A protocol for whole-brain Ca2+ imaging in Astyanax mexicanus, a model of comparative evolution.

In this protocol, we describe a comparative approach to study the evolution of brain function in the Mexican tetra, Astyanax mexicanus. We developed surface fish and two independent populations of cavefish with pan-neuronal expression of the Ca2+ sensor GCaMP6s. We describe a methodology to prepare samples and image activity across the optic tectum and olfactory bulb.

Changes in local interaction rules during ontogeny underlie the evolution of collective behavior.

Collective motion emerges from individual interactions which produce group-wide patterns in behavior. While adaptive changes to collective motion are observed across animal species, how local interactions change when these collective behaviors evolve is poorly understood. Here, we use the Mexican tetra, Astyanax mexicanus, which exists as a schooling surface form and a non-schooling cave form, to study differences in how fish alter their swimming in response to neighbors across ontogeny and between evolutionarily diverged populations. We find that surface fish undergo a transition to schooling mediated by changes in the way fish modulate speed and turning relative to neighbors. This transition begins with the tendency to align to neighbors emerging by 28 days post-fertilization and ends with the emergence of robust attraction by 70 days post-fertilization. Cavefish exhibit neither alignment nor attraction at any stage of development. These results reveal how evolution alters local interactions to produce striking differences in collective behavior.

A brain-wide analysis maps structural evolution to distinct anatomical module.

The vertebrate brain is highly conserved topologically, but less is known about neuroanatomical variation between individual brain regions. Neuroanatomical variation at the regional level is hypothesized to provide functional expansion, building upon ancestral anatomy needed for basic functions. Classically, animal models used to study evolution have lacked tools for detailed anatomical analysis that are widely used in zebrafish and mice, presenting a barrier to studying brain evolution at fine scales. In this study, we sought to investigate the evolution of brain anatomy using a single species of fish consisting of divergent surface and cave morphs, that permits functional genetic testing of regional volume and shape across the entire brain. We generated a high-resolution brain atlas for the blind Mexican cavefish Astyanax mexicanus and coupled the atlas with automated computational tools to directly assess variability in brain region shape and volume across all populations. We measured the volume and shape of every grossly defined neuroanatomical region of the brain and assessed correlations between anatomical regions in surface fish, cavefish, and surface × cave F2 hybrids, whose phenotypes span the range of surface to cave. We find that dorsal regions of the brain are contracted, while ventral regions have expanded, with F2 hybrid data providing support for developmental constraint along the dorsal-ventral axis. Furthermore, these dorsal-ventral relationships in anatomical variation show similar patterns for both volume and shape, suggesting that the anatomical evolution captured by these two parameters could be driven by similar developmental mechanisms. Together, these data demonstrate that A. mexicanus is a powerful system for functionally determining basic principles of brain evolution and will permit testing how genes influence early patterning events to drive brain-wide anatomical evolution.

Novel Husbandry Practices Result in Rapid Rates of Growth and Sexual Maturation Without Impacting Adult Behavior in the Blind Mexican Cavefish.

Animal model systems are dependent on the standardization of husbandry protocols that maximize growth and reduce generation time. The Mexican tetra, Astyanax mexicanus, exists as eyed surface and blind cave dwelling populations. The opportunity for comparative approaches between independently evolved populations has led to the rapid growth of A. mexicanus as a model for evolution and biomedical research. However, a slow and inconsistent growth rate remains a major limitation to the expanded application of A. mexicanus. Fortunately, this temporal limitation can be addressed through husbandry changes that accelerate growth rates while maintaining optimal health outcomes. Here, we describe a husbandry protocol that produces rapid growth rates through changes in diet, feeding frequency, growth sorting and progressive changes in tank size. This protocol produced robust growth rates and decreased the age of sexual maturity in comparison to our previous protocol. To determine whether changes in feeding impacted behavior, we tested fish in exploration and schooling assays. We found no difference in behavior between the two groups, suggesting that increased feeding and rapid growth will not impact the natural variation in behavioral traits. Taken together, this standardized husbandry protocol will accelerate the development of A. mexicanus as a genetic model.

Changes in local interaction rules during ontogeny underlie the evolution of collective behavior.

Collective motion emerges from individual interactions which produce groupwide patterns in behavior. While adaptive changes to collective motion are observed across animal species, how local interactions change when these collective behaviors evolve is poorly understood. Here, we use the Mexican tetra, A. mexicanus, which exists as a schooling surface form and a non-schooling cave form, to study differences in how fish alter their swimming in response to neighbors across ontogeny and between evolutionarily diverged populations. We find that surface fish undergo a transition to schooling during development that occurs through increases in inter-individual alignment and attraction mediated by changes in the way fish modulate speed and turning relative to neighbors. Cavefish, which have evolved loss of schooling, exhibit neither of these schooling-promoting interactions at any stage of development. These results reveal how evolution alters local interaction rules to produce striking differences in collective behavior.

Convergence on reduced aggression through shared behavioral traits in multiple populations of Astyanax mexicanus.

BACKGROUND: Aggression is observed across the animal kingdom, and benefits animals in a number of ways to increase fitness and promote survival. While aggressive behaviors vary widely across populations and can evolve as an adaptation to a particular environment, the complexity of aggressive behaviors presents a challenge to studying the evolution of aggression. The Mexican tetra, Astyanax mexicanus exists as an aggressive river-dwelling surface form and multiple populations of a blind cave form, some of which exhibit reduced aggression, providing the opportunity to investigate how evolution shapes aggressive behaviors. RESULTS: To define how aggressive behaviors evolve, we performed a high-resolution analysis of multiple social behaviors that occur during aggressive interactions in A. mexicanus. We found that many of the aggression-associated behaviors observed in surface-surface aggressive encounters were reduced or lost in Pachón cavefish. Interestingly, one behavior, circling, was observed more often in cavefish, suggesting evolution of a shift in the types of social behaviors exhibited by cavefish. Further, detailed analysis revealed substantive differences in aggression-related sub-behaviors in independently evolved cavefish populations, suggesting independent evolution of reduced aggression between cave populations. We found that many aggressive behaviors are still present when surface fish fight in the dark, suggesting that these reductions in aggression-associated and escape-associated behaviors in cavefish are likely independent of loss of vision in this species. Further, levels of aggression within populations were largely independent of type of opponent (cave vs. surface) or individual stress levels, measured through quantifying stress-like behaviors, suggesting these behaviors are hardwired and not reflective of population-specific changes in other cave-evolved traits. CONCLUSION: These results reveal that loss of aggression in cavefish evolved through the loss of multiple aggression-associated behaviors and raise the possibility that independent genetic mechanisms underlie changes in each behavior within populations and across populations. Taken together, these findings reveal the complexity of evolution of social behaviors and establish A. mexicanus as a model for investigating the evolutionary and genetic basis of aggressive behavior.

Course-based undergraduate research experiences (CURES) as a pathway to diversify science.

There is widespread recognition of the need to increase research opportunities in biomedical science for undergraduate students from underrepresented backgrounds. Here, we describe the implementation of team-based science combined with intensive mentoring to conduct a large-scale project examining the evolution of behavior. This system can be widely applied in other areas of STEM to promote research-intensive opportunities in STEM fields and to promote diversity in science.

Characterizing the genetic basis of trait evolution in the Mexican cavefish.

Evolution in response to a change in ecology often coincides with various morphological, physiological, and behavioral traits. For most organisms little is known about the genetic and functional relationship between evolutionarily derived traits, representing a critical gap in our understanding of adaptation. The Mexican tetra, Astyanax mexicanus, consists of largely independent populations of fish that inhabit at least 30 caves in Northeast Mexico, and a surface fish population, that inhabit the rivers of Mexico and Southern Texas. The recent application of molecular genetic approaches combined with behavioral phenotyping have established A. mexicanus as a model for studying the evolution of complex traits. Cave populations of A. mexicanus are interfertile with surface populations and have evolved numerous traits including eye degeneration, insomnia, albinism, and enhanced mechanosensory function. The interfertility of different populations from the same species provides a unique opportunity to define the genetic relationship between evolved traits and assess the co-evolution of behavioral and morphological traits with one another. To define the relationships between morphological and behavioral traits, we developed a pipeline to test individual fish for multiple traits. This pipeline confirmed differences in locomotor activity, prey capture, and startle reflex between surface and cavefish populations. To measure the relationship between traits, individual F2 hybrid fish were characterized for locomotor behavior, prey-capture behavior, startle reflex, and morphological attributes. Analysis revealed an association between body length and slower escape reflex, suggesting a trade-off between increased size and predator avoidance in cavefish. Overall, there were few associations between individual behavioral traits, or behavioral and morphological traits, suggesting independent genetic changes underlie the evolution of the measured behavioral and morphological traits. Taken together, this approach provides a novel system to identify genetic underpinnings of naturally occurring variation in morphological and behavioral traits.

Blind cavefish retain functional connectivity in the tectum despite loss of retinal input.

Sensory systems display remarkable plasticity and are under strong evolutionary selection. The Mexican cavefish, Astyanax mexicanus, consists of eyed river-dwelling surface populations and multiple independent cave populations that have converged on eye loss, providing the opportunity to examine the evolution of sensory circuits in response to environmental perturbation. Functional analysis across multiple transgenic populations expressing GCaMP6s showed that functional connectivity of the optic tectum largely did not differ between populations, except for the selective loss of negatively correlated activity within the cavefish tectum, suggesting positively correlated neural activity is resistant to an evolved loss of input from the retina. Furthermore, analysis of surface-cave hybrid fish reveals that changes in the tectum are genetically distinct from those encoding eye loss. Together, these findings uncover the independent evolution of multiple components of the visual system and establish the use of functional imaging in A. mexicanus to study neural circuit evolution.

Long lasting anxiety following early life stress is dependent on glucocorticoid signaling in zebrafish.

Chronic adversity in early childhood is associated with increased anxiety and a propensity for substance abuse later in adulthood, yet the effects of early life stress (ELS) on brain development remain poorly understood. The zebrafish, Danio rerio, is a powerful model for studying neurodevelopment and stress. Here, we describe a zebrafish model of ELS and identify a role for glucocorticoid signaling during a critical window in development that leads to long-term changes in brain function. Larval fish subjected to chronic stress in early development exhibited increased anxiety-like behavior and elevated glucocorticoid levels later in life. Increased stress-like behavior was only observed when fish were subjected to ELS within a precise time window in early development, revealing a temporal critical window of sensitivity. Moreover, enhanced anxiety-like behavior only emerges after two months post-ELS, revealing a developmentally specified delay in the effects of ELS. ELS leads to increased levels of baseline cortisol, and resulted in a dysregulation of cortisol receptors' mRNA expression, suggesting long-term effects on cortisol signaling. Together, these findings reveal a 'critical window' for ELS to affect developmental reprogramming of the glucocorticoid receptor pathway, resulting in chronic elevated stress.

Kinematic analysis of social interactions deconstructs the evolved loss of schooling behavior in cavefish.

Fish display a remarkable diversity of social behaviors, both within and between species. While social behaviors are likely critical for survival, surprisingly little is known about how they evolve in response to changing environmental pressures. With its highly social surface form and multiple populations of a largely asocial, blind, cave-dwelling form, the Mexican tetra, Astyanax mexicanus, provides a powerful model to study the evolution of social behavior. Here we use motion tracking and analysis of swimming kinematics to quantify social swimming in four Astyanax mexicanus populations. In the light, surface fish school, maintaining both close proximity and alignment with each other. In the dark, surface fish no longer form coherent schools, however, they still show evidence of an attempt to align and maintain proximity when they find themselves near another fish. In contrast, cavefish from three independently-evolved populations (Pachón, Molino, Tinaja) show little preference for proximity or alignment, instead exhibiting behaviors that suggest active avoidance of each other. Two of the three cave populations we studied also slow down when more fish are present in the tank, a behavior which is not observed in surface fish in light or the dark, suggesting divergent responses to conspecifics. Using data-driven computer simulations, we show that the observed reduction in swimming speed is sufficient to alter the way fish explore their environment: it can increase time spent exploring away from the walls. Thus, the absence of schooling in cavefish is not merely a consequence of their inability to see, but may rather be a genuine behavioral adaptation that impacts the way they explore their environment.

Specialized neurons in the right habenula mediate response to aversive olfactory cues.

Hemispheric specializations are well studied at the functional level but less is known about the underlying neural mechanisms. We identified a small cluster of cholinergic neurons in the dorsal habenula (dHb) of zebrafish, defined by their expression of the lecithin retinol acyltransferase domain containing 2 a (lratd2a) gene and their efferent connections with a subregion of the ventral interpeduncular nucleus (vIPN). The lratd2a-expressing neurons in the right dHb are innervated by a subset of mitral cells from both the left and right olfactory bulb and are activated upon exposure to the odorant cadaverine that is repellent to adult zebrafish. Using an intersectional strategy to drive expression of the botulinum neurotoxin specifically in these neurons, we find that adults no longer show aversion to cadaverine. Mutants with left-isomerized dHb that lack these neurons are also less repelled by cadaverine and their behavioral response to alarm substance, a potent aversive cue, is diminished. However, mutants in which both dHb have right identity appear more reactive to alarm substance. The results implicate an asymmetric dHb-vIPN neural circuit in the processing of repulsive olfactory cues and in modulating the resultant behavioral response.

Trans-inhibition of axon terminals underlies competition in the habenulo-interpeduncular pathway.

Survival of animals is dependent on the correct selection of an appropriate behavioral response to competing external stimuli. Theoretical models have been proposed and underlying mechanisms are emerging to explain how one circuit is selected among competing neural circuits. The evolutionarily conserved forebrain to midbrain habenulo-interpeduncular nucleus (Hb-IPN) pathway consists of cholinergic and non-cholinergic neurons, which mediate different aversive behaviors. Simultaneous calcium imaging of neuronal cell bodies and of the population dynamics of their axon terminals reveals that signals in the cell bodies are not reflective of terminal activity. We find that axon terminals of cholinergic and non-cholinergic habenular neurons exhibit stereotypic patterns of spontaneous activity that are negatively correlated and localize to discrete subregions of the target IPN. Patch-clamp recordings show that calcium bursts in cholinergic terminals at the ventral IPN trigger excitatory currents in IPN neurons, which precede inhibition of non-cholinergic terminals at the adjacent dorsal IPN. Inhibition is mediated through presynaptic GABAB receptors activated in non-cholinergic habenular neurons upon GABA release from the target IPN. Together, the results reveal a hardwired mode of competition at the terminals of two excitatory neuronal populations, providing a physiological framework to explore the relationship between different aversive responses.

Cavefish brain atlases reveal functional and anatomical convergence across independently evolved populations.

Environmental perturbation can drive behavioral evolution and associated changes in brain structure and function. The Mexican fish species, Astyanax mexicanus, includes eyed river-dwelling surface populations and multiple independently evolved populations of blind cavefish. We used whole-brain imaging and neuronal mapping of 684 larval fish to generate neuroanatomical atlases of surface fish and three different cave populations. Analyses of brain region volume and neural circuits associated with cavefish behavior identified evolutionary convergence in hindbrain and hypothalamic expansion, and changes in neurotransmitter systems, including increased numbers of catecholamine and hypocretin/orexin neurons. To define evolutionary changes in brain function, we performed whole-brain activity mapping associated with behavior. Hunting behavior evoked activity in sensory processing centers, while sleep-associated activity differed in the rostral zone of the hypothalamus and tegmentum. These atlases represent a comparative brain-wide study of intraspecies variation in vertebrates and provide a resource for studying the neural basis of behavioral evolution.

Evolution of the acoustic startle response of Mexican cavefish.

The ability to detect threatening stimuli and initiate an escape response is essential for survival and under stringent evolutionary pressure. In diverse fish species, acoustic stimuli activate Mauthner neurons, which initiate a C-start escape response. This reflexive behavior is highly conserved across aquatic species and provides a model for investigating the neural mechanism underlying the evolution of escape behavior. Here, we characterize evolved differences in the C-start response between populations of the Mexican cavefish, Astyanax mexicanus. Cave populations of A. mexicanus inhabit an environment devoid of light and macroscopic predators, resulting in evolved differences in various morphological and behavioral traits. We find that the C-start is present in river-dwelling surface fish and multiple populations of cavefish, but that response kinematics and probability differ between populations. The Pachón population of cavefish exhibits an increased response probability, a slower response latency and speed, and reduction of the maximum bend angle, revealing evolved differences between surface and cave populations. Analysis of the responses of two other independently evolved populations of cavefish, revealed the repeated evolution of reduced angular speed. Investigation of surface-cave hybrids reveals a correlation between angular speed and peak angle, suggesting these two kinematic characteristics are related at the genetic or functional levels. Together, these findings provide support for the use of A. mexicanus as a model to investigate the evolution of escape behavior.

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.

Manipulation of Gene Function in Mexican Cavefish.

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, including eye degeneration, albinism, sleep loss, hyperphagia, and sensory processing. Species of cavefish from around the world display a convergent evolution of morphological and behavioral traits due to shared environmental pressures between different cave systems. Diverse cave species have been studied in the laboratory setting. The Mexican tetra, Astyanax mexicanus, with sighted and blind forms, has provided unique insights into biological and molecular processes underlying the evolution of complex traits and is well-poised as an emerging model system. While candidate genes regulating the evolution of diverse biological processes have been identified in A. mexicanus, the ability to validate a role for individual genes has been limited. The application of transgenesis and gene-editing technology has the potential to overcome this significant impediment and to investigate the mechanisms underlying the evolution of complex traits. Here, we describe a different methodology for manipulating gene expression in A. mexicanus. Approaches include the use of morpholinos, Tol2 transgenesis, and gene-editing systems, commonly used in zebrafish and other fish models, to manipulate gene function in A. mexicanus. These protocols include detailed descriptions of timed breeding procedures, the collection of fertilized eggs, injections, and the selection of genetically modified animals. These methodological approaches will allow for the investigation of the genetic and neural mechanisms underlying the evolution of diverse traits in A. mexicanus.