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RNA-Seq of the rat pineal transcriptome, with in-vivo and in-vitro samples, under various treatment and surgical conditions

ABSTRACT: Pineal function follows a 24-hour schedule, dedicated to the conversion of night and day into a hormonal signal, melatonin. In mammals, 24-hour changes in pineal activity are controlled by a neural pathway that includes the central circadian oscillator in the suprachiasmatic nucleus and the superior cervical ganglia (SCG), which innervate the pineal gland. In this study, we have generated the first next-generation RNA sequencing evidence of neural control of the daily changes in the pineal transcriptome. We found over 3000 pineal transcripts that are differentially expressed (p <0.001) on a night/day basis (70% of these genes increase at night, 376 with fold change >4 or <1/4), the majority of which had not been previously identified as such. Nearly all night/day differences were eliminated by neonatal removal or decentralization of the SCG, confirming the importance of neural input for differential night/day changes in transcript abundance. In contrast, very few non-rhythmic genes showed evidence of changes in expression due to the surgical procedure itself, which is consistent with the hypothesis that post neonatal neural stimulation is not required for cell fate determination and maintenance of phenotype. Many of the transcripts that exhibit marked differential night/day expression exhibited similar changes in response to in vitro treatment with norepinephrine, the SCG neurotransmitter which mediates pineal regulation. Similar changes were also seen following treatment with an analog of the norepinephrine second messenger, cyclic AMP.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE63309 | GEO | 2015/09/02

SECONDARY ACCESSION(S): PRJNA267246

REPOSITORIES: GEO

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Project description:Pineal function follows a 24-hour schedule, dedicated to the conversion of night and day into a hormonal signal, melatonin. In mammals, 24-hour changes in pineal activity are controlled by a neural pathway that includes the central circadian oscillator in the suprachiasmatic nucleus and the superior cervical ganglia (SCG), which innervate the pineal gland. In this study, we have generated the first next-generation RNA sequencing evidence of neural control of the daily changes in the pineal transcriptome. We found over 3000 pineal transcripts that are differentially expressed (p <0.001) on a night/day basis (70% of these genes increase at night, 376 with fold change >4 or <1/4), the majority of which had not been previously identified as such. Nearly all night/day differences were eliminated by neonatal removal or decentralization of the SCG, confirming the importance of neural input for differential night/day changes in transcript abundance. In contrast, very few non-rhythmic genes showed evidence of changes in expression due to the surgical procedure itself, which is consistent with the hypothesis that post neonatal neural stimulation is not required for cell fate determination and maintenance of phenotype. Many of the transcripts that exhibit marked differential night/day expression exhibited similar changes in response to in vitro treatment with norepinephrine, the SCG neurotransmitter which mediates pineal regulation. Similar changes were also seen following treatment with an analog of the norepinephrine second messenger, cyclic AMP. For the in vivo data, there were 8 biological conditions: day and night time points for each of four surgical groups: Control (Ctrl) Sham-surgery (Sham), Decentralized (DCN), and Ganglionectomized (SCGX). Samples were pooled into three biological replicates for each biological condition. For the in vitro data there were 3 biological conditions: Untreated control (CN), DBcAMP-treated (DB), and Norepinephrine-treated (NE). For the pineal enrichment comparison, three samples (i.e. no biological replicates) were used: pineal-day, pineal-night and mixed-tissue. For the mixed tissues sample, the following tissues from three rats sacrificed at ZT7 were used: cortex, cerebellum, midbrain, hypothalamus, hindbrain, spinal cord, retina, pituitary, heart, liver, lung, kidney, skeletal muscle, small intestine, adrenal gland. Total RNA was extracted from each tissue, and then equal amounts of each of the 15 tissues were combined for the final pooled sample.

Project description:Chick pinealocytes exhibit all the characteristics of a complete circadian system, comprising photoreceptive inputs, molecular clockworks and an easily measured rhythmic output, melatonin biosynthesis. We used microarray analysis to investigate the expression of approximately 8000 genes within cultured pinealocytes subjected to both LD and DD cycles. We report that a reduced subset of genes were rhythmically expressed in vitro compared to those previously published in vivo, and that gene expression rhythms were lower in amplitude, although the functional distribution of the rhythmic transcriptome was largely similar. We also investigated the effects of 6-hour pulses of light or of norepinephrine on gene expression in free-running cultures during both subjective day and night. As expected, both light and norepinephrine inhibited melatonin production; however, the two treatments differentially up- or down-regulated specific sets of genes in a fashion that was dependent upon time of day. Our combined approach of utilizing a time of day study and a light/NE pulse microarray experiment allowed us to identify novel genes linking clock input to clock function within the pineal. We identified approximately 30 rhythmic, light-responsive, NE-insensitive genes with no previously known clock function, which may play a role in circadian regulation of the pineal. These are candidates for future functional genomics experiments to elucidate their potential role in pineal physiology. Keywords: circadian; avian; pineal; light; norepinephrine Microarray analysis was performed on pineal samples subjected to one of four experimental groups: cultures placed within a 12-hour light:12-hour dark cycle (LD); cultures placed in continuous darkness (DD); cultures subjected to a 6-hour light pulse beginning at either CT0 or CT12 (i.e., zero or 12 hours after the beginning of subjective day) or that received no light pulse; and cultures subjected to 6 hours of NE treatment beginning at either CT0 or CT12 or that received similarly administered vehicle. For LD and DD groups, time-course sampling was performed such that 6 samples were collected over the course of a day at 4-hour intervals. For the light pulse and NE administration groups, 2 samples were collected over the course of a day, either at CT6 or CT18 (i.e., 6 or 18 hours after the beginning of subjective day). Four biological replicates and two technical replicates (for each biological repeat) were performed for each sample. For the LD and DD groups, time-points ZT18 and CT18 served as the control reference for the time-course analysis. For the light pulse experiment, cultures that did not receive light served as the control. For the NE administration experiment, cultures that received vehicle served as the control. Dye swaps for performed for experimental samples from the light pulse and NE administration experiments.

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RNA-Seq of the rat pineal transcriptome, with in-vivo and in-vitro samples, under various treatment and surgical conditions

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