A multipronged approach to understanding how genes contribute to social behavior in threespine stickleback

Understanding how genes contribute to behavior requires a two-pronged approach – identifying what genes underlie the behavior and characterizing their molecular mechanisms. Naturally, the behavior under study must be heritable. It should ideally be reliably assayable, a difficult provision to satisfy for ecologically important social behaviors which have large but consistent individual differences. Non-traditional model systems offer a limited molecular toolkit, often constraining behavioral genetics to mainly correlative methods rather than direct manipulation. Therefore, to enable establishing a causal relationship between genes and social behavior in the emerging model system threespine stickleback (G. aculeatus), we needed to develop molecular techniques and simplify the already robust behavioral assays. Threespine sticklebacks are a classic system for the study of behavior, ecology, and evolution. A growing number of quantitative trait loci (QTL) and gene expression studies are identifying genes related to ecologically-important social behaviors in sticklebacks, such as parenting and aggression. In order to visualize the expression of these candidate genes, we developed a fluorescence in situ hybridization (FISH) protocol. The FISH protocol resulted in specific labeling under all combinations of dissection (fresh vs. frozen) and embedding (paraffin vs. cryo) conditions. Paraffin embedding preserved morphology better than cryo-embedding. We provide representative results showing the expression of three genes related to social behavior – glial fibrillary acidic protein (GFAP), oxytocin receptor (OXTR) and tyrosine hydroxylase (TH) in the brain. To enable direct manipulation of genes for social behavior, we focused on aggression. Male sticklebacks demonstrate stereotypical aggressive behaviors during an easily-induced territorial defense. Previous studies in stickleback have shown that aggression is heritable, and that hundreds of genes are differentially expressed in the brain following territorial intrusion. However, the traditionally ethology focused territorial-intrusion assay is typically synchronized to the reproductive cycle rather than being yoked in time. Given the seasonality and high dropout rate of non-nesters, this methodology would require a prohibitively large sample size during molecular characterization. Therefore, we first sought to evaluate to what degree territorial aggression is moderated by nesting. Neither nest presence, timing of construction, nor nesting outcome were associated with differences in behavioral measures of territorial aggression. Assessed behaviors were robust, repeatable, and intercorrelated. We conclude territorial aggression is neither predictive of nor altered by nesting in threespine stickleback fish and could therefore synchronize aggression assays based on timing rather than nesting state. Finally, we developed a method for viral-mediated transgenesis in the brain to directly test the effects of increased expression of candidate genes, monoamine oxidase (MAOA) and arginine vasopressin (AVP), on territorial aggression in the sticklebacks. This method is flexible, fast, and amenable to statistically powerful within-subject experimental designs, making it practical for use in natural populations. Fish transfected with either AVP or MAOA constructs were more aggressive in response to a territorial intruder, unlike control animals transfected with a fluorescent protein. Our success demonstrates that widely available mammalian plasmids work with this method, lowering the barrier of entry to the technique. It further enhances the growing molecular toolkit in threespine stickleback, a classic ethological system, and is the first step toward using chemogenetics and optogenetics