Experiment 1: AP5 infusion and acquisition and

G . Chen, J.E. Steinmetz Brain Research 887 2000 144 –156 145 another competitive NMDA antagonist, gamma-LGLA, part of the memory trace for eyeblink conditioning is impaired the retention of the temporal component but not localized within the cerebellum. the spatial discrimination component of a Y-maze active It is generally agreed that neurons in the cerebellar deep avoidance task with mice. The involvement of NMDA nuclei express NMDA receptors [1,3,4,45] but whether or receptors has also been demonstrated for step-through not Purkinje cells express NMDA receptors is somewhat inhibitory avoidance tasks [26,29], step-down inhibitory controversial. Expression analysis by in situ hybridization learning [23], discriminative approach response learning has revealed that Purkinje cells express NMDA receptor [7], and taste-potentiated odor conditioning [21]. subunit NR1 but may not express, or express in relatively It has been demonstrated that systemic administration of low levels, the NR2 family subunits [1,45]. However, the non-competitive NMDA antagonist, MK-801 or PCP, pharmacological and electrophysiological studies failed to and the competitive NMDA antagonist, CGP-39551, im- observe functional evidence of NMDA receptors in Pur- paired acquisition of classical eyeblink conditioning in kinje cells [3,4]. These observations may be reconciled if rabbits [37,42] and rats [39]. In all cases, the NMDA one considers that the functional properties of the antagonists appeared to have no effect on retention and heteromeric NMDA receptor-channel complex are critical- performance of previously acquired eyeblink conditioned ly determined by the constituting NR2 subunits. responses, nor did they appear to affect sensory reactivity In cerebellum, there are cell type-specific expressions of or unconditioned responses. While the above studies did NMDA receptor subunits [1,45]. In adult rats or mice, the not ascertain the target of NMDA antagonists, one study cerebellar granule cells express subunit NR2A and, more showed that memantine, a non-competitive NMDA re- abundantly, NR2C. Deep nuclei express subunit NR2A but ceptor antagonist with higher affinity to cerebellar tissue little NR2C. And, Purkinje cells are thought to express than forebrain tissue, also impaired eyeblink conditioning neither of those subunits. This information combined with in humans [38], suggesting the involvement of cerebellar the gene knockout technique provide some clues of the NMDA receptors. Interestingly, in Robinson’s study [37], functions of NMDA receptors in specific cells. Kishimoto MK-801 appeared to have no effect on conditioning- et al. [27] showed that mutant mice lacking NMDA related potentiation of perforant path-granule cell re- receptor subunit NR2A acquired eyeblink conditioning sponses in hippocampus, suggesting its effective target more slowly than wild-type animals but could attain the might be elsewhere. same asymptotic performance as the wild-type. In contrast, A number of permanent and reversible lesion studies mutants lacking subunit NR2C did not exhibit significant have demonstrated that the cerebellum, especially the impairment. This evidence suggests an important role of interpositus nucleus, is critical for classical eyeblink NMDA receptors in the deep nuclei in the acquisition of conditioning [11,15,28,36,41]. For example, Krupa et al. eyeblink conditioning. [28] showed that infusion of the GABA agonist muscimol This study was an attempt to assess the role of cerebellar A into the region of the interpositus nucleus abolished NMDA receptors in classical eyeblink conditioning. As in conditioned responses that were established before infu- a number of previous studies, we microinjected AP5 sion. More importantly, muscimol infusion during acquisi- solution into the brain region of interest, i.e. the cere- tion training prevented the formation of conditioned re- bellum, and examined its effect on the acquisition and sponses and no evidence of learning i.e. savings could be performance of conditioned responses. In brief, our data discerned when training was instituted after the infusion. A show that AP5 impaired conditioned response acquisition, recent study by Bracha et al. [6] provided additional and in some animals, had a temporary effect on con- evidence that cellular mechanisms in the cerebellum are ditioned response timing once learning had occurred. Some important for learning the eyeblink conditioned response of these results have been presented elsewhere in prelimin- CR. Microinjections of anisomycin, a protein synthesis ary form [14]. inhibitor, into the intermediate cerebellum near the inter- positus nuclei impaired the acquisition of the conditioning and appeared to have no effect on the expression of CRs in

2. Experiment 1: AP5 infusion and acquisition and

well-trained rabbits. In addition, we recently reported that performance of classical eyeblink conditioning cerebellar protein kinases are important for the acquisition, but not retention, of classically conditioned eyeblink NMDA receptors have been shown to be involved in a responses [13], a result that was also demonstrated in a variety of learning and memory tasks including eyeblink study by Gomi et al. [18] who showed that eyeblink conditioning in rabbits [37,42], rats [39] and humans [38]. conditioning induced a CDC2-related protein kinase, And, NMDA receptors appear to be expressed in both KKIAMRE, in the interpositus nucleus. These data argue cerebellar cortex and deep nuclei [1,3,4,45], but little is strongly that important cellular processes in the deep known regarding their function. We reasoned that if cerebellar nuclei are critical for the establishment and synaptic plasticity in the interpositus nucleus during con- maintenance of classical eyeblink conditioning. Overall, ditioning involved NMDA receptors, blocking cerebellar these data provide solid evidence that at least an important NMDA receptors should interfere with the learning pro- 146 G cess, and thus behaviorally retard or prevent conditioning. cannula electrode assembly was cemented into place on Here we used AP5, a specific NMDA receptor antagonist, the skull with dental acrylic along with a bolt for holding which has been widely used in a number of previous the air puff hose and an infrared device that measured studies. eyelid movement during behavioral training. 2.1. Materials and methods 2.1.3. Behavioral training and drug administration After at least 1 week was allowed for recovery from 2.1.1. Subjects surgery, rabbits received two adaptation sessions 15 min Fifteen male New Zealand white rabbits 1.6–2.5 kg at and 30 min while restrained in a Plexiglas box and placed surgery time were used in experiment 1. Six rabbits were inside a sound-attenuating chamber. From the third day on, assigned to an AP5 group. Nine rabbits were initially rabbits received one session of standard delay conditioning assigned to a saline control group with six eventually per day. During the training, a 350 ms, 85 dB SPL, 1 kHz included in subsequent data analyses. Before the experi- tone was used as the conditioned stimulus CS, and a 100 ment and between the training sessions, the rabbits were ms, 3 psi co-terminating air puff was used as the un- individually housed in cages and provided ad lib access to conditioned stimulus US. The tone CS was delivered via food and water. A 12 12 h light dark cycle was main- a speaker mounted |30 cm above the rabbit. The air puff tained in the animal housing area. US was delivered via a 3-mm air nozzle positioned 1 cm from the middle of the rabbit’s eye. Movement of the 2.1.2. Surgery external eyelids was measured as the unconditioned re- Surgeries were performed under aseptic conditions. sponse UR and the conditioned response CR using an Rabbits were deeply anesthetized with injections of 6 infrared emitter detector located 4–5 mm in front of the mg kg xylazine and 60 mg kg ketamine, and maintained rabbit’s cornea that measured changes in diffraction of a throughout the surgery with intramuscular injections of a beam of infrared light [43]. These measured changes were mixture of xylazine 3 mg kg and ketamine 30 mg kg amplified to reflect millimeters of external eyelid closure in delivered every 45 min. During the surgery, the skull over response to the CS or the US. Movement of the external the cerebellum was removed and a cannula–electrode eyelids was not restricted by eyelid clips during training. assembly was implanted into the region between the Each session consisted of six blocks of 10 trials one interpositus nucleus and dentate nucleus. The cannula was CS-alone test trial and nine CS–US paired trials. The constructed from 22 gauge stainless steel tubing. An inter-trial interval ITI varied pseudorandomly from 20 to epoxy-insulated, 00 stainless steel electrode |1 MV 30 s with an average of 25 s. impedance was glued to the cannula with its tip located All rabbits in the saline and AP5 groups were trained for about 1.5 mm lower than the cannula tip. The skull was 20 sessions of standard delay conditioning, with drug positioned so that lambda was 1.5 mm below bregma. The treatment given as shown in Table 1. The first period of electrode tips were stereotaxically implanted at 5.3 mm injections in phase 1 sessions 1–5 was designed to lateral from midline and a maximum of 14.5 mm below examine the effect of AP5 or saline infusion on the lambda depending on the observation of characteristic acquisition of eyeblink conditioning. Phase 2 of training interpositus nucleus activity recorded from the electrode sessions 6–10 allowed us to examine post-injection when it was lowered. The anterior–posterior coordinate performance before rabbits reached asymptotic perform- referenced to lambda was calculated with an equation: 4.8 ance. We then examined the asymptotic performance in mm20.31 mm3D, where D was the distance measured phase 3 sessions 11–15. The second period of injections between bregma and lambda with posterior positive, in phase 4 sessions 16–20 tested the effects of AP5 or anterior negative. This correction formula was created saline infusion on the performance of well-learned re- using regression analyses involving previous interpositus sponses. nucleus recording and lesion data e.g. Ref. [13]. A DL-AP5 solution Sigma Chemical, St. Louis, MO was stainless steel stylet was inserted into the cannula after dissolved in sterile physiological saline to a concentration surgery and was replaced between sessions to prevent the of 2.5 mg ml and the pH of the solution was adjusted to cannula from clogging. After the hole in the skull sur- about 7.0 with NaOH. During each infusion session, a 26 rounding the cannula was filled with bone wax, the gauge needle was inserted into the guide cannula with its Table 1 Drug treatment arrangement in experiment 1 Group Phase 1 Phase 2 Phase 3 Phase 4 sessions 1–5 sessions 6–10 sessions 11–15 sessions 16–20 Saline Saline No injection No injection Saline AP5 AP5 No injection No injection AP5 G . Chen, J.E. Steinmetz Brain Research 887 2000 144 –156 147 tip positioned 0.75 mm below the cannula tip. Using an 2.2. Results infusion pump, each rabbit received 2-ml injections at a rate of 8 ml h total duration 15 min via a Teflon tube Twelve of the 15 rabbits met our criteria for inclusion in connected to a 10-ml Hamilton syringe. The training the study and their data were analyzed. Three rabbits in the procedure started about 5 min after the infusion began to saline group were excluded; one did not reach the 75 CR allow the infusate to diffuse. criteria, another failed to pass the muscimol test, and a third one learned very slowly. Although the third rabbit reached the 75 CR rate criteria on the last day of 2.1.4. Histology and implant location verification training, histology showed a partial lesion to the inter- After completing all the training sessions, rabbits were positus nucleus due to the cannula placement. Therefore, returned to the conditioning chamber for one additional six AP5 rabbits and six saline rabbits were included in the session during which they were infused with 2 ml of the statistical analyses. GABA agonist, muscimol Sigma Chemical, dissolved in A saline to 400 ng ml, to verify the effectiveness of the 2.2.1. Histology implant. The muscimol was infused via the pump 15 min Fig. 1 depicts the cannula tip placements of all rabbits before the test session. A series of paired CS–US trials included in the analysis. Most cannula placements were were then delivered and the effects of muscimol on immediately dorsal to the area of the interpositus nucleus conditioned responding were noted. If muscimol did not identified in past studies as critical for eyeblink con- effectively reduce the CR rate of a rabbit to less than 10 ditioning e.g. Refs. [32,41]. A few placements were CRs on two consecutive blocks of training, that rabbit was somewhat distal from the interpositus nucleus, but the excluded from future data analysis as we assumed that muscimol test indicated that the drug was still able to failing this test indicated that the cannula was not diffuse to the critical area. While some gliosis was positioned correctly in the interpositus nucleus. observed around the cannula and electrode tips in some When this test was completed, the rabbits were over- rabbits, when the tissue was examined under a light dosed with an i.v. injection of 150 mg kg pentobarbitol microscope, cell bodies were clearly seen in the area of the and perfused intra-aortically with 0.9 saline followed by left interpositus and dentate nucleus, and the tissue did not 10 formalin. The brains were then removed and fixed in differ from the other side of the cerebellum. Cell bodies 30 sucrose 10 formalin solution. After a week, the could be clearly seen just beneath the cannula tips sug- brains were embedded, frozen sectioned at a thickness of gesting that the infusion did not cause neuronal death. 40 mm, and stained with cresyl violet. Cannula locations Because it was hard to identify lesions caused by cannula and the conditions of neurons in the interpositus nuclei and electrode implant solely with microscopic histology, were examined under a light microscope. the behavioral criteria described above were also used to exclude animals with lesions caused by cannula placement or volume injection. 2.1.5. Data analysis For the rabbit excluded from statistical analysis because Session-wide averages of behavioral response parame- of failing the muscimol test, the cannula placement was ters, including percent CRs, CR amplitudes, latencies to found to be too posterior and somewhat high. In rabbits response onset and peak, and UR amplitudes were ana- that did not reach the 75 CR criteria, either the cannula lyzed with mixed-design ANOVAs. In these analyses, tip touched the dorsolateral region of the interpositus group AP5 or control was the between factor and session nucleus or the electrode went through it, causing notice- 1–20 was the repeated measure on subjects. Behavioral able damage to the nucleus. responses on the CS-alone test trials and CS–US paired trials were analyzed separately. Rabbits that did not reach 75 CRs on any of the 20 sessions were excluded from 2.2.2. Behavioral analyses statistical analysis as there was a possibility that those Fig. 2 shows the learning curves of the AP5 and saline rabbits sustained damage to the interpositus nucleus due to groups over the 20 training sessions. The data from CS– the cannula and recording electrode that were implanted. US paired trials and CS-alone trials were analyzed separ- Rabbits that showed no reductions in CRs after muscimol ately. infusion were also excluded because the cannulas were likely misplaced. Next, to examine the drug effects on CR characteristics, 2.2.2.1. Phase 1 sessions 1 –5. Phase 1 represented the individual CRs on CS-alone trials were gathered and five initial drug treatment days. For analysis purposes, analyzed. Conditioned responses with onset latency shorter these sessions provide a good sample of acquisition than 50 ms were considered as artifacts and thus excluded. training. Because very few CRs were present in the AP5 or Mixed-design ANOVAs were performed to compare the the saline rabbits on sessions 1 and 2, two ANOVAs were groups. conducted on the sessions 1–5 data; one analysis on 148 G Fig. 1. Schematics of coronal sections through the rabbit cerebellum and brainstem showing locations of cannula tips for experiment 1 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. session 1 and 2 data and one analysis on sessions 3 to 5 in the AP5 group see Fig. 2A. Next, the CR amplitudes data. of the AP5 group were significantly smaller than the saline No group or session effects were found on any of the controls F1,1055.03, P,0.05 see Fig. 2B. In con- measures taken when session 1 and 2 data were analyzed. trast, there was no difference in UR amplitudes between This was expected because very few CRs were displayed the two groups, suggesting that the drug treatments had no by either group of animals on these days. Analyses of the effect on general motor responses. Significant session data from sessions 3 to 5, however, revealed significant effects were noted for all variables analyzed thus indicat- difference between groups. Firstly, analysis of percent CRs ing that learning was taking place all Ps,0.01. showed that the AP5 group produced significantly fewer Analysis of onset latencies revealed that the AP5 rabbits CRs than the saline group on each day F1,10513.91, had significantly longer response onset latencies M5257 P,0.005, indicating a general impairment in acquisition ms than the saline control M5202 ms F1,1057.30, G . Chen, J.E. Steinmetz Brain Research 887 2000 144 –156 149 though AP5 infusions were discontinued after session 5 see Fig. 2. Compared to control rabbits, AP5 rabbits still had significantly lower percentage CRs F1,1055.44, P,0.05 and lower CR amplitudes F1,1055.38, P, 0.05. The AP5 rabbits also displayed significantly longer CR onset latencies F1,1058.75, P,0.015 during phase 2; the mean onset latencies for the AP5 and saline rabbits were 210 ms and 157 ms, respectively. Additional acquisi- tion occurred in both groups, however, as significant session effects were noted for percentage CRs, CR am- plitudes and onset latencies all Ps,0.001. No differences between UR amplitudes were observed, however. 2.2.2.3. Phase 3 sessions 11 –15. Based on a number of previous studies, we expected that the rabbits would reach asymptotic performance levels by sessions 11 to 15. Results of ANOVAs conducted on the percent CR data showed, however, that the AP5 group generated fewer CRs than the saline group F1,1058.03, P,0.05, although the relative difference in the group averages saline vs. AP5 as 94.4 vs. 87.9 was much smaller than that in phase 1 see Fig. 2A. The analysis also revealed that the CR amplitudes of the AP5 group were significantly smaller than the saline controls F1,10512.25, P,0.001 and that the AP5 rabbits M5172 ms had significantly longer response onset latencies than the saline control M5143 ms F1,1059.69, P,0.05. These results indicate that the impairment of conditioning seen in AP5 rabbits lasted well into training when no AP5 was delivered. That is, there appears to be a lack of a savings effect in the AP5 rabbits once infusion of the NMDA antagonist was dis- continued. Again, there was no difference in UR am- plitudes between the two groups. Fig. 2. Learning curves for rabbits trained across 20 sessions in experi- ment 1. A Percent CRs and B CR amplitudes recorded from saline 2.2.2.4. Phase 4 sessions 16 –20. We expected that by squares and AP5 triangles rabbits. Sessions during which drug sessions 16–20, all rabbits would have most certainly infusions occurred are indicated by the I-labeled arrows. Error bars5 reached asymptotic responding levels and that this would S.E.M. be a good time to test the effects of AP5 on well-learned CRs. Analysis of the sessions factor showed few changes in responding between session 16 and session 20 as P,0.05. It should be noted, however, that in calculating significant session effects were not observed when per- the measures for each session, when a CR was absent, the centage CRs, CR amplitudes or CR onset latencies were CR amplitude was assigned zero and the onset latency was analyzed. However, analysis of phase 4 training data assigned a default value of 500 ms i.e. the end of the suggest that the AP5 rabbits reached slightly lower asymp- trial. The smaller average CR amplitudes of the AP5- totic responding levels: the AP5 rabbits showed a sig- treated animals and the longer average onset latencies nificantly lower percentage CR [F1,1058.01, P,0.05] could therefore be due to fewer CRs being executed by and longer onset latencies [F1,1057.82, P,0.05] than these rabbits as well as a prolonged acquisition during control rabbits. The mean onset latency for the AP5 rabbits which lower amplitude and later responses were executed. was 185 ms while the mean onset latency for the control To address this issue, additional analyses on the CRs rabbits was 149 ms. No differences in CR or UR am- observed during CS-alone trials are described below. plitudes were found. 2.2.2.2. Phase 2 sessions 6 –10. Analysis of data from 2.2.2.5. CR characteristics. To characterize better the phase 2 of training revealed that deficits in CR acquisition CRs executed by rabbits in different groups and during seen in the AP5 group during drug infusion sessions were different periods of training, the frequency, onset latencies, still present during sessions 6–10 of paired training even peak latencies and amplitudes of those CRs observed on 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