Seasonal changes in patterns of gene expression in avian song control brain regions.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Photoperiod and hormonal cues drive dramatic seasonal changes in structure and function of the avian song control system. Little is known, however, about the patterns of gene expression associated with seasonal changes. Here we address this issue by altering the hormonal and photoperiodic conditions in seasonally-breeding Gambel's white-crowned sparrows and extracting RNA from the telencephalic song control nuclei HVC and RA across multiple time points that capture different stages of growth and regression. We chose HVC and RA because while both nuclei change in volume across seasons, the cellular mechanisms underlying these changes differ. We thus hypothesized that different genes would be expressed between HVC and RA. We tested this by using the extracted RNA to perform a cDNA microarray hybridization developed by the SoNG initiative. We then validated these results using qRT-PCR. We found that 363 genes varied by more than 1.5 fold (>log(2) 0.585) in expression in HVC and/or RA. Supporting our hypothesis, only 59 of these 363 genes were found to vary in both nuclei, while 132 gene expression changes were HVC specific and 172 were RA specific. We then assigned many of these genes to functional categories relevant to the different mechanisms underlying seasonal change in HVC and RA, including neurogenesis, apoptosis, cell growth, dendrite arborization and axonal growth, angiogenesis, endocrinology, growth factors, and electrophysiology. This revealed categorical differences in the kinds of genes regulated in HVC and RA. These results show that different molecular programs underlie seasonal changes in HVC and RA, and that gene expression is time specific across different reproductive conditions. Our results provide insights into the complex molecular pathways that underlie adult neural plasticity

Activation-dependent regulation of galanin gene expression in gonadotropin-releasing hormone neurons in the female rat [see comments]

In rats, galanin is colocalized in GnRH neurons, and galanin mRNA in GnRH neurons is increased coincidentally with the preovulatory gonadotropin surge. Whether the induction of galanin mRNA in GnRH neurons at proestrus reflects the action of sex steroids is unknown. We tested this hypothesis by challenging ovariectomized rats (n = 7) with estrogen and progesterone (E/P) to induce a LH surge and measuring galanin mRNA in GnRH neurons to determine whether there was an associated induction of galanin message in these cells. We used single and double label in situ hybridization and image analysis to compare among groups the levels of both galanin mRNA and GnRH mRNA in GnRH neurons. We found that steroid-primed animals showed an approximately 400% induction of galanin mRNA signal in GnRH neurons over that in vehicle-treated animals. Second, we hypothesized that steroid-dependent events which induce the expression of galanin mRNA in GnRH neurons depend on transsynaptic input to GnRH neurons. We tested this hypothesis by examining the effect of a pharmacological blockade of the steroid-induced activation of GnRH neurons on levels of galanin mRNA in these cells. We killed groups of ovariectomized adult female rats at the peak of a E/P-primed LH surge (n = 7) and after steroid priming followed by blockade of the LH surge with either the general anesthetic pentobarbital (n = 7) or the specific alpha-adrenergic receptor blocker phenoxybenzamine (n = 7). When we examined signal levels representing galanin mRNA content in GnRH neurons, we observed a 4-fold increase in signal for galanin mRNA in the GnRH neurons of steroid-primed (E/P surge) animals compared with that in oil-treated controls (P < 0.0004). This increase in galanin mRNA was prevented when the LH surge was blocked by treatment with either pentobarbital or phenoxybenzamine (P < 0.03 and P < 0.0001 vs. E/P surge controls, respectively). Cellular levels of GnRH mRNA were not different among control, E/P, and E/P plus pentobarbital groups (P > 0.2). These observations suggest that an increase in galanin mRNA levels in GnRH neurons is tightly coupled to the occurrence of a LH surge. By inference, induction of galanin mRNA in GnRH neurons reflects their activation, possibly via afferent neurons that transduce the steroid signal to GnRH neurons

Heatmap illustrating change in expression of genes related to cell growth in RA.

<p>The scale for expression is illustrated on the right. FDR-adjusted significance levels are also included (*>0.1, **>0.05, ***>0.01, ****>0.001).</p

Heatmap illustrating change in expression of genes related to electrophysiology in HVC and RA.

<p>The scale for expression is illustrated on the right. FDR-adjusted significance levels are also included (*>0.1, **>0.05, ***>0.01, ****>0.001).</p

Many genes showed correlated pattern of expression between HVC and RA.

<p>(A) A frequency histogram (bin size = 0.02) of correlation coefficients for each spot on the array comparing HVC to RA is positively-skewed, indicating that genes that were correlated between HVC and RA were positively correlated. (B) Venn diagram illustrating the proportion of genes that were significantly correlated between HVC and RA that also appeared in the HVC and/or RA gene of interest lists that vary in expression across seasonal conditions. The vast majority of genes that showed correlated expression in HVC and RA were not represented in either list. (C) Expression of EGR1 in HVC was correlated with expression of EGR1 in RA. (D) SCGN is an example of a gene that had a very high Pearson's coefficient (r² = 0.740) that was entirely dependent upon a single outlier. (E) Expression of ARFGAP1 did not vary significantly across breeding conditions in (F) RA or HVC but was positively correlated in expression between HVC and RA. (G) Expression of DIO2, a gene critical for thyroid hormone metabolism, varied significantly across breeding conditions in (H) HVC and RA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035119#pone.0035119.s002" target="_blank">Table S2</a>) and was positively correlated in expression between HVC and RA. Expression values are illustrated relative to the universal reference sample.</p

Scatter plot illustrating correlations of array results to qRT-PCR validation.

<p>The expression of 19 genes from five time points relative to SD-long term comparing results using qRT-PCR to results from the microarray are illustrated. Each point represents the group average at a given time point for a particular gene. Results assessing change in expression from the microarray were significantly correlated with results using qRT-PCR in (A) HVC (p<10⁻⁵) and (B) RA (p<10⁻⁵).</p

Changes in expression following the transition from breeding to nonbreeding conditions.

<p>The number of genes upregulated or downregulated in HVC and RA over SD Day 1 and SD Day 2 relative to LD+T Day 21.</p