Gene expression profiles underlying aggressive behavior in the prefrontal cortex of cattle

Aggressive behavior is an ancient and conserved trait considered habitual and essential for most animals in order to eat, protect themselves from predators and also to compete for mating and defend their territories. Genetic factors have shown to play an important role in the development of aggression both in animals and humans, displaying moderate to high heritability estimates. However, although such types of conducts have been studied in different animal models, the molecular architecture of aggressiveness remains poorly understood. This study compared gene expression profiles of 16 prefrontal cortex (PFC) samples from aggressive and non-aggressive cattle breeds: Lidia, selected for agonistic responses, and Wagyu, specialized on meat production and selected for tameness. RNA sequencing was used to identify 918 up and 278 down-regulated differentiated expressed genes (DEG). The functional interpretation of the up-regulated genes in the aggressive cohort revealed enrichment of pathways such as the Alzheimer disease-presenilin, integrins or the ERK/MAPK signaling cascade, all implicated in the development of abnormal aggressive behaviors and neurophysiological disorders. Moreover, gonadotropins, leading to testosterone release, are also up-regulated as natural mechanisms enhancing aggression. Concomitantly, heterotrimeric G-protein pathways, associated with low reactivity mental states, and the GAD2 gene, a repressor of agonistic reactions at PFC, are down-regulated, guaranteeing the development of the adequate responses required by the aggressive Lidia cattle. We also identified six upstream regulators, whose functional activity fits with the etiology of abnormal behavioral responses associated with aggression. These results provide valuable insights into the complex architecture that underlie naturally developed agonistic behaviors.


Introduction
Aggressive behavior, an evolutionary well-conserved trait in animals, is part of the general conducts repertoire, as most animals need this skill in order to eat, protect themselves and their family against predators, compete for mating, as well as acquire resources and territory (Tremblay and Nagin, 2005). In contrast, aggressive behaviors in humans often refer to abnormal manifestations of aggressiveness such as violence, and are associated with a broad spectrum of neuropsychiatric disorders such as dementia, manic depression, A large number of preclinical studies using different animal species as models has been encouraged on the reasoning that molecular correlates of animal aggressive behavior resemble biological mechanisms in human pathological aggression (Blanchard and Blanchard, 2003). Several attempts to mold abnormal forms of aggressiveness using mainly murine models, and to a lesser extent dogs and semi-domesticated species such as the silver fox, have been performed to display a contrast between docile or tame behaviors and escalated levels of aggressiveness (de Boer et al. 2003). However, relating these mechanisms to the human condition is not simple, since aggressive behaviors are very diverse. In animals, aggressive responses consist of a combination of fight, chase, bite and ram, whereas aggression in humans involves both verbal and physical forms. Despite this, it is possible to look for similarities between species in the components of aggression to better understand its etiology and to further improve its diagnosis, prognosis and intervention strategies, which currently lack in effectiveness (McGuire, 2008).
In the bovine species, the Lidia breed may constitute a useful tool for studying the genomic However, no studies on gene expression differences for behavioral features have been conducted so far in cattle.
The genetic expression of behavior takes part in the brain, where the frontal cortical region, in particular the prefrontal cortex (PFC), has shown to play a crucial role in the regulation of aggressive behavior (Miczek et al., 2007;Siever, 2008). The PFC has been studied on different species, e.g. it has been suggested that PFC lesions result in impulsive and antisocial behaviors in humans (Brower et al. 2001) and offensive aggression in rodents (Craig and Halton, 2009). Moreover, a catalogue of gene-specific sequence variants was detected as differentially expressed between an aggressive-selected strain of silver fox Thus, the goal of our study is to seek for genes that are differentially expressed in the PFC of aggressive and non-aggressive bovines using as models the Lidia and the Wagyu breeds for each cohort respectively. The two breeds differ significantly in their agonistic responses, the Lidia breed known as one of the most aggressive bovine breeds, whereas Wagyu bovines are calm and docile animals, selected and bred by farmers with the aim of easing their handling (Takanishi et al. 2015). This makes our studied populations highly suitable for investigating the biological underpinnings that may contribute to control naturally developed aggressive behavior in animals.

Methods
No special permits were required to conduct the research. All animals were sacrificed for reasons other than their participation in this study. The access to the brains of the animals was allowed in the cutting room, after the "corrida" in the case of Lidia animals and after slaughter with the Wagyu breed animals, following standard procedures approved by the Spanish legislation applied to abattoirs and "corrida" festivities (Reglamento General de Mataderos/BOE-A-1997-3081).

Cross-species comparative analysis (CSCA)
Because no other differential expression analysis using cattle as animal model for aggressive behaviors has been conducted before, we performed a comparison among our DEG and a cross-species compendium of genes associated with aggressiveness previously To homogenize the compendium gene-list with our DEG, gene official names from cattle were converted to its human orthologues using biomaRt (Durinck et al. 2005). In order to establish a ranking according to the total occurrence of each gene in the different sets we assigned a weight (weighted ranking, WR) to each of our DEG in common with the compendium gene list applying the same conditions proposed by Zhang-James et al.

Gene ontology and KEGG pathway enrichment analyses
To examine the relationships between differences in PFC gene expression among groups and its biological function, we first separated the results of DEG in two independent gene-

Sequencing and read assembly
The RNA-sequencing of the sixteen PFC samples generated an average of 78.  Table 2). The mapped reads were processed with Cufflinks toolkits for differential expression analysis, revealing a total of 16,384 DEG between the aggressive and non-aggressive groups; of those genes, 1196 were statistically significant, producing 10,640 isoforms (8.86 transcripts per gene) (Table 1, Figure 1A and Supplementary Figure   1). Gene expression differences of the up-regulated DEG (log 2 FC ≥ 0.1) were higher, involving 918 genes, than the down-regulated 278 DEG (log 2 FC ≤ 0.1) (Figure 1B and C).
For the complete list of up and down-regulated DEG see Supplementary Table 3.

Genes in common with the cross-species comparative analysis (CSCA)
The up and down-regulated DEG were compared with the compendium genes-list associated with aggressive behavior (Supplementary Table 1). This comparison yielded 31 genes, 17 up and 14 down-regulated in the aggressive group of Lidia individuals ( Table 2).
The expression bar plot in Figure 2 shows the common subset of DEGs in relation to their standard deviation FPKM values and their WR. Most of the overlapping genes from the upregulated DEG coincide with GWAS on aggressive behaviors in humans. Instead, the down-regulated DEG greater coincidences were detected with differential expression analyses and KO studies in mice.  Table 4). The Panther Pathway enrichment analysis retrieved five significant pathways: blood coagulation, integrin signaling, Alzheimer disease-presenilin, angiogenesis and gonadotropin-releasing hormone receptor pathways (Table 3A).

Functional annotation and biological pathway analysis
Within the down-regulated DEGs in the aggressive cohort, the GO biological processes included 260 genes as uniquely mapped IDs implicated in 243 processes (FDR ≤ 0.05), the higher significant values, being dendritic cell cytokine production, trans-synaptic signaling by endocannabioid, trans-synaptic signaling by lipid, negative regulation of renin secretion into blood stream and melanocyte adhesion, all with 84.4 fold enrichment and two genes associated with each process (Supplementary table 5). The Panther enrichment pathway analysis retrieved two significant down-regulated pathways in the aggressive Lidia breed, both involved in two different types of Heterotrimeric G-protein signaling (Table 3B).

Signaling networks and upstream regulators enrichment analysis
We used the IPA software to identify pathways to which DEGs in common with the CSCA belong to, as well as to explore the existence of signaling networks connecting the DEGs.
Sixty-eight categories with P-values ≤ 0.05 were associated with different diseases or function annotations significantly enriched in the 31 common genes from the CSCA (Supplementary Table 6). This information should be interpreted cautiously because most pathways were represented by a small number of genes (from 1 to 5).  Table 6).
Finally, the upstream analysis tool of the IPA package was used to identify the potential upstream regulators that may explain the differential patterns of expression between the up and down regulated DEGs in common with the CSCA in the aggressive cohort. Regarding the down-regulated DEG detected in the group of aggressive animals, we found the heterotrimeric G-protein pathways strongly suppressed (Table 2B) The analysis of the data with the IPA upstream enrichment tool retrieved one regulatory network related with behavior, cellular movements and embryonic development functions.
In the network shown in Figure 3 Finally, six upstream regulators were predicted to be major transcriptional regulators of the set of four DEG detected, COL13A1, IGF2, ADCYAP1 and BDNF (Figure 4). The modulator effect of these molecules appears to increase the up-regulation of biological processes such as hyperactive behavior and anxiety, which are often associated with aggressiveness, and Alzheimer disease, a concordance feature with the above findings. We also found that the upstream regulators promote an increased nociception ability. Although it has never been implicated in aggression, it makes sense for Lidia cattle to display an enhancement of the capacity to respond to potentially damaging stimuli, and hence, display aggressive behaviors.
In conclusion, this is the first time a comparison of the differences in genomic expression between aggressive and non-aggressive selected cattle breeds at PFC has been performed, identifying 918 up and 278 down-regulated genes. We have also undertaken a cross-species comparison analysis to identify genes in common implicated in aggressiveness and investigate their regulatory networks. Our results include the up-regulation in the aggressive cohort of animals of pathways such as the Alzheimer disease-presenilin, integrins or the ERK/MAPK signaling cascade, all routes implicated in the development of abnormal aggressive behaviors and neurophysiological disorders, as well as normal mechanisms enhancing aggression such us the up-regulation of gonadotropins and, hence, testosterone, whose levels have been widely linked with agonistic reactions. On the contrary, heterotrimeric G-protein pathways, previously associated with low reactivity mental states like those involved in major depression, or the GAD2 gene, with a pivotal role in the control systems deployed by the PFC to repress agonistic reactions, are both downregulated, guaranteeing the development of the adequate combative responses needed during a "corrida" festivity. However, despite PFC is a key region for the modulation of aggressive behavior, it may not be representative of other brain regions reported also to play important roles in aggression, such as the hippocampus or the hypothalamus.
Nevertheless, this constitutes the first important step towards the identification of the genes that impulse aggression in cattle and, by doing so, we are providing a novel species as model organism for disentangling the mechanisms underlying variability in aggressive behavior.