Conclusion
Looking to the future, elucidating the cellular and molecular changes that occur between reproductively active and inactive neuroendocrine tissues of resident and migrant birds respectively will require additional approaches, such as localization of proteins or ultrastructure imaging of the hypothalamus and pituitary. The observed differentially expressed proteins involved in calcium ion channel activity, synapse, neuron differentiation, dendrite formation, axon guidance all raise a possibility of seasonal changes in the morphology of nerve terminals in the hypothalamus or in endocrine secretory cells in the pituitary. Cellular localization of specific proteins that play a role in GnRH synthesis, stability, and release in residents, or that play a role in migratory processes such as increased food intake or synaptogenesis in migrants are needed and will provide added insight into the seasonal regulation of key life history stages in birds. Seasonal neurogenesis in the hypothalamus and other brain areas has been reported in diverse species ranging from zebrafish to hamsters, sheep, and birds (Sousa-Ferreira, de Almeida, & Cavadas 2014; Pozner, Vistoropsky, Moaraf, Heiblum, & Barnea, 2018). By considering the potential role of proteins in neurogenesis, synapse formation, and cell proliferation, we found a few key molecules such as PURA, MAP2K1, DPYSLL, PAFAH1B1, STONIN 2, and Hypoxanthine-guanine phosporibosyltransferase that are either directly or indirectly involved in neuron, dendrite morphology and synapse formation. The proteomics analysis of the brain regions controlling the neuroendocrine system could be helpful in introducing neuro-proteomics as a new area of study, enabling the improved understanding of the molecular mechanisms and correlates associated with the underlying differential brain response depending on the time of the day, duration of the day length or other environmental factors.
Declaration: Authors have no conflict of interest.
Data Accessibility: The proteomics data are available to the Proteome Xchange Consortium via the MassIVE partner repository with the dataset identifier ftp://MSV000085662@massive.ucsd.edu and password: Junco. Data are available in the Dryad Digital Repository:  https://doi.org/10.5061/dryad.f1vhhmgv4
Authors’ contribution : DS and EDK conceived the idea. DS and AMF collected field samples. DS carried out the data collection and analysis. TS performed the hydrogen stable isotope analyses and geographic assignments. DS wrote the manuscript with the help of all authors. All authors approved the final draft.
Funding. The funds were provided by Indiana University Grand Challenge Initiative, Prepared for Environmental Change to EDK and TMS, National Science Foundation (IOS- 1856423) and, IU Faculty Research Support Program, (FRSP, account number # 2224640) to AMF and EDK.
Acknowledgements: We thank Douglas B. Rusch and Ram Podicheti from the Center for Genomics and Bioinformatics (CGB), IU Bloomington for providing junco protein database generated from recently annotated junco genome. We thank Jonathan C. Trinidad for performing proteome sequencing and access to the Biological Mass Spectrometry Laboratory, IU Bloomington. Mountain Lake Biological Station provided access to field sites. We are grateful to Kathryn Evans for help with sample preparation and Peter Sauer at the Indiana University Stable Isotope Research Facility for assistance with isotopic analysis.
Figure 1. Schematic showing geographical distribution of resident (J. h. carolinesis ; red circle, year-round) and migrant (J. h. hyemalis ; orange as breeding area, pale blue as wintering area; based on IUCN, 2019) dark-eyed juncos on the North America map (a). The middle left panel showing the difference in the early breeding resident juncos gonad development than migrants in early spring (b). The middle right panel showing schematic for the isobaric tandem mass tag (TMT) labeling of individual hypothalamic protein samples pooled to run Liquid Chromatography with tandem mass spectrometry (LCMS/MS) (c). The flow-chart outline steps of protein sequencing, differentially expressed protein (DEPs) analysis, enriched gene ontology (GO) terms, and significant kyoto encyclopedia of genes and genomes (KEGG) pathway analysis (d).