Paternally induced transgenerational environmental reprogramming of metabolic gene expression in mammals.

Epigenetic information can be inherited through the mammalian germline and represents a plausible transgenerational carrier of environmental information. To test whether transgenerational inheritance of environmental information occurs in mammals, we carried out an expression profiling screen for genes in mice that responded to paternal diet. Offspring of males fed a low-protein diet exhibited elevated hepatic expression of many genes involved in lipid and cholesterol biosynthesis and decreased levels of cholesterol esters, relative to the offspring of males fed a control diet. Epigenomic profiling of offspring livers revealed numerous modest (∼20%) changes in cytosine methylation depending on paternal diet, including reproducible changes in methylation over a likely enhancer for the key lipid regulator Ppara. These results, in conjunction with recent human epidemiological data, indicate that parental diet can affect cholesterol and lipid metabolism in offspring and define a model system to study environmental reprogramming of the heritable epigenome.

Behavioral traits that define social dominance are the same that reduce social influence in a consensus task.

Dominant individuals are often most influential in their social groups, affecting movement, opinion, and performance across species and contexts. Yet, behavioral traits like aggression, intimidation, and coercion, which are associated with and in many cases define dominance, can be socially aversive. The traits that make dominant individuals influential in one context may therefore reduce their influence in other contexts. Here, we examine this association between dominance and influence using the cichlid fish Astatotilapia burtoni, comparing the influence of dominant and subordinate males during normal social interactions and in a more complex group consensus association task. We find that phenotypically dominant males are aggressive, socially central, and that these males have a strong influence over normal group movement, whereas subordinate males are passive, socially peripheral, and have little influence over normal movement. However, subordinate males have the greatest influence in generating group consensus during the association task. Dominant males are spatially distant and have lower signal-to-noise ratios of informative behavior in the association task, potentially interfering with their ability to generate group consensus. In contrast, subordinate males are physically close to other group members, have a high signal-to-noise ratio of informative behavior, and equivalent visual connectedness to their group as dominant males. The behavioral traits that define effective social influence are thus highly context specific and can be dissociated with social dominance. Thus, processes of hierarchical ascension in which the most aggressive, competitive, or coercive individuals rise to positions of dominance may be counterproductive in contexts where group performance is prioritized.

Behavior-related gene regulatory networks: A new level of organization in the brain.

Neuronal networks are the standard heuristic model today for describing brain activity associated with animal behavior. Recent studies have revealed an extensive role for a completely distinct layer of networked activities in the brain-the gene regulatory network (GRN)-that orchestrates expression levels of hundreds to thousands of genes in a behavior-related manner. We examine emerging insights into the relationships between these two types of networks and discuss their interplay in spatial as well as temporal dimensions, across multiple scales of organization. We discuss properties expected of behavior-related GRNs by drawing inspiration from the rich literature on GRNs related to animal development, comparing and contrasting these two broad classes of GRNs as they relate to their respective phenotypic manifestations. Developmental GRNs also represent a third layer of network biology, playing out over a third timescale, which is believed to play a crucial mediatory role between neuronal networks and behavioral GRNs. We end with a special emphasis on social behavior, discuss whether unique GRN organization and cis-regulatory architecture underlies this special class of behavior, and review literature that suggests an affirmative answer.

Neural activity patterns differ between learning contexts in a social fish.

Learning and decision-making are greatly influenced by context. When navigating a complex social world, individuals must quickly ascertain where to gain important resources and which group members are useful sources of such information. Such dynamic behavioural processes require neural mechanisms that are flexible across contexts. Here we examine how the social context influences the learning response during a cue discrimination task and the neural activity patterns that underlie acquisition of this novel information. Using the cichlid fish, Astatotilapia burtoni, we show that learning of the task is faster in social groups than in a non-social context. We quantify the neural activity patterns by examining the expression of Fos, an immediate-early gene, across brain regions known to play a role in social behaviour and learning (such as the putative teleost homologues of the mammalian hippocampus, basolateral amygdala and medial amygdala/BNST complex). We find that neural activity patterns differ between social and non-social contexts. Taken together, our results suggest that while the same brain regions may be involved in the learning of a cue association, the activity in each region reflects an individual's social context.

Vasopressin mediates nonapeptide and glucocorticoid signaling and social dynamics in juvenile dominance hierarchies of a highly social cichlid fish.

Early-life social experience can strongly affect adult behavior, yet the behavioral mechanisms underlying developmental trajectories are poorly understood. Here, we use the highly social cichlid, Burton's Mouthbrooder (Astatotilapia burtoni) to investigate juvenile social status and behavior, as well as the underlying neuroendocrine mechanisms. We placed juveniles in pairs or triads and found that they readily establish social status hierarchies, with some group structural variation depending on group size, as well as the relative body size of the group members. Next, we used intracerebroventricular injections to test the hypothesis that arginine vasopressin (AVP) regulates juvenile social behavior and status, similar to adult A. burtoni. While we found no direct behavioral effects of experimentally increasing (via vasotocin) or decreasing (via antagonist Manning Compound) AVP signaling, social interactions directed at the treated individual were significantly altered. This group-level effect of central AVP manipulation was also reflected in a significant shift in whole brain expression of genes involved in nonapeptide signaling (AVP, oxytocin, and oxytocin receptor) and the neuroendocrine stress axis (corticotropin-releasing factor (CRF), glucocorticoid receptors (GR) 1a and 1b). Further, social status was associated with the expression of genes involved in glucocorticoid signaling (GR1a, GR1b, GR2, mineralocorticoid receptor), social interactions with the dominant fish, and nonapeptide signaling activity (AVP, AVP receptor V1aR2, OTR). Together, our results considerably expand our understanding of the context-specific emergence of social dominance hierarchies in juveniles and demonstrate a role for nonapeptide and stress axis signaling in the regulation of social status and social group dynamics.

Social status mediates behavioral, endocrine, and neural responses to an intruder challenge in a social cichlid, Astatotilapia burtoni.

Most animals encounter social challenges throughout their lives as they compete for resources. Individual responses to such challenges can depend on social status, sex, and community-level attributes, yet most of our knowledge of the behavioral and physiological mechanisms by which individuals respond to challenges has come from dyadic interactions between a resource holder and a challenger (usually both males). To incorporate differences in individual behavior that are influenced by surrounding group members, we use naturalistic communities of the cichlid fish, Astatotilapia burtoni, and examine resident dominant male responses to a territorial intrusion within the social group. We measured behavior and steroid hormones (testosterone and cortisol), and neural activity in key brain regions implicated in regulating territorial and social dominance behavior. In response to a male intruder, resident dominant males shifted from border defense to overt attack behavior, accompanied by decreased basolateral amygdala activity. These differences were context dependent - resident dominant males only exhibited increased border defense when the intruder secured dominance. Neither subordinate males nor females changed their behavior in response to a territorial intrusion in their community. However, neural activity in both hippocampus and lateral septum of subordinates increased when the intruder failed to establish dominance. Our results demonstrate how a social challenge results in multi-faceted behavioral, hormonal, and neural changes, depending on social status, sex, and the outcome of an intruder challenge. Taken together, our work provides novel insights into the mechanisms through which individual group members display context- and status-appropriate challenge responses in dynamic social groups.

Molecular profiling of single neurons of known identity in two ganglia from the crab Cancer borealis.

Understanding circuit organization depends on identification of cell types. Recent advances in transcriptional profiling methods have enabled classification of cell types by their gene expression. While exceptionally powerful and high throughput, the ground-truth validation of these methods is difficult: If cell type is unknown, how does one assess whether a given analysis accurately captures neuronal identity? To shed light on the capabilities and limitations of solely using transcriptional profiling for cell-type classification, we performed 2 forms of transcriptional profiling-RNA-seq and quantitative RT-PCR, in single, unambiguously identified neurons from 2 small crustacean neuronal networks: The stomatogastric and cardiac ganglia. We then combined our knowledge of cell type with unbiased clustering analyses and supervised machine learning to determine how accurately functionally defined neuron types can be classified by expression profile alone. The results demonstrate that expression profile is able to capture neuronal identity most accurately when combined with multimodal information that allows for post hoc grouping, so analysis can proceed from a supervised perspective. Solely unsupervised clustering can lead to misidentification and an inability to distinguish between 2 or more cell types. Therefore, this study supports the general utility of cell identification by transcriptional profiling, but adds a caution: It is difficult or impossible to know under what conditions transcriptional profiling alone is capable of assigning cell identity. Only by combining multiple modalities of information such as physiology, morphology, or innervation target can neuronal identity be unambiguously determined.

Conserved transcriptomic profiles underpin monogamy across vertebrates.

Social monogamy, typically characterized by the formation of a pair bond, increased territorial defense, and often biparental care, has independently evolved multiple times in animals. Despite the independent evolutionary origins of monogamous mating systems, several homologous brain regions and neuropeptides and their receptors have been shown to play a conserved role in regulating social affiliation and parental care, but little is known about the neuromolecular mechanisms underlying monogamy on a genomic scale. Here, we compare neural transcriptomes of reproductive males in monogamous and nonmonogamous species pairs of Peromyscus mice, Microtus voles, parid songbirds, dendrobatid frogs, and Xenotilapia species of cichlid fishes. We find that, while evolutionary divergence time between species or clades did not explain gene expression similarity, characteristics of the mating system correlated with neural gene expression patterns, and neural gene expression varied concordantly across vertebrates when species transition to monogamy. Our study provides evidence of a universal transcriptomic mechanism underlying the evolution of monogamy in vertebrates.

Social network dynamics predict hormone levels and behavior in a highly social cichlid fish.

Group living confers many benefits while simultaneously exposing group members to intense competition. An individual's rise to prominence within a group may conflict with the overall functioning of the group. There is therefore a complex and dynamic relationship between the behavioral displays that directly benefit an individual, the consequences of these actions for the community, and how they feed back on individual-level fitness. We used a network analysis approach to study the link between behavior, social stability, and steroid hormone levels in replicate communities of the cichlid fish, Astatotilapia burtoni, which live in social groups with a dominance hierarchy. We demonstrate that individual behavior can have direct and indirect effects on the behavior of others while also affecting group characteristics. Our results show that A. burtoni males form stable social networks, where dominant individuals act as hubs for social interactions. However, there was variation in the temporal stability in these networks, and this variation in stability impacted hormone levels. Dominant males had higher testosterone levels, however, the differences in testosterone levels between dominant and subordinate males were greatest in stable communities. In sum, our analyses provide novel insights into the processes by which individual and community properties interact.

Equal performance but distinct behaviors: sex differences in a novel object recognition task and spatial maze in a highly social cichlid fish.

Sex differences in behavior and cognition can be driven by differential selection pressures from the environment and in the underlying neuromolecular mechanisms of decision-making. The highly social cichlid fish Astatotilapia burtoni exhibits dynamic and complex social hierarchies, yet explicit cognitive testing (outside of social contexts) and investigations of sex differences in cognition have yet to be fully explored. Here we assessed male and female A. burtoni in two cognitive tasks: a novel object recognition task and a spatial task. We hypothesized that males outperform females in a spatial learning task and exhibit more neophilic/exploratory behavior across both tasks. In the present study we find that both sexes prefer the familiar object in a novel object recognition task, but the time at which they exhibit this preference differs between the sexes. Females more frequently learned the spatial task, exhibiting longer decision latencies and quicker error correction, suggesting a potential speed-accuracy tradeoff. Furthermore, the sexes differ in space use in both tasks and in a principal component analysis of the spatial task. A model selection analysis finds that preference, approach, and interaction duration in the novel object recognition task reach a threshold of importance averaged across all models. This work highlights the need to explicitly test for sex differences in cognition to better understand how individuals navigate dynamic social environments.

Effects of air pollution exposure on social behavior: a synthesis and call for research.

BACKGROUND: There is a growing literature from both epidemiologic and experimental animal studies suggesting that exposure to air pollution can lead to neurodevelopmental and neuropsychiatric disorders. Here, we suggest that effects of air pollutant exposure on the brain may be even broader, with the potential to affect social decision-making in general. METHODS: We discuss how the neurobiological substrates of social behavior are vulnerable to air pollution, then briefly present studies that examine the effects of air pollutant exposure on social behavior-related outcomes. RESULTS: Few experimental studies have investigated the effects of air pollution on social behavior and those that have focus on standard laboratory tests in rodent model systems. Nonetheless, there is sufficient evidence to support a critical need for more research. CONCLUSION: For future research, we suggest a comparative approach that utilizes diverse model systems to probe the effects of air pollution on a wider range of social behaviors, brain regions, and neurochemical pathways.

SArKS: de novo discovery of gene expression regulatory motif sites and domains by suffix array kernel smoothing.

MOTIVATION: We set out to develop an algorithm that can mine differential gene expression data to identify candidate cell type-specific DNA regulatory sequences. Differential expression is usually quantified as a continuous score-fold-change, test-statistic, P-value-comparing biological classes. Unlike existing approaches, our de novo strategy, termed SArKS, applies non-parametric kernel smoothing to uncover promoter motif sites that correlate with elevated differential expression scores. SArKS detects motif k-mers by smoothing sequence scores over sequence similarity. A second round of smoothing over spatial proximity reveals multi-motif domains (MMDs). Discovered motif sites can then be merged or extended based on adjacency within MMDs. False positive rates are estimated and controlled by permutation testing. RESULTS: We applied SArKS to published gene expression data representing distinct neocortical neuron classes in Mus musculus and interneuron developmental states in Homo sapiens. When benchmarked against several existing algorithms using a cross-validation procedure, SArKS identified larger motif sets that formed the basis for regression models with higher correlative power. AVAILABILITY AND IMPLEMENTATION: https://github.com/denniscwylie/sarks. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Neural and molecular mechanisms underlying female mate choice decisions in vertebrates.

Female mate choice is a dynamic process that allows individuals to selectively mate with those of the opposite sex that display a preferred set of traits. Because in many species males compete with each other for fertilization opportunities, female mate choice can be a powerful agent of sexual selection, often resulting in highly conspicuous traits in males. Although the evolutionary causes and consequences of the ornamentation and behaviors displayed by males to attract mates have been well studied, embarrassingly little is known about the proximate neural mechanisms through which female choice occurs. In vertebrates, female mate choice is inherently a social behavior, and although much remains to be discovered about this process, recent evidence suggests the neural substrates and circuits underlying other fundamental social behaviors (such as pair bonding, aggression and parental care) are likely similarly recruited during mate choice. Notably, female mate choice is not static, as social and ecological environments can shape the brain and, consequently, behavior in specific ways. In this Review, we discuss how social and/or ecological influences mediate female choice and how this occurs within the brain. We then discuss our current understanding of the neural substrates underlying female mate choice, with a specific focus on those that also play a role in regulating other social behaviors. Finally, we propose several promising avenues for future research by highlighting novel model systems and new methodological approaches, which together will transform our understanding of the causes and consequences of female mate choice.

The genomic substrate for adaptive radiation in African cichlid fish.

Cichlid fishes are famous for large, diverse and replicated adaptive radiations in the Great Lakes of East Africa. To understand the molecular mechanisms underlying cichlid phenotypic diversity, we sequenced the genomes and transcriptomes of five lineages of African cichlids: the Nile tilapia (Oreochromis niloticus), an ancestral lineage with low diversity; and four members of the East African lineage: Neolamprologus brichardi/pulcher (older radiation, Lake Tanganyika), Metriaclima zebra (recent radiation, Lake Malawi), Pundamilia nyererei (very recent radiation, Lake Victoria), and Astatotilapia burtoni (riverine species around Lake Tanganyika). We found an excess of gene duplications in the East African lineage compared to tilapia and other teleosts, an abundance of non-coding element divergence, accelerated coding sequence evolution, expression divergence associated with transposable element insertions, and regulation by novel microRNAs. In addition, we analysed sequence data from sixty individuals representing six closely related species from Lake Victoria, and show genome-wide diversifying selection on coding and regulatory variants, some of which were recruited from ancient polymorphisms. We conclude that a number of molecular mechanisms shaped East African cichlid genomes, and that amassing of standing variation during periods of relaxed purifying selection may have been important in facilitating subsequent evolutionary diversification.

Hippocampal transcriptomic responses to enzyme-mediated cellular dissociation.

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 affects neural gene expression has not been determined. This information is necessary for interpreting the results of experimental manipulations that affect neural function such as learning and memory. The goal of this research was to determine the impact of cellular dissociation on brain transcriptomes. We compared gene expression of microdissected samples from the dentate gyrus (DG), CA3, and CA1 subfields of the mouse hippocampus either prepared by a standard tissue homogenization protocol or subjected to enzymatic digestion used to dissociate cells within tissues. We report that compared to homogenization, enzymatic dissociation alters about 350 genes or 2% of the hippocampal transcriptome. While only a few genes canonically implicated in long-term potentiation and fear memory change expression levels in response to the dissociation procedure, these data indicate that sample preparation can affect gene expression profiles, which might confound interpretation of results depending on the research question. This study is important for the investigation of any complex tissues as research effort moves from subfield level analysis to single cell analysis of gene expression.

Experimentally induced variation in neuroendocrine processes affects male reproductive behaviour, sperm characteristics and social interactions.

While extensive research has focused on how social interactions evolve, the fitness consequences of the neuroendocrine mechanisms underlying these interactions have rarely been documented, especially in the wild. Here, we measure how the neuroendocrine mechanisms underlying male behaviour affect mating success and sperm competition in the ocellated wrasse (Symphodus ocellatus). In this species, males exhibit three alternative reproductive types. "Nesting males" provide parental care, defend territories and form cooperative associations with unrelated "satellites," who cheat by sneaking fertilizations but help by reducing sperm competition from "sneakers" who do not cooperate or provide care. To measure the fitness consequences of the mechanisms underlying these social interactions, we used "phenotypic engineering" that involved administering an androgen receptor antagonist (flutamide) to wild, free-living fish. Nesting males treated with flutamide shifted their aggression from sneakers to satellite males and experienced decreased submissiveness by sneaker males (which correlated with decreased nesting male mating success). The preoptic area (POA), a region controlling male reproductive behaviours, exhibited dramatic down-regulation of androgen receptor (AR) and vasotocin 1a receptor (V1aR) mRNA following experimental manipulation of androgen signalling. We did not find a direct effect of the manipulation on male mating success, paternity or larval production. However, variation in neuroendocrine mechanisms generated by the experimental manipulation was significantly correlated with changes in behaviour and mating success: V1aR expression was negatively correlated with satellite-directed aggression, and expression of its ligand arginine vasotocin (AVT) was positively correlated with courtship and mating success, thus revealing the potential for sexual selection on these mechanisms.

Early-life social environment alters juvenile behavior and neuroendocrine function in a highly social cichlid fish.

Early-life experiences can shape adult behavior, with consequences for fitness and health, yet fundamental questions remain unanswered about how early-life social experiences are translated into variation in brain and behavior. The African cichlid fish Astatotilapia burtoni, a model system in social neuroscience, is well known for its highly plastic social phenotypes in adulthood. Here, we rear juveniles in either social groups or pairs to investigate the effects of early-life social environments on behavior and neuroendocrine gene expression. We find that both juvenile behavior and neuroendocrine function are sensitive to early-life effects. Behavior robustly co-varies across multiple contexts (open field, social cue investigation, and dominance behavior assays) to form a behavioral syndrome, with pair-reared juveniles towards the end of syndrome that is less active and socially interactive. Pair-reared juveniles also submit more readily as subordinates. In a separate cohort, we measured whole brain expression of stress and sex hormone genes. Expression of glucocorticoid receptor 1a was elevated in group-reared juveniles, supporting a highly-conserved role for the stress axis mediating early-life effects. The effect of rearing environment on androgen receptor α and estrogen receptor α expression was mediated by treatment duration (1 vs. 5 weeks). Finally, expression of corticotropin-releasing factor and glucocorticoid receptor 2 decreased significantly over time. Rearing environment also caused striking differences in gene co-expression, such that expression was tightly integrated in pair-reared juveniles but not group-reared or isolates. Together, this research demonstrates the important developmental origins of behavioral phenotypes and identifies potential behavioral and neuroendocrine mechanisms.

Multimodal stimuli regulate reproductive behavior and physiology in male túngara frogs.

Unlike in terrestrial animals, the boundary between internal (e.g., hormones) and external (e.g., social) stimulation can be blurred for aquatic and amphibious species. When chemicals such as hormones and glandular secretions leach into the water, they can further interact with other signaling systems, creating multimodal stimuli. It is unclear, however, whether water-borne chemical secretions from courting male frogs affect the physiology and behavior of their rivals. In order to address this question we first established non-invasive, continuous sampling methods for simultaneously measuring both hormones and behavior in amphibious species. Then, we examined whether interactions between water-borne chemical secretions and conspecific calls affect reproductive behavior and physiology (testosterone and corticosterone) of courting male túngara frogs. Our results demonstrate that conspecific acoustic stimulation alone increases locomotor activity, decreases latency to call, and increases calling behavior but does not alter the amount of hormones excreted. In response to water containing chemical secretions from rivals, but in the absence of calls from other males, males excrete more testosterone. Interestingly, the combined acoustic and chemical stimulus causes a multiplicative increase in both calling behavior and hormonal excretion. Taken together, our results suggest that a multimodal chemical-acoustic stimulus physiologically primes males for aggressive behavior.

Real or fake? Natural and artificial social stimuli elicit divergent behavioural and neural responses in mangrove rivulus, Kryptolebias marmoratus.

Understanding how the brain processes social information and generates adaptive behavioural responses is a major goal in neuroscience. We examined behaviour and neural activity patterns in socially relevant brain nuclei of hermaphroditic mangrove rivulus fish (Kryptolebias marmoratus) provided with different types of social stimuli: stationary model opponent, regular mirror, non-reversing mirror and live opponent. We found that: (i) individuals faced with a regular mirror were less willing to interact with, delivered fewer attacks towards and switched their orientation relative to the opponent more frequently than fish exposed to a non-reversing mirror image or live opponent; (ii) fighting with a regular mirror image caused higher expression of immediate-early genes (IEGs: egr-1 and c-Fos) in the teleost homologues of the basolateral amygdala and hippocampus, but lower IEG expression in the preoptic area, than fighting with a non-reversing mirror image or live opponent; (iii) stationary models elicited the least behavioural and IEG responses among the four stimuli; and (iv) the non-reversing mirror image and live opponent drove similar behavioural and neurobiological responses. These results suggest that the various stimuli provide different types of information related to conspecific recognition in the context of aggressive contests, which ultimately drive different neurobiological responses.

Neural activity in the social decision-making network of the brown anole during reproductive and agonistic encounters.

Animals have evolved flexible strategies that allow them to evaluate and respond to their social environment by integrating the salience of external stimuli with internal physiological cues into adaptive behavioral responses. A highly conserved social decision-making network (SDMN), consisting of interconnected social behavior and mesolimbic reward networks, has been proposed to underlie such adaptive behaviors across all vertebrates, although our understanding of this system in reptiles is very limited. Here we measure neural activation across the SDMN and associated regions in the male brown anole (Anolis sagrei), within both reproductive and agonistic contexts, by quantifying the expression density of the immediate early gene product Fos. We then relate this neural activity measure to social context, behavioral expression, and activation (as measured by colocalization with Fos) of different phenotypes of 'source' node neurons that produce neurotransmitters and neuropeptides known to modulate SDMN 'target' node activity. Our results demonstrate that measures of neural activation across the SDMN network are generally independent of specific behavioral output, although Fos induction in a few select nodes of the social behavior network component of the SDMN does vary with social environment and behavioral output. Under control conditions, the mesolimbic reward nodes of the SDMN actually correlate little with the social behavior nodes, but the interconnectivity of these SDMN components increases dramatically within a reproductive context. When relating behavioral output to specific source node activation profiles, we found that catecholaminergic activation is associated with the frequency and intensity of reproductive behavior output, as well as with aggression intensity. Finally, in terms of the effects of source node activation on SDMN activity, we found that Ile8-oxytocin (mesotocin) populations correlate positively, while Ile3-vasopressin (vasotocin), catecholamine, and serotonin populations correlate negatively with SDMN activity. Taken together, our findings present evidence for a highly dynamic SDMN in reptiles that is responsive to salient cues in a social context-dependent manner.