Experiment 2. Further verification of the effect of

150 G CS-alone trials were analyzed. These responses were not many sessions after AP5 infusion. This suggests that contaminated by the presence of URs and data analyses blocking NMDA receptors disrupted critical cellular plas- included only trials on which CRs were executed. ticity mechanisms that were responsible for establishing During sessions 3 to 5 of phase 1 of training, the AP5 eyeblink CRs. When the rabbits were trained to asymptotic group showed significantly fewer CRs than saline control performance levels and AP5 was infused a second time, group F1,1057.07, P,0.05. The AP5 rabbits also had the NMDA antagonist appeared to have a somewhat lesser, longer onset latencies than the control group Ms5408 ms but nonetheless significant, effect on CR characteristics. versus 289 ms F1,10511.88, P,0.01. Similarly, there These data suggest that activity at NMDA receptors in the was also a significant difference between group means cerebellum may be important for the initial establishment 352 ms versus 283 ms for the AP5 and control rabbits, and subsequent maintenance or performance of well- respectively when CR peak latencies were computed learned classically conditioned eyeblink responses. F1,10516.33, P,0.005. However, analyses did not reveal statistical difference in CR amplitudes between the groups. These results are in agreement with data from

3. Experiment 2. Further verification of the effect of

paired trials that showed that AP5 rabbits had significantly AP5 on retention of eyeblink CRs longer response latencies than the saline control thus suggesting that early in training, infusion of the NMDA In experiment 1, infusion of AP5 into the cerebellum antagonist influenced response timing as well as the was found to impair the acquisition of classical eyeblink number of CRs executed. conditioning in rabbits. And, the same infusion procedures Analysis of CS-alone trials during phase 2 of training appeared to have much lesser effects on the retention or sessions 6–10 revealed significantly longer onset laten- expression of the conditioned responses in well-trained cies for AP5-infused rabbits M5242 ms compared to animals. However, in experiment 1, AP5 retention tests control rabbits M5180 ms F1,1056.59, P,0.025, were conducted on the same rabbits that received AP5 however significant peak latency, percent CRs, and CR treatment during the first 5 days of the acquisition period. amplitude differences were not observed. It is possible that compensation mechanisms might have Significant differences in onset latencies Ms5216 ms developed in these animals such that they were immune and 175 ms for AP5 and control rabbits, respectively and from the later action of AP5. Experiment 2 was performed peak latencies Ms5260 ms and 291 ms for AP5 and to verify the effect of AP5 on the retention of eyeblink control rabbits, respectively, and CR amplitude were conditioning. In this experiment, two groups of rabbits observed in phase 3 of training sessions 11–15 were trained until CRs were well-established then treated F1,10511.38, P,0.01, F1,1059.43, P,0.05, and with either saline or AP5 and CR performance was F1,10517.52, P,0.005 for onset latency, peak latency compared. and CR amplitude, respectively. No significant difference in percentage CRs was found. 3.1. Materials and methods During phase 4 of training sessions 16–20, although significant differences between the AP5 and control rabbits were not found when percentage CRs and CR amplitudes 3.1.1. Subjects and surgery were analyzed, differences were noted when response Fourteen male rabbits of the same type and maintained latencies were examined. The AP5 rabbits displayed longer in the same condition as those included in experiment 1 onset latencies Ms5211 ms versus 167 ms F1,105 were used in this experiment. Of these, based on the 6.78, P,0.05 and longer peak latencies Ms5248 ms inclusion criteria that were described for experiment 1, versus 279 ms F1,1055.63, P,0.05 than control nine were eventually included in the data analyses. Sub- rabbits. jects were housed, fed and surgically prepared as described Overall, analysis of the characteristics of CRs displayed in experiment 1. on CS-alone trials indicated that the effects of NMDA antagonist infusion during early sessions of paired CS–US training had somewhat long-lasting effects on CR per- 3.1.2. Behavioral training, drug administration and formance. Differences between AP5 rabbits and saline histology control rabbits in CR characteristics such as response All the experimental procedures were identical to those timing could be detected 15 days after initial training. in experiment 1, except the drug treatments were delivered as shown in Table 2. That is, rabbits were trained for 10 2.2.2.6. Summary of experiment 1 results. The results of days phases 1 and 2 using paired tone CS and air puff experiment 1 demonstrated that infusions of AP5 retarded US trials, received infusion of AP5 or saline during phase the rate of acquisition and affected response timing of the 3 of paired training sessions 11–15, then received an classically conditioned eyeblink response. Furthermore, the additional 5 days of post-infusion training during phase 4 observed impairments of conditioning were apparent on sessions 16–20. G . Chen, J.E. Steinmetz Brain Research 887 2000 144 –156 151 Table 2 Drug treatment arrangement in experiment 2 Group Phase 1 Phase 2 Phase 3 Phase 4 sessions 1–5 sessions 6–10 sessions 11–15 sessions 16–20 Saline No injection No injection Saline No injection AP5 No injection No injection AP5 No injection 3.2. Results indicating that for the most part, all rabbits had reached asymptotic responding levels by session 10 see Fig. 4. Five rabbits were excluded in this experiment because they did not reach the 75 CR criteria. Three of them gave 3.2.2.3. Phase 3 sessions 11 –15. During the drug infu- no more than 10 of CRs in the first 10 sessions and thus sion sessions session 11 to session 15, no significant were excluded immediately before the injection sessions. differences between the groups on any measures were Hence, four rabbits were included in the saline group and found with ANOVA although the learning curves Fig. 4 five rabbits were included in the AP5 group. appear to show that the AP5 group had some temporary decrements in both percent CRs and CR amplitudes. The 3.2.1. Histology ANOVAs did indicate a significant session effect in onset Fig. 3 illustrates the cannula tip placements of all rabbits latencies F4,2852.85, P,0.05 and a trend of session included in the analyses for experiment 2. As in experi- effect in CR amplitudes F4,2852.18, P,0.1. Mean ment 1, in the left infusion side of the cerebellum, cell onset latencies for the AP5 and control groups were 201 bodies were clearly seen under light microscope in the area and 180 ms, respectively. Examination of the phase 3 data of interpositus and dentate nucleus. The number of cells of individual rabbits revealed that the lower percent CRs, observed did not appear to be different from the other side smaller CR amplitudes and longer onset latencies were of the cerebellum. Limited gliosis was observed around the present in only two of the five AP5 rabbits. The other three cannula and or electrode tips in some of the rabbits, but rabbits showed little or no effect of the AP5 infusion. The the nuclei appeared to be intact. temporary effect on CR performance seemed not to be In the rabbits that did not reach the 75 CR criterion, related to the relative placement of the infusion; one rabbit the electrode tips penetrated into either the interpositus or had a cannula placement just dorsal to the interpositus the dentate nucleus and caused clear damage. Thus, it is nucleus while the second rabbit had a cannula placement likely that poor learning was due to cerebellar damage just beneath the cerebellar cortex. It should be noted that caused by the implantation of the cannulae. all five rabbits showed abolition of responding when muscimol was infused after training thus indicating that all 3.2.2. Behavioral analyses cannula placements were effective in delivering the AP5 to Fig. 4 illustrates the learning curves of the two groups the critical region of the interpositus nucleus known to be over the 20 training sessions that were given. As in involved in eyeblink conditioning. Alternatively, previous experiment 1, the averages of CS–US paired trials and research has suggested that inactivation of select regions of CS-alone trials were analyzed separately. To be consistent cerebellar cortex significantly affects CR performance. For and to allow for cross-experiment comparisons, the same example, Attwell et al. [2] demonstrated that reversible phases of sessions were examined with ANOVAs as in inactivation of cerebellar cortex with CNQX, which experiment 1. blocked cortical AMPA-kainate receptors, effectively blocked the performance of previously established CRs. 3.2.2.1. Phase 1 sessions 1 –5. ANOVAs conducted on data collected during sessions 1 and 2 and sessions 3 to 5 3.2.2.4. Phase 4 sessions 16 –20. During the last five revealed no significant differences between groups on any no-injection sessions session 16 to session 20, no group measures. As expected, there was a strong session effect effects were revealed and no consistent session effects in P,0.0001 for sessions 3 to 5 involving all measures the data were noted see Fig. 4. except UR amplitude, indicating a solid CR acquisition effect but no learning-related changes in the UR am- 3.2.2.5. CR characteristics. As in experiment 1, the onset plitudes see Fig. 4. latencies, peak latencies, percentage CRs, and amplitudes of the CRs observed during CS-alone trials were analyzed. 3.2.2.2. Phase 2 sessions 6 –10. Analyses conducted on ANOVA with repeated measures did not reveal significant data collected during sessions 6–10 reveal no group differences between group means for CR onset latency, differences in CR rate, CR amplitude, onset latency, peak frequency, or amplitude during phase 1, 2 or 4 of training. latency or UR amplitudes. A significant session effect was However, significant effects were noted when CS-alone noted only when onset latencies were analyzed P,0.005 trials from phase 3 the drug infusion phase were ana- 152 G Fig. 3. Schematics of coronal sections though the rabbit cerebellum and brainstem showing locations of cannula tips for experiment 2 rabbits. Numbers represent distance in millimeters of the section relative to the lambda skull landmark. Squares depict cannula tips for saline control rabbits and triangles depict cannula tips for AP5 rabbits. ANS, ansiform lobe; ANT, anterior lobe; IC, inferior colliculus; icp, inferior cerebellar peduncle; IO, inferior olive; PF, paraflocculus lobe; VCN, ventral cochlear nucleus; VN, vestibular nucleus. G . Chen, J.E. Steinmetz Brain Research 887 2000 144 –156 153 trained to asymptotic performance levels, infusions of AP5 appeared to have a temporary effect on CR production and topography in some rabbits. These data are, for the most part, in agreement with the results of experiment 1 that suggest that the NMDA antagonist consistently affects acquisition and has a transitory effect on the timing of classically conditioned eyeblink responses.

4. Discussion