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Effects of chronic lead administration on acquisition and performance of serial position sequences by pigeons
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
47, 377-384 (1979)
Effects of Chronic Lead Administration on Acquisition and Performance of Serial Position Sequences by Pigeons D. D. DIETZ, D. E. MCMILLAN,’
AND P. MUSHAK
Department of Pharmacology and Department of Pathology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514 Received March
30, 1978; accepted July 24, I978
Effects of Chronic Lead Administration on Acquisition and Performance of Serial Position Sequences by Pigeons. DIETZ, D. D., MCMILLAN, D. E., AND MUSHAK, P. (1979). Toxicol. Appl. Pharmacol. 47,377-384. Male White Carneaux pigeons were trained to peck three illuminated response keys in a predetermined four-step position sequence. For one group of birds the correct sequence was changed each day (repeated acquisition group) and for the other group of birds the sequence remained the same each day (performance group). Errors committed during periods of chronic intragastric sodium acetate (initial and recovery control) were compared to those errors recorded during chronic intragastric 6.25 mg/kg lead acetate intubation. Errors by birds in the performance group increased soon after lead intubations were begun while birds in the repeated acquisition group failed to demonstrate visible increases in errors, although a split middle trend analysis detected a statistically significant error increase during lead intervention in both groups. Peak blood concentrations for the performance group ranged between 588 and 1099 pg/dl and between 876 and 2320pg/dl for the repeated acquisition group. Decreases in blood lead concentrations were seen approximately 5 weeks after termination of lead exposure. Only one performance bird showed a satisfactory recoveryduring this nonlead period. These data suggest that the more stable error rates maintained under the performance schedule are more sensitive to the effects of lead than the less stable error rates maintained under the acquisition schedule.
Studies of lead-induced alterations in behavior have utilized measuresof activity levels (Silbergeld and Goldberg, 1974, ! 975), avoidance behavior (Sobotka et al., 1975, Sobotka and Cook, 1974; Overmann, 1977; Driscoll and Stegner, 1976; Weir and Hine, 1970), learning and memory (Brown, 1975; Overmann, 1977; Snowdon, 1973; Sobotka et al., 1975; Avery and Cross, 1974) and behavior controlled by schedulesof food presentation (Barthalmus et al., 1977; Padich and Zenick, 1977; Van Gelder et al., 1973; Carson et al., 1974). Focus has been on the subclinical, asymptomatic, insidious, or nonspecific 1 Please send reprint requests to: Dr. D. E. McMillan, Univ. of Arkansas for Medical Sciences, Dept. of Pharmacology, 4301 W. Markham, Little Rock, Arkansas 72201. 377
signs of lead intoxication (Overmann, 1977; Sobotka et al., 1975; Van Gelder et al., 1973). Theseconcentrations were considerably below those producing encephalopathy, colic, or blood disorders. In addition, most attention has been focused on chronic exposure of mice and rats in utero or perinatally as a sensitive model to produce behavioral alterations (Sauerhoff and Michaelson, 1973; Silbergeld and Goldberg, 1974, 1975). Recently, Barthalmus et al. (1977) studied the effects of intragastric administration of lead acetate on behavior of pigeons maintained under a multiple fixed-ratio fixedinterval schedule of food presentation. Decreasesin rate of responding and increased variability of responding were observed as a function of the dose. In addition, when lead All
0041-008X/79/020377-08802.00/0 Copyright 0 1979 by Academic Press, Inc. rights of reproduction in any form reserved. Printed in Great Britain
378
DIETZ,
MCMILLAN,
intubations were discontinued, responding sometimes continued to be disrupted for many days. In experiments such as those of Barthalmus et al. (1977) behavior is maintained at a steady state for a considerable period of time before toxic agents are administered. In an effort to study transitional behavior states, such as learning, Thompson (1973, 1974, 1977) has adapted procedures developed by Boren and Devine (1968) for determining drug effects on the repeated acquisition of serial position sequences by pigeons. This model requires the pigeon to peck three response keys in a predetermined order in a four-response chain that is changed from session to session. After a few months of training, the bird acquires the new response chain each session with a fairly constant number of errors. The repeated acquisition of response chains can be compared to a performance condition under which birds perform the same fourresponse sequence day after day (Thompson, 1974, 1977). Leander et al. (I 977) have used Thompson’s repeated acquisition procedure to study the effects of the chronic administration of mercuric chloride. The present experiments used a variation (Harting and McMillan, 1976a,b) of Thompson’s procedure to study the effects of the chronic administration of a low dose of lead acetate. Because Thompson’s data (1974, 1977), as well as those from our own laboratory (Harting and McMillan, 1977b; Barthalmus, 1978), suggest that behavior under certain acquisition schedules is more sensitive to drug effects than behavior under performance schedules used in these laboratories, we used the dose of lead acetate (6.25 mg/kg) that Barthalmus et al. (1977) found to be a threshold dose for producing effects on schedule-controlled responding by pigeons. METHODS Animals. Eight adult male White Carneaux pigeons (Palmetto Pigeon Plant, Sumpter, S.C.) served as subjects. An equal number of birds (four) were used in
AND
MUSHAK
both a repeated acquisition and a performance study. These pigeons were maintained at SO:, of their freefeeding weight (375-480 g) throughout all behavioral testing. The diet consisted of RalstonPurina mixed grain with crushed oyster shell supplied as a source of gizzard grit. Body weight was maintained by food delivered during test sessions with supplementary feeding occasionally after testing and on weekends, Dosing procedures. Solutions of lead acetate in water were prepared to give 6.25 mg/ml lead free from its salt form. An equivalent ionic strength of acetate prepared from sodium acetate served as a control solution. Intragastric lead administration occurred 7 days/week by passing a feeding needle down the esophagus, and past the crop and into the proventriculus. Administration on weekdays followed behavioral test sessions. Blood lead assay. Blood was collected each Wednesday by puncture of the radial vein. An anticoagulant, 5 y0 (w/v) sodium citrate in 0.9 % saline, was used to precoat syringes and acid-washed Teflon-sealed storage vials. All samples were stored at 4 to 5’C before lead determinations were made on a PerkinElmer Model 306 double beam spectrometer. The method of Delves (1970) as modified by Ediger and Coleman (1972) was used. Prior to analysis, the samples were diluted with Triton-X solution (low lead) and ultrasonicated to dispense aggregates and small clots. Quantitation was performed by the method of additions to internally compensate for matrix effects. Behavioral testing apparatus. The experimental chambers were designed after those developed by Ferster and Skinner (1957). They were sound attenuating and ventilated with three translucent response keys, 2 cm in diameter, mounted horizontally 2.5 cm apart on a false wall and 19 cm above the chamber floor. These keys were designated right (R), left (L), and center (C). A minimum force of I5 g was necessary to operate the keys whose closure defined a pecking response. Yellow, green, red, and blue 7.5-W lamps were used to transilluminate the keys. About 10 cm below the center response key and 4 cm above the chamber floor, a rectangular opening was located through which the pigeon could be given access to a Gerbrands feeder containing grain. The chamber was illuminated by a 15-W bulb. Conventional relay programming and recording apparatus were employed. Behacioraf procedures. Test sessions were started with the onset of a houselight and transillumination of the response keys, I5 min after a pigeon was placed in the test chamber. After the keys were transilluminated, the pigeon was required to make four pecks in a fixed sequence. Each correct response within a four-response sequence changed the key color in order from yellow to green to red to blue. A
CHRONIC LEAD EFFECTS ON PIGEON BEHAVIOR peck of one of the two incorrect keys resulted in a 2-set timeout signaled by all light in the chamber being off and responses having no programmed consequences. Each incorrect response also reset the four-response sequence to the initial step again. The completion of the four-response sequence activated the feeder for 0.4 set, a period too short to allow the pigeon to eat. Five correct repetitions of the four-response sequence were followed by activation of the feeder for 4 set, allowing sufficient time to eat. During the magazine cycle and presentation of food, the houselight and key lights were off and the grain hopper was illuminated by two 7.5-W bulbs. Sessions were terminated after thirty 4-set food deliveries. Thus a pigeon was required to make 600 correct responses (4 x 5 x 30) within a session. If the pigeon did not respond, the session was terminated after 6000 set had elapsed. Under the repeated acquisition schedule, the sequence of correct key positions changed from session to session. No position sequence was used in which a pigeon could make two successive correct responses on the same key (LL, CC, or RR). The sequences used were those of Harting and McMillan (1976b). Under the performance schedule, the requirements were the same as under the repeated acquisition schedule except that the same four-response sequence (RLCL) was maintained from session to session. Studies involving baseline responding and lead intervention were conducted during identical times for both schedules. Dam analysis. Dependent variables included the average rate of responding (responses/set) based on correct plus incorrect responses, the total errors made during a session, and the number of errors made in each successive block of 100 correct responses during the session. Time and responses accumulated during timeout did not enter into these calculations. A split middle method of trend estimation and analysis was used to determine the statistical significance of lead effects (Kazdin, 1976).
RESULTS Figure 1 shows the blood lead concentrations and the number of errors made under the performance schedule for three birds. Prior to lead administration, the blood concentrations were lessthan 5 pg/dl. Within 7 days after lead intubations were begun, blood concentrations were found to range between 101 and 340 pg/dl. Generally, these well-trained birds made fewer than 10 errors per day under the performance schedule prior to lead adminis-
379
tration and they never made more than 15 errors. When lead was administered, the number of errors made by bird 9847 gradually increased and reached a peak on the 53rd day. A split middle trend estimation indicated, at the ~~0.05 level of significance, an increased trend in errors during the first 67 days of lead treatment followed by a decreased trend in errors during the next 99 days even though lead continued to be administered. When lead administration was discontinued, further error reduction did not occur and blood lead levels remained high for about 5 weeks. Bird 3696 showed a fairly similar pattern although the effects of lead were much greater. After about 5 weeks of lead this bird was making about four times as many errors as under the control condition. Split middle trend analysis showed at the p < 0.05 level of significance an increased trend in errors during the first 49 days of lead administration followed by a decreased trend in error rate during the following 46 days of lead intervention. After the termination of lead administration the blood lead levels decreased gradually and trend analysis revealed a reduction in error rate at the p < 0.05 level of significance. Error levels during the recovery control period, however, remained above initial control levels. Bird 2066 failed to demonstrate statistically significant error increases during lead administration. More careful examination of the data however, did reveal eight lead sessionswith more than four errors, while only one control sessionapproached such a high value. These small effects did not disappear after the termination of lead administration even though the blood lead level gradually decreasedto the low values seen after about 5 weeks, as seen in birds 9847 and 3696. None of these birds ever developed any sign of gross toxicity, such as weight loss or increasesin crop fluid as observed by Barthalmus et al. (1977) after higher doses of lead, nor were there consistent changes in overall
380
DIETZ,
. .
MCMILLAN,
AND
MUSHAK
CHRONIC
LEAD
EFFECTS
ON PIGEON
BEHAVIOR
381
382
DIETZ,
MCMILLAN,
rates of responding. The criteria for termination of lead exposure was that an effect of lead on errors had been observed. The remaining bird studied under the performance schedule, bird 9649, died suddenly on the sixth day of lead administration. The bird was apparently healthy up until that time and a routine pathological examination failed to demonstrate the cause of death. The bird showed no signs of gross toxicity prior to death. Figure 2 shows the blood lead levels and the number of errors for the four birds made under the repeated acquisition schedule. Although the blood lead levels in the birds in the acquisition group were as high or higher (1360-2320 pg/dl) than the levels in the performance group, the absolute error increases as seen in the performance group during lead administration were not apparent in the birds of the acquisition group. Split middle trend analysis, however, did indicate significantly (p < 0.05) increased errors during lead intervention in birds 2781 and 6885 during the first lead intervention and in bird 2654 during the second lead intervention. Errors were significantly decreased during lead administration in bird 372. Three of the birds in the acquisition group showed evidence of gross toxicity during lead administration. Bird 2781 showed a considerable weight loss, developed excessive crop fluid, and stopped responding after 56 days of lead administration. Although lead administration was discontinued, the bird died 3 days later. Bird 2654 showed a large decrease in the rate of responding after 35 days of the first series of lead administrations, so lead administration was discontinued at Day 45 and behavior gradually recovered although excessive crop fluid was observed on Day 20 of the second control period. Lead dosing was reintroduced to attain a chronic level of lead equal to or greater than the two surviving birds of this group (6885 and 372). Bird 6885 began to decrease responding after 67 days of lead administration, showed excessive crop fluid after 68
AND
MUSHAK
days, and stopped all responding by Day 74. At that time lead administration was discontinued and responding gradually recovered. Bird 372 never exhibited signs of gross toxicity, although during lead administration the latency to respond increased dramatically, so that during 32’/, of the sessions the bird failed to obtain 30 food deliveries in 6000 sec. This never happened under the original control condition. The effect of lead on the progression of error elimination under the acquisition schedule was investigated. Since the same problem was repeated every 14th session, it was possible to compare performance on a given problem before, during, and after lead administration, Data on the within session error reduction is shown in Fig. 3 for birds 6885 and 372 for the sequence LCLR during
BIRD 68% 120
0
loo
l 1
80 60-
: OA
402Qo-
;:tt; 8 1
1
2
I
Ei
loo-
l ’
3
. ’
4
’
5
’
6
BIRD 372
80-
T
60 40.
P A .
123456
BLOCKS FIG. 3. Number of errors made during each block of 100 correct responses during individual sessions under the sequence LCLR. Filled circles are for sessions prior to lead exposure, open circles are for sessions during lead exposure, and filled triangles are for sessions after lead exposure.
CHRONIC
LEAD
EFFECTS
three sessions before lead, three or four sessions during lead administration, and two sessions after lead administration. Bird 2781 was not studied because his untimely death prohibited an analysis of error elimination during recovery, while bird 2654 was not considered because the lead intervention period was broken up into two shorter periods. Sessions during lead administration were chosen from periods during which the birds completed the session and no signs of gross toxicity were observed. Each successive time block represents 100 correct responses. During the early blocks of the session when most of the errors were being eliminated, there were no consistent differences between lead and control conditions. During the later blocks of the session, when the number of errors had stabilized, there tended to be a slightly greater number of errors made during lead administration. DISCUSSION The chronic administration of a low dose of lead increased the number of errors under the performance schedule as seen in Fig. 1. The baseline error rates were so stable that even very small increases in the number of errors, perhaps as few as five per session, could be detected. In contrast, the chronic administration of the low dose of lead did not produce visibly detectable increases in the less stable number of errors under the acquisition schedule as seen in Fig. 2. Figure 3 showed that early in the repeated acquisition session the effects of lead could not be observed, but later in the session when the number of errors had become less variable, increases in errors during lead administration could be observed. These data differ from some previous reports where behavior maintained by acquisition schedules was found to be more sensitive to the effects of drugs than behavior maintained by performance schedules (Thompson, 1974; 1977; Barthalmus, 1978). It is possible that the baseline rate of errors was a determinant of the lead effect. When
ON PIGEON
BEHAVIOR
383
the baseline rate of errors was low (under the performance schedule, or late in the session under the acquisition schedule), lead increased the errors. When the baseline rate of errors was high (early in the session under the acquisition schedule), lead had no reliable effects. It is also possible that the behavioral processes underlying performance are more sensitive to lead than those underlying acquisition. The performance schedule detected effects of lead on total errors, but the aquisition schedule did not. Furthermore, it might be argued that during the early blocks of the acquisition schedule, the acquisition process is taking place. Later in the session, when lead produces its effects, the sequence has been acquired and it is an effect on performance that is being measured. At any rate, behavior under the performance schedule appeared to be more sensitive to the effects of lead than behavior under the acquisition schedule, or under the multiple schedule used by Barthalmus et al. (1977). Analysis of trends in error rates using a split middle trend estimation technique, however, revealed a small but significant increase in errors for three out of four repeated acquisition birds during lead intervention. Thus lead induced increases in error trends are seen in spite of baseline variability or the type of schedule. Three of the four birds in the acquisition group showed signs of gross toxicity, but none of the birds in the performance group showed gross toxicity, although one bird in this group died from unknown causes. The reason for the gross toxicity in these three birds is unclear. In previous experiments (Barthalmus et al., 1977 ; Leander and Dietz, unpublished observations) we have dosed approximately 18 birds chronically with 6.25 mg/kg lead and have seen only one instance of gross toxicity. As can be seen in Fig. 1, lead levels determined in the whole blood of the birds are quite high, as we have also seen in earlier studies (Barthalmus et al., 1977). Separate studies are presently underway to study the underlying physiological mechanisms giving
384
DIETZ,
MCMILLAN,
rise to these levels, which are considerably above those encountered in other lead intoxication animal models. ACKNOWLEDGMENTS The authors would like to thank Ms. Peggy Hansen and Ms. Bonita Vines for their help in preparation of this manuscript and Ms. Cedonia Edwards for her
technicalassistance. This research was supported from the NIEHS.
AVERY,
D.
D.,
by Grant ES.01104
REFERENCES AND CROSS, H. A. (1974).The effects
of tetraethyl lead on behavior in the rat. Pharmacol. Bioehem. Behac. BARTHALMUS, G.
2, 473-479. LEANDER,
T.,
J. D., AND MCMILD. E. (1978). Combined effects of ethanol with diazepam on performance and acquisition of serial position sequences by pigeons. Psychopharmacology, 59, 101-102. BARTHALMUS, G. T. LEANDER, J. D., MCMILLAN, I,AN,
D.
E., MUSHAK,
P., AND KRIGMAN,
M. R. (1977).
Chronic effects of lead on schedule-controlled pigeon behavior. Toxicol. Appl. Pharmacol. 42, 271-284. BOREN, J. J., AND DEVINE, D. D. (1968). The repeated acquisition of behavioral chains. J. Exp. Anal. Behau. 11, 651-660. BROWN, D. R. (1975). Neonatal lead exposure in the rat: Decreased learning as a function of age and blood lead concentrations. Toxicol. Appl. Phar-
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MUSHAK
J., AND MCMILLAN, D. E. (1976b). Effects of pentobarbital and d-amphetamine on the repeated acquisition of response sequences by pigeons. Psychopharmacology 49, 245-248. KAZDIN, A. E. (1976). Statistical analyses for singlecase experimental designs. In Single Case Experi-
HAHTING,
mental Change
Designs:
Strategies
for
Studying
Behavior
(M. Hersen and D. H. Barlow, eds.), pp. 265-316. Pergamon, New York. LEANDER, J. D., MCMILLAN, D. E., AND BARLOW, T. S. (1977). Chronic mercuric chloride: Behavioral effects in pigeons. Environ. Res. 14, 424435. OVERMANN,
S. R. (1977). Behavioral effects of asymptomatic lead exposure during neonatal development in rats. Toxicol. Appt. Pharmacol. 41, 459-471. PADICH, R., AND ZENICK, H. (1977). The effects of developmental and/or direct lead exposure on FR behavior in the rat. Pharmacol. Biochem. Behac. 6, 371-375. SAUERHOFF,
M. W., AND MICHAEUON, I. A. (1973). Hyperactivity and brain catecholamines in leadexposed developing rats. Science 182, 1022-1024.
SILBERGELD,
E. K.,
Hyperactivity: Environ. Health SILBERGELD, E.
AND GOLDBERG,
A lead-induced Perspect
A. M. (1974).
behavior disorder.
7, 227-232.
K., AND GOLDBERG, A. M. (1975). Pharmacological and neurochemical investigations of lead-induced hyperactivity. Nearopharmacology
14,431-444. SNOWDON,
injected
C. T. (1973). Learning deficits in leadrats. Pharmacol. Biochem. Behav. 1,
599-603. SOBOTKA, T. J., BRODIE, R. E., AND COOK, M. P. macol. 32, 628-637. (1975). Psychophysiologic effects of early lead CARSON, T. L., VAN GELDER, G. A., KARAS, G. G., exposure. Toxicology 5, 175-191. AND BUCK, W. B. (1974).Developmentof behavioral tests for the assessment of neurologic effects of SOBOTKA, T. J., AND COOK, M. P. (1974).Postnatal lead acetate exposure in rats: Possible relationship lead in sheep. Environ. Health Perspect. 7,233-237. to minimal brain dysfunction. Amer. J. Ment. Def. DELVES, H. T. A. (1970). Micro-sampling method for the rapid determination of lead in blood and urine 79, 5-9. THOMPSON, D. M. (1973). Repeated acquisition as a by atomic absorption spectrometry. Analyst (London) 95, 43 I. behavioral baseline for studying drug effects. DRISCOLL, J. W., AND STEGNER, S. E. (1976). BehaJ. Pharmacol. Exp. Ther. 184, 506-514. THOMPSON, D. M. (1974). Repeated acquisition of vioral effects of chronic lead ingestion on laboratory behavioral chains under chronic drug conditions. rats. Pharmacol. Biochem. Behav. 4, 41 I-417. EDIGER, R. D., AND COLEMAN, R. S. (1972). Modified J. Pharmacol. Exp. Ther. 188, 700-713. Delves cup atomic absorption procedure for the THOMPSON, D. M. (1977). Development of tolerance to determination of lead in blood. At. Absorp. News/. the disruptive effects of cocaine on repeated 11, 33. acquisition and performance of response sequences. FERSTER, C. B., and SKINNER, B. F. (1957). Schedules J. Pharmacol. Exp. Ther. 203, 294-302. of Reinforcemen?. Appleton-Century-Crofts, New VAN GELDER, G. A., CARSON, T., SMITH, R. M., AND York. BUCK, W. B. (1973). Behavioral toxicologic assessHARTING, J., AND MCMILLAN, D. E. (1976a). Rement of the neurologic effect of lead in sheep. peated acquisition of response sequences by pigeons Clin. Toxicol. 6, 405-418. under chained and tandem schedules with reset and WEIR, P. A., AND HINE, C. H. (1970).Effectsof various non-reset contingencies. Psycholog. Rec. 26, metals on behavior of conditioned goldfish. Arch. 361-367. Environ. Health 20, 45-5 I.
AND
APPLIED
PHARMACOLOGY
47, 377-384 (1979)
Effects of Chronic Lead Administration on Acquisition and Performance of Serial Position Sequences by Pigeons D. D. DIETZ, D. E. MCMILLAN,’
AND P. MUSHAK
Department of Pharmacology and Department of Pathology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514 Received March
30, 1978; accepted July 24, I978
Effects of Chronic Lead Administration on Acquisition and Performance of Serial Position Sequences by Pigeons. DIETZ, D. D., MCMILLAN, D. E., AND MUSHAK, P. (1979). Toxicol. Appl. Pharmacol. 47,377-384. Male White Carneaux pigeons were trained to peck three illuminated response keys in a predetermined four-step position sequence. For one group of birds the correct sequence was changed each day (repeated acquisition group) and for the other group of birds the sequence remained the same each day (performance group). Errors committed during periods of chronic intragastric sodium acetate (initial and recovery control) were compared to those errors recorded during chronic intragastric 6.25 mg/kg lead acetate intubation. Errors by birds in the performance group increased soon after lead intubations were begun while birds in the repeated acquisition group failed to demonstrate visible increases in errors, although a split middle trend analysis detected a statistically significant error increase during lead intervention in both groups. Peak blood concentrations for the performance group ranged between 588 and 1099 pg/dl and between 876 and 2320pg/dl for the repeated acquisition group. Decreases in blood lead concentrations were seen approximately 5 weeks after termination of lead exposure. Only one performance bird showed a satisfactory recoveryduring this nonlead period. These data suggest that the more stable error rates maintained under the performance schedule are more sensitive to the effects of lead than the less stable error rates maintained under the acquisition schedule.
Studies of lead-induced alterations in behavior have utilized measuresof activity levels (Silbergeld and Goldberg, 1974, ! 975), avoidance behavior (Sobotka et al., 1975, Sobotka and Cook, 1974; Overmann, 1977; Driscoll and Stegner, 1976; Weir and Hine, 1970), learning and memory (Brown, 1975; Overmann, 1977; Snowdon, 1973; Sobotka et al., 1975; Avery and Cross, 1974) and behavior controlled by schedulesof food presentation (Barthalmus et al., 1977; Padich and Zenick, 1977; Van Gelder et al., 1973; Carson et al., 1974). Focus has been on the subclinical, asymptomatic, insidious, or nonspecific 1 Please send reprint requests to: Dr. D. E. McMillan, Univ. of Arkansas for Medical Sciences, Dept. of Pharmacology, 4301 W. Markham, Little Rock, Arkansas 72201. 377
signs of lead intoxication (Overmann, 1977; Sobotka et al., 1975; Van Gelder et al., 1973). Theseconcentrations were considerably below those producing encephalopathy, colic, or blood disorders. In addition, most attention has been focused on chronic exposure of mice and rats in utero or perinatally as a sensitive model to produce behavioral alterations (Sauerhoff and Michaelson, 1973; Silbergeld and Goldberg, 1974, 1975). Recently, Barthalmus et al. (1977) studied the effects of intragastric administration of lead acetate on behavior of pigeons maintained under a multiple fixed-ratio fixedinterval schedule of food presentation. Decreasesin rate of responding and increased variability of responding were observed as a function of the dose. In addition, when lead All
0041-008X/79/020377-08802.00/0 Copyright 0 1979 by Academic Press, Inc. rights of reproduction in any form reserved. Printed in Great Britain
378
DIETZ,
MCMILLAN,
intubations were discontinued, responding sometimes continued to be disrupted for many days. In experiments such as those of Barthalmus et al. (1977) behavior is maintained at a steady state for a considerable period of time before toxic agents are administered. In an effort to study transitional behavior states, such as learning, Thompson (1973, 1974, 1977) has adapted procedures developed by Boren and Devine (1968) for determining drug effects on the repeated acquisition of serial position sequences by pigeons. This model requires the pigeon to peck three response keys in a predetermined order in a four-response chain that is changed from session to session. After a few months of training, the bird acquires the new response chain each session with a fairly constant number of errors. The repeated acquisition of response chains can be compared to a performance condition under which birds perform the same fourresponse sequence day after day (Thompson, 1974, 1977). Leander et al. (I 977) have used Thompson’s repeated acquisition procedure to study the effects of the chronic administration of mercuric chloride. The present experiments used a variation (Harting and McMillan, 1976a,b) of Thompson’s procedure to study the effects of the chronic administration of a low dose of lead acetate. Because Thompson’s data (1974, 1977), as well as those from our own laboratory (Harting and McMillan, 1977b; Barthalmus, 1978), suggest that behavior under certain acquisition schedules is more sensitive to drug effects than behavior under performance schedules used in these laboratories, we used the dose of lead acetate (6.25 mg/kg) that Barthalmus et al. (1977) found to be a threshold dose for producing effects on schedule-controlled responding by pigeons. METHODS Animals. Eight adult male White Carneaux pigeons (Palmetto Pigeon Plant, Sumpter, S.C.) served as subjects. An equal number of birds (four) were used in
AND
MUSHAK
both a repeated acquisition and a performance study. These pigeons were maintained at SO:, of their freefeeding weight (375-480 g) throughout all behavioral testing. The diet consisted of RalstonPurina mixed grain with crushed oyster shell supplied as a source of gizzard grit. Body weight was maintained by food delivered during test sessions with supplementary feeding occasionally after testing and on weekends, Dosing procedures. Solutions of lead acetate in water were prepared to give 6.25 mg/ml lead free from its salt form. An equivalent ionic strength of acetate prepared from sodium acetate served as a control solution. Intragastric lead administration occurred 7 days/week by passing a feeding needle down the esophagus, and past the crop and into the proventriculus. Administration on weekdays followed behavioral test sessions. Blood lead assay. Blood was collected each Wednesday by puncture of the radial vein. An anticoagulant, 5 y0 (w/v) sodium citrate in 0.9 % saline, was used to precoat syringes and acid-washed Teflon-sealed storage vials. All samples were stored at 4 to 5’C before lead determinations were made on a PerkinElmer Model 306 double beam spectrometer. The method of Delves (1970) as modified by Ediger and Coleman (1972) was used. Prior to analysis, the samples were diluted with Triton-X solution (low lead) and ultrasonicated to dispense aggregates and small clots. Quantitation was performed by the method of additions to internally compensate for matrix effects. Behavioral testing apparatus. The experimental chambers were designed after those developed by Ferster and Skinner (1957). They were sound attenuating and ventilated with three translucent response keys, 2 cm in diameter, mounted horizontally 2.5 cm apart on a false wall and 19 cm above the chamber floor. These keys were designated right (R), left (L), and center (C). A minimum force of I5 g was necessary to operate the keys whose closure defined a pecking response. Yellow, green, red, and blue 7.5-W lamps were used to transilluminate the keys. About 10 cm below the center response key and 4 cm above the chamber floor, a rectangular opening was located through which the pigeon could be given access to a Gerbrands feeder containing grain. The chamber was illuminated by a 15-W bulb. Conventional relay programming and recording apparatus were employed. Behacioraf procedures. Test sessions were started with the onset of a houselight and transillumination of the response keys, I5 min after a pigeon was placed in the test chamber. After the keys were transilluminated, the pigeon was required to make four pecks in a fixed sequence. Each correct response within a four-response sequence changed the key color in order from yellow to green to red to blue. A
CHRONIC LEAD EFFECTS ON PIGEON BEHAVIOR peck of one of the two incorrect keys resulted in a 2-set timeout signaled by all light in the chamber being off and responses having no programmed consequences. Each incorrect response also reset the four-response sequence to the initial step again. The completion of the four-response sequence activated the feeder for 0.4 set, a period too short to allow the pigeon to eat. Five correct repetitions of the four-response sequence were followed by activation of the feeder for 4 set, allowing sufficient time to eat. During the magazine cycle and presentation of food, the houselight and key lights were off and the grain hopper was illuminated by two 7.5-W bulbs. Sessions were terminated after thirty 4-set food deliveries. Thus a pigeon was required to make 600 correct responses (4 x 5 x 30) within a session. If the pigeon did not respond, the session was terminated after 6000 set had elapsed. Under the repeated acquisition schedule, the sequence of correct key positions changed from session to session. No position sequence was used in which a pigeon could make two successive correct responses on the same key (LL, CC, or RR). The sequences used were those of Harting and McMillan (1976b). Under the performance schedule, the requirements were the same as under the repeated acquisition schedule except that the same four-response sequence (RLCL) was maintained from session to session. Studies involving baseline responding and lead intervention were conducted during identical times for both schedules. Dam analysis. Dependent variables included the average rate of responding (responses/set) based on correct plus incorrect responses, the total errors made during a session, and the number of errors made in each successive block of 100 correct responses during the session. Time and responses accumulated during timeout did not enter into these calculations. A split middle method of trend estimation and analysis was used to determine the statistical significance of lead effects (Kazdin, 1976).
RESULTS Figure 1 shows the blood lead concentrations and the number of errors made under the performance schedule for three birds. Prior to lead administration, the blood concentrations were lessthan 5 pg/dl. Within 7 days after lead intubations were begun, blood concentrations were found to range between 101 and 340 pg/dl. Generally, these well-trained birds made fewer than 10 errors per day under the performance schedule prior to lead adminis-
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tration and they never made more than 15 errors. When lead was administered, the number of errors made by bird 9847 gradually increased and reached a peak on the 53rd day. A split middle trend estimation indicated, at the ~~0.05 level of significance, an increased trend in errors during the first 67 days of lead treatment followed by a decreased trend in errors during the next 99 days even though lead continued to be administered. When lead administration was discontinued, further error reduction did not occur and blood lead levels remained high for about 5 weeks. Bird 3696 showed a fairly similar pattern although the effects of lead were much greater. After about 5 weeks of lead this bird was making about four times as many errors as under the control condition. Split middle trend analysis showed at the p < 0.05 level of significance an increased trend in errors during the first 49 days of lead administration followed by a decreased trend in error rate during the following 46 days of lead intervention. After the termination of lead administration the blood lead levels decreased gradually and trend analysis revealed a reduction in error rate at the p < 0.05 level of significance. Error levels during the recovery control period, however, remained above initial control levels. Bird 2066 failed to demonstrate statistically significant error increases during lead administration. More careful examination of the data however, did reveal eight lead sessionswith more than four errors, while only one control sessionapproached such a high value. These small effects did not disappear after the termination of lead administration even though the blood lead level gradually decreasedto the low values seen after about 5 weeks, as seen in birds 9847 and 3696. None of these birds ever developed any sign of gross toxicity, such as weight loss or increasesin crop fluid as observed by Barthalmus et al. (1977) after higher doses of lead, nor were there consistent changes in overall
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rates of responding. The criteria for termination of lead exposure was that an effect of lead on errors had been observed. The remaining bird studied under the performance schedule, bird 9649, died suddenly on the sixth day of lead administration. The bird was apparently healthy up until that time and a routine pathological examination failed to demonstrate the cause of death. The bird showed no signs of gross toxicity prior to death. Figure 2 shows the blood lead levels and the number of errors for the four birds made under the repeated acquisition schedule. Although the blood lead levels in the birds in the acquisition group were as high or higher (1360-2320 pg/dl) than the levels in the performance group, the absolute error increases as seen in the performance group during lead administration were not apparent in the birds of the acquisition group. Split middle trend analysis, however, did indicate significantly (p < 0.05) increased errors during lead intervention in birds 2781 and 6885 during the first lead intervention and in bird 2654 during the second lead intervention. Errors were significantly decreased during lead administration in bird 372. Three of the birds in the acquisition group showed evidence of gross toxicity during lead administration. Bird 2781 showed a considerable weight loss, developed excessive crop fluid, and stopped responding after 56 days of lead administration. Although lead administration was discontinued, the bird died 3 days later. Bird 2654 showed a large decrease in the rate of responding after 35 days of the first series of lead administrations, so lead administration was discontinued at Day 45 and behavior gradually recovered although excessive crop fluid was observed on Day 20 of the second control period. Lead dosing was reintroduced to attain a chronic level of lead equal to or greater than the two surviving birds of this group (6885 and 372). Bird 6885 began to decrease responding after 67 days of lead administration, showed excessive crop fluid after 68
AND
MUSHAK
days, and stopped all responding by Day 74. At that time lead administration was discontinued and responding gradually recovered. Bird 372 never exhibited signs of gross toxicity, although during lead administration the latency to respond increased dramatically, so that during 32’/, of the sessions the bird failed to obtain 30 food deliveries in 6000 sec. This never happened under the original control condition. The effect of lead on the progression of error elimination under the acquisition schedule was investigated. Since the same problem was repeated every 14th session, it was possible to compare performance on a given problem before, during, and after lead administration, Data on the within session error reduction is shown in Fig. 3 for birds 6885 and 372 for the sequence LCLR during
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BLOCKS FIG. 3. Number of errors made during each block of 100 correct responses during individual sessions under the sequence LCLR. Filled circles are for sessions prior to lead exposure, open circles are for sessions during lead exposure, and filled triangles are for sessions after lead exposure.
CHRONIC
LEAD
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three sessions before lead, three or four sessions during lead administration, and two sessions after lead administration. Bird 2781 was not studied because his untimely death prohibited an analysis of error elimination during recovery, while bird 2654 was not considered because the lead intervention period was broken up into two shorter periods. Sessions during lead administration were chosen from periods during which the birds completed the session and no signs of gross toxicity were observed. Each successive time block represents 100 correct responses. During the early blocks of the session when most of the errors were being eliminated, there were no consistent differences between lead and control conditions. During the later blocks of the session, when the number of errors had stabilized, there tended to be a slightly greater number of errors made during lead administration. DISCUSSION The chronic administration of a low dose of lead increased the number of errors under the performance schedule as seen in Fig. 1. The baseline error rates were so stable that even very small increases in the number of errors, perhaps as few as five per session, could be detected. In contrast, the chronic administration of the low dose of lead did not produce visibly detectable increases in the less stable number of errors under the acquisition schedule as seen in Fig. 2. Figure 3 showed that early in the repeated acquisition session the effects of lead could not be observed, but later in the session when the number of errors had become less variable, increases in errors during lead administration could be observed. These data differ from some previous reports where behavior maintained by acquisition schedules was found to be more sensitive to the effects of drugs than behavior maintained by performance schedules (Thompson, 1974; 1977; Barthalmus, 1978). It is possible that the baseline rate of errors was a determinant of the lead effect. When
ON PIGEON
BEHAVIOR
383
the baseline rate of errors was low (under the performance schedule, or late in the session under the acquisition schedule), lead increased the errors. When the baseline rate of errors was high (early in the session under the acquisition schedule), lead had no reliable effects. It is also possible that the behavioral processes underlying performance are more sensitive to lead than those underlying acquisition. The performance schedule detected effects of lead on total errors, but the aquisition schedule did not. Furthermore, it might be argued that during the early blocks of the acquisition schedule, the acquisition process is taking place. Later in the session, when lead produces its effects, the sequence has been acquired and it is an effect on performance that is being measured. At any rate, behavior under the performance schedule appeared to be more sensitive to the effects of lead than behavior under the acquisition schedule, or under the multiple schedule used by Barthalmus et al. (1977). Analysis of trends in error rates using a split middle trend estimation technique, however, revealed a small but significant increase in errors for three out of four repeated acquisition birds during lead intervention. Thus lead induced increases in error trends are seen in spite of baseline variability or the type of schedule. Three of the four birds in the acquisition group showed signs of gross toxicity, but none of the birds in the performance group showed gross toxicity, although one bird in this group died from unknown causes. The reason for the gross toxicity in these three birds is unclear. In previous experiments (Barthalmus et al., 1977 ; Leander and Dietz, unpublished observations) we have dosed approximately 18 birds chronically with 6.25 mg/kg lead and have seen only one instance of gross toxicity. As can be seen in Fig. 1, lead levels determined in the whole blood of the birds are quite high, as we have also seen in earlier studies (Barthalmus et al., 1977). Separate studies are presently underway to study the underlying physiological mechanisms giving
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rise to these levels, which are considerably above those encountered in other lead intoxication animal models. ACKNOWLEDGMENTS The authors would like to thank Ms. Peggy Hansen and Ms. Bonita Vines for their help in preparation of this manuscript and Ms. Cedonia Edwards for her
technicalassistance. This research was supported from the NIEHS.
AVERY,
D.
D.,
by Grant ES.01104
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