Unravelling the neurophysiological basis of aggression in a fish model

Abstract

Background: Aggression is a near-universal behaviour with substantial influence on and implications for human and animal social systems. The neurophysiological basis of aggression is, however, poorly understood in all species and approaches adopted to study this complex behaviour have often been oversimplified. We applied targeted expression profiling on 40 genes, spanning eight neurological pathways and in four distinct regions of the brain, in combination with behavioural observations and pharmacological manipulations, to screen for regulatory pathways of aggression in the zebrafish (Danio rerio), an animal model in which social rank and aggressiveness tightly correlate.

Results: Substantial differences occurred in gene expression profiles between dominant and subordinate males associated with phenotypic differences in aggressiveness and, for the chosen gene set, they occurred mainly in the hypothalamus and telencephalon. The patterns of differentially-expressed genes implied multifactorial control of aggression in zebrafish, including the hypothalamo-neurohypophysial-system, serotonin, somatostatin, dopamine, hypothalamo-pituitary-interrenal, hypothalamo-pituitary-gonadal and histamine pathways, and the latter is a novel finding outside mammals. Pharmacological manipulations of various nodes within the hypothalamo-neurohypophysial-system and serotonin pathways supported their functional involvement. We also observed differences in expression profiles in the brains of dominant versus subordinate females that suggested sex-conserved control of aggression. For example, in the HNS pathway, the gene encoding arginine vasotocin (AVT), previously believed specific to male behaviours, was amongst those genes most associated with aggression, and AVT inhibited dominant female aggression, as in males. However, sex-specific differences in the expression profiles also occurred, including differences in aggression-associated tryptophan hydroxylases and estrogen receptors.

Conclusions: Thus, through an integrated approach, combining gene expression profiling, behavioural analyses, and pharmacological manipulations, we identified candidate genes and pathways that appear to play significant roles in regulating aggression in fish. Many of these are novel for non-mammalian systems. We further present a validated system for advancing our understanding of the mechanistic underpinnings of complex behaviours using a fish model.

Figure 1

Simplified schematic of regulatory neurological pathways of aggression in mammals targeted for this study in fish. Study genes are highlighted in red. Abbreviations: 5-HIAA, 5-hydroindoleacetic acid; 5-HT, 5-hydroxytryptamine (serotonin); 5-HTP, 5-hydroxytryptophan; AANAT, arylalkylamine N-acetyltransferase; ACTH, adrenocorticotropic hormone; ALD-DH, acetaldehyde dehydrogenase; AR, androgen receptor; AVT, arginine vasotocin; COMT, catechol-O-methyl transferase; CRH, corticotrophin releasing hormone; DAO, diamine oxidase; DAT, dopamine transporter; DOPA, dihydroxyphenylalanine; ER, estrogen receptor; FSH, follicle-stimulating hormone; GH, growth hormone; GHRH, growth hormone releasing hormone; GnRH, gonadotropin releasing hormone; GR, glucocorticoid receptor; HIOMT, hydroxyindole-O-methyltransferase; HMT, histamine-N-methyltransferase; HNS, hypothalamo-neurohypophysial system; HPI, hypothalamo-pituitary-interrrenal; HPG, hypothalamo-pituitary-gonadal; IL1β, interleukin 1β; LH, luteinizing hormone; MAO, monoamine oxidase; MR, mineralocorticoid receptor; NOS, nitric oxide synthase; NPY, neuropeptide Y; SERT, 5-HT transporter; TSH, thyrotropin-stimulating hormone. Plus (+) and negative (-) symbols indicate the proposed action of a gene/neurotransmitter/pathway on aggression (i.e. stimulatory or inhibitory, respectively).

Figure 2

Correlation between social rank and aggressiveness in male and female zebrafish. The number of aggressive acts performed by each fish towards the other fish of the same sex in colonies with 2 males and 2 females (n = 16 fish per group) was quantified during a 5 min observation on the day of the sampling. Statistically significant differences in aggression were observed between social ranks for both sexes and between sexes (P < 0.001; Two way ANOVA followed by Holm-Sidak post hoc test).

Figure 3

Regions of the brain associated with differences in aggressiveness in zebrafish. Heat maps showing all consistently expressed genes in four regions of the brain studied in dominant males (high aggression; n = 8) compared to subordinate males (low aggression; n = 8) sampled on day 1 of social interaction. Genes found to be differentially expressed (P < 0.05) are denoted with an asterisk.

Figure 4

Aggression of zebrafish following pharmacological manipulations of various nodes within neurological pathways proposed to regulate aggressive behaviour. A. The hypothalamo-neurohypophysial system (HNS) pathway in males. B. The serotonin (5-HT) pathway in males. C. The HNS pathway in females. Specific agents known to agonise or antagonise particular receptors or inhibit transporters within the identified axes were administered and the effect on aggression determined (treated) compared to a sham administration (sham) with n = 6-9 fish. AVT, arginine vasotocin acetate salt; Manning, Manning compound; FL, fluoxetine HCl; WAY100,635, WAY100,635 maleate salt. Significant changes in aggression (number of aggressive acts per 5 min observation) are indicated by asterisks (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 5

Comparisons of the roles of sex and social rank as determinants of the brain gene expression profiles. A. Hierarchical cluster analysis of all consistently expressed genes in (i) hypothalamus and (ii) telencephalon of dominant (high aggression) and subordinate (low aggression) males and females (n = 7-8 fish per group) and the corresponding PCAs performed on the profiles of the same individual fish in (iii) hypothalamus and (iv) telencephalon) (the dashed lines were drawn arbitrarily to aid the visualisation of clustering of the samples). B. Hierarchical cluster analysis of genes identified as differentially expressed (P < 0.05) between (i) dominants (high aggression) and subordinates (low aggression) irrespective of their sex, and (ii) between males and females irrespective of their social rank. Fish are shown individually. In (i), individual male fish are indicated by numbers in blue and individual female fish by numbers in pink (all genes were overexpressed in dominant fish). In (ii), individual dominant fish are indicated by numbers in red and individual subordinate fish by numbers in black. In (i)-(ii), genes shown in red were overexpressed in females while genes shown in green were overexpressed in males. Grey colouration indicates no data.

Figure 6

Temporal changes in gene expression correlated with changes in aggression. A. Phenotypic anchoring of (i) temporal changes in the total number of genes that were differentially-expressed between social ranks, which differed in aggressiveness (across hypothalamus and telencephalon) and (ii) parallel changes identified in the expression levels of individual genes within ranks over the same time with (iii) temporal changes in aggressiveness of zebrafish between day 1 and day 5. B. Hierarchical cluster analysis of all genes in (i) hypothalamus and (ii) telencephalon of dominant (high aggression) and subordinate (low aggression) males and females and the corresponding PCAs performed on the profiles of the same individual fish (n = 7-8 per group) in (ii) hypothalamus and (iv) telencephalon (the dashed lines drawn arbitrarily indicate clustering of the samples) on day 5. Statistically significant changes (P < 0.05) are indicated by an asterisk.