How long does it take for a zebrafish to stop attacking its own reflection in the mirror? How quickly does the fish attack what it supposes to be an opponent; how long does it snap at it and pursue it with typical swimming movements over an interval of time? The longer the time span, the more “fighting spirit” the fish has. For Florian Reichmann, pharmacologist at the Medical University of Graz, the time measured in the “mirror test” was the yardstick for his investigations into the genetic influences on aggressive behaviour in zebrafish. A Schrödinger Fellow of the Austrian Science Fund FWF, Reichmann conducted research for two years in Will Norton's research group at the University of Leicester in England. The mirror test offers the advantage that all zebrafish, young or adult, male or female, can be tested without risking injury to the animals.
Aggression and histamine regulation
Zebrafish (Danio rerio) belong to the minnow family and are not only popular ornamental fish, but also a favourite of geneticists. Around 70 percent of the zebrafish genes also occur in humans in a similar form. Living in shoals, zebrafish with their high level of fecundity and rapid growth also prove to be a suitable model for behavioural research. With support from the FWF, Florian Reichmann pursued two different research approaches in his project. Firstly, in Will Norton's laboratory using the CRISPR Cas9 tool (“gene scissors”), he created a fish line that does not form an H3 receptor in the brain. This histamine receptor had been identified in previous studies as a candidate for regulating aggressive behaviour.
In order to describe possible behavioural changes in the H3 mutants or to compare them with the wild type (with H3 receptor), Reichmann then filmed the fish with high-resolution cameras. This is what he observed: “At the age of four days, the individuals were already less agile and more fearful when swimming freely. When presented with a threatening video stimulus, the fish lacking the H3 receptor also showed anxious reactions, with the adult H3 mutants being even less aggressive than the young ones.” Given that the young zebrafish are transparent, the researcher was able to measure their brain activity under the microscope using a luminescent marker. Collaboration with the University of Exeter was a crucial factor in this – as was transporting the young fish across England. In the adult fish with and without the H3 receptor, the researcher detected differences in activity in numerous brain areas. Immediately after the encounter with the mirror test, the brain tissue of both lines was examined to identify those areas that are activated by aggression.
Genetic differences in peaceful and belligerent fish
In the second research approach, Reichmann bred very “peaceful” and very “belligerent” fish lines over several generations. This enabled him to prove, among other things, that aggressive behaviour can also be inherited in fish: “After our mirror tests, we mated the most aggressive and the most peaceful fish, respectively, over four generations. Afterwards, we used RNA sequencing to look for differences in genetic activation in the genome, i.e. the entirety of the genetic material.” He found that the two lines displayed pronounced differences – in roughly 500 genes. The analysis suggests that genes related to the immune system are relevant and are more pronounced in aggressive fish. A gene for arsenic metabolism, which is still poorly characterised, was the most significant. In humans, this gene was also found to be associated with schizophrenia.
The results Reichmann and his research colleagues achieved represent an important step forward in behavioural research. They have shown that aggression in zebrafish has a strong genetic basis and that the H3 receptor plays an important role in this context. This may ultimately help to replace the tranquilizers that have been widely used up to now with tailor-made drugs to treat highly aggressive behaviour. There is, however, still a long way to go. Florian Reichmann will use the time to establish a zebrafish group back at the Medical University of Graz.
Florian Reichmann studied human medicine and completed a PhD in neuroscience at the Medical University of Graz. He completed his postdoc at the Institute of Experimental and Clinical Pharmacology and, from 2017 to 2019, in the context of an Erwin Schrödinger Fellowship, conducted research at the University of Leicester (UK). Since August 2020, he has been investigating the genetic and neurobiological basis of emotional-affective, social and cognitive behaviour in the context of neuropsychiatric disorders at the Otto Loewi Research Center at Medical University of Graz using various model organisms. The project “Genetic and environmental basis of aggression in zebrafish” received EUR 170,000 in funding from the Austrian Science Fund FWF.
Reichmann F., Rimmer N., Tilley CA et al.: The zebrafish histamine H3 receptor modulates aggression, neural activity and forebrain functional connectivity, in: Acta Physiologica (Oxford, England) 2020
Reichmann F., Pilic J., Trajanoski S., Norton WHJ: Fighting the mirror: brain transcriptome response of high and low mirror aggression zebrafish. (in review)