Supplementary Figure S1

Supplementary Figure S1

“Unusual Retinal Layer Organization in HPC-1/syntaxin 1A Knockout Mice”

Journal of Molecular Histology

Yuko Kaneko, Rie Suge, Tomonori Fujiwara, Kimio Akagawa, Shu-Ichi Watanabe

Corresponding author: Yuko Kaneko, Department of Physiology, Faculty of Medicine, Saitama Medical University

E-mail:

Supplementary Figure S1. Visual discrimination task in HPC-1/syntaxin 1A knockout (KO) and heterozygous mutant (HT) mice. a. Mean correct reaction ratios of HT and KO mice. Results are expressed as means, and error bars indicate the SEM. Blank discs represent HT, and gray discs represent KO mice. The correct reaction ratio in each block was calculated as the ratio of correct responses to the total number of trialsusing data summed from three sessions. Mice from both strains were able tolearn the discrimination task, and no significant difference was observed in learning scores between the strains (effect of strain: F1, 253 = 0.28, P = 0.596, interaction between strain and block: F11, 253 = 0.71, P = 0.732). Additionally, no difference was observed in the time taken to establish panel key-touching behavior (average number of sessions for pre-training, HT: 8.83 ± 1.11, KO: 8.84 ± 1.01). b. Mean reaction times in HT and KO mice. Reaction times were significantly shorter in HT mice than in KO mice (F1, 251 =11.83, P = 0.001). *P < 0.01, compared to HT. Thus, KO mice showed a slight abnormality in visual discrimination learning, although they were able to learn the task as well as HT mice. (For precise methods, see Supplementary Materials and Methods).

Materials and Methods.

Male HPC-1/syntaxin 1A (STX1A) knockout (KO, n = 13) and littermate control heterozygous mutant (HT, n = 12) mice were used. Each animal was kept in an individual cage in an air-conditioned room (23 ± 1°C) with a constant light:dark cycle (12:12-h). Animals maintained 85% of their ad libitum weight from 10-weeks of age and throughout the experiments. A 2-week period (from 8 to 10 weeks) was used for adaptation to the apparatus and for weight control of the animals.

Learning sessions were conducted in operant chambers (15 × 22 × 15 cm) with two touch-panel keys (Kobayashi et al. 2005). Each chamber was situated in a light- and sound-attenuated box with a ventilation fan. General illumination was provided by a house light (24 V, DC) situated at the center of the back panel, 2 cm from the ceiling. The touch-panel key was placed on the front panel of the chamber, 1 cm from the floor, on either side of a pellet receptacle into which 20-mg food pellets (Bio Serve, Frenchtown, NJ, USA) could be delivered by a pellet dispenser (ENV-203, Med Associate Inc., Georgia, VT, USA). A green light-emitting diode (LED) was located 1 cm above each touch panel key and was used as a visual cue.

Visual discrimination training was performed as described previously (Suge et al. 2007). Ten-week-old mice were trained to touch the panel keys for food pellets, as in pre-training. The animals were trained for the visual discrimination task after establishing stable key-touching behavior. At the start of a trial, the left or right LED was randomly turned on, and if the mice responded to the same side of the key as the illuminated LED within 10 s after the start of the trial, the reaction was defined as “correct” and reinforced. When mice touched the other side of the key, all lights including the house light were turned off for 5 s, and the reaction was defined as “incorrect”. When no response to the panel keys was observed within 10 s, the trial was defined as “no reaction”. Each daily session consisted of 40 trials, and experiments were conducted 6 days per week. Experiments were performed from 9:00 to 12:00 noon. The correct reaction ratio and reaction time were used as indices of behavior and learning. The correct reaction ratio was calculated as the ratio of correct responses to the total number of trials (40 trials), and the reaction time was defined as the duration from the start of the trial to touching one of the panel keys, excluding the data from the “no-reaction” trials. Statistical analysis was performed on summed data from three sessions (3 days) as one block. In this experiment, we regarded a correctreaction ratio of >80% for the second consecutive day as the criterion for acquisition of the task.

All data are presented as group means ± SEMs. Statistical significance was based on an analysis of variance and t-tests (Fisher's LSD test). The criterion for significance was P < 0.05 for two-tailed tests in all cases.

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