Abstract
Behavioral genetics in non-model organisms is currently gated by technological limitations. However, with the growing availability of genome editing and functional genomic tools, complex behavioral traits such as social behavior can now be explored in diverse organisms. Here we present a minimally invasive neurosurgical procedure for a classic behavioral, ecological and evolutionary system: threespine stickleback (Gasterosteus aculeatus). Direct brain injection enables viral-mediated transgenesis and pharmaceutical delivery which bypasses the blood-brain barrier. This method is flexible, fast, and amenable to statistically powerful within-subject experimental designs, making it well-suited for use in genetically diverse animals such as those collected from natural populations. Developing this minimally invasive neurosurgical protocol required 1) refining the anesthesia process, 2) building a custom surgical rig, and 3) determining the normal recovery pattern allowing us to clearly identify warning signs of failure to thrive. Our custom-built surgical rig (publicly available) and optimized anesthetization methods resulted in high (90%) survival rates and quick behavioral recovery. Using this method, we detected changes in aggression from the overexpression of either of two different genes, arginine vasopressin (AVP) and monoamine oxidase (MAOA), in outbred animals in less than one month. We successfully used multiple promoters to drive expression, allowing for tailored expression profiles through time. In addition, we demonstrate that widely available mammalian plasmids work with this method, lowering the barrier of entry to the technique. By using repeated measures of behavior on the same fish before and after transfection, we were able to drastically reduce the necessary sample size needed to detect significant changes in behavior, making this a viable approach for examining genetic mechanisms underlying complex social behaviors.