Research aimed to discover the ADS relate to gut microbiota, with FMT to differentiate the sample group, and determine their level of ADS by behavior tests.

Experiment sample:

The sample used in the research is mice, where only male mice are used. EphB6-deficient mice were generated using the embryonic stem (ES) cells with inserting with mutated form. These mice were crossed with normal mice and their off springs until further generation. Then, as a result of  offspring, only EphB6+/+mice and EphB6−/− mice are obtained. (Li et al.)

FMT

Fecal microbiota transplantation (FMT) experiments is a process where transfer one individual’s fecal bacteria into another individual.  This was done by give the supernatant of feces orally to each mouse.The feces are collected freshly from normal healthy EphB6+/+ mice and EphB6−/− mice every day before the experiment. And feed to healthy normal mice and then to EphB6+/+ mice and EphB6−/−. Thus there will be four groups of samples, normal mice, normal with knockout feces, the EphB6 deficient and Eph deficient with wild type feces.

Behavior tests:

Behavior test are also done on the mice. To test how severe their ASD is compare to group to group. The tested group are naïve and male mice. Different behavioral tests were done with an interval of at least 2 days. There are 8 behavior tests, they are self-grooming test, olfactory habituation/dishabituation test, three-chambered social approach task, marble burying test, open field test, social partition test, elevated plus maze, and Morris water maze.(Li et al.)

Self-grooming test

In this test, in an empty crystal cage is need, the mouse was place into it and habituate the cage for 10 min, to record the time each mouse spent self-grooming during the next 10 min. The recording was done by a double-blind experienced experimenter. The classification of self-grooming included face-wiping, scratching/rubbing of head and ears, and full-body grooming. (Li et al.)

Olfactory habituation/dishabituation test

Before the test, ouse was placed into a clean usual animal cage with thin bedding for 30 min. Give the mouse three times the swab saturated with water for 2 min interval, where need to be quick when changing the swab saturated with water. The other odors were presented to mouse similarly. The three odors are water, almond extract, imitation banana flavor the odors for given to mice as same as listed. (Li et al.) These odors were regarded as unsocial odors, and for social odors, it is present as soiled bedding with the similar sex and age of unfamiliar mice. (Li et al.) This is prepared by placing the mice in the cage for at least 3 days. As the record for this testing, the time each mouse spent sniffing the odorant swabs in every 2-min trial was recorded, and it is done by a double-blind experienced experimenter. (Li et al.)

Three-chambered social approach task

to divide the apparatus into 3 chamber, two walls with removable doors are placed. This allow the mice to move freely between chambers. First of all, the mice is placed into the middle chamber for 5 min, and then can move freely in the three chamber for 10 min. To do the sociability test, an age- and sex-matched mouse was placed in one chamber while other chambers are empty. Then the dividers were removed, and the experimental mouse can freely explore for 10 min. Social novelty test was presented with placing another age- and sex-matched mouse in the empty chamber, with the experimental mouse was originally placed in the center of the chamber. And can move freely for 10 min after doorways were removed. The recording is done on the time spent in each chamber and mouse trajectory were calculated. (Li et al) And the time for sniffing the cages was calculated because it shows the social approach behaviour of mice.

Social partition test

The mice were keep individually for four days before the test. On the test day, a sex- and age-matched mice to experimental mice were placed on the opposite side of the transparent partition. This mouse is the familiar mouse. For the first trial, the record is done on partition during the 5 min, then the familiar mouse is replaced with replaced with another sex- and age-matched unfamiliar mouse, then the record is done on partition during the 5 min. All three trials are recorded by a double-blind experienced experimenter.

Marble burying test

In the cage filled with fresh wood chip bedding, there are regular glass marbles, and placed with 4cm away from each. The mouse is placed into the cage for 30 min, and the records is based on how many marble are buried during the time.

Open field test

In an open field chamber, the mouse is placed in, for 30 min. the records is done on the exploratory behaviour and the total distance it moved.

Elevated plus maze

Maze is built as have two opposing open, two enclosed arms and a center area. The mouse is placed in the center of the maze for 5 min, and the record is done on the time spend for each arm and their entries.

Morris water maze

The build for the maze is that it has a water area and a platform. In total there are four trails every day for 5 days, where for each trail, the searching time for the mouse is less than 1 min, and the stay on the platform is 15sec. The time in between each trial is no more than 1 min. The record is done on the sixth day, where the platform is removed and the swimming time is calculated.