Donepezil-Enhanced Learning Performance In Selected Adult Zebrafish – A Pilot Study
Donepezil is a reversible and highly centrally selective inhibitor of AChE enhancing cognitive functioning for patients with mild to moderate Alzheimer's disease and in rodents as well. Zebrafish have been successfully used to investigate neurobehavioral bases of neuropsychiatric conditions, such as AD. Models in zebrafish can serve as early-stage screening tool for the discovery of CNS drugs by providing increased throughput of in vivo analysis of novel compounds for neurological disorders. Although Donepezil has already been tested in zebrafish to study its effects on AD-like disorders, it has been understudied in this species. The passive avoidance paradigm is based on the natural photophobia of rodents. Similarly to rodents zebrafish has a natural preference for dark environment also when examined in laboratory. The aim of the present study was to examine the memory enhancer effects of Donepezil in selected groups of zebrafish using black&white tank as an apparatus for passive avoidance task testing. Selected adult zebrafish with lower learning performance were found to be suitable for testing memory enhancer drugs like Donepezil by applying passive avoidance task test protocol. Effective experimental design and dose of drug were found to be comparable to those used in rats.
This study confirms that the zebrafish (Danio rerio) which has become a widely used vertebrate model organism for drug discovery because of its high fecundity, transparent embryos and larvae, morphological and hysiological similarity to mammals in the past few decades is also suitable for testing memory enhancer drugs like Donepezil by applying passive avoidance task test protocol similar to those used in rodents.
Introduction
Someone in the world develops dementia every 3 seconds. There are about 50 million people worldwide living with dementia and this number will almost double every 20 years, reaching 75 million in 2030 and 131.5 million in 2050. There are many different types of dementia, including e.g. Alzheimer's disease dementia, and Lewy body-, Frontotemporal-, Vascular-, Parkinson's disease dementias, etc. Alzheimer's disease (AD), however, is the most well-known and common form of dementia. Donepezil is a reversible and highly centrally selective inhibitor of AChE that delays the breakdown of acetylcholine released into synaptic clefts, thereby enhancing cholinergic transmission . Donepezil among other cholinesterase inhibitor drugs has been found to be effective in enhancing cognitive functioning for patients with mild to moderate AD.
In AD research, the rat has for decades been a very important model, for instance in studies on cholinergic dysfunction and memory impairment which played a crucial role in the development of the cholinesterase inhibitor drugs that are currently in use. Donepezil can enhance cognitive function recovery in AD model rats as well. Short-term memory is severely affected early on the course of Alzheimer's disease as well as in the normal aging process, and it is reported that short-term memory is preferentially impaired in aged animals. Orally administered Donepezil (0.5 mg/kg) significantly ameliorated the aged-related short-term memory impairment in rats. Zebrafish possess a great deal of similarity to mammals and are an extremely useful model for screening compounds at several stages of the drug discovery process. Efficacy models in zebrafish can serve as early-stage screening tool for the discovery of CNS drugs by providing increased throughput of in vivo analysis of novel compounds for neurological disorders.
Zebrafish have been successfully used to investigate neurobehavioral bases of various neurological and neuropsychiatric conditions, such as epilepsy, AD, PD, schizophrenia, affective disorders and drug-related disorders. Donepezil has been used in zebrafish to study its effects on AD-like disorders and symptoms, however it has been understudied in this species. Zebrafish have detectable acetylcholinesterase in many primary neurons that is highly conserved to that found in humans. By examining the startle response of larval zebrafish to noise signals, it was found that fish exposed to donepezil increased their startle response and decreased habituation, a similarly to rodents. The innate aversion to brightly illuminated areas is well-known in rodents. Moreover the inhibitory avoidance paradigm is based on inhibition of natural photophobia in rodents. Similarly to rodents zebrafish has a natural preference for dark environment when examined in laboratory using e.g. black& white test tank. Thus zebrafish can also be trained and tested in such passive avoidance apparatus in which the fish are able to cross to the dark side of the tank when the partition is raised. A pulsed electric shock is administered to animals as part of the passive avoidance training (conditioning). The training protocol is repeated during test session (extinction) except that no shock is administered.
The aim of the present study was to examine the memory enhancer effects of Donepezil in selected groups of zebrafish using black& white tank as an apparatus for passive avoidance task testing.
Method
Experimental animal and test apparatus
Adult (8 month old) healthy wild-type commercial zebrafish of both sexes were used for experiments (strain was kindly provided by Dr. Ferenc Baska, University of Veterinary Medicine, Budapest, Hungary). Fish were kept in glass tanks of 100 l using commercial food for daily feeding and 14 h :10 h light-dark cycle prior to the experiments. Water temperature was maintained at 24°C. For the analysis of learning performance, a group of randomly sampled adult zebrafish (30 individuals of both sexes) were tested. Zebrafish were trained and tested individually for passive avoidance task in a glass tank (19 cm L, 10 cm W, 7,5 cm H) with black and white compartments separated by a sliding partition. The two compartments of the glass tank were covered with plastic self-adhesive film of black or white colors, covering walls, floor and the corresponding sides as well. Two wire electrodes extending through each opposing side wall on the black compartment were attached to an electric stimulator.
Baseline test of dark preference
Two groups of naive adult zebrafish (30 individuals/each group) were tested in the above described black & white tank without partition wall in order to measure baseline dark preference on Day1. Zebrafish were placed and tested one by one in the glass tank. After 1 min of habituation time span of staying in the black compartment was recorded in seconds up to 300 seconds (5 min) and the percentage time span in black compartment was calculated as baseline data for statistical analysis. Conditioning for passive avoidance taskFor conditioning zebrafish were placed individually into the white compartment of the glass tank while the partition between compartments was closed. After 1 min of habituation the partition was raised, allowing fish to cross to the black side of the tank through the opening. A pulsed electric shock (2V) was administered each time when the tested fish entered the black side with its entire body. Time span of staying in the black compartment was recorded in seconds up to 300 seconds (5 min) and the percentage of time span in black compartment was calculated data for statistical analysis. Conditioning for passive avoidance task was carried out 3 times on consecutive days.
Extinction and selection of bad learners
Passive avoidance task tests on Day4 were carried out similarly to conditioning protocol but without the administration of electric shock. The effects of extinction were recorded so that the percentage of time span in black compartment was calculated as described above. Individuals showing 20% or higher ratio of time spent in black compartment upon extinction were considered as “bad learners” and were selected for further studies. Learning performance of tested zebrafish individuals was ranked so that dark preference data were sorted in ascending order and selection criteria was set thereafter.
Passive avoidance task tests within bad learners group
Selected group zebrafish (“bad learners”) showing dark preference higher than 20% in extinction period (on Day4) was used for further studies. Zebrafish individuals were tested according to the experimental protocol described above either with or without pre-treatment using Donepezil (Donepezil hydrochloride, MW: 415.95, Merck). Donepezil was dissolved in aquarium-treated H2O (0,5 mg/L). Zebrafish were treated by placing into a pre-treatment tank containing Donepezil for 20 minutes (at 60 minutes prior to each conditioning). Passive avoidance task tests were carried out in triplicate.Statistical analysisData were analyzed using GraphPad Prism 5 software and are shown as mean ± SEM. Time span ratios (%) in black compartment during testing period of 5 min (300 sec) in recorded in baseline (BL) and extinction (E) experiments were compared using independent t-test with Welch’s correction. Data derived from extinction experiments (E groups: untreated and Donepezil-treated) were compared using two-way ANOVA. In all comparisons, p < 0.05 was considered to indicate statistical significance.
Results
Zebrafish group with lower learning performance (termed as “bad learners”) could be identified and set up so that dark preference data derived from individuals showing 20% or higher ratio of time spent in black compartment upon extinction were sorted in ascending order and were selected for further passive avoidance tests. Control group (unselected) and Donepezil-treated group (“bad learners”) showed lower dark preference (time span in black compartment) in extinction (E) period on Day4 when compared to baseline (BL) data. Differences were found to be highly significant in both cases (P < 0.0001, Figure 1). Dark preference results of untreated group of “bad learners” recorded in extinction period was not significantly different from corresponding baseline data (Figure 1). Conditioned and Donepezil-treated zebrafish individuals of “bad learners” group showed significantly lower dark preference in extinction period when compared untreated ones from the same group (P < 0.01, Figure 2).
Discussion
Alzheimer's disease (AD) is the most well-known and common form of dementia. Donepezil among other cholinesterase inhibitor drugs has been found to be sufficiently effective in improving cognitive functioning for patients with mild and moderate AD. Donepezil can promote cognitive function recovery in AD model rats. Orally administered Donepezil (0.5 mg/kg) significantly ameliorated the aged-related short-term memory impairment in rats. Zebrafish have been successfully used to investigate neurobehavioral bases of various neurological and neuropsychiatric conditions, such as epilepsy, AD, PD, schizophrenia, affective disorders and drugrelated disorders. Donepezil has been used in zebrafish to study its effects on AD-like pathology and symptoms, but it has been understudied in the species, with only one study currently published. The innate aversion to brightly illuminated areas is well-known in rodents. Moreover the inhibitory avoidance paradigm is based on inhibition of natural photophobia in rodents.
Similarly to rodents zebrafish has a natural preference for the black compartment when examined in laboratory using e.g. black& white test tank. Thus zebrafish can also be trained and tested in such passive avoidance apparatus in which the fish are able to cross to the dark side of the tank when the partition is raised. A pulsed electric shock is administered to animals as part of the conditioning. Selected zebrafish group with lower learning performance (termed hereby as “bad learners”) could be identified and set up successfully so that dark preference data derived from individuals showing 20% or higher ratio of time spent in black compartment upon extinction were sorted in ascending order and were selected for further passive avoidance tests. By using selected individuals with lower learning performance for memory enhancement studies the co-application of drug-induced learning impairment (using e.g. Scopolamine as suggested by Kim, Lee, Kim, Jung & Lee, 2010 and others) can be avoided. Orally administered Donepezil (0.5 mg/kg) significantly ameliorated the aged-related short-term memory impairment in rats.
According to the above described results Donepezil-treatment in similar dose (0,5 mg/l, dissolved in the pretreatment aquarium water as described by Parker, Brock, Sudwarts, Teh, Combe & Brennan, 2015) could significantly enhance the learning performance of selected, so called “bad learners” zebrafish comparing either to unselected or to untreated and selected individuals. Further and larger serials of experiments are needed for the optimization of each steps of the above described protocol in order to set up an appropriate procedure based on selected zebrafish which is suitable for the screening and the discovery of CNS therapeutics allowing increased throughput of in vivo analysis of novel compounds for neurological disorders as Best & Alderton (2008) suggested. In order to achieve appropriate rate of reproducibility the author of the current study suggests the application of well-characterized zebrafish populations as bases of selection in further studies which are available e.g. at “Zebrafish International Resource Center.
Conclusion
Selected adult zebrafish with lower learning performance are suitable for testing memory enhancer drugs like Donepezil by applying passive avoidance task test protocol similar to those used in rodents. Effective experimental design and dose of drug were found to be comparable to those used in rats. Larger studies and the involvement of further reference drugs together with well-characterized zebrafish lines are necessary for establishing appropriately validated zebrafish test protocols for CNS drug discovery and preclinical evaluation in case of cognitive enhancer drug candidates. AcknowledgementsThe author wishes to express his gratitude to Dr. Ferenc Baska (University of Veterinary Medicine, Budapest, Hungary) for providing the zebrafish broodstock.
