Minimally invasive brain injections for viral-mediated transgenesis: New tools for behavioral genetics in sticklebacks

Abstract

24Major Findings 25 Forced expression of MAOA or AVP resulted in more attacks, showing a causal link between genes and 26 behavior.Abstract 35 Establishing a causal relationship between genes and social behavior is challenging. To enable 36 direct manipulation of candidate genes and thereby examine how their expression contributes to 37 behavior, we developed a neurosurgical method to deliver pharmacological agents or transgenic 38 elements directly into the threespine stickleback (Gasterosteus aculeatus) brain. Threespine sticklebacks 39 are a classic system for the study of behavior, ecology, and evolution. Male sticklebacks defending 40 nesting territories are highly aggressive toward intruders. Previous studies in stickleback have shown 41 that aggression is heritable, and that hundreds of genes are differentially expressed in the brain following 42 territorial intrusion. 43 We use viral-mediated transgenesis to test the effects on territorial aggression of overexpression 44 of candidate genes, monoamine oxidase (MAOA) and arginine vasopressin (AVP), in the stickleback 45 brain. Male sticklebacks received transcranial injections of mammalian homolog cDNA packaged in a 46 replication-deficient Herpes Simplex Virus 1 carrier. Animals transfected with either AVP or MAOA 47 constructs were more aggressive in response to a territorial intruder, unlike control animals transfected 48 with a fluorescent protein. 49 Viral-mediated transgenesis is a promising method to examine genetic underpinnings of 50 behaviors. Our success demonstrates that widely available mammalian plasmids work with this method, 51 lowering the barrier of entry to the technique. This method is flexible, fast, and amenable to statistically 52 powerful within-subject experimental designs, making it practical for use in natural populations. It 53 further enhances the growing molecular toolkit in threespine stickleback, a classic ethological system, 54 and is the first step toward using chemogenetics and optogenetics.55 56 57 Complex behaviors have been repeatedly shown to be heritable (reviewed in Dochtermann et al., 58 2019), yet establishing a causal relationship between genes and social behavior remains challenging. 59Partially, this difficulty arises from limitations of the primarily correlative methods for examining the 60 interplay between genes and behavior (Charney 2017), such as QTL, GWAS, and RNAseq studies. 61These types of studies are certainly a necessary step generating many candidate genes. However, to fully 62 characterize how a gene contributes to behavior, it is necessary to consider not just sequence differences, 63 but also regulatory and epigenetic influences. Therefore, to demonstrate and fully characterize a causal 64 relationship between a gene and behavior, it is crucial to have a method for manipulating gene 65 expression at a specific time and location. We developed one such method, viral-mediated transgenesis, 66 for the classic ethological system of threespined stickleback (Gasterosteus aculeatus).

Threespine Stickleback: A Model System For Evolutionary Genomics

“…The threespine stickleback's natural history, coupled with high-quality genomic resources, has massively facilitated these insights due to the ease of generating huge amounts of DNA sequence and transcriptomic data that can be leveraged across diverse fields, from evolution to behavior to host-parasite and host-microbiome interactions. As a result, the threespine stickleback system is now strongly positioned to exploit quickly emerging transgenic tools, such as CRISPR/Cas9 (66,168,169) and neurosurgical methods (80), to forge strong links between specific genetic variants and phenotypes and provide insights into the molecular basis for heritable human pathology.…”

“…Genome-wide gene expression studies using microarray and RNA-seq transcriptome techniques are frequently used to quantify common changes in specific tissue types, such as regions of the brain that modulate transcriptional regulatory networks to various stimuli (30,59,116,140). Recently, virus-mediated transgenesis tools (such as brain microinjections) have been developed to facilitate modification of gene regulation directly in relevant tissues, allowing individuals to act as their own control and provide a more detailed mechanistic analysis of behavior (80). Threespine sticklebacks exhibit a range of behaviors that are divergent among ecotypes as well as variable within populations (21).…”