Gene expression analysis indicates CB1 receptor upregulation in the hippocampus and neurotoxic effects in the frontal cortex 3 weeks after single-dose MDMA administration in Dark Agouti rats

Abstract

Background: 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") is a widely used recreational drug known to impair cognitive functions on the long-run. Both hippocampal and frontal cortical regions have well established roles in behavior, memory formation and other cognitive tasks and damage of these regions is associated with altered behavior and cognitive functions, impairments frequently described in heavy MDMA users. The aim of this study was to examine the hippocampus, frontal cortex and dorsal raphe of Dark Agouti rats with gene expression arrays (Illumina RatRef bead arrays) looking for possible mechanisms and new candidates contributing to the effects of a single dose of MDMA (15 mg/kg) 3 weeks earlier.

Results: The number of differentially expressed genes in the hippocampus, frontal cortex and the dorsal raphe were 481, 155, and 15, respectively. Gene set enrichment analysis of the microarray data revealed reduced expression of 'memory' and 'cognition', 'dendrite development' and 'regulation of synaptic plasticity' gene sets in the hippocampus, parallel to the upregulation of the CB1 cannabinoid- and Epha4, Epha5, Epha6 ephrin receptors. Downregulated gene sets in the frontal cortex were related to protein synthesis, chromatin organization, transmembrane transport processes, while 'dendrite development', 'regulation of synaptic plasticity' and 'positive regulation of synapse assembly' gene sets were upregulated. Changes in the dorsal raphe region were mild and in most cases not significant.

Conclusion: The present data raise the possibility of new synapse formation/synaptic reorganization in the frontal cortex three weeks after a single neurotoxic dose of MDMA. In contrast, a prolonged depression of new neurite formation in the hippocampus is suggested by the data, which underlines the particular vulnerability of this brain region after the drug treatment. Finally, our results also suggest the substantial contribution of CB1 receptor and endocannabinoid mediated pathways in the hippocampal impairments. Taken together the present study provides evidence for the participation of new molecular candidates in the long-term effects of MDMA.

Figure 1

Venn-diagram representation of significantly altered mRNAs. Venn-diagrams showing the number of significantly (minimum probability of positive log ratio (MinPplr) < 0.001) up- or downregulated genes in the hippocampus (HC), frontal cortex (FC) and dorsal Raphe (DR) of Dark Agouti (DA) rats 3 weeks after a single-dose of 3,4-methylenedioxymethamphetamine (MDMA) (15 mg/kg, intraperitoneally). Altogether 615 genes’ showed altered expression, 481, 155, 14 genes in the HC, FC, DR regions, respectively. Among them only 1 genes’ expression altered significantly in all three regions and 36 genes differed significantly compared to saline controls in both the FC and HC groups. See text for further details.

Figure 2

Network analysis of the GSEA results in the Hippocampus. The network shows significantly (nominal p < 0.05, false discovery rate < 0.25) enriched gene ontology (GO) terms in the gene set enrichment (GSEA) analysis in the hippocampus (HC) of Dark Agouti rats 3 weeks after a single-dose (15 mg/kg, intraperitoneal) 3,4-methylenedioxymethamphetamine (MDMA) administration. Blue circles represent the downregulation of the associated terms, while red circles would represent upregulations. The size of the nodes is proportional with the number of genes in the GO term and the thickness of the edges between lines represents the similarity coefficient. All processes in the HC are downregulated, and two well characterized groups of processes emerge. On one hand the processes related to cognition and memory, e.g. regulation of synaptic plasticity, dendrite development and regulation of glutamatergic synaptic transmission gene sets form diverse groups. On the other hand the processes related to kinase activity form another network. See text for further details.

Figure 3

A schematic representation of MDMA’s sites of action in the Hippocampus. This figure summarizes the effects of a single-dose (15 mg/kg, intraperitoneal) of 3,4-methylenedioxymethamphetamine (MDMA) 3 weeks earlier on hippocampal neurons in Dark Agouti rats. Three distinct processes were identified marked by text boxes on the figure, phosphorylation- and dendrite development-related gene sets and gene sets involved in the proper synaptic functioning were downregulated (signed with red crosses at possible sites of action). These processes can be bound to memory and cognition gene sets (downregulated alike) through a single mediator, namely the type 1 cannabinoid receptor (CB1). Accordingly, single-dose MDMA administration 3 weeks earlier resulted in elevated CB1 mRNA levels in the hippocampus. It has to be noted that compared to the frontal cortical region (Figure  5), where rather intracellular signaling mechanisms were influenced, in the HC gene sets related to the connectivity of neurons were damaged. See text for further details.

Figure 4

Network analysis of the GSEA results in the frontal cortex. The network shows significantly (nominal p < 0.05, false discovery rate < 0.25) enriched gene ontology (GO) terms in the gene set enrichment (GSEA) analysis in the frontal cortex (FC) of Dark Agouti rats 3 weeks after a single-dose (15 mg/kg, intraperitoneal) 3,4-methylenedioxymethamphetamine (MDMA) administration. Blue and red circles represent down- and upregulation of the associated GO terms, respectively. The size of the nodes is proportional with the number of genes in the GO term and the thickness of the grey edges represents the number of common genes between two GO terms, if any. Most of the processes in the FC were downregulated. The cellular biosynthetic process and establishment of cellular localization GO terms are in central positions of two different groups while chromosome and chromatin organization, and transmembrane transport processes form the third and fourth group of tightly related, downregulated gene sets. These alterations suggest a general neurotoxic effect while the upregulation of gene sets related to synapse formation raises the possibility of a parallel running recovery process. The expression of the only immunologically related GO term was attenuated. See text for further details.

Figure 5

A schematic representation of MDMA’s sites of action in the frontal cortex. This figure summarizes the effects of a single-dose (15 mg/kg, intraperitoneal) 3,4-methylenedioxymethamphetamine (MDMA) administration 3 weeks earlier on the frontal cortex (FC) neurons of Dark Agouti rats. The text boxes mark the selected, grouped processes while red crosses represent the possible sites of action. Almost all of the genes involved in different levels of protein synthesis were downregulated, including nucleocytoplasmic transport, ribosome functioning, protein folding and localization and members in the transport of amino acids and lipids, basic constituents of the cells. These changes strongly suggest a wide-scale impairment of the cellular machinery. However, the formation of new neuronal connections can compensate for these effects (not depicted) on a different scale. See text for further details.

Figure 6

Correlation between the microarray and the Fluidigm GEx PCR array data. The figure summarizes the correlation between the logarithmic microarray data and the validation results for both the 200 ng and 500 ng samples. For validation purposes we used the polymerase chain reaction-based (PCR-based) Fluidigm GEx array with 19 genes and upon receiving the results, the numerical analysis was performed with the inbuilt corr.test function of the R statistical computing program. The results are in agreement with the commonly observed decreasing accuracy of the fold change values of the microarray method at the higher and lower ends. The correlation coefficients were 0.619 and 0.610 for the 200 ng and 500 ng samples, respectively. Red line represents the regression line.

Figure 7

Correlation between the microarray and the Fluidigm GEx PCR array data. The figure summarizes the correlation between the logarithmic microarray data and the validation results for both the 200 ng and 500 ng samples. For validation purposes we used the polymerase chain reaction-based (PCR-based) Fluidigm GEx array with 19 genes and upon receiving the results, the numerical analysis was performed with the inbuilt corr.test function of the R statistical computing program. The results are in agreement with the commonly observed decreasing accuracy of the fold change values of the microarray method at the higher and lower ends. The correlation coefficients were 0.619 and 0.610 for the 200 ng and 500 ng samples, respectively. Red line represents the regression line.