Schematic of the behavioral procedures for the NOL and NOR tasks.

A. Experimental timeline. Dams were fed one of three diets for mating, gestation, and offspring consumed the diet until weaning at 25-30 days of age. At 1 month-old, offspring were implanted with electrodes for EEG and recorded for a 24 hr period at 1.2, 2, 3, 4, 5, and 6 months of age. At 3 and 6 months of age behavior was tested. At 6 months of age, after behavioral testing, mice were perfused, brains were sectioned, and sections were processed with antibodies against NeuN and ΔFosB.

B. Prior to NOL or NOR, animals were acclimated to the testing arena. There were three acclimation sessions separated by 24hrs during which animals were allowed to freely explore for 5 min.

C. In the NOL task, animals were placed in a cage with two identical objects and allowed to freely explore for 5 min (Training). After one hour they were brought back to the cage, where one object was displaced, and allowed to freely explore for 5 min (Testing).

D. In the NOR task. animals were placed in a cage with two identical objects and allowed to freely explore for 5 min (Training). After one hour they were brought back to the cage, where one object was replaced, and allowed to freely explore for 5 min (Testing).

Choline enrichment reduced spatial memory deficits.

A. Three months-old mice.

1. WT and Tg2576 mice exposed to the low choline diet showed spatial memory deficits.

2. Tg2576 exposed to the intermediate diet showed spatial memory deficits but not WT.

3. Spatial memory was improved in animals exposed to the high choline diet.

B. Six months-old mice.

1. WT and Tg2576 mice exposed to the low choline diet showed spatial memory deficits.

2. WT and Tg2576 mice exposed to the intermediate diet showed spatial memory deficits.

3. WT and Tg2576 mice exposed to the high choline diet had improved spatial memory.

WT and Tg2576 mice showed recognition memory regardless of diet.

A. Three months-old mice.

1-3. WT and Tg2576 mice treated performed the NOR task.

B. Six months-old mice.

1-3. WT and Tg2576 mice performed the NOR task.

Tg2576 mice treated with the choline supplemented diet spent more time with the objects at each approach.

A-B. NOL.

There was no effect of diet on time exploring or approaches to the novel object in the NOL task at 3 months of age (A) but there were significant differences at 6 months of age (B). Mice exposed to the high choline diet spent more time exploring than mice that had been fed the low choline or the intermediate diet.

C-D. NOR.

There was no effect of diet on time exploring or approaches to the novel object in the NOR task at 3 months of age (C) but there were significant differences at 6 months of age (D). Mice that had been fed the high choline diet spent more time exploring than mice that had been fed the low choline or the intermediate diet.

Choline supplementation improves NeuN immunoreactivity (ir) in hilar cells in Tg2576 animals.

A. A sagittal view of the brain illustrating the coronal sectioning plane.

B. Representative images of NeuN-ir staining in the dorsal dentate gyrus of Tg2576 animals from each treatment group.

C. Quantification methods. Representative images demonstrating the thresholding criteria established to quantify NeuN-ir.

1. A NeuN-stained section shows an inset (arrow) expanded at the top left to show 3 hilar cells. NeuN-ir cells are marked by blue arrows. NeuN-ir that was too close to background staining to be considered positive is marked by a green arrow.

2. After converting the image to grayscale, a threshold was set so that all cells stained well with NeuN antibody were red. The inset shows that the two cells that were darkest met the threshold while the weakly stained cell did not.

D. Six months-old Tg2576 mice treated with the high choline diet had significantly more NeuN-ir cells in the hilus in the dorsal dentate gyrus (1) compared to other treatment groups but not ventral dentate gyrus (2).

Choline supplementation reduced ΔFosB expression in dorsal GCs of Tg2576 mice.

A. A sagittal view of the brain illustrating the coronal sectioning plane.

B. Representative images of ΔFosB staining in the anterior dentate gyrus of Tg2576 animals from each treatment group.

C. Quantification methods. Representative images demonstrating the thresholding criteria established to quantify ΔFosB.

1. A ΔFosB -stained section shows strongly-stained cells (white arrows).

2. A strict thresholding criteria was used to make only the darkest stained cells red.

D. Six months-old Tg2576 mice treated with the choline supplemented diet had significantly more ΔFosB -ir pixels above threshold in the dorsal dentate gyrus (1) compared to other treatment groups but not ventral dentate gyrus (2).

E. Methods are shown using a threshold that was less strict.

1. Some of the stained cells that were included are not as dark as those used for the strict threshold (white arrows).

2. All cells above the less conservative threshold are shown in red.

F. With the less conservative threshold, the results were the same as D. Mice that were fed the high choline diet had less ΔFosB-ir pixels than the mice that were fed the other diets. However, this was true for dorsal (1) but not ventral (2) sections.

The high choline diet reduced IIS frequency in Tg2576 animals.

A. 1. Representative example of an IIS (red arrow). IIS were defined as occurring in all four channels (generalized) to distinguish them from focal spikes that can often be artifact. 2-3. The IIS shown in A1 is expanded.

B. 1. Scatter plot of IIS frequency at each age of recording.

1. Means and sem are plotted. The high choline diet group had fewer IIS than the intermediate diet group at ages 1-3 months ($, p<0.05) and the low choline group had less IIS than the intermediate diet at ages 1-4 months (@, p<0.05). The high and low choline diet groups were significantly different at ages 3 and 4 months (*, p<0.05).

Comparison of the three diets used in this study.

Details of behavioral tasks.

A standard mouse cage is diagrammed with the locations of objects inside it. For NOL, there were two pineapple-like objects and one was moved across the cage for the test session. For the NOR test session, one pineapple-like object was removed and an object made of LEGO was placed in the location where the original pineapple-like object had been.

Mortality was high in mice treated with the low choline diet.

A. Survival curves are shown for mice fed the low choline diet and mice fed the high choline diet.

B. A photo of a mouse after sudden unexplained death. The mouse was found in a posture consistent with death during a convulsive seizure.

IIS frequency before and after seizures.

A. Representative EEG trace of a seizure in a 5 months-old Tg2576 mouse.

B. IIS frequency was quantified from continuous video-EEG for 4 days. During the first 24 hrs, IIS frequency was low. The next day a seizure occurred (arrow), and IIS frequency increased approximately 5 fold, and for the next 24 hr period. On day 4, IIS frequency returned to lower levels.

IIS frequency was similar for each sex.

A. At 1.2 months-old, female and male Tg2576 mice were not significantly different in IIS frequency for the low and high choline diets.

B-C. There were no significant sex differences at 2 (B) and 3 (C) months of age.