A Drosophila systems model of pentylenetetrazole induced locomotor plasticity responsive to antiepileptic drugs
© Mohammad et al; licensee BioMed Central Ltd. 2009
Received: 18 September 2008
Accepted: 21 January 2009
Published: 21 January 2009
Rodent kindling induced by PTZ is a widely used model of epileptogenesis and AED testing. Overlapping pathophysiological mechanisms may underlie epileptogenesis and other neuropsychiatric conditions. Besides epilepsy, AEDs are widely used in treating various neuropsychiatric disorders. Mechanisms of AEDs' long term action in these disorders are poorly understood. We describe here a Drosophila systems model of PTZ induced locomotor plasticity that is responsive to AEDs.
We empirically determined a regime in which seven days of PTZ treatment and seven days of subsequent PTZ discontinuation respectively cause a decrease and an increase in climbing speed of Drosophila adults. Concomitant treatment with NaVP and LEV, not ETH, GBP and VGB, suppressed the development of locomotor deficit at the end of chronic PTZ phase. Concomitant LEV also ameliorated locomotor alteration that develops after PTZ withdrawal. Time series of microarray expression profiles of heads of flies treated with PTZ for 12 hrs (beginning phase), two days (latent phase) and seven days (behaviorally expressive phase) showed only down-, not up-, regulation of genes; expression of 23, 2439 and 265 genes were downregulated, in that order. GO biological process enrichment analysis showed downregulation of transcription, neuron morphogenesis during differentiation, synaptic transmission, regulation of neurotransmitter levels, neurogenesis, axonogenesis, protein modification, axon guidance, actin filament organization etc. in the latent phase and of glutamate metabolism, cell communication etc. in the expressive phase. Proteomic interactome based analysis provided further directionality to these events. Pathway overrepresentation analysis showed enrichment of Wnt signaling and other associated pathways in genes downregulated by PTZ. Mining of available transcriptomic and proteomic data pertaining to established rodent models of epilepsy and human epileptic patients showed overrepresentation of epilepsy associated genes in our PTZ regulated set.
Systems biology ultimately aims at delineating and comprehending the functioning of complex biological systems in such details that predictive models of human diseases could be developed. Due to immense complexity of higher organisms, systems biology approaches are however currently focused on simpler organisms. Amenable to modeling, our model offers a unique opportunity to further dissect epileptogenesis-like plasticity and to unravel mechanisms of long-term action of AEDs relevant in neuropsychiatric disorders.
Epileptogenesis is poorly understood in cellular and molecular terms [1, 2]. A systems level understanding of epileptogenesis – a network problem that may involve molecular, structural, and functional alterations in the brain – is expected to facilitate development of novel antiepileptogenic, disease-modifying, and neuroprotective agents . Besides epilepsy, AEDs are also used in treating various other neurological and psychiatric conditions [4–6]. Overlapping pathophysiological mechanisms may underlie epilepsy and these other nonepileptic conditions [4, 7]. Kindling is a model of brain plasticity in which recurrent activation of neural pathways results in an increased susceptibility to evoked seizures that ultimately progresses to spontaneous seizures . Kindling in rodents is widely used to model epileptogenesis [1, 2] – process whereby structural and functional changes occur following an insult that in some cases result in epilepsy and processes that contribute to the progression observed in some epilepsies including post-seizure cognitive and emotional deficits – and to test AEDs [9–13]. Not surprisingly, kindling-like phenomena is also considered relevant in various neuropsychiatric conditions [14–16]. Only a limited understanding exists at present as to how the initial electrographic seizure-induced changes in synaptic transmission and gene expression relate to permanent alteration in brain function induced by kindling .
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 and seizure-susceptibility has clearly been demonstrated [18–23]. Drosophila system has also been found useful in anticonvulsant drug screening and drug target identification [24–27]. The ultimate goal of systems biology is to delineate and to comprehend the functioning of complex biological systems in such details that predictive models of human diseases could be developed . However, due to immense complexity of higher organisms, systems biology approaches are currently focused on developing simple organisms as integrative models [29, 30]. For example, inherent complexity of mammalian brain however does not render the available rodent epilepsy models as amenable to systems modeling . Given the above, we selected Drosophila for developing a first ab initio systems level model of brain plasticity that might be relevant in understanding mechanisms underlying epileptogenesis and AEDs' long-term therapeutic action in these neuropsychiatric conditions.
Rodent kindling induced by PTZ is the most popular pharmacologically induced kindling model that provides an acceptable approach for quantifying epileptogenesis and for testing AEDs . In rodents, PTZ kindling is induced by repeated injection of a subconvulsant dose of the GABA antagonist over several weeks, resulting 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 [14, 32]. Increased seizure susceptibility is known to occur via excitatory GABAergic signaling in Drosophila . Moreover, chronic exposure to PTZ, a GABA antagonist, has been reported to cause seizure phenotypes in Drosophila larvae . We thus selected PTZ for systems modeling. Altered locomotor activity is one of the kindling and postkindling behavior in rats and mice [33, 34]. Also, seizure-like activity in Drosophila adults is known to be associated with loss of motor coordination, and altered locomotor activities . Locomotor activity is a complex behavior and different neural systems may influence it in fly . Locomotor behavior of Drosophila adults is used to model neuropsychiatric conditions . Given the above, we selected Drosophila adults and focused on developing a locomotor behavior based model.
Fly culture and harvesting
D. melanogaster wild type Oregon-R strain was used. Routine cultures were maintained at 24 + 1°C, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hrs dark cycle. Standard fly medium consisting of agar-agar, maize powder, brown sugar, dried yeast and nipagin was used. Standard methods of fly handling and manipulation were followed. Stringency required in behavioral studies was strictly adhered to at various levels, conditions of housing, exposure to anesthetic agent, light intensity, for example. Three to four days old unmated male flies were used to begin all experiments except dsRNA microinjection. For microinjection, 5–6 day old flies were used.
Drug dosage and treatment
LC50, defined as concentration in normal fly media causing 50% lethality in seven days, was determined for PTZ and the AEDs (results not presented). Unless mentioned otherwise, half of LC50, 8, 3.48, 0.33 and 5 mg/ml for PTZ, ETH, NaVP (all from Sigma-Aldrich), and LEV (Levesam 500, Nicholas Piramal), respectively, was used. As GBP and VGB (both from Sigma-Aldrich) did not cause lethality up to 16 and 24 mg/ml respectively, the same concentrations were used in all the experiments. Thirty flies were housed in each treatment vial. Flies were maintained at 24 + 1°C, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hrs dark cycle.
Climbing speed was measured using an indigenously developed semi-manual method that was validated by DIAS (v. 3.4.2, Soll Technologies). In semi-manual 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 vertically placed column for 90 seconds. The fly was thereafter 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 and, in semi-manual method, a "dot/comma" recording, explained below, was applied the moment fly crossed a height of 5 cm. In "dot/comma" recording, the locomotor activity of a single fly was monitored by keeping pressed 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 activity and rest period respectively, using the cursor speed. Climbing speed was calculated using the following formula, s = h/t, where s = climbing speed, h = height climbed in cm, and t = activity period in sec. One fly each from different treatment groups was first scored as described below, discarded, and the exercise repeated. Measurements were taken at room temperature, between 9 AM to 9 PM. 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. Comma was applied only when the fly took rest. 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 keeping pressed the dot key or the comma key of a personal computer, to record a moving or resting fly respectively. 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 was obtained by dividing the lines (cm) 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, 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, distance walked and walking speed. The semi-manual climbing assay described above was validated using DIAS. TD was measured using DIAS, by video recording flies in the treatment vials with media containing 16 mg/ml PTZ. TD 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.
Microarray expression profiling
Total cellular RNA was isolated from fly heads belonging to five biological replicates, four for microarray profiling, and one for real-time PCR. Each replicate represented four independent sets of control and treated flies. Each independent set consisted of eight vials, four each of NF and PTZ, treated in parallel. Similar design was followed for control NF-NF control microarray experiment. Thirty flies were treated in each vial. Heads of 120 flies were pooled for isolating each RNA sample. 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 -80°C. Total RNA was isolated from frozen fly heads using TRI REAGENT (Sigma), according to the manufacturer's protocol. RNA was quantified using spectrophotometer. RNA quality was checked by running 1% agarose gel.
Microarray -cDNA Synthesis Kit, -Target Purification Kit, and -RNA Target Synthesis Kit (Roche) were used to generate labeled antisense RNA. Starting with 10 μg of total cellular RNA, Eberwine method (kits from Roche) was used to generate cDNA and thereafter Cy3 and Cy5 (Amersham) labeled antisense RNA. The Cy3 and Cy5 labeled aRNAs (control and treated) were pooled together and precipitated, washed, air-dried, and dissolved in 18 MΩ RNAase free water (Sigma). The recovery of labeled aRNAs was checked using spectrophotometer and agarose gel electrophoresis. A total of 16 microarrays (12Kv1, CDMC) were hybridized, four each for NF-NF control (0 hr); NF-PTZ, 12 hrs; NF-PTZ, 2nd day; NF-PTZ, 7th day. Out of four, two slides were dye-swaps. Hybridization solution was prepared by mixing hybridization buffer (DIG Easy Hyb, Roche), 10 mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10 mg/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. The slides were covered by lowering down a 24 × 60 mm coverslip (ESCO, Portsmouth, USA). Hybridization was allowed to take place in hybridization chamber (Corning) at 37°C for 16 hrs. After hybridization, coverslips were removed by submerging the slides in a solution containing 1× SSC and 0.1% SDS at 50°C. Slides were washed (three times for 15 minutes each) in a coplin jar at 50°C with occasional swirling and then transferred to 1× SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Finally, slides were washed in 0.1× SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes. Slides were scanned at 10 μm resolution using GenePix 4000A Microarray Scanner (Molecular Devices), and the images preprocessed and quantified using Gene Pix Pro 6.0 (Molecular Devices).
Unless mentioned otherwise, pair-wise Student's t-test, two-tailed, heteroscedastic, uncorrected, was performed for behavioral analysis. In microarray gene expression analysis, ratio based data normalization and selection of features were performed using Acuity 4.0 (Molecular Devices). All Spots with raw intensity less then 100 U 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 SAM 3.0 (Excel Add-In) , under the conditions of one class response and 100 permutations. Wherever available, non-CG number genes were converted to CG numbers mainly using BDGP http://www.flybase.org. Gene IDs were converted to FBgn numbers using GeneMerge http://genemerge.bioteam.net/convertgenenames.html, before GOTool Box  was used to retrieve overrepresented biological processes in up- or down- regulated genes, under the settings, ontology, biological process; mode, all terms; reference, genome; evidence, all-all evidence; species, D. melanogaster; GO-stats; statistic test, hypergeometric http://burgundy.cmmt.ubc.ca/GOToolBox/. DAVID  was used for pathway enrichment analysis http://david.abcc.ncifcrf.gov/home.jsp. P values were obtained with or without correction for multiple hypotheses testing, as indicated in the results section. Hypergeometric distribution probabilities for genes and GO processes were calculated assuming population sizes of 10000, approximately the number of unique genes in the arrays, and 4041, a previously estimated number for Drosophila , respectively.
Results and Discussion
Chronic PTZ locomotor model and its validation by AEDs
NaVP and LEV, not ETH, GBP and VGB, were found to be effective in our chronic PTZ model of locomotor plasticity. NaVP and LEV are known to suppress both kindling development as well as kindled seizure in rodent models [42, 43]. Clinical trials however suggest that LEV, not NaVP, possesses antiepileptogenic activity [9, 44, 45]. Overall, the profiles of NaVP and LEV in the fly model were consistent with their therapeutic potential. Ineffectiveness of other AEDs in the fly model however suggested that our model is only partly predictive of therapeutic potential. Clinical relevance of the model is also limited by the fact that the drug doses used in developing the model were selected either empirically or based on toxicity.
Time series of transcriptomic changes underlying chronic PTZ
We first examined if 'seizure' genes already described in Drosophila show enrichment in our PTZ induced downregulated genes. Evidence suggests conserved mechanisms between Drosophila bang-sensitive mutants and human seizure disorders . Molecular defects in five such mutants involve genes encoding mitochondrial ribosomal protein (CG7925), ADP/ATP translocase (CG16944), the citrate synthase (CG3861), an ethanolamine kinase (CG3525), and an aminopeptidase (CG5518). Prevalence of mitochondrial function disrupting defects in bang-sensitive mutants suggests that impaired energy metabolism may underlie the affected behavior . 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 . Our microarrays had all the above bang-sensitive genes spotted except CG7925. Notably, all four spotted seizure genes were downregulated by PTZ. This overlap was statistically significant (hypergeometric distribution, p = 0.004).
Overrepresented GO biological processes in genes downregulated on 2nd day of PTZ treatment
transmission of nerve impulse
nervous system development
regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process
regulation of neurotransmitter levels
cell projection organization and biogenesis
generation of neurons
cytoskeleton organization and biogenesis
RNA metabolic process
protein modification process
regulation of transcription
post-translational protein modification
synaptic vesicle transport
photoreceptor cell differentiation
eye photoreceptor cell differentiation
regulation of cell size
Enriched GO biological processes in genes downregulated on 7th day of PTZ treatment
carboxylic acid metabolic process
glutamine family amino acid metabolic process
glutamate metabolic process
glutamate catabolic process
Interacting partners of 12 hrs genes
Overrepresented GO biological processes among CG7583 (CtBP) and its direct interacting partners
negative regulation of transcription from RNA polymerase II promoter
negative regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process
regulation of transcription from RNA polymerase II promoter
negative regulation of transcription, DNA-dependent
transcription from RNA polymerase II promoter
regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process
regulation of transcription
cell fate commitment
RNA metabolic process
RNA biosynthetic process
nucleobase, nucleoside, nucleotide and nucleic acid metabolic process
Enriched pathways in CG7583 (CtBP) direct and indirect interacting partners
dme04310:Wnt signaling pathway
dme04630:Jak-STAT signaling pathway
dme04350:TGF-beta signaling pathway
dme04320:Dorso-ventral axis formation
dme04010:MAPK signaling pathway
We further examined the relevance of fly model in epilepsy and related neurological and psychiatric conditions through mining reported transcriptomic and proteomic data related to available animal models and human patients. For this, we retrieved rat, mouse and human homologs (FLIGHT, homologene; http://www.flight.licr.org/) of above mentioned 1001 genes which were downregulated by PTZ and were part of self-interacting network (for homolog gene lists, see additional file 7). The homologs were then examined to find out if they overrepresent genes/proteins reportedly downregulated in transcriptomic and proteomic studies on rodent models or human epilepsy (for gene/protein lists, see additional file 8). Comparison with individual studies did not reveal statistically significant overlap. Since overlap was also not observed even among reported studies, we pooled down- and up-regulated genes therein separately and matched with our fly gene list. Notably, downregulated genes in fly showed significant match with reported downregulated (p = 0.017) but not upregulated (p = 0.082) genes. This direction specific overlap demonstrated that our fly model is relevant in understanding epileptogenesis.
The ultimate goal of systems biology is to delineate and to comprehend the functioning of complex biological systems in such details that predictive models of human diseases could be developed . However, due to immense complexity of higher organisms, systems biology approaches are currently focused on simpler organisms [29, 30]. We have described here a behavioral and functional genomic model of chemoconvulsant-induced brain plasticity in Drosophila. Although two among five AEDs tested are effective in ameliorating locomotor alteration induced by convulsant drug, flies in our model do not exhibit seizure-like behavior. However, gene expression alterations induced by the convulsant drug show some similarity with expression alterations reported in established rodent models and epileptic patients. Together, these findings suggest that the brain plasticity involved in the fly model may be potentially relevant in understanding mechanisms underlying epileptogenesis to some extent. Chiefly, our model shows downregulation of gene expression and inhibition of neurogenesis/axonogenesis/axon guidance as system level perturbations underlying epileptogenesis-like plasticity. In terms of biochemical pathways, our analysis supports a role of Wnt signaling and other associated pathways in the pathogenesis. Besides epilepsy, AEDs are widely used in treating various neuropsychiatric disorders. Mechanisms of AEDs' long-term action in these disorders are poorly understood. Overlapping pathophysiological mechanisms may underlie epileptogenesis and other neuropsychiatric conditions. Rodent kindling induced by PTZ is a widely used model of epileptogenesis and AED testing. As our fly PTZ model is validated by AEDs, it most importantly offers a unique opportunity for deciphering the mechanisms of long-term action of AEDs that is relevant in various neuropsychiatric conditions. Due to amenability for systems level modeling, candidate gene silencing, small molecule interventions and dietary modifications, for example, can be readily used in our model for generating and testing hypotheses. In brief, the Drosophila systems model is expected to be valuable in identifying disease, drug target, biomarker and pharmacogenomic candidates, and in screening of potential therapeutic agents.
Dynamic Image Analysis System
false discovery rate
The research was supported by Animal Model and Animal Substitute Technologies (NWP0034) grant of Council of Scientific and Industrial Research (CSIR), Government of India, to A.S. Junior and Senior Research Fellowships to F.M. and P.S. from CSIR is duly acknowledged. We thank Mainak Majumder (LabIndia) for help with RT-PCR data acquisition.
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