Inhibitory Avoidance Learning in Zebrafish (Danio Rerio): Effects of Shock Intensity and Unraveling Differences in Task Performance

Abstract

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.

Unpredictable chronic stress decreases inhibitory avoidance learning in Tuebingen Long-Fin zebrafish (Danio rerio Hamilton): stronger effects in the resting phase than in the active phase

“…Interestingly, grβ, mr, bdnf and cnr1 loaded onto the same component in the PCA. In our earlier experiments, concerning the effects on inhibitory avoidance learning of different shock intensities, grβ and bdnf also loaded onto the same component (Manuel et al, 2014), supporting the present data. The loading of these genes onto a single component, and their similar changes following 7 nights of UCS, suggest that they may be associated in the context of a high stress load as deduced from the strong increase in whole-body cortisol following 7 nights of UCS.…”

“…We observed no behavioural differences between groups during exposure to the shock: all groups showed erratic movements, seeking to escape or jumping out of the water when the shock was applied (Manuel et al, 2014). In addition, we did not observe any abnormal behaviour, such as periods of freezing behaviour (Kalueff et al, 2013), on any of the test days.…”

“…In this paradigm, fish learn to avoid swimming from a white to a black compartment to avoid an electric shock (Blank et al, 2009;Ng et al, 2012). Recently, we studied the effects of different shock intensities on inhibitory avoidance learning in an in-house-reared Tuebingen long-fin (TLF) zebrafish strain and showed associated changes in the regulation of a number of genes involved in anxiety, fear, learning and memory when measured 2 h following the task, including grα (Manuel et al, 2014). In rats it has been shown that UCS has a stronger impact on physiology and behaviour when given during the resting phase than during the active (or awake) phase (Aslani et al, 2014).…”

“…We predicted that the UCS protocol applied during the night-time would have a stronger impact compared with application during the active phase [based on studies on rats (Aslani et al, 2014)]. Further, we extended our inhibitory avoidance protocol (Manuel et al, 2014) with a second training session as multiple training sessions may improve the learning performance of zebrafish (Arthur and Levin, 2001;Williams et al, 2002). We therefore predicted that an additional training session would provide Unpredictable chronic stress decreases inhibitory avoidance learning in Tuebingen long-fin zebrafish: stronger effects in the resting phase than in the active phase a better resolution of the effects of UCS on inhibitory avoidance learning.…”

“…As in an earlier study (Manuel et al, 2014), we assessed an array of genes in the telencephalon. Genes were selected on the basis of their relation to the stress response, anxiety and fear, as well as fear conditioning (de Kloet et al, 1999;Liu et al, 2004;Maximino et al, 2013;Upadhya et al, 2013) and include: brain derived neurotrophic factor (bdnf), serotonin receptor 1ab (htr1ab), cocaine-and amphetamine-regulated transcript 4 (cart4), corticotropin-releasing factor (crf), crf-binding protein (crf-bp), cannabinoid receptor 1 (cnr1), grα, grβ and mineralocorticoid receptor (mr).…”