Lead exposure potentiates the effects of NMDA on repeated learning

Neurotoxicology and Teratology, Vol. 16, No. 5, pp. 455-465, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0892-0362/94 $6.00 + .00

Pergamon

0892-0362(94)E0028-B

Lead Exposure Potentiates the Effects of N M D A on Repeated Learning J E F F R E Y C O H N 1 A N D D E B O R A H A. C O R Y - S L E C H T A 2

Department o f Environmental Medicine, University o f Rochester School o f Medicine & Dentistry, Rochester, N Y 14642 Received 13 A u g u s t 1993; Accepted 29 M a r c h 1994 COHN, J. AND D. A. CORY-SLECHTA. Lead exposure potentiates the effects of NMDA on repeated learning. NEUROTOXICOL TERATOL 16(5) 455-465, 1994.-Several studies now suggest that Pb exposure disrupts NMDA receptor complex function, findings which may have implications for understanding the basis of Pb-induced learning impairments. To further evaluate this possibility, the behavioral properties of the glutamate agonist NMDA were compared in rats that had been chronically exposed to 0, 50, or 250 ppm Pb acetate in drinking water from weaning. Acute administration of NMDA (20-50 mg/kg IP) decreased accuracy in both the repeated acquisition (RA) and performance (P) components of this multiple schedule with a selectiveeffect on the learning component in the second half of the session. Analyses of error patterns revealed that the disruption of RA accuracy derived from initial perseverative errors followed by errors of skipping forward and backwards in the 3-member response sequence. Response rates in both RA and P were suppressed by NMDA. Pb-exposure potentiated the accuracy-impairing effects of NMDA by further increasing the frequencies of these error classes, and likewise potentiated the rate-suppressing effects of NMDA. These findings add further support to the possible involvement of Pb with the NMDA receptor complex. Lead

Learning

Repeatedacquisition

NMDA Receptor

THE IMPORTANCE OF the glutamatergic N-methyl-D-Aspartate (NMDA) receptor complex activity to learning processes has been argued from studies showing that the administration of noncompetitive NMDA antagonists, such as dizocilpine (MK-801), impair performance on a variety of learning tasks, including the repeated acquisition of response chains (10,11,13,20), and spatial learning tasks, such as the radial arm maze (7), water maze learning (28,43), and spatial alternation (44). In addition, the NMDA receptor complex has been associated with the long-term potentiation (LTP) of excitatory synaptic transmission, a potential neurobiological substrate of learning and memory processes (7,14,32,41). The detrimental effects of even very low levels of lead (Pb) exposure on learning and memory processes have been demonstrated in several prospective epidemiological studies of pediatric populations (e.g., 5,8,37). Corresponding effects have also been demonstrated at comparable blood lead levels in experimental animal studies, both in rodents (e.g., 12) and nonhuman primates (e.g., 39). Pb-induced changes in the NMDA receptor complex as a potential basis for Pb-induced

N-methyl-D-aspartate

learning impairments has appeal for several reasons. First, if noted at relevant blood Pb concentrations, such changes could conceivably account for behavioral effects that occur following Pb exposure either early in development or after maturity, as noted in occupationally Pb-exposed populations. This is in contrast to many of the developmental morphological explanations which have been proposed. Secondly, NMDA-based changes could help to explain the fact that acquisition of behavior is more vulnerable to disruption by Pb than is performance of a previously learned response (12,15). Finally, NMDA receptor complex changes would provide a mechanism for the mental retardation and cognitive deficits noted with Pb poisoning (8) that apparently occur in the absence of morphological or pathologic changes. There is accumulating evidence, in fact, to indicate that NMDA receptor complex activity is modified by Pb. For example, Pb exposure has been shown to selectively block the NMDA receptor complex in pyramidal neurons of rat hippocampus (1,42). Pb exposure has also been recently reported to inhibit the formation and maintenance of LTP in hippocam-

t Current address: U.S.E.P.A., HERL/NTD (MD-74B), Research Triangle Park, NC 27711. 2 Requests for reprints should be addressed to Deborah A. Cory-Slechta, P.O. Box EHSC, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642. 455

456

COHN AND CORY-SLECHTA

pal slices from perinatally Pb-exposed rats (2,34). Pre- and perinatal Pb exposures of rats have been found to decrease [aH]MK-801 binding to the NMDA receptor complex in vivo and in vitro (23). In addition, we have recently shown that both the accuracy-impairing and response rate-altering properties of the noncompetitive NMDA antagonist MK-801 are attenuated by Pb exposure (11) in rats working on a multiple schedule of repeated acquisition and performance, and that Pb exposure produces subsensitivity to the stimulus properties of MK-801 (26). A possible consequence of a prolonged inhibition at the MK-801 site of the NMDA channel, as suggested in particular by the studies of Guilarte and Miceli (23) and by subsensitivity to the behavioral effects of MK-801 (11,26), might be an upregulation at the glutamate binding site in an attempt to homeostatically overcome this inhibition. Under such conditions, Pb exposure might be associated with an enhanced sensitivity to compounds acting at the glutamate binding site on the NMDA receptor complex. To assess that possibility, the current study compared, in control versus Pb-exposed rats, the effects of acute administration of the glutamate agonist NMDA itself on a multiple schedule of repeated acquisition (learning) and performance, including assessment of the patterns of errors underlying any changes in overall accuracy. Its results, namely a potentiation of both the accuracy-impairing and response-rate suppressing effects of NMDA on this baseline in the Pb-exposed group, add further support to the accumulating evidence for an involvement of Pb with the NMDA receptor complex. METHOD

Animals Sixteen male Long-Evans rats were obtained from Blue Spruce Farms (Altamont, NY), at 21 days of age and randomly assigned to a Pb exposure group of either 0 (n = 6), 50 (n = 4), or 250 (n = 6) ppm Pb acetate dissolved in distilled deionized water. They were then group-housed under a 12L : 12D cycle until 35 days of age at which time they were individually housed. Rats had unrestricted access to semipurified rat chow (Purina, St. Louis, MO) from the time of arrival until 55 days of age when behavioral procedures were implemented. From that time until they reached 300 g body weights, rats were provided enough food to gain 1-5 g/day. After they reached 300 g, body weights were maintained at this level by caloric regulation. Prior to the current study, this sample of rats was part of a larger population of 31 rats with a behavioral history consisting of 75 experimental sessions (5 days/week, 1 h/day) on the multiple RA and P schedule (12), and a subsequent assessment (11) of the effects of acute administration of MK-801 (0.050.30 mg/kg) on this baseline. In examining the data of individual rats in the original study of Pb effects on the RA and P schedule (12), there was an obvious dichotomous population of "learners" and "nonlearners" within the control and the Pb-exposed groups, with a trend toward increasing numbers of nonlearners in the Pb-exposed groups. Learners and nonlearners could be differentiated by the fact that learners exhibited increases in RA2 accuracy across sessions, while nonlearners exhibited essentially chance levels of accuracy during the RA components across the 75 sessions. Because the evaluation of the effects of drugs on this schedule obviously required baseline accuracy levels from which either increases or decreases in accuracy could be detected, only learners (defined as those rats that reached an overall RA

accuracy level of at least 40% by the 75th session of Cohn et al. (12) were included in studies assessing the impact of drugs. Because of the consequently smaller sample sizes this selection produced and because of the lack of any differences between the two Pb exposure groups (50 and 250 ppm) in the magnitude of accuracy impairment or response rate changes on the multiple RA and P schedule in Cohn et al. (12), the two Pb exposure groups were combined to form one Pb group for these drug studies. As only learners from the original study were used, group mean accuracy levels of control and Pbexposed rats were actually comparable at the start of the drug studies, thus also precluding differences in stimulus control (accuracy levels) between Pb treatment groups that could otherwise have totally confounded the interpretation of drug effects. BEHAVIORAL PROCEDURES The behavioral procedure consisted of a multiple threemember repeated acquisition and performance baseline. Both the RA and P components required completion of a threemember sequence of responses for reinforcement. In the RA component, the correct sequence changed with each successive experimental session and was derived from the following list: L (Left) R (Right) C (Center), RCL, RLC, CLR, and CRL. No lever was used more than once within a correct sequence, and sequences requiring a repetitive series of responses, e.g., LLL or LLR, were excluded to preclude any reinforced history of response perseveration. In addition, sequences were ordered such that no lever occupied the same position in a sequence from the preceding day. The correct sequence for the P component was constant across sessions. The sequence LCR was randomly selected to serve as the correct P sequence. Within a session, completion of the correct sequence of responses without error was required for reinforcement delivery. Any error initiated a 2-s timeout period during which the houselight was turned off. Responses occurring during the timeout further extended this period until 2 s had elapsed without a response. The schedule then required reinitiation of the sequence. In addition, a fixed ratio (FR) contingency was operative such that an error occurring either during or between a sequence increased the ratio from 1 to 2 consecutive errorless sequences for food presentation. The FR value was reset to 1 at reinforcement and remained at 1 unless an error occurred which then again increased the FR value to 2. The RA and P components of the schedule alternated twice during the daily 60-min experimental session. This resulted in two P components and two RA components; the first of each of these is hereafter referred to as P1 or RA1, the second presentation as P2 or RA2. Each component lasted until either 25 reinforcers had been delivered or 15 rain elapsed, whichever came first. The component (RA or P) which began each session alternated across sessions. Other details of these procedure have been described elsewhere (12,16).

Selection of Sequences and Drug Administration In Cohn et al. (12), a clear sequence bias was detected in Pb-exposed but not in control rats. Specifically, Pb-exposed rats exhibited significantly higher accuracy levels in the presence of the two RA sequences CRL and RLC, both of which involved the same left-to-right lever-to-lever transition as did the P component sequence LCR. Accuracy levels were significantly lower in the presence of the non-P-like RA sequences, CLR, RCL, and LRC. To maximize the capability of assessing drug effects on acquisition processes, NMDA effects were

NMDA, LEAD, A N D REPEATED ACQUISITION studied only in sessions in which the non-P-like sequence CLR (randomly chosen) served as the correct RA sequence and during which the session began with the RA component (RAI). This strategy reduced the total number of N M D A injections to 6 per animal and assured a consistency of the baseline on which the effects of N M D A were examined. It was considered preferable to attempting to generate N M D A doseeffect curves for each RA sequence or to using different sequences in different sessions and assuming comparability of NMDA's effects across different sequences. Rats were injected IP with either physiological saline, or 20, 35, or 50 mg/kg of N M D A (Research Biochemicals Inc., Natick, MA; prepared in equimolar solution with NaOH at pH 7.0) diluted in physiological saline 15 min prior to the session. Each dose was tested at least twice in each rat. Drug doses and saline were administered in random order and at a constant volume of 0.3 ml. At least one session with no injection occurred between all drug sessions, thus minimizing any carryover effects. No drug or saline sessions were administered on Mondays. It was not possible to maintain a typical Tuesday-Friday injection schedule because drug effects were studied only when the RA sequence was CLR (see above), which did not occur on a regular basis.

457 i.e., perseveration (P), or skipping to the final member of the sequence (S). If the first member of the response pair was incorrect (I), it could be followed by: a correct reinitiation of the sequence (R), a perseverative response (P), a skipping response (S), or a nominally correct response except that reinitiation of the sequence was required following an error (C). These comparisons were based on proportions relative to the total occurrences of the first member of the pair. A minimum of five occurrences of a particular response pair was deemed necessary for inclusion in statistical analyses, to prevent description of spurious drug effects. Explicit posthoc statistical comparisons were not carried on these response pairs given the exploratory nature of the analyses and the large number of tests that would have been required. Suppression of responding in the presence of the 50 mg/kg dose of NMDA necessitated dropping these data from the statistical analyses of the C3-based response pairs, resulting in three dose levels for that analysis. The resulting 50 mg/kg values are depicted in Fig. 3 (see later), however, for comparative purposes.

Blood Lead Concentrations After 60 baseline sessions on the multiple RA and P schedule, blood lead concentrations were determined using anodic stripping voltammetry as described previously (17).

Data Analyses and Statistical Evaluation Overall accuracy was defined as the number of responses on the correct lever in the proper order, and in correct relationship to timeout periods/total number of responses per component x 100. Overall response rate was defined as the total number of responses per component/component length. Repeated measures analyses of variance (ANOVA; BMDP Statistical Software Package, Los Angeles, CA) were used to examine the effects of Pb and N M D A on these measures, with Pb as a between-group and N M D A as a within-group variable. These analyses were carried out separately for components RA1, RA2, P1, and P2 because Cohn et al. (12) revealed that baseline response rates differed significantly by component (declining across components), as did accuracy levels during RA1 and RA2 (lower in RA1). When significant main effects or interactions were detected, posthoc analyses were conducted using one-way ANOVAs. To evaluate the extent to which N M D A preferentially altered accuracy of learning (RA) versus performance (P), RANOVAs were carried out for RA1 versus P1, and for RA2 versus P2 using data from the control group only, with component serving as the between-group variable and N M D A as a within-group variable. Within-session changes in accuracy were examined using A N O V A in which each component was divided into three equal bins based on time to complete that component. Accuracy within each bin was then calculated. In these analyses, Pb exposure was a between-group variable and N M D A and bin were within-group variables. For R A I , suppression of responding at 50 mg/kg necessitated dropping these data from the within-session statistical analysis. In all analyses, an orlevel of 0.05 was defined as the criterion for significance. To determine the behavioral processes underlying changes in RA accuracy, error patterns were examined by calculating the percentage of responses on each lever given a previous response on a particular lever. ANOVAs (Pb as between, NMDA as within) were conducted on pairs o f responses in which the first member o f the pair was identified as either correct (C) or incorrect (I). If the first member of the response pair was correct, the three possible second responses were: a correct second response (C), a repeat o f the first response,

RESULTS

Effects o f lab Exposure on Overall Accuracy N M D A administration significantly reduced accuracy during components R A I , RA2, and P1 of the multiple RA and P schedule (Fig. 1) to levels ranging from approximately 40% (RA1) to 60 (RA2)-70% (P1) of saline control session values. Only overall accuracy during the P2 component was unaffected by NMDA administration. This decline in overall accuracy was potentiated by Pb exposure in the RA2 component as indicated by the significant interaction between Pb and NMDA, primarily due to the more pronounced decrease in accuracy of the Pb-exposed group at the 35 m g / k g dose (0.006). Similar, albeit nonsignificant, Pb-related trends were evident in both RA1 and PI. In the Pb-exposed group, a U-shaped NMDA dose-effect function was noted, as indicated by significantly higher accuracy levels at 50 mg/kg compared to 35 mg/kg in both the RAI and RA2 components ( p < 0.04) with a similar though marginally significant effect in P1 (p = 0.O6).

Effects o f Pb Exposure on Response Rate NMDA administration significantly decreased response rate in all four components of the schedule (Fig. 2) relative to saline values. These decrements in response rate were greater in the Pb-exposed group in RA1, with a similar though marginally significant effect in P2 and with similar trends in RA2 and PI. Posthoc analyses indicated a more pronounced suppression of RA2 response rates in the Pb-exposed group at the 35 mg/kg dose (p = 0.06) but no other statistically significant differences at any dose were found in R A I , P1, or P2. In contrast to overall accuracy, suppression of response rate was not significantly reversed in the Pb-exposed group at the 50 mg/kg N M D A dose.

Analyses o f Error Patterns Response pairs beginning with a correct response. As expected, N M D A administration decreased correct progression through the sequence, with the most pronounced effect fol-

458

COHN AND CORY-SLECHTA RA 1

RA 2

120

80

._1 0

4O

rr l-Z

Drug p < .001 Pb x Drug p < .001

0 0 LL

0

I

I

I

I

I

)

I

0 O3 < ~'-

P1

P2

120-

0 < rc 0 (,~

<

80-

40

Drug p < .001

]

2o 35 5'0

s

20 3'5 50

NMDA (mg/kg) FIG. 1. Group mean overall accuracy + SEM expressed as percent of saline values for the control (O) and Pb-exposed groups (means of combined 50 and 250 ppm rats; (O)) for each of the four components of the multiple repeated acquisition and performance schedule. The absolute percentage values for saline sessions for the control group from this study were 69 ± 3.4, 81 ± 3.5, 80 + 2.1, and 82 + 2.8 for RAI, RA2, PI, and P2 components, respectively; corresponding values for the Pb-exposed group of this study were 69 + 3.0, 81 ± 1.8, 81 ± 3.5, and 82 ± 3.7. The box insert within each plot describes the significant outcomes of the statistical analysis.

lowing a first correct response (C1-C2; Fig. 3). These effects were potentiated by Pb exposure, particularly at the low and intermediate dose of NMDA. Specifically, while doses of 20 and 35 mg/kg NMDA had little effect on C1-C2 percentages in the control group, they decreased accuracy by 41./o-77./0 in the Pb-exposed group. There was again a tendency for this potentiated effect to be reversed in the Pb-exposed group at the 50 mg/kg dose, as the percent C1-C2 pairs increased from 41./0 at 35 mg/kg up to 59% at 50 mg/kg.

This decrease in correct CI-C2 progressions was primarily attributable to an increase in perseverative errors following a first correct response ( C I - E I ) and this effect was also significantly potentiated by Pb exposure. The proportion of C1E1 pairs increased in controls from 6*/0 during saline control sessions to 29*/0 at 50 mg/kg, a 500*/0 increase. In the Pbexposed group, CI-E1 response pairs had already increased by 390*/0 at 35 mg/kg, before declining to 240*/0 of control at the 50 mg/kg dose. Posthoc analyses confirmed a group

NMDA, LEAD, AND REPEATED ACQUISITION

459

RA 1

RA 2

150Drug p < .001 Pb x Drug p = .039

100'

T .._1

O nl--

Z 0 o U_ 0

5o

Drug p

0

I

O3 <

P1

Z

<

.001

)

I

I

)

I

P2

150.

O3 LLI O3 Z O

13.. 09 LU n"

Drug p < .001

]

100

50

Drug p < .004 Pb x Drug p = .077

2'o 3'5 s'o

2b as so

NMDA (mg/kg) FIG. 2. Group mean =t: SEM rate of responding (responses/min) for control (Q) and Pbexposed groups (means of combined 50 and 250 ppm rats; (O)) expressed as percent of saline session values. Group mean absolute response rate values for controls from this study during saline sessions were 22 + 4.3, 8 + 1.3, 8 + 1.2, and 3 + 0.5 responses/rain for the P A l , RA2, P1, and P2 components, respectively; corresponding values for the Pb-exposed group from the current study were 24 + 1.2, 7 :t: 0.4, 7 + 0.5, and 4 + 0.3 responses/rnin. The box insert within each plot describes the significant outcomes of the statistical analysis.

difference in C1-E1 response pairs at the 35 m g / k g N M D A dose. The decline in correct C1-C2 progressions also derived, although to a lesser extent, from an increase in skipping errors

(C1-E3), and again, this change was marginally potentiated by Pb exposure, particularly at NMDA doses of 20 and 35 mg/kg. N M D A also moderately reduced the frequency of correctly progressing from a correct second to a correct third member o f the sequence (C2-C3), and from a correct third member to correct reinitiation of the sequence (C3-C1), an effect that,

again, appeared to arise in both cases from a corresponding increase in perseverative responding (C2-C2; C3-E3). In neither case were these effects altered by Pb exposure. Response pairs beginning with an incorrect response. N M D A administration had no effect on correct reinitiation o f the sequence following an error on the first member of the sequence (El-C1; Fig. 4) but significantly decreased correct sequence reinitiation following an incorrect response E2 or E3 (E3-C1, E2-CI). Although there was a significant interaction between Pb and NMDA in the case o f E 3 - C I ,

460

COHN AND CORY-SLECHTA

Correct

Perseverative

C1-C2

Skip

C1 -El

C1-E3

30--D

100-

PbD

75-

30-~

50-

20-

25-

10-

Pb D PbD

r.f) rr

0 I

GO

~ I

16-

~

10- .

.

0 I

t

20-

8-

O00n 5°I

104-

25

I:E

0

I

I

W

Pb (.08) PbD

i

q

C3-E3

C3-C1 175 0 0 I ~

I

i

30-

D

Z

LU

r

C2-E1

12-

75

20- _

C2-E2

c2-c3

< 1°°T 0_

I

(.08)

i

I

i

C3-E2

1281510-

50-25

0

S

4-

J I I 2O 35 5O

0

s 5'o NMDA (mg/kg)

o$

25

FIG. 3. Group mean frequencies of response pairs beginning with a correct response expressed as a percent of saline session values and plotted as a function of dose of NMDA. For each response pair, the saline session value was calculated as a percentage of total response pairs with the same first member (e.g., CI-EI as a proportion of total response pairs beginning with CI). For all panels, the control group data are indicated by • and the Pb-exposed group by © . The left column depicts response pairs comprising correct progression through the sequence, the center column depicts perseverative response pairs and the right column presents skipping error response pairs. Within each panel, Pb signifies a main effect of Pb in the statistical analysis, D indicates a significant main effect of NMDA, and PbD indicates a significant interaction between Pb and NMDA. Error bars were omitted for clarity.

no systematic differences related to P b in these effects were evident. The decline in correct reinitiation o f the sequence f o l l o w i n g an incorrect response derived from a corresponding increase in skipping errors. A l l three such response pairs ( E l - E 3 , E 2 E 1, and E 3 - E 2 ) increased significantly with increasing dose o f

N M D A . Moreover, for all three o f these response pairs, these increases in frequency were more pronounced in the Pbexposed group, as confirmed by either a main effect o f P b ( E l - E 3 ) or by an interaction between P b and N M D A ( E 2 - E I , E3-E2). Within-session changes in accuracy. W i t h i n - c o m p o n e n t

461

NMDA, LEAD, AND REPEATED ACQUISITION

Perseverate

Correct Transition

El-C1

Skip

E3-C1

E2-C1

50

40

0|

i

S

I

20

~

35

I

30

01 ~bD

,

,

,

0 D

,

,

,

S

20

35

so

S

20

35

so

50

El-E1

Or) CE < Q_ U.J 03 Z

o;

El-E2

2'0 a;

©

s

E2-E2

O_ 03 UJ O~

S

20

35

El-E3

2o 3s so

E2-E3

50

S

E3-E3

20

35

20

35

50

S

E3-E1

50

0

NMDA

20

20

35

50

E3-E2

30/

s

2o 3s so

E2-E1

15

s

s

35

50

0

PbD / ~ s

20

35

50

(mg/kg)

FIG. 4. Group mean frequencies of response pairs beginning with an incorrect response expressed as a percent of saline session values and plotted as a function of dose of NMDA. Other details as described in Fig. 3, except that the top row depicts response pairs consisting of correctly beginning the sequence anew after an incorrect lever press; from the second row down, the left column shows perseverative response pairs and the middle column depicts nominally correct lever-to-lever transitions. The latter are considered incorrect response pairs because one or both members occurred during time-out. The right column depicts skipping errors. Error bars were omitted for clarity.

changes in accuracy are plotted for successive thirds of each component in Fig. 5. Accuracy levels increased across successive thirds of RAI in both the control and Pb-exposed groups as confirmed by a significant effect of bin. Whereas NMDA administration decreased overall accuracy levels in a doserelated fashion (main effect of drug), accuracy levels, nevertheless, still increased over the course of RA1 in controls. Pb exposure resulted in a more pronounced decline in accuracy

levels in response to NMDA, and also prevented the increase in accuracy levels across successive thirds of RA1 under drug conditions (Pb by drug; Pb by bin). Although overall accuracy values were already relatively high by the beginning of RA2, they nonetheless increased significantly across the course of RA2, albeit with a shallow trend (main effect of bin). Overall accuracy levels were again decreased by NMDA to a greater extent in Pb-exposed rats,

462

COHN AND CORY-SLECHTA

Controls

Pb-Exposed

RA 1

"° l 100#

/ 50

01

RA 1

~ .- - O . . . . . . 42~'[3--" I

~

~)

eb 0 0 ......... ~ .......... D

q

RA 2

Drug PbD Bin PbB

RA2

150

o

,ol.

Z133

oI

......

• .......... ~ -

....... ,

[3 . . . . . . .

,

•

.......... O

43 . . . . . . . . . ~

Pb (.09) Drug PbD Bin

, P1

PI

<

>0 < n0 0 <

100150 t

lSlt

• -~- - : : ' - i _ i ..... [9 . . . . . . . . --[3. . . . . . .

P2

• @

Pb Drug PbD PbB (.06

P2

100

PbB

2

3

i

BIN (1/3 COMPONENT) FIG. 5. Group mean accuracy values during each successivethird of each component of the multiple repeated acquisition and performance schedule, shown for the control group in the right column and for the Pb-exposed group in the left column. Data for bins 2 and 3 are plotted as a percent of bin 1 values. Within each plot, • depict data from saline sessions, © depict data for 20 mg/kg NMDA, open squares for 35 mg/kg NMDA and open triangles for 50 mg/kg NMDA. The outcomes of the statistical analysis comparing control to Pb-exposed rats for each component are indicated in the far right column, with Pb signifying a main effect of Pb, D a main effect of N M D A , Bin a main effect of bin, PbD an interaction between Pb and N M D A and PbB an interaction between Pb and bin.

with doses of 20 and 35 mg/kg NMDA effective in the Pbexposed group but having little evident impact in the control group. There was no significant increase in accuracy over the course of P1 or P2, as might be expected. NMDA administration decreased P1 accuracy only in the Pb group (Pb by drug interaction). Moreover, these decrements in accuracy in the Pb-exposed group appeared to decline even further over the course of P1, particularly at the 35 mg/kg dose level (Pb by bin). Although there was no main effect of Pb or any interaction of Pb and NMDA in P2, a Pb by bin interaction was

detected, indicative of differential changes in accuracy across the course of the component in control versus Pb-exposed groups.

Comparative effects o f NMDA on learning and performance. To address the extent to which NMDA administration exerted selective effects on learning as compared to performance, overall accuracy in P A l versus P I , and RA2 versus P2 was compared in the control group (Fig. 1, solid circles). These analyses revealed the clear importance of the time of evaluation to the determination of selectivity. Specifically, the comparison of P A l to P1 revealed a highly significant main

NMDA, LEAD, A N D R E P E A T E D ACQUISITION effect of component ( p = 0.008) and of drug ( p = 0.026) but no interaction of drug by component (p = 0.559), such as would be expected if N M D A administration had differential effects in the two components. In contrast, the RA2 and P2 comparison found main effects of component (p < 0.004) and drug (p < 0.001) as well as a significant component by drug interaction (p < 0.007), consistent with the more pronounced decline in RA2 than P2 accuracy. Blood Pb concentrations. Median + SE blood Pb concentrations values increased with the level of Pb exposure and were 2.8 ± 1.0, 25.1 ± 4.1 and 73.5 ± 5.7 #g/dl for the 0, 50, and 250 ppm groups, respectively. DISCUSSION

Administration o f N M D A at doses ranging from 20 to 50 mg/kg resulted in decrements in accuracy as well as in rate o f responding in both components of a multiple schedule of repeated acquisition and performance. The declines in accuracy in the repeated acquisition component were caused by a pattern of errors that to date appears unique to N M D A (10,13). It consisted of an initial perseverative error (repeated response on the same lever), which was then followed by errors of skipping backward and forward through the sequence rather than by a correct re-initiation of the sequence. Pb exposure significantly potentiated these effects of NMDA, including its alterations of accuracy and response rate, and did so in the case of accuracy by increasing the frequency of the pattern of perseverative followed by skipping errors. This Pb-induced potentiation of NMDA's effects did not occur uniformly across the schedule, i.e., did not achieve statistical significance in all cases. But even where conventional levels o f statistical significance were not achieved, similar trends were evident. Two questions arise with respect to the basis of the potentiated effects of N M D A resulting from Pb exposure. The first is whether these effects arise from CNS properties of N M D A or from nonspecific effects of the compound. That N M D A clearly has CNS activity at the dose ranges used in this study is suggested by the results of previous drug discrimination studies (3,4,6,21,22,30,31,45). Those studies reveal that a dose of 30 mg/kg N M D A administered IP functions as a discriminative stimulus and that its stimulus properties appear to be mediated via the N M D A receptor complex. Specifically, competitive NMDA antagonists acting at the glutamate site, as does NMDA, dose-dependently block the stimulus properties of N M D A and do so with relative potencies corresponding to their affinity for the N M D A receptor and at doses that do not influence response rate. Glycine site antagonists have also been shown to block the stimulus properties of NMDA. In contrast, noncompetitive antagonists acting at the PCP (phencyclidine) site rather than the glutamate site produce less consistent effects. The specificity of N M D A for the N M D A receptor complex, moreover, is indicated by the fact that a wide variety of CNS compounds, including morphine, caffeine, amphetamine, pentylenetetrazol, and pricrotoxin fall to substitute for NMDA, and cholinergic compounds such as nicotine, physostigmine, arecoline, and mecamylamine produce only partial responding and this occurs at doses which also suppress response rate. Moreover, another potent glutamate agonist, LY 285265 has recently been shown to substitute completely for N M D A (22). Taken together, these studies suggest that at this dose range, N M D A can have CNS effects that are selectively mediated via the N M D A receptor complex. The second question is whether the potentiated effects of N M D A on the repeated acquisition and performance baseline

463 observed in Pb-exposed animals in this study represent direct effects on N M D A systems or are secondary to Pb-induced changes in other neurotransmitter systems. Pb exposure is well known to impact a variety of neurotransmitter systems, inchiding opiates, GABA, cholinergic systems and dopamine systems (19,29,40). What has become increasingly clear over the past several years is the extensive interactions of these various neurotransmitter systems, modulating and actually regulating aspects of each other's functions. Potentiation of the behavioral properties of NMDA by Pb exposure, could, then, arise through such interactions. In that regard, dopamine systems are well known to interact with the N M D A receptor complex (9,25,35), and dopaminergic systems are also perturbed by lead (e.g., 19,33). In addition, although lead has been reported to inhibit long-term potentiation, this may not occur solely through its actions on the NMDA receptor complex (24). This study did not specifically address this second question, but it would be interesting to determine whether N M D A antagonists or perhaps dopaminergic antagonists would have blocked the observed potentiated effects of NMDA in the Pb-exposed group. Such neurotransmitter interactions may, in fact, be suggested by the reversal of NMDA effects noted at the highest NMDA dose, 50 mg/kg, in the Pb-exposed groups under several conditions. One conceivable explanation of these findings is that higher doses of the drug recruit additional physiological/biochemical processes that somehow modulate the effects o f lower doses of the drug. If so, one might expect to see similar reversals in the control group at higher doses of NMDA. Alternatively, this reversal of effects might reflect interactions of the drug with other neurochemical systems or alterations in those neurotransmitter systems induced by Pb exposure, as posed above with respect to direct versus indirect mediation of NMDA potentiation. An intriguing aspect of the current findings with NMDA are their correspondences to what would be expected based on what is currently known about the effects of Pb exposure on N M D A receptor complex function. In addition to reports of Pb-induced inhibition of binding of the noncompetitive antagonist MK-801 to the PCP site, and inhibition of NMDA channel activity (1,23,42), Pb exposure is also associated with subsensitivity to behavioral and stimulus properties (11,26) of MK-801. Such effects suggest an inhibition of N M D A receptor complex function and the consequent possibility of an upregulation at the glutamate site to overcome this inhibition. In fact, Petit et al. (38) reported an increase in NMDA receptors in Pb-exposed rats. The current potentiation of N M D A effects in Pb-exposed rats is consistent with these reports and adds further support to the involvement of Pb with the N M D A receptor complex. The fact that NMDA administration resulted in an impairment in accuracy rather than a facilitation of learning might seem somewhat surprising, because activation of the N M D A receptor complex has been associated with processes such as long-term potentiation that are thought to serve as substrates of learning and memory functions. However, a survey of the literature indicates that NMDA agonists do not uniformly enhance performance. For example, Jones et al. (27) reported that systemically administered N M D A actually impaired acquisition o f a passive avoidance response. Nor do NMDA antagonists uniformly impair behavior. Consider the recent report by Mondadori and Weiskrantz (36) that although N M D A antagonists impaired performance in a step-through dark avoidance paradigm, these compounds actually facilitated retention performance in the step-down passive avoid-

464

COHN AND CORY-SLECHTA

ance paradigm. These types of findings remind us of the importance of the behavioral paradigm and the variables controlling behavior in modulating drug effects. They also suggest that neurochemical mediation of behavioral processes must indeed be dynamic rather than static processes. It may also seem unexpected that both the glutamate agonist N M D A (this study) and the noncompetitive antagonist MK-801 (10,11,13) produced apparently corresponding effects on the RA baseline, namely, decreases in overall accuracy. Note, however, that overall accuracy is a relatively gross measure of behavior in such a paradigm. Analyses of underlying error patterns quite clearly demonstrates the differential effects of N M D A and MK-801. The former altered behavior through initial perseverative responding followed by skipping errors. In contrast, MK-801 is primarily associated with perseverative behavior (10,11,13). Thus, the underlying behavioral processes produced by these two compounds are quite distinct. Another issue related to the nature of the effects of N M D A on this baseline is whether this compound produces selective effects on learning processes or whether impairments of accuracy are indirect effects of nonspecific behavioral changes. This question was assessed by comparing the extent of accuracy changes in R A I versus P1 and RA2 versus P2. The present study does not provide a clear answer to that question.

Comparisons of RAI to P 1 accuracy failed to find any significant interaction between NMDA and component, such as would be expected if NMDA produced a more pronounced effect on the RA component. An interaction of N M D A by component was found in the comparisons of RA2 to P2 overall accuracy, consistent with a more pronounced effect of N M D A administration on learning. However, this could have reflected the cessation of drug effect during the P2 component of the session, because drug effects were studied under constant conditions where CLR always served as the correct RA sequence, and where the session always began with an RA rather than a P component, yielding the constant component • order of RA1, PI , RA2, P2. Nevertheless, the fact that significant decrements in rate of responding occurred in the P2 component in the absence of any corresponding impact on overall accuracy in P2 does provide evidence for a separation of these outcome variables, and could be consistent with the possibility of selective effects of NMDA on learning processes at doses lower than those required to produce nonspecific disruption of behavior. Further evaluation of this possibility will be necessary to resolve this question. ACKNOWLEDGEMENT Supported by NIEHS Grants ES07026, ES05017, ES05903, and ES02047.

REFERENCES 1. Alkondon, M.; Costa, A. C. S.; Radharkrishnan, V.; Aronstam, R. S.; Albuquerque, E. X. Selective blockade of NMDA-activated channel currents may be implicated in learning deficit caused by lead. FEBS 261:124-130; 1990. 2. Altmann, L.; Weinsberg, F.; Sveinsson, K.; Lilienthal, H.; Weigand, H.; Winneke, G. Impairment of long-term potentiation and learning following chronic lead exposure. Toxicol. Lett. 66:105112; 1993. 3. Amrick, C. L.; Bennett, D. A. N-MethyI-D-aspartate produces a discriminative stimuli in rats. Life Sci. 40:585-591; 1987a. 4. Amrick, C. L.; Bennett, D. A. NMDA discriminative stimuli are antagonized by competitive NMDA antagonists, PCP-type compounds and kappa agonists. Soc. Neurosci. Abst. 13:1561; 1987b. 5. Bellinger, D.; Leviton, A.; Waternaux, C.; Needleman, H.; Rabinowitz, M. Longitudinal analyses of prenatal and postnatal lead exposure and earlycognitive development. New Eng. J. Med. 316: 1037-1043; 1987. 6. Bennett, D. A.; Bernard, P. S.; Amrick C. L. A comparison of PCP-like compounds for NMDA antagonism in two in vivo models. Life Sci. 42:447-454; 1988. 7. Butelman, E. R. A novel NMDA antagonist, MK-801, impairs performance in a hippocampal-dependent spatial learning task. Pharmacol. Biochem. Behav. 34:13-16; 1989. 8. Byers, R. K.; Lord, E. E. Later effects of lead poisoning on mental development. Am. J. Disabled Child 66:417-494; 1943. 9. Carrozza, D. P.; Ferroar, T. N.; Golden, G. T.; Reyes, P. F.; Hare, T. A. In vivo modulation of excitatory aminoacid receptors: microdialysis studies on N-methyl-D-aspartate-evoked striatal dopamine release and effects of antagonists. Brain Res. 574: 42-48; 1992. 10. Cohn, J.; Cory-Slechta, D. A.: Differential effects of MK-801, NMDA, and scopolamine on rats learning a four-member repeated acquisition paradigm. Behav. Pharmaeol. 3(4):403-413; 1992. 11. Cohn, J.; Cory-Slechta, D. A. Subsensitivity of lead-exposed rats to the accuracy-impairing and rate-altering effects of MK-801 on a multiple schedule of repeated acquisition and performance. Brain Res. 600:208-218; 1993. 12. Cohn, J.; Cox, C.; Cory-Slechta, D. A. The effects of lead on

13.

14. 15. 16. 17.

18. 19. 20.

21. 22. 23. 24.

learning under a multiple schedule of repeated acquisition and performance. Neurotoxicol. 14:329-346; 1993. Cohn, J.; Ziriax, J. M.; Cox, C.; Cory-Slechta, D. A. Comparison of error patterns produced by scopolamine and MK-801 on repeated acquisition and transition baselines. Psychopharmacol. 107:243-254; 1992. Collingridge, G. L. Long-term potentiation in the hippocampus: Mechanisms of initiation and modulation by neurotransmitters. Trends in Pharmacol. Sci. 6:407-411; 1985. Cory-Slechta, D. A. Exposure modifies the effect of low-level lead on fixed interval performance. Neurotoxicol. 11:427-442; 1990. Cory-Slechta, D. A.; Weiss, B.; Cox, C. Performance and exposure indices of rats exposed to low concentrations of lead. Toxicol. Appl. Pharmacol. 78:291-299; 1985. Cory-Slechta, D. A.; Weiss, B.; Cox, C. Mobilization and redistribution of lead over the course of calcium disodium ethylenediamine tetraacetate chelation therapy. J. Pharmacol. Exp. Therap. 243:804-813; 1987. Cory-Slechta, D. A.; Widzowski, D. V. Low-level lead exposure increases sensitivity to the stimulus properties of dopamine O 1 and D2 agonists. Brain Res. 553:65-74; 1991. Cory-Slechta, D. A.; Widzowski, D. V.; Pokora, M. J. Functional alterations in dopamine systems assessed using drug discrimination procedures. Neurotoxicol. 14:105-114; 1993. France, C. P.; Moerschbacher, J. M.; Woods, J. H. MK-801 and related compounds in monkeys: Discriminative stimulus effects and effects on a conditioned discrimination. J. Pharmacol. Exp. Ther. 257:727-734; 1991. Grech, D. M.; Willetts, J.; Balster, R. L. Pharmacological specificity of N-methyI-D-aspartate discrimination in rats. Neuropharmacol. 32:349-354; 1993a. Grech, D. M.; Li, H.; Lunn, W. H. W.; Balster, R. L. The discriminative stimulus effects of excitatory amino acid agonists in NMDA-tralned rats. Soc. Neurosc. Abst. 19:1354; 1993b. Guilarte, T. R.; Miceli, R. C. Age dependent effects of lead on 3H-MK-801 binding to the NMDA receptor-gated ionophore: In vitro and in vivo studies. Neurosci. Lett. 148:27-30; 1992. Hori, N.; Busseiberg, D.; Matthews, M. R.; Parsons, P. J.;

NMDA, LEAD, AND REPEATED ACQUISITION

25. 26.

27.

28. 29. 30.

31. 32.

33. 34.

Carpenter, D. O. Lead blocks LTP by an action not at NMDA receptors. Exp. Neurol. 119:192-197; 1993. Imperato, A.; Scrocco, M. G.; Bacchi, S.; Angelucci, L. NMDA receptors and in vivo dopamine release in the nucleus accumbens and candatus. Eur. J. Pharmacol. 187:555-556; 1990. Johnson, S. C.; Cory-Slechta, D. A. Postnatal (PN) and postweaning (PW) lead exposures differentially affect sensitivity to the noncompetitive NMDA antagonist MK-801. Toxicol. 13:166; 1993. Jones, K. W.; Schaeffer, C. L.; DeNoble, V. J. Systemically administered N-methyl-D-aspartate interferes with acquisition of a passive avoidance response in rats. Pharmacol. Biochem. Behay. 34:181-185; 1989. Kant, G. J.; Wright, W. L.; Robinson, T. N., III; D'Angelo, C. P. Effects of MK-801 on learning and memory as assessed using a novel water maze. Pharmacol. Biochem. Behav, 39:479-485; 1991. Kitchen, I. Lead toxicity and alterations in opioid systems. Neurotoxicol. 14:115-124; 1993. Koek, W.; Colpaert, F. C. N-methyl-D-aspartate antagonism and phencyclidine like activity: Behavioral effects of glycine site ligands. In: Kamenka, J.-M.; Domino, E., eds. Multiple sigma and PCP receptor ligands: Mechanisms for neuromodulation and neuroprotection? Ann Arbor, MN: NPP Books; 1992: 655-671. Koek, W.; Woods, J. H.; Colpaert, F. C. N-methyl-D-aspartate antagonism and phencyclidine-like activity: A drug discrimination analysis. J. Pharmacol. Exp. Therap. 253:1017-1025; 1990. Laroche, S.; Doyere, V.; Bloch, V. Linear relation between the magnitude of long-term potentiation in the dentate gyrus and associative learning in the rat. A demonstration using commissural inhibition and local infusion of an N-methyl-D-aspartate receptor antagonist. Neurosci. 28:375-386; 1989. Lasley, S. M. Regulation of dopaminergic activity, but not tyrosine hydroxylase, is diminished after chronic inorganic lead exposure. Neurotoxicol. 13:625-636; 1992. Lasley, S. M.; Armstrong, D. L.; Polan-Curtain, J. In vivo induction of hippocampal long-term potentiation (LTP) in rats is impaired by chronic exposure to environmental levels of lead (Pb). Abst. Ninth Intern. Neurotoxicol. Conf. 42; 1991.

465 35. McCullough, L. D.; Salamone, J. D. Increases in extracellular dopamine levels and locomotor activity after direct infusion of phencyclidine into the nucleus accumbens. Brain Res. 577:1-9; 1992. 36. Mondadori, C.; Weiskrantz, L. NMDA receptor blockers facilitate and impair learning via different mechanisms. Behav. Neural Biol. 60:205-210; 1993. 37. Needleman, H. L.; Gunnoe, C.; Leviton, A.; Reed, R.; Peresie, H.; Maher, C.; Barrett, P. Deficits in psychologic and classroom performance of children with elevated dentine lead levels. N. Eng. J. Med. 300:689-695; 1979. 38. Petit, T. L.; LeBoutillier, J. C.; Brooks, W. J. Increased sensitivity to NMDA following chronic lead exposure. Soc. Neurosc. Abst. 16:1186, #490.18; 1990. 39. Rice, D. C. Chronic low-lead exposure from birth produces deficits in discrimination reversal in monkeys. Toxicol. Appl. Pharmacol. 77:201-210; 1985. 40. Shellengerger, M. K. Effects of early lead exposure on neurotransmitter systems in the brain. A review with commentary. Neurotoxicol. 5:177-212; 1984. 41. Skelton, R. W.; Scarth, A. S.; Wilkie, D. M.; Miller, J. J.; Phillips, A. G. Long-term increases in dentate granule cell responsivity accompanying operant conditioning. J. Neurosc. 7:30813087; 1987. 42. Ujihara, H.; Albuquerque, E. X. Developmental change of the inhibition by lead of NMDA-activated currents in cultured hippocampal neurons. J. Pharmacol. Exp. Therap. 263:868-875; 1992. 43. Upchurch, M.; Wehner, J. M. Effects of N-methyl-D-aspartate antagonism on spatial learning in mice. Psychopharmacol. 100: 209-214; 1990. 44. Walker, D. L.; Gold, P. E. Effects of the novel NMDA antagonist, NPC 12626, on long-term potentiation, learning and memory. Brain Res. 549:213-221; 1991. 45. Willetts, J.; Balster, R. L. Effects of competitive and noncompetitive N-methyl-D-aspartate (NMDA) antagonists in rats trained to discriminate NMDA from saline. J. Pharmacol. Exp. Therap. 251: 627-633; 1989.