Differential effects and rates of normal aging in cerebellum and hippocampus

Abstract

Cognitive functions show many alternative outcomes and great individual variation during normal aging. We examined learning over the adult life span in CBA mice, along with morphological and electrophysiological substrates. Our aim was to compare cerebellum-dependent delay eyeblink classical conditioning and hippocampus-dependent contextual fear conditioning in the same animals using the same conditioned and unconditioned stimuli for eyeblink and fear conditioning. In a subset of the behaviorally tested mice, we used unbiased stereology to estimate the total number of Purkinje neurons in cerebellar cortex and pyramidal neurons in the hippocampus. Several forms of synaptic plasticity were assessed at different ages in CBA mice: long-term depression (LTD) in both cerebellum and hippocampus and NMDA-mediated long-term potentiation (LTP) and voltage-dependent calcium channel LTP in hippocampus. Forty-four CBA mice tested at one of five ages (4, 8, 12, 18, or 24 months) demonstrated statistically significant age differences in cerebellum-dependent delay eyeblink conditioning, with 24-month mice showing impairment in comparison with younger mice. These same CBA mice showed no significant differences in contextual or cued fear conditioning. Stereology indicated significant loss of Purkinje neurons in the 18- and 24-month groups, whereas pyramidal neuron numbers were stable across age. Slice electrophysiology recorded from an additional 48 CBA mice indicated significant deficits in LTD appearing in cerebellum between 4 and 8 months, whereas 4- to 12-month mice demonstrated similar hippocampal LTD and LTP values. Our results demonstrate that processes of aging impact brain structures and associated behaviors differentially, with cerebellum showing earlier senescence than hippocampus.

Fig. 1.

Eyeblink classical conditioning at five ages in CBA mice. (A) Percentage of conditioned responses (CRs) over 10 sessions of 90 paired trials in 8–10 male CBA mice per age group. The 24-month group was significantly impaired in the acquisition of CRs. (B) Mean total percentage of CRs in the five age groups of CBA mice in A above. The 24-month group produced significantly fewer CRs. An asterisk indicates a significant difference (*P < 0.05). Error bars are SE of the mean.

Fig. 2.

Fear conditioning at five ages in CBA mice. Performance was assessed by percentage of time freezing in 1-min time intervals in cued and contextual fear conditioning. There were 8–10 male CBA/J mice tested in three sessions. Session 1 consisted of a 2-min pre-conditioned stimulus (pre-CS) baseline period and a 2-min period after pairing of the CS and unconditioned stimulus (US). Twenty-four hours later, the context test was carried out in session 2. Mice were placed for 5 min in the same chamber where they experienced the CS-US pairing. Session 3 occurred 1 h later in a different chamber distinguished by novel auditory, olfactory, tactile, and visual cues. There was a 3-min pre-CS period of no stimulation and a 3-min post-CS period after the tone CS sounded. There were no age differences in freezing.

Fig. 3.

Stereological neuron counts in cerebellum (A) and hippocampus (B). (A) Mean estimated number of Purkinje neurons in the entire cerebellar cortex of CBA mice aged 4, 8, 12, 18, and 24 months. Stereological counts were carried out on six mouse cerebella per age group. (B) Mean estimated number of pyramidal neurons in the hippocampus of CBA mice aged 4, 8, 12, 18 and 24 months. Stereological counts were made on two mouse hippocampi per age group. Bars represent SE of the mean.

Fig. 4.

Cerebellar LTD recorded from 4-, 8-, and 12-month CBA mouse slices. A histogram is shown of EPSP slope from 4-, 8-, and 12-month CBA male mouse slices following low-frequency stimulation designed to induce cerebellar LTD. The increased amount of LTD in the 4-month group is statistically significant (P < 0.0001).

Fig. 5.

Varying stimulation frequencies (25, 100, and 200 Hz) used to induce hippocampal LTP (and LTD) in 4- and 12-month male CBA mouse slices. A histogram is shown of fEPSP slope values (percentage of baseline) from 4- and 12-month CBA male mouse slices following low-frequency stimulation designed to induce hippocampal long-term depression (LTD) and long-term potentiation (LTP). Although there were appreciable amounts of both hippocampal LTD and hippocampal LTP (compared to baseline) across all stimulation frequencies tested, there were no significant differences found between the 4- and 12-month age groups in any of the stimulation conditions that were tested.