Seasonal differences of gene expression profiles in song sparrow (Melospiza melodia) hypothalamus in relation to territorial aggression

Abstract

Background: Male song sparrows (Melospiza melodia) are territorial year-round; however, neuroendocrine responses to simulated territorial intrusion (STI) differ between breeding (spring) and non-breeding seasons (autumn). In spring, exposure to STI leads to increases in luteinizing hormone and testosterone, but not in autumn. These observations suggest that there are fundamental differences in the mechanisms driving neuroendocrine responses to STI between seasons. Microarrays, spotted with EST cDNA clones of zebra finch, were used to explore gene expression profiles in the hypothalamus after territorial aggression in two different seasons.

Methodology/principal findings: Free-living territorial male song sparrows were exposed to either conspecific or heterospecific (control) males in an STI in spring and autumn. Behavioral data were recorded, whole hypothalami were collected, and microarray hybridizations were performed. Quantitative PCR was performed for validation. Our results show 262 cDNAs were differentially expressed between spring and autumn in the control birds. There were 173 cDNAs significantly affected by STI in autumn; however, only 67 were significantly affected by STI in spring. There were 88 cDNAs that showed significant interactions in both season and STI.

Conclusions/significance: Results suggest that STI drives differential genomic responses in the hypothalamus in the spring vs. autumn. The number of cDNAs differentially expressed in relation to season was greater than in relation to social interactions, suggesting major underlying seasonal effects in the hypothalamus which may determine the differential response upon social interaction. Functional pathway analyses implicated genes that regulate thyroid hormone action and neuroplasticity as targets of this neuroendocrine regulation.

Figure 1. Aggressive behavior of sparrows subjected to simulated territorial intrusion during breeding and non-breeding season.

Male song sparrows (n = 8/group) in their territory were exposed to a caged conspecific male decoy in addition to a recorded male song playbacks in breeding season, spring (SE) and non-breeding season, autumn (AE). As controls (AC, SC), a white-crowned sparrow decoy and recorded songs were used. Behavior of a subject was recorded for number of songs, number of flight, closest approach to the decoy (meters; m), and time spend within 5-m of the decoy. Kruskal-Wallis 1-way ANOVA followed by Mann-Whitney U test; significant at * p<0.05, ** p<0.01 between experimental conditions of the same season, or at † p<0.05 between seasons under same experimental conditions. Results are shown in mean±SEM.

Figure 2. Venn-diagram of regulated cDNA spots in different seasons and with simulated territorial intrusions.

A) Number of regulated cDNA spots in autumn compared to spring in either control or STI groups (AC vs. SC, 262 total; AE vs. SE, 283 total), and those that showed interaction effect of season and STI (88 total). B) Number of regulated cDNA spots in STI compared to controls in each season (SE vs. SC, 67 total; AE vs. AC, 173 total), and those that showed interaction effect of season and STI. The numbers within the overlapped circle indicates an overlap of differentially expressed cDNAs. Cell-means model was used for statistics, significant at p<0.01.

Figure 3. Hierarchical clustering of all differentially expressed cDNA spots.

All cDNA spots that passed the cut-off (p<0.01) in the cell-means model (total of 727 genes) were clustered using Pearson correlation and shown as a heatmap. Up-regulated cDNAs are shown in red, down-regulated cDNAs are shown in blue compared to a mean of all four groups. Examples of two distinct groups are seen in AC vs. SC comparison (blue and red line; down- and up-regulation, respectively). Those cDNA spots affected by interactions are also shown as sections (A, B, C). Portion of ACTTR and CRYM) shows consistency of expression patterns among these cDNAs.

Figure 4. Representative gene network discovered by Ingenuity Pathway Analysis.

A) A network affected in AC vs. SC comparison. Red represents up-regulation of the gene in autumn control (AC) vs. spring control (SC), whereas blue represents down-regulation. Color intensity correlates with a degree of fold change. Thyroid hormone (L-triiodothyronine, T₃) is highlighted by yellow. White represents genes that were either not differentially expressed, not been annotated, or not in the custom-made array. B&C) A network affected by interaction (season×STI). The same network shown with expression fold-changes for SE vs. SC (B) or AE vs. AC (C). Red represents up-regulation of the gene in STIs (SE in B, AE in C) when compared with controls (SC in B, AC in C), whereas blue represents down-regulation. Same as (A) for the white. When there were multiple cDNA clone spots per gene, fold change values that were of most significance were selected.

Figure 5. Validation of microarray results by real-time PCR.

Expression of A) glycoprotein hormone alpha chain precursor (CGA), B) transthyretin (TTR), C) μ-Crystallin (CRYM), D) heat shock 70 KDa protein (HSPA2), E) zinc finger and BTB domain containing 16 (ZBTB16), F) growth arrest and DNA-damage-inducible, beta (GADD45B), G) vasoactive intestinal peptide (VIP), and H) c-fos (FOS) were determined. Results were calculated in relative RNA amounts using β-actin (ACTB) as internal control and shown as mean fold-change to group with lowest expression value. n = 5−7/group, * p<0.05 by ANOVA followed by Tukey's test as posthoc pair-wise comparisons. Results are shown in mean±SEM.

Figure 6. Schematic diagram showing the regulation of thyroid hormone action in pars tuberalis of pituitary and hypothalamus.

Blue indicates genes that were significantly affected in the current microarray study: common glycoprotein alpha subunit (CGA), transthyretin (TTR), μ-crystallin (CRYM), and heat shock 70 KDa protein (HSPA2), and amyloid-β precursor protein (APP). TSH: thyroid stimulating hormone, DIO2: type-2 deiodinase, LH: luteinizing hormone, FSH: follicular-stimulating hormone, 3 V: third ventricle, T₃: L-triiodothyronine, T₄: thyroxine.