Differential balance of prefrontal synaptic activity in successful versus unsuccessful cognitive aging

Abstract

Normal aging is associated with a variable decline in cognitive functions. Among these, executive function, decision-making, and working memory are primarily associated with the prefrontal cortex. Although a number of studies have examined the structural substrates of cognitive decline associated with aging within this cortical area, their functional correlates remain poorly understood. To fill this gap, we aimed to identify functional synaptic substrates of age-associated frontal-dependent deficits in layer 2/3 pyramidal neurons of medial prefrontal cortex of 3-, 9-, and ≥ 23-month-old Fischer 344 rats. We combined, in the same animals, novelty recognition and exploratory behavioral tasks with assessment of structural and functional aspects of prefrontal synaptic properties. We found that subsets of aged animals displayed stereotyped exploratory behavior or memory deficits. Despite an age-dependent dendritic spine loss, patch-clamp recording of synaptic activity revealed an increase in miniature EPSC frequency restricted to aged animals with preserved exploratory behavior. In contrast, we found a strong positive relationship between miniature IPSC frequency and the occurrence of both stereotyped exploratory behavior and novelty-related memory deficits. The enhanced miniature inhibitory tone was accompanied by a deficit in activity-driven inhibition, also suggesting an impaired dynamic range for modulation of inhibition in the aged, cognitively impaired animals. Together, our data indicate that differential changes in the balance of inhibitory to excitatory synaptic tone may underlie distinct trajectories in the evolution of cognitive performance during aging.

Figure 1.

Lack of change in synaptic properties during aging in the prefrontal cortex. Representative traces of spontaneous synaptic events (sEPSCs, A; sIPSCs, B) in layer 2/3 pyramidal cells obtained from young (blue), adult (gray), and aged (black) rats are shown. No significant changes were observed in the mean frequency or mean amplitude of sEPSCs or sIPSCs. The recording condition is represented in the inset.

Figure 2.

Lack of change in miniature synaptic activity during aging in the prefrontal cortex. Representative traces of miniature synaptic events (mEPSCs, A; mIPSCs, B) recorded in layer 2/3 pyramidal cells in the presence of TTX in the same group of rats as in Figure 1, A and B, are shown. No significant changes were observed in the mean frequency or mean amplitude of mEPSCs or mIPSCs (p > 0.05, Sidak-Holm pairwise multiple comparisons; n = 14–55 cells from N = 9–32 rats). The recording condition is represented in the inset.

Figure 3.

A subset of aged rats displayed stereotyped exploratory behavior. A, Representative paths for young (Y, blue), adult (Ad, gray), Aged-GE (brown), and Aged-BE (green) rats in the modified hole-board arena diagram (inset) and the distribution of ICE obtained for each group. In the young and adult groups, the distribution of ICE was well fit with single Gaussian functions, whereas the aged group was better fit with a double Gaussian function. We thus isolated two aged subgroups, an Aged-GE group with ICE comparable with that of younger groups and an Aged-BE group with higher ICE indicative of a stereotyped exploratory behavior. The Aged-BE animals were defined as those with an ICE > mean of young + 2 × SD. B, Average score of ICE of young, adult, aged good explorer, and aged bad explorer groups. C, The total distance traveled during the 10 min test in the hole-board arena was significantly lower for Aged-BE. D, The Aged-BE did not display thigmotaxis as shown by the lack of differences in the average distance from the arena border. E, We did not observe a significant difference in the maximum speed of displacement during the test. Data shown are means ± SEM. *p < 0.05; ***p < 0.001, Sidak-Holm pairwise multiple comparisons.

Figure 4.

Novelty-based memory deficits occur in a subgroup of aged rats. A, Mnemonic performances were assessed by a NOR test. Data are presented as the ratio of time spent exploring the new object at 1 h (NOR index). B, During the training phase (sample phase), the rats explored to the same extent the two identical objects. At 1 h, the aged group explored less the new object compared with young rats, confirming a disruption in the memorization process. C, Aged animals explored for a significantly shorter time the two objects at 1 h but for the same duration during the sample phase. D, E, We isolated as Aged-BL (BL) animals with a NOR index lower than that of the young group (Y, blue dotted line) by 2 × SD (purple dotted line). F, The aged group explored less the objects; however, there was no significant difference between the Aged-GL (GL) and Aged-BL (BL) in total time of exploration. **p < 0.01; ***p < 0.001, Sidak-Holm pairwise multiple comparisons.

Figure 5.

Exploratory behavior is not associated with spontaneous synaptic activity. A, Representative traces of sEPSCs and sIPSCs recorded in layer 2/3 prefrontal pyramidal neurons of Aged-GE (red) and Aged-BE (green) rats. B, There were no changes in sEPSC frequency in layer 2/3 pyramidal neurons from the Aged-GE group when compared with the young (Y) and Aged-BE groups. There was also no change in mean sEPSC amplitude (N = 6–10 rats per group). C, No changes in sIPSC frequency in Aged-BE rats compared with the other groups. There was also no change in mean sIPSC amplitude (N = 6–9 rats per group). D, To evaluate the relationship between excitatory synaptic activity and behavioral performance, the ICE of each rat was plotted against the sEPSC frequency. No significant correlation could be established across the four age groups or within the aged group. E, No relationship between the ICE and the sIPSC frequency across the four groups or within the aged group. Data shown are means ± SEM; Sidak-Holm pairwise multiple comparisons.

Figure 6.

Exploratory behavior is associated with specific change in the balance of action potential-independent synaptic transmission in pyramidal neurons of the medial prefrontal cortex. A, Representative traces of mIPSC and mEPSC recorded in layer 2/3 prefrontal pyramidal neurons of Aged-GE (red) and Aged-BE (green) rats. B, We observed a significant increase in mEPSC frequency in layer 2/3 pyramidal neurons from the Aged-GE group when compared with the young (Y) and Aged-BE groups. There was no change in mean mEPSC amplitude (N = 4–8 rats per group). C, On the other hand, our analysis revealed a significant increase in mIPSC frequency in Aged-BE rats compared with the other groups. There was no change in mean mIPSC amplitude (N = 6–9 rats per group). D, To evaluate the relationship between excitatory synaptic activity and behavioral performance, the ICE of each rat was plotted against the mEPSC frequency. No significant correlation could be established across the four age groups; however, we observed a significant correlation within the aged group (p < 0.05). E, A linear positive correlation was established between the ICE and the mIPSC frequency across the four groups (gray text; p < 0.001; r = 0.63) as well as within the aged group (red text and red line; p < 0.05; r = 0.49). Data shown are means ± SEM. *p < 0.05; ***p < 0.001, Sidak-Holm pairwise multiple comparisons.

Figure 7.

Memory deficits are not correlated with spontaneous synaptic activity. A, Representative traces of sEPSC and sIPSC recorded in layer II/III prefrontal pyramidal neurons of Aged-GL (red) and Aged-BL (purple) rats. B, No change in sEPSC frequency or amplitude across age groups (N = 6–12 rats per group). C, No changes in sIPSC frequency or amplitude was observed across age groups (N = 6–11 rats per group). Y, Young; Ad, adult. D and E, No correlation between excitatory or inhibitory synaptic activity and the mnemonic performance at 1 h could be established across the four age groups or within the aged group. Sidak-Holm pairwise multiple comparisons were conducted.

Figure 8.

Memory deficits are significantly correlated with an imbalance in synaptic activity toward inhibition. A, Representative traces of mIPSC and mEPSC recorded in layer II/III prefrontal pyramidal neurons of Aged-GL (red) and Aged-BL (purple) rats. B, No change in the mean excitatory synaptic frequency or amplitude across age groups (N = 4–8 rats per group). C, In contrast, a significant increase in mIPSC frequency was observed in the Aged-BL group (N = 6–10 rats per group. Y, Young; Ad, adult. D, No correlation between excitatory synaptic activity and memory performances. E, A significant correlation between the inhibitory synaptic activity and the mnemonic performance at 1 h could be established across the four age groups (p < 0.05; r = −0.38) as well as within the aged group (p < 0.05; r = −0.52). Data shown are means ± SEM. ***p < 0.001, Sidak-Holm pairwise multiple comparisons.

Figure 9.

Age-dependent changes in dendritic spine density. A, Three examples of pyramidal neurons from layer II/III drawn after injection of Lucifer yellow and followed by anti-Lucifer yellow immunodetection conjugated with DAB-Ni-based reaction. B, No correlation between the dendritic length and behavioral performance. C, Illustration of the methods used to analyze spine density of layer II/III pyramidal neurons: the grayscale image scale was reversed and deconvolved, and a semiautomated analysis of dendritic spine density was performed, yielding a separation of thin and mushroom spines. The other spines, presumably stubby spines, were discarded from analyses. D, Significant age-dependent spine loss. E, Scatter plots of thin spine densities versus exploratory or novel object recognition performance. The thin spine density appears to be predictive of behavioral performance across all groups of age but not within the aged group. F, No relation between mushroom spines density and behavioral performance. **p < 0.01 Student's t test.

Figure 10.

Exploratory behavior correlates with memory performance and inhibitory synaptic activity. A, We found a significant linear correlation between the performances of the animals in the NOR test (NOR index) and the modified hole-board arena test (ICE) (red line; p < 0.01; r = −0.38). The black dotted lines represent the cutoff limits between the impaired and unimpaired status for each test. The majority of animals displayed optimal behavioral performance (yellow region), whereas a subset of aged animals was impaired on both tests (gray region) B, Contour map where frequency (in hertz) of mIPSCs is plotted using color coding, superimposed on the values plotted in A. Animals with the most severe behavioral impairments also displayed an enhanced GABA_A receptor-mediated synaptic activity (bottom right quadrant), whereas, at the opposite end, animals with a lower inhibitory synaptic activity displayed the best performance during the behavioral tests (top left quadrant).