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

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.

Insights into adaptive behavioural plasticity from the guppy model system

“…Isotocin and vasotocin have been shown to play a role in guppy grouping using intracerebroventricular administration techniques that could be used to study the impact of other hormones on behavioural plasticity [85]. Intracranial administration can also be used to manipulate gene expression using viral-mediated transgenesis [86]. Additionally, guppies have greatly expanded their native and non-native range, even into heavily polluted environments [87], creating new challenges that require integrated endocrinological and behavioural adaptations and providing unique opportunities for research.…”

Anemonefishes: A model system for evolutionary genomics

“…James and Bell (2021) used virus-mediated transgenesis (through direct brain injection) to study how overexpression of certain genes affected aggressive behaviors in the fish model Gasterosteus aculeatus. 202 Indeed, species such as the threespine stickleback and zebrafish have long been the subject of behavioral genetics, [202][203][204][205][206] providing valuable insights that can potentially be transferred to anemonefish.…”

Anemonefishes: A model system for evolutionary genomics

“…James and Bell (2021) used virus-mediated transgenesis (through direct brain injection) to study how overexpression of certain genes affected aggressive behaviors in the fish model Gasterosteus aculeatus. 213 Indeed, species such as the threespine stickleback and zebrafish have long been the subject of behavioral genetics, [213][214][215][216][217] providing valuable insights that can potentially be transferred to anemonefish.…”