Erno Vreugdenhil

Deep sequencing-based expression analysis shows major advances in robustness, resolution and inter-lab portability over five microarray platforms

The hippocampal expression profiles of wild-type mice and mice transgenic for δC-doublecortin-like kinase were compared with Solexa/Illumina deep sequencing technology and five different microarray platforms. With Illuminas digital gene expression assay, we obtained ∼2.4 million sequence tags per sample, their abundance spanning four orders of magnitude. Results were highly reproducible, even across laboratories. With a dedicated Bayesian model, we found differential expression of 3179 transcripts with an estimated false-discovery rate of 8.5%. This is a much higher figure than found for microarrays. The overlap in differentially expressed transcripts found with deep sequencing and microarrays was most significant for Affymetrix. The changes in expression observed by deep sequencing were larger than observed by microarrays or quantitative PCR. Relevant processes such as calmodulin-dependent protein kinase activity and vesicle transport along microtubules were found affected by deep sequencing but not by microarrays. While undetectable by microarrays, antisense transcription was found for 51% of all genes and alternative polyadenylation for 47%. We conclude that deep sequencing provides a major advance in robustness, comparability and richness of expression profiling data and is expected to boost collaborative, comparative and integrative genomics studies.

Downregulation of BDNF mRNA and protein in the rat hippocampus by corticosterone.

Previously, we showed that corticosterone regulates BDNF mRNA levels in the hippocampus. In the present study, we have investigated the time course and dose-dependency of this effect at both the mRNA and the protein level. Corticosterone was administered in doses of 30 and 1000 microgram/kg b.w. subcutaneously to adrenalectomized animals. At 3, 6, 12 and 24 h after administration BDNF and trkB mRNA levels in hippocampal subfields were measured by in situ hybridization. Our results show a dose-dependent decrease in BDNF mRNA in dentate gyrus and CA1 at 3 h. After the high dose, this decrease was 70% and 40% respectively. In addition, ELISA was performed to study if this downregulation is also detectable at the protein level. Hippocampal tissue was used from adrenalectomized animals which had received 1000 microgram/kg b.w. corticosterone 4 and 6 h before decapitation. At both time points, a decrease in BDNF protein was observed; 17% at 4 h and 14% at 6 h after corticosterone, as compared to the vehicle injected controls. TrkB mRNA levels were not affected by corticosterone. However, between 6 and 24 h after treatment, increases in trkB mRNA were observed. In conclusion, we have found a transient, dose-dependent decrease in BDNF mRNA and protein in the hippocampus, which may underly changes in neuronal plasticity in the hippocampus after short-term changes in corticosterone concentrations.

Corticosterone Effects on BDNF Expression in the Hippocampus Implications for Memory Formation

The adrenal steroid corticosterone has profound effect on the structure and function of the hippocampus. Probably as a result of that, it modulates memory formation. In this review, the question is addressed if the corticosterone effects on memory processes are mediated by alterations in the expression of the neurotrophin Brain-Derived Neurotrophic Factor (BDNF) in the hippocampus. First, studies are described investigating the effect of corticosterone on BDNF expression in the rat hippocampus. It appears that corticosterone suppresses the BDNF expression at the mRNA and protein level in a subfield-specific way. Second, a model for the mechanism of action is proposed. In this model, activated mineralocorticoid and glucocorticoid receptors repress transcriptional activity of the BDNF promoter site-specifically via interaction with other transcription factors. Third, the implications for learning and memory are discussed. Studies show that during water maze training, corticosterone levels rise significantly, but the BDNF expression is not suppressed in any hippocampal subfield. Furthermore, high BDNF expression levels in specific subfields correlate with a good memory performance. Therefore, we suggest that the resistance of the hippocampal BDNF expression to suppression by corticosterone, as seen after water maze training, may contribute to an optimal memory performance.

Identification of corticosteroid-responsive genes in rat hippocampus using serial analysis of gene expression.

Adrenal corticosteroids (CORT) have a profound effect on the function of the hippocampus. This is mediated in a coordinated manner by mineralocorticoid (MR) and glucocorticoid receptors (GR) via activation or repression of target genes. The aim of this study was to identify, using serial analysis of gene expression (SAGE), CORT‐responsive hippocampal genes regulated via MR and/or GR. SAGE profiles were compared under different conditions of CORT exposure, resulting in the identification of 203 CORT‐responsive genes that are involved in many different cellular processes like, energy expenditure and cellular metabolism; protein synthesis and turnover; signal transduction and neuronal connectivity and neurotransmission. Besides some previously identified CORT‐responsive genes, the majority of the genes identified in this study were novel. In situ hybridization revealed that six randomly chosen CORT‐responsive genes had distinct expression patterns in neurons of the hippocampus. In addition, using in situ hybridization, we confirmed that these six genes were indeed regulated by CORT, underscoring the validity of the SAGE data. Comparison of MR‐ and GR‐dependent expression profiles revealed that the majority of the CORT‐responsive genes were regulated either by activated MR or by activated GR, while only a few genes were responsive to both activated MR and GR. This indicates that the molecular basis for the differential effects of activated MR and GR is activation or repression of distinct, yet partially overlapping sets of genes. The putative CORT‐responsive genes identified here will provide insight into the molecular mechanisms underlying the differential and sometimes opposing effects of MR and GR on neuronal excitability, memory formation and behaviour as well as their role in neuronal protection and damage.

Corticosterone regulates expression of BDNF and trkB but not NT‐3 and trkC mRNA in the rat hippocampus

Corticosterone has profound effects on growth, differentiation, and synaptic transmission of hippocampal neurons by activation of mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs). In the present study we tested if neurotrophins can be implicated in these effects. For this purpose we injected 30, 300, and 1,000 μg corticosterone s.c. (per kg body weight) in adrenalectomized rats and measured the mRNA levels of brain‐derived neurotrophic factor (BDNF), tyrosine receptor kinase (trk)B, neurotrophin (NT)‐3, and trkC in hippocampal cell fields at 6 hr after steroid administration by in situ hybridization. NT‐3 and trkC mRNA did not show significant changes in any hippocampal region after the various doses of conticosterone. BDNF mRNA decreased after corticosterone administration dose dependently, resulting in a maximal suppression of 35, 20, and 50% in dentate gyrus, CA3, CA1, respectively. Interestingly, trkB responded to corticosterone in an inverted U‐shaped fashion in CA3 and dentate gyrus: the low dose of corticosterone increased trkB mRNA expression in both regions by approximately 30%, while the effect of the two higher doses was not different from the vehicle injected controls. In conclusion, we found differential effects of low and high doses of corticosterone on BDNF and trkB expression in hippocampus, which suggests involvement of a coordinated MR‐ and GR‐mediated action. J. Neurosci. Res. 48:334–341, 1997.

Evaluation of Affymetrix Gene Chip sensitivity in rat hippocampal tissue using SAGE analysis

DNA microarrays are a powerful tool for monitoring thousands of transcript levels simultaneously. However, the use of DNA microarrays in studying the central nervous system faces several challenges. These include the detection of low‐abundance transcripts in highly complex tissue as well as estimating relatively low‐magnitude changes in transcript levels in response to experimental manipulation. Many transcripts important to brain function have low expression levels or are expressed in relatively few cells, making them difficult to detect in the complex background of brain tissue. The aim of the present study is to evaluate the sensitivity of Gene Chip detection of transcripts in brain by using results from serial analysis of gene expression (SAGE) studies. The results of this comparison indicate that Affymetrix Gene Chips, like SAGE, only reliably detect medium‐ to high‐abundance transcripts and that detection of low‐abundance transcripts, many of which have great relevance to biological function in brain, is inconsistent. Specifically, we estimate that Gene Chips reliably detect no more than 30% of the hippocampal transcriptome when using a gross hippocampal dissection as the source tissue. This report provides the first broad evaluation of Affymetrix Gene Chip sensitivity relevant to studying the brain.

Glucocorticoid signaling and stress-related limbic susceptibility pathway: about receptors, transcription machinery and microRNA.

BACKGROUND Stress is essential for health, but if coping with stress fails, the action of the stress hormones cortisol and corticosterone (CORT) becomes dysregulated, precipitating a condition favorable for increased susceptibility to psychopathology. We focus on the question how the action of CORT can change from protective to harmful. APPROACH CORT targets the limbic brain, where it affects cognitive processes and emotional arousal. The magnitude and duration of the CORT feedback signal depends on bio-availability of the hormone, the activity of the CORT receptor machinery and the stress-induced drive. If CORT action becomes dysregulated, we postulate that this is linked to compromised receptor regulation in the limbic brains susceptibility pathway. RESULTS CORT action on gene transcription is mediated by high affinity mineralocorticoid (MR) and 10 fold lower affinity glucocorticoid (GR) receptors that also can mediate fast non-genomic actions. MR and GR operate a feedback loop that involves access and binding to the receptors, activation and shuttling of the CORT receptor complexes, which require interaction with coregulators and transcription factors for transcriptional outcome. CORT modulates the expression of gene transcripts encoding specific chaperones, motor proteins and transcription factors as well as its own receptors. The emerging evidence of microRNAs operating translational control points to further fine-tuning in receptor signaling. CONCLUSION Imbalance in MR:GR-mediated actions caused by receptor variants and epigenetic modulations have been proposed as risk factor in stress-related disease. We here provide key regulatory steps in the activation, transport and regulation of CORT receptors that may sensitize susceptibility pathways underlying psychopathology.

Knockdown of the glucocorticoid receptor alters functional integration of newborn neurons in the adult hippocampus and impairs fear-motivated behavior

Glucocorticoids (GCs) secreted after stress reduce adult hippocampal neurogenesis, a process that has been implicated in cognitive aspects of psychopathology, amongst others. Yet, the exact role of the GC receptor (GR), a key mediator of GC action, in regulating adult neurogenesis is largely unknown. Here, we show that GR knockdown, selectively in newborn cells of the hippocampal neurogenic niche, accelerates their neuronal differentiation and migration. Strikingly, GR knockdown induced ectopic positioning of a subset of the new granule cells, altered their dendritic complexity and increased their number of mature dendritic spines and mossy fiber boutons. Consistent with the increase in synaptic contacts, cells with GR knockdown exhibit increased basal excitability parallel to impaired contextual freezing during fear conditioning. Together, our data demonstrate a key role for the GR in newborn hippocampal cells in mediating their synaptic connectivity and structural as well as functional integration into mature hippocampal circuits involved in fear memory consolidation.

Altered hippocampal gene expression prior to the onset of spontaneous seizures in the rat post‐status epilepticus model

Neuronal loss, gliosis and axonal sprouting in the hippocampal formation are characteristics of the syndrome of mesial temporal sclerosis (MTS). In the post‐status epilepticus (SE) rat model of spontaneous seizures these features of the MTS syndrome can be reproduced. To get a global view of the changes in gene expression in the hippocampus we applied serial analysis of gene expression (SAGE) during the early phase of epileptogenesis (latent period), prior to the onset of the first spontaneous seizure. A total of 10 000 SAGE tags were analyzed per experimental group, resulting in 5053 (SE) and 5918 (control group) unique tags (genes), each representing a specific mRNA transcript. Of these, 92 genes were differentially expressed in the hippocampus of post‐SE rats in comparison to controls. These genes appeared to be mainly associated with ribosomal proteins, protein processing, axonal growth and glial proliferation proteins. Verification of two of the differentially expressed genes by in situ hybridization confirmed the changes found by SAGE. Histological analysis of hippocampal sections obtained 8 days after SE showed extensive cell loss, mossy fibre sprouting and gliosis in hippocampal sub regions. This study identifies new high‐abundant genes that may play an important role in post‐SE epileptogenesis.

Circadian variation in BDNF mRNA expression in the rat hippocampus

BDNF mRNA levels in the hippocampus were studied during the circadian cycle by in situ hybridization. These levels display a circadian pattern, which may be due to regulation by corticosterone. This may have consequences for hippocampal functioning at different time points of the circadian cycle.