Hyperactivity and impulsiveness in rats fed diets supplemented with either Aroclor 1248 or PCB-contaminated St. Lawrence river fish

Behavioural Brain Research 126 (2001) 1 – 11 www.elsevier.com/locate/bbr

Research report

Hyperactivity and impulsiveness in rats fed diets supplemented with either Aroclor 1248 or PCB-contaminated St. Lawrence river fish David F. Berger a,*, John P. Lombardo a, Peter M. Jeffers a, Anne E. Hunt b, Brian Bush b, Ann Casey b, Fred Quimby c a

Department of Psychology, State Uni6ersity of New York at Cortland, PO Box 2000, Cortland, NY 13045, USA b State Uni6ersity of New York at Albany, Albany, NY USA c Cornell Uni6ersity, Albany, NY USA Received 15 June 2000; received in revised form 23 March 2001; accepted 23 March 2001

Abstract We examined whether exposure to polychlorinated biphenyls (PCBs) around puberty would produce hyperactivity and impulsiveness in adult Sprague–Dawley rats. Randomly assigned groups consumed food containing environmental concentrations of Aroclor 1248, PCB-contaminated St. Lawrence River carp, or corn oil (control). All received operant training to a final multiple (mult) 120-s, fixed interval (FI), 5-min extinction (EXT) schedule. Pressing rates of both exposed groups for drops of water averaged more than 1.5 times that of controls, especially toward the end of the 120-s interval. This overactivity included bursts with short ( 50.5 s) interresponse times (IRTs), behavior characteristic of hyperactive boys and genetically hyperactive rats. The exposed groups also overreacted to the decreases in reinforcement density associated with transition to the final schedule. The results were interpreted in terms of the possible alterations in the animals’ reinforcement mechanisms and the possible neurotoxic effects of PCB exposure. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Attention Deficit Disorder; Hyperactivity; Impulsiveness; Attention; Animal model; Reinforcement schedule; AD/HD; Polychlorinated biphenyls; PCBs; Dopamine

1. Introduction Polychlorinated biphenyls (PCBs) are a family of 209 man made compounds that were once manufactured in large quantities in the US, Federal Republic of Germany, France, UK, Japan, Spain, and Italy. They are still being manufactured in North Korea and Russia. According to the World Health Organization [50] production of PCBs, by the end of 1980, totaled 1,054,800 tons. Production of these substances was banned in the US in 1977 because of widespread concern that they were associated with health risks. Large amounts of * Corresponding author. Tel.: + 1-607-7534217; fax: +1-6077535738. E-mail address: [email protected] (D.F. Berger).

PCBs continue to be found in bodies of water in the US including the Great Lakes, the St. Lawrence and Hudson Rivers. These substances are highly stable and bioaccumulate and because of their stability they have entered the food chain. Unfortunately, when ingested, PCBs are readily absorbed and because they are lipophilic and resistant to metabolism, they can persist in fat tissue [47]. For example, PCB congener 153 is persistent in the environment, in the human body, and because it is resistant to degradation, it is one of the three dominant congeners found in human adipose tissue [15]. It is also the congener with the highest concentration in breast milk [20]. According to the World Health Organization [50], PCBs ‘‘were reported as contaminants in almost every component of the global ecosystem’’ (p. 149).

0166-4328/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 3 2 8 ( 0 1 ) 0 0 2 4 4 - 3

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Rice [23] stated that ‘‘The congruence of findings between studies in humans and animal models, as well as the similarity of body burden at which neurotoxic effects are observed, support the hypothesis that PCB exposure produces neurotoxicity in the developing human being at environmentally relevant levels’’ (p. 228). If, as Rice suggested, en6ironmentally rele6ant le6els produce neurotoxic effects, the importance of studying the behavioral effects associated with the ingestion of PCBs is readily apparent. Several lines of research with human beings and animal models suggest the possibility of a relationship between ingestion of PCBs and behaviors associated with Attention Deficit Hyperactivity Disorder (AD/ HD) and Attention Deficit Disorder (ADD). Animals that were prenatally, perinatally or postnatally exposed to PCBs showed many of the behavioral characteristics indicative of AD/HD. For example, in utero exposure of mice led to increased motor activity and impaired learning [48], pre- and post-natal exposure led to higher activity levels and poorer visual discrimination learning in rats [1]. Holene et al. [18] showed that male rats exposed to PCB congener 153 during lactation became hyperactive and impulsive. Rhesus monkeys exposed throughout gestation and lactation showed locomotor hyperactivity during their first year of life [4]. According to Tilson et al. [49] ‘‘The most common finding in the animal studies was that developmental exposure to PCBs results in behavioral hyperactivity’’ (p. 245). The offspring of women who ingested PCB-contaminated rice-bran cooking oil in Taiwan and Japan were prenatally exposed to PCBs. The Japanese children were hypoacti6e [16]. The Taiwanese children were more active and had more behavior problems compared with unexposed controls [9]. When tested at ages between 7 and 12, the exposed Taiwanese children also had significantly lower verbal and full-scale IQs. They also showed prolongation of, and a significant reduction in the amplitude of auditory event evoked potentials (P300) [8]. Low P300 amplitude has been shown to be related to AD/HD [8]. Rice [27] recently articulated the similarities in children with AD/HD and monkeys exposed to PCBs. Rice [27] indicated both have problems learning from the consequences of their behavior, and that both show an inability to organize the temporal sequencing of behavior. The present research was designed to determine if exposure to environmental concentrations of PCBs around puberty would produce hyperactive and impulsive behavior in adult male rats, using the same protocol that was used by Sagvolden and associates to measure the behavior of the genetically hyperactive SHRs. The operant conditioning procedures used have provided a sensitive measure of activity level, impulsiveness and visual discrimination behavior [3,31– 34]. The same schedules produce quite similar behavior in ani-

mals and humans [30], there is a shared terminology, and the control of reinforcers is superior to other often-used tasks. Sagvolden et al. [30] compared the rates of pressing the nose of a clown apparatus [2] for coins and plastic trinkets by Norwegian boys diagnosed with AD/HD to a normal comparison group, using a multiple (mult) 30 s fixed interval (FI), 120 s extinction (EXT) schedule of reinforcement. This protocol was a modification of that developed to study the behavior of spontaneously hypertensive rats (SHR), a strain that has been found to be hyperkinetic and to show increased behavioral variability [31–34]. The children with AD/HD produced relatively more responses, especially toward the end of the FI 30-s period, which can be characterized as impulsiveness. A substantial amount of that higher response rate consisted of bursts of responses, that is those with short (B 0.33 s) interresponse times (IRTs). The comparison-group boys emitted almost no response bursts. The AD/HD group also produced more than twice as many responses during EXT. These findings are consistent with observations of the SHR [3], demonstrating the external validity of the animal model. Berger and Sagvolden [3] compared the rates of lever pressing of male and female SHRs with male and female Wistar–Kyoto (WKY), the normotensive progenitor strain, for drops of water. They utilized a two-component mult 2-min FI, 5-min EXT schedule of reinforcement; and replicated earlier studies showing SHRs to be overactive and impulsive compared with WKYs. The SHR males produced more response bursts, and pressed more during the EXT components than SHR females. Responding during EXT and response bursts by WKYs of both sexes were rare. Sagvolden and associates [3,30] accounted for the overactivity and impulsiveness of SHRs and AD/HD boys as due to an altered reinforcement mechanism. That alteration may result from genetically based hypofunctioning of their dopamine system [29,35]. As they acquire the operant response their proposed steeper gradients of reinforcement will produce higher response rates towards the end of the FI period (steeper scallop) because the reinforcing effect is relatively stronger on responses closer in time to the subsequent reinforcers. Catania et al. [7] had explained that relations between responses are also conditioned and maintained by reinforcers. Thus a steeper delay gradient will also generate bursts of responses, because the development and reinforcement of a series of responses with long IRTs is unlikely. Holene et al. [18] reported that rats exposed to the di-ortho- substituted PCB congener 153 and the coplanar PCB congener 126 both pressed the lever more than controls during FI. However, only the PCB congener 153 group produced response bursts similar to those of

D.F. Berger et al. / Beha6ioural Brain Research 126 (2001) 1–11

AD/HD boys in Sagvoldens et al. [30] study, and to male SHRs [3]. Rice [24] found that monkeys exposed to PCBs from birth through 20 weeks of age (at a dose typically found in human breast milk) responded with shorter IRTs in a FI schedule. Additional studies by Rice [25,26] again found that monkeys exposed to PCBs were significantly different from untreated controls on FI and differential reinforcement of low rate (DRL) schedules. For example, Rice [25] found that PCB-treated animals had more nonreinforced responses and received fewer reinforcements than the untreated controls with a DRL 30-s schedule. Rice [26] replicated the DRL findings and also showed that treated animals had shorter IRTs in a FI schedule, and that the treated animals made many more responses per reinforcement than control animals. She concluded that animals treated with PCBs demonstrate an inability to inhibit inappropriate responding. The similarity of the findings of Berger and Sagvolden [3] with SHRs, Sagvolden et al. [30] with AD/HD children, and Rice’s findings with monkeys exposed to PCBs is striking and suggests that all are deficient in their ability to inhibit responding. Animals exposed to PCBs also exhibit a behavior that is common to boys diagnosed with AD/HD; an exaggerated response to frustration of anticipated rewards. Douglas and Parry [13] reported that children with AD/HD responded more intensely than the normal comparison group when expected rewards were not delivered. Daly [10] and Daly et al. [11] found that rats pre- or post-natally exposed to PCB-contaminated Lake Ontario Salmon reacted with greater magnitude of frustration than unexposed controls to the reduction in frequency of reinforcements. We examined the effects of ingesting food mixed either with fish from a portion of the St. Lawrence river that is contaminated with Aroclor 1248, or with environmental concentrations of that industrial mixture of PCBs alone, by male Sprague– Dawley rats between ages 35 and 65 days (starting around puberty), on their subsequent operant behavior between ages 80 and 117 days (adults). It was hypothesized that exposed animals would become relatively overactive and impulsive. Based upon the findings of Daly and associates [10,11], the exposed groups were also expected to overreact to the decreases in reinforcement density associated with the lever press training prior to the final schedule of reinforcement, and to the EXT periods during those transition training sessions.

2. Method

2.1. Participants A total of 42 Sprague– Dawley, experimentally naive

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male rats (Rattus Norvegicus) were obtained from Zivic–Miller Laboratories, Belinope, PA. All were 30day-old and weighed 110–139 g upon arrival in the colony at the State University of New York College at Cortland. They were maintained on a 12-h light:12-h dark cycle, at 20–22 °C, with a relative humidity of 60–63%. They were housed in pairs from the same group in 24× 18× 18 cm (height) cages. The rats were randomly assigned to one of three groups (n=14). Eight rats from each group were run from May through August, and the other six from September through December.

2.2. Apparatus A total of six operant chambers were used; three were BRS-Foringer series 900, and three were BRS/ LVE Model 143. All chambers are surrounded by sound-attenuating enclosures to minimize extraneous noise. In addition, a 72-dB masking noise is produced in each by individual ventilating fans. The house lights in the BRS-Foringer chambers are two 14-V (GE 1893) lights, wired in series and centered above the chambers. In the BRS/LVE chambers they are a 28-V (GE 1820) bulb located at the top of the lever panel. All are operated at 20 V from an isolated AC source. The BRS-Foringer chambers each have a cylindrical-shaped response lever measuring 1.5 cm in diameter, and the BRS/LVE chambers a paddle-shaped lever measuring 3 cm in width. All are located 8 cm from the grid floor, and require 1.2× 104 dynes of vertical force (equivalent to the application of a mass of 12 g) to depress. Response-contingent drops of water are delivered to each chamber by liquid dippers, which lower to pick up the water and then raise back up for the rat to collect it, with no time limit. A 28-V (GE 1820), clear cue light located 5 cm above the levers in each chamber is associated with each water delivery. The water troughs are centered at the bottom of the lever panel. A BRS/ LVE electromechanical system scheduled the reinforcers and recorded the behavior. The interior surfaces of all the chambers were washed daily with a soap and water solution.

2.3. Procedure 2.3.1. Exposure phase After a 1-week acclimation period following their arrival, the rats were weighed, randomly assigned to the groups, and marked for identification. During the next 30-day exposure period all had free access to water, but were placed on a 23-h food deprivation schedule so that they would learn to eat their respective diets in one large meal during the same hour, rather than several small ones throughout the day. All were given 30-g (wet weight) daily portions of the diets, in round glass

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7.5-cm-diameter sponge cups in new cages. The food cups were placed in the cages for 1 h after which they were removed and weighed to determine the amounts eaten. The PCB-food group was fed a mash consisting of Purina Laboratory Rodent Diet 5001 (meal) augmented with 1 ml corn oil containing 0.5 mg/g Aroclor 1248, an amount approximately equal to that found in the contaminated fish. The fish-food group was fed a mash consisting of 27.50 g Purina Laboratory Rodent Diet 5001, 0.95 g corn oil, 0.40 g water, and 1.15 g ground fish. The carp was caught in the St. Lawrence River near the Akwesasne Reservation. However, during the September replication of the procedure we were only able to obtain a 10-day supply of the fish. Therefore, the diet for the last six animals in the fish-food group contained Purina Laboratory Rodent Diet 5001 and 1-ml corn oil only on days 11–30 of the exposure phase. The control group was fed the same mash consisting of Purina Laboratory Rodent Diet 5001 and 1-ml corn oil only. This group’s home cages were moved to a separate cage rack on the other side of the room to avoid possible contamination from the other two groups. Due to the requirements of another procedure taking place in the same room, the fronts of cage racks for each group were draped with a loosely fitted 8-mil plastic sheet that isolated the rats visually; and all were exposed to the noise from five portable electrical air pumps that varied between 75 and 80-dB (SPL), depending upon location in the room. These conditions were present only during the 30-day exposure phase. After that the plastic sheeting and pump noise were removed, and all the rats were returned to a single rack, still housed in pairs in their home cages, but now with free access to Purina Laboratory Rodent Diet 5001 (pellets) and water for 6 days. During the subsequent lever press training for drops of water all groups had free access to the uncontaminated Purina Laboratory Rodent Diet 5001, but were placed on a 22-h water deprivation schedule (see below).

2.3.2. Operant training phase The training sessions were balanced so that equal numbers of animals from each group were run throughout the day. Each group was assigned to two of the six chambers, one of each type, which the rats occupied during all training sessions. Individual animals were always tested in the same order. Following each session the animals were returned to their home cages where they were given water for 1 h. The training took place from Mondays through Saturdays between 10:00 and 18:00 h. On the first day of operant training each rat was placed in his respective chamber for a 30-min habituation session after which the 22-h water deprivation schedule began. During the next 4 days each was given

30-min sessions of dipper training during which water was delivered on a variable time 30-s schedule, independent of the rat’s behavior. Then six 20-min sessions were run. In the first of these, lever pressing was shaped using the method of differential reinforcement of successive approximations [6]. This was followed by five continuous reinforcement (CRF) sessions to stabilize responding during which each lever press operated the dipper. The house lights were on during the dipper training, shaping, and CRF sessions. All groups were then run through a series of 40-min sessions with mult FI, EXT schedules of reinforcement. A schedule is termed multiple when two or more schedule components operate in alternation, each in the presence of a different stimulus. The house light was on during the FI components, but off during the 5-min EXT components. The series started with two sessions of mult 30-s FI, 5-min EXT. Next were two sessions with mult 1-min FI, 5-min EXT. These were followed by the final mult 2-min FI 5-min EXT schedule. During that FI component, the first lever press after the 2-min interval had elapsed was reinforced by a drop of water. During the EXT component there was no house light, and no water was ever presented. Each session was divided into four parts: (a) a 2-min FI component in which a maximum of seven reinforcers was delivered; (b) a 5-min EXT component; (c) a new 2-min FI component with the same parameters as the one above, and finally; (d) a 5-min EXT component that ended the session. During the 2-min FI components the seventh reinforcer can be obtained in as little as 14 min if the animal responds without delay. However, a maximum of 15 min was allowed, after which the FI components of the schedule were terminated and the EXT component introduced. Therefore, individual rats could take longer to respond and may not receive all the reinforcers. For each rat the numbers of lever presses and reinforcements delivered were recorded daily. In addition, response bursts, that is presses with short (50.5 s) IRTs, indicative of hyperactivity [3], were recorded for each rat during all CRF and FI components of sessions. During the final schedule the 2-min FI component was divided into four consecutive 30-s segments, and the 5-min EXT component was divided into five consecutive 1-min segments [3]. The numbers of lever presses per segment during FI and during EXT were recorded separately. A summary of the experimental protocol is presented in Table 1.

2.3.3. Tissue sampling The animals were euthanized with an overdose of isofluorane on the day after the last operant session. Samples of abdominal fat were taken for PCB analysis to confirm the effectiveness of the diets. To minimize possible cross contamination the control group was

D.F. Berger et al. / Beha6ioural Brain Research 126 (2001) 1–11 Table 1 Experimental protocol Procedure

Duration

Exposure phase

30 days — 23 h per Age of rats —35–65 day days 1 day —30-min session House-lights off

Habituation to apparatus Dipper training

Lever press training Continuous reinforcement FI 30 s-EXT 5 min FI 60 s-EXT 5 min FI 120 s-EXT 5 min

4 days— 30-min sessions 2 days 5 days — 40-min sessions 2 days— 40-min sessions 2 days— 40-min sessions Until behavior stabilized—40-min sessions

Comments

Begin 22 h water deprivation — house-lights on Shaping procedure — house-lights on House-lights on FI-lights on, EXT— house-lights off FI-lights on, EXT— house-lights off FI-lights on, EXT— house-lights off

dissected first, followed by the PCB-food group and then the fish-food group. Samples were stored at − 20 °C until analyses.

2.3.4. PCB analyses The PCB concentrations in adipose tissue samples taken from several animals in each group were determined by the procedures developed at the New York Public Health Laboratory, with minor modifications [5]. A 1-g tissue sample was ground in a ceramic mortar and pestle with 3-g anhydrous Na2SO4, then extracted with three 10-ml aliquots of 1:1 hexane– acetone. The combined extract was evaporated to near dryness, the evaporation Erlenmeyer flask was carefully rinsed with a few ml hexane, and the solution was transferred with a 14 cm× 6 mm disposable pipette to a 4% deactivated Florisil (US Silica, PA) column prepared in another 14 cm× 6 mm disposable pipette. The column was eluted with about 5 ml hexane, the eluent was diluted to precisely 10 ml with hexane and was stored in a screwcap vial over about 1 g of fresh Florisil. The gas chromatograph (GC) analysis utilized either a 0.25 mm ×30 m RTX-5 column (Restek) in a Varian 3300 GC, programmed from 120 to 190 °C at 1.2° per min, then to 280 °C at 5° per min, or a Hewlett-Packard 5890 GC with 0.53 mm×15 m RTX-1701 column and the same temperature program. Electron capture detectors were used. A series of pure PCB congeners and congener mixes were used to calibrate the analyses. The congener profiles in tissue samples did not match any commercial Aroclor, probably because of biodegradation, differing volatility of the congeners with degree of chlorination, and differences in bioaccumulation. The closest match of tissue sample profiles was with

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Aroclor 1254, although Aroclor 1248 was the material added to food. We chose to report quantitative amounts of PCB by measuring four peaks eluting from the RTX-1701 column at 26.67, 28.66, 30.31, and 32.32 min, tentatively identified as PCB numbers 168, 153, 149, and 138. Each of these peaks was observed in samples of Aroclors 1242, 1248, 1254, and 1260 in significant amounts, and were among the most abundant peaks in most tissue samples. A total PCB value was calculated from the summed area of these four peaks and a linear calibration curve established with Aroclor 1254. Concentrations, sample sizes, and volumes were used to report final values as mg PCB per kg tissue (ppm).

2.4. Statistics The data were analyzed using univariate analysis of variance (ANOVA) or Student’s t-test. Statistica for the Macintosh, Release 4.0 [46], was used for the statistical calculations. Error terms for the ANOVA were not pooled, significant effects were followed up by ANOVA for simple effects or by Newman–Keuls comparisons [19,21]. The alpha level was set at 0.05 for all statistical tests. Three of the rats in the PCB-food group died during the 6-day period immediately following the exposure phase. In addition, inspection of the data showed that the stable-state total FI responding of one animal in the fish-food group (with only 10 days of exposure to the fish), and one in the control group, were more than 2.6 standard deviations (S.D.) above their respective group means. Therefore, their data were excluded from the subsequent analyses.

3. Results

3.1. PCB le6els The mean of the total PCB concentrations in adipose tissue of animals randomly selected from each group (numbers of rats in parentheses) were 1.05 (S.E.=0.32) ppm for PCB-food group (n= 8), 1.54 (S.E.=0.39) ppm for the fish-food group (n=6), and 0.10 (S.E.= 0.05) ppm for the control group (n= 7). A one-way ANOVA using the data from the subgroups showed a significant treatment effect, F(2, 18)= 6.14, P=0.009. Follow-up comparisons showed that the PCB-food and fish-food groups were both higher than the control group (PB 0.05), but did not differ from each other.

3.2. Stable-state beha6ior The final mult 2-min FI EXT schedule was run for 22 sessions. The mean for each animal over the last six

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D.F. Berger et al. / Beha6ioural Brain Research 126 (2001) 1–11

Fig. 1. The mean number of lever presses ( 9 S.E.M.) by each group during the fixed-interval (FI) component of the final multiple 120-s FI, 5-min extinction schedule as a function of 30-s segments of the FI, when behavior had stabilized.

sessions, when the behavior had stabilized [3], were used for the statistical analyses. The data from the FI and EXT components were evaluated with separate ANOVA [3,31 –34]. Examination of the fish-food group data suggested that the behavior of the two subgroups (30 vs. 10 day on fish-supplemented diet) did not differ. An two-sample t-test comparing their mean stable-state total FI responding was not significant (P \0.05). This finding was confirmed by two separate ANOVA of their mean stable-state responding, similar to those reported in more detail below. A 2× 4 mixed ANOVA (subgroups vs. 30-s segments) of the FI data, and 2× 5 mixed ANOVA (subgroups vs. 1-min segments) of the EXT data showed no significant differences between the 30 and 10-day fish-food subgroups, and no interactions with segments (P \ 0.05). Therefore, the data from these two subgroups were combined into a single fish-food group (n = 13) for all the analyses reported below.

3.2.1. Fixed inter6al presses The lever pressing rate of the PCB-food (n = 11) and fish-food groups averaged more than one and a half times that of the control group (n =13). Fig. 1 shows the mean number of lever presses by each group represented as a function of 30-s segments of the FI. The hyperactivity of the two exposed groups was particularly pronounced at the end of every FI. The data were evaluated with a 3×4 mixed ANOVA. The between variable was groups (PCB-food vs. fish-food vs. control), and the within variable was consecutive 30-s segments of FI. The results showed a main effect of segments, F(3, 102) =61.50, P B 0.05. Pair-wise comparisons showed that the rats pressed more during the fourth 30-s segment (M = 79.9,

S.E.= 9.3), than the first (M=5.5, S.E.= 0.8), the second (M= 14.9, S.E.=1.9), or the third (M=42.2, S.E.= 4.8), PB 0.05. There were fewer presses during the first, than during the second and third 30-s segments; and fewer during the second than the third, P= 0.0001. There also was a significant Groups× Segments interaction, F(6, 102)=2.26, P= 0.044. Simple effects ANOVA of the interaction [19,21] revealed that the pressing rates of the PCB-food, F(3, 120)= 30.87; fishfood, F(3, 120)=24.24; and control, F(3, 120)=9.35; groups all increased over the 120-s interval; PB0.05. Pair-wise mixed ANOVA (Groups×Segments) showed that only the PCB-food and control groups increased at different rates, F(3, 102)= 4.11, PB 0.05. Newman– Keuls comparisons of the interaction mean revealed that both the PCB-food and the fish-food groups differed from the control group by 120 s (P= 0.0001), but did not differ from each other. At 90 s only the PCB-food and control groups differed (P= 0.0001). There were no differences among the three groups at 30 and 60 s (P\ 0.05).

3.2.2. Response bursts The two exposed groups produced more response bursts than the control group. The mean numbers of lever presses with IRTs5 0.5 s during the last six sessions were 29.83 (S.E.= 10.59) for the PCB-food group, 54.45 (S.E.= 19.04) for the fish-food group, and 11.68 (S.E.= 4.82) for the control group. A one-way ANOVA showed that the apparent differences were not significant, F(2, 34)=2.81, P=0.074. Inspection of the above data indicated that the rats’ mean numbers of response bursts over the last six sessions were quite variable (M.S.E.= 2131.80); however, the mean during the last three sessions were slightly less variable (M.S.E.=1822.77). A one-way ANOVA of the latter data was significant, F(2, 34)= 3.30, P= 0.049. The mean for the PCB-food, fish-food, and control groups were 29.09 (S.E.= 10.83), 53.85 (S.E.=17.12), and 11.00 (S.E.= 4.64), respectively. Only the fish-food and control groups differed significantly (P= 0.039). 3.2.3. Reinforcements deli6ered The mean numbers of reinforcements delivered during the last six sessions by each animal were used to compute a one-way ANOVA. There was no significant effect of groups, FB1. The grand mean was 11.88 (S.E.= 0.25). 3.2.4. Extinction presses Each group’s mean numbers of lever presses over 1-min segments of EXT are represented in Fig. 2 [3]. The mean rates of responding for all the groups were

D.F. Berger et al. / Beha6ioural Brain Research 126 (2001) 1–11

relatively low, and their patterns across the 5-min EXT period were different. The numbers of responses by control group remained relatively steady; while those of the PCB-food group increased, and the fish-food group decreased. Each rat’s mean numbers of presses was used to compute a 3×5 mixed ANOVA [3,31– 34]. The between variable was groups and the within variable was consecutive 1-min segments of EXT. Only the interaction was significant, F(8, 136)=2.02, P = 0.049. Simple effects ANOVA, computed as above, indicated that only the fish-food group’s numbers of presses changed significantly across EXT segments, F(4, 136)=2.83, PB 0.05. The mixed ANOVA (Groups × Segments) of the groups in pairs showed that only the PCB-food and fish-food groups had a significantly different pattern of responding over the EXT period, F(4, 136)=3.56, PB0.05.

3.3. Transition training effects Although our primary focus was on stable-state behavior during the final mult 2-min FI, 5-min EXT schedule [3,31– 34]; informal observations indicated that the groups behaved differently during the series of schedules on which they were trained in transition to the final mult schedule. As the reinforcement density was decreased, rats in the two exposed groups often tore up the paper that lined the pans under the grid floor of their operant chambers. At the end of these transition training sessions many rats ran in circles, jumped, became harder to handle, and squealed when they were being removed from the chambers. This behavior did not occur in the control group. To evaluate the transition training effects more formally we compared the groups’ numbers of lever presses and response bursts during the last two CRF, the two FI-30 s, the two FI-60 s, and the first two

Fig. 2. The mean number of lever presses by each group ( 9 S.E.M.) during the extinction (EXT) component of the final multiple 120-s FI, 5-min EXT schedule as a function of 1-min segments of the EXT period, when behavior had stabilized.

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FI-120 s training sessions. We also compared the numbers of presses during the EXT component of the three transition mult schedules in the aforementioned sessions. These EXT periods accompanied the FI 30, 60 and 120 s components of the mult FI-EXT schedules during the training period (see Table 1). The animals tended to press more, and to produce more response bursts, when the density of reinforcement was reduced relative to the previous session (i.e. on the first session of a new mult schedule). They also lever pressed more often during the EXT components associated with FI 30 s than with the subsequent FI 60 s and FI 120 s. These patterns of behavior were most conspicuous in the fish-food group.

3.3.1. Transition presses Each rat’s total number of presses during the transition CRF and FI sessions were used to compute a 3× 4×2 mixed ANOVA. The between variable was groups (PCB-food vs. fish-food vs. control), the first within variable was schedule of reinforcement (CRF vs. FI 30 s vs. FI 60 s vs. FI 120 s), and the second within variable was transition schedule session (first vs. second). The main effect of schedule of reinforcement was significant, F(3, 102)= 12.28, PB0.05. The mean for the CRF, FI-30 s, FI-60 s and FI-120 s schedules (with S.E. in parentheses) were 334.4 (20.3), 336.3 (32.5), 263.5 (33.7), and 190.4 (17.9), presses, respectively. The groups pressed significantly more during CRF than during FI 60 s and FI 120 s. There were also more presses during FI 30 s than FI 60 s and FI 120 s, and more during FI 60 s than FI 120 s (all PB 0.05). The main effect of schedule session was also significant, F(1, 34)= 31.27, PB0.05. The mean were 310.8 (S.E.=23.6) presses for the first sessions, and 251.5 (S.E.=19.3), for the second. These effects were qualified by a Groups × Schedule Session interaction, F(2, 34)= 4.00, P= 0.029. The interaction mean are presented in Fig. 3. Pair-wise comparisons confirmed that both the PCB-food and the fish-food groups lever pressed more often during the first session of a new schedule than during the second (PB 0.05), while the control group did not (P\0.05). 3.3.2. Transition bursts A similar 3× 4× 2 mixed ANOVA was computed with the response burst data. The main effect of groups was significant, F(2, 34)= 38.37, PB0.05. The overall mean numbers of bursts were 41.3 (S.E.= 14.6) for PCB-food group, 94.2 (S.E.= 13.4) for fish-food group, and 12.3 (S.E.= 13.4) for control group. The fish-food differed from the PCB-food and control groups (PB 0.05), which did not differ from each other. There was also a main effect of schedule of reinforcement, F(3, 102)= 9.51, PB0.05. The mean number of responses bursts (S.E. in parentheses) across schedules

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D.F. Berger et al. / Beha6ioural Brain Research 126 (2001) 1–11

Fig. 3. The Groups × Schedule Session interaction found with lever presses as the density of reinforcements was decreased during the operant transition training. Each group’s mean numbers of lever presses ( 9 S.E.M.) during the last two continuous reinforcement and fixed-interval sessions are shown for the first versus second of these sessions.

were 26.3 (4.0) during CRF, 69.8 (12.8) during FI 30 s, 72.9 (15.3) during FI 60 s, and 28.2 (6.8) during FI 120 s. There were more response bursts during FI 30 s and FI 60 s than during CRF and FI 120 s (P B 0.05). Again, there was a main effect of schedule session, F(1, 34)= 7.48, P= 0.01. The mean of the first and second sessions were 54.6 (S.E.=8.3) and 44.0 (S.E.= 8.0) bursts, respectively. These results were qualified by a Groups × Schedules interaction, F(6, 102)= 7.04, P B 0.05. The interaction mean are presented in Fig. 4. The fish-food group had more bursts than the control group during both FI 30 s and FI s 60s, and more than the PCB-food group during FI 60 s (PB 0.05). There were no group differences during CRF and FI 120 s (P \0.05). In addition, only the fish-food showed differences across schedules. They had more bursts during FI 30 s than CRF and FI

Fig. 4. The Groups ×Schedules interaction found with the response bursts (presses with interresponse times 5 0.5 s) during the operant transition training as the density of reinforcements was decreased. Each group’s mean numbers of bursts ( 9 S.E.M.) are shown collapsed over the first and second sessions with each schedule.

Fig. 5. The triple interaction (Groups ×Associated Schedule ×Schedule Session) found with lever presses during the extinction (EXT) components of the transition schedules as the density of reinforcements for the associated fixed-interval (FI) components was decreased. Each group’s mean numbers of presses ( 9 S.E.M.) are shown for the first (a) and second (b) session with each multiple schedule.

120 s, and more during FI 60 s than the other three schedules (PB 0.05).

3.3.3. Transition extinction presses A 3× 3×2 mixed ANOVA (Groups× Associated Schedule× Schedule Session) was computed using each animal’s total numbers of lever presses during the EXT components of the transition training sessions. The main effect of groups was significant, F(2, 34)=5.37, P=0.009. The fish food (M= 43.6, S.E.= 5.7) pressed more than both the PCB-food (M= 19.7, S.E.=6.2) and control (M= 21.0, S.E.= 5.7) groups (PB0.05), which did not differ. There were also main effects of schedule sessions, F(1, 34)= 10.98, P= 0.002; and of associated schedule, F(2, 68)= 19.81, P B0.05. The first session mean was 32.8 (S.E.= 3.9), and the second was 23.3 (S.E.=3.5), presses. The mean over the associated transition schedules were 51.4, 18.3, and 14.5 presses (S.E.=8.0, 2.7, and 2.4) for FI 30 s, FI 60 s, and FI 120 s, respectively. Animals pressed more during the EXT components associated with FI 30 s than both FI 60 s and FI 120 s (PB0.05). However, there were significant Groups× Schedule, F(4, 68)=2.67, P= 0.039; and Groups× Associated Schedule× Schedule Session F(4, 68)=2.98, P=0.025, interactions. The triple interaction means are depicted in Fig. 5. Simple effects analyses of the triple interaction revealed that the numbers of EXT presses by the PCB-food, F(2, 68)= 16.6; fish-food, F(2, 68)=88.58; and control, F(2, 68)= 8.30; groups all decreased across the transition schedules, PB0.05. Pair-wise mixed ANOVA (Groups× Associated Schedule× Schedule Session) showed that the pattern of change for only the PCB-food and fish-food groups was different across transition sessions, F(2, 68)= 4.84, PB 0.05.

D.F. Berger et al. / Beha6ioural Brain Research 126 (2001) 1–11

4. Discussion The higher total PCB concentrations in the adipose tissue samples obtained from animals that ingested diets mixed with either environmental levels of Aroclor 1248 or contaminated St. Lawrence River fish, compared with those that ate uncontaminated food, provide a manipulation check on the exposure procedure. The levels in the control animals were barely detectable. The hypothesis that the exposed groups would become overactive and impulsive was supported. Their overactivity was shown by their tendency, at stablestate, to press the lever more often than unexposed control animals during the FI component of the mult schedule. The fact that these increases in pressing occurred primarily during the 90, and 120-s segments of the 2-min FI periods shows their impulsiveness by responding prematurely in anticipation of the end of the time interval prior to the next reinforcer. The exposed animals, particularly in the fish-food group, also produced relatively more response bursts than the control rats, which is another component of hyperactive/impulsive behavior [3]. These differences were observed even though response bursts were operationally defined as lever presses with IRTs5 0.5 s, which is half the size of the 1-s window used by Berger and Sagvolden [3]. That could account for the variability of the present data. The hyperactivity produced in the present study is in line with that earlier reported in animals exposed to PCBs [1,24–26]. For example, Rice [24– 26] found that monkeys exposed to PCBs performed less efficiently; that is, made two to three times more nonreinforced responses than reinforced responses. Like animals in the present study, PCB-treated monkeys also had shorter IRTs relative to controls. The results of the present study are also similar to those of Holene et al. [18]. Those authors exposed rat pups to PCB congeners 153 and 126 through mother’s milk and tested the pups later using the mult 2-min FI, 5-min EXT schedule of reinforcement. Animals in both congener groups made more lever presses during the 2-min FI component. However, only the animals exposed to PCB 153 (a di-ortho-substituted PCB) exhibited response bursts. The hyperactivity and bursting behavior of the PCB 153 group resembled the behavior of male SHRs reported by Berger and Sagvolden [3], Sagvolden et al. [31–34] and Sagvolden et al. [30] in AD/HD boys. Sagvolden et al. [30] hypothesized the existence of an altered reinforcement mechanism in AD/HD children (and SHRs) in which the reinforcement gradient is shorter and steeper. Research has indicated that AD/ HD children consistently choose small immediate rewards while non-diagnosed (comparison) children prefer larger rewards after a delay [13,45]. Catania et al. [7] had theorized that reinforcers act retroactively to

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increase the probability of operant responses that preceeded them. The assumption is that the effect is largest with immediate delivery of the reinforcer, and it is diminished by delay. This relationship between the reinforcing effect and delay time is known as the delay of reinforcement gradient [30]. The effect would be to make reinforcers closer in proximity to a response more effective in children with AD/HD and it would also produce response bursts. It is possible that exposure to PCBs, and the reductions in dopamine that tend to accompany exposure to ortho-substituted PCBs [37–41] produce such an altered reinforcement mechanism in rats that are not genetically hyperactive. The hypothesis that the exposed animals would have difficulty discriminating between the signaled FI and EXT components of the mult schedule, that is would lever press more often during the 5-min EXT periods than the control group, was not supported. Responding during EXT was infrequent in all three groups, indicating stimulus control. The absence of an effect of PCB exposure on this operant discrimination procedure agrees with the findings of Holene et al. [18]; and both findings are in contrast to the operant discrimination difficulties observed by Berger and Sagvolden [3], Sagvolden et al. [31–34], with SHRs. For example Berger and Sagvolden [3] reported that although the response rates of their male SHRs were relatively low during the first minute of the EXT periods, they increased conspicuously during the last 3 min. Holene et al. [17] did find poorer visual discrimination in male rats following pre- and post-natal exposure to PCB congener 126. However, they used a different operant procedure from that used both by Holene et al. [18] and in the present study. Further research is needed to determine the role of differences in operant procedures, routes of PCB exposure, or both, in accounting for these contradictory findings. Finally, the prediction that the exposed groups would overreact to the decreases in reinforcement density associated with the lever press training prior to the final mult 2-min FI, 5-min EXT schedule, compared with the control group, was supported. During the transition training the PCB-food and fish-food groups both pressed more often and produced more response bursts than the control group following the schedule changes. This reaction, reminiscent of the frustration effect [2], was also reflected in lever presses during the EXT components, and was particularly apparent in the fishfood group. Inspection of Figs. 4 and 5 suggests that these animals also took relatively longer to adapt to the reductions in reinforcement across the transition schedules than the PCB-food group, suggesting that another contaminant found in the fish may be involved. For example, the offspring of rats fed on Lake Ontario salmon had significant elevations of PCBs, DDE and Mirex [12]. Rice [22] also showed that the tissue levels

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D.F. Berger et al. / Beha6ioural Brain Research 126 (2001) 1–11

of both PCBs and methylmercury in Great Lakes fish were high enough to be neurotoxic. Both pre and postnatal exposure were associated with overreaction to reductions in the magnitude of reinforcement [12]. Animals in the present study that were exposed to fish responded prematurely in the FI, and like the animals in Daly et al. [12], they also overreacted to the reduction in reinforcement density (e.g. transition bursts). The difference between the PCB food group and the fish group in the present study suggests that the combination of toxicants in the fish could be influencing behavior by affecting different areas of the brain. The transition data of animals exposed to PCBs in the present study also replicated, with a different measure, earlier studies [10,11] with rats, and by Douglas and Parry [14] with AD/HD diagnosed boys. That is, animals exposed to PCBs in both of the Daly studies were very sensitive to reductions in the magnitude of reinforcement and overreacted (compared with control animals) to the reductions. The AD/HD boys were also sensitive to a decrease in the frequency of reinforcements, reacting with increased responding. According to Tilson et al. [49], hyperactivity is a common finding in animals exposed to PCBs, and the link between ortho-substituted PCBs and hyperactivity may be related to PCBs’ ability to alter dopamine levels. For example, in vitro studies [36,39,42] found reduced levels of cellular dopamine in PC 12 cells that were exposed to PCBs. In vivo studies with mice [1], monkeys [38] and rats [40] also found reduced levels of dopamine in the brains of exposed animals. The link between reduced levels of dopamine and hyperactivity in rats was well established by Shaywitz and associates [43,44]. In both studies, 5-day-old rat pups were treated with 6-hydroxydopamine (6-OHDA) which produced a rapid and profound reduction in brain dopamine. Shaywitz et al. [44] indicated that the increase in activity following reduction of brain dopamine is related to its function as a modulator of excitatory noradrenergic activity, and that the reduction of dopamine by 6OHDA ‘‘allows activity to occur unchecked’’ (p. 307). Shaywitz et al. [43] also showed that the increased activity levels of animals treated with 6-OHDA were reduced when the animals were treated with amphetamine. Exposure to PCBs may lead to alterations in behavior through mechanisms not related to dopamine depletion. For example, Holene et al. [18], found increased rates of responding during the FI in rats exposed to the coplanar 126 and the di-ortho substituted 153. The co planar PCBs are structurally similar to polychlorinateddibenzo-p-dioxins, bind to the Ah receptor and are highly toxic [28]. The coplanar PCBs also do not reduce dopamine [42]. The fact that rats exposed to PCB congeners 126 and 153 by Holene et al. showed increased responding during the FI, but only the 153

group produced response bursts, suggests that the congeners have different neurobehavioral effects. Another possible explanation of the present findings is that the exposure conditions could have changed the hedonistic value of the water reinforcer, that is made them thirstier. This could explain the higher rate of responding during the FI periods, but not the graduated increases over time seen in the present results. In addition, it would not explain the treated animals’ tendency to produce response bursts, similar to the genetically hyperactive SHRs. In light of the Holene et al. findings one would also have to assume that PCB congeners 126 and 153 differently affect thirst. Acknowledgements This research was supported by a grant from the Great Lakes Research Consortium. The authors would like to thank the following research assistants: Kim Krafczek, Joann Lammano Jefferson Lu, and Joseph Pizza, for their help with the present study. References [1] Agrawal AK, Tilson HA, Bondy SC. 3, 4, 3%, 4% tetrachlorobiphenyl given to mice prenatally produces long term decreases in striatal dopamine and receptor binding sites in the caudate nucleus. Toxicol Lett 1981;7:417 – 24. [2] Berger DF. Alternative interpretations of the frustration effect. J Exp Psychol 1969;81:475 – 83. [3] Berger DF, Sagvolden T. Sex differences in operant discrimination behavior in an animal model of Attention-Deficit Hyperactivity Disorder. Behav Brain Res 1998;94:73 – 82. [4] Bowman RE, Heironimus MP, Barsotti DA. Locomotor hyperactivity in PCB-exposed rhesus monkeys. Neurotoxicology 1981;2:251 – 68. [5] Casey AC, Berger DF, Lombardo JP, Hunt AE, Quimby F. Aroclor 1242 inhalation and ingestion by Sprague – Dawley rats. J Toxicol Environ Health 1999;56:101 – 32. [6] Catania AC. Learning. Englewood Cliffs, NJ: Prentice-Hall, 1992. [7] Catania AC, Sagvolden T, Keller KJ. Reinforcement schedules: retroactive and proactive effects of reinforcers inserted into fixed-interval performance. J Exp Anal Behav 1988;49:49 –73. [8] Chen YJ, Hsu CC. Effects of prenatal exposure to PCBs on the neurological function of children: a neuropsychological and neurophysiological study. Dev Med Child Neurol 1994;36:312 –20. [9] Chen YC, Yu ML, Rogan WJ, Gladen BC, Hsu CC. A 6-year follow-up of behavior and activity disorders in the Taiwan Yu-cheng children. Am J Public Health 1994;84:415 – 21. [10] Daly H. Reward reductions found more aversive by rats fed environmentally contaminated salmon. Neurotoxicol Teratol 1991;13:449 – 53. [11] Daly HB, Hertzler DR, Sargent DM. Ingestion of environmentally contaminated Lake Ontario salmon by laboratory rats increases avoidance of unpredictable aversive nonreward and mild electric shock. Behav Neurosci 1989;103:1356 – 65. [12] Daly HB, Stewart PW, Lunkenheimer L, Sargent D. Maternal consumption of Lake Ontario salmon in rats produces behavioral changes in the offspring. Toxicol Ind Health 1998;14:25 – 39.

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