Aging in the cerebellum and hippocampus and associated behaviors over the adult life span of CB6F1 mice

Abstract

In the present study we examined the effects of normal aging in the hippocampus and cerebellum, as well as behaviors associated with these substrates. A total of 67 CB6F1 hybrid mice were tested at one of five ages (4, 8, 12, 18 or 25 months) on the context pre-exposure facilitation effect (CPFE) modification of fear conditioning, rotorod, Barnes maze, acoustic startle, Morris water maze (MWM) and 500-ms trace eyeblink classical conditioning (EBCC). Behavioral tasks were chosen to increase the ability to detect age-related changes in learning, as trace EBCC is considered a more difficult paradigm (compared to delay EBCC) and the CPFE has been found to be more sensitive to hippocampus insults than standard contextual fear conditioning. To assess the effects of age on the brain, hippocampus volume was calculated and unbiased stereology was used to estimate the number of Purkinje neurons in the cerebellar cortex. A significant, age-related loss of Purkinje neurons was found-beginning at 12 months of age-and hippocampus volume remained stable over the adult life span. Age-related impairment was found, beginning at 12-18 months in the rotorod, and mice with fewer Purkinje neurons showed greater impairment in this task. CB6F1 mice retained auditory acuity across the life span and mice aged 25 months showed significant age-related impairment in the EBCC task; however, deficits were not associated with the loss of Purkinje neurons. Although the CPFE task is considered more sensitive to hippocampus insult, no age-related impairment was found. Spatial memory retention was impaired in the Barnes maze at 25 months, but no significant deficits were seen in the MWM. These results support the finding of differential aging in the hippocampus and cerebellum.

Keywords: ANOVA; CB6F1; CPFE; CRs; EBCC; EMG; MWM; Morris water maze; SD; SMART; Spontaneous Motor Activity Recording and Tracking; age sensitivity; aging; analysis of variance; cerebellum; conditioned responses; context pre-exposure facilitation effect; electromyography; eyeblink classical conditioning; hippocampus; learning; standard deviation.

Figure 1

Age-related deficits in cerebellar cortical Purkinje neurons. Total estimated number of Purkinje neurons in mice aged 4–25 months is shown. Significant loss of neurons was found in mice aged 12, 18, and 25 months. Values are mean ± SE. *p < .05 compared to 4 mo, ** p < .01 compared to 4 mo, ^ p < .05 compared to 8 mo.

Figure 2

CPFE fear conditioning across the life span. Percentage of time freezing during pre-exposure (Phase 1) and context test (Phase 3) in mice aged 4–25 months is shown. 25 month-old mice froze significantly more during the initial five minute pre-exposure, but no differences were found during the six minute context test.

Figure 3

Age-related deficits in motor learning on the rotorod. Latency to fall from the rotating beam in mice aged 4–25 months is shown. Main effects of Age were found at both rotation speeds (15 and 25 RPM). Post-hoc tests determined that mice aged 18 (p < .05) and 25 (p < .01) months displayed significant impairment relative to mice aged 4 months at 15 RPM. At 25 RPM, mice aged 12 (p = .05), 18 (p < .05), and 25 months (p < .05) displayed significant impairment compared to mice aged 4 months.

Figure 4

Barnes maze retention across the life span: Primary latency Day 2, Trial 1. Latency to the first visit to the escape hole during Trial 1 of Day 2 in mice aged 4–25 months is shown. Mice aged 25 months took significantly longer to first visit the escape hole than mice aged 4 months. Values are mean ± SE. *p < .05 compared to 4 mo.

Figure 5

Auditory acuity is maintained across the adult life span in CB6F1 mice. Average startle intensity (Vmax) to stimuli ranging from 80–90 dB in mice aged 4–25 months is shown. Mice of all ages had similar startle responses, which confirmed that older mice retained hearing ability.

Figure 6

MWM acquisition across the life span: Latency. Latency to escape the MWM in mice aged 4–25 months is shown. Learning is expressed by all groups, as latency decreases across Training Sessions. Overall, mice aged 25 months took significantly longer to find the platform than did 8 month-old mice (p < .05). The Age x Training Session interaction revealed differences in performance between 25 month-old mice and younger mice at Training Sessions 1, 8 and 9. Values are mean ± SE. *p < .05 compared to 4 mo, ** p < .01 compared to 4 mo, ^ p < .05 compared to 8 mo, ^^ p < .01 compared to 8 mo, + p < .05 compared to 18 mo.

Figure 7

Age-related impairment in 500 ms trace EBCC. A. The percentage of conditioned responses across baseline and five training sessions in mice aged 4–25 months is shown. Overall, mice aged 25 months produced significantly fewer conditioned responses (ps < .001). B. The mean total percentage of conditioned responses during training in mice aged 4–25 months is shown. Mice aged 25 months were significantly impaired on this measure (p < .001) as well.