Understanding the physiological mechanisms that limit animal thermal tolerance is crucial in predicting how animals will respond to increasingly severe heat waves. Despite their importance for understanding climate change impacts, these mechanisms underlying the upper thermal tolerance limits of animals are largely unknown. It has been hypothesized that the upper thermal tolerance in fish is limited by the thermal tolerance of the brain and is ultimately caused by a global brain depolarization. In this study, we developed methods for measuring the upper thermal limit (CT max ) in larval zebrafish ( Danio rerio ) with simultaneous recordings of brain activity using GCaMP6s calcium imaging in both free-swimming and agar-embedded fish. We discovered that during warming, CT max precedes, and is therefore not caused by, a global brain depolarization. Instead, the CT max coincides with a decline in spontaneous neural activity and a loss of neural response to visual stimuli. By manipulating water oxygen levels both up and down, we found that oxygen availability during heating affects locomotor-related neural activity, the neural response to visual stimuli, and CT max . Our results suggest that the mechanism limiting the upper thermal tolerance in zebrafish larvae is insufficient oxygen availability causing impaired brain function.

Understanding animal thermal tolerance is crucial to predict how animals will respond to increasingly warmer temperatures, and to mitigate the impact of the climate change on species survival. Yet, the physiological mechanisms underlying animal thermal tolerance are largely unknown. In this study, we developed a method for measuring upper thermal limit (CTmax) in larval zebrafish (Danio rerio) and found that it occurs at similar temperatures as in adult zebrafish. We discovered that CTmax precedes a transient, heat-induced brain-wide depolarization during heat ramping. By monitoring heart rate, we established that cardiac function is sub-optimal during the period where CTmax and brain depolarization occur. In addition, we found that oxygen availability affects both locomotor neural activity and CTmax during a heat stress. The findings of this study suggest that neural impairment due to limited oxygen availability at high temperatures can cause CTmax in zebrafish.HighlightsLarval zebrafish reach their critical thermal limit (CTmax) at similar temperature as adult zebrafishAcute heat stress causes a brain-wide spreading depolarization near the upper thermal limitCTmax precedes brain-wide depolarizationHeart rate declines at high temperatures but is maintained during CTmax and brain depolarizationNeural activity is impaired prior to CTmax and brain-wide depolarizationOxygen availability in the water affects both CTmax and neural activity