Caloric restriction and age affect synaptic proteins in hippocampal CA3 and spatial learning ability

Abstract

Caloric restriction (CR) is a daily reduction of total caloric intake without a decrease in micronutrients or disproportionate reduction of any one dietary component. CR can increase lifespan reliably in a wide range of species and appears to counteract some aspects of the aging process throughout the body. The effects on the brain are less clear, but moderate CR seems to attenuate age-related cognitive decline. Thus, we determined the effects of age and CR on key synaptic proteins in the CA3 region of the hippocampus and whether these changes were correlated with differences in behavior on a hippocampal-dependent learning and memory task. We observed an overall, age-related decline in the NR1, N2A and N2B subunits of the N-methyl-d-aspartate (NMDA)-type and the GluR1 and GluR2 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA)-type ionotropic glutamate receptors. Interestingly, we found that CR initially lowers the glutamate receptor subunit levels as compared to young AL animals, and then stabilizes the levels across lifespan. Synaptophysin, a presynaptic vesicle protein, showed a similar pattern. We also found that both CR and ad libitum (AL) fed animals exhibited age-related cognitive decline on the Morris water maze task. However, AL animals declined between young and middle age, and between middle age and old, whereas CR rats only declined between young and middle age. Thus, the decrease in key synaptic proteins in CA3 and cognitive decline occurring across lifespan are stabilized by CR. This age-related decrease and CR-induced stabilization are likely to affect CA3 synaptic plasticity and, as a result, hippocampal function.

Figure 1. Representative Western Blots for Synaptic Proteins in the CA3 Region of Hippocampus

Western blot analysis was performed to assess CA3 protein levels of the NMDA subunits NR2A, NR1, and NR2B; the AMPA subunits GluR1 and GluR2; and the essential synaptic protein, synaptophysin (Syn). Actin was assessed on each blot to insure equal protein loading. Experimental groups were young (10-12 months), middle-aged (MA; 18-20 months), and old (29-32 months) Fischer 344 × Brown Norway rats that were caloric restricted (CR) from 4 months of age or fed ad libitum (AL).

Figure 2. Densitometric Analysis of Synaptic Protein Levels in CA3

Protein levels of each subunit examined declined significantly with age in AL animals. In contrast, there were no age-related changes in CR animals. Although subunit protein levels in young animals were lower in the CR than in the AL group, those levels in CR animals were stabilized across lifespan. By old age, each subunit in the AL group decreased to a level significantly lower than that in the CR group. Levels of the synaptic protein synaptophysin (Syn) in AL and CR groups across lifespan demonstrate a similar pattern. Experimental groups were young (10-12 months), middle-aged (MA; 18-20 months), and old (29-32 months) F344 × BN rats that were caloric restricted (CR) from 4 months of age or fed ad libitum (AL). All measurements are reported as optical density units. An asterisk indicates an effect of age and # indicates an effect of diet. All significant differences are p < 0.05.

Figure 3. Morris Water Maze Performance during Training Trials in AL and CR Rats across Lifespan

(A) The total distance to platform on the Morris water maze (MWM) increased between young and middle age for both AL (ad libitum fed) and CR (caloric restricted) rats, indicating a decline in performance (p<0.001). Between middle and old age, this measure increased significantly for AL rats (p<0.001), but the performance of CR rats did not change. At old age, AL rat swam significantly further than CR rats to reach the platform (p<0.001). (B-D) The improved performance of young, middle age, and old rats across training blocks on the total distance measure demonstrated that the animals in each group learned the task. (E) The path length to platform increased significantly for both AL and CR rats between young and middle age (p<0.001), but did not change between middle and old age. (F) Escape latency increased significantly for both AL and CR rats between young and middle age (p<0.001) and for AL rats between middle and old age (p<0.001). Escape latency did not change for CR rats between middle and old age, and old CR rats had a significantly smaller escape latency than did old AL rats (p<0.001). (G) Although AL and CR groups differed significantly on mean swim velocity across training trials at young, middle, and old age, this difference cannot explain the superior performance of old CR rats on the total distance to platform and escape latency measures (see text).

Figure 4. Morris Water Maze Probe Trial Performance in AL and CR Rats across Lifespan

(A) The mean distance to platform on the probe trial of the Morris water maze (MWM) did not differ across life span or between AL (ad libitum fed) and CR (caloric restricted) groups. (B) Platform crossings, or the number of times the rats crossed the platform’s previous location, did not differ across life span or between AL and CR groups. (C-E) The percent of time young (C), middle age (MA, D), and old (E) rats spent in the target quadrant in which the platform was previously located (quadrant 4) did not differ between AL and CR groups. Each group spent significantly more time in quadrant 4 than in the other three quadrants (p<0.001).

Figure 5. Morris Water Maze Performance during Visible Platform Testing

Average swimming velocity differed with age, but not diet during the visible platform testing phase of the Morris water maze (MWM; p <0.001). Post-hoc tests revealed that this effect of age is significant in AL (ad libitum fed; p<0.05), but not CR (caloric restricted) rats.