WO2008041244A2 - A method for the identification of potential biomarkers and drug targets in brain plasticity related disorders - Google Patents

Abstract

The invention relates to identification of potential biomarkers and drug targets in brain plasticity related disorders. In particular, the invention describes a system level model of brain plasticity related disorders in the fruit fly Drosophila melanogaster, thereby identifying potential biomarkers and drug targets in epileptogenesis and associated conditions. The invention also provides a means of screening therapeutic agents against epilepsy and related disorders using gene expression profiling in Drosophila.

Description

"A METHOD FOR THE IDENTIFICATION OF POTENTIAL BIOMARKERS AND DRUG TARGETS IN BRAIN PLASTICITY RELATED DISORDERS"

FIELD OF THE INVENTION The invention relates to identification of potential biomarkers and durg target candidates using a systems model of Drosophila melanogaster for the brain plasticity related disorders. Invention provides microarray gene expression profiles associated with kindling epileptogensis-like plasticity in fly. The fly genes that are up and down-regulated by convulsant and antiepileptic drugs have been identified. Potential biomarkers of epileptogenesis and related conditions are identified on the basis of expression profiling in the fly model. Potential drug targets for developing therapeutic agents against epilepsy and related disorders have been identified based on transcriptomic analysis. A method for screening antiepeileptic, antiepileptogenic, disease modifying, and neuroprotective agents using expression profiling is provided.

BACKGROUND OF THE INVENTION AND DESCRIPTION OF PRIOR ART

In general, the antiepileptic drugs (AEDs) being currently used are symptomatic in that they inhibit seizure but do not modify the course of or prevent epileptogenesis (McNamara et al. 2006; Garriga- Canut et al. 2006). A system-level understanding of epileptogenesis is expected to facilitate development of novel antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents (Gorter et al. 2006). Inherent complexity of mammalian brain however does not allow the available rodent models of epileptogenesis to be subjected to the reiterative methods of systems modeling (Gorter et al. 2006). It is this gap of enabling invention that we used a systems biology approach to identify potential biomarkers and drug targets in neurological and neuropsychiatric diseases. Kindling is an animal model of brain plasticity in which repeated activation of neural pathways through administration of a subconvulsant electrical or chemical stimulus produces a gradually increasing electroencephalographic and behavioral response which, after withdrawal of the stimulus, eventually progresses to spontaneous seizures (Goddard et al. 1969; Pavlova et al 2006). Kindling in rodents is widely used to model epileptogenesis (McNamara et al. 2006; Garriga-Canut et al. 2006). Seizure in Drosophila and man have several similarities, and the utility of the fruit fly as a genetic model system for studying human seizure disorders, seizure-susceptibility, and AED screening has clearly been demonstrated (Hekmat-Scafe et al. 2006; Stilwell et al. 2006; Fergestad et al. 2006; Sharma & Kumar, 2001). Behavioral model of drug-induced neural plasticity in Drosophila has also been described (Sharma, 2005). However, no attempt has been made so far to develop a systems model of kindling in fly. In the present invention, a Drosophila model of kindling epileptogenesis-like plasticity has been used to discern system-level characteristics of kindling acquisition, kindled state, and therapeutic action of drugs used in the treatment of epilepsy and other central nervous system (CNS) disorders. In an inventive step, potential biomarkers and drug targets in such diseases have been identified using the model so developed. It is important to note here that the above discussion or citation of references shall not be construed as an admission that any reference is prior art to the present invention.

OBJECTS OF THE INVENTION

The main object of the invention is to provide a method for detecting potential biomarkers for brain plasticity related disorders.

Another object of the invention is to provide a method for identifying potential drug targets for developing antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents.

Yet another object of the invention is detection of potential biomarkers for brain plasticity related disorders.

Still another object of the invention is identification of potential drug targets for developing antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents.

Yet another object of the invention is to provide a method for screening of potential antiepileptic, antiepileptogenic, disease-modifying, and neuroprotective agents based on gene expression profiling in Drosophila.

SUMMARY OF THE INVENTION

The invention provides a method for identifying potential biomarkers and drug targets in brain plasticity related disorders. The said method is based on systems modeling of kindling epileptogenesis-like plasticity in the fruit fly Drosophila melanogaster. Using the systems model so developed, the invention describes detection of potential biomarkers in epileptogenesis and associated disorders. Further, the invention describes identification of drug targets for developing antiepileptic, antiepileptogenic, disease- modifying, and neuroprotective agents.

Accordingly, the present invention provides a method for identification of potential biomarkers for epileptogenesis and related brain plasticity disorders, said method consisting the steps of:

(a) treating Drosophila flies with either convulsant or antiepileptic drugs in a chronic manner;

(b) extracting RNA from the heads of treated flies of step [a] at several time-points secondary to drug treatments; (c) generating microarray gene expression profiles specific for convulsant and antiepileptic drugs respectively using the RNA extracted from step [b];

(d) identifying genes that are up- and down-regulated by convulsant and antiepileptic drugs from the microarray of step [c]; (e) identifying genes which are down- and up-regulated respectively by convulsant and antiepileptic drugs in the fly model and the homologs downregulated in established rodent models of epilepsy development as candidate biomarkers of epileptogenesis.

Another aspect of the invention is to provide a method for identification of potential drug targets against epileptogenesis and related brain plasticity disorders, said method comprising the steps of:

[i] comparing fly genes that are regulated by different antiepileptic drugs in Drosophila head, in terms of biological processes they over-represent; [ii] identifying fly genes that are similarly regulated by different antiepileptic drugs, as potential drug targets; [iii] confirming the candidature of the potential drug targets by verifying the biological processes they represent.

In an embodiment of the invention, the products of Drosophila genes Aldolase (CG6058) and Glutathione S transferase D4 (CGl 1512) are identified as biomarkers of kindling-epileptogenesis like plasticity in the fly model.

In still another embodiment of the invention, the products of homologs of fly genes CG6058 and CGl 1512 are identified as potential biomarkers of epileptogenesis and associated disorders.

In yet another embodiment of the invention, the products of Drosophila genes Glutamate oxaloacetate transaminase 2 (CG4233) and Sorbitol dehydrogenase 1 (CG1982) are identified as targets of antiepileptic drugs in fly model of kindling-epileptogenesis like plasticity.

In another embodiment of the invention, the products of homologs of fly genes CG4233 and CG1982, are identified as potential targets for developing drugs against epilepsy and related disorders.

The invention further provides a means of using products of human homologs of fly genes CG6058 and CGl 1512 as biomarkers of epileptogenesis in at-risk individuals.

The invention also provides a means of using products of human homologs of fly genes CG4233 and CGl 982 as drug targets for developing therapeutic agents against epilepsy and related brain disorders. The invention also provides a method of screening antiepileptic, antiepileptogenic, disease modifying and neuroprotective agents using Drosophila gene expression profiles.

5 DETAILED DESCRIPTION OF THE FIGURES:

Figure 1. Climbing speed in cm/sec using DIAS. Speed was measured after seven days of chronic PTZ treatment and seven days after PTZ withdrawal i.e. on 7^th day and 14^th day from the beginning of the treatment, respectively. The difference in the speeds of the two groups compared was found to be significant (n = 16, 7^th day; n — 9, 14^th day). Asterisk indicates significant difference between NF and

10 PTZ; /rvalue is provided over asterisk). As observed for the semi-manual method used in the experiments, DIAS also showed decreased and increased speed at the two time points, in that order. It is notable here that absolute speed between the two methods may differ because of difference in the assays. Whereas semi-manual method calculates climbing speed based on the time spent in activity (dots), DIAS analysis considered the entire traceable path, including momentary rest or jumps, and the

15 time spent.

Figure 2. Reverse northern hybridization. We selected CGl 1064, as expression of this gene was found altered at all three time points of PTZ kindling (downregulated), for validation of microarray results using reverse northern analysis. Note that CGl 1064 is downregulated at all time points •20

Figure 3. Validation of microarray gene expression profiling by Real Time PCR. Downregulation and upregulation of genes in PTZ kindling and AEDs respectively were all validated except that Cyp28 was found to be downregulated in NaVP, in contrast to upregulation in microarray profiling.

25 DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the development of a fly model of kindling epileptogenesis. Kindling- like phenomena is considered relevant in various neuropsychiatric conditions (Mortazavi et al. 2005; Becker et al. 2006; Bob et al. 2006). Further, in addition to treatment of epilepsy, NaVP, an AED used in developing the present model, is used in treating various other disorders and diseases such as acute

30 mania, bipolar disorder, panic disorder, alcoholism, aggression, personality disorder, migraine headache, delirium, neuromuscular syndrome, cancer etc. (Yatham et al., 2005; Van Ameringen et al., 2004; Ichikawa et al., 2005; Book et al., 2005; Hollander et al., 2005; Reiter et al., 2005; Bourgeois et al., 2005; Vasconcelos and Dalakas, 2003; Blaheta et al., 2005). Similarly, LEV, another AED used in developing the present model, is known to be of use in various neurological and psychiatric conditions

35 such as mood disorders, posttraumatic stress disorders, social anxiety disorder, neuropathic pain, startle disease, drug withdrawal etc. (Davidson, 2006; Kinrys et al., 2006; Luef and Loscher, 2007; Matsuura, 2007; Muralidharan and Bhagwagar, 2006; Lamberty et al., 2002). We describe below, in sequence, various parts of our invention.

Development of fly systems model PTZ kindling in rodents is induced by repeated injection of a subconvulsant dose of the GABA antagonist over several weeks. The kindling results in partially and fully kindled animals with the latter group showing clonic-tonic seizures. Once kindled, the state of behavioral hyperexcitability persists for up to several weeks after discontinuation of chemoconvulsant treatment (De Sarro et al. 2000; Becker et al. 2006). We simulated PTZ induced kindling-like plasticity in Drosophila by empirically determining a regime in which seven days of chronic PTZ treatment and seven following days of PTZ discontinuation respectively- caused a decrease and increase in startle-induced climbing speed in flies (Table 1). The startle-induced vertical locomotor effect of PTZ was specific because spontaneous horizontal locomotor activities and various non-locomotor characteristics, namely, courtship duration, fertility, and body weight, were not found altered after either chronic PTZ phase or withdrawal period (Table 2). Moreover, although horizontal locomotor speed was decreased by chronic PTZ, any long term effect on this aspect of locomotor behavior was not observed after drug withdrawal. Climbing speed was thus found to be the behavior of choice for further evaluation. Kindling and postkindling behaviors in rats and mice include alteration in locomotor activity (Beekman et al. 1998; Franke et al.2001; Mortazavi et al. 2005). Seizure-like activity in fly is also known to be associated with loss of motor coordination, and altered locomotor activities (Wang et al. 2004). Further, increased seizure susceptibility is known to occur via excitatory GABAergic signaling in Drosophila (De Sarro et al. 2000). Therefore, notwithstanding our failure in observing explicit seizure-like symptoms during either chronic PTZ treatment or withdrawal period, we considered our locomotor behavior model promising enough for further validation using AEDs.

We reasoned that if seven day long chronic PTZ treatment indeed parallels kindling acquisition and the locomotor alteration on 7^th day to kindled-seizure, then AEDs should be able to rescue flies from PTZ induced climbing deficiency. Sodium valproate (NaVP) and levetiracetam (LEV) are known to suppress both kindling development as well as kindled seizure in rodent models (Loscher 2002). To test prophylactic activity in our model, we concomitantly treated the flies with PTZ and either AED for first four days followed by only PTZ treatment for next three days, and then measured the climbing speed on 7^th day (Table 3). To test symptomatic activity, we treated with PTZ alone for six days followed by one day of concomitant treatment with PTZ and either AED, and then measured the climbing speed on 7^th day (Table 4). On the basis of j?-values, LEV showed strong antiepileptogenic and weak antiepileptic activities, whereas NaVP showed just the reverse. Whereas both LEV and NaVP have been identified as having anti-kindling activity in rodent models (Loscher 2002), the latter has not shown an antiepileptogenic activity in clinical trials (Temkin et al. 2001; Walker et al. 2002). Though clinical trials evaluating antiepileptogenic potential of LEV are yet to be reported, evidence suggests that the drug might possess a prophylactic effect (Walker et al. 2002; Ji-qun et al. 2005). Since the AED profiles predicted by our model were in consonance with the ones known from reports on rodent kindling and clinical trials, our model became established as a model of epileptogenesis.

We produced genome-level expression profiles of fly heads at three time points during the course of seven day long chronic PTZ treatment - 12 hrs, the beginning phase; 2^nd day, the latent phase; and 7^th day, the behaviorally expressive phase. All three time points exhibited only down-, not up-, regulated genes (Table 5). The three time points showed extremely small, extremely large, and moderate number of genes, in that order. Statistically significant overlap of genes between adjacent time points was observed (Hypergeometric distribution, assuming a total of 10,000 genes, approximately the number of genes represented on the microarray used;/? = 5.3xlO^"6 for 12 hrs and 2^nd day genes, p = 1.9xlO^'22 for 2^nd and 7^th day genes; p = 0.34 for 12 hrs and 7^th day genes). This demonstrated the usefulness of our approach. Because our objective was to dissect epileptogenesis at biological systems level, we here onwards focused mainly on gene ontology (GO) biological processes which the differentially expressed genes overrepresented. Whereas no biological process was significantly overrepresented in the beginning phase, the latent and expressive phases showed various but distinct categories after adjusting for multiple comparisons using Bonferroni correction (see below for further details on overrepresented GO processes). The latent phase exhibited alterations in a wide variety of signaling, developmental, structural, and metabolism related processes. In contrast, glutamate metabolism was most characteristic of the behavioral phase. Many of the processes that we identified, were consistent with our present understanding of the pathophysiology of epilepsy and with transcriptomic and proteomic profiles in different rodent epilepsy models (Gorter et al. 2006; Lukasiuk et al. 2003, 2006; Lukasiuk and Pitkanen, 2004; Hunsberger et al. 2005; Scimemi et al. 2006). This established the utility of our model in unraveling system-level alterations in epileptogenesis.

Correction for multiple statistical testing, like Bonferroni correction, may be overly conservative to the point of being counterproductive in interpreting microarrays with genetic knowledgebases such as GO (Osier et al. 2004). We therefore retrieved biological processes without adjusting for multiple corrections (Table 6). These processes showed significant overlaps between adjacent as well as extreme time points, signifying their involvement in epileptogenesis (Hypergeometric distribution, considering total number of categories of GO biological processes represented by all the genes in fly genome as 4041, Mi et al. 2003, and 129, 593, and 231 processes showing significant enrichment for 12 hrs, 2^nd day, and 7^th day genes respectively; p = 1.9xlO^"19 for 12 hrs and 2^nd day GO processes, p = 4.2x10^~36 for 2^nd and 7^th day GO processes, p = 0.008 for 12 hrs and 7^th day GO processes). It was striking that whereas there was no significant overlap between 12 hrs and 7^th day genes, as mentioned above, the overlap between GO processes between these same time points was highly significant. This finding underscores the notion that biological processes, not genes per se, are better indicators of similarities, and supports our current approach towards developing the fly model. Processes common to all the three time points, 11 specific processes (Table 7), may suggest their causal role in development and behavioral expression of epilepsy. We simply reasoned that if downregulation of these processes are indeed causal in epileptogenesis then AEDs must upregulate them. To test this prediction, we generated head gene expression profiles after treating the flies with either LEV or NaVP. Both the drugs only up-, not down-, regulated gene expression (Table 8), with enriched GO processes mostly belonging to metabolic category (Table 9). There was statistically significant overlap of genes as well as GO processes between LEV and NaVP (hypergeometric distribution as mentioned above, 4 common genes and 19 common GO processes between LEV and NaVP, total processes in NaVP and LEV being 115 and 33 respectively; p = 3.5xlO^~5,/? = 5.4x10^~22, in that order). Processes such as glutamate and aspartate metabolism were common to both AEDs. Among unique categories, LEV affected extracellular structure organization and biogenesis, besides others. On the other hand, uniquely altered processes included aerobic respiration and protein biosynthesis in case of NaVP. Remarkably, both LEV and NaVP upregulated (Table 10) statistically significant number of the total 11 specific processes which were downregulated (Table 7) at all the three time points in PTZ kindling (Hypergeometric distribution, considering total number of categories of GO biological processes represented by all the genes in fly genome as 4041, Mi et al. 2003, and 33 and 115 GO processes which were found significantly enriched in LEV and NaVP gene sets respectively; p = 8.2xlO^"u for 11 specific GO processes common to all the three kindling time points and 6 shared processes in LEV, p = 1.8xlO^"7 for 11 specific GO processes common to all the three kindling time points and 6 shared processes in NaVP). Overlap between GO processes upregulated by LEV and downregulated by PTZ at each of the three time points was also significant (Hypergeometric distribution, considering total number of categories of GO biological processes represented by all the genes in fly genome as 4041, Mi et al. 2003 and 129, 593, 231, and 33 enriched GO processes obtained for 12 hrs, 2^nd day, 7^th day, and LEV respectively; p ~ 4.8xlO^"7 for 12 hrs and LEV, p = 1.2xlO^"9 for 2^nd day and LEV, p = 4.5x10^"16 for 7^th day and LEV). Similarly, overlap between GO processes upregulated by NaVP and downregulated by PTZ at each of the three time points was also significant (Hypergeometric distribution, considering total number of categories of GO biological processes represented by all the genes in fly genome as 4041, Mi et al. 2003 and 129, 593, 231, and 115 enriched GO processes obtained for 12 hrs, 2^nd day, 7^th day, and NaVP respectively; p = 0.002 for 12 hrs and NaVP, p = 4xlO^"31 for 2^nd day and NaVP, p = 1.4xlO^"50 for 7^th day and NaVP). While LEV upregulated processes point towards synaptic remodeling and energy metabolism, NaVP upregulated transport and energy metabolism related processes. Since LEV showed strong antiepileptogenic and weak antiepileptic activities and NaVP the reverse, evidence of perturbations in synaptic remodeling and transport emerged as extremely causal in development and behavioral expression of epilepsy, in that order, and energy metabolism as having an intermediate role. Finally, we argued that if deficiency in energy generation is indeed causal in epilepsy, then an increased supply of dietary sugar must show only a neuroprotective effect and result in less pronounced behavioral deficit in flies. Conversely, a decreased supply of dietary sugar must exaggerate the behavioral deficit. Indeed, high sugar diminished the locomotor abnormality induced by chronic PTZ in flies (Table 11). In contrast, a lower dietary sugar caused lethality. This established our systems model and allowed us to identify most promising biomolecular targets for development of therapeutic agents against a wide range of brain disorders owing to established relevance of rodent kindling model and the AEDs used in various neurological and neuropsychiatric conditions including epilepsy, epileptogengsis, drug addiction, mood disorders, migraine etc.

Injection of gene specific dsRNA in the thorax of adult Drosophila is known to cause inhibition of expression of the target gene in the brain through cell non-autonomous RNAi (Goto et al. 2003). We therefore injected dsRNA against one of the candidate epilepsy gene that we identified on the basis of showing altered expression at the earliest of the three time points, CG8428, which is under synaptic remodeling category, in flies' thorax, maintained the injected flies on normal media for seven days, and then measured climbing speed of flies. Remarkably, the injected flies showed climbing speed deficit as shown by PTZ kindled animals (Table 12). This deficiency was ameliorated by LEV. Candidacy of the targets that our model identified was thus established.

We described above a systems model of epileptogenesis. Our approach has been to first evolve a behavioral model of rodent kindling in Drosophila, identify systems level perturbations underlying kindling acquisition to predict the causal biological processes, and then test the predictions by identifying systems effect of AEDs. That our results are indeed relevant to epilepsy is exemplified by the following instance. A class of Drosophila mutants called bang-sensitive paralytics exhibit seizures and paralysis upon mechanical stimulation (Fergestad et al. 2006). Behavioral, biochemical, electrophysiological, pharmacological, and molecular characterization of bang-sensitive mutants suggests existence of conserved mechanisms between bang-sensitive mutants and human seizure disorders (Fergestad et al. 2006). Molecular defects identified in five such mutants involve genes encoding a mitochondrial ribosomal protein (tko, CG7925), the ADP/ATP translocase (sesB, CGl 6944), the citrate synthase (kdn, CG3861), an ethanolamine kinase {eas, CG3525), and an aminopeptidase [sda, CG5518). Prevalence of mitochondrial function disrupting defects in bang-sensitive mutants suggests that impaired energy . metabolism may underlie the affected behavior (Fergestad et al. 2006). Importantly, human seizure disorders have been linked to mutations in genes encoding pyruvate carboxylase, and pyruvate dehydrogenase, the two enzymes upstream of citrate synthase, as well as to mutations affecting various steps in the TCA cycle (Fergestad et al. 2006). Our microarrays represented all the above bang-sensitive genes except CG7925. Remarkably, each of the four genes represented on the array showed downregulation on either 2^nd or 7^th day. This overrepresentation of bang-sensitive genes (Hypergeometric distribution, assuming a total of 10,000 genes, approximately the number of genes represented on the microarray used, and considering a total of 2567 genes in 2^nd and 7^th day time points; p = 0.004), along with enrichment of various biological processes related to energy metabolism, in our expression profiles in consistent with the above literature.

Identification of potential biomarkers If a significant overlap is found between genes downregulated at all three kindling time points and genes upregulated by LEV and NaVP then downregulation of the common genes would be potential markers for epileptogenesis. Out of 2574 and 178 genes which were down- and up-regulated, in that order, 87 were common. This overlap was highly significant (Hypergeometric distribution, assuming a total of 10,000 genes, approximately the number of genes represented on the microarray used; p = 1.3 x 10^'u). The common genes are listed in Table 13a. The human counterparts (FLIGHT, a Drosophila database mining site; mParanoid and Homologene options; http://www.flight.licr.org) of these genes are listed in Table 13b. Downregulation of these human genes in patients' brain sample may thus potentially be used as biomarkers of epileptogenesis and related disorders. Several genes have been found to be downregulated in rat traumatic brain injury (TBI) model of epileptogenesis (Matzilevich et al. 2002). Importantly, fly homologs of two of the downregulated TBI genes Aldoa and Gsta3, namely, CG6058 and CGl 1512, in that order, are present in Table 13a. The human homologs of CG6058 and CGl 1512 are ALDOA, ALDOB, ALDOC and GSTAl. These four genes and their products are therefore identified here as the most promising biomarker candidates in epileptogenesis and related disorders.

Identification of potential drug targets

Downregulation of a total of 14 gene ontology (GO) biological processes were identified as common to all the three time points in PTZ kindling, 12 hrs, 2^nd day, and 7^th day. Out of these 14 processes, the following 11 are specific; GO:0005975, GO:0006091, GO:0007416, GO:0030198, GO:0043062, GO:0006810, GO:0009628, GO:0015980, GO:0051179, GO:0051234, and GO:0050808 (Table 7). Downregulation of the 11 specific processes mentioned above thus represent process-level signature of epilepsy. Among these 11 processes, following six are the ones which are upregulated by LEV, an AED with strong antiepileptogenic and weak antiepileptic activities; GO:0005975, GO:0006091, GO:0007416, GO:0030198, GO:0043062, and GO:0050808 (Table 10). Similarly, among the above 11 processes, following six are upregulated by NaVP, an AED with weak antiepileptogenic and strong antiepileptic activities; GO:0005975, GO:0006091, GO:0006810, GO:0015980, GO:0051179, and GO:0051234 (Table 10). The first two categories are common for both the AEDs. Since LEV has both symptomatic as well as prophylactic activities, the 6 GO categories upregulated by LEV represent minimal and yet sufficient processes upregulation of which can ameliorate both development as well as behavioral expression of epilepsy. LEV upregulated genes under the above six GO categories, four in number, are listed in Table 14a. Agents that can upregulate these four genes would be expected to be of therapeutic value in epilepsy. The human counterparts exist for all four genes (FLIGHT, a Drosophila database mining site; InParanoid and Homologene options; http://www.flight.licr.org). The human homologs, five in number, are listed in Table 14b. All annotate genes are enzymes, ideal as drug targets. We thus identify here the above genes as potential drug targets for treating epilepsy and related disorders. The fly genes and their products, or their counterparts in other organisms, may be used to identify agents that enhance their activity or expression. Agent(s) that induce these enzyme(s) would be expected to be of therapeutic value in epilepsy and related brain disorders. Fly homologs of two of the genes shown in Table 14b, namely, GOT2 and SORJD, are upregulated by both LEV and NaVP. These two genes and their products therefore become most promising drug target candidates in epileptogenesis and related disorders. Two other genes that were upregulated by both LEV and NaVP, CG10833 and CG8345, could also have served as potential drug targets but their human homologs are not known to exist.

Once we identified potential drug targets, we searched for chemical agent(s) described in the literature to affect expression of the target gene(s). We reasoned that agents that upregulate such'gene(s) will be a potential therapeutic agent in neurological and neuropsychaitric disorders. It was found that expression of mammalian counterpart of one such gene, CG4233, is known to be upregulated by potassium chloride (KCl), N-methyl-d-aspartate (NMDA), prolactin, 12-O-tetra-decanoylphorbol 13 -acetate (TPA), testosterone etc. (Caballero-Benitez et al., 2004; Gorski et al, 1999; Juang et al, 1995). Given this, we tested KCl in our fly behavioral model of kindling induced by chronic PTZ. Remarkably, KCl ameliorated climbing deficit caused by chronic PTZ in our model (Table 15). This established the candidacy of the potential targets that we identified using fly systems model.

In a data-mining approach, we searched relevant literature (Lukasiuk et al. 2006) to find description of complete set of genes showing altered expression in an established model of brain plasticity related to epileptogenesis. Such a description was found for traumatic brain injury (TBI) in rat, at two time points (Matzilevich et al. 2002). We retrieved the overrepresented (based on unadjusted p values) GO processes in the downregulated genes at both time points and found a significant overlap (hypergeometric distribution, p = 4.5 x 10^"24) between the two sets. Importantly, significant overlap was observed between the common GO processes in TBI and the 11 specific processes common in fly kindling (hypergeometric distribution, p = 2.4 x 10^"13). Among seven overlapping processes found, four are related to synaptic remodeling and three to transport. Although the two energy metabolism related processes identified by us as central in epileptogenesis were missing in the processes overlapping between TBI and fly kindling, they were nonetheless overrepresented at earlier of the two TBI time points. The above observations support the portability of biomarkers and drug targets identified through systems modeling in fly to human disorders.

Next, we examined if Drosophila downregulated genes belonging to biological process categories that we identified as causal in fly kindling are overrepresented among genes implicated in seizure phenotype in mouse, using phenotype ontology of Mouse Genome Informatics (MGI, http://www.informatics.jax.org/mgihome/other/citation.shtml; Eppig et al. 2005). With 22,751 total genotypes and 377 'seizure' genotypes described in MGI, and 491 total fly causal GO biological process genes (PTZ fly kindling associated genes belonging to synaptic remodeling, energy metabolism, and transport related processes described above) of which 24 were homologs of the mouse 'seizure' genes, the fly genes belonging to causal processes were found to be overrepresented in seizure related genes (hypergeometric distribution, P = 0.0000007). The unique mouse genes described under "seizure" in MGI were Acp2, Adam22, Adamts4, Adarbl, Aifml, Akp2, Aldh5al, Amph, Ank3, Ap3b2, Ap3m2, Ascl, Atcay, Atoxl, Atrn, Atxn7, Avprlb, Bakl, Bax, Bckdha, Bdnf, Bisl, Bis4, Bmil, Bsn, Cacnala, Cacna2d2, Cacnb4, Cacng2, Cacng4, Cdknlb, Cdkn2d, Chraa4, Chrna5, Chrna7, Chrnb4, Cit, Ckb, Cln6, Cnp, Cnrl, Cntn2, Cntn5, Col2al, Coszl, Cosz2, Cosz3, CpIxI₃ Cstb, Ctnnbl, Ctsd, Dbp, Dgke, Dmtfl, Dst, Edg5, Ell, E12, E13, E14, E15, E16, Elnv, Emxl, Epm2a, Exql, Faim2, Fgfl4, Fosb, Foxa2, Fyn, Fzd9, G6pc, Gabbrl, Gabbr2, Gabrb3, Gabrd, Gabrg2, Gad2, Gan, Gfap, Gfra2, Gjal2, Gjbl, Glral, Glra3, Gnaol, Gng3, Gpr98, Gria2, Gria3, Gria4, Grik2, Grik5, Grinl, Grin2a, Grm7, Hcn2, Hcrtrl, Hdh, HeIt, Hexa, Hexb, HIf, Hprtl, Hrh2, Htrla, Htr2c, Htr4, Htra2, Itprl, Jun, Kcnal, Kcna4, Kcnab2, Kcncl, Kcnc2, Kcnc3, Kcnj6,^'Kcnj9, Kcnk2, Kcnmb4, Kcnq2, Kif5a, Mafg, Mafk, MapklO, Mcoln3, Mecp2, Msx2, Mt3, Myo5a, Ncdn, Neurod2, Nidi, Nmfl34, NmGl, Npy, Nr4a3, Ntrk2, Numb, Numbl, Nur37, Oprdl, Otxl, Pcmtl, Pdgfra, Pdyn, Pisl, Pis2, Pitpna, Plaur, Plcbl, PIcIl₅ Pldn, PIpI₅ Pmp22, Ppargcla, Ppplr9b, Pptl, Ppt2, Psap, Pten, Ptpro, Pura, Qk, Scg5, Scnla, Scnlb, Scn2b, Scn8a, Serpine2, Sgce, Shh, Sicdl, Slcl2a5, Slcl2a6, SIcDaI₅ Slcla2, Slcla3, Slc25al2, Slc2al, Slc37a4, Slc4a3, Slcδal l, Slc7a8, Slc9al, Slc9a6, Snap25, Sod2, Soxl, Sox2, Stam, Sumfl, Sv2a, Sv2b, Synl, Syn2, Synjl, Szsl, SzslO, Szsl l, Szsl2, Szsl3, Szs2, Szs5, Szs6, Szs7, Szsδ, Szs9, Sztl, Szvl, Szv2, Szv3, Tal2, Tcfl2, Tef, Tef, Thrb, Tppl, Ty, TyI, Ube3a, UncDb, Usf2, Usplδ, aspl, asp2, asp3, gt, hpc, nmf391, nmf88, nur33, nur8, psrt, spkwl, spkw2, tmgcβl, and to. Among 491 fly genes, following were found to be homologous to mouse seizure genes; CG12055, CG12348, CG13907, CG14741, CG1522, CG16935, CG17884, CG3159, CG3168, CG3747, CG3937, CG3979, CG4684, CG5594, CG6058, CG6562, CG6703, CG6747, CG7535, CG7765, CG8585, CG8604, CG9071, and CG9995. Significant overlap of fly kindling genes with an unbiased set of mammalian seizure and epilepsy genes demonstrated that our kindling model is relevant in epilepsy. In Genetic Association Database (GAD, http://geneticassociationdb.nih.gov/cgi-bin/index.cgi; Becker et ah. 2004), a total of 2568 genes have been associated with human diseases. We queried GAD using key words 'epilepsy' and 'seizure' and retrieved 25 genes that showed association with these phenotypes. The 491 causal process associated genes in fly kindling represented three homologs of GAD 'epilepsy' and 'seizure' genes (CG1522, CG8585, and CG9071). This overlap was insignificant. Similarly, we retrieved 108 genes that showed association with 'seizure' and 'epilepsy' in Online Mendelian Inheritance in Man (OMlM) Morbid Map

(http://www.ncbi.nlm.nih.gov/Omim/getraorbid.cgi). With human homologs of only two of the 491 causal biological process genes present in OMDVI genes (CG10693 and CG9071), the fly kindling genes did not show any enrichment in 'seizure' and 'epilepsy' genes. Unlike mouse knock outs, GAD and OMTM genes are not an unbiased set because of bias in reported association studies. An insignificant overlap between fly kindling and human epilepsy and seizure genes may not be therefore considered as an evidence against the relevance of fly model in epilepsy.

EXAMPLES

The invention is illustrated by the following examples, which are provided to illustrate the invention and therefore should not be construed to limit the scope of the present invention.

EXAMPLE 1

Unless otherwise mentioned, standard methods of fly manipulation were followed. Standard fly medium consisting of agar-agar, maize powder, brown sugar, dried yeast and nipagin was used. Flies were cultured at 24 ± I⁰C, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. D. melanogaster wild type Oregon-R strain, a stock of which was maintained by the corresponding author , for around eight years in his laboratory, was used. To obtain flies used in the experiments, individuals from identical cultures were first allowed to lay eggs in milk bottles containing normal fly media. Flies were shifted to fresh bottle every 12 hr. First 4 sets of bottles were discarded. Flies that emerged in subsequent bottles were only used. Those that emerged in the beginning were first discarded and then flies were collected within 1 day, at 4 hr interval. Flies were anesthetized with diethyl ether and males and females were separated immediately after collection using a stereomicroscope. The two sexes were kept separately in a single bottle - for each set of parallel treatments that was to be carried out. Flies so harvested were used for control or drug treatment 2-3 days later. In brief, flies used in all the experiments were unmated and were in the age group of either 3-4 days (all behavioral experiments and microarray experiments on PTZ) or 10-11 days (microarray experiments on AEDs) in the beginning of the treatments. EXAMPLE 2

PTZ (for all experiments except that used for inducing convulsive and associated behavior by treating flies for around one day, where double the concentration was used; Sigma-Aldrich), NaVP (Sigma- Aldrich), and LEV (Levesam 500; manufactured by Hetero Drugs Ltd. and marketed by Nicholas Piramal India Ltd.) were mixed in the media at a final concentration of 8, 0.33, and 5 mg/ml, respectively for all the experiments described in the result section. The drug doses were selected mainly on the basis of LC₅₀ experiment. Preliminary observations using arbitrarily chosen dosage had suggested 7 days period to be effective in inducing long-term behavioral change in flies' behavior. Lethality in flies was therefore recorded for up to 7 days. Age may have an effect on drug-induced behavior and, therefore, same age group of flies that was used for behavioral examination had been treated with drugs in lethality experiment. Drugs in general caused more deaths in males compared to females. Sex- differences in drug induced behavior may exist and, therefore, average of male and female death was used to determine an uniform dosage applicable to both sexes.

EXAMPLE 3

Drugs were first dissolved in distilled water at 1OX concentration. One-tenth volume of freshly made drug solutions were then poured in molten fly media, and mixed thoroughly. For control, i.e., normal food (NF), one-tenth volume of distilled water was added in the medium and mixed. Following this, the molten media was dispensed in glass (Borosil) vials, stored overnight at 4⁰C and then used in the experiment. Flies harvested in the manner described earlier were anesthetized using diethyl ether and 30 of them, aged 3-4 days, were shifted to each of the treatment vials. Flies were maintained at 24 + I⁰C, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle.

EXAMPLE 4

Various methods have previously been used in an attempt to measure locomotor activity in Drosophila (Martin, 2004). Different neural systems may influence various locomotor activities in fly (Martin, 2004). We developed a manual method, described below, to measure both vertical locomotor activities of individual flies. Measurements were taken at room temperature, between 9 AM to 9 PM. They were all performed at a specified area in a room, where the same light sources was always used. While measuring locomotor activities, extreme care was taken to ensure that the room is quiet, and the table onto which assays were performed undisturbed and vibration free. Utmost care was taken to ensure identical handling of flies, in minutest details. The vials were coded to hide the treatment identity. A single fly was randomly selected at a time for measurement. This was achieved by trapping all the flies in a treatment vial in separate empty vials individually, using flies' negative geotactic behavior. Appropriate number of vials, each containing a single fly, was then randomly picked-up and the flies pooled in the original treatment vial. One fly each from different treatment groups was first scored and then the same exercise was repeated to score other sets of flies treated in parallel. Once a fly was scored for locomotor activities, it was discarded. Equal number of flies from two replicate treatment vials was scored. Flies were particularly checked for intact legs and wings, before they were used in locomotion assay. A glass column of 2 cm internal diameter and 30 cm internal length, i.e., length between the two cotton plugs, was used in the assays. The column was marked with lines at every cm along the length. Each fly was First familiarized in the column by keeping it for 90 sec in vertically placed column, before vertical locomotion assays, respectively, were performed.

In manual vertical locomotion assay, a single fly was trapped inside the column. The fly was brought to the bottom of the tube, by tapping the tube on a piece of packing foam. As soon as the fly had fallen on the cotton plug at the bottom, the tube was as such placed vertically on the surface of the work table and the dot/comma recording described below was applied the moment a fly crossed a height of 5 cm. m climbing assay, movement of a single fly was monitored by keep pressing the dot key or the comma key of a personal computer, to record a moving or resting fly respectively. Dots and commas were subsequently transformed in rest and activity period respectively, using the cursor speed. Occasionally, a fly fell down before achieving a height more than the 5 cm mark. In such a case, the fly was given another startle and then measured. Climbing was considered complete when the fly reached the cotton plug at the top, fell to the bottom after climbing a certain height beyond 5 cm mark, or stopped after climbing to a certain height for more than 10 sec. The number of lines a^' fly climbed was noted down. While climbing, flies sometimes, rarely though, jumped and/or took flights upwards or downwards. All these activities were recorded as dots. Spiral movement, uncommon though, during climbing was also recorded as dots. Downward movement, rare though, during climbing was also recorded as dots. Comma was applied only when the fly took rest. Only one locomotor parameter, the climbing speed, was finally considered to represent the vertical locomotor activity. Climbing speed was calculated using the following formula, s = hit, where s = climbing speed, h - height climbed in cm, and t - activity period in sec.

EXAMPLE 5 The vertical locomotor assay was adapted to measure horizontal (spontaneous) locomotor activities. A single fly was first brought to the middle of the column by gentle shaking and then the fly movement was constantly monitored for 90 sec by keep pressing the dot key or the comma key of a personal computer, to record a moving or resting fly respectively. Any single jump, short or long, was recorded as dots. Usually flies walked straight along the upper surface towards one end of the tube, explored there by moving around the inner periphery for some time, and then moved towards the other end, and so on. Though uncommon, they also walked along the lower surface, moved in a spiral fashion and explored much at one end. All these variations were recorded as dots. The comma was applied whenever the fly stopped. The total number of lines that a fly crossed in the 90 sec assay time was counted and noted down at the end of the dot/comma recording. The data was normalized for 90 sec and the dots and commas were subsequently transformed in rest and activity period respectively, using the cursor speed. Walking speed (s) was obtained by dividing the cm lines a fly crossed (distance walked, d) by time, in sec, it spent in activity (activity period, a), during the 90 sec assay period. For some flies, value of both d and a were found to be zero. Such individuals were excluded while calculating population mean of s. Finally, the horizontal assay was used to extract four locomotor parameters, namely, activity period, rest period, distance walked and walking speed. The rest period is not presented here because it is invertionally proportional to the activity period.

EXAMPLE 6

Software-based vertical locomotor activity was measured by first video recording the individual flies, housed in culture vials and then analyzing various locomotor parameters. Movies were captured in Sony DCR-VX2100-E and transferred from the camera to the inbuilt frame grabber iMovie in an Apple system (PowerMac G5) and compressed for QuickTime using iMovie settings. Movies were opened in Dynamic Image Analysis system (DlAS 3.4.2, Soil Technologies, Inc., 321 Lexington Ave. Iowa City, IA 52246 USA) at 25 fps (frames per second) for analysis. First of all, a scale factor was calculated for the movies using a known length. This scale factor (0.018 cm/pixel) was then applied to all the movies. Tracing method used was 'autotrace by threshold'. Threshold value entered as 150 to eliminate all background and highlight only the object, i.e. fly. For tracing, those frames were selected in which the object traveled the longest continuous distance, i.e., without any path breakage because of jumps or rest. The path flies thus generated were saved with unique names and speed of each object was calculated using the 'compute parameters' command in DIAS. Now, in the 'Edit path file header' window, the scale factor was entered, frame rate was given as 1 frame per second (equivalent to default value of 60 frames per minute), and the time unit taken as second. All the results computed by DIAS were saved as tab delimited files and used for further analysis.

EXAMPLE 7 To examine if 16 mg/ml PTZ induces convulsive and associated locomotor alterations, video recording of flies' groups in the treatment vials were carried out after around one day of shifting 3-4 days old unmated males in the vials containing normal or drug mixed media. Movies were later on examined to see if PTZ flies exhibit spontaneous hyperkinetic and convulsive behavior. DIAS 3.4.2 was used to obtain the locomotor measure "total directionality". Video recording of flies treated with 16 mg/ml PTZ clearly showed hyperkinetic and convulsive behavior that was absent in NF controls. Mean + S. E of "total directionality" was found to be 0.39 ± 0.07 (« = 11) and 0.19 ± 0.05 (n = 11) for control and PTZ respectively. The difference was significant (p = 0.04). 'Total Directionality' is the net path length divided by the total path length. This gives 1.0 for a completely straight path and a smaller value for a meandering path. PTZ thus causes flies to take a circuitous path. The above observations therefore demonstrated an association between convulsive and locomotor activity, hi view of this, further experiments were all carried out with a lower dose of PTZ (8 mg/ml). Also, because of relative ease in quantifying and richness of attributes, locomotor activities were used as the behavior of choice in all the experiments to follow, i.e., for developing fly model of kindling-like plasticity.

EXAMPLE 8 A single randomly selected unmated control or treated male was first shifted to an empty vial. A single virgin female of same age, not exposed to any drug at any time, was then introduced in the vial housing the single male. Time at which the female was introduced (Q was noted down. Flies were observed throughout courtship. Time at which the male successfully mounted the female (4) was recorded. Courtship duration was obtained as t_/, - t_a.

EXAMPLE 9

In a vial containing NF, a single, randomly selected, unmated control (NF) or drug exposed (PTJZ) male was housed along with a single virgin female, not exposed to any drug at any time, of same age. The parents were discarded after one day and the total number of Fl flies that emerged in each vial was counted as a measure of fertility of the PTZ exposed male flies.

EXAMPLE 10

Flies were over-anesthetized and weighed individually using an analytical balance with a readability of 0.01 mg (Sartorius, model CP225D). Precaution was taken to ensure uniformity in applying the anesthetic diethyl ether.

EXAMPLE 11

For measurements/counts related to locomotor activity, body weight, courtship duration, and fertility, two-sample Student's t-tsst, heteroscedastic, was performed to check for equality of the population means underlying the control and the treated group. The ^-values were calculated for two-tailed tests.

EXAMPLE 12

Flies were frozen in 50 ml falcon tubes in liquid nitrogen. Two cooled sieves were arranged such that the larger sieve (mesh size 850 mm) was placed on top of the smaller one (mesh size 355 mm). Frozen flies were shaken 4-5 times in the falcon and poured onto the top sieve. The flies were brushed gently with a paint brush till all heads were sieved out on the bottom sieve. Bodies that remained on the top sieve were discarded and heads were collected in cryovials and kept frozen at -8O⁰C till use. Total RNA was isolated from frozen fly heads using TRI REAGENT (Sigma) according to the manufacturer's protocol. Extracted RNA was dissolved in 18MΩ RNase free water (Sigma) at 55⁰C. RNA was quantified using spectrophotometer and its purity was checked on 1% Agarose gel. RNA was diluted to a final concentration of 1 μg/μl and was kept at -8O⁰C until use.

EXAMPLE 13

Double stranded cDNA was synthesized from 10 μg of total RNA using Microarray cDNA Synthesis Kit (Roche). The cDNA was purified using Micorarray Target Purification Kit (Roche), according to the manufacturer's protocol. Purified cDNA was used for labeling with Cy3/Cy5 dyes (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche) and labeled product was purified by Microarray Target Purification Kit (Roche). Each experiment was replicated in four microarrays, where two were standard and two were dye-swap arrays. Control was labeled with Cy3 and treatment with Cy5. However in dye-swap experiments, labeling was reversed. In Table 1-4, slides 3 and 4 are dye-swaps. The labeled cRNAs (control and treated) were pooled together and precipitated, washed and air-dried. The dried pellet was dissolved in 18MΩ RNAase free water (Sigma). Hybridization solution was prepared by mixing hybridization buffer (DIG Easy Hyb; Roche)), lOmg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma) and added to the labeled product. This mixture was denatured at 65°C and applied onto cDNA microarray slides (D12Kvl, CDMC, Toronto). The slides were covered by lowering down a 24X60 mm coverslip (ESCO, Portsmouth, USA). Hybridization was allowed to take place in hybridization chamber (Corning) at 37⁰C for 16 hrs. Coverslips were removed by submerging the slides in a solution containing IX SSC and 0.1% SDS at 5O⁰C. Slides were washed (three times for 15 minutes each) in a coplin jar at 5O⁰C with occasional swirling and then transferred to IX SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Finally, slides were washed in O.lX SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes.

EXAMPLE 14

Microarray slides were scanned at lOμm resolution in GenePix 4000A Microarray Scanner (Molecular Devices), using both green and red lasers. The 16 bit TIFF images were preprocessed and quantified using Gene Pix Pro 6.0 software (Molecular Devices). Data normalization was performed using Acuity

4.0 software (Molecular Devices). Ratio based normalization was used for all slides. All Spots with raw intensity less then IOOU and less then twice the average background was ignored during normalization.

Normalized data was filtered for the selection of features before further analysis. Only those spot were selected which contained only a small percentage (<3) of saturated pixels, were not flagged bad or found absent (flags > 0), had relatively uniform intensity and uniform background (Rgn R2 (635/532) > 0.6) and were detectable above background (SNR > 3). Analyzable spots in at least three of four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 3.0, Excel Add-In, Stanford; Tusher et. al. 2001) under the conditions of one class response and 100 permutations. Differentially expressed genes were retrieved at False Discovery Rate (FDR; q-value) of less than or equal to 26%.

EXAMPLE 15

Non-CG number genes were converted into CG numbers mainly using BDGP (flybase). Thereafter, gene numbers were converted to FBgn numbers using GeneMerge (http://www.geneontology.com; Castillo and Hartl, 2003; gene name converter). Next, the FBgn numbers were fed in the GOTool Box (http://www.geneontology.com; Martin et al., 2004) using the following options - ontology, molecular function/biological process; mode, all terms; reference, genome; evidence, all-all evidence; species, D. melanogaster; GO-stats; statistic test, hypergeometric; either no correction or Bonferroni correction for multiple testing. ^'

EXAMPLE 16

We selected CGl 1064, as expression of this gene was found altered at all the three time points (downregulated) and after NaVP treatment (upregulated), for validation of microarray results using reverse northern analysis. CGl 1064 DNA fragment was amplified with PCR using gene specific primers (Fp: GCCAAATACTAAGCGGGAAGAAGA and Rp: TCGGCCACCAGCAGCAACA). Amplified and purified product of -745 bp was blotted on a N⁺ Nylon membrane (Hybond XL, Amersham Pharmacia) along with pSPT DNA as a loading control. The Blotted DNA was cross linked to the membrane with a UV-cross linker (Amersham Pharmacia). cDNA synthesis from 5 μg of total cellular RNA each of control and treatment pertaining to 12 hrs, 2^nd day, and 7^th day PTZ, and from 0.5 ng mRNA of pSPT were carried out using cDNA synthesis kit (Roche) using random primer with attached T7 promoter sequence, as per manufacturer's recommendations. ³²P-labelled cRNA were synthesized and purified using target synthesis kit (Roche) and spin mini prep kit (Qiagen) respectively, as per manufacturers' recommendations. Labeled probes were hybridized separately to identical multiple blots. Each blot represented two gel lanes, each having equal quantities of PCR fragment amplified from genomic DNA using the above mentioned CGl 1064 primers and pSPT DNA. After post hybridization washing, blots were analyzed using phosphor imager (Kodak). Signal intensities of control and treatment spots of CGl 1064 bands were normalized using pSPT control.

EXAMPLE 17 The templates for synthesis of dsRNAs corresponding to spinster (CG8428) and nee (CGl 857) were prepared by using gene specific primers (Table 16) containing T7 polymerase sites, Drosophila nee (CGl 857) was taken as a positive control for the methodology of RNAi. Inhibition of nee is expected to produce necrotic patches over the body of Drosophila. PCR product of each sense and antisense template were purified using Qiagen PCR purification kit and were transcribed to generate sense and antisense RNA using MEGAscript T7 transcription Kit (Ambion, Austin, TX) according to the manufacture's instructions. After annealing dsRNA, concentrations were measured (Abs₂60₎ and the quality of dsRNA were checked at 2% Agarose gel. Annealed dsRNAs were ethanol precipitated and dissolved in injection buffer (IB, 0.1 mM sodium phosphate, pH 6.8; 5 mM KCl). Using an automated injector (FemtoJet Express, Eppendorf) and manually pulled needle (internal diameter of 0.58mm), 32 ηl (nanolitre) of 3 μg/μl (microgram/microlitre) dsRNA or the vehicle, i.e., IB, were injected into the thorax of 5-6 day old, unmated, anesthetized male flies. Injection of CGl 857 dsRNA indeed caused necrotic patches, demonstrating the success of our methodology.

EXAMPLE 18

To examine if LC₅₀ dose of PTZ, i.e., 16 mg/ml, induces convulsive and associated locomotor alterations, video recording of flies' groups in the treatment vials were carried out after 12 hours of shifting 3-4 days old unmated males in the vials containing normal or drug mixed media. Movies captured in Sony DCR-VX2100-E were examined to see if PTZ flies exhibit spontaneous hyperkinetic and convulsive behavior. Dynamic Image Analysis System (DIAS 3.4.2) was used to obtain the locomotor measure "total directionality". Video recording of flies treated with 16 mg/ml PTZ clearly showed hyperkinetic and convulsive behavior that was absent in NF controls. 'Total Directionality' is the net path length divided by the total path length. This gives 1.0 for a completely straight path and a smaller value for a meandering path. Mean + S.E of "total directionality" was found to be 0.39 + 0.07 (n

= 11) and 0.19 + 0.05 (n = 11) for control, i.e., NF, and PTZ respectively. The difference was significant

(p = 0.04). PTZ was therefore found to causes a circuitous path. Assuming this behavior as convulsion- like, we used 8 mg/ml of PTZ, half of the dose used above, in all subsequent experiment.

EXAMPLE 19

The semi-manual method of locomotor speed measurements was verified using an automated method. Software-based vertical locomotor activity was measured by first video recording the individual flies housed in culture vials and then analyzing various locomotor parameters. Movies were captured in Sony DCR-VX2100-E and transferred from the camera to the inbuilt frame grabber iMovie in an Apple system (PowerMac G5) and compressed for QuickTime using iMovie settings. Movies were opened in Dynamic Image Analysis System (DIAS 3.4.2, Soil Technologies) at 25 fps (frames per second) for analysis. First of all, a scale factor was calculated for the movies using a known length. This scale factor (0.018 cm/pixel) was then applied to all the movies. Tracing method used was 'autotrace by threshold'. Threshold value entered as 150 to eliminate all background and highlight only the object, i.e. fly. For tracing, those frames were selected in which the object traveled the longest continuous distance, i.e., without any path breakage because of jumps or rest. Appropriate entries for scale factor, frame rate and time unit were made in the 'Edit path file header' window and speed/directionality of each object was calculated by using 'compute parameters' option in DIAS.

The alteration in fly speed after seven days of chronic PTZ treatment and seven days after PTZ withdrawal i.e. on 7^th day and 14^th day, respectively, was observed using DIAS (Figure 1). Flies were introduced one by one in the same glass column used for manual measurements, and the vertical climbing for a total length of 8 cm (due the limitation of recording adjustments, 30cm could not be exactly focused in the camera) was video recorded. Path-tracing and the speed calculation were done as explained above, using the 'compute parameters' command and frame-by- frame values were averaged to obtain a single value for each object. DIAS analysis thus confirmed the behavioral observations made using semi-manual method.

EXAMPLE 20

To check the efficiency of differential gene expression detection in our microarrays, we performed a control gene expression profiling analysis in which RNA isolated from different pools of heads of wild- type flies grown on normal food (NF) were compared between themselves. We performed four biological replicates of array, following procedures which were used in all later profiling. Downstream analysis using methods described in the method section did not reveal any gene as differentially expressed in NF versus NF below a false discovery rate (FDR) of 96.6 %. It was therefore concluded that our experimental design is robust enough to reliably compare control versus treated samples.

EXAMPLE 21 Detection of genes which are expressed at a relatively low level in fly brain (Posey et al, 2001) diss (CG4211), dnc (CG32498), dco (CG2048), per (CG2647), for example, as differentially expressed genes in our microarrays demonstrated that our expression profiling was efficient.

EXAMPLE 22 We selected CGl 1064, as expression of this gene was found altered at all the three time points of PTZ kindling (downregulated), for validation of microarray results using reverse northern analysis. CGl 1064 DNA fragment was amplified with PCR using gene specific primers (Fp: GCCAAATACTAAGCGGGAAGAAGA and Rp: TCGGCCACCAGCAGC AACA). Amplified and purified product of -745 bp was blotted on a N⁺ Nylon membrane (Hybond XL, Amersham Pharmacia) along with pSPT DNA as a loading control. The Blotted DNA was cross linked to the membrane with a UV-cross linker (Amersham Pharmacia). cDNA synthesis from 5 μg of total cellular RNA each of control and treatment pertaining to 12 hrs, 2^nd day, and 7^th day PTZ, and from 0.5 ng mRNA of pSPT were carried out using cDNA synthesis kit (Roche) using random primer with attached T7 promoter sequence, as per manufacturer's recommendations. ³²P-labelled cRNA were synthesized and purified using target synthesis kit (Roche) and spin mini prep kit (Qiagen) respectively, as per manufacturers' recommendations. Labeled probes were hybridized separately to identical multiple blots. Each blot represented two gel lanes, each having equal quantities of PCR fragment amplified from genomic DNA using the above mentioned CGl 1064 primers and pSPT DNA. After post hybridization washing, blots were analyzed using phosphor imager (Kodak). Signal intensities of control and treatment spots of CGl 1064 bands were normalized using pSPT control. Gel photograph and phosphor imager images (Figure 2) confirmed CGl 1064 downregulation at all three time points in PTZ kindling.

EXAMPLE 23

A total of 11 genes were selected for validation of expression profiles representing the three PTZ time- points (12 hrs., 2^nd day, 7^th day), LEV, and NaVP (Figure 3). PCR amplification reactions were carried out in an ABI Prism 7700 sequence detection system (Applied Biosystems). RNA of each sample was. reverse transcribed into cDNA using High capacity cDNA Archive kit (Applied Biosystems) following manufacturer's recommendations. All reactions were performed in duplicates using a total of ~50 ng of total RNA per reaction, using custom based gene expression assays, in a 384 well plate. Each assay consisted of two sequence-specific PCR primers and a TaqMan assay-FAM™ dye-labeled MGB probe. 18S rRNA was used as an endogenous control. The specific primers and probe set to measure 18S rRNA were present in for each sample. Data was generated using software SDS 2.1 and C_T values were calculated. All genes were detectable under the detection thresholds recommended by Applied Biosystems (Cx < 36). To compare 18S rRNA and target gene, relative quantification was performed using comparative C_τ method. Briefly, this comparative C_τ method involved averaging duplicate samples of each target and endogenous control in both calibrator (i.e. control) and treatment samples (i.e. ΔC_T (absolute C_τ value — endogenous control C_T value) and ΔΔ C_T (ΔC_T for each gene — ΔC_T for a common reference gene). The fold change was calculated according to the formula 2^~(ΛΔCT), where ΔΔCT was the difference between ΔC_T target and the ΔC_T calibrator value. ABI gene expression assay IDs used were as follows: Dm01803245_ml (GRHR, CGl 1325), Dm01805173_ml (CG9238), Dm01806642_gl (CG9619), Dm02148936_ml (Men, CG10120), Dm01825396_ml (PhKgamma, CG1830), Dm01846862_ml (CG33138), DmO1834182_ml (CG7766), Dm01842786_ml (Pdk, CG8808), Dm01804635_gl (Cyp28, CG10833), Dm01846045_ml (Got2, CG4233), Dm01817100_ml (CG9485). Table 1: Kindling results in PTZ kindled animals

Note (Table 1): NF, normal food; PTZ, normal media containing PTZ. Climbing speed was measured at three time .points, 12 hrs, 2^nd day, and 7^th day, during seven days of chronic PTZ treatment. Subsequently, flies were maintained in normal media for next seven days. Climbing speed was measured at the end, i.e., on 14^th day from the beginning of PTZ treatment. Details regarding measurements and statistics are mentioned in the text.

Table 2: Mean and S. E. of body weight (in mg), number of offspring produced, and courtship duration (in sec) pertaining to (a) flies treated with PTZ, and (b) flies treated with PTZ for seven days and then treated with normal media for the next seven days

(a)

Note (Table 2): NF; normal food; PTZ, normal media containing PTZ. Details regarding measurements and statistics are mentioned in the text. Table 3: Mean and S. E. of climbing speed in cm/sec, in flies examined to test the prophylactic activities in AEDs

Note (Table 3): Treatments were as follows: (i) normal media for 4 days and 3 days (NF-NF); (ii) PTZ containing media for 4 days and 3 days (PTZ-PTZ); (iii) PTZ and LEV containing media for 4 days, and PTZ containing media for 3 days, in that order (PTZ+LEV-PTZ); (iv) PTZ and NaVP containing media for 4 days, and PTZ containing media for 3 days, in that order (PTZ+NaVP-PTZ); (v) LEV containing media for 4 days, and normal media for 3 days, in that order (LEV-NF); (vi) NaVP containing media for 4 days, and normal media for 3 days, in that order (NaVP-NF). Details regarding measurements and statistics are mentioned in the text. Both unadjusted as well as adjusted (Bonferroni correction) p-values are given. All the vials used to shift flies for the last three days were pre-conditioned by keeping unmated male flies, which were discarded later on, in them for four days.

Table 4: Mean and S.E. of climbing speed in cm/sec, in flies examined to test the symptomatic activities in AEDs

Note (Table 4): Treatments were as follows: (i) normal media for 6 days and 1 day (ΝF-ΝF); (ii) PTZ containing media for 6 days and 1 day (PTZ-PTZ); (iii) PTZ containing media for 6 days, and LEV and PTZ containing media for 1 day, in that order (PTZ-LEV+PTZ); (iv) PTZ containing media for 6 days, and NaVP and PTZ containing media for 1 day, in that order (PTZ-ΝaVP+PTZ); (v) normal media for 6 days and LEV containing media for 1 day, in that order (ΝF-LEV); (vi) normal media for 6 days and NaVP containing media for 1 day, in that order (ΝF-ΝaVP). Details regarding measurements and statistics are mentioned in the text. Both unadjusted as well as adjusted (Bonferroni correction) p-vahiβs are given. AU the vials used to shift flies for the last one day were pre-conditioned by keeping unmated male flies, which were discarded later on, in them for six days.

Table 5

(a) Genes showing altered expression (downregulation) at 12 hrs time point during PTZ kindling: CG10078, CG3407, CG9619, CG5707, CGl 1094, CG3241, CG9448, CGl 1064, CGl 1963, CG10593, CG7110, CG7314, CG8428, CG9238, CG18769, CGl 1325, CG1600, CGl 1958, CG4799, CGl 8660, CG6730,, CG14032, CG7583

(b) Genes showing altered expression (downregulation) at 2 days time point during PTZ kindling:

CG10026, CG13237, CG12262, CG12289, CG9435, CGl 828, CG32455, CG33235, CG31025, CG3330, CG6184, CG6333, CG12550, CG13597, CG2127, CQ9871, CG10019, CG10026, CG10033, CG10037, CG10047, CG10063, CG10068, CG1007, CG10071, CG10073, CG10103, CG10106, CG10113, CG10118, CG10120, CG10124, CG10143, CG10146, CG10161, CG1017, CG10176, CG10185, CG10192, CG10194, CG10197, CG10198,CG10200, CG10207, CG10208, CG10214, CG10226, CG10237, CG10242, CG10253, CG10267, CG10272, CG10277, CG1028, CG10281, CG10287, CG10289, CG10293, CG10295, CG10297, CG10308, , CG1031, CG10311, CG10318, CG10326, CG10348, CG10354, CG10359, CG10371, CG10373, CG10377, CG10379, CG10390, CG10395, CG10423, CG10433, CG10435, CG1049, CG10494, CG10521, CG10527, CG10528, CG10536, CG10542,CG10545, CG10553, CG1056, CG10561, CG10562, CG10576, CG10578, CG10582, CG10594, CG10596, CG10598, CG10600, CG10605, CG10620, CG10621, CG10626, CG10631, CG10635, CG10639, CG10640, CG10641, CG10645, , CG10648, CG10658, CG1066, CG10664, CG10693, CG10697, CG10698, CG10701, CG10706, CG10710, CG10711, CG10719, CG10722, CG10737, CG10742, CG10743, CG10746, CG10757, CGI0778, CG1078, CG1079, CG10798, CG10804, CG10808, CG1081, CG10810, CG10811, CG10816, CG10840, CG10842, CG10845, CG10849, CG10851, CG10863, CG1088, CG10888, CG10895, CG10899, CG1090, CG10908, CG1092, CG10922, CG10936, CG10939, CG10966, , CG10967, CG10970, CG10971, CG10973, CG10987, CG109S8, CG10989, CG10992, CGl 1008, CGl 102, CGl 1024, CGl 1.035, CG1104, CG1105, CG11059, , CG11062, CG11063, CG11064, CG11066, CG11071, CG11081, CG11094, CG11095, CG11099, CGlI lOO, CG11111, CG11132, CG11138, CG11139, CG11140, , CGl 1147, CGl 1148, CG11151, CG11155, CGl 1173, CGl 1177, CGl 1180, CG11181, CGl 1184, CG11190, CG11191, CG11203, CG11218, CG11227, CG11242, , CG11247, CG11249, CG11259, CGl 1266, CGl 1271, CGl 1278, CGl 129, CGl 1290, CGl 1298, CGl 1299, CGl 1306, CGl 1307, CG11315, CGl 1319, CG11329, , CG11331, CG11334, CG11348, CG11352, CG11357, CG1136, CG11368, CGl 137, CG11371, CG11375, CG11388, CG11390, CG11399, CG11401, CG11407, , CGl 1414, CGl 1419, CGl 1430, CGl 1444, CGl 1454, CGl 1462, CGl I486, CGl 1502,CGl 1509, CG11512, CG1152, CG11523, CG11526, CG11533, CG11538, , CG1155, CG11550, CG11561, CGl 1567, CGl 157, CGl 1584, CGl 1586, CGl 1597, CGl 1598, CGl 1614, CGl 1630, CGl 1650, CGl 1655, CGl 1661, CGl 1663, , CGl 1665, CGl 167, CGl 1676, CGl 1678, CGl 168, CGl 1694, CGl 1723, CGl 1727, CGl 1739, CGl 1760, CGl 1771, CGl 1784, CGl 1786, CGl 1791, CGl 1793,, CGl 1798, CGl 1799, CGl 1804, CGl 1825, CGl 1844, CGl 1857, CGl 1870, CGl 1876, CGl 1888, CGl 1907, CGl 1908, CGl 1920, CGl 1924, CGl 1937, CGl 1940, CGl 1956, CGl 1958, CGl 1981, CGl 1983, CG12004, CG12005, CG12016, CG12018, CG12020, CG12025, CG12052, CG12054, CG12075, CG12081, CG12085, , CG12090, CG12106, CG12108, CG12110, CG12111, CG12116, CG12118, CG12128, CG1213, CG12131, CG12142, CG12154, CG12171, CG12175, CG12178, , CG12184, CG12204, CG12206, CG12207, CG12214, CG12217, CG12223, CG12233, CG12234, CG12238, CG12263, CG12267, CG12283, CG12290, CG12295, CG1231, CG12311, CG12316, CG12333, CG12345, CG12347, CG12348, CG12350, CG12358, CG12363, CG12366, CG12369, CG12372, CG12377, CG12384, CG12391, CG12393, CG12404, CG12428, CG12437, CG1244, CG12449, CG12464, .CG12488, CG1249, CG12512, CG12534, CG12598, CG12656, CG12664, CG12665, CG12676, CG12690, CG12723, CG12730, CG12736, CG12737, CG12740, CG12749, CG12758, CG12772, CG12781, CG12784, CG12789, CG12792, CG12802, CG12806, CG12819, CG12838, CG12855, CG12862, CG12864, CG1288, CG12921, CG12923, CG12944, CG12954, CG12956, CG1299, CG13025, CG13029, CG13030, CG13037, CG13039, CG13041, CG13061, CG13067, CG13078, CG13081, CG13095, CG13109, CG13116, CG13124, CG13126, CG13154, CG13186, CG13189, CG13213, CG13243, CG13316, CG1332, CG1333, CG13335, CG13344, CG13349, CG13363, CG13367, CG13380, CG13387, CG13388, CG13389, CG13390, CG13397, CG13405, CG1341, CG13418, CG13423, CG13429.CG13431, CG13444, CG13476, CG13499, CG13502, CG13503, CG13567, CG13575, CG1358, CG13585, CG13586, CG13594, CG13623, CG13624, CG13631, CG13688, CG13705, CG1371, CG1372, CG1373, CG13739, CG13741, CG13746, CG13758, CG13777, CG13778, CG13784, CG13830, CG13848, CG13850, CG13855, CG13868, CG13873, CG13874,CG13887, CG13888, CG13906, CG13907, CG13920, CG13960, CG13969, CG13983, CG13995, CG14000, CG14017,CG14025, CG14026, CG14028, CG14032, CG1404, CG14040, CG14041, CG14045, CG1406, CG14066, CG14073, CG14080, CG14154, CG14162, CG1417, CG14207, CG14213, CG14214, CG14216, CG14217, CG14253, CG14271, CG14275, CG14277, CG1429, CG14290, CG14291, CG14302, CG14305, CG14322, CG14327, CG14363, CG14375, CG14401, CG1443, CG14437, CG14450, CG14472, CG14477, CG1449, ^• CG14500, CG14537, CG14548, CG1458, CG14605, CG14644, CG14648, CG14666, CG14685, CG14687, CG1469, CG14691, CG14757, CG14762, CG14763, CG14764, CG14777, CG14779, CG14791, CG14866, CG14872, CG1488, CG14887, CG14889, CG14896, CG14904, CG14906, CG14919, CG14938, CG14939, CG14941, CG14948, CG14967, CG14989, CG14991, CG14992, CG14994, CG14995, CG14996, CG14998, CG15006, CG1501, CG15012, CG15098, CG15102, CG1511, CG15138, CG15141, CG15144, CG15145, CG15151, CG15162, CG15175, CG15189, CG15201, CG15202, CG15209, CG1522, CG15251, CG15270, CG15304, CG15365, CG1537, CG15382, CG15387, CG15434, CG15435, CG15436, CG15438, CG15444, CG15457, CG15478, CG15509, CG15525, CG1553, CG15532, CG15538, CG1554, CG15543, CG1558, CG15580, CG1560, CG15611, CG15669, CG15675, CG15676, CG15678, CG15706, CG1572, CG15731, CG15745, CG1575, CG15760, CG15767, CG15770, CG15771, CG1578, CG15812, CG15819, CG15824, CG15845, CG15861, CG15862, CG1587, CG15883, CG15893, CG15897, CG15916, CG15926, CG1599, CG1600, CG1624, CG1630, CG1634, CG1640, CG1643, CG1646, CG1658, CG1659, CG1660, CG1662, CG1668, CG16705, CG16712, CG16720, CG16721, CG16725, CG16743, CG16747, CG16757, CG16758, CG16765, CG1677, CG16782, CG16784, CG16793, CG16799, CG1681, CG16838, CG16853, CG16863, CG1688, CG16884, CG16886, CG16898, CG16901, CG1691, CG16910, CG16926, CG16935, CG16936, CG16941, CG16944, CG16952, CG16960, CG16974, CG16982, CG16983, CG16996, CG17018, CG17034, CG17041, CG17046, CG17052, CG17054, CG17084, CG1710, CG17122, CG17124, CG17131, CG17136, CG17142, CG17144, CG17161, CG17168, CG17176, CG17187, CG17199, CG17205, CG1721, CG17216, CG17228, CG17244, CG17248, CG17255, CG17256, CG17258, CG17259, CG17266, CG17271, CG17273, CG17278, CG17294, CG17298, CG17299, CG17309, CG17332, CG17336, CG17360, CG17378, CG17390, CG1740, CG1743, CG1746, CG1749, CG17494, CG17520, CG17523, CG17533, CG17540, CG17566, CG17569, CG17571, CG17594, CG17610, CG1762, CG17645, CG17646, CG1765, CG17680, CG17686, CG17697, CG17715, CG1772, CG17724, CG17736, CG17737, CG17739, CG17759, CG1776, CG17762, CG17766, CG17800, CG17816, CG17836, CG17838, CG17841, CG1785, CG17870, CG17888, CG17896,CG17903, CG17907, CG17922, CG1793, CG17932, CG17938, CG17944, CG17950, CG17960, CG17964, CG17975, CG17977, CG17985, CG1799, CG17994, CG17999, CG18000, CG18009, CG1801, CG18011, CG18026, CG18069, CG18076, CG18090, CG18102, CG18111, CG1812, CG18131, CG18135, CG18140, CG1815, CG18156, CG18176, CG1819, CG18214, CG1824, CG18250, CG18255, CG1829, CG18292, CG1830, CG18314, CG18316, CG18319, CG18358, CG1838, CG18397, CG18402, CG18405, CG18408, CG18418, CG18445,CG18473, CG18495, CG18497, CG18506, CG18542, CG18559, CG1856, CG18599, CG18617, CG18619, CG1862, CG18622, CG18631.CG18638, CG18646, CG18647, CG18660, CG18743, CG18768, CG18769, CG18777, CG18778, CG18783, CG18788, CG18809, CG18811, CG18815, CG18817, CG1884, CG1886, CG1893, CG1897, CG1901, CG1902, CG1912, CG1915, CG1919, CG1921, CG1938, CG1945, CG1954, CG1973, CG1977, CG1981, CG1987, CG2005, CG2009, CG2028, CG2041, CG2048, CG2050, CG2056, CG2060, CG2063, CG2075, CG2082, CG2083, CG2086, CG2087, CG2096, CG2099, CG2116, CG2129, CG2135, CG2140, CG2147, CG2157, CG2161, CG2162, CG2165, CG2171, CG2179, CG2189, CG2201, CG2204, CG2207, CG2209, CG2210, CG2213, CG2219, CG2227, CG2233, CG2239, CG2244, CG2245, CG2248, CG2254, CG2257, CG2262, CG2272, CG2304, CG2321, CG2336, CG2342, CG2457, CG2534, CG2555, CG2595, CG2608, CG2617CG2647, CG2678, CG2682, CG2702, CG2712, CG2723, CG2727, CG2736, CG2765, CG2767, CG2774, CG2781, CG2811, CG2813, CG2830, CG2837, CG2841, CG2845, CG2852, CG2865, CG2867, CG2875, CG2914, CG2928, CG2934, CG2938, CG2947, CG2957, CG2958, CG2969, CG2972, CG2977, CG2985, CG2991, CG2995, CG2998, CG2999, CG30007, CG3001, CG30011, CG30017, CG30019, CG3002, CG30020, CG30035, CG30052, CG3OO55, CG30069, CG30092, CG30093, CG30118,CG30122, CG30132, CG3014, CG3018, CG3019, CG30197, CG30270, CG30296, CG3032, CG3036, CG30362, CG30363, CG30365, CG30369, CG30372, CG30394, CG30404, CG30418, CG30421, CG30427, CG30442, CG30445, CG30450, CG30472, CG30483, CG30492, CG3050, CG3059, CG3065, CG3095, CG31010, CG31012, CG31022, CG31038, CG31052, CG31057, CG31075, CG31084, CG31088, CG31092, CG31095, CG31099, CG31120, CG31134, CG31140, CG31145, CG31148, CG31163, CG31175, CG31181, CG31182, CG3119,CG31207, CG31209, CG3121, CG31216, CG31224, CG31226, CG3123, CG31236, CG31240, CG31243, CG31248, CG31264, CG3129, CG31293CG31295, . CG31313, CG31320, CG31326, CG31345, CG31352, CG3136, CG31361, CG31365, CG31367, CG3139, CG31414, CG3143, CG31451, CG31459, CG31472, CG31475CG31483, CG31522, CG31528, CG31530, CG31531, CG31534, CG31536, CG31551, CG31555, CG3159, CG31605, CG31607, CG3161, CG31635, CG31642, CG31651, CG31660CG31666, CG31672, CG31678, CG3168, CG31689, CG31690, CG31704, CG31705, CG31714, CG31716, CG31719, CG31732, CG31743, CG31762, CG31764, CG31793, CG31795.CG3181, CG31811, CG31814, CG31815, CG31818, CG31826, CG31846, CG31847, CG31851, CG31856, CG31872, CG31908, CG31912, CG31916, CG31918, CG31919, CG3192, CG31924, CG31935, CG3194, CG31973, CG31992, CG31998, CG31999, CG32000, CG32006, CG3201, CG32016, CG32019, CG32037, CG32038, CG32043, CG32052, CG32056, CG32062, CG32066, CG32067, CG32082, CG3209,CG32100, CG32105, CG32130, CG32146, CG32148, CG32149, CG32150, CG32156, CG32158, CG32164, CG3217, CG32171, CG32177,CG32179, CG32180, CG3219, CG32209, CG32217, CG3222, CG32230, CG3224, CG32245, CG3225, CG32266, CG32276, CG32280, CG32284, CG32299, CG32316, CG32343, CG32344, CG32346, CG32355, CG32365, CG32381, CG3241, CG32412, CG32418, CG32423,CG32432, CG32434, CG32435, CG3244, CG32443, CG32444, CG3246, CG32464, CG32475, CG32479, CG3249, CG32498, CG32509, CG32512, CG32521, CG32527, CG32529, CG32530, CG32542, CG32553, CG32555, CG32562, CG32564, CG32573, CG32594, CG32599, CG32613, CG32626, CG3263, CG32631, CG32632, CG32634, CG32638, CG32662, CG32663, CG32672, CG32676, CG3269, CG32698, CG32703, CG32709, CG32717, CG32737, CG32743, CG32758, CG32767, CG32772, CG32776, CG32791, CG32795, CG32796, CG32809, CG32810, CG32811, CG32835, CG3284, CG32849, CG32919, , CG32920, CG32940, CG32954, CG3299, CG33005, CG3301, CG3303, CG33041, CG33048, CG3306, CG33080, CG33087, CG33106, CG33107, CG33113, CG33126, CG33129, CG33130, CG33138, CG33143,CG3315, CG33171, CG33175, CG3318, CG33184, CG33188, CG33197, CG33207, CG3321, CG3325, CG3327, CG33278, CG3329, CG3346, CG33472, , CG3360, CG3376, CG3403, CG3425, CG3430, CG3448, CG3454, CG3458, CG3460, CG3461, CG3466, CG3473, CG3479, CG3481, CG3488, CG3497, CG3501, CG3504, CG3523, , CG3529, CG3567, CG3572, CG3576, CG3579,CG3589,CG3593, CG3594, CG3604, CG3612, CG3625, CG3630, CG3652, CG3654, CG3661, CG3664, CG3682, CG3692, CG3696, , CG3698, CG3705, CG3713,CG3725, CG3732, CG3736, CG3747, CG3751, CG3757, CG3761, CG3777, CG3779, CG3812, CG3820, CG3825, CG3829, CG3830, CG3850, CG3851, CG3860, CG3861, CG3875, CG3880, CG3902, CG3917, CG3920, CG3937, CG3945, CG3954, CG3955, CG3960, CG3961, CG3967, CG3971, CG3973, CG3975, CG3979, CG3981, CG3988, CG3991, CG3995, CG3996, CG3998, CG4001, CG4003, CG4004, CG4005, CG40127, CG4013,

CG40146, CG40188, CG4019, CG40218, CG4022, CG4035, CG4036, , CG4039, CG40411, CG4042,

CG4043, CG4057, CG4070, CG4073, CG4083, CG4098, CG4114, CG4122, CG4123, CG4128,

CG4145, CG4148, CG4152, CG4161, CG4168, CG4170, CG4173, CG4192, CG4202, CG4206, CG4217, CG4239, CG4260, CG4265, CG4268, CG4274, CG4287, CG4294, CG4302, CG4306,

CG4313, CG4314, CG4316, CG4317, CG4321, CG4334, CG4351, CG4353, CG4354, CG4364,

CG4376, CG4389, CG4394, CG4405, CG4409, CG4413, CG4420,- CG4428, CG4435, CG4449,

CG4452, CG4453, CG4462, CG4466, CG4485, CG4494, CG4501, CG4502, CG4511, CG4533,

CG4539, CG4553, CG4556, CG4558, CG4586, CG4590, CG4591, CG4600, CG4602, CG4605, CG4609, CG4612, CG4630, CG4636, CG4643, CG4658, CG4660, CG4662, CG4676, CG4684,

CG4688, CG4691, CG4695, CG4696, CG4698, CG4700, CG4712, CG4717, CG4721, CG4722,

CG4723, CG4726, CG4729, CG4746, CG4750, CG4756, CG4759,CG4760, CG4761, CG4764,

CG4771, CG4798, CG4799, CG4806, CG4807, CG4817, CG4832, CG4838, CG4841, CG4843,

CG4845, CG4861, CG4867, CG4879, CG4881, CG4889, CG4893, CG4894, CG4898, CG4905, CG4911, CG4912, CG4918, CG4922, CG4928, CG4930, CG4931, CG4938, CG4945, CG4963,

CG4966, CG4972, CG4978, CG4979, CG4993, CG4999, CG5000, CG5014, CG5020, CG5023,

CG5027, CG5034, CG5055, CG5059, CG5064, CG5065, CG5073, CG5075, CG5076, CG5081,

CG5098, CG5116, CG5119, CG5137, CG5148, CG5155, CG5156, CG5162, CG5165, CG5166,

CG5167, CG5170, CG5184, CG5185, CG5187, CG5195, CG5201,CG5203, CG5210, CG5214, CG5216, CG5241, CG5248, CG5249, CG5269, CG5280, CG5292, CG5317, CG5320, CG5323,

CG5325, CG5341, CG5343, CG5364, CG5373, CG5385, CG5394, CG5397, CG5403, CG5405,

CG5409, CG5410, CG5411, CG5421, CG5431, CG5439, CG5441, CG5444, CG5452, CG5454,

CG5455, CG5462, CG5467, CG5472, CG5478, CG5482, CG5484, CG5486, CG5499, CG5500,

CG5518, CG5522, CG5532, CG5537, CG5538, CG5543, CG5546, CG5571, CG5594, CG5597, CG5605, CG5620, CG5629, CG5639, CG5650, CG5654, CG5657, CG5659, CG5664, CG5670,

CG5674, CG5675, CG5677, CG5683, CG5684, CG5688, CG5694, CG5708, CG5722, CG5726,

CG5727, CG5728, CG5735, CG5738, CG5739, CG574I; CG5753, CG5758, CG5760, CG5776,

CG5784, CG5785, CG5787, CG5803, CG5808, CG5813, CG5820, CG5821, CG5823, CG5830,

CG5851, CG5855, CG5857, CG5864, CG5867, CG5869, CG5880, CG5884, CG5889, CG5902, CG5905, CG5907, CG5915* CG5916, CG5919, CG5935, CG5938, CG5945, CG5958, CG5965,

CG5968, CG5973, CG5986, CG5994, CG6007, CG6008, CG6013, CG6016, CG6023, CG6024,

CG6027, CG6030, CG6036, CG6040, CG6050, CG6056, CG6058, CG6072, CG6073, CG6074,

CG6084, CG6091, CG6097, CG6105, CG6119, CG6121, CG6122, CG6138, CG6144/ CG6145,

CG6148, CG6151, CG6169, CG6174, CG6190, CG6192, CG6193, CG6203, CG6213, CG6214, CG6219, CG6231, CG6236, CG6238, CG6259, CG6264, CG6272, CG6290, CG6291, CG6302,

CG6311, CG6315, CG6321, CG6322, CG6329, CG6332, CG6335, CG6339, CG6340, CG6341,

CG6347, CG6354, CG6356, CG6357, CG6359, CG6363, CG6369, CG6372, CG6376, CG6383,

CG6394, CG6395, CG6398, CG6407, CG6409, CG6413, CG6422, CG6428, CG6438, CG6440,

CG6445, CG6450, CG6459, CG6463, CG6467, CG6474, CG6476, CG6490, CG6494, CG6496, CG6501, CG6503, CG6513, CG6520, CG6535, CG6540, CG6547, CG6549, CG6556, CG6562,

CG6567, CG6571, CG6575, CG6579, CG6588, CG6606, CG6619, CG6622, CG6623, CG6627,

CG6631, CG6647, CG6652, CG6654, CG6656, CG6668, CG6671, CG6680, CG6682, CG6686,

CG6689, CG6691, CG6693, CG6694, CG6702,CG6703, CG6707, CG6713, CG6726, CG6729,

CG6730, CG6733, CG6741, CG6745, CG6747, CG6749, CG6790, CG6794, CG6803, CG6806, CG6808, CG6815, CG6822, CG6834, CG6836, CG6838, CG6845, CG6851, CG6863, CG6864, CG6870, CG6871, CG6889, G6896, CG6903, CG6913, CG6917, CG6919, CG6920, CG6931, CG6936,

CG6937, CG6946, CG6948, CG6954, CG6959, CG6967, CG6971, CG6987, CG6992, CG6998,

CG7001, CG7009, CG7010, CG7013, CG7018, CG7023, CG7034, CG7035, CG7061, CG7067,

CG7082, CG7085, CG7088, CG7093, CG7097, CG7102, CG7103, CG7107, CG7110, CG7115, CG7120, CG7123, CG7140, CG7145, CG7146, CG7149, CG7154, CG7156, CG7162, CG7163,

CG7181, CG7184, CG7185, CG7206^ CG7220, CG7224, CG7231, CG7239,CG7265, CG7272,

CG7282, CG7292, CG7298, CG7301, CG7313, CG7321, CG7323, CG7324, CG7331, CG7354,

CG7359, CG7360,CG7362, CG7366, CG7376, CG7393, CG7421, CG7425, CG7434, CG7437,

CG7448, CG7459, CG7461, CG7478, CG7479, CG7503,CG7523, CG7525, CG7528, CG7529, CG7530, CG7532, CG7533, CG7535, CG7544, CG7555, CG7563, CG7571, CG7574, CG7576, CG7580, CG7583, CG7590, CG7594, CG7605, CG7607, CG7611, CG7614, CG7627, CG7628, CG7635, CG7641, CG7643, CG7644, CG7646, CG7654, CG7656, CG7672, CG7685, CG7693, CG7697, CG7698, CG7700, CG7708, CG7722, CG7730, CG7734, CG7739, CG7740, CG7757, CG7758, CG7765, CG7766, CG7772, CG7776, CG7778, CG7781, CG7811, CG7828, CG7830, CG7832, CG7834,CG7843, CG7850, CG7853, CG7861, CG7866, CG7869, CG7888, CG7891, CG7892, CG7893, CG7895, CG7896, CG7899, CG7904,CG7905, CG7913, CG7919, CG7920, CG7921, CG7922, CG7924, CG7931, CG7935, CG7939, CG7943, CG7945, CG7951, CG7958, CG7971, CG7978, CG7985, CG7993, CG7997, CG8001, CG8002, CG8003, CG8012, CG8014, CG8026, CG8031, CG8032, CG8048, CG8050, CG8055, CG8058, CG8066, CG8091, CG8092, CG8097, CG8098, CG8100, CG8103, CG8104, CG8116, CG8127, CG8129, CG8135, CG8144, CG8153, CG8161, CG8167, CG8174, CG8176, CG8177, CG8182, CG8186, CG8192, CG8195, CG8199, CG8200, CG8209, CG8211, CG8221, CG8226, CG8233, CG8235, CG8251, CG8272, CG8273, CG8276, CG8277, CG8280, CG8282, CG8284, CG8287, CG8293, CG8301, CG8312, CG8318, CG8320, CG8326, CG8334, CG8353, CG8359, CG8376, CG8378, CG8385, CG8388, CG8416, CG8417, CG8421, CG8426, CG8428, CG8434, CG8446, CG8448, CG8449, CG8451, CG8464, CG8472, CG8474, CG8478, CG8483, CG8484, CG8485, CG8493, CG8502, CG8505, CG8529, CG8532, CG8536, CG8538, CG8544, CG8545, CG8547, CG8556,CG8573, CG8580, CG8582, CG8588, CG8595, CG8600, CG8602, CG8604, CG8610, CG8613, CG8624, CG8631, CG8637, CG8649,CG8668, CG8669, CG8676, CG8677, CG8680, CG8683, CG8710, CG8717, CG8726, CG8735, CG8736, CG8739, CG8740, CG8774, CG8776, CG8787, CG8791, CG8798, CG8804, CG8808, CG8814, CG8815, CG8817, CG8824, CG8825, CG8827, CG8846, CG8863, CG8873, CG8884, CG8909, CG8950, CG8962, CG8977, CG8996, CG9006, CG9007, CG9009, CG9022, CG9036, CG9042, CG9045, CG9047, CG9056, CG9057, CG9071, CG9075, CG9083, CG9093, CG9098, CG9114, CG9119, CG9126, CG9131, CG9133, CG9159, CG9163, CG9172, CG9173, CG9175, CG9193, CG9194, CG9195, CG9212, CG9214, CG9218, CG9224, CG9238, CG9256, CG9259, CG9265, CG9267, CG9273, CG9281, CG9285, CG9291, CG9293, CG9296, CG9297, CG9299, CG9306, CG9310, CG9311, CG9313, CG9316, CG9317, CG9325, CG9328, CG9333, CG9335, CG9336, CG9338, CG9339, CG9345, CG9350, CG9364, CG9375, CG9381, CG9386, CG9388, CG9390, CG9393, CG9396, CG9399, CG9401, CG9412, CG9413, CG9415, CG9424, CG9426, CG9427, CG9434, CG9441, CG9445, CG9448, CG9453, CG9471, CG9473, CG9475, CG9517, CG9523, CG9528, CG9537, CG9540, CG9553, CG9575, CG9578, CG9581, CG9588, CG9603, CG9609, CG9610, CG9619, CG9636, CG9638, CG9641, CG9648, CG9654, CG9674, CG9691, CG9702, CG9748, CG9749, CG9771, CG9775, CG9796, CG9825, CG9828, CG9829, CG9847, CG9853, CG9854, CG9858, CG9868, CG9878, CG9889, CG9893, CG9894, CG9896, CG9901, CG9908, CG9916, CG9922, CG9924, CG9928, CG9954, CG9986,, CG9990, CG9995, CR30068, CR31429, CR31808, CR31969, CR32028, CR32218, CR32314, CR32489, CR32646, CR32735, CR32777, CR32957,CR33327, GH01566, CG10618, CG9075, CG12740, CG6486, GH05702, CG7776, CG2668, CG8639, CG7192, CG4013, CG6895, CG2931, CG9665, CG15281, CG15316, CG33558, CG13057, CG1594, CG4429, CG15889, CG5215, CG14782,

GH20809, GH22170, CG7180, GH23165, CG12262, CG3857, CG5748, CGl 1079, GH25573,

GH26506, GH26692, CG18531, CG6216, CG4602, GM01267, CG17941, GM02923, GM03003,

GM03761, GM03914, GM04363, GM04458, GM04775, GM05777, CG8146, CG9266, GM06790, CG32434, CG17632, GM07660, CG3333, CG15161, GM08821, GM09444, GM09534, CG8585, CG5281, CG14741, CG1362, HL01242, CG16757, CG5670, CG9008, CG15293, HL03512, CG4658, HL03793, CGl 1711, CG3620, CG5694, LD02060, CG4212, CG17484, CGl 116, CG31605,CG40293, LD07701, CGlOl 17, CG30032, CG12526, CG4059, CGl 1990, CG40280, CG4021, LD14406, CG8648, LD18978, CG12498, CG2875, CG9244, CG17454, CG3880, CG40285, CG10624, CG2505, CG17484, CG18124, CG7156, CG5720, CG6487, CG7985, CG32264,CG10023, LD46618, CG6762, CG1091, CG7586, CG14686, CG5214, CG1863, CG14206, CG3887, CG7836, LP07922, CG15489, CGl 1943, CG9533, CG8280, CG14802, CG5674, CG4362, CG12927, CGl 1147, CG7130, CG9265, CG9952, CG12543, CG6087, CG31085, CG17291, CG10618, CG3446, CG17866, CG5835, CG10497, CG7999, CG32169, CG1722, CG10118, CG17399, CG11206, CG18001, CG14904, CG1560,CG6531, CG4443, CG30483, CG3186, CG5038, CG3780, CG16876, CG12297, CG10746, CG12758, CG17299, CG15331, CG13841, CG40006, CG15519, CG3340, CG3340, CG11056, CG6847, CG11390, CG14806, CG13176, CG5407, CG12660, CG17725, CG10794,CG13143, CG3747, CG17534, CG3252, CG15295, CG7016, CG9894, CG1344, CG17052, CG11224, CG6426, CG8495, CG11895, CG13872, CG18001, CG17420, CG6949, CG17884, CG17866, CG12055, CG4211, CG1810, CG5344, CG6345, CG12704, CG5596, CG2960, CG7525, CG7361, CG8455, CG17131, CG18490, CG4392, CG10231, CG7662, CG2239, CG15565, CG12405, CG7781.CG10108, CG3446, CG1345, CG7787, CG5163, CG15162, CG12038, CG1651, CG14668, CG7998, CG11793, CG15483, CG5659, CG14336, CG4042, CG12992, CG6794, CG17018, CG7836, CG12563, CG5151, CG9777, SD03311, CG32850, SD04448, CG12473, CG3984, SD05379, CG12473, SD05785, SD06908, CG40153, CG8007, CG5929, CG15118, CG17474, CG7354, CG13872, CG14505, CG17018, CG12220, CG13086, TE20317

(c) Genes showing altered expression (downregulation) at 7 days time point during PTZ kindling: CG10019, CG1004, CG10045, CG10091, CG10106, CG10120, CG10126, CG10155, CG10214, CG10233, CG10433, CG10444, CG10553, CG10596, CG10640, CG1065, , CG1078, CG10812, CG10833, CG10863, CG1088, CGl 102, CG11064, CG11064, CGl 107, CGl 112, CG11147, CG11154, CGl 1218, CGl 1218, CGl 1368, CGl 1390, CGl 1440, , CGl 1665, CGl 1804, CGl 1853, CG12005, CG12054, CG12055, CG12120, CG12262, CG12393, CG12403, CG1274, CG13037, CG13421, CG13431, CG13848, CG13928, CG13933, CG14375, CG1449, CG1468, CG14872, CG14994, CG14996, CG1507, CG15093, CG1511, CG15201, CG15209, CG15261, CG15381, CG15845, CG1633, CG1668, CG1668, CG16747, CG16926, CG16944, CG16982, CG17108, CG17205, CG17280, CG17369, CG1743, CG1746, CG17521, CG17533, CG17759, CG17759, CG1783, CG17870, CG1793, CG17944, CG17945, CG18180, CG18285, CG18319, CG1838, CGl 8444, CG18778, CG1982, CG2060, CG2082, CG2096, CG2185, CG2222, CG2254, CG2297, CG2736, CG2827, CG2855, CG2993, CG30011, CG30052, CG30059, CG3036, CG3059, CG31207, CG3136, CG3139, CG3153, CG31535, CG31536, CG31605, CG31764, CG3192, CG31998, CG32000, CG32100, CG32112, CG32245, CG32412, CG3244, CG32444, CG32498, CG32521, CG32553, CG32568, CG32638, CG32653, CG32767, CG32820, CG3305, CG3314, CG3454, CG3488, , CG3511, CG3525, CG3572, CG3593, CG3625, CG3644, CG3692, CG3747,CG3752, CG3780, CG3860, CG3979, CG3982, CG4257, CG4261, CG4264, CG4347, CG4389, CG4409, , CG4475, CG4551, CG4553, CG4716, CG4729, CG4769, CG4800, CG4914, CG5061, CG5125, CG5194, CG5201, CG5263, CG5279, CG5315, CG5320, CG5428, CG5482,CG5499, CG5680, CG5708, CG5711, CG5735, CG5778, CG5809, CG5820, CG5830, CG5855, CG5867, CG5887, CG5935, CG5945, CG6033, CG6092, CG6311, CG6329, CG6343, CG6357, , CG6357, CG6416, CG6503, CG6513, CG6518, CG6554, CG6783, CG6917, CG6948,CG6983, CG6998, CG7077, CG7145, CG7235, CG7291, CG7331, CG7478, CG7540, CG7584, , CG7623, CG7738, CG7781, CG7929, CG7997, CG8137, CG8229, CG8261, CG8295, CG8430, CG8444, CG8643, CG8680, CG8732, CG8770, CG8808, CG8882, CG8933, CG8996, , CG9078, CG9218, CG9291, CG9326, CG9363, CG9396, CG9400, CG9441, CG9754, CG9847, CG9882, CR32777, CG6486, GH05702, CG14277, GM01267, HL03474, CG4551, CG4877, CG11153, LD22017, LD32791, LP04961, CG2043, CGl 1147, CG3525, CG1722, CG2254, CG4994, CGl 1390, CG4264, CG10045, CG3747, CG17399, RH37635, CGl 1556, RH38929, RH40310, RH48810, RH57045, RH59970, RH61355, CGl 1315, SD04448, SD06908

Table 6

(a) Overrepresented (based on unadjusted p-value) GO biological processes at 12 hrs time point during PTZ kindling: GO:0043062, GO:0048589, GO.0007300, GO:0006607, GO:0048086, GO:0019102, GO:0045496, GO:0030238, GO:0045497, GO:0000122, GO:0007619, GO:0040008, GO:0046701, GO:0042178, GO:0007486, GO:0030540, GO:0009407, GO:0009113, GO:0048071, GO.0006810, GO:0048070, GO:0035263, GO:0045477, GO:0030539, GO:0016457, GO:0015904, GO:0007485, GO:0042714, GO:0006091, GO:0007617, GO:0019098, GO:0019101, GO:0048062, GO:0042332, GO:0009629, GO:0045498, GO:0007487, GO:0044262, GO:0040007, GO:0009047, GO:0035193, GO:0035112, GO:0007484, GO:0046112, GO:0006188, GO:0046040, GO:0009987, GO:0051234, GO:0009886, GO:0045570, GO:0005977, GO:0006073, GO:0017143, GO:0009410, GO:0009404, GO:0006805, GO:0051179, GO:0015980, GO:0007483, GO:0046661, GO:0015893, GO:0006564, GO:0045476, GO:0045433, GO:0007301, GO:0008582, GO:0016545, GO:0007303, GO:0016482, GO:0030237, GO:0006563, GO:0050875, GO:0030725, GO:0043063, GO:0051124, GO:0048065, GO:0007582, GO:0007549, GO:0007276, GO:0009628, GO:0009070, GO:0045924, GO:0006112, GO:0007417, GO:0035215, GO:0019953, GO:0046660, GO:0017085, GO:0045892, GO:0000059, GO:0006839, GO:0048232, GO:0007283, GO:0018993, GO:0009116, GO:0009167, GO:0009127, GO:0009168, GO:0048542, GO:0007528, GO:0009069, GO:0009126, GO:0042493, GO:0007291, GO:0050795, (30:0016481, GO:0016542, GO:0009161, GO:0009156, GO:0000003, GO:0045944, GO:0009123, GO:0009124, GO:0045934, GO:0042445, GO:0005975, GO:0051093, GO:0035265, GO:0046620, GO:0007292, GO:0031324, GO:0007416, GO:0007446, GO:0009892, GO:0048513, GO:0006816, GO:0050808, GO:0007224, GO:0030198

(b) Overrepresented (based on unadjusted p-value) GO biological processes at 2 days time point during PTZ kindling: GO:0009987, GO:0050875, GO:0007582, GO:0007154, GO:0016043, GO:0050789, GO:0050794, GO:0051179, GO:0050791, GO:0007165, GO:0007242, GO:0007010, GO:0051244, GO:0051234, GO:000,6996, GO:0000902, GO:0019226, GO:0019222, GO:0008152, GO:0031323, GO:0006139, . GO:0006810, GO:0007267, GO:0044237, GO:0044238, GO:0019219, GO:0001505, GO:0050874, GO:0045449, GO:0007268, GO:0006350, GO:0008104, GO:0006351, GO:0006355, GO:0046907, GO:0051649, GO:0051641, (30:0048731, GO:0006886, GO:0045055, GO:0007269, (30:0015031, GO:0050877, GO:0002009, GO:0045184, GO:0016192, GO:0043283, GO:0043412, GO:0006464, GO:0006366, GO:0006357, GO:0006897, GO:0007626, GO:0000904, GO:0007163, GO:0006796, GO:0006793, GO:0030154, GO:0048468, GO:0000165, GO:0030198, GO:0050808, GO:0007264, GO:0045045, GO:0048489, GO:0007155, GO:0007243, GO:0043062, GO:0007610, GO:0007409, GO:0040007, GO:0007399, GO:0001738, GO:0046903, GO:0016265, GO:0048667, GO:0048666, GO:0031175, GO:0008283, GO:0008361, GO:0030029, GO:0030036, GO:0007411, GO:0016318, GO:0007467, GO:0030182, GO:0007416, GO:0016477, GO:0007275, GO:0006936, GO:0009056, GO:0016049, GO:0042067, GO:0044248, GO:0040008, GO:0009653, GO:0016310, GO:0006468, GO:0008345, (30:0016071, GO:0048732, GO:0016271, GO:0007559, GO:0016079, GO:0007015, GO:0040011, GO:0006898, GO:0051674, GO:0006928, GO:0001751, GO:0048518, GO:0007622, GO:0048522, GO:0006397, GO:0048102, GO:0035071, GO:0035070,GO:0007431, GO:0035272, GO:0006376, GO:0009628, GO:0048512, GO:0015980, GO:0012501, GO:0048511, GO:0008219, GO:0046530, GO:0006887, GO:0045494, GO:0007166, GO:0016334, GO:0045197, GO:0007164, GO:0001736, GO:0048519, GO:0016333, GO:0006090, GO:0051052, GO:0008105, GO:0007619, GO:0043119, GO:0043037, GO:0008069, GO:0045475, GO:0035088, GO:0001754, GO:0007623, GO:0042063, GO:0008360, GO:0051242, GO:0006605, GO:0016331, GO:0006066, GO:0048598, GO:0006811, GO:0009888, GO:0016542, GO:0006457, GO:0048523, GO:0007254, GO:0031098, GO:0016070, GO:0006268, GO:0048488, GO:0002165, GO:0007017, GO:0042592, GO:0006812, GO:0006092, GO:0008334, GO:0007568, GO:0008340, GO:0007391, GO:0051239, GO:0009791, GO:0007519, GO:0043118, GO:0006396, GO:0007422, GO:0042133, GO:0009057, GO:0042051, GO:0042462, GO:0007617, GO:0007517, GO:0051243, GO:0008594, GO:0006091, GO:0050801, GO:0007528, GO:0000074, GO:0008039, GO:0019933, GO:0050953, GO:0007601, GO:0050793, GO:0044265, GO:0051017, GO:0016350, GO:0044262, GO:0005996, GO:0051246, GO:0042461, GO:0016055, GO:0007498, GO:0006006, GO:0042445, GO:0008038, GO:0008037, GO:0019932, GO:0031326, GO:0035295, GO:0009889, GO:0019098, GO:0016200, GO:0001672, GO:0006493, GO:0045011, GO:0006950, GO:0006417, GO:0007167, GO:0009605, GO:0048065, GO-.0019935, GO:0006915, GO:0001709, GO:0006913, GO:0007286, GO:0051169, GO:0048515, GO:0017038, GO:0007222, GO:0001558, GO:0035239, GO:0009892, GO:0048589, GO:0009266, GO:0000122, GO:0009310, GO:0044270, GO:0045165, GO:0006445, GO:0009826, GO:0042332, GO:0009629, GO:0006094, GO:0048062, GO:0016335, GO:0007394, GO:0006096, GO:0017157, GO:0006606, GO:0009123, GO:0009124, GO:0045216, GO:0009586, GO:0007424, GO:0019318, GO:0006261, GO:0051049, GO:0015674, GO:0030030, GO:0051170, GO:0043170, GO:0042221, GO:0009117, GO:0009790, GO:0030537, GO:0009187, GO:0008016, GO:0016332, GO:0008015, GO:0000003, GO:0030707, (30:0007611, GO:0006512, GO:0043297, GO:0051301, GO:0000245, ⁽30:0006901, GO:0016183, GO:0031324, GO:0007178, GO:0008356, GO:0042330, GO:0006206, GO:0006082, GO:0019752, GO:0009314, GO:0051128, GO:0048513, GO:0009966, GO:0009416, GO:0044255, GO:0007318, GO:0016056, GO:0016540, GO:0009065, GO:0035089, GO:0006817, GO:0007043, GO:0007291, GO:0030005, GO:0007365, GO:0046777, GO:0035073, GO:0050807, GO:0035210, GO:0046528, GO:0006538, GO:0015977, GO:0046529, GO:0007050, GO:0042428, GO:0007276, GO:0008355, GO:0042052, GO:0019438, GO:0048545, GO:0035075, GO:0050896, GO:0016545, GO:0008062, GO:0008582, GO:0045433, GO:0045186, GO:0030001, GO:0007417, GO:0045927, GO:0051276, GO:0007459, GO:0006413, GO:0007033, GO:0007040, GO:0006816, GO:0007156, GO:0042048, GO:0001700, GO:0006629, GO:0019725, GO:0009165, GO:0009063, GO:0009161, GO:0009156, GO:0005975, GO:0007398, GO:0046552, GO:0006403, GO:0043067, GO:0007265, GO:0016081, GO.-.0035152, GO:0031589, GO:0002168, GO:0007160, GO:0007476, GO:0009725, GO:0019827, GO:0050829, GO:0046365, GO:0019320, GO:0006007, GO:0046164, GO:0042981, GO:0007423, GO:0045934, GO:0002164, GO:0000184, GO:0016476, GO:0019953, GO:0016337, GO:0007612, GO:0043087, GO:0051124, GO:0007392, GO:0030307, GO:0006874, GO:0006873, GO:0006875, GO:0030003, GO:0007041, GO:0035150, GO:0007472, GO:0009408, GO:0007028, GO:0042706, GO:0001763, GO:0001752, GO:0043285, GO:0007283, GO:0048232, GO:0048469, GO:0042127, GO:0009611, GO:0043068, GO:0035220, GO:0007389, GO:0007186, GO:0007297, GO:0044260, GO:0007016, GO:0000059, GO-.0030111, GO:0019538, GO:0009129, GO:0035209, GO:0016486, GO:0006348, GO:0035320, GO:0045313, GO:0006165, GO:0031509, GO:0009130, GO:0007000, GO:0051247, GO:0035188, GO:0006587, GO:0035268, GO:0035269, GO:0042135, GO:0046939, GO:0042326, GO:0044267, GO:0007507, GO:0006869, GO:0006461, GO:0045333, GO:0016358, GO:0046356, GO:0006099, GO:0009060, GO:0007001, GO:0009583, GO:0007218, GO:0016044, GO:0007602, GO:0006354, GO:0007034, GO:0045810, GO:0006885, GO:0007210, GO:0010001, GO:0046693, GO:0006835, GO:0015976, GO:0015698, GO:0006820, GO:0006470, GO:0015672, GO:0009719, GO:0019220, GO:0042559, GO:0035222, GO:0051174, GO:0009109, GO:0006084, GO:0009112, GO:0016481, GO:0000226, GO:0006073, GO:0048056, GO:0005977, GO:0007632, GO:0042136, GO:0007525, GO:0006221, GO:0006536, GO:0007464, GO:0030031, GO:0007292, GO:0030097, GO:0009168, GO:0009127, GO:0009126, GO:0009167, GO:0006259, GO:0007552, GO:0030163, GO:0008544, GO:0007613, GO:0006520, GO:0051187, GO:0016051, GO:0035114, GO:0048737₅ GO:0006519, GO:0045595, GO:0031325, GO:0009893, GO:0007530, GO:0006725, GO:0009792, GO:0007179, GO:0046698, GO:0009058, GO:0007350, GO:0006858, GO:0016360, GO:0045793, GO:0006112, GO:0043413, GO:0006486, GO:0006401, GO:0035151, GO:0042493, GO:0045926, GO:0006378, GO:0006732, GO:0048736, GO:0035107, GO:0006807, GO:0009968, GO:0006260, GO:0012502, GO:0048534, GO:0007281, GO:0044249, GO:0009582, GO:0007298, GO:0006402, GO:0046364, GO:0006220, GO:0030010, GO:0001737GO:0006302, GO:0019319, GO:0046165, GO:0006275, GO:0009993, GO:0009101, GO:0044257, GO:0051603, GO:0007049, GO:0044275, GO:0016052, GO:0009308, GO:0030534, GO:0045198, GO:0030859, GO:0007415, GO:0030381, GO:0006907, GO:0007414, GO:0008590, GO:0045936, GO:0045317, GO:0006611, GO:0009887, GO:0009880, GO:0046843, GO:0010033, GO:0009309, GO:0044271, GO:0019941, GO:0007169, GO:0007307, GO:0035317, GO:0040014, GO:0035264, GO:0000160, GO:0045892, GO:0048477, GO:0007635, GO:0045935, GO:0009953, GO:0007306, GO:0007018, GO:0007560, GO:0006164, GO:0009260, GO:0030705; GO:0048565, GO:0009100, GO:0006643, GO:0007293, GO:0007444, GO:0009259, GO:0006163, GO:0000278, GO:0006323, GO:0006325, GO:0007420, GO:0006118, GO:0009967, GO:0048627, GO:0048628, GO:0016311, GO:0007157, GO:0030855, GO:0016059, GO:0009607, GO:0006952, GO:0017145 (c) Overrepresented (based on unadjusted p-value) GO biological processes at 7 days time point during PTZ kindling:

GO:0006536, GO:0009064, GO:0006538, GO :0006082, GO:0019752, GO:0007582, GO:0009586, GO:0009583, GO:0007602, GO:0009065, GO :0008152, GO:0006091, GO:0007202, GO:0007610, GO:0009582, GO:0009581, GO:0009987, GO :0006119, GO:0016059, GO:0042048, GO:0006725, GO:0050875, GO:0051606, GO:0045213, GO :0043112, GO:0008345, GO-.0006793, GO:0006796, GO:0009628, GO:0009063, GO:0044237, GO :0009112, GO:0009310, GO:0044270, GO:0016062, GO:0007635, GO:0007154, GO:0009416, GO -.0042398, GO:0006519, GO:0046693, GO:0050896, GO:0006520, GO:0009314, GO:0044248, GO :0016310, GO:0007438, GO:0042026, GO:0051179, GO:0009605, GO:0007626, GO:0051234, GO :0008355, GO:0030537, GO:0006810, GO:0051084, GO:0001742, GO:0016056, GO:0009056, GO :0044238, GO:0019395, GO:0007613, GO:0050953, GO:0007601, GO:0007612, GO:0006092, GO :0009308, GO:0046483, GO:0009084, GO:0007167, GO:0007242, GO:0006807, GO:0007416, GO :0042136, GO:0044271, GO:0009309, GO:0015980, GO:0006144, GO:0050874, GO:0050877, GO :0046692, GO:0006732, GO:0050808, GO:0030198, GO:0009117, GO:0007611, GO:0007267, GO :0030154, GO:0007165, GO:0030097, GO:0030718, GO:0051186, GO:0001505, GO:0006542, GO :0006646, GO:0015988, GO:0046337,GO:0018987, GO:0007258, GO:0015858, GO:0007185, GO :0006580, GO:0035313, GO:0006588, GO:0009449, GO:0046358, GO:0046335, GO:0015860, GO :0009755, GO:0006754, GO:0015986, GO:0015985, GO:0006753, GO:0050876, GO:0007320, GO :0048609, GO:0046034, GO:0048488, GO:0006631, GO:0008103, GO:0048015, GO:0035223, GO :0007479, GO:0007200, GO:0042133, GO:0009206, GO:0009201, GO:0009145, GO:0007010, GO :0048534, GO:0009142, GO:0009199, GO:0009205, GO:0009144, GO:0042775, GO:0006090, GO :0042401, GO:0007432, GO:0019438, GO:0042773, GO:0009141, GO:0007309, GO-.0006752, GO :0006120, GO:0007281, GO:0019827, GO:0009165, GO:0046530, GO:0007308, GO:0043062, GO :0019226, GO:0048599, GO:0007498, GO:0006118, GO:0006139, GO:0007449, GO-.0009954, GO :0006818, GO:0048111, GO:0015992, GO:0048110, GO:0015865, GO:0051503, GO:0006862, GO :0015866, GO:0009588, GO:0006103, GO:0042811, GO:0007174, GO:0019551, GO:0015867,GO: 0048129, GO:0006587, GO:0006531, GO:0042787, GO:0035202, GO:0009994, GO:0009612, GO :0009060, GO:0006099, GO:0046356, GO:0045333, GO-.0009109, GO:0006084, GO:0007166, GO :0019932, GO:0051187, GO:0007169, GO:0006575, GO:0009072, GO:0006952, GO-.0009152, GO :0006100, GO:0006635, GO:0030952, GO:0016325, GO:0030951, GO:0009150, GO:0009607, GO :0006164, GO:0009260, GO:0005975, GO:0009259, GO:0006163, GO:0006457, GO:0007600, GO :0044255, GO:0042427, GO:0044404, GO:0006939, GO:0044419, GO:0009399, GO:0006573, GO -.0006572, GO:0046219, GO:0009448,GO:0006541, GO:0035247, GO:0019919, GO-.0018195, GO :0018216,GO:0045742, GO:0046958, GO:0035246, GO:0044403, GO:0009405, GO:0019605, GO :0007467, GO:0048468, GO-.0009108, GO:0048489

Table 7: GO biological processes (downregulated) common to all the three time points during PTZ kindling - 12 hrs, 2^nd day, and 7^th day

Note (Table 7): GO:0007582, GO:0009987, and GO:0050875 may be considered too broad. This leaves 11 specific processes perturbed consistently during kindling. Further details are mentioned in the text.

Table 8

(a) Genes upregulated by LEV:

CG12591, CG1021, CG8345, CG33473, CG4233, CG3534, CG9485, CG10833, CG13181, CG1982, CG7065, CG1112

(b) Genes upregulated by NaVP:

CG1004, CG10120, CG10186, CG10360, CG10527, CG10652, CG10742, CG10833, CG10863, CG10888, CG10944, CG10960, CG11064, CG11089, CG11303, CG11315, CG11390, CG11400, CG11739, CG11843, CG12055, CG12393, CG13037, CG13279, CG13517, CG13868, CG13928, CG14109, CG14290, CG14648, CG1475, CG14757, CG15096, CG1516, CG15365, CG1561, CG1633, CG16747, CG16926, CG16936, CG17051, CG17143, CG17273, CG1750, CG17533, CG17534, CG17841, CG17896, CG17903, CG17977, CG18240, CG18319, CG1865, CG1902, CG1927, CG1973, CG1982, CG2060, CG2076, CG2233, CG2674, CG2698, CG2718, CG2736, CG2746, , CG2827, CG2846, CG2934, CG3050, CG31159, CG31689, CG31764, CG3203, CG3214, CG32306, CG32444, CG32653, CG32920,CG32954, CG3301, CG33060, , CG33106, CG3314, CG33187, CG3320, CG3395, CG3403, CG3644, CG3661,CG3665, CG3752, CG3811, CG3979, CG3989, CG4067, CG4233, CG4347, CG4527, CG4572, CG4600, CG4634, CG4692, CG4716, CG4769, CG4897, CG4914, CG5192, CG5330, CG5362, CG5384, CG5493, CG5502, CG5827, CG6020, CG6058, CG6119, CG6180, CG6287, CG6341, CG6467, CG6647, CG6950, CG7053, CG7109, CG7176, CG7470, CG7490, CG7576, CG7584, CG7592, CG7686, CG7726, CG7834, CG7997, CG8036, CG8223, CG8229, CG8309, CG8345, CG8430, CG8636, CG8759, CG8808, CG8882, CG8922, CG9140, CG9212, CG9258, CG9282, CG9282, CG9390, CG9396, , CG9445, CG9691, CG9734, CG9916, CG6486, CG14933, GH13704, HL03474, CG12526, CG14439, CG14219, CG9360, CG8317, CG10045, CG12055, CG7390, CG7662, , CGl 1793, CG1548, CG15118

Table 9

(a) GO biological processes overrepresented in LEV upregulated gene set: (30:0006531,- GO:0045213, GO:0009250, GO:0005978, GO:0043112, GO:0006537, GO:0005975, (30:0006091, GO:0009066, GO:0009084, GO:0006073, GO:0043284, GO:0006536, GO:0005977, GO:0000271, GO:0006112, GO:0009064, GO:0006520, GO:0007416, GO:0006519, GO:0050808, GO:0030198, GO:0006118, GO:0043062, GO:0009308, GO:0019752, GO:0006082, GO:0006807, GO:0016051, GO:0044249, GO:0009058, GO:0006418, GO:0043039

(b) GO biological processes overrepresented in NaVP upregulated gene set:

(30:0006091, GO:0044249, GO:0019752, GO:0006082, GO:0009058, GO:0007582, GO:0008152,

GO:0006092, GO:0015980, GO:0009064, GO:0008652, GO:0006531, GO:0044237, GO:0044271,

GO:0009309, GO:0006118, GO:0044248, GO:0006090, GO:0050875, GO:0009056, GO:0009059,

GO:0006537, GO:0006412, GO:0009060, GO:0045333, GO:0006099, GO:0046356, GO:0009987,

GO:0006084, GO:0009109, GO:0006414, GO:0051187, GO:0006520, GO:0009066, GO:0009084, GO:0006006, GO:0006536, GO:0006519, GO:0005975, GO:0043037, GO:0006119, GO:0006108,

GO:0006810, GO:0042775, GO:0042773, GO:0044262, GO:0006120, GO:0044238, GO:0019318,

GO:0015671, GO:0046500, GO:0006797, GO:0019058, GO:0006097, GO:0015669, , GO:0016032,

GO:0006556, GO:0046487, GO:0006798, GO:0006732, GO:0006164, GO:0051186, GO:0006163,

GO-.0046483, GO:0046365, GO:0046164, GO:0019320, GO:0006007, GO:0009308, GO:0006807, GO:0005996, GO:0006725, GO:0035202, GO:0006081, GO:0009231, GO:0007174, GO:0042727,

GO:0042726, GO:0019430, GO:0006771, GO:0006100, GO:0009117, GO:0006767, GO:0035272,

GO:0007431, GO:0051234, GO:0006793, GO:0006796, GO:0009165, GO:0048732, GO:0043170,

GO:0044267, GO:0043112, GO:0045213, GO:0009399, GO:0045742, GO:0006573, GO:0006541,

GO:0019538, GO:0035070, GO:0048102, GO:0035071, GO:0007559, GO:0044275, GO:0016052, GO:0016271, GO:0051179, GO:0044260, GO:0007602, GO:0009583, GO:0000097, GO:0006561, GO:0007176, GO:0009112, GO:0016310

Table 10: GO biological processes upregulated by LEV and NaVP, compared to specific processes downregulated at all the three time points during PTZ kindling

Note (Table 10): Further details are mentioned in the text.

Table 11: Mean and S. E. of climbing speed in cm/sec, in flies examined to test the effect of dietary sugar on PTZ induced kindling.

Note (Table 11): NF, normal media; PTZ, normal media containing PTZ. * sugar deficient media, with 50% of sugar, caused lethality; ** sugar rich media, with 200% of sugar. Further details are mentioned in the text.

Table 12: Mean and S.E. of climbing speed in cm/sec, in flies examined to test the behavioral effect of

CG8428 dsRNA injection.

Note (Table 12): IB, injection buffer; LEV, levetiracetam; dsKNA, CG8428 dsRNA in IB. Further details are mentioned in the text.

Table 13

(a) Eighty seven Drosophila genes identified as enriched source of potential biomarkers::- CG1004, CG10045, CG10120, CG10527, CG10742, CG10833, CG10863, CG10888, CGl 1064, CGl 112, CG11315, CG11390, CG11739, CG11793, CG12055, CG12393, CG12526, CG13037, CG13868, CG13928, CG14290, CG14648, CG14757, CG15118, CG15365, CG1633, CG16747, CG16926, CG16936, CG17273, CG17533, CG17534, CG17841, CG17896, CG17903, CG17977, CG18319, CG1902, CG1973, CG1982, CG2060, CG2233, CG2736, -CG2827, CG2934, CG3050, CG31689, CG31764, CG32444, CG32653, CG32920, CG32954, CG3301, CG33106, CG3314, CG3403, CG3644, CG3661, CG3752, CG3979, CG4347, CG4600, CG4716, CG4769, CG4914, CG6058, CG6119, CG6341, CG6467, CG6486, CG6647, CG7576, CG7584, CG7662, CG7834, CG7997, CG8229, CG8430, CG8808, CG8882, CG9212, CG9390, CG9396, CG9445, CG9691, CG9916, and CG15293.

(b) Eighty two human genes which are counterparts of fly genes listed in (a) - ACAA2, ACSS2, ADSSLl, ALDHlBl, ALDH2, ALDH6A1, ALDOA, ALDOB, ALDOC, ANKRD13A, ANKRD13B, ANKRD17, ATP6V0D1, BRP44, CESl, CES2, CYCl, CYCS, EEF1B2, EIF3S2, ENSG00000060762, ENSGOOOOO 108272, ENSG00000134717, ETFB, FAM109B, FAM57A, FAM57B, FLJ20487, GALM, GAPDH, GLA, GOTl, GOT2, GSTAl, LIN7C, LOC441034, LOC442239, LOC645296, LOC645846, LOC653381, MEl, MTHFSD, NAGA, NIPSNAPl, OVCHl, OVCH2, PDKl, PDK2, PDK3, PDK4, PEX7, PPIF, PPILl₅ PRDX2, PRDX5, PREI3, RAB3A, RAB3B, RAB3C, RHO, RHOA, RHOB, RPL23, RPL7A, SCYLl, SFXNl, SLC13A1, SLC13A2, SLC13A3, SLC18A2, SODl, SORD, SRAl, TALDOl, TMPRSSl IB, TSPAN33, TSPAN5, TTK, UBE2N, UBE2NL, UGP2, and VDAC2

Table 14

(a) LEV up regulated genes under the six GO categories - CG4233, CG1982, CG3534, and CG9485.

(b) Human counterparts of genes listed in (a) - SORD, GOT2, XYLB, AGL, and LOC653381.

Table 15: Mean and S.E. of climbing speed in cm/sec, in flies examined to test the behavioral effect of KCl in PTZ kindling.

Table 16: PCR primers used in dsRNA preparation

ADVANTAGES OF THE INVENTION:

1. There is a pressing need to identify new therapeutic targets in epileptogenesis. Potential drug targets identified in the present invention fulfills, this need. Small molecules which are already known to act upon these targets can be readily screened using the behavioral module of the fly systems model described. This will provide a rapid path to drug development.

2. The present invention provides gene expression signatures of epileptogenesis and antiepileptic drug action in a fly model. A gene expression-based approach can be readily applied in screening of approved drugs, to identify those which produce antiepileptogenic expression signatures. This would provide a more rapid path to clinical application.

3. By providing transcriptomic changes associated with kindling epileptogenesis and therapeutic action of antiepileptic drugs, the invention allows prediction of therapeutic as well as side effect profiles of existing or novel therapeutic agents. The existing antiepileptic drugs, for example, can be rapidly screened to uncover these profiles at systems level.

4. By providing gene expression profiles underlying epileptogenesis and antiepileptic drug action, the invention allows selection of promising pharmacogenetic candidates. Single nucleotide polymorphisms (SNPs) in these candidate genes, for example, may be used to examine if they contribute to differential side effect profiles and adverse drug reactions in different groups of patients. Successful identification of such genetic markers would facilitate realization of personalized medicine.

5. Epileptic activity in patients's brain can potentially be identified using gene expression-based methods. The potential gene expression biomarkers provided in the invention may be used to detect the process of epileptogenesis in at-risk individuals.

6. Chronic models of epilepsy currently available for screening of antiepileptogenic drugs are difficult to prepare, expensive and time-intensive. Developing simpler, faster and less expensive models are urgently required for drug screening. The behavioral phenotype based screening method described here is most simple, rapid and inexpensive, and hence of tremendous advantage.

7. Identification of bimolecular targets for discovering antiepileptogenic, disease-modifying and neuroprotective drugs is highly required at present. It is expected to be of great value in developing not only high throughput methods of screening small molecules but also biopharmaceuticals such as protein therapeutics and antibodies. The present description of such targets is therefore of obvious advantage.

8. Potential biomarkers identified here may provide means of disease diagnosis by analysis of urine, cerebrospinal fluid, and plasma samples.

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Claims

1. A method for identification of potential biomarkers for epileptogenesis and related brain plasticity disorders, said method consisting the steps of: [a] treating Drosophila flies with either convulsant or antiepileptic drugs in a chronic manner;

[b] extracting RNA from the heads of treated flies of step [a] at several time-points secondary to drug treatments;

[c] generating microarray gene expression profiles specific for convulsant and. antiepileptic drugs respectively using the RNA extracted from step [b];

[d] identifying genes that are up- and down-regulated by convulsant and antiepileptic drugs from the microarray of step [c];

[e] identifying genes which are down- and up-regulated respectively by convulsant and antiepileptic drugs in the fly model and the homologs downregulated in established rodent models of epilepsy development as candidate biomarkers of epileptogenesis.

2. A method for identification of potential drug targets against epileptogenesis and related brain plasticity disorders, said method consisting the steps of:

(a) comparing fly genes that are regulated by different antiepileptic drugs in Drosophila head, in terms of biological processes they over-represent;

(b) identifying fly genes that are similarly regulated by different antiepileptic drugs as potential drug targets;

(c) confirming the candidature of the potential drug targets by verifying the biological processes they represent.

3. A method according to claim 1, wherein products of Drosophila genes Aldolase (CG6058) and Glutathione S transferase D4 (CGl 1512) are identified as biomarkers of kindling- epileptogenesis like plasticity in the fly model.

4. A method according to claim 1, wherein the products of homologs of fly genes CG6058 and CGl 1512 are identified as potential biomarkers of epileptogenesis and associated disorders.

5. A method according to claim 2, wherein products of Drosophila genes Glutamate oxaloacetate transaminase 2 (CG4233) and Sorbitol dehydrogenase 1 (CG1982) are identified as targets of antiepileptic drugs in fly model of kindling-epileptogenesis like plasticity.

6. A method according to claim 2, wherein products of homologs of fly genes CG4233 and CGl 982 are identified as potential targets for developing drugs against epilepsy and related disorders.

7. Use of products of human homologs of fly genes CG6058 and CGl 1512 as biomarkers of epileptogenesis in at-risk individuals.

8. Use of products of human homologs of fly genes CG4233 and CGl 982 as drug targets for developing therapeutic agents against epilepsy and related brain disorders.