Duboué, Erik

Animals display a remarkable variety of social behaviors that are necessary for survival. Despite the importance of social behaviors, the neurobiological mechanisms underlying the evolution of such behaviors are largely unknown. The Mexican tetra, Astyanax mexicanus, is a powerful model for studying how behaviors evolve, including social behavior. A. mexicanus exists as a schooling surface form and a non-schooling cave form. Here we have utilized this model in order to investigate how differences in the behavior of individuals result in differences at the level of emergent group social behaviors. We begin by reviewing how fish have contributed to the study of social behavior in Chapter 1, then continue to dissect differences in the schooling and shoaling behavior of adult surface and cave fish in Chapter 2, and finally address ontogenic differences that result in these differences in Chapter 3. All-in-all this, work reveals how evolution may act on the behavior of individuals to produce differences in relevant group behaviors.

A conserved and important form of variability within and across species is behavioral individuality or the inter-individual variation in behavioral responses to similar stimuli. Although there is extensive research on the neurobiological basis of individuality, little is known about how individualistic behaviors are imposed on the brain and the mechanisms by which they change through evolution. A considerable impediment to addressing this question has been the lack of a suitable model organism with naturally occurring differences in behavior and physiology which can be manipulated experientially. Astyanax mexicanus is an emerging vertebrate model in evolutionary biology comprising two same-species morphological types. This system shares neuronal and molecular homology with zebrafish in which turning bias, an individualistic behavior, has been established. Accordingly, A. mexicanus can be used to determine how individuality is imposed on the brain and how the evolution of distinct brain loci alters individuality at the behavioral and neurological levels.

Fluorescent in-situ hybridization chain reaction (HCR-RNA FISH) is a technique that allows for the simultaneous labeling of multiple target mRNA molecules, signal quantification, and high-resolution oligonucleotide visualization. While this method has been employed to study several common laboratory model systems, its application to non-traditional models is lacking. With the goals of circumventing this limitation and introducing Hybridization Chain Reaction (v3.0) (HCR), the latest version of a new technique for high resolution in situ hybridization, to the Astyanax mexicanus model, we have optimized a novel protocol for this robust evolutionary system. The optimization of HCR in Astyanax mexicanus strengthens the reproducibility of this staining method across a larger number of model organisms while opening the doors to the molecular physiology and genetic study of an insufficiently explored yet promising biological system.

The Mexican cavefish, Astyanax mexicanus, exists as a surface-dwelling form and multiple cave-dwelling populations that have converged on a series of behavioral and morphological changes. Previous studies identifying differences in hypothalamic neuropeptides in cave populations compared to surface fish call for further investigation of neuropeptides within the cavefish hypothalamus. Galanin is a hypothalamic neuropeptide that has been implicated in multiple behaviors across the teleost genus and has yet to be investigated in A. mexicanus. This study aimed to label and quantify galanin in the brain of 6 dpf Pachón cavefish and surface fish. The number of galanin-positive neurons in the hypothalamus is significantly decreased in Pachón cavefish compared to surface fish. These findings allow for further studies investigating the role of galanin in modulating behavior in A. mexicanus.

Dopamine is an essential component in the neural pathway for attractive and aversive behavior. Dopaminergic (DA) neurons are known to have a key role in neurotransmission which can result in the modulation of different behaviors as well as the manifestation of different mental health disorders. Drosophila share similar genetics that are associated with several neurodegenerative diseases and disorders in humans. Furthermore, previous studies have shown conservation of DA neurons between humans and Drosophila which facilitate research using Drosophila as a model organism. In this study, we initially developed and tested a novel optogenetics system, which targeted neurons with spatial specificity, that activated or inhibited neurons through channelrhodopsin microbial opsins that are sensitive to red light. This system was then used to investigate the DA subsets that mediate attractive and aversive behavior. The activation of PPL1 clusters mostly resulted in aversive behavior as aligned with the literature, however activation of clusters with output neurons (PPL1 & PAM) concluded with different results.

Humans subjected to childhood trauma are more likely to develop anxiety disorders such as post-traumatic stress disorder, yet how Early-Life-Stress (ELS) impacts the function, or the development of the nervous system remains poorly understood. We developed a zebrafish model of ELS. Because of the powerful genetics and accessibility to the brain, the zebrafish is an excellent system to explore how ELS alters brain development and function. The neuroendocrine stress system in zebrafish is mediated in part by the hypothalamic–pituitary–interrenal (HPI) axis, which is analogous to mammalian hypothalamic—pituitary—adrenal (HPA) axis. When perceiving stress, the zebrafish hypothalamus releases corticotropin-releasing hormone, which signals to the pituitary gland to release adrenocorticotropic releasing hormone (acth). Acth then signals down to the interrenal gland, which in-turn secretes cortisol. Cortisol then binds to glucocorticoid and mineralocorticoid receptors in the brain, to mediate stress responses and inhibit the HPA/HPI. We hypothesize that in zebrafish larvae that the ELS may alter the relative expression of genes in the HPI pathway. We generated ELS zebrafish larvae and measured the relative expression levels of corticotropic releasing hormone (crh), glucocorticoid receptor (gr), and mineralocorticoid receptor (mr). We find that the level of expression of crh is modestly higher in ELS zebrafish, which is consistent with higher levels of stress. Moreover, the expression level of gr is higher in ELS zebrafish, while no significant differences in the expression level of mr were found. This data suggests that ELS may disrupt the normal gr:mr ratio in zebrafish subjected to ELS. We are following up on these studies by localizing which neuronal regions are most affected via in situ hybridization.

Stress responses are conserved reactions exhibited by most animal taxa. Enduring stress can lead to changes in behaviors such as sleep and feeding. We have developed the larval zebrafish as a model for investigating neuronal mechanisms underlying stress. A mild electric shock in larval zebrafish induces changes in behavior, particularly affecting feeding. We found that when larvae are exposed to random period of shock over a 5-day period during early life (2-6 days post fertilization), they display increased feeding later in life as juveniles. We have quantified the total brine shrimp consumption of juveniles and found that those subjected to chronic stress have a 2-fold increase in total intake. To probe the neuronal circuits underlying these changes, we have focused on the hypothalamus. We are currently testing the hypothesis that increased feeding may be caused by altered activity of neurons producing agouti-related protein (agrp).

Brain atlases have been created across species from flies to humans in order to obtain a better understanding of neuroanatomical morphology. Although these brain atlases allow for analysis of neuroanatomy they do not give insight about how the morphology adapt to fit challenges brought on by unique environments. Here I developed a brain atlas for Astyanax mexicanus, a species known to have populations that differ in various behaviors, to gain a better understanding about how populations of the same species, derived from different environments, evolve to be best suited for the challenges they face. By creating a brain atlas for adult surface fish and three populations of cavefish I was able to examine differences in neuroanatomical structures implicated in regulating behavior. My findings show significant differences in neuroanatomical regions known to regulate behavior. Along with these findings, the brain atlases created are a tool for researches to use and expand on in the future.

Behaviors are complex processes that are constantly evolving. Although little is known about the genetic and neuronal mechanisms underlying the evolution of behavior, studying the brain may be useful in addressing this. The Mexican Cavefish, Astyanax mexicanus, is a species with populations that inhabit surface rivers as well as caves. A. mexicanus populations are excellent models for convergent and parallel evolution. Using morphometric analysis software, we investigated the differences in brain morphology between the two morphs. The cave populations exhibit a smaller optic tectum volume, which is consistent with their loss of eyes, a larger hypothalamus, and an increased size of the olfactory bulb volume. Each of these regions have known roles in different behaviors, which differ between cave and surface fish. These findings provide insight into how evolution can affect behavior by changing neuroanatomical structure.