Age-dependent impairment of eyeblink conditioning in prion protein-deficient mice

Abstract

Mice lacking the prion protein (PrP(C)) gene (Prnp), Ngsk Prnp (0/0) mice, show late-onset cerebellar Purkinje cell (PC) degeneration because of ectopic overexpression of PrP(C)-like protein (PrPLP/Dpl). Because PrP(C) is highly expressed in cerebellar neurons (including PCs and granule cells), it may be involved in cerebellar synaptic function and cerebellar cognitive function. However, no studies have been conducted to investigate the possible involvement of PrP(C) and/or PrPLP/Dpl in cerebellum-dependent discrete motor learning. Therefore, the present cross-sectional study was designed to examine cerebellum-dependent delay eyeblink conditioning in Ngsk Prnp (0/0) mice in adulthood (16, 40, and 60 weeks of age). The aims of the present study were two-fold: (1) to examine the role of PrP(C) and/or PrPLP/Dpl in cerebellum-dependent motor learning and (2) to confirm the age-related deterioration of eyeblink conditioning in Ngsk Prnp (0/0) mice as an animal model of progressive cerebellar degeneration. Ngsk Prnp (0/0) mice aged 16 weeks exhibited intact acquisition of conditioned eyeblink responses (CRs), although the CR timing was altered. The same result was observed in another line of PrP(c)-deficient mice, ZrchI PrnP (0/0) mice. However, at 40 weeks of age, CR incidence impairment was observed in Ngsk Prnp (0/0) mice. Furthermore, Ngsk Prnp (0/0) mice aged 60 weeks showed more significantly impaired CR acquisition than Ngsk Prnp (0/0) mice aged 40 weeks, indicating the temporal correlation between cerebellar PC degeneration and motor learning deficits. Our findings indicate the importance of the cerebellar cortex in delay eyeblink conditioning and suggest an important physiological role of prion protein in cerebellar motor learning.

Figure 1. Eyeblink conditioning in 16-week-old Ngsk Prnp ^0/0 mice.

(A) CR development was calculated using 100 trials/day for each animal during a 7-day session in 16-week-old control (open circle, n = 14) and Ngsk Prnp ^0/0 (experimental mice; closed circle, n = 14) mice. The top inset shows individual response topographies of averaged electromyographs (EMGs) until US onset in the control and experimental mice on days 3 and 7. (B) The averaged EMG amplitudes on day 7 of the acquisition session in control (n = 14) and experimental (n = 14) mice. All EMG amplitudes obtained in 1 day (100 trials) were summed, representing the average of the eyelid responses. The CR components of 250–350 ms from CS onset were masked by artifacts caused by the US. (C) The averaged EMG amplitudes were evaluated using only 10 CS-only trials on day 7 of the acquisition session. These analyses enable us to indicate EMG patterns in the whole CS period without the US artifacts. Values are mean ± SEM. *p<0.05.

Figure 2. CR timing is altered in 16-week-old Ngsk Prnp ^0/0 mice.

(A) Histogram showing the normalized frequency of the peak CR plotted as a function of its latency. The CR in 100 trials on day 7 was binned into time windows of 52–102, 102–152, 152–202, and 202–252 ms measured from CS onset in control mice (open column) and Ngsk Prnp ^0/0 mice (experimental mice; closed column). (B) Frequency histogram of the CR peak latencies in 10 CS-only trials on day 7 in the time window until the CS end (50-ms widths) for control (open column) and experimental mice (closed column). The adaptive components during US period (252–302 and 302–352 ms from CS onset) of CRs were significantly less in experimental mice. Data points represent the mean ± SEM. *p<0.05.

Figure 3. Impaired eyeblink conditioning in 40-week-old Ngsk Prnp ^0/0 mice.

(A) CR development during a 7-day acquisition session in control (open circle, n = 10) and Ngsk Prnp ^0/0 (experimental mice; closed circle, n = 10) mice aged 40 weeks. The top inset shows individual response topographies of averaged EMGs until US onset in control and experimental mice on day 7. (B) Averaged EMG amplitudes during the acquisition session on day 7. All EMG amplitudes obtained in 1 day were summed, representing the average of the eyelid responses. (C) Frequency histogram showing the peak CR latencies. The CR on day 7 was binned into time windows of 52–102, 102–152, 152–202, and 202–252 ms measured from CS onset in experimental mice (closed column) and control mice (open column). Data points represent the mean ± SEM. *p<0.05.

Figure 4. Impaired eyeblink conditioning in 60-week-old Ngsk Prnp ^0/0 mice.

(A) Eyeblink conditioning performance during the 7-day acquisition training in Ngsk Prnp ^0/0 mice (experimental mice; closed circle, n = 8) and control mice (open circle, n = 8) aged 60 weeks. The top inset shows individual response topographies of averaged EMGs until US onset in control and experimental mice on day 7. (B) Averaged EMG amplitudes during the acquisition session on day 7. All EMG amplitudes obtained in 1 day were summed, representing the average of the eyelid responses. (C) Frequency histogram showing the peak CR latencies. The CR on day 7 was binned into time windows of 52–102, 102–152, 152–202, and 202–252 ms measured from CS onset in experimental mice (closed column) and control mice (open column). Data points represent the mean ± SEM. Data points represent the mean ± SEM. *p<0.05, **p<0.01.

Figure 5. Eyeblink conditioning in ZrchI Prnp ^0/0 mice.

(A) Development of the CR during 7-day acquisition training in ZrchI Prnp ^0/0 mice (closed circle, n = 9), FVB/Nj mice (open circle, n = 7), 129/Sv mice (open triangle, n = 5), and C57BL/6J mice (open square, n = 6) aged 18–22 weeks. (B) Histogram showing the normalized frequency of the peak CR plotted as a function of its latency. Histograms showing the frequency of the CR plotted as a function of its latency. The CR in 100 trials on day 7 was binned into time windows of 52–102, 102–152, 152–202, and 202–252 ms measured from CS onset in the three control strains and ZrchI Prnp ^0/0 mice (closed column). (C) Frequency histogram of the CR peak latencies in 10 CS-only trials on day 7 in extended time windows (52–102, 102–152, 152–202, 202–252, 252–302, and 302–352 ms from CS onset) for the three control strains and ZrchI Prnp ^0/0 mice (closed column). Data points represent the mean ± SEM. *p<0.05.