000;55:B242–8. Continued
Seizures _ Seizure-Induced Gene Expression
Table 1
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Continued
Gene
Model
Author
cyclophilin HSP73 (heat shock protein 73) Hsc70 (heat shock cognate protein 70) pannexin1
Hilus lession ECS, kindling ECS
Yount et al. Mol Brain Res 1992;14:139–42. Wong et al. Mol Brain Res. 1992 Mar;13(1–2):19–25. Kaneko et al. Neurosci Lett 1993;157:195–8.
4-aminopyridineinduced seizures Hilus lesion Metrazol
Zappala et al. Neuroscience 2006;141:167–78.
agrin MAP 1B (microtubule-associated protein 1B) synapsin I ferritin heavy chain glycerol phosphate dehydrogenase sialyltransferase isoenzymes ST3Gal IV, ST6GalNAc II, ST3Gal I, ST8Sia IV cyclooxygenase-2 G protein signaling protein subtypes-2,-4,-7,-8 and -10 Claudin 8 orphanin FQ/nociceptin calbindin-D28k GAD (glutamate decarboxylase)
Kindling Absence seizures Metrazol Kindling
O’Connor et al. Mol Brain Res 1995;33:277–87. Popa-Wagner et al. Exp Neurol 1997;148:73–82. Popa-Wagner et al. Neuroscience. 1999;94(2):395–403. Morimoto et al. Seizure 1998;7:229–35. Lakaye et al. Exp Neurol 2000;162:112–20. Link et al. J Neurochem 2000;75:1419–28. Okabe et al. J Neurochem 2001;77:1185–97.
Kindling ECS
Tu et al. Exp Neurol 2003;179:167–75. Gold et al. J Neurochem 2002;82:828–38.
Kindling Kindling Kindling Kindling
Lamas et al. Mol Brain Res 2002;104:250–4. Bregola et al. Epilepsia 2002;43 S5:18–9. Sonnenberg et al. Mol Brain Res 1991;9:179–90. Sonnenberg et al. Mol Brain Res 1991;9:179–90.
global analyses of gene expression have been published (see Further Reading section). In the coming sections, we summarize some of the genes that are most studied. Transcription Factors Seizures not only influence the activity of preexisting transcription factor proteins but also act by inducing their mRNA transcription. A number of seizure-induced transcription factors are immediate early genes. By definition, induction of immediate-early genes (IEGs) mRNA expression is rapid and does not depend on protein synthesis. Kindling, electroconvulsive seizures and chemoconvulsants have all been shown to markedly induce the expression of transcription factor mRNAs in the hippocampus and/or cerebral cortex. Among the best studied are components of AP-1 transcription factor (c-fos, c-jun, c-myc, jun-B, fra-1), krox-20, erg-1, erg-2, erg-3, Nurr 1 or CREM/ICER. Genes coding for transcription factors, following translation, can regulate the expression of other sets of genes. Targets genes for some transcription factors have been identified. For example, ATF4 and CREB regulate the expression of GABABR1a and GABABR1b isoforms of GABAB receptors, thus influencing neuronal excitability. The DREAM transcription factor regulates genes related to neuronal plasticity such as c-fos and preprodynorphin. The AP-1 transcription factor has been shown to regulate the expression of nerve growth factor (NGF), proenkephalin, and prodynorphin. Changes in the expression of such transcription factors can have long-lasting and pronounced consequences.
Neurotransmitter Receptors, Transporters and Ion Channels Alterations in the expression of proteins related to the ligand and voltage-gated ion channels would influence the electrophysiological properties of neurons and glia, and therefore represent obvious candidates for studies of network excitability. Many changes in the expression of mRNAs coding for neurotransmitter receptors and ion channels have been described. For example, changes in mRNA expression for selected GABAA receptor (ligand-gated chloride channels that mediate fast inhibitory synaptic transmission) subunits have been reported following kainic acid, pilocarpine, or electrically induced status epilepticus as well as following kindling-induced seizures. Changes in the expression of mRNAs encoding metabotropic GABAB receptors occur following chemically or electrically induced status epilepticus or kindling. Expression of genes coding for the glutamate receptors, the major excitatory receptors in the brain, has been extensively described. Seizures induce alteration in the expression of several glutamate-gated ion channels, including NMDA, AMPA, and kainate receptor subunits, as well as metabotropic mGLUR5 and mGLUR1 glutamate receptors. Finally, expression of genes coding for sodium channels, calcium channels, hyperpolarization-activated cyclic nucleotide-gated cation channels, and glutamate transporters can be regulated by seizures. All alterations in the gene expression of neurotransmitter receptors and ion channels will lead to changes in the quantity or subunit composition of receptors and channels, at the synapse or
1308 Seizures _ Seizure-Induced Gene Expression
on other parts of cell membranes, and thus, influence synaptic function. Alteration in neurotransmitter transporter will modify neurotransmitter metabolism and the availability of neurotransmitter for synaptic transmission. Thus, such seizure-induced changes can have pronounced effects on neuronal excitability, and these changes can lead to functional abnormalities.
proteins like chromogranin, secretogranin or clathrin; structural synaptic proteins like dystrophin; or extracellular proteins like tenascin or extracellular metaloproteinases and their inhibitors.
Neuromodulators
Recent technologic developments allow the analysis of gene expression at the level of nearly the whole transcriptome, an approach that provides an unbiased insight into molecular events that occur in the brain following seizures or status epilepticus. Data coming from such experiments have shed new light on molecular phenomena and metabolic pathways involved in determining the effect of seizures on brain function. Studies using animal models (of both seizures and status epilepticus) have confirmed previous results about the effects of seizures on the expression of genes coding for neurotrophic factors, growth factors, ion channels and receptors, a number of plasticity-related genes, and other genes mentioned earlier. In addition, several new groups of genes have been identified, including those involved in angiogenesis, cell cycle and proliferation, protein synthesis, and chemokine signaling. Advanced bioinformatics tools have been used for data mining in order to group these genes into meaningful functional classes, and have been useful for highlighting the most prominent metabolic pathways.
In addition to channels and receptors, many other factors – such as neurotrophins or growth factors – can affect neuronal excitability. Since neurotrophins can influence neuronal network excitability, their expression following seizures has been extensively studied. For example, seizures can induce neuropeptide Y (NPY) expression, which in turn can have a significant role in suppression of seizure activity. Seizures can also regulate the expression of BDNF (increase or decrease), NGF or NT-3, as well as neurotrophin receptors. Seizures have also strong potential for regulating expression of neuropeptides like dynorphin, somatostatin or prohormone convertase. All these changes in expression levels can contribute to seizures-induced plastic changes in the neuronal circuits.
Defense Proteins Upregulation of the expression of genes coding for immune and defense proteins has been described repeatedly, mainly following status epilepticus. The proteins most often identified include interleukin-1 and-6 (IL-1, Il-6), tumor necrosis factor (TNF-a), leukemia inhibiting factor (LIF), and numerous heat shock proteins (Hsp) or heat shock cognate proteins (Hsc). These expression changes may be caused by status epilepticus-induced neurodegeneration, may reflect cell death, or may reflect the cell’s attempt to switch on tissue repair mechanisms. Some of the immune proteins, like IL-6, have been shown to have a direct influence on neuronal excitability. Structural Proteins Seizures can influence the expression of number of genes which, by different means, can influence structural features of neurons, including structure of the synapse. A few of the many such genes include homer-1 (which is induced as an immediate early gene and codes for a scaffold protein that anchors metabotropic glutamate receptors to the cytoskeleton and regulates pyramidal neuron excitability) and immediate early gene Arc (an activity-regulated cytoskeletal-associated gene; Arc mRNA is targeted to recently activated synapses and locally translated into protein). Seizures also regulate cytoskeletal protein like a-tubulin or GFAP; vesicle
Insight from Global Analysis of Gene Expression Studies
Future Goals The work of several laboratories over few past decades has uncovered a large number of genes that are induced in the brain by seizures or status epilepticus. These genes belong to a variety of functional classes, and can have effects on many aspects of cell metabolism/function. Despite this knowledge, we still lack a unifying theory about the role of seizure-induced gene expression in the brain. Especially now, in this ‘‘genomic era,’’ we are faced with many new genes with unknown roles in the brain. Before we can understand the outcome of changed expression of the whole assembly of genes, we have to address several questions for each gene. Given the phenotypic cell diversity in the brain, the expression of a given gene of interest in neuron versus microglia versus astroglia versus endothelium can have quite a different outcome from the point of view of brain function. Further, neurons and glia display great diversity, and can be divided into a large number of subclasses. In addition to cellular localization of the transcription, one should consider localization within the brain (e.g., hippocampus, cortex) or localization within brain structures (e.g., cortical layer, hippocampal division).
Seizures _ Seizure Initiation and Termination
In the case of many novel seizure-induced genes, it is still a challenge to define the consequences of altered expression because of the lack of information about the function of protein products. Even if we know the localization of transcription, in many cases the only other available information is gene sequence. The information available about the function of a gene (or gene product) in other tissues or cell lines cannot always be easily translated to the brain. Therefore, much basic research is needed before we understand the net effect of global alterations of gene expression in the brain, or even attempt to determine if a given alteration in gene expression is beneficial of harmful. See also: Epileptogenesis: Epileptogenesis and Plasticity; Gene Expression in Immature and Mature Hippocampus After Status Epilepticus; Patterns of Gene Expression during Epileptogenesis: Micro-Array Studies in Rats; Surrogate Markers for Epileptogenesis; Plasticity: Proteomic Approaches to the Analysis of Protein Alterations at the Synapse in Kindling; Post-Traumatic Epilepsy: Posttraumatic Models.
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Further Reading Crino PB (2007) Gene expression, genetics, and genomics in epilepsy: Some answers, more questions. Epilepsia 48(supplement 2): 42–50. Lodish H, Berk A, Zipursky SZ, Matsudaira P, Baltimore D, and Darnell JE (2000) Molecular Cell Biology. New York: W. H. Freeman. Lukasiuk K and Pitka¨nen A (2004) Large scale analysis of gene expression in epilepsy research: Is synthesis already possible? Neurochemisty Research 29: 1169–1178. Lukasiuk K, Dabrowski M, Adach A, and Pitkanen A (2006) Epileptogenesis-related genes revisited. Progress in Brain Research 158: 223–241. Lukasiuk K, Dingledine R, Lowenstein DH, and Pitkanen A (2007) Gene expression underling changes in network exccitability. In: Engel J and Pedley TA (eds.) Epilepsy: A Comprehensive Textbook, 2nd edn. Philadelphia, PA: Lippincott Williams & Wilkins. Majores M, Schoch S, Lie A, and Becker AJ (2007) Molecular neuropathology of temporal lobe epilepsy: Complementary approaches in animal models and human disease tissue. Epilepsia 48(supplement 2): 4–12. Sambrook J and Russell DW (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbour Laboratory Press. Pitkanen A, Schwartzkroin PA, and Moshe SL (2006) Models of Seizures and Epilepsy. Oxford: Elsevier Academic Press. Pitkanen A, Kharatishvili I, Karhunen H, et al. (2007) Epileptogenesis in experimental models. Epilepsia 48(supplement 2): 13–20. Zagulska-Szymczak S, Filipkowski RK, and Kaczmarek L (2001) Kainate-induced genes in the hippocampus: Lessons from expression patterns. Neurochemisty International 38: 485–501.
Seizure Initiation and Termination J L Stringer, Baylor College of Medicine, Houston, TX, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction A seizure is defined as an episode of excessive, synchronized neuronal activity associated with disturbance of sensation, loss of consciousness, convulsive movements, or combinations of these clinical signs. The precise combination is thought to reflect the brain regions that are synchronized in the discharge. Ever since electrical activity has been recorded in the brain, there has been reference to ‘spontaneous’ activity, the most marked form of which is seizure activity. Normally, brain activity does not appear to be highly synchronized. However, during a seizure, there is abnormal synchronization of brain circuits. Importantly, however, the occurrence of a seizure does not mean that the brain is abnormal. For example, seizures can be triggered in ‘normal’ brain by metabolic abnormalities such as hyponatremia and hypoglycemia, or by infection or inflammation. Every brain has a capacity to express a seizure.
The brain produces a variety of transient oscillations – synchronized activity in populations of neurons – that do not initiate seizures. This observation can be viewed in two ways. One is that there is a specific type of ‘trigger’ necessary to initiate a seizure. This trigger – such as an electrical shock – may itself produce excessive synchronization or may lower some threshold for seizure initiation. Alternatively, one could view the brain as having mechanisms that control the degree of synchronization; if these limiting factors fail, a seizure may begin. An example of such a process would be loss of inhibitory function in the cortex. Interestingly, if both excitation and inhibition are blocked, neurons tend to fire at a higher rate and in a more rhythmic pattern compared with activity during baseline conditions. This finding supports the idea that there are intrinsic mechanisms that determine neuronal excitability in brain networks, and that these intrinsic mechanisms could limit seizure initiation. Certainly, an active input (a trigger) could interact with loss of an