The serotonin transporter (5-HTT) plays an important role in regulating serotonergic transmission via removal of serotonin (5-HT) from synaptic clefts. Alterations in 5-HTT expression and subsequent 5-HT transmission have been found to be associated with changes in behaviour, such as fearfulness or activity, in humans and other vertebrates. In humans, alterations in 5-HTT expression have been suggested to be able to lead to better learning performance, with more fearful persons being better at learning. Similar effects of the variation in the 5-HTT on fearfulness have been found in chickens, and in this study, we investigated effects on learning. Therefore, we tested 52 adult laying hens, differing in their functional 5-HTT genotype (W/W, W/D and D/D) in an operant learning paradigm in three different phases (initial learning, reversal learning and extinction) and in a tonic immobility test for fearfulness. We found that the 5-HTT polymorphism affects the initial learning performance of laying hens, with homogeneous wild-type (W/W) hens being the slowest learners, and the most fearful birds. W/W hens, showed significantly more choices to solve the initial learning task (LME, p = 0.031) and had the highest latencies in a tonic immobility test (p = 0.039), indicating the highest fearfulness. Our results provide interesting first insights into the role of 5-HTT in chickens and its sensitive interaction with the environment. We further suggest that the 5-HTT gene can be an interesting target gene for future breeding strategies as well as for further experimental studies.

General locomotor activity is a highly variable phenotypic trait of animals. In domestic chickens it is different within and between breeds. The general locomotor activity is a substantially heritable trait and has been shown to be correlated with several other behavioural traits, such as for example feather pecking and anxiety in chickens. However, whether there is a relation between different levels of general locomotor activity and behavioural changes remained unclear. Therefore, a selection line model system has been established, where hens from the same founder population were selected over eight generations for either high or low general locomotor activity. The selection led to significant increases, respectively decreases in general locomotor activity and differences in growth. We here tested 128 hens of the 8th generation in three behavioural tests. We assumed fearfulness to be affected from selection on general locomotor activity, which we tested in a tonic immobility test. Socio-positive and socio-negative behaviours were tested in respective test paradigms. Fearfulness was higher in hens selected for high general locomotor activity. Social behavioural traits and feather pecking were not affected by selection for general locomotor activity. Evolutionary mechanisms that link fear and general locomotor activity are discussed and also why social behaviours and feather pecking seems not to be affected from selection on general locomotor activity. Our results provide interesting new insights on how selection on one trait, general locomotor activity, affects the behavioural phenotype in other dimensions too.

The serotonin transporter gene (5-HTT) is involved in the regulation of the neural serotonin. Polymorphisms in the 5-HTT gene have been described in many species to be involved in physiological processes and emotions. A functional polymorphism in the 5´-flanking region of the 5-HTT gene is known from chickens, with a deletion-allele (D), which is associated with an increased 5-HTT expression, in comparison to the wild-type-allele (W). In domestic populations, the majority of hens carry the W-allele. The regulatory changes of the 5-HTT are accompanied in chickens, as in humans, by modulations of fear. Beside these effects on fear, the understanding of potential functional consequences on the social behaviour in the gregarious chicken is lacking. Thus, we here investigated whether the 5-HTT polymorphism with three genotypes (WW, WD, DD), is not only linked to fear-related behaviour, but affects also socio-positive and -negative behaviours of adult hens. Our data confirmed the effects on fear-related behaviour. WW hens showed highest levels of fear. Interestingly, no differences in the social behaviours were present between the hens of the different 5-HTT genotypes. We further discuss implications for potential evolutionary pathways via natural selection and / or artificial selection through domestication of the 5-HTT polymorphism, which might have enabled a stable social lifestyle in the wild ancestors of modern chickens.

The serotonin transporter (5-HTT) plays a key role in regulating serotonergic transmission via removal of serotonin (5-hydroxytryptamine, 5-HT) from synaptic clefts. Alterations in 5-HTT expression and 5-HT transmission have been shown to cause changes to adult behavior including fear. The objective of the present study was to investigate the 5-HTT role in fear in birds at the very early stages of post-hatching life. Using an avoidance test with an elevated balance beam, which was based on depth perception and the respective fear of heights, we assessed fear-related avoidance behaviors of newly hatched chicks of the three functional 5-HTT genotypes W/W, W/D and D/D. Newly hatched chicks of the genotype D/D, which was linked to high 5-HTT expression, showed less intensive avoidance responses as measured by decreased latency to jump than W/W and W/D chicks. Further, significantly fewer D/D hens than W/W hens showed fear-like behavior that resembled a freezing response. Furthermore, in an arousal test the arousal reaction of the chicks in response to an acute short-term visual social deprivation in the home compartment was assessed 5 weeks after hatching, which also revealed that D/D chicks exhibited decreased arousal reaction, compared to W/W chicks. Thus, the results indicate that fear responses differ in D/D chicks in the early post-hatching periods, possibly due to the different expression of 5-HTT respectively 5-HT levels in this strain.

The neural serotonin (5-HT)/serotonin transporter (5-HTT) system is involved in the regulation of physiological processes and emotional states. In humans, the short (S) allele in the 5-HTT gene-linked polymorphic region, which decreases 5-HTT expression, has been shown to be associated with behavioral changes including an increased level of anxiety. Also in birds a polymorphism in the 5-HTT gene is described, a deletion (D) has been found to have functional consequences on growth and locomotion. Furthermore, the D-allele leads to an increased 5-HTT expression compared to the wild type (W), a feature which is linked to lower levels of fear in mammalian species. Thus, we aimed here to test whether the polymorphism in the chicken 5-HTT gene also leads to respective alternations of fear-related behaviors. We tested 268 hens of three genotypes (W/W, W/D, D/D) in two behavioral paradigms (open field, light dark test) to assess fear-related behavior. Both tests revealed that hens possessing the D-allele showed lower levels of fear than those having the W-allele. These similar outcomes in fear-related behaviors in an avian and a mammalian species are associated with an increased 5-HIT expression. In the human 5-HTT gene, the long (L) allele is linked to such increased expression, whereas in chickens it is the D-allele. Thus, increased 5-HTT expression causing decreased fear may be a general mechanism in vertebrates.

The serotonergic system has been shown to be implicated in the regulation of mood and feeding behavior. Previous studies have identified a polymorphism in the 5′-flanking region of the serotonin transporter ( 5 - HTT ) gene of Lohmann Brown (LB) laying hens. The deleted variant D was found to be associated with increased body weight. The objective of this study was to address whether the increased body weight may be due to an increased feed intake. After hatching, hens were kept under ad libitum feeding conditions, and their body weight and feed intake were weekly determined. From 5 weeks of age, the body weight of hens with the D/D and W/D genotypes was significantly greater than that of W/W carrying hens. Interestingly, we found that the feed intake of D/D carrying hens, relative to body weight, was transiently increased only between 4 and 7 weeks of age ( p < 0.05), leading to a higher growth rate ( p < 0.05), compared with that of W/W carrying hens. These results suggest that the presence of variant D may be correlated with a transiently increased appetite of D/D carrying hens.

Serotonin (5-HT) has been reported to be involved in cancer progression by stimulating angiogenesis and cell growth. In this study, we examined the expression of the serotonin transporter (5-HTT) and the role of histone deacetylases (HDACs) in regulating the 5-HTT gene in tumor cells. The 5-HTT gene expression was almost silenced in chicken lymphoma DT40, myelomonocytic tumor HD11 and hepatoma DU249 cells, compared to their physiological counterpart. In contrast, HDAC1 mRNA expression was increased in these cell lines. Indeed, the pan-HDAC inhibitor trichostatin A (TSA) enhanced the 5-HTT mRNA expression in several tumor cell lines including the human cell lines HepG2 and THP-1 and increased the 5-HT uptake in HD11 cells. In addition, treatment with parthenolide, which is capable of depleting HDAC1, and knockdown of HDAC1 using siRNA resulted in increased 5-HTT mRNA expression, confirming the role of HDAC1 in the down-regulation of 5-HTT in the tumor cells. Deletion analysis of the 5-HTT promoter and site-directed mutagenesis revealed that the transcription factor CCAAT/enhancer binding protein beta (C/EBPß), in interacting with the 5-HTT promoter, mediated both the inhibition of the 5-HTT expression by HDAC1 and the activation by CREB-binding protein (CBP). Using a chromatin immunoprecipitation assay, we found increased acetylation of histone H4 associated with the 5-HTT promoter in cells treated with TSA. Our results suggest that the 5-HTT gene is epigenetically downregulated by HDAC1 in several types of cancer.

Serotonin (5-hydroxytryptamine; 5-HT) transporter (5-HTT) is involved in inflammation and the stress response. In this study, we examined the regulation of 5-HTT expression in macrophage HD11 cells in response to bacterial LPS. Long-term exposure of cells to LPS (6–18 h) produced a decrease in 5-HTT mRNA expression. Accordingly, reduced 5-HTT activity measured by 5-HT uptake was also observed in LPS-treated HD11 cells. Moreover, LPS treatment, as well as co-transfection with an expression vector encoding the chicken CCAAT/enhancer binding protein beta (C/EBPβ), resulted in inhibition of 5-HTT promoter activity. Indeed, sequence analysis revealed several C/EBPβ binding motifs in the upstream region of the 5-HTT gene, which specifically interacted with C/EBPβ both in an in vitro band shift assay and in living HD11 cells. The C/EBPβ binding was activated in cells treated with LPS. The role of C/EBPβ in LPS inhibition of 5-HTT expression was further confirmed by small interfering RNA interference, which demonstrated that knockdown of endogenous C/EBPβ attenuated the inhibition of 5-HTT expression in LPS-treated cells. Taken together, the results suggest that C/EBPβ plays a critical role in regulating the 5-HTT gene in macrophages in response to pro-inflammatory stimuli.

In this study, we identified a polymorphism in the 5-flanking region of the chicken serotonin transporter (5-HTT) gene. Sequencing analysis revealed that in comparison with the wild-type variant (W), a deleted variant (D) is generated by deletion of four nucleotides (5-AATT-3) and a single nucleotide change (AT). Using a polyacrylamide gel electrophoresis system, we found that the 360-bp DNA fragment containing the W variant with the wild-type sequence 5-AATTAATT-3 shows intrinsic DNA curvature while the 356-bp fragment containing the D variant lacking the four base pairs AATT is not curved. Quantitative real-time RT-PCR and ELISA demonstrated that the expression of 5-HTT in D/D chickens was higher than that in W/W and W/D chickens. In addition, transient transfection experiments with chloramphenicol acetyltransferase reporter gene constructs revealed increased 5-HTT promoter activity mediated by the D variant and a silencer activity of the W variant. Interestingly, females and males with D/D genotype showed significant greater increase in body weight from 6weeks and 16weeks of age, respectively, and higher body mass index. Moreover, we found that D/D chickens of both genders were physically more active than W/W and W/D chickens.

Most known DNA replication origins in vertebrate genomes have been found to occur close to transcriptional promoters. The origin of bidirectional DNA replication of the chicken lysozyme GAS41 locus was identified in a CpG island covering the GAS41 gene promoter. In this study, we generated an α-Orc2 antibody from rabbits immunized with the C-terminal half of Orc2 for studying in vivo Orc2 binding to the lysozyme-GAS41 origin. Using the chromatin immunoprecipitation technique and quantitative real-time polymerase chain reaction, we were able to map the Orc2-binding site to a region of the lysozyme GAS41 origin that contains multiple Sp1/Sp3-binding sites co-mapping with two DNase I hypersensitive sites. Further, knockdown of endogenous Sp1 by RNA interference reduced specific Orc2 binding to the lysozyme GAS41 origin. These results suggest that Sp1 participates in recruiting Orc2 to the origin.

The 5'-flanking region of the chicken glioma-amplified sequence (GAS) 41 gene is close to the 3' end of the lysozyme gene and contains no typical TATA box, but several GC boxes. In this study, we have localized the GAS 41 promoter to this narrow region. Electrophoretic mobility shift assays and chromatin immunoprecipitation analyses revealed that Sp1 and Sp3 bind to this promoter. Mapping by a technique of indirect end labeling demonstrated that the Sp1-binding sites contained in this region exactly co-map with two previously identified DNase I hypersensitive (HS) sites, which suggests the important role of Sp1 binding in maintaining an open chromatin structure of the GAS41 promoter. We further found that Sp1 and Sp3 strongly activate CAT expression controlled by the putative GAS41 promoter in Drosophila Schneider S2 cells and that deletion of the Sp1 sites resulted in a loss of promoter activity in chicken HD11 cells. The results indicate that transcription factors of the Sp family play an important role in the transcriptional regulation of the chicken GAS41 gene.

We used a rapid and simple protocol using lysolecithin for mapping HS sites in vivo. The protocol is based on partial digestion with DNase I of exponentially growing cells following permeabilization by short treatment with lysolecithin. Using this protocol, we analyzed the chromatin structure of the region surrounding two overlapping elements, an origin of bidirectional DNA replication and the GAS41 promoter, in chicken myelomonocytic HD11 cells arrested in G0, G1 and S phases as well as at the G1/S border. The results show that the chromatin of this region became more nuclease sensitive when cells were arrested in G1 phase and that this change in chromatin structure was reversible after the cells began to enter S phase.