Neither the main effects nor the interaction was significant. Data from young and old mice indicate that young mice show ceiling performance on the first day, whereas old mice do not perform at a comparable level until the second day of training. A post hoc analysis using the Tukey honestly significant difference test HSD indicated that 4-month-old mice significantly outperformed both and month-old mice on the first day of acquisition Fig. Given this significant interaction, we performed a one-way repeated-measures analysis for the 4-month-old group, comparing percentage of CRs over the 10 training blocks.
Block-by block analysis for training session 2 indicated that there were no group differences on this subsequent training day Fig.
Block-by-block CR acquisition on the first day of training reveals that older mice acquire CRs considerably more slowly than do young mice. B Percentage of CRs on session 2 in the same mice over 10 nine-paired trial blocks in the msec delay eyeblink classical conditioning procedure as assessed by computer scoring of CRs. The second day of acquisition shows that age-related differences in percent CR do not extend beyond the first training session.
Eyeblink data from 4-month-old mice shows that mice in the paired CS-US procedure produce significantly more responses than do mice in the explicitly unpaired procedure. Responses during the CR period decreased from day 1 to day 5 in the explicitly unpaired procedure. Two dependent measures, taken from the rotorod task that are often examined as indicators of motor coordination and motor learning are walk time and latency to fall.
Walk time is a measure of the time that the mouse is actively walking on the rotating beam.
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Latency to fall is a measure of the amount time the mouse is able to avoid falling. Because mice may have a tendency to grip the rotorod and passively rotate rather than actively locomote, these two dependent measures may differ.
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Paired samples t tests were conducted between the daily mean for walk time and latency to fall at each rotation speed. The absence of significant differences indicated that mice spent their time on the rotorod actively walking rather than passively rotating. Because no difference was revealed between walk time and latency to fall, the latter measure was used in the present analyses to assess motor abilities.
Examining the main effect of training session within each age group, post hoc tests using the Bonferroni adjustment for multiple comparisons indicated that learning occurred in all groups. The month-old group showed a significant difference in latency to fall between the training sessions, showing longer latencies each session.
The 4- and month-old groups both showed significant motor learning between the first and second training sessions, and no difference between sessions 2 and 3. This result most likely indicates ceiling performance in the rpm rotorod task by session 2 in young and middle-aged mice.
A Tukey HSD post hoc test determined that the 4-month-old group outperformed the month-old group in session 1, as well as the month-old group in sessions 1 and 2 of the rpm rotorod task. There were no differences between and month-old rpm rotorod performance in any training session Fig. Learning in the cerebellar-dependent rotorod task is impaired in older mice at both 15 and 25 rpm. The rpm procedure is sensitive to deficits found in month-old mice. These mice do not perform similarly to 4 month olds until the third day of training. The rpm procedure is sensitive to deficits found in both and month-old mice.
Learning, nonetheless, is expressed by all age groups in both procedures. Examining the main effect of training session within each age group, simple effects tests revealed the degree to which repeated training was effective in improving rpm rotorod performance in each age group. Post hoc tests using the Bonferroni adjustment for multiple comparisons found that learning occurred in all groups. The and month-old groups showed a significant difference in latency to fall between the training sessions, showing longer latencies each session.
The 4-month-old group showed significant motor learning between the first and second test session, and no difference between sessions 2 and 3. This most likely indicates ceiling performance in the rpm rotorod task by session 2 in young mice.
Neural Substrates of Eyeblink Conditioning: Acquisition and Retention
A Tukey HSD post hoc test determined that the 4-month-old group outperformed the month-old group on sessions 1 and 2, as well as the month-old group on all training sessions of the rpm rotorod task. There were no differences between and month-old rpm rotorod performance on any training session Fig. A Pearson product-moment correlation was performed between percentage of CRs on the first training day for the 46 mice in the age range of 4 to 18 months that were tested with the msec eyeblink classical conditioning procedure and rotorod.
Percentage of CRs was not correlated at significant level with dependent measures from the acoustic startle, prepulse inhibition, or Morris water maze assessments. The intensity of a startle response is measured according to the amount of stabilimeter deflection that a mouse's body movement causes when it is presented with an auditory stimulus. The deflection is transduced into a congruent electrical pulse, which is read into the computer in millivolts.
Response intensity is expressed as Vmax, the measure of maximum voltage transduced when movement occurs in the startle chamber. It is possible that the weight of an animal may contribute to the intensity of the response, and therefore, the effect of weight on response intensity must first be considered. Correlations between weight and Vmax were conducted within the three age groups to determine to what extent an animal's weight has contributed to the response intensity evoked by the startle stimuli.
None of the correlations approached significance, indicating that weight had little effect on Vmax. Post hoc comparisons using the Tukey HSD test revealed differences in responding between groups at all dB levels. The only nonsignificant difference was between the 4- and month-old startle responses to a dB stimulus Fig. Within subjects post hoc analyses using the Bonferroni test revealed that mice in the 4-month-old group showed a significant difference in Vmax between the decibel levels, showing larger responses to louder sounds.
In all other age groups, however, response intensity did not necessarily increase as stimulus intensity increased. Although there were large startle responses shown by the month-old group, no significant differences in Vmax were detected between the decibel levels. The month-old group showed no difference in startle between 95 and dB, but a significant increase in Vmax was detected at dB. This indicates a deficit in responding to lower decibel levels in older mice. Young mice produce larger startle responses to louder stimuli.
click here This is not necessarily true for older mice. Although month-old mice show strong reactions to the three dB levels, there is no difference between these reactions. Mice aged 18 months produce small startle responses to and dB stimuli, but a significant increase in startle is not observed until the loudest stimulus is presented. The low Vmax values produced by month old mice at 95 and dB may indicate that the mice were not hearing the stimuli well. To examine the possibility that older mice did not hear the stimulus at 95 and dB, paired samples t tests were used to examine differences in Vmax between no-stimulus trials which record baseline chamber activity without presenting stimuli and the lower decibel levels.
In each case, the stimulus trials evoked a significantly higher Vmax than the no-stimulus trials, indicating that month-old mice heard the stimuli and produced startle responses. Post hoc comparisons using the Tukey HSD test examined differences in percent inhibition between age groups. These analyses revealed significant differences between both the 4- and month-old groups and the month-old group for the dB prepulse stimulus and the dB prepulse stimulus. The dB prepulse stimulus produced significant differences between the month-old and the month-old groups only.
There were no differences between the 4- and month-old groups at any decibel level of the prepulse stimulus. A high percent PPI indicates that the prepulse inhibited the startle response.
Mice aged 4 and 12 months perform at a similar rate, but month-old mice are impaired. Because startle was not inhibited with an dB prepulse stimulus, the hearing threshold for month-old mice is most likely between 80 and 85 dB. Impairment in attention and sensory gating may be to blame for low inhibition at 85 and 90 dB. Examining the main effect of prepulse intensity within each age group, simple effects tests revealed the degree to which the prepulse decibel level is differentially effective at inhibiting the startle response in each age group.
Post hoc tests using the Bonferroni adjustment for multiple comparisons revealed significant differences between all prepulse intensities except 85 and 90 dB in month-old mice, and between all prepulse intensities in month-old mice. Although 4-month-old mice showed inhibition of the startle response across all prepulse intensities, there were no significant differences.
The change in PPI across prepulse intensities did approach significance, however, and a general trend was that louder prepulse decibel levels produced greater inhibition. This trend was observed across all age groups. The lack of any response inhibition at the dB level in the month-old mice likely indicates that the oldest mice were unable to hear the prepulse stimulus. During acquisition, or hidden platform training, mice were repeatedly introduced to a pool filled with aversively cool water and were required to learn the location of a hidden escape platform using stationary contextual cues.
The dependent measure of learning was latency to escape from the water by climbing onto the platform. There was no main effect for age and no interaction between age group and training session. This indicates that all age groups learned at equal rates, producing shorter latencies to escape over the 3 days of acquisition. Post hoc tests using the Bonferroni adjustment for multiple comparisons found that the 4-month-old group showed significant differences between sessions 1 and 3, the month-old group showed significant differences between all training sessions, and the month-old group showed differences between session 1 and sessions 2 and 3.
TS indicates training session. Young mice outperform older mice on session 1 of spatial learning, and group differences disappear by session 2. This is most likely a result based on the observation that young mice tend to show thigmotaxis, an inclination to circle the pool in search of an external escape rather than an internal one. After hidden platform training, the escape platform is removed from the pool.
Probe trials are used to measure place learning retention. One measure taken from probe trials is the number of times the mouse crosses over the former platform area. This measure is taken from each quadrant, and it is expected that mice will cross over the area in the trained quadrant more often than the same area in the other three untrained quadrants. In the Morris water maze probe trials, all mice crossed the area in the platform trained quadrant more times than they crossed the same area in untrained adjacent and opposite quadrants.
Mice generally spent more time swimming in the trained platform quadrant than they did swimming in the untrained adjacent and opposite quadrants. Another measure of retention is the time spent swimming in each quadrant, regardless of whether or not mice pass over the platform area.
Generally, the age group means indicated that mice preferred the former platform quadrant. In this condition, a flag was attached to the platform to mark its location below the surface of the water. A Bonferroni post hoc test determined that mice aged 4 and 12 months old performed significantly better from session 1 to session 2 of cued training, whereas month-old swimming performance did not change between the two sessions. Early in the development of a mouse model of eyeblink classical conditioning in our laboratory, we heard from one experienced colleague that female mice conditioned more poorly than did male mice.
To reduce costs, we aged only male mice, and our older age groups have few female mice. The only older female mice we tested at the age of 12 months were older breeders, and their data were hand-scored. The 4-month-old computer-scored group had eight males and eight females, but they were dispersed through the paired and explicitly unpaired conditions. The same result occurred in the analysis of the five males and three females tested in the explicitly unpaired condition.
Low power may have contributed to our inability to detect sex differences in eyeblink conditioning.