The ability to detect and avoid predators is essential to survival. Various animals, from sea urchins to damselfly larvae, use injury of conspecifics to infer the presence of predators. In many fish, skin damage causes the release of chemicals that elicit escape and fear in members of the shoal. The chemical nature of the alarm substance ("Schreckstoff" in German), the neural circuits mediating the complex response, and the evolutionary origins of a signal with little obvious benefit to the sender, are unresolved. To address these questions, we use biochemical fractionation to molecularly characterize Schreckstoff. Although hypoxanthine-3 N-oxide has been proposed to be the alarm substance, it has not been reliably detected in the skin and there may be other active components. We show that the alarm substance is a mixture that includes the glycosaminoglycan (GAG) chondroitin. Purified chondroitins trigger fear responses. Like skin extract, chondroitins activate the mediodorsal posterior olfactory bulb, a region innervated by crypt neurons that has a unique projection to the habenula. These findings establish GAGs as a new class of odorants in fish, which trigger alarm behavior possibly via a specialized circuit.

Animals quickly learn to avoid predictable danger. However, if pre-exposed to a strong stressor, they do not display avoidance even if this causes continued contact with painful stimuli [1, 2]. In rodents, lesioning the habenula, an epithalamic structure that regulates the monoaminergic system, has been reported to reduce avoidance deficits caused by inescapable shock [3]. This is consistent with findings that inability to overcome a stressor is accompanied by an increase in serotonin levels [4]. However, other studies conclude that habenula lesions cause avoidance deficits [5, 6]. These contradictory results may be caused by lesions affecting unintended regions [6]. To clarify the role of the habenula, we used larval zebrafish, whose transparency and amenability to genetic manipulation enables more precise disruption of cells. We show that larval zebrafish learn to avoid a light that has been paired with a mild shock but fail to do so when pre-exposed to inescapable shock. Photobleaching of habenula afferents expressing the photosensitizer KillerRed causes a similar failure in avoidance. Expression of tetanus toxin in dorsal habenula neurons is sufficient to prevent avoidance. We suggest that this region may signal the ability to control a stressor, and that its disruption could contribute to anxiety disorders.

These data provide physiological and functional evidence that the habenula functions as a higher center in zebrafish olfaction and suggest that activity in the right dorsal subdomain gates innate attraction to specific odors.

The alarm response is an antipredator behavior displayed by many fish species and was first described 70 years ago. It is triggered through the olfactory system by substances released from injured skin and is characterized by dramatic, measurable changes in locomotion as well as physiology. We propose that this is an ideal time to revisit this response and to utilize it as an assay for understanding how neural circuits mediate innate fear. A suitable organism for these studies is the zebrafish, a genetic model with a rapidly expanding toolkit for molecular manipulation of the nervous system. Individual neurons mediating the response, ranging from receptor neurons to those in higher brain centers, should first be identified. New tools, specifically transgenic lines that allow spatial and temporal control of neural activity, provide a way to define and test the role of specific neurons, while genetic screens provide a route to identifying individual molecules essential for a normal response. Optical recording, which has proven successful in studies of information processing in the bulb, will provide valuable insights into neural circuitry function during the alarm response. When carried out on mutants, physiological analysis can provide insight into aspects of signal processing that are essential for normal behavior. The alarm response thus provides a paradigm to examine innate fear in a vertebrate system, enabling analysis at multiple levels from genes to the entire neural circuit. Additionally, the context dependency of the response can be utilized to investigate attention and decision making.