Zebrafish larvae were observed in individual wells of a 12-well plate for one hour a day at 4, 5, 6 and 7 dpf in two experiments. In Experiment 1, the same larvae were observed for four consecutive days beginning on post-fertilization day 4; in Experiment 2, different larvae from the same egg collection were observed at each of the 4 ages tested. Automated images collected every 6 s were analyzed for information about larval location, orientation and general activity. Three principal findings emerged from our studies. First, both experiments found significant behavioral differences in resting, strength of preferred quadrant and to a lesser extent in activity between 4 dpf and older larvae. Second, in both experiments, there were behavioral biases for the edge region and an outward facing orientation across all four ages tested. Third, there were differences between the two experiments in space use patterns of older larvae and in visual lateralization of 7 dpf larvae. Collectively, these findings reflect both maturational and experiential effects on larval zebrafish behavior.
Regarding maturational factors, our data suggest two ways in which the behavioral profile of 4 dpf larvae is clearly distinguishable from that of 5 to 7 dpf larvae. In both experiments, 4 dpf larvae rested significantly more than the 5, 6, and 7 dpf larvae, and they were located significantly more often in their highest use quadrant than older larvae. These results are consistent with other reports of high rates of resting in larvae prior to inflation of the swim bladder and the emergence of spontaneous swimming at 5 dpf (Granato et al., 1996
; Thirumalai and Cline, 2008). The novel finding that this high rate of resting is associated with a reduction in overall spatial distribution has important implications both for the presentation of stimuli to larvae younger than 5 dpf and for the assessment of their effects in studies of learning, memory and cognition.
We also found evidence in line with previous reports that younger larvae are less active than older larvae. In both experiments, 4 dpf larvae moved less than older larvae although not all comparisons were statistically significant. Increasing the size of the testing arena might enhance detection of statistically significant age-related changes in activity. The small testing arenas used in our experiments may have limited the opportunity for exploratory behavior in the older larvae, especially when coupled with their extensive exposure and potential habituation to the wells in Experiment1 where we found no statistically significant age-related effect in amount of movement. Development of the larvae in Experiment 1 may also have been affected by the size of the testing arena which in turn could have affected activity levels. The resolution of our imaging system was insufficient to provide precise measurement of larval length because of the transparent tail tip. Future research will need to address this issue. Alternatively, it is possible that features of our testing arenas artificially increased activity in the 4 dpf larvae as they tried to inflate their swim bladders. If the agarose ring was not sufficiently rigid to support climbing and the water in the wells was not deep enough for the larvae to hang naturally, the 4 dpf larvae may have made frequent lateral movements as they struggled to migrate vertically to the surface. Recent observations in our laboratory confirm that agarose is a more challenging surface for a newly hatched larva to navigate than the smooth plastic wall of a multiwell plate.
Complicating statistical comparisons further is the possibility that activity in 4 dpf larvae may be inherently more variable than at any other age. Larvae at 4 dpf are making the transition from an inactive swim bladder to a fully inflated one. The timing of this transition is not precisely synchronized but is complete by 5 dpf. Swimming is topographically distinct in the periods before and after inflation of the swim bladder as the transition is made from intact touch-evoked swimming responses but negligible spontaneous swimming to a free swimming, self-feeding larva with a functional beat and glide motor system and the ability to maneuver in all three dimensions (Brustein et al., 2003
; Buss and Drapeau, 2001
; Granato et al., 1996
; Thirumalai and Cline, 2008). Despite identical egg collection and rearing protocols, there were fairly large behavioral differences between the 4 dpf larvae in our two experiments. For example, we had higher percentages of resting and lower rates of movement in Experiment 1 than in Experiment 2. Almost 20% of the 4 dpf larvae in Experiment 1 scored rest rates of 100% whereas this was true for only 5% of the 4 dpf larvae in Experiment 2. Among the factors known to affect speed of embryonic development are temperature and light (Laale, 1977
). However, even when these external factors are controlled and egg fertilization is synchronous, there are still variations in time of hatching and larval development. In future studies, whenever feasible, it would be useful to screen 4 dpf zebrafish larvae after testing to stage inflation of the swim bladder. This can be done by immobilizing the larvae on their side in 2% methylcellulose or by anesthetizing the larvae in tricaine and examining them under a stereo microscope.
Two clear behavioral biases emerged in our studies that were shared by larvae of all four ages tested. One was a preference for an outward orientation and the other was a consistently strong preference for the edge of the well; both were significantly above chance. The edge preference we observed confirms and extends a previous report of an edge effect in the swimming trajectories of 5 dpf but not of 3 dpf WT larvae (Thirumalai and Cline, 2008). Observations of wall seeking behavior (thigmotaxis) have also been reported for adult zebrafish (Anichtchik et al., 2004
; Peitsaro et al., 2003
). For example, Peitsaro et al. (2003)
found that individually tested adult zebrafish tended to avoid the center of an unfamiliar circular tank (22 cm in diameter with water 8 cm deep) and to swim in a circular pattern by following the wall of the tank. For 4 dpf larvae, the observed behavioral biases may be related exclusively to their drive to reach the air-water boundary to inflate their swim bladder. On the other hand, we cannot discount the possibility that an explanation for thigmotactic behavior in adult zebrafish and larvae with fully inflated swim bladders may also apply to the 4 dpf larvae.
Three functional explanations that could account for thigmotactic behavior in older larvae and adult zebrafish are shelter seeking, foraging, and predator avoidance. Two results, however, suggest that wall seeking is unlikely to be either a shelter seeking strategy or a foraging strategy. One result is our novel finding that the edge effect was attenuated in larvae after 24 hours exposure to the test wells in Experiment 1. Specifically, we found that the mean percent observations in the center of the well showed a two-fold increase from approximately 10 to 20 per cent between 4 and 5 dpf in Experiment 1 but were consistently around 10 per cent at all ages tested in Experiment 2. The other result is Lockwood et al.'s (2004)
finding that wall seeking was not observed in the presence of a group of 10 conspecifics. There is no obvious reason why shelter seeking or foraging would be attenuated by exposure to the test well or by the presence of other larvae. Instead, the effect of these manipulations favors a widely held view that thigmotactic behavior is indicative of anxiety and may function as a defensive strategy to avoid detection by predators. Familiar environments provide security and social groups afford protection against predation.
Thigmotactic behavior in rodents has generally been interpreted as evidence of anxiety because it is increased by anxiogenic drugs (dexamphetamine, pentylenetetrazole, yohimbine, idazoxan), reduced by anxiolytic drugs (buspirone, phenobarbital), and attenuated by familiarization to the novel testing arena or context (Choleris et al., 2001
; Simon et al., 1994
; Treit and Fundytus, 1989
). Related effects have been observed in adult zebrafish. Peitsaro et al. (2003)
found that adult zebrafish spent more time in the center of a tank after injection with α-fluoromethylhistidine, a chemical that lowers histamine levels in the brain. Reduced histamine production has been linked to an increase in anxiolytic behaviors in mammals (Frisch et al., 1998
; Yanai et al., 1998
). Peitsaro et al. (2003)
also found that when control fish, treated with saline, were reintroduced to the tanks 3 days after their first exposure, they spent more time in the center of the tank than they did on the first day. They suggested that the adult fish recognized the environment and were less anxious because of reduced neophobia. Additional studies are needed to determine if that interpretation or one involving generalized state changes such as sensitization applies to our related finding that zebrafish larvae also spent more time in the center of a well with which they were familiar.
Prolonged exposure to the testing environment had one other effect aside from its influence on exploration of the center of the well and levels of overall activity. In Experiment 2, 7 dpf larvae unfamiliar with the wells displayed a significant preference for a clockwise orientation whereas 7 dpf larvae in Experiment 1 that had been exposed to the wells since 4 dpf did not show any such preference. A clockwise orientation of 7 dpf zebrafish larvae has previously been observed in a two-fish assay and was thought to reflect a right-eye preference in social interactions (Creton, 2009
). However, the present finding of a similar clockwise orientation with just one fish per well suggests an alternative possibility that 7 dpf larvae may simply have a left eye preference for evaluating the novel agarose edge, with or without another fish in the well. It will be of interest to evaluate the connection between visual lateralization and learning by examining the consequence of a left-eye preference for viewing the agarose ring on subsequent exploratory behavior in the well.
The results of our studies have three broad implications for zebrafish behavioral studies. First, based on our data, we strongly recommend using 6 or 7 dpf larvae for examining the effects of drug treatments and chemical mutagenesis on locomotor activity. In both experiments, between-subject variability was relatively minimal at these ages on all of our behavioral measures including activity. Second, our analysis suggests multiple ways to expand behavioral measures of space use that could be used to detect more subtle differences among pharmacological compounds in this simple testing situation. Third, our data suggest a relatively simple but powerful method and behavioral index for studying motivational and cognitive processes in individual larval zebrafish. Vertebrate studies of classical conditioning processes have used modulation of overall activity as an index of motivational states and cognitive expectancies (Bouton and Bolles, 1980
; Grau and Rescorla, 1984
; Mustaca et al., 1991
). By supplementing activity measures with those of space use developed in the current studies, we have been able to develop medium- and high-throughput assays for measuring avoidance behavior (Pelkowski et al., in preparation
) and recognition memory for contextual stimuli (Colwill et al., in preparation
). Studies aimed at understanding the development of learning and memory in both wild-type and mutant zebrafish will be able to exploit the behavioral repertoire that we have established.