The function of the glutamate-nitric oxide-cGMP pathway in brain in vivo and learning ability decrease in parallel in mature compared with young rats

Abstract

Aging is associated with cognitive impairment, but the underlying mechanisms remain unclear. We have recently reported that the ability of rats to learn a Y-maze conditional discrimination task depends on the function of the glutamate-nitric oxide-cGMP pathway in brain. The aims of the present work were to assess whether the ability of rats to learn this task decreases with age and whether this reduction is associated with a decreased function of the glutamate-nitric oxide-cGMP pathway in brain in vivo, as analyzed by microdialysis in freely moving rats. We show that 7-mo-old rats need significantly more (192 +/- 64%) trials than do 3-mo-old rats to learn the Y-maze task. Moreover, the function of the glutamate-nitric oxide-cGMP pathway is reduced by 60 +/- 23% in 7-mo-old rats compared with 3-mo-old rats. The results reported support the idea that the reduction in the ability to learn the Y-maze task (and likely other types of learning) of mature compared with young rats would be a consequence of reduced function of the glutamate-nitric oxide-cGMP pathway.

Figure 1.

The ability to learn the Y-maze conditional discrimination task is lower in mature rats than in young rats; 3-mo-old and 7-mo-old male and female rats were subjected to the conditional discrimination learning test in the Y-maze as described in Materials and Methods. Values are the mean ± SEM from seven rats per group and are given as the number of trials needed to learn. Data of different sex and age were analyzed using two-way ANOVA. There were no significant differences between sexes, but the effect of age is significant (P < 0.001). Values that are significantly different from young (3-mo-old) rats are indicated by asterisks: ***P < 0.001 (two-way ANOVA). Pooled data were analyzed using Student’s t-test to compare performance of young and mature rats. Significant difference from young (3-mo-old) rats is indicated by asterisks: ***P < 0.001.

Figure 2.

The function of the glutamate–NO–cGMP pathway in rat cerebellum in vivo is reduced in mature rats compared with young rats. The function of the Glu–NO–cGMP pathway was assessed by in vivo brain microdialysis. NMDA 0.3 mM was infused at the times indicated, and extracellular cGMP was determined. Data are presented as percentage of basal values. Values are the mean ± SEM from seven rats per group: (A) male rats, (B) female rats, and (C) pooled data of males and females. Values that are significantly different from young (3-mo-old) rats are indicated by asterisks: *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test).

Figure 3.

The basal concentration of extracellular cGMP in rat cerebellum is lower in mature rats than in young rats. The basal level of extracellular cGMP was measured in brain microdialysis samples (fractions 1–4 of Fig. 2). Values are mean ± SEM from seven rats per group. Data of different sex and age were analyzed using two-way ANOVA. There were no significant differences between sexes, but the effect of age is significant (P < 0.001). Values that are significantly different from young (3-mo-old) rats are indicated by asterisks: **P < 0.01, ***P < 0.001 (two-way ANOVA). Pooled data were analyzed using Student’s t-test. Significant difference from young (3-mo-old) rats is indicated by asterisks: ***P < 0.001.

Figure 4.

The amount of NMDA receptors in cerebellum is lower in mature male rats than in young male rats. Thirty micrograms of protein from homogenates of the cerebellum from 3-mo-old or 7-mo-old rats was subjected to SDS–polyacrylamide gel electrophoresis using an 8% acrylamide gel. The NR1 subunit was visualized by immunoblotting as indicated in Materials and Methods. Typical immunoblotting is shown for males (B) and females (C). The intensities of the bands were quantified and are shown in A. Values are the mean ± SEM of five rats per group and are expressed as percentage of NR1 amount in 3-mo-old rats. Data of different sex and age were analyzed using two-way ANOVA. There were no significant differences between sexes, but the effect of age is significant (P < 0.05). Values that are significantly different from young (3-mo-old) rats are indicated by asterisks: *P < 0.05 (two-way ANOVA). Pooled data were analyzed using Student’s t-test. Significant difference from young (3-mo-old) rats is indicated by asterisks: *P < 0.05.

Figure 5.

There is a good correlation between NMDA-induced increase in extracellular cGMP and the ability to learn the Y-maze task. Individual data for 7-mo-old males and females are plotted. The function of the glutamate–NO–cGMP pathway is expressed as the increase in cGMP induced by administration of NMDA, i.e., the increase in cGMP in fractions 7–9 of Figure 2. There is a negative and significant (r = −0.769; P = 0.0013) correlation between the function of the pathway and the number of trials required to learn the task. A higher number of trials means a lower learning ability. This means that there is a direct correlation between the function of the pathway and learning ability: A higher function of the pathway correlates with higher learning ability.