intra-cerebellar infusion of nmda receptor antagonist ap5 disrupts

Brain Research 887 (2000) 144–156www.elsevier.com/ locate /bres

Research report

Intra-cerebellar infusion of NMDA receptor antagonist AP5 disruptsclassical eyeblink conditioning in rabbits

a a,b ,*Gengxin Chen , Joseph E. SteinmetzaProgram in Neural Science, Indiana University, Bloomington, IN 47405-7007, USA

bDepartment of Psychology, Indiana University, 1101 E. 10th Street, Bloomington, IN 47405-7007, USA

Accepted 19 September 2000

Abstract

Rabbits were infused with AP5, an NMDA receptor antagonist, into the region of the cerebellar interpositus nucleus during classicaleyeblink conditioning with a tone conditioned stimulus and an air puff unconditioned stimulus. Acquisition of the conditioned eyeblinkresponse was delayed in rabbits infused with AP5 but the NMDA receptor antagonist had little effect on conditioned responses when thesesame rabbits were infused a second time after reaching asymptotic responding levels. Some rabbits that received AP5 infusions for thefirst time after the conditioned response was well learned showed temporary alterations in response timing. These data indicate thatNMDA receptor activity is involved in the acquisition of classically conditioned eyeblink response and may also be involved in regulatingcellular processes involved in response timing and other aspects of conditioned response execution. 2000 Elsevier Science B.V. Allrights reserved.

Theme: Neural basis of behavior

Topic: Learning and memory: systems and functions

Keywords: Plasticity; Learning and memory; Interpositus nucleus; Cerebellar cortex; Response timing

1. Introduction early training phase (e.g. see Ref. [8]). Somewhat con-sistent, Caramanos and Shapiro [10] found that systemic

It has been well established that the NMDA receptor injections of MK-801, another NMDA antagonist, andplays a critical role in the induction of long-term potentia- intracerebral ventricular infusions of APV impaired spatialtion (LTP) (for review see Refs. [5,16,35]). Administration working memory or reference memory of the radial mazeof selective NMDA receptor blockers, such as APV, tasks depending on the drug dose, familiarity with theprevents the induction of LTP but appears to have little environment, and training procedure.effect on normal synaptic transmission [20,22] (but see Other studies have also suggested that NMDA receptorsRefs. [19,44]). In an attempt to associate spatial learning are involved in a variety of learning and memory tasks.with LTP, Morris et al. [34] infused APV into the rat Intra-amygdala infusion of APV blocked the acquisitionhippocampus and found that the same drug dose and and consolidation, but not expression of, auditory con-infusion protocol that blocked LTP in vivo also caused ditioned fear-potentiated startle in rats [9,33]. Similarly,impairment of spatial learning in the Morris water-maze. Kim et al. [24] found that ventricular infusions of APVHowever, their finding is complicated by the observation completely blocked acquisition, but not expression, ofthat APV may have caused sensorimotor disturbance in the Pavlovian fear conditioning, while the same dose of APV

appeared to have no effect on pain sensitivity. Further-more, it has been shown that infusion of APV into thebasolateral amygdala also blocked acquisition of con-*Corresponding author. Tel.: 11-812-855-3991; fax: 11-812-855-textual Pavlovian fear conditioning [17]. Mathis et al. [30]4691.

E-mail address: [email protected] (J.E. Steinmetz). showed that post-training ventricular infusion of AP5 and

0006-8993/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 00 )03005-5

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 isimpaired 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 deepavoidance task with mice. The involvement of NMDA nuclei express NMDA receptors [1,3,4,45] but whether orreceptors has also been demonstrated for step-through not Purkinje cells express NMDA receptors is somewhatinhibitory avoidance tasks [26,29], step-down inhibitory controversial. Expression analysis by in situ hybridizationlearning [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 toand 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 ifrabbits [37,42] and rats [39]. In all cases, the NMDA one considers that the functional properties of theantagonists 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 ofor unconditioned responses. While the above studies did NMDA receptor subunits [1,45]. In adult rats or mice, thenot ascertain the target of NMDA antagonists, one study cerebellar granule cells express subunit NR2A and, moreshowed that memantine, a non-competitive NMDA re- abundantly, NR2C. Deep nuclei express subunit NR2A butceptor antagonist with higher affinity to cerebellar tissue little NR2C. And, Purkinje cells are thought to expressthan forebrain tissue, also impaired eyeblink conditioning neither of those subunits. This information combined within humans [38], suggesting the involvement of cerebellar the gene knockout technique provide some clues of theNMDA receptors. Interestingly, in Robinson’s study [37], functions of NMDA receptors in specific cells. KishimotoMK-801 appeared to have no effect on conditioning- et al. [27] showed that mutant mice lacking NMDArelated potentiation of perforant path-granule cell re- receptor subunit NR2A acquired eyeblink conditioningsponses in hippocampus, suggesting its effective target more slowly than wild-type animals but could attain themight 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 significanthave demonstrated that the cerebellum, especially the impairment. This evidence suggests an important role ofinterpositus nucleus, is critical for classical eyeblink NMDA receptors in the deep nuclei in the acquisition ofconditioning [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 cerebellarA

into the region of the interpositus nucleus abolished NMDA receptors in classical eyeblink conditioning. As inconditioned responses that were established before infu- a number of previous studies, we microinjected AP5sion. 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 andsponses and no evidence of learning (i.e. savings) could be performance of conditioned responses. In brief, our datadiscerned 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. Someimportant 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 conditioningand appeared to have no effect on the expression of CRs in 2. Experiment 1: AP5 infusion and acquisition andwell-trained rabbits. In addition, we recently reported that performance of classical eyeblink conditioningcerebellar protein kinases are important for the acquisition,but not retention, of classically conditioned eyeblink NMDA receptors have been shown to be involved in aresponses [13], a result that was also demonstrated in a variety of learning and memory tasks including eyeblinkstudy 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 bothKKIAMRE, in the interpositus nucleus. These data argue cerebellar cortex and deep nuclei [1,3,4,45], but little isstrongly that important cellular processes in the deep known regarding their function. We reasoned that ifcerebellar 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 cerebellarthese data provide solid evidence that at least an important NMDA receptors should interfere with the learning pro-

146 G. Chen, J.E. Steinmetz / Brain Research 887 (2000) 144 –156

cess, and thus behaviorally retard or prevent conditioning. cannula /electrode assembly was cemented into place onHere we used AP5, a specific NMDA receptor antagonist, the skull with dental acrylic along with a bolt for holdingwhich has been widely used in a number of previous the air puff hose and an infrared device that measuredstudies. eyelid movement during behavioral training.

2.1. Materials and methods 2.1.3. Behavioral training and drug administrationAfter at least 1 week was allowed for recovery from

2.1.1. Subjects surgery, rabbits received two adaptation sessions (15 minFifteen 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 conditioningassigned to a saline control group with six eventually per day. During the training, a 350 ms, 85 dB SPL, 1 kHzincluded in subsequent data analyses. Before the experi- tone was used as the conditioned stimulus (CS), and a 100ment 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 viafood and water. A 12/12 h light /dark cycle was main- a speaker mounted |30 cm above the rabbit. The air pufftained 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 the2.1.2. Surgery external eyelids was measured as the unconditioned re-

Surgeries were performed under aseptic conditions. sponse (UR) and the conditioned response (CR) using anRabbits were deeply anesthetized with injections of 6 infrared emitter /detector located 4–5 mm in front of themg/kg xylazine and 60 mg/kg ketamine, and maintained rabbit’s cornea that measured changes in diffraction of athroughout the surgery with intramuscular injections of a beam of infrared light [43]. These measured changes weremixture of xylazine (3 mg/kg) and ketamine (30 mg/kg) amplified to reflect millimeters of external eyelid closure indelivered every 45 min. During the surgery, the skull over response to the CS or the US. Movement of the externalthe 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 (oneinterpositus nucleus and dentate nucleus. The cannula was CS-alone test trial and nine CS–US paired trials). Theconstructed from 22 gauge stainless steel tubing. An inter-trial interval (ITI) varied pseudorandomly from 20 toepoxy-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 forabout 1.5 mm lower than the cannula tip. The skull was 20 sessions of standard delay conditioning, with drugpositioned so that lambda was 1.5 mm below bregma. The treatment given as shown in Table 1. The first period ofelectrode tips were stereotaxically implanted at 5.3 mm injections in phase 1 (sessions 1–5) was designed tolateral from midline and a maximum of 14.5 mm below examine the effect of AP5 or saline infusion on thelambda (depending on the observation of characteristic acquisition of eyeblink conditioning. Phase 2 of traininginterpositus nucleus activity recorded from the electrode (sessions 6–10) allowed us to examine post-injectionwhen 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 inmm20.31 mm3D, where D was the distance measured phase 3 (sessions 11–15). The second period of injectionsbetween bregma and lambda (with posterior positive, in phase 4 (sessions 16–20) tested the effects of AP5 oranterior 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) wasstainless steel stylet was inserted into the cannula after dissolved in sterile physiological saline to a concentrationsurgery and was replaced between sessions to prevent the of 2.5 mg/ml and the pH of the solution was adjusted tocannula from clogging. After the hole in the skull sur- about 7.0 with NaOH. During each infusion session, a 26rounding the cannula was filled with bone wax, the gauge needle was inserted into the guide cannula with its

Table 1Drug 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 SalineAP5 AP5 No injection No injection AP5


tip positioned 0.75 mm below the cannula tip. Using an 2.2. Resultsinfusion pump, each rabbit received 2-ml injections at arate of 8 ml /h (total duration 15 min) via a Teflon tube Twelve of the 15 rabbits met our criteria for inclusion inconnected to a 10-ml Hamilton syringe. The training the study and their data were analyzed. Three rabbits in theprocedure started about 5 min after the infusion began to saline group were excluded; one did not reach the 75% CRallow the infusate to diffuse. criteria, another failed to pass the muscimol test, and a

third one learned very slowly. Although the third rabbitreached 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 thesession during which they were infused with 2 ml of the statistical analyses.GABA agonist, muscimol (Sigma Chemical, dissolved inA

saline to 400 ng/ml), to verify the effectiveness of the2.2.1. Histologyimplant. The muscimol was infused via the pump 15 min

Fig. 1 depicts the cannula tip placements of all rabbitsbefore the test session. A series of paired CS–US trialsincluded in the analysis. Most cannula placements werewere then delivered and the effects of muscimol onimmediately dorsal to the area of the interpositus nucleusconditioned responding were noted. If muscimol did notidentified 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 wereCRs on two consecutive blocks of training, that rabbit wassomewhat distal from the interpositus nucleus, but theexcluded from future data analysis as we assumed thatmuscimol test indicated that the drug was still able tofailing this test indicated that the cannula was notdiffuse to the critical area. While some gliosis waspositioned correctly in the interpositus nucleus.observed around the cannula and electrode tips in someWhen this test was completed, the rabbits were over-rabbits, when the tissue was examined under a lightdosed with an i.v. injection of 150 mg/kg pentobarbitolmicroscope, cell bodies were clearly seen in the area of theand perfused intra-aortically with 0.9% saline followed byleft interpositus and dentate nucleus, and the tissue did not10% formalin. The brains were then removed and fixed indiffer from the other side of the cerebellum. Cell bodies30% sucrose /10% formalin solution. After a week, thecould be clearly seen just beneath the cannula tips sug-brains were embedded, frozen sectioned at a thickness ofgesting that the infusion did not cause neuronal death.40 mm, and stained with cresyl violet. Cannula locationsBecause it was hard to identify lesions caused by cannulaand the conditions of neurons in the interpositus nucleiand electrode implant solely with microscopic histology,were examined under a light microscope.the behavioral criteria described above were also used toexclude animals with lesions caused by cannula placementor volume injection.2.1.5. Data analysis

For the rabbit excluded from statistical analysis becauseSession-wide averages of behavioral response parame-of failing the muscimol test, the cannula placement wasters, including percent CRs, CR amplitudes, latencies tofound to be too posterior and somewhat high. In rabbitsresponse onset and peak, and UR amplitudes were ana-that did not reach the 75% CR criteria, either the cannulalyzed with mixed-design ANOVAs. In these analyses,tip touched the dorsolateral region of the interpositusgroup (AP5 or control) was the between factor and sessionnucleus or the electrode went through it, causing notice-(1–20) was the repeated measure on subjects. Behavioralable damage to the nucleus.responses on the CS-alone test trials and CS–US paired

trials were analyzed separately. Rabbits that did not reach75% CRs on any of the 20 sessions were excluded from 2.2.2. Behavioral analysesstatistical analysis as there was a possibility that those Fig. 2 shows the learning curves of the AP5 and salinerabbits 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 werelikely misplaced.

Next, to examine the drug effects on CR characteristics, 2.2.2.1. Phase 1 (sessions 1 –5). Phase 1 represented theindividual 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 acquisitionthan 50 ms were considered as artifacts and thus excluded. training. Because very few CRs were present in the AP5 orMixed-design ANOVAs were performed to compare the the saline rabbits on sessions 1 and 2, two ANOVAs weregroups. conducted on the sessions 1–5 data; one analysis on


Fig. 1. Schematics of coronal sections through the rabbit cerebellum and brainstem showing locations of cannula tips for experiment 1 rabbits. Numbersrepresent distance in millimeters of the section relative to the lambda skull landmark. Squares depict cannula tips for saline control rabbits and trianglesdepict 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 amplitudesdata. of the AP5 group were significantly smaller than the saline

No group or session effects were found on any of the controls (F(1,10)55.03, 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 betweenThis was expected because very few CRs were displayed the two groups, suggesting that the drug treatments had noby either group of animals on these days. Analyses of the effect on general motor responses. Significant sessiondata 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 rabbitsCRs than the saline group on each day (F(1,10)513.91, had significantly longer response onset latencies (M5257P,0.005), indicating a general impairment in acquisition ms) than the saline control (M5202 ms) (F(1,10)57.30,


though AP5 infusions were discontinued after session 5(see Fig. 2). Compared to control rabbits, AP5 rabbits stillhad significantly lower percentage CRs (F(1,10)55.44,P,0.05) and lower CR amplitudes (F(1,10)55.38, P,

0.05). The AP5 rabbits also displayed significantly longerCR onset latencies (F(1,10)58.75, P,0.015) during phase2; the mean onset latencies for the AP5 and saline rabbitswere 210 ms and 157 ms, respectively. Additional acquisi-tion occurred in both groups, however, as significantsession effects were noted for percentage CRs, CR am-plitudes and onset latencies (all Ps,0.001). No differencesbetween UR amplitudes were observed, however.

2.2.2.3. Phase 3 (sessions 11 –15). Based on a number ofprevious studies, we expected that the rabbits would reachasymptotic performance levels by sessions 11 to 15.Results of ANOVAs conducted on the percent CR datashowed, however, that the AP5 group generated fewer CRsthan the saline group (F(1,10)58.03, P,0.05), althoughthe relative difference in the group averages (saline vs.AP5 as 94.4% vs. 87.9%) was much smaller than that inphase 1 (see Fig. 2A). The analysis also revealed that theCR amplitudes of the AP5 group were significantly smallerthan the saline controls (F(1,10)512.25, P,0.001) andthat the AP5 rabbits (M5172 ms) had significantly longerresponse onset latencies than the saline control (M5143ms) (F(1,10)59.69, P,0.05). These results indicate thatthe impairment of conditioning seen in AP5 rabbits lastedwell into training when no AP5 was delivered. That is,there appears to be a lack of a savings effect in the AP5rabbits 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 certainlyinfusions occurred are indicated by the I-labeled arrows. Error bars5

reached asymptotic responding levels and that this wouldS.E.M.

be a good time to test the effects of AP5 on well-learnedCRs. Analysis of the sessions factor showed few changesin 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 wereCR amplitude was assigned zero and the onset latency was analyzed. However, analysis of phase 4 training dataassigned 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 [F(1,10)58.01, P,0.05]could therefore be due to fewer CRs being executed by and longer onset latencies [F(1,10)57.82, P,0.05] thanthese rabbits as well as a prolonged acquisition during control rabbits. The mean onset latency for the AP5 rabbitswhich lower amplitude and later responses were executed. was 185 ms while the mean onset latency for the controlTo 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 thephase 2 of training revealed that deficits in CR acquisition CRs executed by rabbits in different groups and duringseen 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


CS-alone trials were analyzed. These responses were not many sessions after AP5 infusion. This suggests thatcontaminated 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 asymptoticgroup showed significantly fewer CRs than saline control performance levels and AP5 was infused a second time,group (F(1,10)57.07, 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) (F(1,10)511.88, P,0.01). Similarly, there These data suggest that activity at NMDA receptors in thewas 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.(F(1,10)516.33, P,0.005). However, analyses did notreveal statistical difference in CR amplitudes between thegroups. These results are in agreement with data from 3. Experiment 2. Further verification of the effect ofpaired trials that showed that AP5 rabbits had significantly AP5 on retention of eyeblink CRslonger response latencies than the saline control thussuggesting that early in training, infusion of the NMDA In experiment 1, infusion of AP5 into the cerebellumantagonist influenced response timing as well as the was found to impair the acquisition of classical eyeblinknumber 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-trainedcies for AP5-infused rabbits (M5242 ms) compared to animals. However, in experiment 1, AP5 retention testscontrol rabbits (M5180 ms) (F(1,10)56.59, P,0.025), were conducted on the same rabbits that received AP5however 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 immuneand 175 ms for AP5 and control rabbits, respectively) and from the later action of AP5. Experiment 2 was performedpeak latencies (Ms5260 ms and 291 ms for AP5 and to verify the effect of AP5 on the retention of eyeblinkcontrol rabbits, respectively), and CR amplitude were conditioning. In this experiment, two groups of rabbitsobserved in phase 3 of training (sessions 11–15) were trained until CRs were well-established then treated(F(1,10)511.38, P,0.01, F(1,10)59.43, P,0.05, and with either saline or AP5 and CR performance wasF(1,10)517.52, P,0.005 for onset latency, peak latency compared.and CR amplitude, respectively). No significant differencein percentage CRs was found.

3.1. Materials and methodsDuring phase 4 of training (sessions 16–20), althoughsignificant differences between the AP5 and control rabbitswere not found when percentage CRs and CR amplitudes 3.1.1. Subjects and surgerywere analyzed, differences were noted when response Fourteen male rabbits of the same type and maintainedlatencies were examined. The AP5 rabbits displayed longer in the same condition as those included in experiment 1onset latencies (Ms5211 ms versus 167 ms) (F(1,10)5 were used in this experiment. Of these, based on the6.78, P,0.05) and longer peak latencies (Ms5248 ms inclusion criteria that were described for experiment 1,versus 279 ms) (F(1,10)55.63, 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 NMDAantagonist infusion during early sessions of paired CS–UStraining had somewhat long-lasting effects on CR per- 3.1.2. Behavioral training, drug administration andformance. Differences between AP5 rabbits and saline histologycontrol rabbits in CR characteristics such as response All the experimental procedures were identical to thosetiming 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 102.2.2.6. Summary of experiment 1 results. The results of days (phases 1 and 2) using paired tone CS and air puffexperiment 1 demonstrated that infusions of AP5 retarded US trials, received infusion of AP5 or saline during phasethe rate of acquisition and affected response timing of the 3 of paired training (sessions 11–15), then received anclassically conditioned eyeblink response. Furthermore, the additional 5 days of post-infusion training during phase 4observed impairments of conditioning were apparent on (sessions 16–20).


Table 2Drug 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 injectionAP5 No injection No injection AP5 No injection

3.2. Results indicating that for the most part, all rabbits had reachedasymptotic responding levels by session 10 (see Fig. 4).

Five rabbits were excluded in this experiment becausethey 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 significantwere excluded immediately before the injection sessions. differences between the groups on any measures wereHence, 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. The3.2.1. Histology ANOVAs did indicate a significant session effect in onset

Fig. 3 illustrates the cannula tip placements of all rabbits latencies (F(4,28)52.85, P,0.05) and a trend of sessionincluded in the analyses for experiment 2. As in experi- effect in CR amplitudes (F(4,28)52.18, P,0.1). Meanment 1, in the left (infusion) side of the cerebellum, cell onset latencies for the AP5 and control groups were 201bodies were clearly seen under light microscope in the area and 180 ms, respectively. Examination of the phase 3 dataof 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 wereof the cerebellum. Limited gliosis was observed around the present in only two of the five AP5 rabbits. The other threecannula and/or electrode tips in some of the rabbits, but rabbits showed little or no effect of the AP5 infusion. Thethe 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 rabbitthe electrode tips penetrated into either the interpositus or had a cannula placement just dorsal to the interpositusthe dentate nucleus and caused clear damage. Thus, it is nucleus while the second rabbit had a cannula placementlikely that poor learning was due to cerebellar damage just beneath the cerebellar cortex. It should be noted thatcaused by the implantation of the cannulae. all five rabbits showed abolition of responding when

muscimol was infused after training thus indicating that all3.2.2. 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 beover the 20 training sessions that were given. As in involved in eyeblink conditioning. Alternatively, previousexperiment 1, the averages of CS–US paired trials and research has suggested that inactivation of select regions ofCS-alone trials were analyzed separately. To be consistent cerebellar cortex significantly affects CR performance. Forand to allow for cross-experiment comparisons, the same example, Attwell et al. [2] demonstrated that reversiblephases of sessions were examined with ANOVAs as in inactivation of cerebellar cortex with CNQX, whichexperiment 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 ondata collected during sessions 1 and 2 and sessions 3 to 5 3.2.2.4. Phase 4 (sessions 16 –20). During the last fiverevealed no significant differences between groups on any no-injection sessions (session 16 to session 20), no groupmeasures. 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 acquisitioneffect but no learning-related changes in the UR am- 3.2.2.5. CR characteristics. As in experiment 1, the onsetplitudes (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 significantdata 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-alonenoted only when onset latencies were analyzed (P,0.005) trials from phase 3 (the drug infusion phase) were ana-


Fig. 3. Schematics of coronal sections though the rabbit cerebellum and brainstem showing locations of cannula tips for experiment 2 rabbits. Numbersrepresent distance in millimeters of the section relative to the lambda skull landmark. Squares depict cannula tips for saline control rabbits and trianglesdepict 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.


trained to asymptotic performance levels, infusions of AP5appeared to have a temporary effect on CR production andtopography in some rabbits. These data are, for the mostpart, in agreement with the results of experiment 1 thatsuggest that the NMDA antagonist consistently affectsacquisition and has a transitory effect on the timing ofclassically conditioned eyeblink responses.

4. Discussion

The primary findings of this study can be summarized asfollows: (1) intracerebellar infusion of AP5, a selectiveNMDA receptor antagonist, during training impaired theacquisition of classical eyeblink conditioning in rabbits.(2) NMDA receptor antagonist effects on conditioningwere still apparent several days after AP5 infusion sug-gesting that AP5 may have blocked critical cellular pro-cesses responsible for neuronal plasticity that underlies CRacquisition. (3) Administration of AP5 to well-trainedrabbits produced an effect on CR performance in a subsetof rabbits given the NMDA antagonist when asymptoticresponding levels were reached.

An important issue addressed with the present data iswhether the AP5 infusions acted on the learning processduring early phases of acquisition training, or merelyaffected the performance or expression of the CRs duringlearning. Two basic lines of evidence in our data suggestthat the effects of AP5 during acquisition period were onthe learning process, per se, and not restricted to CRperformance. Firstly, when the drug treatments stoppedafter Session 5, the drug-treated animals did not immedi-ately perform as well as the saline animals. Instead, theirFig. 4. Learning curves for rabbits trained across 20 sessions in experi-performance gradually improved over the next few ses-ment 2. (A) Percent CRs and (B) CR amplitudes recorded from salinesions (see Fig. 2). In fact, a statistical difference in percent(squares) and AP5 (triangles) rabbits. Sessions during which drug

infusions occurred are indicated by the I-labeled arrow. Error bars5 CRs between the AP5 and control rabbits was still presentS.E.M. during sessions 11 to 15 indicating a reduced savings of

learning effect during the post-infusion training period.lyzed. The AP5 rabbits showed longer peak latencies Secondly, when AP5 was injected into well-trained ani-(M5285 ms) (F(1,7)56.49, P,0.05) than did control mals that were exposed previously to AP5, their CRrabbits (M5232 ms). As was the case for paired trials, two performances were generally not affected (experiment 1).of the five rabbits showed relatively large changes in CR Although two rabbits from experiment 2 showed a pro-characteristics whereas the remaining three rabbits showed nounced decrement in percent CRs and CR amplitudes onslight, if any, changes. Taken together, the results of these paired trials when AP5 was infused for the first time afterCS-alone analyses may provide an explanation for the asymptotic responding was reached, analyses of CS-alonetemporary decrement in percent CRs observed during AP5 trial data showed that these deficits may be attributable toinfusion on sessions 11–15. It appears that the AP5 caused delayed CR onsets. The data strongly suggest that, at most,a temporary impairment in the performance of the CR, i.e. a temporary effect on CR performance occurs in someon some trials, a subset of the AP5 rabbits were executing well-trained rabbits infused with AP5 for the first time.relatively late conditioned responses (as indicated by However, the potential contribution of a performancesignificantly longer CR peak latencies) that may have been deficit in producing the slow acquisition noted in Experi-obscured by the UR present on many of the paired trials ment 1 cannot be unequivocally excluded by the presentdelivered in these sessions. data.

Another important issue is a determination of the3.2.2.6. Summary of experiment 2 results. The results of cerebellar site at which the AP5 acted in the present study.experiment 2 demonstrate that when the rabbits were Specifically, the issue is whether the AP5 infusions were


restricted to the interpositus nucleus or spread to the NMDA receptor activity during paired presentations of theoverlying cerebellar cortex. A comparison with a previous CS and US late in each daily training session. In addition,study by Krupa et al. [28] might shed some light on this it is possible that NMDA receptor activity is important forpoint. In their study with labeled muscimol, Krupa et al. only a portion of the critical cellular processes involved inshowed that the drug diffusion area included the inter- CR acquisition.positus nucleus and portions of the overlying cortex. They Our present results are in agreement with previousobserved no labeling outside of the cerebellum. Our studies involving systemic administration of NMDA re-cannula placements were very similar to those of Krupa et ceptor antagonists [37–39,42], that is, blocking NMDAal. While we injected a larger volume, it was injected at a receptors impaired the acquisition of classical eyeblinkslower rate. We thus expect that our drugs diffused into conditioning. Indeed, our results may help to explain thesimilar areas as the Krupa et al. study. Given the extent of seemingly contradictory results reported by Robinson [37]spread that we suspected to have occurred, it seems likely that systemic infusion of MK-801 retarded acquisition ofthat the AP5 affected the interpositus nucleus and also at the eyeblink CR but did not affect perforant path–granuleleast the ventral portion of cerebellar cortex. Thus, the site cell excitability. If one assumes that a major target of theof action of the NMDA-receptor antagonist could have effects of NMDA blockers is the cerebellum, and not thebeen in the cerebellar cortex, the deep cerebellar nuclei, or hippocampal formation, one would expect the resultsin both regions. observed in the present study and by Robinson [37]. There

Interestingly, some recent models of the involvement of is one discrepancy between our data and the data ofthe cerebellum in eyeblink conditioning have suggested Robinson [37] and Thompson and Disterhoft [42] — thesethat critical learning-related plasticity occurs in both studies did not report alternations in CR timing after drugcerebellar cortex and the interpositus nucleus (e.g. Refs. injection for delay conditioning. By contrast, our data from[12,25,31,40]). In these models, excitability changes at the experiments 1 and 2 showed some alteration in CR timinglevel of the interpositus nucleus have been suggested to be in AP5-treated animals. One obvious difference betweenimportant for providing drive on brainstem motor neurons the present studies and the previous studies is the respec-responsible for CR execution while excitability changes in tive routes of administration of the NMDA antagonists.cerebellar cortical areas have been suggested to be im- Infusing NMDA receptor antagonists directly into theportant for response timing, amplitude gain and, perhaps, cerebellum may produce stronger NMDA receptor an-regulation of plasticity in the deep cerebellar nuclei. Given tagonism in areas of the cerebellum critical for responsethe range of locations of infusions sites used in the present timing than when the antagonists are by systemic adminis-experiments (see Figs. 1 and 3), it seems likely that the tration. It is also possible that systemic administration ofpattern of NMDA receptor antagonist infusions differed NMDA receptor antagonists may produce effects onacross rabbits thus producing varying levels of effects in multiple brain sites such that the overall effects arecerebellar cortex and the deep nuclei. For example, it is different than local administration effects observed in ourpossible that the temporary CR timing deficits noted in the study. And, we cannot rule out that AP5 may have sidetwo rabbits from experiment 2 were due to AP5 effects on effects other than blocking NMDA receptor (see Ref. [44]).timing mechanisms that are generated by activity in areas It would be interesting to repeat our experiment with otherof cerebellar cortex (i.e. AP5 infusions affected predomi- NMDA receptor antagonists, such as MK-801 or PCP, tonantly cortical areas). Further studies involving varying examine their effects on the eyeblink conditioning.amounts of AP5 placed at a variety of locations within In summary, the present data indicate that NMDAcerebellar cortex and the deep nuclei would provide receptor activities within the cerebellum are involved inadditional information concerning the learning-related the learning of classical eyeblink conditioning in rabbits.processes that are dependent on NMDA receptor activity. This finding is consistent with a number of previous

It should also be noted that AP5 does not completely studies that have demonstrated an involvement of NMDAblock acquisition of the eyeblink CR as does muscimol receptors in learning and neural plasticity. The finding alsoinfusion [28] or permanent lesion techniques (e.g. Refs. supports the hypothesis that at least part of the memory[32,41]). We observed a similar effect in a previous study trace for the learned response for this paradigm is locatedwhen a H7, a protein kinase inhibitor, was infused into the within the cerebellum. This observation is compatible withregion of the deep cerebellar nuclei [13]. There are several a host of other lesion and recording experiments implicat-possible reasons why a complete blockade of learning was ing a critical role for the cerebellum in the learning andnot observed. For example, while our muscimol test memory of this simple conditioned response.revealed that the cannula were placed in locations thataffected performance of the eyeblink CR, we cannot becertain that the diffusion of the amount of AP5 that we Acknowledgementsinfused affected the targeted region as effectively as themuscimol. Also, the AP5 effect could have subsided This research was supported by a grant from theduring later portions of training sessions allowing some National Institutes of Mental Health ([MH51178) to


classical conditioned behavior, Behav. Neurosci. 106 (1992) 879–J.E.S. This research formed a portion of the Ph.D. disserta-888.tion written by G.C. who is currently a Research Scientist

[16] Y. Dudai, The Neurobiology of Memory, Oxford University Press,at Cold Spring Harbor Laboratory, Cold Spring Harbor, 1989.New York. [17] M.S. Fanselow, J.J. Kim, Acquisition of contextual Pavlovian fear

conditioning is blocked by application of an NMDA receptorantagonist D,L-2-amino-5-phosphonovaleric acid to the basolateralamygdala, Behav. Neurosci. 108 (1994) 210–212.

[18] H. Gomi, W. Sun, C.E. Finch, S. Itohara, K. Yoshimi, R.F.References Thompson, Learning induces a CDC2-related protein kinase,

KKIAMRE, J. Neurosci. 19 (1999) 9530–9537.[19] J.J. Hablitz, I.A. Langmoen, N-methyl-D-aspartate receptor antago-[1] C. Akazawa, R. Shigemoto, Y. Bessho, S. Nakanishi, N. Mizuno,

nists reduce synaptic excitation in the hippocampus, J. Neurosci. 6Differential expression of five N-methyl-D-aspartate receptor subunit(1986) 102–106.mRNAs in the cerebellum of developing and adult rats, J. Comp.

[20] E.W. Harris, A.H. Ganong, C.W. Cotman, Long-term potentiation inNeurol. 347 (1994) 150–160.the hippocampus involves activation of N-methyl-D-aspartate re-[2] P.J.E. Attwell, S. Rahman, M. Ivarsson, C.H. Yeo, Cerebellarceptors, Brain Res. 323 (1984) 132–137.cortical AMPA-kainate receptor blockade prevents performance of

[21] T. Hatfield, M. Gallagher, Taste-potentiated odor conditioning:classically conditioned nictitating membrane responses, J. Neurosci.impairment produced by infusion of an N-methyl-D-aspartate re-19 (RC45) (1999) 1–6.ceptor antagonist into basolateral amygdala, Behav. Neurosci. 109[3] E. Audinat, B.H. Gahwiler, T. Knopfel, Excitatory synaptic po-(1995) 663–668.

tentials in neurons of the deep nuclei in olivo-cerebellar slice[22] C.E. Herron, R.A. Lester, E.J. Coan, G.L. Collingridge, Frequency-

cultures, Neuroscience 49 (1992) 903–911.dependent involvement of NMDA receptors in the hippocampus: a

[4] E. Audinat, T. Knopfel, B.H. Gahwiler, Responses to excitatorynovel synaptic mechanism, Nature 322 (1986) 265–268.

amino acids of Purkinje cells and neurones of the deep nuclei in [23] D. Jerusalinsky, M.B.C. Ferreira, R. Walz, R.C. Da Silva, M.cerebellar slice cultures, J. Physiol. 430 (1990) 297–313. Bianchin, A.C. Ruschel, M.S. Zanatta, J.H. Medina, I. Izquierdo,

[5] T.V.P. Bliss, G.L. Collingridge, A synaptic model of memory: Amnesia by post-training infusion of glutamate receptor antagonistslong-term potentiation in the hippocampus, Nature 361 (1993) into the amygdala, hippocampus, and entorhinal cortex, Behav.31–39. Neural Biol. 58 (1992) 76–80.

[6] V. Bracha, K.B. Irwin, M.L. Webster, D.A. Wunderlich, M.K. [24] J.J. Kim, J.P. DeCola, J. Landeira-Fernandez, M.S. Fanselow, N-Stachowiak, J.R. Bloedel, Microinjections of anisomycin into the methyl-D-aspartate receptor antagonist APV blocks acquisition butintermediate cerebellum during learning affect the acquisition of not expression of fear conditioning, Behav. Neurosci. 105 (1991)classically conditioned responses in the rabbit, Brain Res. 788 126–133.(1998) 169–178. [25] J.J. Kim, R.F. Thompson, Cerebellar circuits and synaptic mecha-

[7] L.H. Burns, B.J. Everitt, T.W. Robbins, Intra-amygdala infusion of nisms involved in classical eyeblink conditioning, Trends Neurosci.the N-methyl-D-aspartate receptor antagonist AP5 impairs acquisi- 20 (1997) 177–181.tion but not performance of discriminated approach to an appetitive [26] M. Kim, J.L. McGaugh, Effects of intra-amygdala injections ofCS, Behav. Neurol. Biol. 61 (1994) 242–250. NMDA receptor antagonists on acquisition and retention of inhib-

[8] D.P. Cain, D. Saucier, J. Hall, E.L. Hargreaves, F. Boon, Detailed itory avoidance, Brain Res. 585 (1992) 35–48.behavioral analysis of water maze acquisition under APV or CNQX: [27] Y. Kishimoto, S. Kawahara, Y. Kirino, H. Kadotani, Y. Nakamura,contribution of sensorimotor disturbances to drug-induced acquisi- M. Ikeda, T. Yoshioka, Conditioned eyeblink response is impaired intion deficits, Behav. Neurosci. 110 (1996) 86–102. mutant mice lacking NMDA receptor subunit NR2A, NeuroReport 8

[9] S. Campeau, M.J.D. Miserendino, M. Davis, Intra-amygdala infu- (1997) 3717–3721.sion of the N-methyl-D-aspartate receptor antagonist AP5 blocks [28] D.J. Krupa, J.K. Thompson, R.F. Thompson, Localization of aacquisition but not expression of fear-potentiated startle to an memory trace in the mammalian brain, Science 260 (1993) 989–auditory conditioned stimulus, Behav. Neurosci. 106 (1992) 569– 991.574. [29] K.C. Liang, W. Hon, M. Davis, Pre- and post-training infusion of

[10] Z. Caramanos, M.L. Shapiro, Spatial memory and N-methyl-D- N-methyl-D-aspartate receptor antagonists into the amygdala impairaspartate receptor antagonists APV and MK-801: memory impair- memory in an inhibitory avoidance task, Behav. Neurosci. 108ments depends on familiarity with the environment, drug dose, and (1994) 241–253.training duration, Behav. Neurosci. 108 (1994) 30–43. [30] C. Mathis, J. de Barry, A. Ungerer, Memory deficits induced by

[11] P.F. Chapman, J.E. Steinmetz, L.L. Sears, R.F. Thompson, Effects g-L-glutamyl-L-aspartate and D-2-amino-5-phosphonovalerate in aof lidocaine injection in the interpositus nucleus and red nucleus on Y-maze avoidance task: relationship to NMDA receptor antagonism,conditioned behavioral and neuronal responses, Brain Res. 537 Psychopharmacology 105 (1991) 546–552.(1990) 149–156. [31] M.D. Mauk, Roles of cerebellar cortex and nuclei in motor learning:

[12] L. Chen, S. Bao, J.M. Lockard, J.J. Kim, R.F. Thompson, Impaired contradictions or clues?, Neuron 18 (1997) 343–346.classical eyeblink conditioning in cerebellar-lesioned and Purkinje [32] D.A. McCormick, R.F. Thompson, Cerebellum: essential in-cell degeneration ( pcd) mutant mice, J. Neurosci. 16 (1996) 2829– volvement in the classically conditioned eyelid response, Science2838. 223 (1984) 296–299.

[13] G. Chen, J.E. Steinmetz, Microinfusion of protein kinase inhibitor [33] M.J.D. Miserendino, C.B. Sananes, K.R. Melia, M. Davis, BlockingH7 into the cerebellum impairs the acquisition but not the retention of acquisition but not expression of conditioned fear-potentiatedof classical eyeblink conditioning in rabbits, Brain Res. 856 (2000) startle by NMDA antagonists in the amygdala, Nature 345 (1990)193–201. 716–718.

[14] G. Chen, J.E. Steinmetz, Intra-cerebellar infusion of NMDA re- [34] R.G.M. Morris, R.F. Halliwell, N. Bowery, Synaptic plasticity andceptor antagonist AP5 disrupts classical eyeblink conditioning in learning II: do different kinds of plasticity underlie different kinds ofrabbits, Soc. Neurosci. Abs. 23 (1997) 783. learning?, Neuropsychology 27 (1989) 41–59.

[15] R.E. Clark, A.A. Zhang, D.G. Lavond, Reversible lesions of the [35] R.A. Nicoll, J.A. Kauer, The current excitement in long-termcerebellar interpositus nucleus during acquisition and retention of a potentiation, Neuron 1 (1988) 97–103.


[36] A.F. Nordholm, J.K. Thompson, C. Dersarkissian, R.F. Thompson, [41] J.E. Steinmetz, D.G. Lavond, D. Ivkovich, C.G. Logan, R.F.Lidocaine infusion in a critical region of cerebellum completely Thompson, Disruption of classical eyelid conditioning after cerebel-prevents learning of the conditioned eyeblink response, Behav. lar lesions: damage to a memory trace system or a simple per-Neurosci. 107 (1993) 882–886. formance deficit?, J. Neurosci. 12 (1992) 4403–4426.

[37] G.B. Robinson, MK801 retards acquisition of a classically con- [42] L.T. Thompson, J.F. Disterhoft, N-methyl-D-aspartate receptors inditioned response without affecting conditioning-related alterations associative eyeblink conditioning: both MK-801 and phencyclidinein perforant path-granule cell synaptic transmission, Psychobiology produce task- and dose-dependent impairments, J. Pharmacol. Exp.21 (1993) 253–264. Ther. 281 (1997) 928–940.

[38] M.M. Schugens, R. Egerter, I. Daum, K. Schepelmann, T. Klock- [43] L.T. Thompson, J.R. Moyer Jr., E. Akase, J.F. Disterhoft, A systemgether, P. Loschmann, The NMDA antagonist memantine impairs for quantitative analysis of associative learning. Part 1. Hardwareclassical eyeblink conditioning in humans, Neurosci. Lett. 224 interfaces with cross-species applications, J. Neurosci. Methods 54(1997) 57–60. (1994) 109–117.

[39] R.J. Servatius, T.J. Shors, Early acquisition, but not retention, of the [44] D.L. Walker, P.E. Gold, Intrahippocampal administration of both theclassically conditioned eyeblink response is N-methyl-D-aspartate D- and the L-isomers of AP5 disrupt spontaneous alternation(NMDA) receptor dependent, Behav. Neurosci. 110 (1996) 1040– behavior and evoked potentials, Behav. Neurol. Biol. 62 (1994)1048. 151–162.

[40] J.E. Steinmetz, Brain substrates of classical eyeblink conditioning: a [45] M. Watanabe, M. Mishina, Y. Inoue, Distinct spatiotemporal expres-highly localized but also distributed system, Behav. Brain Res. 110 sions of five NMDA receptor channel subunit mRNAs in the(2000) 13–24. cerebellum, J. Comp. Neurol. 343 (1994) 513–519.

intra-cerebellar infusion of nmda receptor antagonist ap5 disrupts

Documents