Microinjection of Interleukin-6 into the Bloodstream of TSC Zebrafish Larvae to Study Neuropsychiatric Disorder-Like Behaviors.

Patients with tuberous sclerosis complex (TSC1/TSC2 mutations) exhibit variable expressivity of autism spectrum disorder (ASD), and maternal immune activation is known to heighten ASD risk. Interleukin-6 (IL-6) is a pleiotropic cytokine with well-established roles in immune regulation, inflammation, and neuroinflammatory processes. It is produced by multiple immune cell types in response to infection or tissue injury, and is commonly characterized as a pro-inflammatory mediator. Notably, IL-6 can cross the blood-brain barrier and has been implicated in neuroinflammation and inflammation-associated neuropsychiatric conditions, including autism spectrum disorder (ASD) and intellectual disability (ID). Here, we describe a protocol to induce systemic inflammatory signaling in zebrafish by delivering IL‑6 into the bloodstream through injection into the duct of Cuvier. This approach enables controlled and reproducible inflammatory responses to study inflammation-driven changes in early development. The protocol details the essential steps for IL-6 preparation, microinjection, and downstream experimental applications in zebrafish models of disease and neuroinflammation, including studies designed to probe gene-environment interactions. By enabling controlled induction of systemic inflammatory signaling, this approach provides a versatile platform for examining how genetic susceptibility and inflammatory exposures intersect to shape developmental outcomes.