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Non-associative learning in larval zebrafish is mediated by the nicotinic and NMDA pathways
Habituation, where a response is reduced when exposed to a continuous stimulus is one of the simplest forms of non-associative learning (1) and has been shown in a number of organisms from sea slugs to rodents (2). However, very little has been reported in the zebrafish, a model that is gaining popularity for high-throughput compound screens (3). Furthermore, since most of the studies involving learning and memory in zebrafish have been conducted in adults (4), we sought to determine if zebrafish larvae could display non-associative learning. Having demonstrated habituation of the acoustic startle response (ASR) in 7 days post fertilisation (d.p.f.) zebrafish larvae (5), we sought to determine if this response could be modulated by compounds used for the treatment of cognitive impairment. All procedures were conducted in accordance with the Animals (Scientific Procedures) Act of 1986 ( UK) and its associated guidelines. All zebrafish experiments were performed following protocols previously described (5). Essentially, zebrafish larvae were placed in a 96-well mesh plate containing 200 µl E3 embryo medium, one fish per well, and exposed to compounds added to the E3 medium. After 24 hours of exposure, larvae in the 96-well plate were placed in a video tracking system (Tracksys Ltd) and Ethovivion software (Noldus) was used to monitor their behaviour. ASR was generated in 7 d.p.f. zebrafish larvae of the WIK strain by exposure to 50 consecutive auditory tones of 200 Hz at a volume of 113 dB. Group data ASR were analysed as area under the curve (AUC) for the consecutive auditory tones, and compared using one-way ANOVA and post-hoc t-tests. Donepezil (10 µM in the E3 medium), an acetylcholinesterase inhibitor, significantly increased the AUC in comparison to the control group (4-fold; p<0.0001). Mecamylamine (30 µM), a nicotinic receptor antagonist, attenuated the effect of donepezil on the habituation response with an AUC not significantly different from the control group. Atropine (100 µM), a muscarinic receptor antagonist, had no effect on the donepezil response with a similar increase of the AUC for the donepezil-atropine combination when compared to control (3.9-fold; p<0.01). Memantine (30 µM), a glutaminergic NMDA receptor antagonist, significantly increased the AUC (2.8-fold; p<0.05) and this was abolished by NMDA. Finally, the nootropic piracetam also had an effect on the zebrafish habituation response with a 3.4-fold increase of the AUC in comparison to the control group (p<0.05). As the habituation response effect by donepezil was abolished by mecamylamine but not atropine, it suggests that the donepezil cholinergic response is mediated by nicotinic rather than muscarinic receptors. The effect observed with memantine, attenuated by NMDA, along with the effect with piracetam suggests a glutamatergic response that will require further investigations using AMPA and CNQX receptors antagonists. Zebrafish larvae possess numerous advantages for medium to high throughput screening; the model described herein therefore offers the potential to screen for additional compounds for further study on cognition function.
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