Lead-induced changes in NMDA receptor complex binding: correlations with learning accuracy and with...

E L S E V I E R Behavioural Brain Research 85 (1997) 161-174

BEHAYIOURAL BRAIN

RESEARCH

Research Report

Lead-induced changes in NMDA receptor complex binding: correlations with learning accuracy and with sensitivity to learning impairments

caused by MK-801 and NMDA administration

Deborah A. Cory-Slechta a,,, Monica Garcia-Osuna b,1, j. Timothy Greenamyre a.b,2 a Department of Neurobiology and Anatomy, Box 603, University of Rochester, School of Medicine and Dentistry, Rochester,

N Y 14642, USA b Department of Neurology, University of Rochester, School of Medicine and Dentistry, Rochester, NY14642, USA

Received 24 May 1996; received in revised form 1 August 1996; accepted 28 August 1996

Abstract

This study sought to further evaluate potential mechanistic relationships between Pb-induced alterations in glutamate neurotransmission and behavioral toxicity. It examined correlations between Pb-induced changes in [3H]MK-801 and [3H]CGP-39653 binding sites in 4 different brain regions (frontal cortex, dentate gyrus, CA1 and striatum) and (1) changes in learning accuracy on a multiple repeated acquisition and performance schedule, and (2) sensitivity to the accuracy-impairing effects of MK-801 and NMDA on this learning baseline. All data were obtained from a single population of rats that had been chronically exposed from weaning to 0, 50 or 250 ppm Pb acetate in drinking water and demonstrated selective learning impairments and altered sensitivity to the effects of MK-801 and NMDA on learning accuracy. Pb exposure decreased MK-801 binding and possibly increased CGP-39653 binding, effects statistically significant in some brain regions, but generally exhibiting similar trends across regions. At 0 ppm, higher levels, particularly of MK-801 binding, were associated with higher accuracy levels in the learning paradigm and with greater decrements in learning accuracy following MK-801 or NMDA administration. These linear correlations were negated and in some cases even reversed by 50 and 250 ppm Pb, an effect that might be attributable to an alteration of NMDA receptor complex subunit composition and thus, ligand binding. Of the 4 brain regions examined, striatal MK-80I binding proved to be the best predictor of learning accuracy levels. These data provide additional support for an involvement of the NMDA receptor complex in Pb-induced learning impairments. The fact that these effects were noted most frequently in striatum also raises the possibility that dopamine-glutamatergic interactions contribute to Pb's effects.

Keywords: Lead; CGP-39653; MK-801; Learning; Cognition; N-Methyl-D-aspartate (NMDA) receptor complex

1. Introduction

Even today, years after its removal from paint and from gasoline, lead (Pb) exposure remains a significant public health problem. The current problem derives from the residual environmental contamination resulting from prolonged use of lead, and from the low exposure levels known to be associated with its adverse effects.

* Corresponding author. Fax: ( 1 ) (716) 256-2591; E-mail: [email protected] ~Present address: Department of Neurology, Emory University School of Medicine, WMRB 6000, PO Drawer V, Atlanta, GA 30322, USA. 2present address: Department of Neurology, Emory University School of Medicine, WMRB 6000, PO Drawer V, Atlanta, GA 30322, USA.

0166-4328/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved Pll S0166-4328 (96) 00174-X

Prospective longitudinal studies in pediatric populations show that blood lead levels as low as 10 lxg/dl can deleteriously impact cognitive functions, measured as group changes in IQ test scores and other psychometric indices [58]. Blood lead concentrations on this order are still widely prevalent in many regions of the world, and it has been estimated that in the US alone, I in 6 children still have blood lead concentrations above the currently defined levels of concern [12].

Experimental animal studies have corroborated the nature of the findings in children and report effects at similar blood Pb concentrations, i.e., l l -15~tg/dl . Impaired discrimination learning, particularly reversal learning, has been described following Pb exposure in

162 D.A. Cory-Slechta et al. / Behavioural Brain Research 85 (1997) 161-174

both rodents and non-human primates [ 10, 37, 44, 49, 57 ]. Such effects can be found even after a change from a food reward to a non-food based reward [10], and, in rodents, acquisition of new response sequences is impaired, even while performance of previously learned response sequences remains intact [16], confirming that these cognitive impairments can occur independently of changes in sensory, motor or motivational function.

The neurobiological mechanisms of Pb-induced cognitive impairments have yet to be elaborated. Based on biochemical evidence, several investigators have advanced the idea that glutamatergic systems, particularly the NMDA receptor complex may be involved. Pb exposure alters NMDA-evoked whole-neuron and single-channel currents, as well as the frequency of NMDA-activated channel openings [1] in addition to binding of ligands at both the ion channel and glutamate binding sites of the NMDA receptor complex [8,24,31,50]. Pb exposure has also been reported to inhibit long-term potentiation, thought to be a cellular substrate of learning and memory processes [2,35], the induction of which, at least in hippocampus, is considered to be dependent upon NMDA receptor activation.

Although such reports are intriguing, they are,-of course, not definitive. It is certainly conceivable that other biochemical changes, e.g., in dopaminergic or cholinergic systems, could contribute to Pb-induced learning impairments; both systems contribute to cognitive function and both are altered by Pb exposure [5, 6,19]. In support of the possibility that glutamatergic system changes might be preferentially involved, however, are recent studies showing that Pb-exposed rats that had exhibited selective deficits in learning [16], also showed differential responses to the acute administration of glutamatergic (MK-801 and NMDA), but not dopaminergic compounds (quinpirole, SKF-38393 and cz-methyl-p-tyrosine) on accuracy levels in this paradigm [11,15,59].

Examination of the relationships between reported cellular/biochemical changes and behavioral impairments is one way to begin to address the extent to which NMDA receptor complex changes might underlie Pb-related cognitive deficits. This study explored the hypotheses that: (1) under normal conditions, NMDA receptor binding would be preferentially correlated with accuracy of learning, but not of performance of an already learned response, and that Pb would alter this correlation; and (2) MK-801- or NMDA-induced disruption of learning, but not performance accuracy, should be inversely related to NMDA binding and this relationship should be altered by Pb exposure. To examine these hypotheses, levels of MK-801 (a non-competitive ion-channel antagonist) and CGP-39653 binding (a competitive antagonist at a site overlapping, but not identical to, that for glutamate and NMDA [36]) in 4 different brain regions (dentate, CA1, frontal cortex and

striatum) were determined autoradiographically in a population of rats that had previously exhibited: (1) selective lead-induced deficits in learning accuracy in a multiple schedule of repeated learning and performance [16]; and (2) altered sensitivity to the effects of acute administration of MK-801 and NMDA on learning accuracy in this paradigm [13,14]. Direct correlations between behavior and binding could then be determined, as well as the alterations in these patterns of correlations in response to Pb exposure.

2. Materials and methods

2.1. Animals

Thirty-one male Long-Evans rats, 21 days of age, from Blue Spruce Farms (Altamont, NY) were ran- domly assigned to a Pb-exposure group and provided drinking solutions containing 0 (n= 10), 50 (n= 10) or 250 (n = 11 ) ppm Pb acetate dissolved in distilled deion- ized water ad libitum. They were group-housed under a 12-h light/dark cycle until 35 days of age at which time they were individually caged. Rats had unrestricted access to semi-purified 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, 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 regula- tion for the duration of the behavioral experiments.

2.2. Behavioral procedures

2.2.1. Assessment of learning All rats were first utilized in an experiment assessing

the effects of Pb on learning in a multiple schedule of repeated acquisition (RA) and performance (P) [16]. One-hour behavioral test sessions were carried out 5 days per week in operant chambers housed in sound- attenuated enclosures ventilated by a fan. Each chamber contained three response levers configured horizontally in a row, designated as left (L), center (C) and right (R). On this multiple schedule, completion of a sequence of 3 responses was required for reinforcement (45-mg food pellets) in both the RA and P components of the schedule. In the RA component, the sequence of 3 responses required for reinforcement changed unpredict- ably with each session thus providing a new learning curve with each session (CRL, RLC, CLR, RCL, LRC), whereas the sequence of 3 responses required for reinforcement in the P component was constant across sessions (LCR) providing a performance control for non-specific effects of Pb. Two presentations each of the RA and P component occurred in each experimental session (RA1, P1, RA2, P2), with the components

D.A. Cory-Slechta et al. / Behavioural Brain Research 85 (1997) 161-174 163

changing after 15 min or after the delivery of 25 rein- forcers, whichever occurred first. The component which began each session (RA1 or P1) alternated across sessions. The P component was cued by the onset of the lights above each lever, while the onset of the RA component was signaled by the offset of these lights. Errors during the completion of sequences resulted in a 2-s time-out period. Responses during the time-out extended its duration until two seconds had elapsed without a response. Rats were then required to begin the sequence over. A titrating fixed ratio schedule was operative in both the RA and P components, such that an error occurring during or between a sequence increased the ratio requirement for reinforcement from one to two consecutive errorless sequences. Overall accuracy was defined as the total number of responses on the correct lever divided by the total number of responses. A total of 75 sessions on this schedule were carried out.

Pb exposure exerted a selective effect on learning as shown in Table 1 for the final blocks of sessions for each condition: decrements in accuracy were noted in the RA, but not the P, component of the schedule. More specifically, the RA decrements were restricted to those R A sequences (CLR, R C L L R C ) that had no overlap in execution with the P component sequence LCR. Thus, considered across the 75 sessions, Pb-associated accuracy deficits in the RA component were specific to the non P-like sequences, while RA accuracy levels in the presence of P-like sequences RLC and C R L as well as in the P components were compara- ble in control and Pb-treated groups.

Subsequent analyses of error patterns demonstrated

Table 1 Group mean + SE overall accuracy levels on the multiple schedule of repeated acquisition and performance a

Component/sequence type b Pb exposure concentration (ppm)

0 50 250

Repeated acquisition/ 57 + 7 36 ___ 3 f 32 --+'4 f

non-P-like sequences ~ Repeated acquisition/ 55 + 6 76 __+ 3 72 + 5

P-like sequences a Performance ~ 82 + 3 84 + 2 84 ___ 2

aData from [16]. bP-like sequences involved the same lever-to-lever transition as did the P component sequence LCR; lever-to-lever transitions required to execute non-P-like sequences did not overlap with that required for completion of the P component sequence LCR. CFinal block of 10 sessions. aFinal block of 5 sessions. eFinal block of 15 sessions. rDiffered significantly from corresponding 0 ppm data as determined using randomization tests of the constant and linear coefficients derived from cubic orthogonal polynomial fits to each individual animal's data across sessions.

that Pb exposure decreased R A accuracy by increasing the frequency of two types of perseverative errors: repeated responses on the same lever and repetitions of the P component sequence LCR. The latter error pattern was the basis for the finding that Pb-exposed rats showed no deficits in RA accuracy in those sessions where P-like sequences were utilized.

2.2.2. Assessment o f MK-801 and N M D A sensitivity Following assessment of Pb-induced changes in learn-

ing on the multiple RA and P schedule, the comparat ive effects of acute administration of MK-801 [13] and of N M D A [14] on this schedule were studied. These experiments were based on the assertion that differential changes in accuracy produced by MK-801 and/or N M D A in control and Pb-exposed rats would provide indirect evidence for glutamatergic system involvement in Pb-induced learning impairments. After 75 sessions on the multiple RA and P schedule, MK-801 and then N M D A (both f rom Research Biochemicals, Inc., Natick, MA) dose-effect curves were collected. Rats were injected i.p. with either physiological saline, or with doses of 0.05, 0.10, 0.20 and 0.30 mg/kg MK-801 maleate diluted in physiological saline 45 min prior to the session. N M D A was prepared in equimolar solution with N a O H at p H 7.0 diluted in physiological saline and administered at doses of 0, 20, 35 and 50 mg/kg 15 rain prior to the session. For both drugs, all doses and saline were administered in random order at a constant volume of 0.3 ml, and each dose was replicated at least twice in each rat. At least one session with no injection occurred between all drug sessions to minimize any carryover effects.

Because of the bias towards P-like sequences in Pb-exposed rats, drug effects were assessed separately on P-like ( R L C ) and non-P-like ( L R C ) R A sequences in the case of MK-801 and only for the non-P-like sequence CLR for NMDA. This was because non-P- like sequence sessions were considered a better indicator of effects of drugs on acquisition than were P-like sequences. Moreover, even though all rats were actually tested, drug effects in those studies [13,14] were reported only for 'learners', i.e., rats in each group who sustained RA2 accuracy levels of at least 40%; accuracy decrements could not be as readily detected in 'non-learners ' due to a floor effect. Thus, drug effects were reported for 6 of the rats in the 0 ppm group, and for a combined total of 10 rats from the 50 and 250 ppm groups.

Both MK-801 and N M D A administration decreased accuracy levels on the multiple RA and P schedule (Table 2; [13,14]). In the case of MK-801, accuracy decreases were noted in both the R A and P components of the schedule suggesting non-specific effects of the drug. Acute N M D A administration at a dose of 50 mg/kg (not at the 35 mg/kg dose shown in Table 2) exerted a selective effect on RA2 vs. P2 accuracy during


Table 2 Group mean + SE accuracy levels on the multiple repeated acquisition and performance schedule following drug treatment calculated as percent of saline control values

Dose, drug and sequence Exposure group Schedule component

RA1 RA2 P1 P2

0.30 mg/kg MK-801 a Control 43 + 11 45 + 15 55 +__ 7 30 ___ 10 LRC (non P-like) Pb 50 + 5 62 +__ 3 ~ 43 + 6 56_+ 5 c

0.30 mg/kg MK-801 a Control 45 + 12 48 + 10 43 + 7 44 +__ 8 RLC (P-like) Pb 63 +__ 8 68 + 7 60 + 7 c 62 + 7

35 mg/kg NMDA b Control 67+ 10 96+ 5 72+ 19 100+ 5 CLR (non P-like) Pb 30 ___ 10 45 _ 13 c 43 + 20 85 + 10

~Data from [13]. bData from [ 14]. cPb-exposed group differed significantly from control as determined by repeated measures analyses of variance based on the full dose--effect curve.

the second half of the experimental session, suggesting a preferential impairment of learning.

The effects of MK-801 administration were attenuated by Pb exposure, while those of N M D A were potentiated by Pb exposure [13,14]. These Pb-induced alterations in the accuracy-impairing properties of MK-801 and N M D A were noted in the RA2 component preferentially for non-P-like sequences. Only non-systematic Pb- related alterations in sensitivity were noted in the P components, with attenuated effects of MK-801 in the P2 component when the non-P-like sequence LRC served as the correct RA sequence, and in the P1 component when the P-like sequence RLC was used in the RA component. Taken together, the pattern of outcomes is suggestive of greater susceptibility of acquisition conditions (non-P-like sequences in RA components) than of performance processes to the drug effects.

2.3. Autoradiographic binding assays

2.3.1. Tissue At completion of the behavioral and drug studies,

rats were decapitated, and their brains were rapidly removed, frozen in dry ice, and stored at -70°C. Horizontal sections (15 ~tm) were cut in a cryostat, thaw-mounted onto subbed slides and stored at - 7 0 ° C until the day of the assay. Four regions were examined. CA1 and dentate regions of hippocampus were included because of the prevalence of N M D A receptors in hippocampal areas and because of prior reports of Pb-induced alterations in MK-801 binding in this area [31]. Frontal cortex also contains a high density of N M D A receptors [22]. While N M D A receptor density in striatum is relatively low, the inclusion of both frontal cortex and striatum was also based on the repeatedly described dopaminergic-glutamatergic interactions in these areas and such interactions may be important to Pb effects

(e.g., [20]). In addition, Pb exposure has been shown to alter parameters of dopaminergic function in striatum [19,45], effects which could be indirectly produced by altered glutamatergic function. MK-801 acts as a non- competitive antagonist by binding inside the ion channel, while CGP-39653 acts as a competitive antagonist at a site overlapping, but not identical to, that at which the agonists glutamate and N M D A act [36,43,48].

2.3.2. MK-801 binding On the day of the assay, sections were removed from

the freezer and allowed to reach room temperature. Following a 30-min prewash in 5 0 m M Tris-acetate buffer at 4°C (pH 7.4), sections were incubated for 2 h at room temperature in 5 0 m M Tris-acetate buffer (pH7 .4 ) containing 5 n M [3H]MK-801 (22-22.5 Ci/mmol; NEN, Boston, MA), 30 BM glutamate and 10 ~tM glycine (4 slide-mounted sections/10 ml). Non- specific binding was defined by 5 ~tM MK-801. At the end of the assay, sections were dipped for 5 s in ice-cold buffer, and then placed for 90 min in cold fresh buffer. The tissue sections were dried and apposed to tritium- sensitive film (Hyperfilm-3H, Amersham) with tritium standards.

2.3.3. CGP-39653 binding [3H]CGP-39653 binding was performed according to

the method of Jaarsma et al. [30]. Briefly, sections were washed three times for 20 min in 50 mM Tris-HC1 (pH 7.4) at room temperature. Then the sections were incubated for 60 min in 50 mM Tris-HC1 (pH 8.0 at 4°C) containing glutamate dehydrogenase (30 U/ml) , N A D ÷ (1 .1mM), 0.03% hydrazine and [3H]CGP- 39653 (10nM) . Non-specific binding was defined by 500 ~M NMDA. After the incubation, sections were washed three times for 20 s in ice-cold 50 mM Tris-HC1 buffer, dried under a stream of hot air, and apposed to tritium-sensitive film with tritium standards.

D.A. Cory-Slechta et al. /Behavioural Brain Research 85 (1997) 161-174 165

2.3.4. Analysis After at least 4 weeks of exposure, autoradiograms

were developed in Kodak D-19, fixed and dried. Films were analyzed using a video-based image analysis system (Imaging Research, Inc., St. Catherine's, Ontario, Canada). Binding was quantified in frontal cortex, striatum, molecular layer of the dentate gyrus and stratum radiatum of the CA1 region of hippocampus by person- nel blinded to Pb treatment group.

2.4. Statistical analyses

2.4.1. Pb-induced changes in MK-801 and CGP-39653 binding sites and correlations between binding sites by brain region

One-way analyses of variance, with Pb as the between- groups factor, were used to determine whether chronic postweaning Pb exposure was associated with changes in MK-801 or CGP-39653 binding (SuperANOVA 1.11, Abacus Concepts, Inc.). In addition, correlation coefficients relating: (I) numbers of MK-801 binding sites in various brain regions; (2) numbers of CGP-39653 binding sites in various brain regions; and (3) numbers of MK-801 vs. CGP-39653 binding sites in different brain regions were examined by correlation analysis with associated P values (Statview 4.5, Abacus Concepts, Inc.).

2.4.2. Correlations between accuracy levels & the RA2 and P2 components of the multiple schedule of repeated learning and performance

To determine if RA2 accuracy levels were related to P2 accuracy levels, forward stepwise regression analyses were used and the partial F-ratio for entering various sequences from the RA2 component as independent variables to predict P2 accuracy levels as the dependent variable was set at the default value of 4.00 (StatView 4.5, Abacus Concepts, Inc.). These analyses were done separately for RA components with P-like and non-P- like sequences (Statview 4.5, Abacus Concepts, Inc.).

2.4.3. Correlations between behavior and binding sites Complete data on the effects of Pb exposure on

accuracy in the multiple schedule of RA and P [16], the effects of MK-801 [13] and NMDA administration [14] on accuracy in the multiple schedule, and autoradiographic analyses of MK-801 and CGP-39653 binding sites were available for 8 rats from the 0 ppm group, 10 from the 50 ppm group, and 8 from the 250 ppm group. Unlike the drug studies where only 'learners' were used to preclude 'floor effects' from impacting the outcome [13,14], data from all animals were used in the regression and correlation analyses in this study to maximize the range of values associated with both the dependent and independent variables; the inclusion of both learners

and non-learners, moreover, was essential to assess the validity of the hypotheses being tested.

The different patterns of relationships examined were: (1) levels of accuracy during saline sessions (i.e., 'normal' accuracy levels) in RA2 with numbers of MK-801 binding sites; (2) levels of accuracy during saline sessions in RA2 with numbers of CGP-39653 binding sites; (3) levels of accuracy in P2 with numbers of MK-801 binding sites; (4) levels of accuracy in P2 with numbers of CGP-39653 binding sites; (5) levels of accuracy in RA2 following the administration of 0.30 mg/kg MK-801 (i.e., MK-801 sensitivity) with numbers of MK-801 binding sites; (6) levels of P2 accuracy following administration of 0.30 mg/kg MK-801 with numbers of MK-801 binding sites; and (7) levels of RA2 accuracy following the administration of 35 mg/kg NMDA (i.e., NMDA sensitivity) with numbers of CGP-39653 binding sites.

For these analyses, accuracy levels from the RA2 component were used in preference to RA1 accuracy levels because effects of Pb were found to be more pronounced during RA2 than RA1 [16] as were differential drug effects in control vs. Pb-exposed rats (see Table 2); to maintain consistency, P2, rather than P1, accuracy levels were utilized. For the assessment of correlations based on changes in learning accuracy following drug administration, the doses of 0.30 mg/kg MK-801 and 35 mg/kg NMDA were chosen because they were generally associated with the maximal differences in accuracy between control and Pb-treated groups. Data for 'normal' accuracy levels in the RA component were derived from the RA2 or P2 components of saline sessions included in the MK-801 and NMDA studies [13,14].

Forward stepwise regression analyses were used to examine these relationships (Statview, 4.5 Abacus Concepts, Inc.). These analyses were carried out separately for each Pb-exposure group and for P-like and non-P-like sequences for a total of 8 such analyses per exposure group. The former was required to determine whether any correlations were altered by Pb exposure; the latter permitted an additional assessment of the significance of the acquisition (learning) process itself (P-like vs. non-P-like) in these correlations. For each regression analyses, the partial F-ratio for entering binding values from specific brain regions as independent variables to predict accuracy levels as the dependent variable was set at the default value of 4.00 (StatView 4.5, Abacus Concepts, Inc.).

2.5. Blood lead concentrations

Group mean blood Pb concentrations, evaluated after approximately 60 sessions on the multiple RA and P schedule were determined using anodic stripping voltam-


metry [21]. Resulting values were 2.8___ 1.0, 25.1__+4.1 and 73.5 + 5.7 gg/dl, for the 0, 50 and 250 ppm groups, respectively, as reported previously [ 13,14,16].

3 . R e s u l t s

3.1. General characteristics o f MK-801 and CGP-39653 binding

More than 80% of MK-801 binding and more than 95% of CGP-39653 binding was specific. For MK-801 binding, the standard deviation was typically about 15-20% of the mean, and for CGP-39653 binding, the standard deviation was approximately 7-12% of the mean. Thus, for each anatomical region within a treatment group, binding of both ligands was highly reproducible.

3.2. Pb-induced changes in MK-801 and CGP-39653 binding

Pb exposure decreased MK-801 binding by approximately 30%, effects statistically significant in the dentate and CA1 region of the hippocampus (Fig. 1, top panels; F=4.34, df=2,24, P=0.025 and F=4.55, df= 2,24, P = 0.02, respectively), but evidencing similar trends in all four brain regions examined. Subsequent post-hoc comparisons based on Bonferroni/Dunnett tests showed this effect to derive primarily from the 250 ppm exposure in both regions. In contrast, Pb exposure suggestively increased CGP-39653 binding although only modestly (approximately 11%). This effect approached statistical

significance only in frontal cortex (Fig. 1, bottom panels; F=3.39, df=2,24, P=0.051). These effects again were primarily attributable to the 250 ppm exposure.

Relationships between levels of MK-801 binding in different brain regions and levels of CGP-39653 binding in different brain regions are shown in Fig. 2, top and bottom panels, respectively. In all three Pb-exposure groups, levels of MK-801 binding were highly correlated across brain regions (top panel) with maximal slope estimates noted for comparisons between frontal cortex and hippocampal regions. Corresponding slope estimates for comparisons involving striatum were much less pronounced, r 2 values for these correlations ranged from 0.54 to 0.97, 0.63 to 0.91 and 0.80 to 0.99 for the 0, 50 and 250 ppm groups, respectively.

Unlike MK-801, CGP-39653 binding levels did not systematically correlate across brain regions. Only one significant correlation was observed in the 0 ppm group (frontal cortex and dentate, r2---0.63, P=0.02), none were detected in the 50 ppm group, and three significant correlations were found in the 250 group (dentate and CA1, r 2 = 0.70, P = 0.009; dentate and striatum, r2=0.60, P=0.025; CA1 and striatum, rZ=0.82, P = 0.002).

As might be predicted from Fig. 2, levels of MK-801 and CGP-39653 binding did not correlate significantly in any brain region; r 2 values ranged from 0.01 to 0.12.

3.3. Correlations between RA2 and P2 accuracy levels

Forward stepwise regression analyses revealed that under normal conditions (0 ppm), accuracy levels in the RA2 and P2 components were highly correlated,

500' Frontal Cortex

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~ 2 . .

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400- Frontal Cortex*

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0 50 250

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800~ CAI T

200

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Pb Exposure Concentration (ppm)

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Fig. 1. MK-801 (top panels) and CGP-39653 binding (bottom panels) as a function of Pb exposure concentration brain regions. Each bar depicts a group mean_SE. Group sizes were n=8, 10 and 8 for the 0, 50 and 250 ppm indicate significant main effect of Pb (P < 0.05) in the one-way analysis of variance.

(ppm) in each of the indicated groups, respectively. Asterisks


[3 H]MK-801 1000

2 0 0 - / / ~ o

~ o i"1 FC-DENT

0 , , f , ~ , ~ a r , O FC-CA1 ~ 100 30o soo 700 900 ~00 ~ 500 700 900 100 30o 5~0 7~0

= 0 FC.STR

'~ A DENT-CAt =o r3 H]CGP-39653

u° ~ 800 t [] DENT-STR

~ O V CAI-STR

° i 200-

0 i i ' I J i i [ t i i t i

200 400 600 800 200 400 600 800 200 400 600 800

NMDA Receptor Binding (fmol/mg tissue)

Fig. 2. N M D A receptor binding (fmol/mg tissue) in each brain region plotted against all other regions. Top row shows correlations for MK-801 binding and bo t tom row shows corresponding plots for CGP-39653 binding for the indicated exposure group and the indicated correlations between various regions. Each symbol shows data for an individual rat within the exposure group.

r ega rd less o f w h e t h e r the R A s e q u e n c e was n o n - P - l i k e

( L R C ) o r P- l ike ( R L C ) (F ig . 3), w i t h the l a t t e r ac tua l ly

s e rv ing as the s t ronges t p r e d i c t o r o f P2 a c c u r a c y levels

( F = 11.72, d f = 1,6, P = 0.019) . Pa r t i a l c o r r e l a t i o n scores

r a n g e d f r o m 0.65 to 0.76. N o such r e l a t i onsh ips were

e v i d e n c e d in the 50 o r 250 p p m g r o u p . F o r these g roups ,

r 2 va lues r a n g e d o n l y f r o m 0 .01-0 .13 , n o n e o f wh ich

e v e n a p p r o a c h e d s ta t i s t ica l s ignif icance.

100

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75 80 85 90 95 100 75 80 85 90 95 60 70 80 90 100

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75 80 85 90 95 100 75 80 85 90 95 65 70 75 80 85 90 95 100

P2 / L C R P e r c e n t A c c u r a c y

Fig. 3. Percent accuracy in the RA2 component plotted against percent accuracy in the P2 component for the indicated Pb-exposure groups. Top row shows correlations when the correct RA2 component sequence was LRC (non-P-like) and the bot tom when the sequence was RLC (P-like). Each circle shows data for an individual rat within the exposure group, r 2- values are provided in the case where a statistically significant or marginally significant effect was found in the forward stepwise regression analysis.


3.4. Correlations of RA2 accuracy levels in the multiple RA and P schedule with numbers of MK-801 and CGP-39653 binding sites

In all three possible forward stepwise regression.analyses examining relationships between MK-801 binding and normal RA2 learning accuracy levels (i.e., for sequences LRC, RLC and CLR following saline administration), striatal MK-801 binding emerged as a significant positive predictor of RA2 accuracy in the control (0 ppm) group (Fig. 4, left panels). These effects reached conventional levels of statistical significance for the non- P-like sequence CLR (F= 6.69, df= 1,6, P=0.049) and were marginally significant for the other non-P-like sequence LRC (F=5.6, dr= 1,7, P=0.056) and for the P-like sequence RLC (F=4.33, d f= 1,7, P=0.086) even with a sample size of only 8 rats.

Corresponding forward stepwise regression analyses for the 50 and 250 ppm groups showed that these correlations were either negated or even reversed by Pb exposures (Fig. 4, middle and right panels). In the case of the non-P-like sequence CLR, striatal MK-801 binding was again found to be the best single predictor of RA2 accuracy levels, but resulted in negative correlations that just missed conventional significance levels in both the 50 (F---4.61, df= 1,9, P=0.06) and the 250 ppm

(F=5.80, df= 1,7, P=0.053) Pb-exposure groups, even with sample sizes of only 10 and 8 rats, respectively. Similar patterns of effects were generally noted for both the non-P-like sequence LRC and for the P-like sequence RLC.

As with MK-801 binding, positive linear correlations between CGP-39653 binding sites and RA2 accuracy levels were observed in the 0 ppm group with this pattern of correlations either negated or reversed by exposure to 50 or 250ppm Pb exposure (data not shown). Forward stepwise regression analyses examining relationships between CGP-39653 binding in the four brain regions evaluated and RA2 learning accuracy levels, however, revealed a pattern of generally non-significant correlations in the 0 ppm group that were not systematically related to brain region and exhibiting lower overall r 2 values (multiple regression analyses: LRC, r2=0.0; RLC, r2=0.51, frontal cortex, F=6.26, df=l ,6 , P=0.047; CLR, r2=0.0).

3.5. Correlations of P2 accuracy levels in the multiple RA and P schedule with numbers of MK-801 and CGP-39653 binding sites

Forward stepwise regression analyses demonstrated that P2 accuracy levels, like RA2 accuracy levels, were

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Fig. 4. Striatal MK-801 binding (fmol/mg tissue) plotted against RA2 accuracy levels (percent) in the multiple schedule of repeated learning and performance in sessions preceded by saline administration (control accuracy levels; [16]) for the indicated Pb-exposure groups, with each circle showing data for an individual rat. The top row shows correlations when the correct RA2 component sequence was LRC (non-P-like), the middle row for the P-like sequence RLC and the bottom row for the non-P-like sequence CLR. r 2 values are provided in the case where a statistically significant or marginally significant effect was indicated in the forward stepwise regression analysis.


also best predicted by striatal MK-801 binding sites (rZ = 0.73; F = 16.09. df= 1,7, P = 0.007) which were posi- tively related to P2 accuracy levels (Fig. 5). As with RA2 accuracy levels, these correlations were negated or even reversed by exposure to 50 and 250 ppm Pb. In fact, significant negative correlations were detected for the 50 ppm group, between dentate MK-801 binding and P2 accuracy (F=12.44, df=l ,7 , P=0.01); no regional MK-801 binding sites significantly predicted 250 ppm P2 accuracy levels. A similar, but non-significant, overall pattern of effects was noted in the forward stepwise regression analyses relating CGP-39653 binding to P2 accuracy (data not shown; multiple regression analyses: r2=0.41, for frontal cortex, F=4.09, df= 1,6, e=0.09) .

3.6. Correlations between the magnitude of RA2 or P2 accuracy deficits following MK-801 administration and numbers of MK-801 binding sites

Effects of acute MK-801 administration were determined in the presence of two sequences, the non-P-like sequence LRC and the P-like sequence RLC. Forward stepwise regression analyses revealed striatal MK-801 binding sites to be the best single predictor of normal (0 ppm) RA2 accuracy for the non-P like LRC (Fig. 6, top left; F=6.54, df=l ,7 , P=0.04). Although striatal MK-801 binding also exhibited the highest correlations with RA2 accuracy when the P-like sequence RLC was evaluated (Fig. 6, middle left), these effects were not of sufficient magnitude to be entered into the regression. Unlike the positive relationships noted under normal (saline administration) conditions at 0 ppm, however, no systematic or significant correlations were observed between MK-801 binding sites and RA2 accuracy levels in the presence of 0.30 mg/kg MK-801 in either the 50 or 250 ppm Pb groups.

Significant negative correlations between MK-801 binding and P2 accuracy levels under normal (0 ppm conditions) were also observed (Fig. 6 bottom left),

although in this case, the best predictor was frontal cortex (F= 11.16, df= 1,7, P=0.016), rather than stria- turn. Exposures to 50 or 250 ppm Pb acetate again negated these correlations and in neither of those cases did MK-801 binding in any brain region significantly predict P2 accuracy levels.

3. 7. Correlations between the magnitude of RA2 accuracy deficits following NMDA administration and numbers of CGP-39653 binding sites

Effects of acute NMDA administration were determined only in the presence of the non-P-like sequence CLR. Forward stepwise regression analyses revealed striatal CGP-39653 binding sites to be the best single predictor of decrements in RA2 accuracy under normal (0 ppm) conditions (left panel of Fig. 7; F=4.92, df= 1,6, P = 0.077) resulting in a marginally significant negative correlation. Again, exposures to 50 or 250 ppm Pb acetate either negated or reversed these correlations, no significant correlations between CGP-39653 binding sites and RA2 accuracy levels following administration of 35 mg/kg NMDA were detected in the stepwise regression analyses for either the 50 or 250 ppm group.

4. Discussion

One objective of the current study was to assess changes in NMDA receptor complex binding in a population of rats that had exhibited selective Pb-induced learning deficits [16] and altered sensitivity to the accuracy-impairing properties of MK-801 [4] and NMDA [ 14] on the same learning paradigm. Chronic postweaning Pb exposure decreased MK-801 binding, particularly in hippocampal regions (~30% in dentate gyrus and CA1 regions), while data were suggestive of an increase in CGP-39653 binding to the glutamate site in frontal cortex, albeit modestly (~ 11%), with similar trends in dentate and striatum. Both the current study and a

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Fig. 5. Striatal MK-801 binding (fmol/mg tissue) plotted against P2 accuracy levels (percent) in the multiple schedule of repeated learning and performance in sessions preceded by saline administration (control accuracy levels; [16]) for the indicated Pb-exposure groups, when the correct sequence for the RA2 component was LRC (non-P-like). Each circle shows data for an individual rat. The corresponding r 2 value from the forward stepwise regression analysis is presented which confirmed striatal MK-801 binding to be the best and a significant predictor of P2 accuracy.


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Fig. 6. Striatal MK-801 binding (fmol/mg tissue) plotted against RA2 (top two rows) or P2 (bot tom row) accuracy levels (percent) in the multiple schedule of repeated learning and performance in sessions preceded by administration of 0.30 mg/kg MK-801 [13] for rats in the indicated Pb-exposure groups. Each circle shows data for an individual rat. The top row shows correlations when the correct RA2 component sequence was LRC (non-P-like), the middle row for the P-like sequence RLC and the bot tom row for the P2 sequence LCR when the non-P-like sequence LRC was the correct RA component sequence, r 2 values are provided in the case where a statistically significant or marginally significant effect was indicated in the forward stepwise regression analysis,

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Fig. 7. Striatal CGP-39653 binding (fmol/mg tissue) plotted against RA2 accuracy (percent) based on the non-P-like sequence CLR in the multiple schedule of repeated learning and performance in sessions preceded by administration of 35 mg/kg N M D A [14] for rats in the indicated Pb-exposure groups. Each circle shows data for an individual rat. The corresponding r 2 value from the forward stepwise regression analysis confirmed striatal CGP-39653 binding to be the best and a significant predictor of RA2 accuracy.

previous one [8] report increases in binding to the glutamate site of the NMDA receptor after Pb treatment. Reported changes in MK-801 binding, however, have been far less consistent, with reports of decreases (this study; [24,32]) as well as increases [25,50,57], presumably due to the numerous methodological differences between these studies, including developmental period of exposure, Pb exposure levels, time at which evaluation of binding was performed, methods of receptor binding (homogenate assays vs. autoradiography)

and the use of stimulated vs. unstimulated binding conditions. Clearly, systematic examinations of such parameters (e.g., see [57] and [55]) is needed to resolve these discrepancies. While Altmann et al. [3] found no effects of postweaning Pb exposure on hippocampal long-term potentiation, which is thought to be dependent upon NMDA receptor activation, this study clearly demonstrates that the postweaning period of development in the rat is one of vulnerability to Pb-induced changes in NMDA receptor complex function.


One particularly interesting aspect of the receptor binding changes was their correspondence to changes in the behavioral effects of MK-801 and NMDA produced by this Pb exposure protocol. Specifically, in this same population of rats, chronic postweaning Pb exposures decreased: (1) MK-801 binding (this study); (2) the degree of accuracy impairment in the multiple RA and P schedule produced by MK-801 administration; and (3) sensitivity to the stimulus properties of MK-801 in a drug discrimination paradigm [ 18]. All of these attenuated behavioral effects of MK-801 could be explained by a Pb-induced decrease in the affinity of the NMDA receptor for this channel blocker, such that less MK-801 bound to the receptor and, as a consequence, less behavioral activity ensued.

In contrast, chronic postweaning Pb exposures may have slightly increased CGP-39653 binding to the glutamate site in frontal cortex in this study, and, correspond- ingly: (1) increased the accuracy-impairing effects of NMDA on the multiple schedule of repeated acquisition and learning [14]; and (2) increased sensitivity to the stimulus properties of NMDA, which acts at the glutamate binding site, in a drug discrimination paradigm [20]. These effects (the increase in binding to the glutamate site and the increased behavioral sensitivity to NMDA) might be explained on the basis of an increase in the affinity of the receptor for glutamate site ligands. While it might seem counter-intuitive that NMDA would decrease, rather than increase accuracy in a learning paradigm, the literature clearly indicates that NMDA does not uniformly enhance behavioral performance [33] nor do NMDA receptor complex antagonists reliably impair such behaviors [17,40].

A primary objective of the current study was to begin to explore the relationships between the NMDA receptor complex binding changes and behavioral consequences resulting from Pb exposure, to begin to determine the extent to which NMDA receptor complex changes might contribute to Pb-induced learning impairments. The current study utilized an indirect approach that explored two specific hypotheses. These included the contentions that learning, but not performance, accuracy levels should be correlated with NMDA receptor binding, and that Pb exposure should alter such correlations. Similarly, MK-801- or NMDA-induced disruption of learning, but not performance accuracy, should be inversely related to NMDA receptor complex binding and this should be selectively altered by Pb exposure.

In that context, RA2 accuracy levels correlated posi- tively with both MK-801 and CGP-39653 binding in control rats, a relationship suggesting that the higher the levels of NMDA receptor binding, the better the learning accuracy. The negative correlations between accuracy levels and binding observed following drug administration suggest that the greater the binding of a ligand to the channel or glutamate site, the greater the effects of MK-801 or CGP-39653, respectively, on accu-

racy levels. These relationships were observed even with a relatively small population of animals, and were more prominent in the case of MK-801 binding.

However, the fact that the same correlations were also observed for P2 accuracy levels would seem to argue against any specific relationship between Pb-induced changes in NMDA receptor complex bio- chemistry and learning impairments. If NMDA receptor complex binding is related both to learning and to performance accuracy levels, then disruption of NMDA receptor complex function would not easily account for a selective effect of Pb exposure on learning ([16] and Table 1). And yet, as was also noted in this study, levels of RA2 accuracy were actually highly predictive of P2 accuracy levels under normal conditions (0 ppm), even though P2 values encompassed a very small range. This predictive relationship between levels of RA2 and P2 accuracy may reflect another type of learning, in that the higher the accuracy levels in each component, the more rapid the behavioral transition between the RA and P components of the multiple schedule. If so, the relationships between P2 accuracy and binding parameters would not necessarily be incompatible with the premise of this study. An incompatibility would be indicated had both RA2 and P2 accuracy levels correlated with NMDA receptor complex binding, but not with each other.

The second component of the hypotheses proposed by this study was also supported by its outcomes: Pb exposure altered virtually all patterns of correlations between behavior and binding as well as the correspondence between levels of RA2 and P2 accuracy seen under normal (0 ppm) conditions, as summarized in Table 3. As it shows, in all but two instances, chronic postweaning Pb exposure eliminated relationships between levels of RA2 and P2 accuracy and between accuracy levels and binding. For the two exceptions, Pb reversed the direction of the correlations.

What must be considered in evaluating these findings, though, is the limited scope of this assessment. Would it be the case, for example, that similar correlations would have been observed if changes in levels of dopaminergic or cholinergic binding had also been examined? Arguing against the former was the fact that dopaminergic agents, unlike glutamatergic agents, failed to differentially alter accuracy levels of control and Pb-treated rats on the multiple schedule of repeated acquisition and performance [ 13-15]. Nevertheless, it seems very likely that other systems might contribute or even be more strongly predictive of accuracy levels. Caution in interpretation must always be practiced when restricted examinations are undertaken; the correlations described here should not be taken to imply specificity. Furthermore, these effects of Pb may not necessarily be mechanistic; i.e., they may be the evidence, rather than the cause, of the observed learning impairments. It is conceivable, for example, that the preferential changes


Table 3 Summary of Pb-related changes in correlations between learning and performance accuracy and between learning accuracy or sensitivity to learning impairments caused by MK-801 and NMDA administration and levels of NMDA receptor complex binding

Correlation Component/sequence Pb exposure concentration (ppm) ~

0 50 250

RA2 by P2 accuracy

RA2 accuracy by MK-801 binding

P2 Accuracy by MK-801 binding

Accuracy after 0.30 mg/kg MK-801 by MK-801 binding

Accuracy after 35 mg/kg NMDA by CGP-39653 binding

RA2/LRC Positive/LRC a None None

LRC Positive/striatum b None None RLC Positive/striatum b None None CLR Positive/striatum ~ Negative/striatum a Negative/striatum b

LCR Positive/striatum a Negative/dentate a None

RA2/LRC Negative/striatum a None None RA2/RLC None None None P2/LCR Negative/frontal cortex ~ None None

R A 2 / C L R Negative/striatum b None None

aP<0.05. bP<0.10. Clndicates nature of observed correlation and brain region associated with significant effect following forward stepwise regression analyses.

in dopaminergic function in mesolimbic systems produced by Pb [46] alter dopamine/glutamate balance in this system and thus underlie the observed effects. Likewise, changes in GABAergic or even cholinergic interneurons in these systems would also ultimately affect glutamatergic function.

Of the four brain regions examined in this study, MK-801 binding in striatum was found to be the best predictor of accuracy levels in 6 of the 8 stepwise regression analyses carried out in control animals (one indicated frontal cortex and the other failed to find evidence of a significant correlation between behavior and binding). These findings emphasize the potential importance of dopamine-glutamate system interactions in Pb effects, interactions which have been reported at virtually all levels of nervous system function [51]. Pb-exposure effects on DA systems are well established, both at the biochemical and behavioral level [19]. Whether Pb effects on dopaminergic or glutamatergic systems are mediated directly or indirectly via interactions in vivo remains to be addressed experimentally.

The molecular basis for the differential pattern of correlations relating N M D A receptor binding to learning accuracy, and to MK-801 and N M D A sensitivity in response to Pb exposure cannot be specifically ascer- tained from this study. One possibility is that Pb exposure alters the subunit composition of the N M D A receptor complex and thus, the efficacy of ligand binding. Cloning studies have revealed the presence of multiple N M D A receptor subunits, each with distinct pharmacological and physiological properties. NR1, required for receptor activation and having an extremely broad

pattern of expression [42], has at least eight splice variants [23,27,52]. There are also at least four NR2 subunits termed NR2A-D with different anatomical distributions [28,29,34,39,41]. It is the NR2 subunits, which do not form functional homomeric receptors in expression systems, but require the presence of NR1 that confer distinct properties on the expressed receptors, such as differential agonist and antagonist selectivity, variable sensitivity to glycine and Mg 2+ , and differing channel kinetics [29, 39,41]. Interestingly, the NR1 subunits have been shown to have different sensitivity for Zn z+ [23], and some [1,26], though not all studies [53,54], have suggested that Pb may act at the Zn 2+ site of the N M D A receptor complex.

Furthermore, there is abundant evidence showing that N M D A receptors of various subunit compositions dis- play marked differences in their affinities for both MK-801 and CGP-39653 [36] and that differences in subunit composition and pharmacological selectivity translate into regional variations in the binding properties of N M D A receptor ligands [7,9,47]. Moreover, it appears that the extent of cognitive impairment caused by N M D A receptor blockade may depend, in part, on the subtypes of receptor that are blocked [47]. Thus, it is tempting to hypothesize that the ~oordinate changes in ligand binding, behavioral sensitivity to N M D A receptor ligands, and learning and behavior seen in the present study reflect Pb-induced alterations in N M D A receptor composition. In support of this possibility, are reports of two MK-801 binding sites in striatum, raising the possibility that distinct N M D A receptor subunits contribute differentially to Pb-related effects [7], and of

D.A. Cory-Slechta et al. / Behavioural Brain Research 85 (1997) 161 174 173

in vitro demonstrations of alterations in N M D A receptor subunit composition following Pb treatment [38, 56 ]. This issue can be addressed in future studies by using subunit-specific molecular reagents in combination with detailed behavioral studies.

Finally, the issue of levels of Pb exposure associated with these effects should be addressed. The fact that group changes in receptor binding per se (Fig. 1) were seen at 250 ppm where blood Pbs averaged 73.5 lag/dl, suggesting that these glutamatergic system changes would only be problematic for occupational Pb-exposure scenarios. However, past studies using this exposure protocol have documented behavioral changes at PbBs as low as 15 ~tg/dl, i.e., just above the current level of concern. Moreover, even the 50 ppm exposure in this study negated the correlations between behavior and binding. The broader and more pernicious implication of these latter findings is that lower levels of Pb exposure may shift glutamatergic function not sufficiently to result in overt behavioral impairment, but enough to serve as a predisposing factor to other conditions with which it might act additively or even synergistically: Pb is by no means the only harmful agent or condition to which populations are routinely and involuntarily exposed.

Acknowledgement

Supported in part by Grants ES05903 and ES01247 (D.A.C.-S.) and a Mallinckrodt Scholar Award (J.T.G.).

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