INGEsT: An Open-Source Behavioral Setup for Studying Self-motivated Ingestive Behavior and Learned Operant Behavior.

Ingestive behavior is driven by negative internal hunger and thirst states, as well as by positive expected rewards. Although the neural substrates underlying feeding and drinking behaviors have been widely investigated, they have primarily been studied in isolation, even though eating can also trigger thirst, and vice versa. Thus, it is still unclear how the brain encodes body states, recalls the memory of food and water reward outcomes, generates feeding/drinking motivation, and triggers ingestive behavior. Here, we developed an INstrument for Gauging Eating and Thirst (INGEsT), a custom-made behavioral chamber which allows for precise measurement of both feeding and drinking by combining a FED3 food dispenser, lickometers for dispensing liquid, a camera for behavioral tracking, LED light for optogenetics, and calcium imaging miniscope. In addition, in vivo calcium imaging, optogenetics, and video recordings are well synchronized with animal behaviors, e.g., nose pokes, pellet retrieval, and water licking, by using a Bpod microprocessor and timestamping behavioral and imaging data. The INGEsT behavioral chamber enables many types of experiments, including free feeding/drinking, operant behavior to obtain food or water, and food/water choice behavior. Here, we tracked activity of insular cortex and mPFC Htr3a neurons using miniscopes and demonstrate that these neurons encode many aspects of ingestive behavior during operant learning and food/water choice and that their activity can be tuned by internal state. Overall, we have built a platform, consisting of both hardware and software, to precisely monitor innate ingestive, and learned operant, behaviors and to investigate the neural correlates of self-motivated and learned feeding/drinking behaviors.

Analysis of stress responses in Astyanax larvae reveals heterogeneity among different populations.

Stress responses are conserved physiological and behavioral outcomes as a result of facing potentially harmful stimuli, yet in pathological states, stress becomes debilitating. Stress responses vary considerably throughout the animal kingdom, but how these responses are shaped evolutionarily is unknown. The Mexican cavefish has emerged as a powerful system for examining genetic principles underlying behavioral evolution. Here, we demonstrate that cave Astyanax have reduced behavioral and physiological measures of stress when examined at larval stages. We also find increased expression of the glucocorticoid receptor, a repressible element of the neuroendocrine stress pathway. Additionally, we examine stress in three different cave populations, and find that some, but not all, show reduced stress measures. Together, these results reveal a mechanistic system by which cave-dwelling fish reduced stress, presumably to compensate for a predator poor environment.

An Adult Brain Atlas Reveals Broad Neuroanatomical Changes in Independently Evolved Populations of Mexican Cavefish.

A shift in environmental conditions impacts the evolution of complex developmental and behavioral traits. The Mexican cavefish, Astyanax mexicanus, is a powerful model for examining the evolution of development, physiology, and behavior because multiple cavefish populations can be compared to an extant, ancestral-like surface population of the same species. Many behaviors have diverged in cave populations of A. mexicanus, and previous studies have shown that cavefish have a loss of sleep, reduced stress, an absence of social behaviors, and hyperphagia. Despite these findings, surprisingly little is known about the changes in neuroanatomy that underlie these behavioral phenotypes. Here, we use serial sectioning to generate brain atlases of surface fish and three independent cavefish populations. Volumetric reconstruction of serial-sectioned brains confirms convergent evolution on reduced optic tectum volume in all cavefish populations tested. In addition, we quantified volumes of specific neuroanatomical loci within several brain regions that have previously been implicated in behavioral regulation, including the hypothalamus, thalamus, and habenula. These analyses reveal an enlargement of the hypothalamus in all cavefish populations relative to surface fish, as well as subnuclei-specific differences within the thalamus and prethalamus. Taken together, these analyses support the notion that changes in environmental conditions are accompanied by neuroanatomical changes in brain structures associated with behavior. This atlas provides a resource for comparative neuroanatomy of additional brain regions and the opportunity to associate brain anatomy with evolved changes in behavior.

Behavioral Approaches to Studying Innate Stress in Zebrafish.

Responding appropriately to stressful stimuli is essential for survival of an organism. Extensive research has been done on a wide spectrum of stress-related diseases and psychiatric disorders, yet further studies into the genetic and neuronal regulation of stress are still required to develop better therapeutics. The zebrafish provides a powerful genetic model to investigate the neural underpinnings of stress, as there exists a large collection of mutant and transgenic lines. Moreover, pharmacology can easily be applied to zebrafish, as most drugs can be added directly to water. We describe here the use of the 'novel tank test' as a method to study innate stress responses in zebrafish, and demonstrate how potential anxiolytic drugs can be validated using the assay. The method can easily be coupled with zebrafish lines harboring genetic mutations, or those in which transgenic approaches for manipulating precise neural circuits are used. The assay can also be used in other fish models. Together, the described protocol should facilitate the adoption of this simple assay to other laboratories.