A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes.

Hundreds of human genes are associated with neurological diseases, but translation into tractable biological mechanisms is lagging. Larval zebrafish are an attractive model to investigate genetic contributions to neurological diseases. However, current CRISPR-Cas9 methods are difficult to apply to large genetic screens studying behavioural phenotypes. To facilitate rapid genetic screening, we developed a simple sequencing-free tool to validate gRNAs and a highly effective CRISPR-Cas9 method capable of converting >90% of injected embryos directly into F0 biallelic knockouts. We demonstrate that F0 knockouts reliably recapitulate complex mutant phenotypes, such as altered molecular rhythms of the circadian clock, escape responses to irritants, and multi-parameter day-night locomotor behaviours. The technique is sufficiently robust to knockout multiple genes in the same animal, for example to create the transparent triple knockout crystal fish for imaging. Our F0 knockout method cuts the experimental time from gene to behavioural phenotype in zebrafish from months to one week.

Whole-brain mapping of socially isolated zebrafish reveals that lonely fish are not loners.

The zebrafish was used to assess the impact of social isolation on behaviour and brain function. As in humans and other social species, early social deprivation reduced social preference in juvenile zebrafish. Whole-brain functional maps of anti-social isolated (lonely) fish were distinct from anti-social (loner) fish found in the normal population. These isolation-induced activity changes revealed profound disruption of neural activity in brain areas linked to social behaviour, social cue processing, and anxiety/stress. Several of the affected regions are modulated by serotonin, and we found that social preference in isolated fish could be rescued by acutely reducing serotonin levels.

Socialising is good for people's mental health and wellbeing. The connections and relationships that we form can make us more resilient and healthier. Researchers also know that prolonged periods of social isolation, and feeling lonely, can be detrimental to our health, especially in early childhood. The paradox is that loneliness often results in an even lower desire for social contact, leading to further isolation. But not everyone craves social contact. Some people prefer to be alone and feel more comfortable avoiding social interaction. Zebrafish display the same social preferences. This, along with their transparent brains, makes them a useful model to study the links between social behaviour and brain activity. Like humans, zebrafish are social animals, with most fish taking a strong liking to social interactions by the time they are a few weeks old. A small number of 'loner' fish, however, prefer to avoid interacting with their siblings or tank mates. And so, if loneliness quells the desire for more social contact, the question becomes, does isolation turn otherwise social fish into loners? Here, Tunbak et al. use zebrafish to study how social isolation changes brain activity and behaviour. Social fish were isolated from others in the tank for a few days. These so-called 'lonely fish' were then allowed back in contact with the other fish. This revealed that, after isolation, previously social fish did avoid interacting with others. With this experimental set-up, Tunbak et al. also compared the brains of lonely and loner fish. When fish that prefer social interaction were deprived of social contact, they had increased activity in areas of the brain related to stress and anxiety. These lonely fish became anxious and very sensitive to stimuli; and their brain activity suggested that social interaction became overwhelming rather than rewarding. Positively, the lonely fish quickly recovered their normal, social behaviour when given a drug that reduces anxiety. This work provides a glimpse into how human behaviour could be affected by lengthy periods in isolation. These results suggest that humans could feel anxious upon returning to normal life after spending a long time alone. Moreover, the findings show the impact that social interaction and isolation can have on the young, developing brain.