Zebrafish (Danio rerio Hamilton) are increasingly used as a model to study the effects of chronic stress on brain and behaviour. In rodents, unpredictable chronic stress (UCS) has a stronger effect on physiology and behaviour during the active phase than during the resting phase. Here, we applied UCS during the daytime (active phase) for 7 and 14 days or during the night-time (resting phase) for 7 nights in an in-house-reared Tuebingen long-fin (TLF) zebrafish strain. Following UCS, inhibitory avoidance learning was assessed using a 3 day protocol where fish learn to avoid swimming from a white to a black compartment where they will receive a 3 V shock. Latencies of entering the black compartment were recorded before training (day 1; first shock) and after training on day 2 (second shock) and day 3 (no shock, tissue sampling). Fish whole-body cortisol content and expression levels of genes related to stress, fear and anxiety in the telencephalon were quantified. Following 14 days of UCS during the day, inhibitory avoidance learning decreased (lower latencies on days 2 and 3); minor effects were found following 7 days of UCS. Following 7 nights of UCS, inhibitory avoidance learning decreased (lower latency on day 3). Whole-body cortisol levels showed a steady increase compared with controls (100%) from 7 days of UCS (139%), to 14 days of UCS (174%) to 7 nights of UCS (231%), suggestive of an increasing stress load. Only in the 7 nights of UCS group did expression levels of corticoid receptor genes (mr, grα, grβ) and of bdnf increase. These changes are discussed as adaptive mechanisms to maintain neuronal integrity and prevent overload, and as being indicative of a state of high stress load. Overall, our data suggest that stressors during the resting phase have a stronger impact than during the active phase. Our data warrant further studies on the effect of UCS on stress axis-related genes, especially grβ; in mammals this receptor has been implicated in glucocorticoid resistance and depression.

Recently, we established an inhibitory avoidance paradigm in Tupfel Long-Fin (TL) zebrafish. Here, we compared task performance of TL fish and fish from the AB strain; another widely used strain and shown to differ genetically and behaviourally from TL fish. Whole-body cortisol and telencephalic gene expression related to stress, anxiety and fear were measured before and 2 h post-task. Inhibitory avoidance was assessed in a 3-day paradigm: fish learn to avoid swimming from a white to a black compartment where a 3V-shock is given: day 1 (first shock), day 2 (second shock) and day 3 (no shock, sampling). Tupfel Long-Fin fish rapidly learned to avoid the black compartment and showed an increase in avoidance-related spatial behaviour in the white compartment across days. In contrast, AB fish showed no inhibitory avoidance learning. AB fish had higher basal cortisol levels and expression levels of stress-axis related genes than TL fish. Tupfel Long-Fin fish showed post-task learning-related changes in cortisol and gene expression levels, but these responses were not seen in AB fish. We conclude that AB fish show higher cortisol levels and no inhibitory avoidance than TL fish. The differential learning responses of these Danio strains may unmask genetically defined risks for stress-related disorders.

The inhibitory avoidance paradigm allows the study of mechanisms underlying learning and memory formation in zebrafish (Danio rerio Hamilton). For zebrafish, the physiology and behavior associated with this paradigm are as yet poorly understood. We therefore assessed the effects of environmental enrichment and fish age on inhibitory avoidance learning. Fish raised in an environmentally enriched tank showed decreased anxiety-like behavior and increased exploration. Enrichment greatly reduced inhibitory avoidance in 6-month (6M)- and 12-month (12 M)-old fish. Following inhibitory avoidance, telencephalic mRNA levels of proliferating cell nuclear antigen (pcna), neurogenic differentiation (neurod), cocaine- and amphetamine-regulated transcript 4 (cart4), and cannabinoid receptor 1 (cnr1) were lower in enriched-housed fish, while the ratios of mineralocorticoid receptor (nr3c2)/glucocorticoid receptor α [nr3c1(α)] and glucocorticoid receptor β [nr3c1(β)]/glucocorticoid receptor α [nr3c1(α)] were higher. This was observed for 6M-old fish only, not for 24-month (24 M) old fish. Instead, 24 M-old fish showed delayed inhibitory avoidance, no effects of enrichment, and reduced expression of neuroplasticity genes. Overall, our data show strong differences in inhibitory avoidance behavior between zebrafish of different ages and a clear reduction in avoidance behavior following housing under environmental enrichment.

The zebrafish (Danio rerio) is increasingly used as a model in neurobehavioral and neuroendocrine studies. The inhibitory avoidance paradigm has been proposed as tool to study mechanisms underlying learning and memory in zebrafish. In this paradigm subjects receive a shock after entering the black compartment of a black-white box. On the next day, latency to enter the black compartment is assessed; higher latencies are indicative of increased avoidance learning. Here, we aimed to understand the effects of different shock intensities (0, 1, 3, and 9 V) and to unravel variation in inhibitory avoidance learning in an in-house reared Tuebingen Long-Fin zebrafish (D. rerio) strain. While median latencies had increased in the 1, 3, and 9 V groups, no increase in median latency was found in the 0 V group. In addition, higher shock intensities resulted in a higher number of avoiders (latency ≥180 s) over nonavoiders (latency <60 s). Both changes are indicative of increased avoidance learning. We assessed whole-body cortisol content and the expression levels of genes relevant to stress, anxiety, fear, and learning 2 h after testing. Shock intensity was associated with whole-body cortisol content and the expression of glucocorticoid receptor alpha [nr3c1(alpha)], cocaine- and amphetamine-regulated transcript (cart4), and mineralocorticoid receptor (nr3c2), while avoidance behavior was associated with whole-body cortisol content only. The inhibitory avoidance paradigm in combination with measuring whole-body cortisol content and gene expression is suitable to unravel (genetic) mechanisms of fear avoidance learning. Our data further show differences in brain-behavior relationships underlying fear avoidance learning and memory in zebrafish. These findings serve as starting point for further unraveling differences in brain-behavior relationships underlying (fear avoidance) learning and memory in zebrafish.

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