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Vol. 286, Issue 2, 794-805, August 1998
Department of Neurobiology and Anatomy, University of Rochester Medical School, Rochester, New York
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Abstract |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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To examine the assertion that changes in nucleus accumbens (NAC) dopamine (DA) activity serve as a mechanism of lead (Pb)-induced disruption of fixed interval (FI) schedule-controlled behavior, the effects of intra-NAC administration of the irreversible DA antagonist EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihyroquinoline) and of dopamine itself on FI performance were compared in rats that had been chronically exposed to 0, 50 or 500 ppm Pb acetate in drinking water from weaning. Pb exposure per se (500 ppm), as in past studies, increased FI response rates, primarily by shortening interresponse times. Although DA, which produced rate-dependent effects, increased FI rates at low doses in the 0 and 50 ppm groups, it did so by decreasing postreinforcement pause times. All DA doses decreased rates in the 500 ppm group. In contrast, the DA antagonist EEDQ suppressed FI response rates, effects that were not strongly rate dependent, by increasing both postreinforcement pause values and mean interresponse times. Pb exposure (500 ppm) delayed the recovery of response rates to control levels at the highest EEDQ dose, raising the possibility of a delay in receptor production rate. Collectively, these data suggest that NAC DA activity may be an important modulator of FI response rates. Enhanced NAC DA activity may contribute to Pb-associated increases in FI rates and may underlie the differential response of control and 500 ppm Pb-treated groups to intra-NAC DA administration. The different processes by which DA and Pb increase FI rates, however, suggests that additional mechanisms are operative in the case of Pb.
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Introduction |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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FI
schedules reinforce behavior on the basis of time. Specifically,
reinforcement delivery follows the first occurrence of a designated
response after a fixed interval of time has elapsed since the previous
reinforcement delivery; responses occurring during the interval itself
have no programmed consequence. These reinforcement contingencies
typically generate a highly characteristic "scalloped" pattern of
responding marked by periods of little or no responding early in the
interval, which is followed by a gradually increasing rate of
responding as the opportunity for reinforcement delivery approaches
(Ferster and Skinner, 1957
). The wide range of species across which
this response pattern is observed attests to the generality of the
underlying behavioral processes (Kelleher and Morse, 1968). The
characteristic scalloped FI performance has sometimes been interpreted
to involve a temporal discrimination, with the very low rates of
responding early in the interval reflecting the absence of
reinforcement availability and the high rates at the end of the
interval ensuring that reinforcement delivery is not delayed.
Different lines of evidence point to an involvement of
catecholaminergic systems in FI performance. Systemic injections of 
-methyl-paratyrosine or NAC administration of 6-OHDA, for example, decrease response rates on the FI schedule. Preferential involvement of
mesolimbic DAergic systems in FI performance was recently indicated by
our finding that microinjections of the nonspecific irreversible DA
antagonist EEDQ into NAC significantly depressed rates of responding, whereas FI performance was virtually unaffected by EEDQ injection into
dorsal STR (Cory-Slechta et al., 1997b). Decreased FI
response rates in the NAC group recovered over the following two or
four experimental sessions, consistent with the turnover of DA receptor proteins.
Low level Pb exposures (i.e., those relevant to current
human environmental conditions) have repeatedly been demonstrated to
increase rates of responding on FI schedules of reinforcement in
experimental animal studies. These increased response rates have been
noted across a wide range of developmental periods of Pb exposure,
including prenatal, postnatal, postweaning and even adult exposures. In
addition, these effects have been observed across several species,
including mouse, rat, pigeon, sheep and nonhuman primates
(Cory-Slechta, 1994). Analyses of the various components of FI
performance reveal that Pb sometimes effects the pause times that
characterize behavior early in the interval (i.e., the PRP)
and generates higher rates of responding later in the interval as the
time of reinforcement availability approaches (Cory-Slechta, 1994).
Collective evidence to date suggests that the increases in FI response
rates produced by chronic postweaning Pb exposures may derive from
increases in mesolimbic (NAC) DA activity. The fact that DA antagonism
(e.g., -methyl paratyrosine, 6-OHDA and EEDQ;
Cory-Slechta et al., 1997b; Robbins et al., 1983;
Schoenfeld and Seiden, 1969) apparently decreases FI response rates,
suggests the converse hypothesis that increases in DA availability
(i.e., DA agonism) might increase FI response rates, thus
mimicking Pb exposure. Furthermore, the fact that alterations in FI
response rate produced by regional EEDQ administration in our recent
study (Cory-Slechta et al., 1997b) were observed selectively
in NAC suggests NAC DA systems/regions as a primary site mediating FI performance.
Two studies provide evidence consistent with the possibility that
chronic postweaning Pb exposure does indeed preferentially enhance NAC
DA activity. In an autoradiographic time course study (Pokora et
al., 1996), chronic postweaning Pb exposure selectively decreased
DA (D1, D2 and DA transporter) binding in NAC, while having no effects
on STR DA binding even after 12 months of exposure. These findings are
consistent with excess mesolimbic DA activity in response to low-level
Pb exposure with consequent receptor downregulation. In addition, a
recently completed in vivo electrochemistry time course
study (Zuch et al., 1998) revealed increases in evoked DA
overflow in NAC at both 11 weeks and 11 months of chronic postweaning Pb exposure, whereas no changes in evoked DA overflow were found in STR
at either time point, even though clearance times were equivalently
increased in both regions.
The study described has further examined the hypothesis of elevated NAC DA as a mechanism of Pb-induced changes in FI performance in two ways. First, it postulated that if blockade of NAC DA receptors decreased FI response rates, then the converse should be true (i.e., NAC DA agonists should increase FI response rates, thereby mimicking the effects of chronic postweaning Pb exposure on FI performance). Second, it hypothesized that if NAC DA alterations serve as the basis of Pb-induced changes in FI performance, then dose-effect curves characterizing NAC DA or EEDQ effects on FI performance should differ in normal compared with Pb-treated rats because base-line DA function would already be altered in response to Pb exposure. Third, the behavioral mechanisms by which DA agonists and Pb alter FI performance might be expected to be similar. These hypotheses were evaluated by comparing the effects of microinjections of DA and EEDQ into NAC on FI performance in control vs. Pb-treated rats.
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Methods |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Animals and Pb exposure. Male Long-Evans rats (n = 41) obtained at 21 days of age (Charles Rivers Breeding Laboratories, Indianapolis, IN) were divided into three groups of 13, 14 and 14 rats of approximately equal average body weight and exposed to 0, 50 or 500 ppm Pb acetate, respectively, drinking solutions from the day of arrival through the duration of the experiment. From 21 to 50 days of age, all rats had unrestricted access to a semipurified diet (Purina Test Diet). Standard rodent diets maintain excessively high levels of the essential metals Ca, Fe, and Zn that decrease oral absorption of Pb. The semipurified diet contains required rather than excess levels of these essential metals, permitting the use of lower Pb acetate drinking water concentrations to achieve target blood Pb concentrations. Beginning at ~50 days of age, rats were provided with a sufficient quantity of semipurified diet to produce a 3- to 5-g b.wt. gain per day until reaching a maximum weight of 300 g, the value at which they were stabilized for the remainder of the experiment. Rats were housed in a colony room maintained at 22°C on a 12-hr light/dark cycle. All procedures were carried out in accord with National Institutes of Health and University of Rochester Animal Use and Care Committee Guidelines.
Apparatus.
Behavioral sessions were conducted in operant
chambers (Coulbourn Instruments, Lehigh Valley, PA; model E10-10) each
of which was housed in a sound-attenuating enclosure ventilated by a
fan as described previously (Cory-Slechta et al., 1996).
These chambers contained three response levers configured horizontally
on the front panel; only the right lever was active in these
experiments.
Behavioral procedures.
Lever press responding was shaped in
automated overnight sessions using procedures described previously
(Cory-Slechta et al., 1985). After the completion of
shaping, an FI 1-min schedule of reinforcement was imposed. On this
schedule, a 45-mg food pellet delivery followed the first lever press
response occurring at least 1 min after the preceding food delivery,
with responses occurring during the 1-min interval itself having no
programmed consequences. Sessions ended after the completion of the
1-min interval that was in progress 30 min after the session began or after a total of 32 min, whichever occurred first. Sessions were conducted 5 to 7 days per week between 9:00 a.m. and 1:00 p.m. A total
of at least 50 sessions were carried out before surgery was undertaken.
FI performance measures. The following performance measures were computed from each session for every animal: (1) overall response rate, or total number of responses divided by total session time; (2) mean running rate (i.e., the rate of responding calculated with the PRP) time (time from food delivery to the first response in the next interval) subtracted out; (3) mean postreinforcement pause time (i.e., the time to the occurrence of the first response in an interval) and (4) mean IRT (i.e., the average of all IRT values).
Surgery.
Rats were given 0.1 ml of 10 mg/ml atropine and 10 min later anesthetized with 60 mg/kg sodium pentobarbital i.p. and
placed in a Kopf stereotaxic frame with the incisor bar set 5 mm above the interaural line. Two stainless steel guide cannula were implanted (22 gauge, 15 mm long) in NAC aimed at coordinates of AP +3.4, ML +1.6
and V 
5.5 from the surface of the skull (Pellegrino et al., 1979). Cannulae were held in place by cranioplasty and
anchored to the skull with three stainless steel skull screws.
Removable obturators were placed in the guide cannulae to retain
patency of the guides.
Schedule of the assessment of DA effects. Rats were allowed to recover for 1 week after surgery, after which FI performance assessment recommenced. Once behavior restabilized, microinjections began. All rats received their first microinjection as a DMSO vehicle injection 30 to 60 min after a behavioral test session (postsession injection). This was done to minimize as much as possible any conditioning effects associated with microinjection procedures. This was followed at least two or three sessions later by a DMSO vehicle injection administered 30 min before the initiation of the FI session (presession DMSO injection). Repeated presession DMSO injections were carried out in the event that a significant change in FI performance occurred. Repeated DMSO injections were required by 5 rats from the 0 ppm group, 3 rats from the 50 ppm group and 1 rat in the 500 ppm group.
Subsequently, microinjections of EEDQ were initiated after at least two or three additional control (noninjection control) sessions, with EEDQ doses administered in a semirandom order. Each such microinjection of EEDQ was followed 20 to 30 min later by the first subsequent behavioral test session (test session 1), and FI performance was assessed for at least three additional sessions at 24-hr intervals (test sessions two to four). At least two or three additional control sessions occurred before any further microinjections of EEDQ. Total bilateral doses of EEDQ examined, in a semirandom order, were 90, 120, 150 and 200 µg. Because of occasional microinjection problems, not all rats necessarily received every dose of EEDQ. Sample sizes for each drug or dose are indicated in the figure legends. Approximately 10 days after the completion of the EEDQ dose-effect curves, assessment of the effects of DA (20, 40 and 80 µg) was initiated, with doses administered in a semirandom order. DA was microinjected immediately before the FI session, and at least two or three noninjection control sessions intervened between each DA dose tested. Not all rats were included in both the EEDQ and DA dose-effect curve determinations. Sample sizes for the DA dose-effect determinations are indicated in the figure legends. In combination with the previous EEDQ microinjections, rats generally received a total of 10 to 12 microinjections (1 rat received 13).Microinjection procedures. For microinjections, rats were placed in a Plexiglas cage for the duration of the procedure. Injection cannulae (17 mm long, 28 gauge) connected via PE-10 tubing to 10-µl Hamilton syringes were inserted through the guide cannulae. These cannulae protruded 2.0 mm below the guide cannulae. Drugs were then infused bilaterally via a syringe pump at a rate of 0.25 µl over a 2-min period with the injection cannulae remaining in place for at least 2 min after the injection. Injection volumes were calculated so as not to exceed 0.25 µl/side. After the removal of the injection cannulae, obturators were placed back in the guide cannulae.
Drugs.
EEDQ and DA were obtained from Research Biochemicals.
EEDQ was dissolved in DMSO vehicle (Giorgi and Biggio, 1990a) and
prepared on the day of injection. DA was dissolved in saline and
prepared on the day of injection.
Blood Pb analysis.
After ~3 months of Pb exposure and at
the completion of the experiment, before perfusion, blood was collected
from a randomly chosen subset of rats within each Pb exposure group
from a tail nick for the measurement of blood Pb (PbB) levels by anodic
stripping voltammetry (model 3010A Trace Metals Analyzer; Environmental Science Associates, Bedford, MA) according to the method of Morrell and
Giridhar (1976).
Data and statistical analyses. To determine the extent to which Pb exposure per se altered FI performance before surgery, median FI overall response rate values were computed for each rat across blocks of five sessions for the 50 sessions before initiation of surgery (base-line). Statistical analyses were then carried out using repeated measures analysis of variance (RMANOVA) with Pb as a between-group variable and session blocks as a within-group variable.
Assessment of the effects of DMSO vehicle were based on percent of control, with DMSO effects calculated as a percent of the two control noninjection sessions preceding it. DMSO vehicle effects were then analyzed using RMANOVA with Pb as a between-groups factor and session (control session 1, control session 2, DMSO vehicle session) as within-group factors. These analyses were carried out separately for each FI performance measure. FI performance during EEDQ and DA sessions was also calculated as a percent of the mean value of the two control sessions preceding the microinjection session, and pre-EEDQ and pre-DA control values and variability (labeled "Pre" in corresponding figures) were defined by calculating the second control session value as a percentage of the first. DMSO vehicle sessions were not used as the basis for percent of control determinations for EEDQ or DA because base-line FI performance could show some drift over time and DMSO sessions were separated to a greater extent in time from DA than from EEDQ microinjection sessions. Because not all rats received all doses of EEDQ (sample sizes for each dose are indicated in the appropriate figure legend), data for EEDQ effects were analyzed separately for each dose by RMANOVA with Pb as a between-group variable and session (pre-EEDQ control and sessions one to four after injection) as a within-group variable. These analyses were carried out separately for each FI performance measure. For analyses of DA, "Pre" values were averaged across the various doses of DA. DA dose-effect curves for overall rate were then analyzed using RMANOVA with Pb as a between-group variable and DA dose ("Pre," 20, 40 and 80 µg) as a within-group variable. Because significant differences in numbers of rats exhibiting rate increases vs. decreases in response to DA occurred within each Pb group, assessment of other FI performance measures were restricted to visual observation of data based on the 0 ppm group for rate increasing effects of DA and of the 500 ppm group for rate-decreasing effects of DA, because these were the primary groups contributing to the differences in response rates. For similar reasons, no statistical assessment of Pb modulation of such effects was carried out. Chi-square tests were used to compare the proportion of rats within each Pb group exhibiting rate increases vs. decreases in response to DA. Simple linear regression analyses were used to determine the extent to which baseline response rates influenced the response to DA and EEDQ. Blood Pb levels were analyzed using two-factor ANOVA with both Pb exposure concentration and time as between-group variables (because the same rats were not tested at the two time points). For all statistical analyses, values of P
.05 were considered
statistically significant; trends (P > .05 and < .10) in
the data are also noted where relevant. In cases of significant main effects or interactions, the nature of the effects were explored using
least-squares mean analyses or one-way ANOVAs as appropriate.
Histology. At the conclusion of the experiments, brains were collected after perfusion with physiologically buffered saline followed by paraformaldehyde/glutaraldehyde solution prepared with 0.2 M PO4. Brains were put into paraformaldehyde/glutaraldehyde solution for 1 hr and then immersed in 30% sucrose until they sank. They were subsequently blocked and sectioned at 40 µm, and every alternate section was mounted onto glass slides and stained with cresyl violet for the determination of accuracy of cannulae placements. Based on these analyses, data for one rat from the 500 ppm group was eliminated due to misplaced cannulae and one rat from the 0 ppm group was eliminated due to an infection on one side of the brain, leaving final total sample sizes of 12, 14 and 13 for the 0, 50 and 500 ppm groups, respectively.
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Results |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Blood Lead Analysis
Blood Pb (PbB) values determined after ~3 months of exposure
averaged 2.1 ± 1.8 µg/dl for the 0 ppm group, 7.2 ± 2.7 µg/dl for the 50 ppm group and 49.1 ± 9.0 µg/dl for the 500 ppm group (n = 7, 7 and 7, respectively). Corresponding
values for the PbB determinations made at the end of the experiment
were 0.5 ± 0.3 µg/dl (n = 8), 9.6 ± 1.0 µg/dl (n = 7) and 49.4 ± 5.2 µg/dl
(n = 8), respectively. Statistical analyses confirmed a
main effect of Pb (F = 81.3, df = 2,59, P = .0001) and no difference in relation to time point. Increases in PbB
values were concentration related with post-hoc
least-squares mean tests indicating that all groups differed
significantly from each other (all Ps < .05). These PbBs are
relatively low compared with those produced by these same exposures in
our past studies, in which values of 15 to 20 µg/dl have been
characteristic of 50 ppm exposures and 80 to 100 µg/dl of 500 ppm
exposures (Cory-Slechta et al., 1985). The lower PbBs obtained here appear to be peculiar to the source/strain of rats obtained for this study from the Indiana facility because drinking water exposure concentrations were determined to be within 1% of the
stated level, and Long-Evans rats obtained from the Maryland facility
housed in the same vivarium rooms and concurrently receiving these same
drinking solutions and diet and consuming the same quantities of water
during this same time period averaged 15.5 to 16 µg/dl at 50 ppm and
25 to 44 µg/dl at an exposure concentration of 150 ppm.
Lead-Induced Changes in FI Performance
As in previous studies, Pb exposure increased overall response rates on the FI schedule (fig. 1), with rate increases emerging after ~20 to 25 sessions (session blocks four and five). Overall response rates of controls reached asymptotic values of ~30 responses per minute, whereas corresponding values for the 500 ppm group were ~40 responses per minute (main effect of Pb, F = 3,58, df = 2.44, P = .036). As might be predicted based on the lower PbBs achieved with these exposures than in past studies, only the 500 ppm exposure group exhibited rate increases, with elevations compared with both controls (P = 0.03) and the 50 ppm group (P = 0.022); the 50 ppm group failed to exhibit any significant increases in FI response rates relative to the 0 ppm group.
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Effects of EEDQ Microinjections into NAC on FI Performance
DMSO vehicle microinjections. As expected, effects of DMSO vehicle microinjections on FI performance were negligible because such injections were repeated if they initially disrupted FI performance (data not shown). DMSO did not statistically affect any of the four measures of FI performance (overall rate, PRP time, mean IRT or run rate), nor did Pb exposure modulate DMSO vehicle effects in any way.
Effects of EEDQ: Rate-dependency analysis. Given the higher overall response rate values of the 500 ppm group on the FI schedule (fig. 1) and the observation of dichotomous response changes in some rats after EEDQ microinjections (see below), rate-dependency analyses using simple linear regression techniques were carried out to determine whether base-line FI response rates might influence the effects of EEDQ microinjection. The results of those analyses are presented in table 1. Computed across all rats, significant correlations were obtained at all EEDQ doses when the maximal change over the four sessions after EEDQ microinjection was used for the analysis, even though corresponding r2 values were extremely low, ranging only from .10 to .18. When data from the few rats exhibiting substantial increases rather than decreases in rates after EEDQ were excluded from these analyses, the correlations were no longer significant for any dose or condition, and the corresponding r2 values for maximal change then ranged from only .001 to .095. Consequently, data for EEDQ effects were analyzed in the absence of those few rats exhibiting the substantial increases in rates given that base-line response rates were generally not a major contributor to the effects of EEDQ on FI performance in the remaining members of these groups.
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Time course and dose-effect for EEDQ. As already mentioned, two distinct responses to EEDQ emerged. A small number of rats within each Pb exposure group exhibited extreme increases in response rates at some doses of EEDQ, and these effects are presented in table 2. These data came from a total of 4 of 12 rats in the 0 ppm group, 3 of 14 rats in the 50 ppm group and 2 of 13 rats in the 500 ppm group, with not all rats displaying this pattern at all EEDQ doses. The increases obtained ranged up to levels as high as 443% of control values and were not analyzed statistically because of the small numbers of animals involved. As also shown in table 2, base-line overall response rate values of rats exhibiting this pattern of increased response rates after EEDQ were generally considerably lower than those of the remainder of rats within each Pb exposure group (i.e., those demonstrating rate decreases in response to EEDQ administration).
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), was characterized by an initial suppression of response rate followed by a return to control levels and, at some
doses, a subsequent rebound increase in FI rates above control response
rate levels. Effects of EEDQ on FI overall response rates over four
sessions after administration for the groups exemplifying the primary
EEDQ response are shown in figure 2. All
four doses of EEDQ resulted in significant decrements in overall
response rates during the first test session after its administration
that ranged from ~21% to 56% of corresponding control values (main effect of test session: 90 µg, F = 9.67, df = 4,20, P = .0001; 120 µg, F = 16.68, df = 4,128, P = .0001; 150 µg, F = 14.89, df = 4,124, P = .0001; 200 µg, F = 22.04, df = 4,40, P = .0001). Least-squares mean analyses showed these effects
to derive from the decline in rates relative to pre-EEDQ session mean
values during the first session post-EEDQ administration (all Ps < .05). For doses of 90 and 200 µg, rates remained below control
levels during the second post-EEDQ administration session (P = .03 and .017, respectively). Response rates had returned to control levels by the third post-EEDQ session at all doses of EEDQ. At the 150-µg dose, there was a suggestion of increases in rates above control levels
four sessions after EEDQ administration (P = .08), with this
effect achieving conventional levels of significance at 200 µg
(P = .01) where response rates exceeded pre-EEDQ control levels by
~25% in both the 0 and the 50 ppm group.
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Pb-induced modulation of EEDQ effects. Pb exposure, in particular, the 500 ppm concentration, appeared to potentiate the effects of EEDQ on FI performance (fig. 2) at least at the 200-µg dose (main effect of Pb, F = 4.56, df = 2,35, P = .017). This effect was not manifest in the initial magnitude of EEDQ-induced suppression during session 1 but instead as a delay in recovery of response rates to control levels, as shown by the decreased rates of the 500 ppm group relative to the 0 ppm group during sessions two to four post-EEDQ (one-way ANOVA main effect of Pb for session 2: P = .038; session 3, P = .022; and session 4, P = .076).
EEDQ effects on FI performance measures and modulation by Pb. As assessed based on results obtained at the 200-µg dose, EEDQ decreased overall response rates by increasing PRP times (F = 5.13, df = 4,84, P = .0009) and mean IRT values (F = 7.14, df = 4,84, P = .0001), consequently decreasing running rates (F = 10.86, df = 4,84, P = .0001), effects that were most pronounced during the first session after EEDQ administration and evidenced a subsequent recovery (data not shown). PRP values of control rats increased by ~30% and mean IRT values by 80% above control levels during session 1, whereas corresponding run rate values had decreased by >40%. The apparent overshoot of overall response rates in the 0 ppm group at session 4 after 200 µg EEDQ (fig. 2) appeared to derive primarily from a decline in mean IRT values even below pre-EEDQ levels by 10% to 15% (RMANOVA, 0 ppm comparing pre-EEDQ with session 4, F = 13.5, df = 11, P = .004).
Pb exposure marginally augmented the increase in postreinforcement pause time at 200 µg EEDQ (F = 3.52, df = 1,21, P = .07), whereas a suggestion of Pb-induced enhancements of changes in both mean IRT values and running rate after EEDQ did not attain conventional levels of statistical significance.Effects of DA Microinjected into NAC
Rate-dependency analysis. Given the higher overall response rate values of the 500 ppm group on the FI schedule as shown in figure 1, as well as the significant differences in the proportion of rats within each Pb group exhibiting rate increases vs. decreases in response to DA (see below), rate-dependency assessments based on simple linear regression analyses were carried out to determine whether base-line FI response rates influenced the effects of DA. Scatterplots depicting the percent change in FI overall response rate after DA microinjection as a function of base-line FI response rate are shown in figure 3. Significant negative linear relationships were found for each DA dose, indicating that low base-line response rates tended to be increased by DA administration while higher response rates tended to be increased to a lesser extent or to be decreased by DA (ANOVA based on simple linear regression analyses: F = 17.58, df = 1,35, P = .0002; F = 20.34, df = 1,35, P < .0001 and F = 13.0, df = 1,35, P = .001 for the 20-, 40- and 80-µg doses, respectively, with corresponding r2 values ranging from .28 to .37).
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Dose-effect curves for DA and modulation by Pb exposure. As might be expected based on the outcome of the rate-dependency analysis shown in figure 3 and the higher response rates of the 500 ppm group relative to the 0 ppm group shown in figure 1, changes in FI overall response rates as a function of the dose of DA depended entirely on Pb exposure concentration (main effect of Pb: F = 5.26, df = 2,33, P = .01), as shown in figure 4. Specifically, DA increased FI response rates on average by ~25% to 35% above control levels in the 0 and 50 ppm groups, particularly at the doses of 20 and 40 µg, whereas overall response rates were depressed by DA in the 500 ppm group across all doses. Not surprisingly, when the data were collapsed across Pb exposure groups, there were only marginal trends indicative of a main effect of DA itself in the overall statistical analysis (F = 2.2.4, df = 3,99, P = .089).
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DA effects on components of FI performance and modulation by Pb. Examination of data from the 0 ppm group only (i.e., the group with a high frequency of rats exhibiting rate increases in response to DA and the one that should reflect "normal" performance) indicated that DA acted to increase FI overall response rates primarily by decreasing PRP times, with decreases averaging between 6% and 25% of control. Mean IRT values were actually increased after DA administration to 30% to 40% above control. For assessment of the basis of rate decreasing effects of DA, only the 500 ppm group had a sufficient sample size to provide information. Inspection of data from this group showed virtually no changes in PRP values but notable increases in mean IRT values (160-270%) with concomitant decreases in run rates by 15% to 55% (data not shown).
Histological assessments. Placements of guide cannulae tips are shown in figure 6 for rats included in the experiment. As can be seen, placements for rats included in the data analyses were confined to the NAC/ventral striatal region and primarily to core regions of NAC.
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Discussion |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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We sought to explore three hypotheses. The first stipulated that
because intra-NAC administration of the DA antagonist EEDQ decreased
overall response rates on the FI schedule (Cory-Slechta et
al., 1997b), the converse would likewise be true (i.e.,
intra-NAC DA agonists should increase FI response rates and thus
potentially mimic Pb exposure effects). The second hypothesis
stipulated that if NAC DA alterations serve as the basis of chronic
low-level postweaning Pb-induced increases in FI response rates, then
dose-effect curves characterizing the effects of intra-NAC DA-based
compounds such as dopamine and EEDQ on FI performance should differ in
control and Pb-treated rats, because base-line DA function would
already differ in response to Pb treatment. Third, the behavioral
mechanisms underlying alterations in overall response rates on the FI
schedule produced by excess DA might be similar to those resulting from Pb exposure. Our findings show that DA agonism can indeed increase FI
response rates in normal rats. Furthermore, the dose-effect curves
relating intra-NAC DA and EEDQ to FI performance exhibit clear
Pb-related differences, differences that were more restricted in the
case of EEDQ. Third, the behavioral mechanisms by which intra-NAC DA
and Pb induce alterations in FI performance show both similarities and
differences, suggesting that additional mechanisms modulate the
specific behavioral processes underlying FI rate increases produced by
Pb exposure.
Intra-NAC DA agonism should increase FI response rates and mimic Pb exposure effects. Intra-NAC DA administration indeed increased FI response rates, as was evidenced in the 0 ppm group, the group that logically serves as the basis from which to examine this particular contention. DA administration in the 0 ppm group exhibiting rate increases (10 of 12 rats) revealed an inverse U-shaped dose-effect function characterizing changes in FI response rates (fig. 5, left), with the 20-µg dose increasing FI rates, 40 µg marginally increasing rates and 80 µg producing rates indistinguishable from control values. Thus, under normal condition, excess DA activity appears to increase FI response rates, while further increases in DA ultimately suppress FI rates, as schematized in figure 7A.
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; Robbins et al., 1983), even when considered across
experimental subjects (Urbain et al., 1978).
As we previously reported (Cory-Slechta et al., 1997b), the
nonspecific DA antagonist EEDQ suppressed FI overall response rates
during the first session after its administration into NAC, with
behavioral recovery occurring over the next two or three sessions.
Evidence for rate-dependent effects of EEDQ were somewhat ambiguous, at
least when considered across all subjects, although dichotomous effects
in different subjects of both DA and EEDQ on FI response rates were
observed, and are certainly consistent with numerous other reports of
individual differences in behaviors mediated by dopamine systems,
particularly by agonists, including drug self-administration (Piazza
et al., 1989) feeding effects of amphetamine (Sills et
al., 1993) and the response to novelty, amphetamine-induced
activity and drug discrimination in rats (Bevins et al.,
1997).
The time course of intra-NAC EEDQ effects on FI performance, moreover,
is consistent with its impact on behavior in other studies, including
amphetamine-induced changes in activity (Crawford et al.,
1994) and apomorphine-induced stereotypy and catalepsy (Hamblin and
Creese, 1983), which generally disappeared within 4 days after the
systemic administration of EEDQ. In addition, the time course of the
observed changes in FI performance parallels those reported for the
turnover of DA receptor proteins after EEDQ administration (Hamblin and
Creese, 1983; Giorgi and Biggio, 1990b; Kula et al., 1992).
Intra-NAC DA agonist and antagonist dose-effect curves should differ in control vs. Pb-treated rats. The second hypothesis of this study asserted that intra-NAC DA agonist or antagonist dose-effect curves should differ in control vs. Pb-treated rats because DA function is already altered by Pb. The most pronounced such difference in relation to Pb treatment was the effect of DA on overall response rates across all subjects in each Pb group (fig. 4), with what might be interpreted, based on a U-shaped dose-effect curve for intra-NAC DA and FI performance (fig. 7), as a right-shift of the DA dose-effect curve at 500 ppm Pb given that response rates were suppressed rather than increased by DA. Such findings, moreover, are consistent with the hypothesis of excess NAC DA availability in the 500 ppm Pb-treated group as the basis of the higher base-line FI response rates of this group compared with 0 ppm controls.
Pb exposure did not modify the magnitude of EEDQ-evoked decreases in overall response rates on the FI schedule but instead delayed the time course and extent of recovery after EEDQ (fig. 2). This effect of Pb was limited, though, because it occurred only at the highest dose of EEDQ (200 µg) and appeared to derive from the net effect of small but statistically insignificant effects across all three measures, including PRP, mean IRT and run rate, rather than any specific measure of FI performance. One possible interpretation of this delayed recovery at 200 µg is a delay in receptor turnover or perhaps, more specifically, in receptor production rate (Kula et al., 1992). EEDQ has been widely used to assess DA receptor turnover and
occupancy, after both peripheral and regional administration (Giorgi
and Biggio, 1990a; Fukuchi et al., 1986; Nowak et
al., 1988), with several such studies repeatedly showing EEDQ to
affect receptor Bmax rather than affinities. The current findings, in fact, show interesting parallels to those described with aging (Giorgi et al., 1992) where a reduction
in the density of D1 DA receptors in striatum, NAC and
substantia nigra of aged rats (23 months of age) (i.e., the
delay in recovery to full Bmax values) was
actually due to larger decreases in the receptor production rate than
in the degradation rate compared with younger counterparts (3 months of
age). Such an effect, which has not previously been described for Pb
exposure to our knowledge, could suggest that the decreases in DA
binding measured autoradiographically in Pokora et al.
(1996) actually reflect a delayed turnover instead of, or in addition
to, enhanced NAC DA availability (Zuch et al., 1998).
Whether a delayed production of DA receptors is in fact selective to
NAC and whether it underlies the apparent preferential vulnerability of
NAC to Pb remain to be determined.
It must also be remembered that EEDQ does not selectively block DA
receptors but can also impact alpha adrenergic and
serotonergic systems (Meller et al., 1992; Gozlan et
al., 1994), systems that also could conceivably contribute to the
observed changes in FI performance. However, the fact that restoration
of the functionality of either D1 or D2
receptors in NAC in our prior study (Cory-Slechta et al.,
1997b) restored normal FI performance suggests that it was the
DA-inactivating properties of this compound that served as the basis of
its alterations in FI schedule-controlled behavior, rather than changes
in any other neurotransmitter system. Another caveat is the possibility
that intra-NAC EEDQ administration may have changed levels of DA and
metabolites, an effect that has been reported after peripheral
administration (Crawford et al., 1992) and that could alter
homeostatic mechanisms of DA receptor regulation. Whether similar
effects follow local EEDQ administration has not been evaluated to the
best of our knowledge and was not measured in this study. Furthermore,
as noted above, EEDQ may have also affected other neurotransmitter
systems which could likewise modulate DA receptor production
parameters. Finally, EEDQ may have acted differentially in control and
Pb-treated rats on DA and/or other neurotransmitter systems to
consequently affect recovery rates. Nevertheless, the possibility that
Pb exposure delays DA receptor production rate merits further study.
Intra-NAC DA and Pb should exert changes in FI response rates
through similar behavioral mechanisms.
A final hypothesis of this
study postulated that the effects of intra-NAC DA administration on FI
performance should mimic those produced by chronic low-level
postweaning Pb exposure. Both intra-NAC DA and Pb can increase FI
response rates, again providing support for the assertion of enhanced
NAC DA activity as a mechanism of Pb-induced increases in FI response
rates. However, additional mechanisms must play a role in the case of
Pb exposure because the behavioral mechanisms by which each treatment
increased FI rates differed. Specifically, intra-NAC DA increased FI
rates primarily by shortening PRP times, thus allowing the accumulation of a greater total number of responses, an effect showing considerable similarity to those ascribed to systemic administration of
d-amphetamine in rats and pigeons (McAuley and Leslie, 1986;
Branch and Gollub, 1974) and interpreted by McAuley and Leslie (1986)
to be most consistent with a disruption of temporal discrimination
processes.
). What the additional
mechanisms may be that differentiate Pb and DA effects on FI
performance cannot be determined from the current study, but one
possibility is changes in other neurotransmitter systems that affect
mesolimbic DA systems, including glutamatergic or GABAergic systems. Pb
clearly alters excitatory amino acid receptors that compose the
afferent input to mesolimbic systems (Cory-Slechta et al.,
1997a; McCoy et al., 1997). Furthermore, it must be kept in
mind that Pb-induced behavioral toxicity reflects the effects of
protracted exposure to multiple brain regions and is compared here with
the effects of the acute administration of DA in a single brain region.
Indirect effects of Pb may occur under typical exposure conditions that
modulate FI performance and a sustained enhancement of DA activity may
likewise yield additional compensatory effects. Thus, enhancement of
NAC DA activity could represent either a final common pathway for
multiple effects or even one of multiple converging mechanisms for FI
performance changes.
A summary hypothesis relating NAC DA activity to FI response
rates.
The changes in FI performance produced by intra-NAC DA and
EEDQ administration confirm again the importance of mesolimbic systems
to the mediation of FI performance. Considered collectively, these
findings and those from our prior study (Cory-Slechta et al., 1997b), suggest that NAC DA activity may be an important modulator of FI response rates. Specifically, the inverse U-shaped function produced by DA in control rats, along with the FI rate decreases observed in response to intra-NAC EEDQ could indicate that
too low of a level of DA activity results in FI rate suppression. This
decreasing limb is also supported by the findings of Robbins et
al. (1983) that intra-NAC 6-OHDA also decreases FI response rates.
A rise in DA activity to moderate and higher levels increases FI
response rates. However, as DA levels increase even further, FI rates
are ultimately suppressed again, as diagrammed schematically in figure
7A. Such a scheme, moreover, reflects the effects of other DA agonists
such as apomorphine and d-amphetamine that would likewise
increase NAC DA activity and produce similar dose-effect functions
(e.g., Zuccarelli and Barrett, 1980; Robbins et
al., 1983).
Health relevance of Pb effects.
Involuntary exposures to low
levels of Pb occur over the lifetime in most of the world's
population. The highest PbBs occur early in development and again in
advancing age (Brody et al., 1994), those two intervals of
the life span that clearly encompass periods of maximal vulnerability
of the central nervous system to insult. Our current findings generate
new concerns about the extent to which Pb body burden might serve as a
risk factor that acts in conjunction with other environmental or
genetic risk factors to influence mesolimbic DA-mediated disturbances
such as schizophrenia, drug abuse and addiction and various cognitive
and attention deficits.
10 µg/dl (Centers for
Disease Control, 1991). What is unclear from the current studies,
however, is the threshold for such effects. PbBs of the 50 ppm group
averaged only 9.6 µg/dl, which appears to be a level below the
effective level in the rodent, which then must lie between 9.6 and 48 µg/dl. However, we have previously shown marked decreases in NAC DA
binding sites and KCl-evoked DA release as well as behavioral changes
in FI performance at PbBs as low as 16 µg/dl in the rat (Pokora
et al., 1996; Cory-Slechta et al., 1985; Zuch
et al., 1998), a species well known for its resistance to Pb
toxicity compared with humans (Scharding and Oehme, 1973; Winder
et al., 1983).
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Footnotes |
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Accepted for publication April 6, 1998.
Received for publication November 6, 1997.
1 This work was supported by NIEHS Grants ES05017, ES05903 and ES01247.
Send reprint requests to: Dr. Deborah Cory-Slechta, Department of Neurobiology and Anatomy, Box 603, University of Rochester Medical Center, Rochester, NY 14642. E-mail: slechta{at}envmed.rochester.edu
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Abbreviations |
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DA, dopamine; NAC, nucleus accumbens; STR, dorsal striatum; Pb, lead; ANOVA, analysis of variance; RMANOVA, repeated measures analysis of variance; EEDQ, N-ethoxycarbonyl-2-ethoxy-1,2-dihyroquinoline; DMSO, dimethylsulfoxide; FI, fixed interval; 6-OHDA, 6-hydroxydopamine; IRT, interresponse time; PbB, blood lead; PRP, postreinforcement pause.
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA) and 6-[3H]cyano-7-nitroquinoxaline-2,3-dione ([3H]CNQX) binding in response to chronic low-level lead exposure: Reversal versus potentiation by chronic dopamine agonist treatment.
J Neurochem
69:
2466-2476[Medline].
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