A search for effective reinforcers in appetitive conditioning for adult zebrafish: Ecologically relevant unconditioned stimuli.

Learning and memory related brain disorders represent a large unmet medical need. Laboratory studies with animals may model brain disorders and facilitate uncovering their mechanisms. The zebrafish has been proposed for such studies. However, numerous factors that influence performance in learning tasks have yet to be understood in zebrafish. One such factor is what motivates zebrafish. Here we introduce a novel reinforcer, an ecologically relevant unconditioned stimulus (US). We placed a photograph of gravel underneath quarter of the bottom of an experimental tank on one side and also positioned artificial plants there, the "natural" US. First, we showed that this stimulus was preferred by zebrafish. Next, we investigated whether this stimulus could serve as US for associative learning. We marked the walls of the tank on the side where the US was presented with red paper, the conditioned stimulus (CS+) we found neutral before, and we also marked the walls on the other side of the tank where no US was placed with blue paper (CS-). In addition to fish receiving this "paired" training, we also ran unpaired training with another group of zebrafish, in which the fish saw the US associated with blue and red in a random manner. After having trained the fish in this manner, we tested the performance of the paired and unpaired group of zebrafish in a memory probe trial during which no US was present, and only the CSs (blue and red walls) were shown. We found the paired group of zebrafish to show significant preference for the CS+, as they spent more time and swam closer to the red side compared to the unpaired group and compared to chance. We conclude that ecologically relevant stimuli can serve as efficient US in appetitive conditioning of zebrafish.

Behavioral Effects of Buspirone in Juvenile Zebrafish of Two Different Genetic Backgrounds.

Anxiety continues to represent a major unmet medical need. Despite the availability of numerous anxiolytic drugs, a large proportion of patients do not respond well to current pharmacotherapy, or their response diminishes with chronic drug application. To discover novel compounds and to investigate the mode of action of anxiolytic drugs, animal models have been proposed. The zebrafish is a novel animal model in this research. It is particularly appropriate, as it has evolutionarily conserved features, and drug administration can be employed in a non-invasive manner by immersing the fish into the drug solution. The first step in the analysis of anxiolytic drugs with zebrafish is to test reference compounds. Here, we investigate the effects of buspirone hydrochloride, an anxiolytic drug often employed in the human clinic. We utilize two genetically distinct populations of zebrafish, ABSK, derived from the quasi-inbred AB strain, and WT, a genetically heterogeneous wild-type population. We placed juvenile (10-13-day, post-fertilization, old) zebrafish singly in petri dishes containing one of four buspirone concentrations (0 mg/L control, 5 mg/L, 20 mg/L or 80 mg/L) for 1 h, with each fish receiving a single exposure to one concentration, a between subject experimental design. Subsequently, we recorded the behavior of the zebrafish for 30 min using video-tracking. Buspirone decreased distance moved, number of immobility episodes and thigmotaxis, and it increased immobility duration and turn angle in a quasi-linear dose dependent but genotype independent manner. Although it is unclear whether these changes represent anxiolysis in zebrafish, the results demonstrate that behavioral analysis of juvenile zebrafish may be a sensitive and simple way to quantify the effects of human anxiolytic drugs.

Behavioral effects of acute ethanol in larval zebrafish (D. rerio) depend on genotype and volume of experimental well.

Ethanol consumption is a worldwide problem. Sensitivity to acute effects of ethanol influences the development of chronic ethanol abuse and ethanol dependence. Environmental and genetic factors have been found to contribute to differential effects of acute ethanol. Animal models have been employed to investigate these factors. An increasingly frequently utilized animal model in ethanol research is the zebrafish. A large proportion of ethanol studies with zebrafish have been conducted with adult zebrafish. However, high throughput drug and mutation screens are particularly well adapted to larval zebrafish. These studies are often carried out using the 96-well-plate that allows monitoring large numbers of fish efficiently. Here, we investigate the effects of acute (30 min long) ethanol exposure in 8-day post-fertilization (dpf) old zebrafish. We compare four genetically distinct populations (strains) of zebrafish, measuring numerous parameters of their swim path in two well sizes, i.e., in the 96-well-plate (small volume wells) and in the 6-well-plate (large volume wells). In general, we found that the highest dose of ethanol (1% vol/vol) reduced swim speed, increased duration of immobility, increased turn angle, and increased intra-individual variance of turn angle, while the intermediate dose (0.5%) had a less strong effect, compared to control. However, we also found that these ethanol effects were strain dependent and, in general, were better detected in the larger volume well. We conclude that larval zebrafish are appropriate for quantification of acute ethanol effects and also for the analysis of environmental and genetic factors that influence these effects. We also speculate that using larger wells will likely increase sensitivity of detection and precision in screening applications.

Exposure of parents to alcohol alters behavior of offspring in zebrafish.

Alcoholism and alcohol abuse represent a significant medical and societal problem, and have been thoroughly investigated in humans as well as using animal models. A less well understood aspect of alcohol related disorders is the possible effect of this drug on offspring whose parents were exposed prior to conception. The zebrafish has been successfully employed in alcohol research, however, the effect of exposing the parents to alcohol before fertilization of the eggs on offspring has not been demonstrated in this species. In this proof of concept study, we attempt to address this hiatus. We exposed both adult male and female zebrafish to 0.0% (control) or 0.5% (vol/vol) alcohol chronically for 7 days, subsequently bred the fish within their respective treatment group, collected the fertilized eggs, allowed them to develop, and tested the behavior of free-swimming offspring at their age of 7-9 days post-fertilization. We conducted the analysis in two genetically distinct quasi-inbred strains of zebrafish, AB and TL. Although gross morphology and general activity of the fish appeared unaffected, we found significant behavioral alterations in offspring of alcohol exposed parents compared to offspring of control parents in both strains. These alterations included robustly increased duration and reduced frequency of immobility, increased turn angle, and increased intra-individual variance of turn angle in offspring of alcohol exposed parents in both strains. The mechanisms underlying these behavioral effects or whether the effects are due to exposure of the father, the mother, or both to alcohol are unknown. Nevertheless, our results now set the stage for future studies with zebrafish that will address these questions.

Fracture Resistance of Anterior CAD/CAM Nanoceramic Resin Endocrowns with Different Preparation Designs / Resistência à Fratura de Endocrowns de Resina Nanocerâmica CAD / CAM Anterior Com Diferentes Projetos de Preparação

Objective: The aim of the study was to evaluate the effect of extension of endocrown in pulp space and the effect of ferrule on the fracture resistance of anterior endocrowns made of nano-ceramic resin blocks. Material and Methods: Twenty-eight freshly extracted human upper central incisor teeth were prepared to receive CAD/CAM nanoceramic resin endocrowns with four different designs. The specimens were divided into two groups (n = 14) according to the extension of the endocrown inside root canal (A:short and B:long), then each group was further subdivided into another two subgroups (n = 7) according to the presence or absence of ferrule effect (1:ferrule and 2: no ferrule). Endocrowns were then fabricated using CEREC in lab and nanoceramic resin blocks (DENTSPLY Sirona, Germany) and LAVA ultimate (3M ESPE, Germany) blocks size 14L. Results: Two-way ANOVA showed that the ferrule (p > 0.0001) had statistically significant effect on the fracture resistance. However, the extension of the endocrown into the canal had no statistical significant effect on the fracture resistance (p = 0.837). The interactions between the independent variables (extension into the canal and ferrule) had statistically significant effect on the fracture resistance (p = 0.029). Load to fracture for group 1A was 439.53 N, 1B was 306.46 N, 2A was 516.29 N and 2B was 242.04 N. Conclusions: Fracture resistance was not improved by the long or short extensions of the endocrowns in the pulp space, however, the ferrule effect shows significant improvement of the fracture resistance of the nanoceramic resin endocrowns. (AU)

Objetivo: O objetivo deste estudo foi avaliar o efeito da extensão da coroa endocrown na câmara pulpar e o efeito férula na resistência à fratura de coroas endocrowns anteriores confeccionadas através de blocos de resina nanocerâmica. Material e Métodos: Vinte e oito dentes incisivos centrais superiores humanos recém-extraídos foram preparados para receber endocrowns de resina nanocerâmica CAD / CAM com quatro diferentes tipos de preparos. Os espécimes foram divididos em dois grupos (n = 14) de acordo com a extensão da endocrown dentro do canal radicular (A: curto e B: longo), então cada grupo foi subdividido em outros dois subgrupos (n = 7) de acordo com presença ou ausência do efeito férula (1: com férula e 2: sem férula). As endocrowns foram usinadas através do CEREC In Lab e blocos de resina nanocerâmica (DENTSPLY Sirona, Alemanha) e blocos de LAVA ultimate (3M ESPE, Alemanha) tamanho 14L. Resultados: ANOVA dois fatores demostrou que a férula (P> 0,0001) teve efeito estatisticamente significativo na resistência à fratura. No entanto, a extensão do endocrown no canal não teve efeito estatisticamente significativo na resistência à fratura (P = 0,837). As interações entre as variáveis independentes (extensão no canal e férula) tiveram efeito estatisticamente significativo na resistência à fratura (P = 0,029). A carga para fratura do grupo 1A foi de 439,53 N, 1B foi de 306,46 N, 2A foi de 516,29 N e 2B foi de 242,04 N. Conclusões: A resistência à fratura não foi melhorada pelas extensões longas ou curtas das endocrowns na câmara pulpar, no entanto, o efeito férula mostrou uma melhora significativa na resistência à fratura das coroas endocrowns de resina nanocerâmica (AU)

The effects of small but abrupt change in temperature on the behavior of larval zebrafish.

The zebrafish has become increasingly utilized in behavioral neuroscience, psychopharmacology, and behavior genetics research. However, little attention has been paid to the potential effects of environmental conditions fish are exposed to before and during behavioral testing. One such important factor is temperature, more specifically, the difference in temperature between holding and test tanks. In the current study, we bred and raised zebrafish of the AB strain in 28 °C water for 7 days until they were free swimming. On the 7th day we placed each experimental subject singly into the testing well with water set to 24 °C, 28 °C or 30 °C for 15 min and recorded the behavior of the subjects. We found significant and behavior-specific idiosyncratic effects of the employed temperature changes. For example, lowering the temperature decreased speed, but increasing the temperature did not alter it. Increased temperature reduced duration of immobility and reduced absolute turn angle, but lowered temperature did not alter these behaviors. While lowered temperature, and to a lesser degree also increased temperature, reduced intra-individual temporal variance of absolute turn angle. Furthermore, we found no change in thigmotaxis and frequency of immobility by either temperature change. Our results demonstrate the importance of temperature in behavioral studies with zebrafish and suggest that equating water temperature between holding and testing tanks is required to enhance reproducibility and replicability of results with this species.

Developmental stage and genotype dependent behavioral effects of embryonic alcohol exposure in zebrafish larvae.

Fetal Alcohol Spectrum Disorders (FASD) represent a worldwide problem. The severity and types of symptoms of FASD vary, which may be due to the genotype of the fetus and the developmental stage at which the fetus is exposed to alcohol. The most prevalent forms of FASD present less severe symptoms, including behavioral and cognitive abnormalities, and arise from exposure to low amounts of alcohol consumed infrequently. Treating or diagnosing FASD patients has been difficult because we do not understand the mechanisms underlying FASD. Animal models, including the zebrafish, have been suggested to answer this question. Here, we present a proof of concept analysis studying the behavioral effects of embryonic alcohol exposure in one-week old juvenile zebrafish. We exposed zebrafish embryos at one of five developmental stages (8, 16, 24, 32, or 40 hour post-fertilization) to 0% (control) or 1% (vol/vol) ethanol for 2 h, and tested the behavior of these fish at their age of 7-9 days post-fertilization. We employed two genetically distinct zebrafish populations, a quasi-inbred AB derivative strain, and a genetically variable WT population. We report significant developmental time and genotype dependent effects of alcohol on certain measures of motor function and/or anxiety-like responses. For example, we found embryonic alcohol exposed AB fish to swim faster, vary their speed more, stop moving more often and turn less compared to control fish, alcohol induced changes that were absent or less robust in WT fish. We conclude that our results open new avenues to the identification of genetic mechanisms that mediate or influence alcohol induced developmental alteration of brain function and behavior, which, on the long run, may allow us to identify diagnostic biomarkers and treatment options for human FASD.