Chronic potassium chloride (KCl) treatment induced gene expression changes in Drosophila head
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ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE10808 | GEO | 2008/03/18
SECONDARY ACCESSION(S): PRJNA107397
REPOSITORIES: GEO
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Project description:We have recently developed a Drosophila behavioral- and transcriptomic- based (systems) model of chronic pentylenetetrazole induced locomotor plasticity. Pharmacological validation at behavioral level and at the level of fly head microarray gene expression shows that the model is of potential value in screening of compounds for detecting antiepileptic, antiepileptogenic, disease-modifying and neuroprotective activities. While screening compounds using this model at behavioral level, potassium chloride (KCl) was identified as having therapeutic potential. Examination of transcriptomic changes induced by KCl in fly head further supports its potential use as a therapeutic agent in neurological and neuropsychiatric conditions. Keywords: Drug Response D. melanogaster Oregon-R wild type flies were grown in standard fly medium consisting of agar-agar, maize powder, brown sugar, dried yeast, and nipagin. The cultures were grown at 24 + 1 C, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Ten to eleven days old unmated adult males were grown in either normal food (NF) or food containing 100 mM KCl for three days. Each treatment vial contained 30 flies. Heads were harvested at the end of seven day treatment. For this, flies frozen in liquid nitrogen were shaken and the heads collected using cooled sieves. Total RNA was isolated from eight pools of frozen heads, every two of which represented a single parallel set of treatment in which four vials contained NF treated control flies, and four KCl treated individuals, using TRI REAGENT (Sigma) according to the manufacturerâs protocol. 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 manufacturerâs protocol. Each of the four sets of control and treated cDNA samples, belonging to the four biological replicates, was used for labeling with either Cy3 or Cy5 dyes (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche). The labeled products were purified by Microarray Target Purification Kit (Roche). The Cy3 and Cy5 labeled two cRNA samples of each biological replicate were pooled together, precipitated, washed, air-dried, and dissolved in 18MΩ RNAase free water (Sigma). Dye swapping was accomplished by hybridizing two arrays with NF control as Cy3- and KCl treated as Cy5- labeled sample, and the rest two as the opposite, NF as Cy5- and drug treated as Cy3- labeled sample. The labeled product was mixed with hybridization solution containing hybridization buffer (DIG Easy Hyb; Roche), 10mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma). The hybridization mixture was denatured at 65ºC and applied onto cDNA microarray slides (D14Kv1, CDMC, Toronto, Canada). The slides were covered by a coverslip (ESCO, Portsmouth, USA) and hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs. Following hybridization, the coverslips were removed in a solution containing 1X SSC and 0.1% SDS at 50ºC, and the slides washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ºC with occasional swirling and then transferred to 1X SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Slides were given a final wash in 0.1X 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 10µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices). The preprocessing and quantification of the 16 bit TIFF images were carried out using Gene Pix Pro 6.0 software (Molecular Devices). Ratio based normalization was performed using Acuity 4.0 software (Molecular Devices). All Spots with raw intensity less then 100U 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 the four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 3.0, Excel Add-In, Stanford) under the conditions of one class response, imputation and 100 permutations.
2010-05-25 | E-GEOD-10808 | biostudies-arrayexpress
Project description:We have recently developed a Drosophila behavioral- and transcriptomic- based (systems) model of chronic pentylenetetrazole (PTZ) induced locomotor plasticity. Pharmacological validation using antiepileptic drugs (AEDs) sodium valproate and levetiracetam (LEV) at behavioral level showed a weaker prophylactic and stronger symptomatic effect of sodium valproate and the opposite, i.e., stronger prophylactic and weaker symptomatic, effect in the model. Microarray gene expression profiling of fly head after sodium valproate and LEV treatment of flies further supported the above therapeutic effect of the two AEDs in the PTZ fly model. Given the strong prophylactic effect of LEV in our model, we hypothesized that time series of transcriptomic alterations caused by PTZ at 12 hrs, 2nd day and 7th day (an earlier GEO submission) may be neutralized by LEV. The present submission relates to microarray expression profiles at 12 hrs, 2nd day and 7th day of a drug regime in which flies were treated with both PTZ and LEV for four days followed by three days of treatment with PTZ alone. Results are consistent with our above hypothesis. Keywords: Drug combination response
2008-03-19 | GSE10850 | GEO
Project description:We recently developed a fly model of pentylenetetrazol (PTZ)-induced long-term behavioral plasticity. Pharmacological validation suggests this model to be kindling-like. Hence, our model is of relevance in understanding the molecular pathogenesis of epileptogenesis as well as in screening potential antiepileptogenic agents. Vigabatrin (VGB) is an antiepileptic drug, and in the process of developing our fly model we generated gene expression profile of flies’ head secondary to treatment of the insects with VGB. Our results provide novel insights in to the drug’s mode of action. Keywords: Dose response
2007-10-05 | GSE7203 | GEO
Project description:We recently developed a fly model of pentylenetetrazol (PTZ)-induced long-term behavioral plasticity. Pharmacological validation suggests this model to be kindling-like. Hence, our model is of relevance in understanding the molecular pathogenesis of epileptogenesis as well as in screening potential antiepileptogenic agents. Gabapentin (GBP) is an antiepileptic drug, and in the process of developing our fly model we generated gene expression profile of flies’ head secondary to treatment of the insects with GBP. Our results provide novel insights in to the drug’s mode of action. Keywords: Dose response
2007-10-05 | GSE7202 | GEO
Project description:We recently developed a fly model of pentylenetetrazol (PTZ)-induced long-term behavioral plasticity. Pharmacological validation suggests this model to be kindling-like. Hence, our model is of relevance in understanding the molecular pathogenesis of epileptogenesis as well as in screening potential antiepileptogenic agents. Sodium valproate (NaVP) is an antiepileptic drug, and in the process of developing our fly model we generated gene expression profile of flies’ head secondary to treatment of the insects with NaVP. Our results provide novel insights in to the drug’s mode of action. Keywords: Dose response
2007-10-05 | GSE7200 | GEO
Project description:We recently developed a fly model of pentylenetetrazol (PTZ)-induced long-term behavioral plasticity. Pharmacological validation suggests this model to be kindling-like. Hence, our model is of relevance in understanding the molecular pathogenesis of epileptogenesis as well as in screening potential antiepileptogenic agents. Ethosuximide (ETH) is an antiepileptic drug, and in the process of developing our fly model we generated gene expression profile of flies’ head secondary to treatment of the insects with ETH. Our results provide novel insights in to the drug’s mode of action. Keywords: Dose response
2007-10-05 | GSE7199 | GEO
Project description:Cultured hippocampal neurons from mice were treated with high concentration KCl for five minutes to depolarise the plasma membrane and model sustained brain activity. A high KCl condition was compared to low KCl using phosphoproteomics. Phosphopeptides were enriched from trypsin digested bio-replicates of the high and low KCl conditions and analysed by mass spectrometry. Approximately 20,000 phosphopeptides were identified enabling a comparison of the relative level of phosphorylation in response to depolarisation.
2023-10-15 | PXD020232 | Pride
Project description:Drosophila melanogaster adult flies fed on food containing 16 mg/ml of pentylenetetrazole (PTZ) in the food show a hyperkinetic behavior within 24 hours. Half of that concentration, i.e., 8 mg/ml, of PTZ, if fed for seven days, though doesn’t cause seizure-like behavior, results in a decreased climbing speed in flies. This change in locomotor behavior is progressive and becomes significant only on seventh day of the treatment. We also examined flies’ locomotor behavior secondary to PTZ withdrawal. Interestingly, an increased climbing speed was found to develop seven days after withdrawal. Importantly, antiepileptic drugs showed effectiveness in the above fly model. Earlier, we submitted in GEO time series of fly head microarray gene expression profiling during chronic PTZ and PTZ withdrawal phase. We also submitted previously expression profiles associated with antiepileptic drug treatment. Here, we have undertaken a different line of work. Having developed a well characterized acquired model of behavioral and gene expression plasticity, we found an opportunity here to investigate if drug exposure to adult males could cause transgenerational effect. To probe this, we carried out a systematic study at both behavioral and microarray gene expression levels. In the latter, we asked the question that do F0 testis, F1 males’ head, F1 females’ head, F1 testis, F2 males’ head and F2 females’ head show gene expression changes if F0 male parents had a history of PTZ exposure? A total of 28 microarray slides were used in this study.
2009-03-26 | GSE15136 | GEO
Project description:Drosophila melanogaster adult flies fed on normal food (NF) containing 16 mg/ml of pentylenetetrazole (PTZ) in the food show a hyperkinetic behavior within 24 hours. Half of that concentration, i.e., 8 mg/ml, of the PTZ, if fed for seven days, though doesn’t cause seizure-like behavior, results in a decreased climbing speed in flies. This change in locomotor behavior is progressive and becomes significant only on seventh day of the treatment. This climbing deficit is ameliorated when flies are treated concomitantly with PTZ and either of the two antiepileptic drugs (AEDs), sodium valproate (NaVP) or levetiracetam (LEV). Further experiments based on different regimes of combination and singular PTZ treatment demonstrated that whereas NaVP show a weaker prophylactic and stronger symptomatic effect, LEV exhibit an opposite effect, i.e., stronger prophylactic and weaker symptomatic action. These observations are consistent with the therapeutic effect of antiepileptic drugs. Time series of microarray gene expression profiling (earlier GEO submission) during chronic PTZ provided evidence of underlying downregulation of three main categories of biological processes, synaptic remodeling, energy metabolism and transport, in that order, in fly head. Transcriptomic analysis secondary to NaVP and LEV (earlier GEO submission) was found to cause statistically significant upregulation of two biological process categories each, energy metabolism and transport in case of NaVP and synaptic remodeling and energy metabolism in case of LEV. Time series of genome wide expression profiling secondary to PTZ and LEV combination treatment (earlier GEO submission) showed neutralizing effect of LEV on PTZ induced expression changes. Mining of published transcriptomic and proteomic data pertaining to epilepsy or established rodent animal models of epileptogenesis supported the relevance of the fly model in understanding the underlying brain pathophysiology. Systems modeling using RNA interference and small molecules demonstrated the robustness of the fly model. In brief, the above results clearly showed that the fly model is valuable not only in screening of antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents, but also in advancing our knowledge of mechanisms of action of drugs used in treating human patients suffering from various neurological and neuropsychiatric conditions, pharmacogenomics of these drugs, identification of potential drug targets, and selection of potential candidate genes for association analysis in epilepsy. Considering the above fly locomotor behavior model as relevant in kindling attainment, it was of obvious interest to study the behavioral and transcriptomic changes post-kindling, i.e., after withdrawing PTZ following seven day long chronic PTZ administration. It was observed that when PTZ is discontinued and flies climbing speed is monitored for next seven days of PTZ discontinuation, climbing speed deficit initially disappears and then a progressive increase in climbing speed is detected. Behavioral pharmacology of AEDs in post-kindling regime, like fly kindling described above, showed its usefulness in screening of potential antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents. The present submission relates to transcriptomic changes in fly head secondary to gabapentin (GBP) treatment following discontinuation of PTZ for seven days (after seven days of chronic PTZ treatment). To delineate possible prophylactic or symptomatic effect of GBP at behavioral level, flies were treated with GBP for first three days and then shifted to NF for next four days (3 + 4 day regime) or flies treated with NF for first four days and then drug treated for next three days (4 + 3 day regime), in that order. At transcriptomic level, we have now examined the effect of GBP in 3 + 4 day regime. Gene expression profiles have been generated at both time points, 3rd and 7th day, i.e., on 10th and 14th day from the beginning of PTZ treatment. Effect of three day long chronic GBP treatment on fly head microarray gene expression profile, in otherwise normally grown flies, has already been determined (earlier GEO submission). Similarly, transcriptomic changes after one day, three days and seven days of PTZ discontinuation (i.e., 8th, 10th and 14th day of the beginning of chronic PTZ treatment) have already been deciphered (earlier GEO submission). Unlike the previous experiment in which flies were maintained in the same container during the post-kindling period of seven days, flies needed to be shifted in fresh vials once in the present experiment because of the demand of 3 + 4 day regime. Since change in housing conditions has the potential to influence head transcriptome, we have also generated microarray expression profile of fly head after treating flies with PTZ for seven days, maintaining the flies to NF containing vials for three days and then shifting and maintaining them in fresh NF containing vials for next four days (submitted in GEO previously). Keywords: Drug Response
2008-05-03 | GSE10985 | GEO
Project description:Drosophila melanogaster adult flies fed on normal food (NF) containing 16 mg/ml of pentylenetetrazole (PTZ) in the food show a hyperkinetic behavior within 24 hours. Half of that concentration, i.e., 8 mg/ml, of the PTZ, if fed for seven days, though doesn’t cause seizure-like behavior, results in a decreased climbing speed in flies. This change in locomotor behavior is progressive and becomes significant only on seventh day of the treatment. This climbing deficit is ameliorated when flies are treated concomitantly with PTZ and either of the two antiepileptic drugs (AEDs), sodium valproate (NaVP) or levetiracetam (LEV). Further experiments based on different regimes of combination and singular PTZ treatment demonstrated that whereas NaVP show a weaker prophylactic and stronger symptomatic effect, LEV exhibit an opposite effect, i.e., stronger prophylactic and weaker symptomatic action. These observations are consistent with the therapeutic effect of antiepileptic drugs. Time series of microarray gene expression profiling (earlier GEO submission) during chronic PTZ provided evidence of underlying downregulation of three main categories of biological processes, synaptic remodeling, energy metabolism and transport, in that order, in fly head. Transcriptomic analysis secondary to NaVP and LEV (earlier GEO submission) was found to cause statistically significant upregulation of two biological process categories each, energy metabolism and transport in case of NaVP and synaptic remodeling and energy metabolism in case of LEV. Time series of genome wide expression profiling secondary to PTZ and LEV combination treatment (earlier GEO submission) showed neutralizing effect of LEV on PTZ induced expression changes. Mining of published transcriptomic and proteomic data pertaining to epilepsy or established rodent animal models of epileptogenesis supported the relevance of the fly model in understanding the underlying brain pathophysiology. Systems modeling using RNA interference and small molecules demonstrated the robustness of the fly model. In brief, the above results clearly showed that the fly model is valuable not only in screening of antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents, but also in advancing our knowledge of mechanisms of action of drugs used in treating human patients suffering from various neurological and neuropsychiatric conditions, pharmacogenomics of these drugs, identification of potential drug targets, and selection of potential candidate genes for association analysis in epilepsy. Considering the above fly locomotor behavior model as relevant in kindling attainment, it was of obvious interest to study the behavioral and transcriptomic changes post-kindling, i.e., after withdrawing PTZ following seven day long chronic PTZ administration. It was observed that when PTZ is discontinued and flies climbing speed is monitored for next seven days of PTZ discontinuation, climbing speed deficit initially disappears and then a progressive increase in climbing speed is detected. Behavioral pharmacology of AEDs in post-kindling regime, like fly kindling described above, showed its usefulness in screening of potential antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents. The present submission relates to transcriptomic changes in fly head secondary to vigabatrin (VGB) treatment following discontinuation of PTZ for seven days (after seven days of chronic PTZ treatment). To delineate possible prophylactic or symptomatic effect of VGB at behavioral level, flies were treated with VGB for first three days and then shifted to NF for next four days (3 + 4 day regime) or flies treated with NF for first four days and then drug treated for next three days (4 + 3 day regime), in that order. At transcriptomic level, we have now examined the effect of VGB in 3 + 4 day regime. Gene expression profiles have been generated at both time points, 3rd and 7th day, i.e., on 10th and 14th day from the beginning of PTZ treatment. Effect of three day long chronic VGB treatment on fly head microarray gene expression profile, in otherwise normally grown flies, has already been determined (earlier GEO submission). Similarly, transcriptomic changes after one day, three days and seven days of PTZ discontinuation (i.e., 8th, 10th and 14th day of the beginning of chronic PTZ treatment) have already been deciphered (earlier GEO submission). Unlike the previous experiment in which flies were maintained in the same container during the post-kindling period of seven days, flies needed to be shifted in fresh vials once in the present experiment because of the demand of 3 + 4 day regime. Since change in housing conditions has the potential to influence head transcriptome, we have also generated microarray expression profile of fly head after treating flies with PTZ for seven days, maintaining the flies to NF containing vials for three days and then shifting and maintaining them in fresh NF containing vials for next four days (submitted in GEO previously). Keywords: Drug Response
2008-05-03 | GSE10987 | GEO