UNLABELLED ABSTRACT: BACKGROUND Antisense oligonucleotide (AON)-mediated exon skipping is a powerful tool to manipulate gene expression. In the present study we investigated the potential of exon... Show moreUNLABELLED ABSTRACT: BACKGROUND Antisense oligonucleotide (AON)-mediated exon skipping is a powerful tool to manipulate gene expression. In the present study we investigated the potential of exon skipping by local injection in the central nucleus of the amygdala (CeA) of the mouse brain. As proof of principle we targeted the splicing of steroid receptor coactivator-1 (SRC-1), a protein involved in nuclear receptor function. This nuclear receptor coregulator exists in two splice variants (SRC-1a and SRC-1e) which display differential distribution and opposing activities in the brain, and whose mRNAs differ in a single SRC-1e specific exon. METHODS For proof of principle of feasibility, we used immunofluorescent stainings to study uptake by different cell types, translocation to the nucleus and potential immunostimulatory effects at different time points after a local injection in the CeA of the mouse brain of a control AON targeting human dystrophin with no targets in the murine brain. To evaluate efficacy we designed an AON targeting the SRC-1e-specific exon and with qPCR analysis we measured the expression ratio of the two splice variants. RESULTS We found that AONs were taken up by corticotropin releasing hormone expressing neurons and other cells in the CeA, and translocated into the cell nucleus. Immune responses after AON injection were comparable to those after sterile saline injection. A successful shift of the naturally occurring SRC-1a:SRC-1e expression ratio in favor of SRC-1a was observed, without changes in total SRC-1 expression. CONCLUSIONS We provide a proof of concept for local neuropharmacological use of exon skipping by manipulating the expression ratio of the two splice variants of SRC-1, which may be used to study nuclear receptor function in specific brain circuits. We established that exon skipping after local injection in the brain is a versatile and useful tool for the manipulation of splice variants for numerous genes that are relevant for brain function. Show less
Introduction. In a marmoset model of hypoactive female sexual function, we have shown that repeated administration of the serotonin (5-HT)-1A agonist R-(+)-8-hydroxy-2-(di-N-propylamino)tetralin (8... Show moreIntroduction. In a marmoset model of hypoactive female sexual function, we have shown that repeated administration of the serotonin (5-HT)-1A agonist R-(+)-8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT) inhibits sexual receptivity in female marmoset monkeys and increases aggression toward the male pairmate. Aim. The aims of this study are to investigate gene expression changes induced by 8-OH-DPAT in laser-microdissected brain areas that regulate female sexual function and to identify genes, functional gene classes, and pathways associated with 8-OH-DPAT-mediated inhibition of female sexual receptivity. Methods. Gene expression was measured in the medial prefrontal cortex (mPFC), medial preoptic area (mPOA), cornu ammonis-1 (CA1) area of the hippocampus (CA1), and dorsal raphe nucleus (DRN) of four 8-OH-DPAT-treated (0.1mg/kg; daily administration for 16 weeks) and four vehicle-treated female marmosets using a marmoset-specific microarray (European Marmoset Microarray [EUMAMA]) and validated by real-time quantitative polymerase chain reaction (RTqPCR). Enriched functional gene classes were determined. In a parallel candidate gene approach, the expression of serotonergic candidate genes, i.e., the 5-HT1A, 5-HT2A, and 5-HT7 receptors and the 5-HT transporter (5-HTT), was measured by RTqPCR. Main Outcome Measures. The main outcome is the differential expression of genes between 8-OH-DPAT- and vehicle-treated marmosets. Results. 8-OH-DPAT affected the gene classes important to neural development (mPFC, mPOA, and DRN), neurotransmission (mPOA), energy production (mPFC and mPOA), learning and memory (CA1), and intracellular signal transduction (DRN). Oxytocin (OXT) in the mPOA and 5-HTT in the DRN were strongly increased by 8-OH-DPAT. 5-HT1A tended to increase in the mPFC, while 5-HT7 was decreased in the CA1. Conclusions. Brain region-specific alterations of gene expression regulating neural circuitries, energy demands, and learning processes are associated with 8-OH-DPAT-induced decrease in female sexual receptivity and increase in pairmate aggression. The role of OXT in the serotonergic regulation of female sexual behavior and partner interactions warrants attention in future studies. Show less
