Free Access Electroconvulsive seizure-induced gene expression profile of the hippocampus dentate gyrus granule cell layer Correction(s) for this article Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract Electroconvulsive shock (ECS) is the most effective treatment for depression, but the mechanism underlying the therapeutic action of this treatment is still unknown. To better understand the molecular changes that may be necessary for the clinical effectiveness of ECS we have combined the technologies of gene expression profiling using cDNA microarrays with T7-based RNA amplification and laser microdissection to identify regulated genes in the dentate gyrus granule cell layer of the hippocampus. We have identified genes previously reported to be up-regulated following ECS, including brain-derived neurotrophic factor, neuropeptide Y, and thyrotrophin releasing hormone, as well as several novel genes. Notably, we have identified additional genes that are known to be involved in neuroprotection, such as growth arrest DNA damage inducible beta (Gadd45beta), and the excitatory amino acid transporter-1 (EAAC1/Slc1A1). In addition, via in situ hybridization we show that EAAC1 is specifically up-regulated in the dentate gyrus, but not in other hippocampal subfields. This study demonstrates the utility of microarray analysis of microdissected subregions of limbic brain regions and identifies novel ECS-regulated genes. Abbreviations used ARC activity regulated cytoskeletal-associated protein BDNF brain-derived neurotrophic factor ECS electroconvulsive shock ELL2 elongation factor RNA polymerase 2 FDR false discovery rate Gadd45beta growth arrest DNA damage inducible beta NARP neuronal activity-regulated pentaxin Depression is a devastating illness that affects 12–15% of people at some point in their lives (Kessler etal. 2003). Current chemical treatments are only partially effective, alleviating symptoms for only approximately 65% of depressed individuals, and require weeks or months of administration to achieve a therapeutic response. Electroconvulsive shock (ECS) is an extremely effective, non-chemical treatment for depression, often efficacious in cases that are resistant to chemical antidepressants. Despite the clinical usefulness of ECS, the mechanisms underlying the therapeutic actions of this treatment are currently unknown. Previous reports have documented gene expression changes in the hippocampus (Newton etal. 2003; Altar etal. 2004) and other brain regions (Yamada etal. 2002) following ECS that could contribute to adaptive changes underlying the therapeutic actions of ECS. Although there has been progress made using these traditional gene expression profiling techniques, these data sets still have shortcomings because they represent detectable changes that occur within large subregions of the brain (e.g. hippocampus or frontal cortex) constituting multiple cell types and independently acting neuronal circuits. This could result in dilution of important gene expression changes, underscoring the importance of utilizing methods that can increase signals from discrete brain regions and/or cell types. Recent advances in laser microdissection methodologies coupled with T7-based RNA amplification and gene expression detection technologies have made it feasible to determine gene expression profiles from discrete regions of the brain as well as from single cells (Bonaventure etal. 2002; Kelz etal. 2002; Kamme etal. 2003; Ginsberg etal. 2004; Glanzer etal. 2004). The dentate gyrus is a relevant hippocampal structure to study as it is well documented that stress suppresses the expression of brain-derived neurotrophic factor (BDNF) and reduces adult neurogenesis of dentate gyrus granule neurons, and, conversely, that antidepressants reverse these effects (Gould etal. 1997; Tanapat etal. 1998; Malberg etal. 2000; Duman etal. 2001; Malberg and Duman 2003; Santarelli etal. 2003; Warner-Schmidt and Duman 2006). In addition, direct infusion of neurotrophic factors into the dentate gyrus produces an antidepressant response in behavioral models of depression, including the forced swim and learned helplessness paradigms (Shirayama etal. 2002; Dow etal. 2005). To perform gene expression profiling from discrete regions of the brain, the shortcoming of limited RNA quantities must be overcome. Conventional gene expression profiling techniques require microgram quantities of RNA, as opposed to the pico- to nanogram quantities of RNA typically obtained from microdissected samples. To overcome this limitation, messenger RNA is amplified using a T7 RNA polymerase-based enzymatic reaction to produce the microgram quantities of RNA necessary for gene expression profiling. Although RNA amplification is absolutely necessary, a potential drawback of this technique is that RNA amplification directly manipulates transcript levels, which in theory can distort the quality of microarray data (Li etal. 2005); this is of significant concern when attempting to detect small changes in gene expression. Here we report the gene expression profile of the dentate gyrus granule cell layer of the adult hippocampus following ECS. This is the first report of gene expression profiling of the dentate gyrus following antidepressant treatment. Although the dentate gyrus is composed of multiple cell types, expression profiling from this discrete region is a vast improvement over traditional expression profiling methods utilizing larger regions. The increase in signal and resolution has made it possible to identify previously unidentified gene expression changes, which should aid our understanding of how ECS exerts an efficacious therapeutic response. Male Sprague Dawley rats (180–220 g) (Charles River Laboratories, Wilmington, MA, USA) were housed, four per cage, under standard illumination parameters (12 h light/dark cycle) and were given free access to water and food. Animal use procedures were in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Yale University Animal Care and Use Committee. Bilateral ECS was administered via moistened spring-loaded ear clip electrodes to rodents once daily for 10 days using a pulse generator (ECT Unit 57800–001; Ugo Basile, Comerio, Italy) (frequency, 100 pulses/ s; pulse width, 0.5 ms; shock duration, 0.5 s; current, 55 mA). Sham-treated animals were handled in the same fashion as the chronic treated animals with ear clips placed on their ears, but no shock was delivered. All animals were killed by decapitation 6 h after the last ECS treatment. For experiments utilizing whole hippocampus, the entire structure was exposed and dissected away from the adjacent cerebral cortex. The dissected hippocampus was immediately frozen on dry ice and stored at − 80°C until RNA purification. For experiments for microdissection, whole brains were dissected, placed in falcon tubes and rapidly frozen in liquid nitrogen. Cryocut sections of 10 microns were placed on PEN-coated slides (Leica Microsystems) followed by ethanol dehydration (75% 45 s, 95% 45 s, 100% 15 s, 100% 45 s, xylene 5 min) Histogene (Arcturus, Mountain View, CA, USA). Laser microdissection was carried out on a Leica AS LMD (Leica Microsystems). Immediately after microdissection, samples were placed on dry ice and then transferred to a − 80°C freezer until RNA purification. Whole hippocampal RNA was purified using RNA Aqueous according to the manufacturer\'s directions (Ambion, Austin, TX, USA). Total RNA was re-purified using the RNeasy Kit (Qiagen, St. Louis, MO, USA) to remove potential contaminating salt carryover. Laser microdissected samples were purified using RNA Aqueous Micro (Ambion). Samples were re-purified via precipitation using Pellet Paint NF (Novagen). All steps were carried out according to manufacturers\' instructions. For comparison of amplified and unamplified RNA, 2 μg of total hippocampal RNA was used in RNA amplification reactions using either the Genisphere SenseAmp Plus Kit (Genisphere, Hatfield, PA, USA) or the Ambion Message Amp II Kit as described by the manufacturers\' directions. RNA amplifications were carried out according to the manufacturers\' recommendations. RNA amplifications utilizing total RNA from laser microdissected tissue was completed using the Message Amp II Kit (Ambion). Each amplification reaction used total RNA purified from 20, 10 micron laser microdissected dentate gyri. All amplification reactions were incubated at 37°C for 14 h. One microarray per ECS/Sham pair was completed for a total of four microarrays analyzing whole hippocampus gene expression and four microarrays analyzing dentate gyrus gene expression. Custom cDNA microarrays with 1200 transcription factors and 1800 neurobiologically significant genes were used in this study. Microarrays analyzing whole hippocampus gene expression were completed essentially as previously described (Newton etal. 2003). Briefly, 3 μg of total RNA from each sample was converted to cDNA utilizing oligo dT-capture sequence primers. cDNA was hybridized in 1× formamide buffer overnight at 46°C followed by posthybridization washes. cDNA was visualized using Array 900 fluorescent Cy5 and Cy3 dendrimer technology (Genisphere). For microarrays analyzing dentate gyrus gene expression, cDNA was generated from 13 μg of amplified RNA generated from each rodent using the Ambion Message Amp II Kit followed by labeling with AminoAllyl UTP using the AminoAllyl cDNA Synthesis Kit (Ambion). This was followed by coupling to Cy3 and Cy5 dyes (Amersham Biosciences) and hybridized overnight at 45°C in the presence of #3 Slide Hyb Buffer (Ambion). Following posthybridization washes, all microarray slides were scanned using a GenePix scanner (Axon Instruments, Union City, CA, USA). Image analysis was performed using genepix Pro 4.0 software (Axon Instruments). Resulting files from Genepix 4.0 (Axon Instruments) analysis were imported into Genespring 7.0 (Silicon Genetics, Redwood City, CA, USA) for data analysis. Per chip intensity dependent Lowess normalization was performed using a cutoff value of 10. Expressed genes lists were created based on the signal intensity of individual genes to be above a cutoff value of 50 on at least three of the four microarrays. Gene regulation was determined by filtering the data for genes that had a t-test p-value of 0.05 with the false discovery rate (FDR) as the multiple testing correction. For comparison of amplified and unamplified RNA, 1000 ng of total RNA or aRNA was converted to cDNA. For experiments with laser microdissected samples, total RNA was purified from 10 10-micron laser microdissected dentate gyri and converted to cDNA. All cDNA synthesis reactions were 20 μL reactions using Genisphere reverse transcriptase with 0.65 μL of 3 μg/μL random hexamers (Invitrogen). qRT-PCR was performed using an ABI 7900 instrument (Applied Biosystems) using the following cycling parameters for 40 cycles: 94 for 2 s, 60 for 30 s, 72 for 30 s. Gene specific primers were designed using Primer3 Software and primers were used at a concentration of 300 nm. The Quantitect SYBR Green PCR kit (Qiagen) was used with reaction volumes of 16 μL. Relative gene concentrations were normalized against cyclophilin. In situ hybridization was performed as previously described (Newton etal. 2002, 2003). Relative gene expression changes were determined using the software imagej (http://rsb.info.nih.gov/ij/). To assess the fidelity of RNA amplification in our hands, we compared transcript profiles from RNA pools that were not amplified vs. RNA that was amplified from the same pools using two commercially available RNA amplification kits (Fig.1). Although the quality of RNA isolated from whole hippocampus may not truly reflect that of RNA extracted from laser microdissected material, the goal of this experiment was to assess the amplification process. Whole hippocampi were dissected from four chronically ECS treated rodents and four sham control rodents. RNA was purified and an aliquot of the RNA was reverse-transcribed to cDNA and another aliquot of RNA was used for RNA amplification and then reverse-transcribed to cDNA. Quantitative real-time PCR was performed from the cDNA samples to compare the relative gene expression changes of the activity regulated cytoskeletal-associated protein (ARC), BDNF, neuronal activity-regulated pentaxin (NARP), and beta-tubulin between sham and ECS treated rodents. ARC, BDNF and NARP are differentially regulated between the ECS and sham control rodents. In addition, both RNA amplification kits tested (Ambion and Genisphere) faithfully reproduced the differential gene expression pattern found with the original unamplified population. These findings are consistent with previous reports (Baugh etal. 2001; Iscove etal. 2002; Zhao etal. 2002; Goff etal. 2004; Rudnicki etal. 2004; Schneider etal. 2004). Assessing the fidelity of RNA amplification. The relative ECS-induced gene expression changes were determined between RNA samples that were not amplified compared to the same RNA samples that were amplified. The genes ARC, BDNF, NARP, and Beta-tubulin (Btub) were analyzed utilizing quantitative real-time PCR. RNA amplification kits from Ambion and Genisphere faithfully reproduced the differential gene expression pattern found with the original unamplified population. Error bars represent standard deviation between four ECS treated rodents and four sham control rodents. To better understand the molecular changes induced by ECS, the gene expression profile of the rodent dentate gyrus granule cell layer was determined following a chronic ECS treatment. Rodents were treated with a 10-day chronic regimen of ECS followed by brain dissection and sectioning on a cryostat. Brain slices were mounted on PEN-coated slides, followed by ethanol dehydration. The dentate gyrus granule cell layer was laser microdissected (Fig.2) from dehydrated sections. We estimate that this dissection consists of approximately 90% granule cell layer, with a small contribution of cells, about 10%, from cells outside this layer. Total RNA was purified from microdissected samples and subjected to T7-based RNA amplification using the Message Amp II kit from Ambion. Amplified RNA (aRNA) was converted to cDNA with aminoallyl modified UTP and indirectly labeled with Cy5 or Cy3. Fluorescently labeled cDNA was hybridized to dual channel custom cDNA microarray chips. Gene spots that appear yellow represent genes that remain unchanged between ECS and sham treated rodents. Gene spots that appear red are up-regulated in the ECS treated rodents (Cy5 labeled). Gene spots appearing green are down-regulated in the ECS treated rodents (Cy3 labeled sham channel). Laser microdissection of the rodent dentate gyrus. (a) Coronal section of rodent brain within the region of the hippocampus before microdissection. (b) One leaflet of the dentate gyrus cut with the laser microdissector. (c) Laser cut of the second leaflet of the dentate gyrus, and removal of other leaflet. (d) Dentate gyrus fully removed. (Magnification 40X). Four microarrays were completed, representing a gene expression profile from each of four ECS treated rodents and four sham control rodents (one microarray per ECS/Sham pair). Table1a represents the genes identified to be up-regulated in the dentate gyrus following ECS, and down-regulated genes are shown in Table1b. All genes pass the 0.05 p-value FDR multiple testing correction. Genes from multiple functional classes were identified, such as transcription factors, receptors, hormones/growth factors, and kinases/enzymes. Most of these genes have previously not been identified to be regulated in the whole hippocampus following ECS treatment, underscoring the value of gene expression analysis from discrete regions of the brain. Genes that have been previously identified to be regulated in the hippocampus following ECS treatment include: NPY (Mikkelsen etal. 1994), TRH (Sattin etal. 1994), BDNF (Nibuya etal. 1995), Wnt2 (Madsen etal. 2003), Grb2 (Newton etal. 2004) and ChgB (Newton etal. 2003). Gene expression changes of selected up-regulated genes were confirmed by quantitative real-time PCR (Table2). Here we show for the first time the up-regulation of Gadd45beta, elongation factor RNA polymerase 2 (ELL2), and excitatory amino acid transporter EAAC1 (Slc1A1/EAAT3) following ECS. Table 1. Genes identified as being differentially regulated in the rodent dentate gyrus following chronic electroconvulsive shock. (a) Genes up-regulated following chronic ECS in the dentate gyrus. (b) Genes down-regulated following chronic ECS. All genes passed the 0.05 p-value FDR. Average fold change found between ECS and sham treated controls is listed next to gene names. Standard deviation (SD) reflects variation between four microarrays Table 2. Quantitative real-time PCR (qRT-PCR) secondary confirmation of selected genes identified to be differentially regulated in the dentate gyrus following electroconvulsive shock. qRT-PCR was performed on cDNA generated from total RNA purified from microdissected dentate gyrus samples The regulation of EAAC1 was also examined by in situ hybridization, which revealed a statistically significant up-regulation in the dentate gyrus following chronic ECS (Fig.3a,b). No changes were detected in other hippocampal subregions, including the CA1, CA2, CA3 pyramidal cell layers or the molecular layer. We examined the possibility that other amino acid transporters may also be up-regulated in the dentate gyrus, such as GLT-1 (Slc1A2/EAAT2) (average fold change = 0.70; t-test p-value 0.265) and GLAST (Slc1A3/EAAT1) (average fold change = 0.66; t-test p-value 0.276), but did not detect a statistical difference between sham control and ECS sections. In situ hybridization of the excitatory amino acid transporter EAAC1. In situ hybridization of cryocut hemisected rodent brains from (a, c) sham control animals and rodents (b, d) chronically treated with electroconvulsive shock. EAAC1/Slc1A1 is up-regulated specifically in the dentate gyrus an average fold change of 2.4 times in ECS treated rodents (t-test p-value = 0.028). Data reflects EAAC1 expression changes from in situ hybridizations completed on sections from four sham control rodents and four ECS treated rodents. Expanded view of the hippocampal subregions (c, d). CA1, CA2, CA3, DG (dentate gyrus), ML (molecular layer), hilus shown. Bar graph of relative EAAC1 gene expression changes within the hippocampal subfields following ECS treatment (e). Combining the technologies of laser microdissection and RNA amplification to perform gene expression profiling from discrete regions of the brain or even single cells has tremendous value for the field of neuroscience. As the brain is a heterogeneous structure, it is necessary to dissect regions or cells of interest to obtain the resolution to detect region or cell specific changes in gene expression. By limiting dilution effects and increasing the signal to noise ratio, subtle changes in gene expression can be detected that may contribute to the cellular phenomenon under study. These approaches thereby aid our characterization of brain function at a cellular and neural network level. Despite the power and promise of gene expression profiling from subregions and single cells, technical hurdles make this approach difficult and caution must be used when interpreting data sets using such technology. For example, as RNA amplification must be used to obtain adequate levels of RNA for current gene expression profiling protocols, transcript levels are directly manipulated, which could produce spurious results. In spite of this caveat, this technology increasingly is being used successfully in the field of neuroscience (Kamme etal. 2003; Telfeian etal. 2003; Zirlinger and Anderson 2003; Gustincich etal. 2004; Mutsuga etal. 2004; Torres-Munoz etal. 2004; Chung etal. 2005; Cristobal etal. 2005; Ginsberg 2005; Grottick etal. 2005; Ivanov etal. 2005; Perrin etal. 2005; Boehm etal. 2006; Peeters etal. 2006). Gene expression profiling from discrete regions of the brain compared to expression profiling from whole regions has the ability to detect changes that would otherwise go undetected due to limiting dilution effects and increasing signal to noise. The detection of EAAC1 regulation upon chronic ECS treatment in the dentate gyrus is a perfect example of this phenomenon. EAAC1 was detected by microarray analysis of the dentate gyrus, but changes were not detected when microarray analysis was conducted with dissections of whole hippocampus (Fig.4a). Notably, this was not a simple matter of the raw signal intensity of the EAAC1 gene being too low for detection on the array. The signal intensity of EAAC1 was above the cutoff to be detected on all arrays and therefore EAAC1 was likely not identified as being regulated in the whole hippocampus due to a dilution effect (Fig.4b). Interestingly, many of the genes that were regulated in the dentate gyrus following ECS were not significantly regulated when whole hippocampus was analyzed, and this effect may have been due to low transcript abundance. A few possibilities that may explain why these transcripts were present at a low level are as follows: Average fold change of genes induced by ECS in the rodent dentate gyrus vs. results found when gene expression analysis was completed on whole rodent hippocampus following ECS treatment (a). Many of the genes regulated in the dentate gyrus following ECS were not detected to be significantly regulated when whole hippocampal gene expression was analyzed. (b) Comparison of average raw signal intensity of genes detected on microarrays. Arrow in lower right hand corner points to minimal average raw signal intensity to be considered present on the array. these genes are enriched in the dentate gyrus vs. other regions of the hippocampus; amplification increased transcript abundance compared to amounts of transcript present during conventional microarrays not utilizing amplification; within the whole hippocampus, the genes are regulated in opposite directions in different cell types. The neurotrophic hypothesis of depression asserts that mood disorders occur when there is ineffective or reduced growth factor signaling in the brain. This hypothesis is based on preclinical studies demonstrating that stress decreases neurotrophic factor expression, most notably BDNF (Duman 2004; Warner-Schmidt and Duman 2006), as well as on postmortem studies reporting decreased levels of BDNF in brains of depressed suicide subjects (Dwivedi etal. 2003; Karege etal. 2005). Decreased expression of BDNF and other neurotrophic factors may also contribute to the hippocampal atrophy reported in brain imaging studies of subjects with depression and post-traumatic stress disorder (Bremner etal. 1995; Gurvits etal. 1996; Sheline etal. 1996, 1999, 2003a, 2003b; Bremner 2001). In contrast, chronic antidepressant treatment increases the expression of BDNF and thereby blocks or reverses the actions of stress and depression (Nibuya etal. 1995, ; Sheline etal. 2003b; Duman 2004). In addition, infusions of BDNF into the hippocampus are sufficient to produce an antidepressant response in behavioral models of depression (Shirayama etal. 2002). The results of the current study are consistent with these findings and demonstrate that expression of BDNF is significantly increased by ECS in the dentate gyrus granule cell layer of hippocampus. A recent study also demonstrates that the antidepressant effects of BDNF are region specific, demonstrating the complexity of neurotrophic actions in the brain (Berton etal. 2006). Stress-induced hippocampal atrophy may be, in part, due to the neurotoxic effects of excessive stress-induced glutamate release (McEwen 1999). The maintenance of physiological levels of extracellular glutamate is controlled largely by a family of amino acid transporters that are differentially expressed in the brain. These transporters are critical for the control and termination of glutamate neurotransmission, and to reduce potential neurotoxic levels of glutamate (O\'Shea 2002). The results of the current study thereby suggest that up-regulation of one of the glutamate transporters, EAAC1, would serve to maintain or control extracellular glutamate levels and protect cells from neurotoxic damage. Five members of the excitatory amino acid transporter family are differentially expressed in varying cells types and brain regions. Glutamate-transporter 1 (GLT-1/EAAT2/Slc1a2) and glutamate-aspartate transporter (GLAST/EAAT1/Slc1a3) are localized primarily to astrocytes. EAAC1 (EAAT3/Slc1a1), EAAT4 (Slc1A6), and EAAT5 (Slc1A7) are localized primarily to neurons, where EAAT4 and EAAT5 are localized to Purkinje cell neurons of the cerebellum and neurons of the retina, respectively (Rothstein etal. 1994; Arriza etal. 1997; Shashidharan etal. 1997; Danbolt 2001). Our results demonstrate that administration of ECS significantly increases the expression of EAAC1 mRNA in the dentate gyrus granule cell layer. Interestingly, ECS did not influence the expression of EAAC1 in the CA1, CA2 or CA3 pyramidal cell layers of the hippocampus (Fig.3). This layer-specific effect is interesting as ECS treatment would be expected to depolarize cells throughout the hippocampus, particularly neurons that receive afferents from the granule cell layer (i.e. CA3 pyramidal cells). This highlights the importance of microarray analysis of subregions of hippocampus in these kinds of studies. Previous studies have examined the expression of GLT-1 and found that this member of the amino acid transporter family is increased in the CA3 region of the hippocampus following chronic restraint stress (Reagan etal. 2004). This effect may represent an adaptive response to increased glutamate neurotransmission during repeated stress. In addition, it was found that the atypical antidepressant tianeptine reversed the effects of stress and decreased GLT-1 expression levels. The authors hypothesized that tianeptine could act as an excitatory amino acid buffer, protecting the CA3 neurons from excitotoxic levels of glutamate and thereby allowing GLT-1 expression to return to baseline. Expression of EAAC1 was not examined in this report. In a recent study we found that GLT-1 expression, determined by in situ hybridization analysis, was increased in the molecular layer of the hippocampus following chronic ECS, whereas no changes were reported in the dentate gyrus, consistent with the RT-PCR findings of the present study (Newton etal. 2006). Lastly, a recent study has reported that administration of a beta-lactam, a treatment that increases GLT-1 (Rothstein etal. 2005), produces an antidepressant response in rodents (Mineur etal. 2006). This finding supports the hypothesis that increased transporter expression is associated with an antidepressant response. Further studies will be required to determine whether increased expression of EAAC1 also produces an antidepressant effect. The differential actions of tianeptine and ECS on transporter expression is likely due to the mechanism of action of the respective antidepressants and the differing roles of these transporters. For example, although glutamate is a substrate for EAAC1, this role of EAAC1 in regulating extracellular glutamate is secondary to GLT-1 and GLAST (Rothstein etal. 1996; Peghini etal. 1997; Tanaka etal. 1997). Recent evidence suggests that EAAC1 contributes to reducing the effects of oxidative stress and age-dependent neurodegeneration due to its ability to shuttle cysteine into neurons, an essential component of glutathione (Aoyama etal. 2006). This action of EAAC1 is consistent with the possibility that this transporter plays a significant role in neuroprotection. We also found that the antiapoptotic protein Gadd45beta was up-regulated in the dentate gyrus by administration of ECS. Gadd45-beta is induced by environmental stressors and protects cells by inhibiting the apoptotic process (Takekawa and Saito 1998; De Smaele etal. 2001). Gadd45-beta is likely indirectly involved in the response to ECS, by protecting neurons from the acute stress that results from ECS treatment. In addition, ELL2 was also identified as being differentially expressed in the dentate gyrus following ECS. ELL2 increases the catalytic rate of RNA polymerase II transcription by suppressing transient pausing of the polymerase (Shilatifard etal. 1997). This effect might be an adaptive response to meet the transcriptional demands induced by ECS. Interestingly, a few genes previously identified to be induced by chronic ECS within the dentate gyrus, Cox2 and Egr3, were not identified in the present study (Newton etal. 2003). This is possibly due to differences in tissue processing which may cause changes in RNA quality, the introduction of other variables such as RNA amplification biases, or differences in the kinetics of hybridization due to differing buffers used. For example, tissue microdissections, although useful and necessary to reduce dilution effects, require additional processing which may effect RNA quality. Lastly, it is well established that variability exists between different microarray platforms, suggesting that caution should be used when interpreting different microarray data sets (Tan etal. 2003). In summary, utilizing laser microdissection and RNA amplification coupled to gene expression profiling has enabled the identification of hippocampal subfield specific gene expression induced by the non-chemical antidepressant ECS. Specifically, we have identified a member of the excitatory amino acid transporter family, EAAC1, as well as several other genes. 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