Several behavioral paradigms were applied to determine the fmr1 KO effects on development of social behavior and possible therapeutic effects of n-3 PUFAs supplement. For example, the locomotor activity was used to analyze the spontaneous motor activity; novel tank was applied to quantify anxiety-like behavior; social score was detected by shoaling and shoaling preference behavior; and inhibitory avoidance task (IA) was also applied for studying the formation of associative memory. The detail procedures of each paradigm were summarized as following.
4.1. Locomotor activity
For larva, animals were placed into a 6 cm petri dish with water depth kept in 0.4 cm height for larva behavioral test. For adult fish, a transparent acrylic cylinder (20 cm in height and 24 cm in diameter) with water depth kept with 3.5 cm was used. EthoVision video tracking system (Noldus Information Technology, Leesburg, VA, USA) was applied for data acquisition and analyzing. The recording has two stages including a
5 min adaptation and following with a 10 minutes recording. The total moving distance within the 10 minutes recording was used for analyzing the locomotor activity of the zebrafish.
4.2. Novel tank test
In this behavioral test, zebrafish was placed into a transparent rectangular tank (6 cm length, 2 cm width and 6 cm in height), which water depth kept with 4 cm for larval fish test or a transparent trapezoidal tank (26 x 22 x 6x 11.5 cm, top x bottom x width x height) and water depth kept with 11.5 cm for adult fish analysis. Furthermore, two tanks were divided into upper and lower water zone. Each zone contained 1/2 of the total volume in the two different tanks. During the experiment, fish were freely swimming in the tank for 15 minutes, and we divided three different 5 minutes from total detect duration. We calculated the duration in upper zone and transferred to percentage, which value indicates anxiety level in fish (Egan et al., 2009).
4.3. Social behavior
To explore the development of social behavior, we followed behavioral procedure from our previous study (Hsu et al., 2014). There were three compartments in the behavior chamber for determining the shoaling and shoaling preference behavior in zebrafish (Fig 2).
Compartments were separated by transparent acrylic plates, and each side compartment was placed with different group of fishes, either zebrafish and/or medaka. Animals were placed into the center area (6.4 x 3.0 x 2.5
x 1.0, length x width x height x depth) and time spent in each side compartment was quantified. Briefly, the center compartment was further divided into three different zones. Both side zones, which closed to both side compartments, occupied 1/5 of the volume of center compartment and the center zone occupied 3/5 of the volume of center area. We analyzed larval fish from 6 days post fertilization (dpf) to 28 dpf. The experimental designs used here including group of zebrafish on both side (ZF-ZF), groups of medaka on both end (MK-MK), or place the zebrafish in one end with medaka in the other end (ZF-MK). The social activity was detected by using an EthoVision video tracking system (Noldus Information Technology, Leesburg, VA, USA) for 10 minutes, and we calculated the duration of swimming time in three different zones and transferred the time to percentage of shoaling “(duration in both side zones)*100 / total duration” and/or shoaling preference score “duration in closing zebrafish zone/ total duration for shoaling”.
The n-3 PUFAs diet supplement studies were aimed to clarify whether supplement had rescue effects on social behavior abnormalities in fmr1 KO zebrafish. The apparatus was designed for testing adult fish, with center area size (24 x 12 x 12 x 4.5 cm, length x width x height x depth). In addition, the duration of experiment was 25 minutes. The first 10 minutes is called acclimation phase, both screens were blocked by white acrylic board for avoiding the visual contact between the subject and other group of fishes. After that, both acrylic boards were removed and the fish could observe the different groups of fishes (ZF-MK) in each end. We detected the time of the zebrafish spend on the three different
zones by EthoVision video tracking system (Noldus Information Technology, Leesburg, VA, USA) The testing time was set for 15 minutes. In the same way for larval study, we calculated the percentage of shoaling and shoaling preference score for qualifying the therapeutic effects of n-3 PUFAs supplement (Engeszer et al., 2004; Engeszer et al., 2007).
4.4. Inhibitory avoidance (IA)
In this research, the apparatus (28 x 12 x 17, length x width x height) was divided into two different zones, a shallow water zone (2 cm in depth) and a deep water zone (8 cm in depth). They were separated with a white, opaque guillotine door between shallow and deep compartment (Fig. 3).
Based on the natural preference of zebrafish for a deep environment comparing to a shadow one (Darland and Dowling, 2001), the procedure contained a training phase and a testing phase. Animals were allowed to acclimate to the behavior chamber via a habituation session. They were placed in the shallow chamber for 5 min; the white, opaque guillotine door was then removed, and the fish were allowed to swim freely between the two compartments for another 5 min. In the training session, the fish were placed in the shallow compartment, allowing them to swim for 1 min before the guillotine door was opened. Once the fish entered the deep compartment, the guillotine door was closed, and a mild electric shock was applied to the deep compartment for 5 s. Animals were then tested for the avoidance learning 24 hours later. During the testing phase, we used the similar procedure with training phase but omitting the electrical shock. The duration of fish moved from shallow to deep
chamber escape latency) was recorded and analyzed. The maximum duration for testing is 300 seconds.
5. Gene expression quantitative analysis