Neurotranscriptomics: The Effects of Neonatal Stimulus Deprivation on the Rat Pineal Transcriptome

<div><p>The term neurotranscriptomics is used here to describe genome-wide analysis of neural control of transcriptomes. In this report, next-generation RNA sequencing was using to analyze the effects of neonatal (5-days-of-age) surgical stimulus deprivation on the adult rat pineal transcriptome. In intact animals, more than 3000 coding genes were found to exhibit differential expression (adjusted-p < 0.001) on a night/day basis in the pineal gland (70% of these increased at night, 376 genes changed more than 4-fold in either direction). Of these, more than two thousand genes were not previously known to be differentially expressed on a night/day basis. The night/day changes in expression were almost completely eliminated by neonatal removal (SCGX) or decentralization (DCN) of the superior cervical ganglia (SCG), which innervate the pineal gland. Other than the loss of rhythmic variation, surgical stimulus deprivation had little impact on the abundance of most genes; of particular interest, expression levels of the melatonin-synthesis-related genes Tph1, Gch1, and Asmt displayed little change (less than 35%) following DCN or SCGX. However, strong and consistent changes were observed in the expression of a small number of genes including the gene encoding Serpina1, a secreted protease inhibitor that might influence extracellular architecture. Many of the genes that exhibited night/day differential expression in intact animals also exhibited similar changes following <i>in vitro</i> treatment with norepinephrine, a superior cervical ganglia transmitter, or with an analog of cyclic AMP, a norepinephrine second messenger in this tissue. These findings are of significance in that they establish that the pineal-defining transcriptome is established prior to the neonatal period. Further, this work expands our knowledge of the biological process under neural control in this tissue and underlines the value of RNA sequencing in revealing how neurotransmission influences cell biology.</p></div

Venn diagrams displaying the overlap between the different differential expression analyses.

<p>Diagrams indicate the concordance in the genes found to display strong (FC > 4 in either direction) and statistically significant (adjusted-p < 0.001) differential expression in multiple separate analyses. (a) Comparison of the (<i>in vivo</i>) Control and Sham night/day differential expression analyses. (b) Comparison of the (<i>in vitro</i>) NE vs untreated and DBcAMP vs untreated differential expression analyses. (c) Comparison of the overlapping region from (a) with the results of the two <i>in vitro</i> analyses from (b).</p

Genes significantly differentially expressed in the pineal gland on a night/day basis in Control and/or Sham groups, <i>in vivo</i> (adjusted-p < 0.001).

<p>*Gene exhibits high relative expression in the pineal gland (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137548#pone.0137548.s010" target="_blank">S2 Table</a>).</p><p>^†Gene has previously been found to be differentially expressed between day and night conditions in microarray experiments.</p><p>Genes that exhibit strong differential expression in both are listed in bold (genes are classified by the least-extreme fold change across the two analyses). Note that some genes are listed twice if the fold changes of the two analyses fall in different intervals. In the SCGX and DCN groups, none of the genes displayed in this table exhibited night/day differential expression at this level (adjusted p < 0.001, FC > 4 in either direction). A complete list of all genes with fold changes, p-values, and normalized expression estimates is available in the SI (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137548#pone.0137548.s002" target="_blank">S1 Dataset</a>).</p

Genes with consistent differential expression in the DCN group relative to the Sham group.

<p>*Gene exhibits high relative expression in the pineal gland (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137548#pone.0137548.s010" target="_blank">S2 Table</a>).</p><p>^†Gene has previously been found to be differentially expressed between day and night conditions in microarray experiments (adjusted-p < 0.05).</p><p>^‡Gene has strong (FC > 4 or FC < 0.25) and statistically significant (adjusted-p < 0.001) differential expression in the Control and/or Sham night/day <i>in vivo</i> analyses (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137548#pone.0137548.t001" target="_blank">Table 1</a>).</p><p>^§Gene has strong and statistically significant differential expression in either or both of the treated vs untreated <i>in vitro</i> analyses (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137548#pone.0137548.t003" target="_blank">Table 3</a>).</p><p>These genes displayed significant (adjusted-p < 0.001) differentials across 2 analyses: DCN-day vs Sham-day and DCN-night vs Sham-night. Genes are classified by the least-extreme fold change across both analyses. The complete results for these analyses are available in the SI (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137548#pone.0137548.s003" target="_blank">S2 Dataset</a>).</p

MA plots for six differential expression analyses.

<p>These plots display the mean normalized read-pair counts (x-axis) versus the estimated fold change (y-axis), on a log-log scale for four <i>in vivo</i> analyses and two <i>in vitro</i> analyses. The blue shading indicates the density of genes, and each red point represents a gene with statistically significant differential expression (adjusted-p < 0.001). Dashed horizontal lines mark 4-fold changes in both directions, dashed vertical line indicates minimum abundance threshold for the statistical tests. The four <i>in vivo</i> analyses compared night and day time points in adult rats for the following groups: (a) no surgery (Control); (b) neonatal sham surgery (Sham); (c) neonatal superior cervical ganglia decentralization (DCN); (d) neonatal superior cervical ganglionectomy (SCGX). The two <i>in vitro</i> analyses compared treated/untreated pineal glands: (e) norepinephrine-treated (NE) vs untreated and (f) dibutyryl-cyclic-AMP-treated (DBcAMP) vs untreated.</p

Genes significantly differentially expressed in pineal gland tissues in response to <i>in vitro</i> treatment with NE or DBcAMP (adjusted-p < 0.001).

<p>*Gene exhibits high relative expression in the pineal gland (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137548#pone.0137548.s010" target="_blank">S2 Table</a>).</p><p>^†Gene has previously been found to be differentially expressed between day and night conditions in microarray experiments.</p><p>Genes that exhibit strong differential expression in both the NE and DBcAMP analyses are listed in bold (genes are classified by the least-extreme fold change across the two analyses). A complete list of all genes with fold changes, p-values, and normalized expression estimates is available in the SI (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137548#pone.0137548.s004" target="_blank">S3 Dataset</a>).</p