Supplementary material for “Chronic Adjunction of 1-Deoxynojirimycin Protects from Age-Related Behavioral and Biochemical Changes in the SAMP8 Mice”

Authors: Gui-Hai Chena,b,d,1*, Jing-Jing Tongc,d,1, Fang Wangd, Xue-Qin Hub, Xue-Wei Lid, FeiTaod, Zhao-Jun Weib*

aDepartment of Neurology, the Affiliated Chaohu Hospital of Anhui Medical University, Chaohu, Hefei 238000, P.R. China

bSchool of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009, P.R. China

c Department of Rheumatism and Immunity, the First Affiliated Hospital of Anhui Medical University, Hefei 230022, P.R. China

dDepartment of Neurology, the First Affiliated Hospital of Anhui Medical University, Hefei 230022, P.R. China

(*Corresponding author: Gui-Hai Chen, PhD, Department of Neurology, the Affiliated Chaohu Hospital of Anhui Medical University, Chaohu, Hefei 238000, P.R. China. E-mail address: .

Zhao-Jun Wei, PhD, School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009, Anhui, PR China. .)

1These authors contributed equally to this work.

Behavioral tests

The apparatuses and procedures of sensorimotor tasks, open field, Novel-object recognition (NOR) and Morris water maze (MWM) had been described in our previous study [1]and provided in this supplemental material.

1. Sensorimotor tasks

1.1. Beam walking.A 110-cm-long steel rod (diameter 10-mm), with both ends connected to a platform (10-cm in diameter), was supported by two vertical poles and elevated 50-cm above the water surface in a tank. Each mouse was given three consecutive trials and perpendicularly placed on the center of the beam. Each trial was maintained for a maximum of 60-s. The balance time, during which the animal did not fall from the rod, was recorded for each of the three trials. If the animal remained on the beam for the duration of the trial or escaped to either of the two platforms, it was recorded as 60-s. The mean time recorded for the three trials was used for statistical analysis.

1.2. Tightrope.The apparatus was a taut, tiny cotton rope (2-mm in diameter) that stretched across a circular tank (100-cm in diameter, 30-cm in height) half-filled with water (at 21 °C). The rope had black ink markers every 5-cm to measure the horizontal movement of the mouse. Each mouse was placed in the water for 5-s, and then raised to grasp the middle of the rope with forepaws before released slowly. The suspension time (during which the animal did not fall from the rope) and the number of markers crossed by forepaws were recorded during a 60-s trial. Once a mouse had fallen into the water, or stayed on the rope for 60-s, it was immediately removed to its home cage to have a rest before the next trial (three subsequent trials on a single day). To distinguish the mice who suspended motionlessly and those move dexterously, we used a transformed score [=(average suspension time)+10 ×(average number of the marker crossed)][2].

2. Open field

The device is an openwooden box (81 cm× 81 cm × 28 cm) with black floor painted with white lines forming 16 equal squares (20 cm× 20 cm each). Illumination was provided with a 40-W white light placed 2.80-m above the center of the field. Each mouse was placed in a corner square of the box, facing the wall, and was observed for 5-min. The following parameters were recorded: latency (time taken by the mouse to depart from the first square), number of squares crossedand peripheral time (the time spent in the 12 peripheral squares). The box was cleaned with water after each test.

3.NOR

The Y-apparatus has three equidistant arms (10-cm wide and 40-cm high), one is a start arm (30-cm long) and the other two are object arms (23-cm long). The start arm contained a guillotine door 10-cm away from the end and provided a square area where the mouse could be confined before each trial. The floor and the inside walls are black. The apparatus was rounded with a black cloth curtain. A video camera was installed above the apparatus to capture the activities of mice. In the acclimation phase, the mice were allowed to freely explore the empty Y-shaped apparatus for 5-min per day for 3 days. In the sample phase, the mice were returned to the home cage and staying there for 2-min following each exploration of the apparatus with no object for 1-min. Two identical familiar (or sample) objects (A1 and A2) were placed in the left and right ends of the object arms, respectively. Each animal was placed at the start arm with the guillotine door open. Once the animal completely entered the object arm(s), it had a planned sample-object exposure time of 5-min in the apparatus with the guillotine door closed, and then it was returned to the colony. After a 10-min interval (choice phase 1；10-min phase), one of the sample objects was replaced with a novel object (B) and the other sample object with A3, and the animal was reintroduced into the apparatus for 5-min period of exploration. After a 24-h delay(choice phase 2; 24-h phase), the animal was reintroduced for a 3-min exploration in the apparatus, inwhich the object B replaced by the sample object A4 and A3 replaced by another novel object (C). The apparatus and objects were cleaned with water following exploration of each animal. The video-image analysis was completedusinga specific software (XNote Stopwatch 1.39) by an operator who was blind to the experimental design. It was considered exploration if an animal touched the objects with its vibrissae, snout or forepaws. The total time (Tt), exploring-time for the familiar (TF) or the novel object (TN) within the first 1-min during choice phase was recorded separately. The preferential index for novel object (PI) in the choice phase was defined as TN/(TF + TN) × 100%.

4. MWM

Place learning trial. A black circular tank (150-cm indiameter, 30-cm in height) was filled with fresh tap water daily (21–22 °C). A black escape platform (10-cm in diameter, 24-cm in height) was fixed 1.0-cm below the water surface in one of the four quadrants of the pool. The tank was placed on a steel rack, and rounded by a white cloth curtain with three pieces of black cardboards in different shapes (circle, triangle and square, respectively) hung equidistantly. A camera that linked to a computer was hung above the center of the pool to monitor swim patterns.

The test lasted 10 days with each mouse receiving four successive trials a day. On each trial, an animal was released into the water, facing the wall, and from a randomly assigned location along the pool’s perimeter (one position per quadrant). The time allowed to find the platform was a maximum of 60-s. Upon locating the platform, each mouse was kept there for 30-s. If the mouse failed to locate the platform within 60-s, it was guided to the platform and kept there for 30-s. Then, it was transferred to its cage until the next trial. After all of the mice completed this trial, the next trial beganfollowing the same order of the mice. The latency (s) and swim distance (cm) and velocity (cm/s) were recorded

Probe trial. On the last day of place learning task, a probe trial was given while the platform waswithdrawn. The mice were placed into the quadrant opposite to the quadrant where the platform was previously located (target quadrant) and swim freelyfor 60-s. The percentage of swim distance spent in the target quadrant was used as a measure of spatial memory.

References

[1] Chen GH, Wang H, Yang QG, Tao F, Wang C, Xu DX. Acceleration of age-related learning and memory decline in middle-aged CD-1 mice due to maternal exposure to lipopolysaccharide during late pregnancy.Behav Brain Res. 2011;218:267-79.

[2] Hengemihle JM, Long JM, Betkey J, Jucker M, Ingram DK. Age-related psychomotor and spatial learning deficits in 129/SvJ mice. Neurobiol Aging 1999;20:9–18.