Harris RM, Kao H-Y, Alarcon JM, Hofmann HA, Fenton AA. Analysis of hippocampal transcriptomic responses to technical and biological perturbations. bioRxiv. 2017.Abstract

Cost-effective next-generation sequencing has made unbiased gene expression analysis possible. Single-neuron gene expression studies may be especially important for understanding nervous system structure and function because of the neuron-specific functionality and plasticity that defines functional neural circuits. Cellular dissociation is a prerequisite technical manipulation for single-cell and single cell-population studies, but the extent to which the cellular dissociation process cells affects neural gene expression has not been determined, nor has it been determined how gene expression is altered by the stress that accompanies many of the behavioral manipulations that are required to study learning and memory and other cognitive functions. Here, we determined to which extent cellular dissociation-induced changes in hippocampal gene expression might confound studies on the behavioral and physiological functions of the hippocampus. We processed tissue punch samples from the dentate gyrus (DG), CA3, and CA1 hippocampus subfields using either a tissue homogenization protocol or a cellular dissociation protocol in preparation for RNA sequencing analysis to evaluate the impact of the tissue preparation. Then, we evaluated the effect of stressful experience and cognitive training on hippocampus subfield specific gene expression and determined to which extent these response overlap with the cellular dissociation response. Finally, we assessed the extent to which the subfield-specific gene expression patterns are consistent with those identified in a recently published hippocampus subfield-specific gene expression database. We report substantial differences in baseline subfield-specific gene expression, that 1% of the hippocampal transcriptome is altered by the process of cellular dissociation, that an even weaker alteration is detected 24 h after stressful experience, and that while these alterations are largely distinct from the subfield specific response of the hippocampus transcriptome to cognitive training, there is nonetheless some important confounding overlap. These findings of the concordant and discordant effects of technical and behavioral manipulations should inform the design of future neural transcriptome studies and thus facilitate a more comprehensive understanding of hippocampal function.

Weitekamp CA, Nguyen J, Hofmann HA. Neuromolecular Regulation of Aggression Differs by Social Role during Joint Territory Defense. Integrative and Comparative Biology. 2017 :1-9.Abstract

In response to a territory intrusion, neighboring males of the African cichlid fish Astatotilapia burtoni engage in aggressive joint territory defense in a manner that depends on their social role. Here, we examine the possible function of several neuroendocrine and neuromodulator pathways previously implicated in the regulation of complex social behavior. We find that the neuromolecular regulation of aggression during joint territory defense is very much dependent on an individual’s role in this context. In neighbors but not in residents, aggression is correlated to gene expression in the medial part of the dorsal telencephalon (area Dm), the putative homolog to the mammalian basolateral amygdala. This correlation is strikingly high for expression of the serotonin receptor 5-HT2c, suggesting the serotonin system is important in regulating context-dependent behavior. Furthermore, by examining candidate gene expression co-variance patterns in area Dm and in the lateral part of the dorsal telencephalon (area Dl), the putative homolog to the mammalian hippocampus, we identify two main patterns: gene expression is co-regulated within, but not across, brain regions, and co-regulation is synergistic rather than antagonistic. Our results highlight the critical effect of social context on both behavior and its neuromolecular basis.

Weitekamp CA, Nguyen J, Hofmann HA. Social context affects behavior, preoptic area geneexpression, and response to D2 receptor manipulationduring territorial defense in a cichlid fish. Genes, Brain and Behavior. 2017.Abstract

Social context often has profound effects on behav-ior, yet the neural and molecular mechanisms whichmediate flexible behavioral responses to different socialenvironments are not well understood. We used theAfrican cichlid fish, Astatotilapia burtoni, to examineaggressive defense behavior across three social contextsrepresenting different motivational states: a reproduc-tive opportunity, a familiar male and a neutral context.To elucidate how differences in behavior across con-texts may be mediated by neural gene expression, weexamined gene expression in the preoptic area, a brainregion known to control male aggressive and sexualbehavior. We show that social context has broad effectson preoptic gene expression. Specifically, we found thatthe expression of genes encoding nonapeptides andsex steroid receptors are upregulated in the familiarmale context. Furthermore, circulating levels of testos-terone and cortisol varied markedly depending on socialcontext. We also manipulated the D2 receptor (D2R) ineach social context, given that it has been implicatedin mediating context-dependent behavior. We foundthat a D2R agonist reduced intruder-directed aggressionin the reproductive opportunity and familiar male con-texts, while a D2R antagonist inhibited intruder-directedaggression in the reproductive opportunity context andincreased aggression in the neutral context. Our resultsdemonstrate a critical role for preoptic gene expression,as well as circulating steroid hormone levels, in encodinginformation from the social environment and in shap-ing adaptive behavior. In addition, they provide furtherevidence for a role of D2R in context-dependent behavior.

Liebeskind BJ, Hofmann HA, Hillis DA, Zakon HH. Evolution of Animal Neural Systems. Annual Review of Ecology, Evolution, and Systematics. 2017. liebeskind_et_al._2017.pdf
Dijkstra PD, Maguire SM, Harris RM, Rodriguez AA, DeAngelis RS, Flores SA, Hofmann HA. The melanocortin system regulates body pigmentation and social behaviour in the cichlid fish Astatotilapia burtoni. Proc R Soc B. 2017. dijkstra_et_al._2017.pdf
Baker MR, Hofmann HA, Wong RY. Neurogenomic mechanisms of behavioral plasticity within and across socioecological contexts. In: Encyclopedia of Life Sciences. Chichester: John Wiley and Sons, Ltd. ; 2017.
Weitekamp CA, Solomon-Lane TK, Del Valle P, Triki Z, Nugent BM, Hofmann HA. A role for oxytocin-like receptor in social habituation in a teleost. Brain, Behavior, and Evolution. 2017.
Harris RM, O'Connell LA, Hofmann HA. Chapter 15: Brain Evolution, Development, and Plasticity. In: Shepherd SV The Wiley Handbook of Evolutionary Neuroscience. 1st ed. John Wiley & Sons, Ltd ; 2017. pp. 422-443. harris_et_al_2017.pdf
Weitekamp CA, Hofmann HA. Neuromolecular correlates of cooperation and conflict during territory defense in a cichlid fish. Hormones and Behavior [Internet]. 2017;89 :145-156. Publisher's VersionAbstract

Cooperative behavior is widespread among animals, yet the neural mechanisms have not been studied in detail. We examined cooperative territory defense behavior and associated neural activity in candidate forebrain regions in the cichlid fish, Astatotilapia burtoni. We find that a territorial male neighbor will engage in territory defense dependent on the perceived threat of the intruder. The resident male, on the other hand, engages in defense based on the size and behavior of his partner, the neighbor. In the neighbor, we find that an index of engagement correlates with neural activity in the putative homolog of the mammalian basolateral amygdala and in the preoptic area, as well as in preoptic dopaminergic neurons. In the resident, neighbor behavior is correlated with neural activity in the homolog of the mammalian hippocampus. Overall, we find distinct neural activity patterns between the neighbor and the resident, suggesting that an individual perceives and processes an intruder challenge differently during cooperative territory defense depending on its own behavioral role.

Weitekamp CA, Hofmann HA. Brain Systems Underlying Social Behavior. In: Kaas J Evolution of Nervous Systems. Vol. 1. 2nd ed. Oxford: Elsevier ; 2017. pp. 327-334.Abstract

Recent progress in animal behavior research, based on the insight that proximate mechanisms both shape and constrain behavioral responses to natural and sexual selection, has reinforced the importance of knowing the neuromolecular basis of social behavior for understanding its evolution. Here, we review the current state of knowledge of the neural substrates of vertebrate social behavior, with an emphasis on the neuroendocrine and neurochemical pathways involved. Using an integrative perspective, we then discuss the evolution of these mechanisms and highlight several challenges that have hampered progress in this area. Finally, we provide a road map for an integrative evolutionary neuroethology.

Renn SCP, O'Rourke CF, Aubin-Horth N, Fraser EJ, Hofmann HA. Dissecting the transcriptional patterns of social dominance across teleosts. Integrative and Comparative Biology [Internet]. 2016;56 :1250-1265. Publisher's VersionAbstract

In many species, under varying ecological conditions, social interactions among individuals result in the formation of dominance hierarchies. Despite general similarities, there are robust differences among dominance hierarchies across species, populations, environments, life stages, sexes, and individuals. Understanding the proximate mechanisms underlying the variation is an important step toward understanding the evolution of social behavior. However, physiological changes associated with dominance, such as gonadal maturation and somatic growth, often complicate efforts to identify the specific underlying mechanisms. Traditional gene expression analyses are useful for generating candidate gene lists, but are biased by choice of significance cut-offs and difficult to use for between-study comparisons. In contrast, complementary analysis tools allow one to both test a priori hypotheses and generate new hypotheses. Here we employ a meta-analysis of high-throughput expression profiling experiments to investigate the gene expression patterns that underlie mechanisms and evolution of behavioral social phenotypes. Specifically, we use a collection of datasets on social dominance in fish across social contexts, sex, and species. Using experimental manipulation to produce female dominance hierarchies in the cichlid Astatotilapia burtoni, heralded as a genomic model of social dominance, we generate gene lists, and assess molecular gene modules. In the dominant female gene expression profile, we demonstrate a strong pattern of up-regulation of genes previously identified as having male-biased expression and furthermore, compare expression biases between male and female dominance phenotypes. Using a threshold-free approach to identify correlation throughout ranked gene lists, we query previously published datasets associated with maternal behavior, alternative reproductive tactics, cooperative breeding, and sex-role reversal to describe correlations among these various neural gene expression profiles associated with different instances of social dominance. These complementary approaches capitalize on the high-throughput gene expression profiling from similar behavioral phenotypes in order to address the mechanisms associated with social dominance behavioral phenotypes.

Nishi R, Castaneda E, Davis GW, Fenton AA, Hofmann HA, King J, Ryan TA, Trujillo KA. The Global Challenge in Neuroscience Education and Training: The MBL Perspective. Neuron [Internet]. 2016;92 :632-636. Publisher's VersionAbstract

The greatest challenge in moving neuroscience research forward in the 21st century is recruiting, training, and retaining the brightest, rigorous, and most diverse scientists. The MBL research training courses Neurobiology and Neural Systems & Behavior, and the Summer Program in Neuroscience, Excellence, and Success provide a model for full immersion, discovery-based training while enhancing cultural, geographic, and racial diversity.

Goppert C, Harris RM, Theis A, Boila A, Hohl S, Ruegg A, Hofmann HA, Salzburger W, Bohne A. Inhibition of Aromatase Induces Partial Sex Change in a Cichlid Fish: Distinct Functions for Sex Steroids in Brains and Gonads. Sexual Development [Internet]. 2016;10 :97-110. Publisher's VersionAbstract

 Sex steroids are major drivers of sexual development and also responsible for the maintenance of the established gender. Especially fishes exhibit great plasticity and less conservation in sex determination and sexual development compared to other vertebrate groups. In addition, fishes have a constant sex steroid production throughout their entire lifespan, which makes them particularly susceptible to interferences with the endogenous sex steroid system. This susceptibility has recently been used to show that inhibition of the key enzyme of estrogen synthesis, aromatase Cyp19a1, can induce functional sex reversal even in adult fish. Here, we investigated the impact of the aromatase inhibitor (AI) fadrozole in adult females of the East African cichlid fish Astatotilapia burtoni. Using gene expression, phenotypic measurements, behavioral experiments, and hormone measurements, we assessed if females treated with fadrozole develop a male-like phenotype. We found that AI treatment has a different effect on gene expression in the gonad compared to the brain, the 2 tissues mostly implicated in sexual development. In contrast to observations in other gonochoristic species, A. burtoni ovaries cannot be transformed into functional testis by AI. However, rapid changes towards a male-like phenotype can be induced with AI in coloration, hormone levels, and behavior. 

Nugent BM, Stiver KA, Alonzo SH, Hofmann HA. Neuroendocrine profiles associated with discrete behavioural variation in Symphodus ocellatus, a species with male alternative reproductive tactics. Molecular Ecology [Internet]. 2016;25 (20) :5212-5227. Publisher's VersionAbstract

The molecular mechanisms underlying phenotypic plasticity are not well understood. Identifying mechanisms underlying alternative reproductive tactics (ARTs) in species for which the behavioural and fitness consequences of this variation are well characterized provides an opportunity to integrate evolutionary and mechanistic understanding of the maintenance of variation within populations. In the ocellated wrasse Symphodus ocellatus, the behavioural phenotypes of three distinct male morphs (sneakers, satellites and nesting males), which arise from a single genome, have been thoroughly characterized. To determine the neuroendocrine and genomic mechanisms associated with discrete phenotypic variation and ARTs in S. ocellatus in their natural environment, we constructed a whole-brain de novo transcriptome and compared global patterns of gene expression between sexes and male morphs. Next, we quantified circulating cortisol and 11-ketotestosterone (11-kt), mediators of male reproductive behaviours, as well as stress and gonadal steroid hormone receptor expression in the preoptic area, ventral subpallial division of the telencephalon and dorsolateral telencephalon, critical brain regions for social and reproductive behaviours. We found higher levels of 11-kt in nesting males and higher levels of cortisol in sneaker males relative to other male morphs and females. We also identified distinct patterns of brain region-specific hormone receptor expression between males such that most hormone receptors are more highly expressed in satellites and nesting males relative to sneakers and females. Our results establish the neuroendocrine and molecular mechanisms that underlie ARTs in the wild and provide a foundation for experimentally testing hypotheses about the relationship between neuromolecular processes and reproductive success.

Ockendon NF, O'Connell LA, Bush, Stephen J, Monzonsandoval J, Barnes H, Szekely T, Hofmann HA, Dorus S, Urrutia AO. Optimization of next-generation sequencing transcriptome annotation for species lacking sequenced genomes. Molecular Ecology Resources [Internet]. 2016;16 :446-458. Publisher's VersionAbstract

Next-generation sequencing methods, such as RNA-seq, have permitted the exploration of gene expression in a rangeof organisms which have been studied in ecological contexts but lack a sequenced genome. However, the efficacyand accuracy of RNA-seq annotation methods using reference genomes from related species have yet to be robustlycharacterized. Here we conduct a comprehensive power analysis employing RNA-seq data from Drosophila melano-gaster in conjunction with 11 additional genomes from related Drosophila species to compare annotation methodsand quantify the impact of evolutionary divergence between transcriptome and the reference genome. Our analysesdemonstrate that, regardless of the level of sequence divergence, direct genome mapping (DGM), where transcriptshort reads are aligned directly to the reference genome, significantly outperforms the widely used de novo andguided assembly-based methods in both the quantity and accuracy of gene detection. Our analysis also reveals thatDGM recovers a more representative profile of Gene Ontology functional categories, which are often used to inter-pret emergent patterns in genomewide expression analyses. Lastly, analysis of available primate RNA-seq datademonstrates the applicability of our observations across diverse taxa. Our quantification of annotation accuracy andreduced gene detection associated with sequence divergence thus provides empirically derived guidelines for thedesign of future gene expression studies in species without sequenced genomes.

Jordan LA, Maguire SM, Hofmann HA, Kohda M. The social and ecological costs of an "over-extended" phenotype. Proceedings of the Royal Society B [Internet]. 2016;283 :20152359. Publisher's VersionAbstract

Extended phenotypes offer a unique opportunity to experimentally manipulate and identify sources of selection acting on traits under natural conditions. The social cichlid fish Neolamprologus multifasciatus builds nests by digging up aquatic snail shells, creating an extended sexual phenotype that is highly amenable to experimental manipulation through addition of extra shells. Here, we find sources of both positive sexual selection and opposing natural selection acting on this trait; augmenting shell nests increases access to mates, but also increases social aggression and predation risk. Increasing the attractiveness of one male also changed social interactions throughout the social network and altered the entire community structure. Manipulated males produced and received more displays from neighbouring females, who also joined augmented male territories at higher rates than unmanipulated groups. However, males in more attractive territories received more aggression from neighbouring males, potentially as a form of social policing. We also detected a significant ecological cost of the ‘over-extended' phenotype; heterospecific predators usurped augmented nests at higher rates, using them as breeding sites and displacing residents. Using these natural experiments, we find that both social and ecological interactions generate clear sources of selection mediating the expression of an extended phenotype in the wild.

Liebeskind BJ, Hillis DM, Zakon HH, Hofmann HA. Complex Homology and the Evolution of Nervous Systems. Trends in Ecology and Evolution [Internet]. 2016;31 (2) :127 - 135. Publisher's VersionAbstract

We examine the complex evolution of animal nervous systems and discuss the ramifications of this complexity for inferring the nature of early animals. Although reconstructing the origins of nervous systems remains a central challenge in biology, and the phenotypic complexity of early animals remains controversial, a compelling picture is emerging. We now know that the nervous system and other key animal innovations contain a large degree of homoplasy, at least on the molecular level. Conflicting hypotheses about early nervous system evolution are due primarily to differences in the interpretation of this homoplasy. We highlight the need for explicit discussion of assumptions and discuss the limitations of current approaches for inferring ancient phenotypic states.

Hofmann HA, Renn SCP, Rubenstein DR. New Frontiers in the Integrative Study of Animal Behavior: Nothing in Neuroscience Makes Sense Except in the Light of Behavior. Integrative and Comparative Biology [Internet]. 2016 :icw127 - icw127. Publisher's Version 2016.hofmann.icb_.pdf
Smith CC, Harris RM, Lampert KP, Schartl M, Hofmann HA, Ryan MJ. Copy number variation in the melanocortin 4 receptor gene and alternative reproductive tactics the swordtail Xiphophorus multilineatus. Environmental Biology of Fishes [Internet]. 2015;98 (1) :23-33. Publisher's VersionAbstract

Alternative reproductive tactics are powerful examples of how variation in genetics and physiology among individuals can lead to striking diversity in phenotype. In the swordtails (genus Xiphophorus), copy number variation (CNV) at the melanocortin 4 receptor (mc4r) locus is correlated with male body size, which in turn is correlated with male mating behavior. We measured the relationship between mc4r CNV, behavior, and 11-ketotesterone (11-KT) in X. multilineatus to determine whether mc4r CNV was associated with other components of male tactics in addition to body size. We confirmed the results of previous studies, showing that male size increases with mc4r CNV and that mating behavior toward females was size-dependent. We also examined agonistic behavior by exposing males to their mirror image and found that male-male displays behavior were size-dependent. Small males were less likely to exhibit an agonistic response, suggesting that alternative reproductive tactics span intrasexual and intersexual contexts. There was no significant association between mc4r CNV and behavior or 11-KT hormone titer. Mc4r CNV is thus associated with the variation in male body size, but not with other traits independent of body size.

Oldfield RG, Harris RM, Hofmann HA. Integrating resource defence theory with a neural nonapeptide pathway to explain territory-based mating systems. Frontiers in Zoology [Internet]. 2015;12(Suppl 1) :S16. Publisher's VersionAbstract

The ultimate-level factors that drive the evolution of mating systems have been well studied, but an evolutionarily conserved neural mechanism involved in shaping behaviour and social organization across species has remained elusive. Here, we review studies that have investigated the role of neural arginine vasopressin (AVP), vasotocin (AVT), and their receptor V1a in mediating variation in territorial behaviour. First, we discuss how aggression and territoriality are a function of population density in an inverted-U relationship according to resource defence theory, and how territoriality influences some mating systems. Next, we find that neural AVP, AVT, and V1a expression, especially in one particular neural circuit involving the lateral septum of the forebrain, are associated with territorial behaviour in males of diverse species, most likely due to their role in enhancing social cognition. Then we review studies that examined multiple species and find that neural AVP, AVT, and V1a expression is associated with territory size in mammals and fishes. Because territoriality plays an important role in shaping mating systems in many species, we present the idea that neural AVP, AVT, and V1a expression that is selected to mediate territory size may also influence the evolution of different mating systems. Future research that interprets proximate-level neuro-molecular mechanisms in the context of ultimate-level ecological theory may provide deep insight into the brain-behaviour relationships that underlie the diversity of social organization and mating systems seen across the animal kingdom.