Hippocampal neurogenesis levels predict WATERMAZE search strategies in the aging brain.

The hippocampus plays a crucial role in the formation of spatial memories, and it is thought that adult hippocampal neurogenesis may participate in this form of learning. To better elucidate the relationship between neurogenesis and spatial learning, we examined both across the entire life span of mice. We found that cell proliferation, neuronal differentiation, and neurogenesis significantly decrease with age, and that there is an abrupt reduction in these processes early on, between 1.5-3 months of age. After this, the neurogenic capacity continues to decline steadily. The initial abrupt decline in adult neurogenesis was paralleled by a significant reduction in Morris Water Maze performance, however overall learning and memory remained constant thereafter. Further analysis of the search strategies employed revealed that reductions in neurogenesis in the aging brain were strongly correlated with the adoption of spatially imprecise search strategies. Overall, performance measures of learning and memory in the Morris Water Maze were maintained at relatively constant levels in aging animals due to an increase in the use of spatially imprecise search strategies

Learning of the MWM task is only modestly affected by age.

<p>C57BL/6J male mice with 1.5, 3, 6, 9, and 12 months of age were tested in the MWM for their ability to learn and recall the location of a hidden platform. (A) Time required to find the hidden platform (i.e., escape latency) in both days 1 and 3 of the MWM training. For all age groups, escape latency on day 3 of training was significantly reduced when compared with escape latency on day 1, indicating that all mice were able to learn the task. (B) When the average time required to locate the hidden platform on day 3 was subtracted from that of day 1, a significant effect of ageing on the ability to learn the MWM task was observed, with older animals taking longer to learn the location of the hidden platform. (<b>C</b>) Distance traveled to find the hidden platform (i.e., path length) in both days 1 and 3 of the MWM training. For all age groups, path length on day 3 of training was significantly reduced when compared with the respective path length on day 1, further indicating that all mice were able to learn the task. However, on day 3 of training, 1.5-month old mice swam significantly less than their oldest counterparts. Data are presented as means ± SEM. ** <i>p</i> < 0.01, when comparing day 1 and day 3 of training for each age group; ^#<i>p</i> < 0.05, when comparing the distance traveled on day 3 of training between 1.5- and 9-month old mice; ^##<i>p</i> < 0.01, when comparing the distance traveled on day 3 of training between 1.5- and 12-month old mice; see text for additional statistical details.</p

Cell proliferation is significantly decreased by age in the mouse hippocampal DG.

<p>POMC-EGFP transgenic mice were sacrificed at 1, 1.5, 2, 2.5, 3, 4, 6, 12, 18, 24 months of age by transcardial perfusion and their brains were processed for immunohistochemistry for the endogenous cell proliferation markers Ki-67 (A-C) and PCNA (D-F). Age induced a significant decline in the expression of both Ki-67 (A-C) and PCNA (D-F). An abrupt reduction in cell proliferation was detected early on (between 1.5 and 3 months of age) with both markers. Data are presented as means ± SEM. (A) *** <i>p</i> < 0.001 between 1 or 1.5 and 2 months, between 2 and 3 months, and between 2.5 and 4, 6, 12, 18 or 24 months. (D) ** <i>p</i> < 0.01 between 2 and 4 months, and between 3 and 6 months, *** <i>p</i> < 0.001 between 1 or 1.5 and 2 months, and between 3 and 12, 18, or 24 months. See text for additional statistical details. Representative sections of the DG processed for Ki-67 (B, C) and PCNA (E, F) immunohistochemistry in 1- (B, E) and 24- (C, F) month old POMC-EGFP mice. Black arrows indicate either Ki-67- (B) or PCNA- (E, F) immunopositive cells in the DG of 1- (B, E) and 24- (F) month old mice. Scale bars = 100 µm.</p

Age-induced reduction in spatially precise search strategies is correlated with levels of adult hippocampal neurogenesis.

<p>(<b>A</b>) Representative examples of the eight distinct types of search strategies employed during training in the MWM that were recognized by the classification algorithm. Strategies are grouped into either spatially precise or spatially imprecise categories. (B) Percentage of trials classified according to each search strategy across training days for the various age groups. All groups showed a progression towards an increase in the use of hippocampal-dependent spatially precise strategies with training. However, this progression clearly decreased with age. (C) Percentage difference in the frequency of each search strategy in the oldest mice (12 months old) relative to their youngest counterparts (1.5 month old). A significant reduction in the use of direct swim and direct search (spatially precise strategies) was accompanied by a significant increase in the use of random search, thigmotaxis, and perseverance (spatially imprecise strategies) in 12 month-old mice. (D) Age resulted in a decrease in the combined use of spatially precise search strategies and a concomitant increase in the combined use of spatially imprecise search strategies. (E) The use of spatially precise search strategies (calculated as the % use of combined spatially precise strategies on day 3 of training – day 1 of training) is strongly correlated with the levels of overall hippocampal neurogenesis (calculated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075125#pone-0075125-g004" target="_blank">Figure 4C</a>). Correlation coefficient <i>r</i>² = 0.965. See text for additional statistical details.</p

Neuronal differentiation is significantly decreased by age in the mouse hippocampal DG.

<p>POMC-EGFP-expressing mice were sacrificed at 1, 1.5, 2, 2.5, 3, 4, 6, 12, 18, 24 months of age by transcardial perfusion and their brains were processed for immunohistochemistry for the immature neuronal marker NeuroD (A-C) and visualized for the expression of EGFP (D-F). Age induced a significant decline in the expression of both NeuroD (A-C) and EGFP (D-F). An abrupt reduction in neuronal differentiation was detected early on (between 1.5 and 3 months of age) with both markers. Data are presented as means ± SEM. (A) *** <i>p</i> < 0.001 between 1 or 1.5 and 2 months, between 2 and 4 months, between 4 and 12 months, and between 6 and 18 or 24 months. (D) ** <i>p</i> < 0.01 between 4 and 12, 18, or 24 months, *** <i>p</i> < 0.001 between 1 or 1.5 and 2 months, and between 2 and 4 months. See text for additional statistical details. Representative sections of the DG processed for NeuroD immunohistochemistry (B, C) and analyzed for EGFP expression (E, F) in 1- (B, E) and 24- (C, F) month old POMC-EGFP mice. Arrows indicate a NeuroD-immunopositive cell (C) and an EGFP-expressing cell (F) in the DG of a 24-month old mouse. Scale bars (B, C) = 100 µm; scale bars (E, F) = 50 µm.</p

Experimental time-line for the BrdU and Morris water maze experiments.

<p>C57BL/6J male mice with 1.5, 3, 6, 9, and 12 months of age received BrdU injections (200 mg/kg i.p.) every 12 hours for 3 consecutive days. On day 12, animals were pre-trained in the MWM (three trials of 60 seconds each) with the platform protruding from the water surface. Between days 13 and 15 mice were trained to locate the hidden platform in the MWM. Each day animals received two training sessions separated by a 4-hour interval. Each session was composed by two blocks (of three 60-second trials each) separated by one hour. Mice were left undisturbed between days 16 and 42 and on day 43 they received a probe test in the MWM by placing them in the water without the platform for 60 seconds and assessing the amount of time they spent in the target quadrant (where the platform used to be located). Mice were sacrificed one hour later by transcardial perfusion and their brains were processed for immunohistochemistry.</p

Neural Mechanisms of Communication

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