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Chronic effects of lead on schedule-controlled pigeon behavior
Chronic
Effects
of Lead
on Schedule-Controlled Behavior’
Pigeon
GEOKGE T. BAKTHAL.ML:S.~ J. DAVII) Lt-ANI)I-I<.’ DONAL.I)E. Mc.MII.I..\N.’ P \I I ML;SHAK.~ANL)MAIWN R. KKIC;MAN~
Chronic Effects of Lead on Schedule-Controlled Pigeon Beha\lor. B \KIII \I XII \. G. T.. Lt .\uI),:K. .I. D.. McM11.1 A\. D. E.. Mu >,I \K. P.. \NI) KICK,\! \N. M. R. ( lY77). Toxicol. ./lppI. Phnr~nacol. 42, 271-284. Pigeons were trained to peck a response key under a multiple tixed ratio 30 response (FR 30) fixed-interval 5-min (Fl 5) schedule of food presentation. When rate\ of responding stabilized, lead acetate (6.25. 12.5. and 25 mg/kg body weight) or sodium acetate solutions were given daily by gastric intubation. Blood-lead concentrations were measure? weekly by atomic absorption spcctrometry. and responding under the multiple schedule wab measured Monday through Friday. Control lntubations of sodium acetate did not atl’ect responding under either component of the schedule. The 25-mg/kg dose of lead decreased rate\ of responding after 3 to IO days of administration and usually was lethal between 18 and 35 Jays. Post mortem examination of the birds ahowed obvious esophageal dilatation. damage to the crop. discontinued but rates
weight loss. in some of responding
and aubarachnoid hemorrhages. Admimstration of the birds after responding had ceased. Responding gradually remained low and variable under both components
25-mg/kg dose was began to recover. of the schedule for
many days. Daily administration of the l2.5-mg/kg dose of lead caused one death. but no obvious signs of intoxication occurred in the other birds. However, three of the four bird, yurbiving daily l2.5-mg/kg doses of lead showed decreased rates of responding under both schedule components within 30 days of the initial intubation. The fourth bird showed a high11 1 ariable rate under the FI component of the schedule during lead administration: however. when lead administration was discontinued. dramatic increases in ra!es of responding above lead pretreatment levels occurred. particularly under the FI component. These rate increase\ persisted for 75 days thereafter. The 6.25-mgikg dose of lead produced only small changes in rates of responding during 70 days of intubation. Blood-lead concentrations were very high ( 150-3470 pgldl) during treatment with all doses of lead. Increasing doses produced increasing blood concentrations which were correlated with increasing behavioral effects. although correlations individual
between birds.
blood-lead
concentrations
’ Supported by Grant No. ES01 104 from ’ Department of Zoology. North Carolina ’ Department of Pharmacology, School Carolina 275 14. To whom reprint requests ’ Department of Pharmacology. School Carolina 275 14. ’ Department of Pathology. School of Carolina 27514.
and
behavioral
the NIEHS. State University. Raleigh, North of Medicine. University of North should be addressed. of Medicine. University of North Medicine.
University
of North
changes
were
low
within
Carolina Carolina,
27607. Chappel
I Hill.
North
Carolina.
Chappel
Hill.
North
Hill.
North
Carolma.
Chappel
272
BARTHALMUS
ET AL
Considerable research has been focused on the pathological, physiological, and neurological consequences of both acute and chronic exposure to lead (Bouldin and Krigman, 1975; Goyer and Rhyne, 1975; Goyer and Wilson, 1975; Krigman et al., 1972, 1974; Mahaffey et al., 1973). However, there are few controlled experiments on the behavioral effects of lead ingestion. Lead-induced hyperactivity in mice has been suggested as a model for studying drug responses in children with minimal brain dysfunction hyperactivity (Silbergeld and Goldberg, 1974, 1975). Deficits in the learned behavior of animals including visual discrimination (Carson et al., 1974). conditioned avoidance responding (Weir and Hine, 1970), and maze learning (Brown, 1975: Bullock ef al., 1966; Snowdon, 1973) have been reported, but there have been few attempts to use schedule-controlled behavior to assess the effects of chronic exposure to lead. The present experiments were performed to examine the effects of chronic oral lead administration on responding maintained by schedules of food presentation to the pigeon.
METHODS
Animals Adult male White Carneaux pigeons (Palmetto Pigeon Plant, Sumter, South Carolina) were maintained at 80% of their free-feeding weights throughout all experiments. Their 80% body weights ranged from 410 and 480 g. Birds were fed mixed grain (Ralston-Purina pigeon diet), and crushed oyster shell was freely available in home cages as a source of gizzard grit. Water was freely available both in home cages and test chambers. Materials and Dosing Lead solutions were prepared weekly for administration as 6.25, 12.5, and 25.0 mg of lead (as lead acetate)/kg body weight. Solutions of sodium acetate served as a control intubation solution. Birds were given lead or sodium acetate 7 days/week by oral intubation into the proventriculus by passing a vinyl catheter down the esophagus and past the crop. Intubations followed the test sessions on weekdays.’ Determination
of Blood-Lead
Concentrations
Blood samples from the radial vein were obtained each Wednesday after the test sessions. Syringes and acid-washed, Teflon-sealed storage vials were precoated with anticoagulant consisting of 5% w/v sodium citrate in 0.9% saline. Samples were frozen immediately and stored until lead determinations were made by atomic absorption spectrometry employing a Perkin-Elmer Model 306 double-beam instrument. The method of Delves (1970) as modified by Ediger and Coleman (1972) was used. Ten- to twenty-microliter samples of titrated whole blood. with or without prior dilution, were delivered in lead-free nickel crucibles, preignited in the cool portion of the flame and then directly inserted into the flame for lead atomization. An aligned deuterium arc background correction accessory minimized signal errors. Levels of total lead in the oyster shell and pigeon gram also were determined.
LEAD
AND
PIGEON
BEHAVIOR
273
Histology Tissues removed at necropsy were fixed in formalin for light microscopy and some tissues, peripheral nerve, kidney, liver, and lung, were selected for electron microscopy. Tissues for electron microscopy were initially fixed in 10% formalin, postfixed in 1% osmium tetraoxide in 0.1 M phosphate buffer, dehydrated in alcohol and propylene oxide, and embedded in an Epon (D.E.R. 732) plastic. Thin sections were stained with many1 acetate. Apparatus for Behavioral
Testing
Behavioral testing was conducted in sound- and light-attentuated chambers similar to those described by Ferster and Skinner (1957). The chamber was illuminated by a 7.5. W light (ac) and contained a translucent plastic response key (2.0 cm in diameter) which was centrally mounted in the chamber wall 22 cm above a wire-mesh floor. The response key could be transilluminated by red or blue lights. The minimal force required to operate the key was about 0.15 N. Centrally below the key and 6 cm above the floor was a rectangular opening through which the pigeon could be given access to mixed grain. Relay programming and recording apparatus housed in the adjacent room controlled the delivery of grain and recorded the pattern of key pecking. A masking noise was supplied by a speaker mounted inside the chamber. Procedure Pigeons performed Monday through Friday under a multiple fixed-ratio, fixedinterval schedule (mult FR FI) of food presentation. In the presence of a blue light, the 30th key peck (FR 30) produced 4-set access to grain. In the presence of the red light, the first response after 5 min (FI 5) produced 4-set access to grain. The FR and FI components alternated after each food presentation. If a bird failed to respond within 40 set after 5 min had elapsed during the FI component, the schedule changed to the FR component. If the bird failed to make 30 responses within 40 set during the FR component, the schedule changes to the FI component. Sessions were started in the FR component 10 min after the pigeon was placed in the chamber and were terminated the first time the schedule component changed after an hour of testing had elapsed. Birds were weighed before each session and were weighed and dosed immediately following each session except Wednesday when dosing followed the blood sampling. Evaluation Average rates of responding for the I-hr sessions were computed separately for the FR and FI components from data recorded on digital counters and elapsed-time meters. The distribution of responses within the FI component was obtained by dividing the interval into 10 30.set segments and recording the number of responses in each segment on counters. These data were used to calculate the quarter-life value, a statistic which is independent of response rate and is used to describe quantitatively the positively accelerated pattern of responding that occurs under the FI schedule component. The quarterlife is defined as the percentage of the FI taken to emit 25% of all responses in the FI (Gollub, 1964). All pigeons were tested for 40-50 nontreatment sessions to establish a stable control baseline of behavior. Then they began the experiment, with some birds remaining as
214
BARTHALMUS
ET AL
untreated controls, whereas others were treated with either sodium acetate or lead. Data points on the figures which fell outside the range of the untreated controls and/or the sodium acetate control ranges were considered to be behavioral effects of lead. RESULTS Figures l-5 show the response rate data of 14 birds. The quarter-life no trends and, therefore, are not shown in the figures.
values showed
0
A FR-30
BIRD
5610
FR-30
BIRD
1469
FI-5
SUCCESSIVE
DAYS
SUCCESSIVE
C BIRD
FR- 30 3r
25
ACET.
;
DAYS
4756
25 LEAD
t OL
1
5
’
IO I5
8’
SUCCESSIVE
6
5
’
IO
’
15
’
20
sL
25
’
30
35
DAYS
FIG. I Effects of 3-5 mg/kg of lead on the average rate of responding FR 30 FI 5 schedule of food presentation. Bird 1469 (B) was given times the volume of water given to bird 56 10 (A) and bird 4758 (C). lead, Abscissa. successive ahsociatcd with the FR micrograms per deciliter.
days: ordinate. 30 rates of
mean responding
rate
under each component of the mult on 2S-mg/kg lead intubation in t&o All pigeons died from treatment with
of responding during indicate Wednesday
a complete blood-lead
session. Numbers concentrations in
Figure 1 shows the response rates under the FR 30 and FI 5 schedule of food presentation fcr three pigeons that died following 17-33 days of 25-mg/kg intubations. Rates of responding decreased for all birds within 7 days of exposure to lead. Gross effects. observed 8-20 days following the response-rate decreases, included the copious
LEAD
AND
PIGEON
275
BEHAVIOR
accumulation of crop fluid, esophageal dilatation, and a large weight loss. At mortem examination, the crop of each bird was distended with grain; however, the had not entered the gizzard. The brains of all three birds were covered with subarachnoid hemorrhage. Gross effects were seen first in birds 5610 and 4758, and since these effects may resulted from the high concentration of lead acetate dissolved in distilled water,
FIG. 2. Effects of 25 mg/kg of lead on the average FR 30 Fl 5 xhedule of food presentation. Bird 110 Icad. Other details as in Fig. 1.
rate (A),
of responding under 3933 (B). and 4619
post grain thin have bird
each component of the mult (C) survived treatment with
1469 received the 25.mg/kg dose in twice the volume of water. Again, response-rate decreases under both the FR and FI components were soon followed by gross signs and death. Figure 2 shows the data for three pigeons which did not die from the repeated administration of 25 mg of Pb/kg. Birds 110, 3933, and 4619 were dosed for 52, 43, and 13 days, respectively. Within 2 days, birds 110 and 3933 showed obvious changes in the rates of responding under both the FR and FI components. Throughout the period of lead dosage, bird 110 exhibited a moderate decrease in rates under the FR component. while rates under the Fl tended to increase slightly, but with wide daily
276
BARTHALMUS ET.4l..
variation. When lead intubations were discontinued, the rates of responding under the FR component continued to fall and then partially recovered. However, the FR rates remained below the baseline rates for 75 days after lead administration was discontinued. In contrast, the rates under the FI tended to remain within the control range. The rates of responding under the FR and FI components of bird 3933 declined during lead exposure until responding was abolishedafter about 3.5days of exposure. A
FIG. 3. Effects of 12.5 mg,/kg of lead on the average rate of responding under each component of the mult FR 30 and FI 5 schedule of food presentation. Bird 5775 (A) died during treatment with lead; birds 4522 (B) and 3594 (C) survived treatment with lead. Other details as in Fig. I.
few days later, lead intubations were discontinued. After 31 days without any appreciable responding, key pecking began to recover under both FR and Fl components. Rates under the FR returned to and remained at baselinefor 37 days but then dropped to a consistent subbaselinelevel for the remaining 48 days. However. when responding under the FI returned, it was highly variable for about 25 days after which the rates fell below baseline for the remaining 60 days. Excessive crop fluid was observed in bird 3933 during the period when responding had ceased;however, bird 110 and 46 19 showedno grosssigns. Bird 46 19. who received control intubations for 55 days, showed no change in either
ILAD
AND
PIGEON
BEHAVIOK
277
schedule component during this time. nor during the following 13 days of lead intubations and the 36.day period without intubations. Three pigeons treated with 12.5 mg/kg of lead are shown in Fig. 3. Bird 5715. who died after 42 days, showed decreased rates of responding under both schedules after 15 days of exposure. The autopsy showed evidence of subarachnoid hemorrhage but excessive crop fluid. esophageal dilatation, and weight loss did not occur.
Y r
FIG. 4. Effects of 12.5 mg/kg of lead on the average rate of responding under each component of the mult FR 30 FI 5 schedule of food presentation. Bird 7773 (A) and bird 5397 (B) were given lead: bird 2923 (C)was given only sodium acetate. All birds survived the treatment. Other details as in Fig. I.
After 27 days of lead intubation, the rate of responding under the FI component of bird 4522 increased, whereas the response rate under the FR component was un changed. Continued lead administration led to rate decreasesunder both schedule components, although rates were near normal during some sessionsduring the last 30 days of lead administration. Rates of responding by bird 4522 gradually recovered under both schedulecomponentsduring the 42 days following lead intubation. The rates of responding by bird 3594 decreasedunder both the FR and FI schedule components after 2 1 days of lead intubations. The rates of responding decreasedabout 30% over a 19-day span and then remained relatively constant for the remaining 41 days of treatment. When lead administration was discontinued, 25 days elapsedwithout signsof a return to baselineperformance.
278
t.7’ -II..
HAKTH.4LMUS
Figure 4 shows the data from two additional birds given 12.5 mg of Pb/kg. A third bird. bird 2923, received sodium acetate as a control for 90 days without behavioral disruptions. Bird 7773 showed a temporary decrease in the rates under the FR schedule between 7 and IO days of exposure, a return to baseline rates between Days 11 and 34. and then the rates of responding decreased or were abolished for the remaining 24 days. When lead was discontinued, the rates of responding under the FR schedule recovered quickly, and most of the subsequent sessions were characterized by rates above the baseline rate.
FIG. mult
5. Effects
of 6.25
FR 30 FI 5 schedule
mg/kg
of Lead
of food
presentation.
on
the average Both
birds
rate
of responding
survived
under
the treatment.
each Other
component details
of the
as in Fig.
I.
The rates of responding under the FI component by bird 7773 were less affected than the FR rates, although the FI rates did decrease after 40 and 48 days of lead intubation. The discontinuation of lead produced no further changes in rates of responding under the FI component over 55 days of testing. Pigeon 5397 showed no consistent changes in rates under the FR after 71 days exposure to 12.5 mg of Pb/kg, whereas the rates under the FI became more variable and were often higher than control rates. After 22 days without lead intubations. rates of responding under the FR component exceeded control levels and remained elevated for 73 days of testing. After discontinuation of lead administration, rates under the FI exceeded baseline values almost immediately. maintaining levels nearly double those of the control sessions. These high rates were maintained for more than 90 days. Figure 5 shows two pigeons treated with 6.25 mg of Pb/kg for 72 days. In bird 855 the rates under the FI component were decreased immediately after lead intubation started. but the rates were only slightly below baseline. The rate under the FR component was unaffected. In bird 1764, there was a slight increase in the rates under the FR component. whereas the rate under the FI was largely unaffected.
LEAD
AND
PIGEON
279
BEHAVIOK
Table 1 summarizes the effects of lead on the schedule-controlled behavior of the pigeon. Both rate increases and rate decreases were observed under both schedule components, although rate decreases were the predominant effect in 80% of the cases where rate changes were observed regularly. There was no evidence that behavior controlled by one schedule component was more sensitive to the effects of lead than the other. The mean number of intubation days elapsed to produce a behavioral effect at the 25. 12.5. and 6.25 mg of Pb/kg doses were 3.8. 15.8 and 17.0 days, respectively, suggesting that the behavioral effects of lead were dose-dependent. TABLE
1
SUMMAKY OF EFFECTS OF LEAD ON RATI’ OF RI~SPONDING OF PIGEONS
Initial effect observed Dose Bird
hdkd
Day effect established
FR
FI
Predominanteffect over days -~
FR
FI
” Eventually died. ” +. Rate increases: I rate decreases. ’ No effect. ” Given sodium acetate only.
The weekly blood-lead concentrations are shown in Figs. l-5 above the response data for the FR component. Three of the birds receiving 25 mg of Pb/kg in Figs. 1 and 2 exceeded 100 Ergof Pb/dl of whole blood, with bird 110 reaching a peak blood level of 3400 pugof Pb/dl. The blood-lead concentrations appeared to be dose-related since lower levels were found in the birds receiving 12.5 and 6.25 mg of Pb/kg. Nevertheless. the two birds receiving 6.25 mg of Pb/kg still attained peak blood-lead levelsof 285 and 4 I2 ,ugof Pb/dl. Table 2 shows that there was not a strong correlation between blood concentration and behavioral effects within individual birds, despite the fact that both the blood concentration and the behavioral effect were dose-relatedacrossbirds. Neither the initial behavioral effect nor the maximum behavioral effect occurred at the time of peak blood lead concentrations for most birds. Histologic examination revealed no specific organ changesexcept in the kidney. The sciatic nerves were within normal limits. The brains showed no changes except for
280
BARTHALMUS
TABLE COMPARISON
OF BLOOD-LEAD
ET .4L.
2
CONCENTRATIONS AND RESPONDING IN PIGEONS
Blood-lead
Bird 5610” 1469” 4758” 110
3933 1113 5397 5715” 4522 3594 855 I164 2923”
Dose (w/kg)
At time of initial behavioral effect
25 25 25 25 25 12.5 12.5 12.5 12.5 12.5 6.25 6.25 0
” Eventually died. h Given sodium acetate only. C Range of observations (no behavioral
503 491 IO1 408 339 427 429 414 353 280 285 311 -
CHANGES
IN RATES
concentrations
At time of maximum behaviorial effect .--~ 923 380 918 608 269 175 71 513 409 145 285 193
OF
@g/dl) Highest concentration observed 1235 624 918 3415 1147 441 848 569 409 398 285 412 17-64k’
change).
FIG. 6. Electron photomicrograph depicts two red ceils in a pulmonary capillary, one of which containb a nuclear lead-inclusion body (arrow). Formalin-fixed necropsy tissue. postfixed with osmium tetraoxide. and the ultrathin sections were stained with uranyl acetate. Magnification Y 1.98 x IO’.
LEAD
AND
PIGEON
BEHAVIOR
281
congestion and recent subarachnoid hemorrhage in the three pigeons that died on the 25-mg/kg dose schedule. Light microscopy examination revealed typical lead-inclusion bodies in the proximal convoluted tubules of the kidney, and by electron microscopy, inclusion bodies were also identified in erythrocyte nuclei. The erythrocytic nuclear inclusions (Fig. 6) were circular to oval, electron-dense bodies, which were distinctly separate and different from the heterochromatin, nucleolus, and nuclear hemoglobin. Occasionally a red-cell nucleus contained two contiguous inclusion bodies. Further details of the histological changes will be reported elsewhere. Analysis of two samples from different sacks of pigeon grain yielded values of 0.3 1 and 0.38 pg of lead/g of grain, and analysis of two samples of oyster shell yielded values of 0.19 and 0.16 pg of lead/g of shell. Since the daily grain intake of these birds was about 20 g/day, the birds would be consuming less than 8 ,uglday, or about 0.02 mg/kg/day. This dose from the diet is a very small fraction of the dose delivered by intubation. Although exact values on the ingestion of oyster shell are not available, smaller amounts of lead would be available from this source, since 25 g of shell lasts for several weeks, and the concentration of lead in the shell is lower than in the grain. DISCUSSION
This study shows that schedule-controlled responding of the adult male White Carneaux pigeon is sensitiveto the effects of chronic low-level exposure to lead. A dose of 6.25 mg/kg produced marginal behavioral effects. A dose of 12.5 mg/kg usually disrupted rates of schedule-controlledresponding in four of five birds without producing either ataxia or other grosssignsof lead toxicosis. A doseof 25 mg/kg of lead produced both gross toxicity (marked dilatation of the esophagusand marked weight loss) and effects on schedule-controlledbehavior. It was difficult to characterize precisely the behavioral effects of lead on the schedulecontrolled behavior of the pigeon, since both rate increases and rate decreaseswere observed. although the latter predominated. The increasesand decreasesusually were observed under both schedulecomponents, although one bird (56 10 in Table 1) showed effects during only the FI component, and two birds (4522 and 855 in Table I) showed effects only in the FR component. On the basisof these data no case can be made that one schedulecomponent was more sentitive to the effects of lead than the other. Several studieshave failed to find behavioral changes after exposure to lead or lead compounds. in some casesat doseswhich produce grosstoxicity. For example. 15-20 mg/kg of tetraethyl lead (5-mg/day cumulative doses)produced toxicosis and death but did not affect the ability of 150-g rats to learn a water T-maze task before the gross effects occurred (Bullock et al., 1966). and neonatal and young adult rats given 100 m&g of lead acetate (ip) initiated at 8. 21. or 35 days of age showed no deficits in learning or memory in a water escape maze (Brown et al., 1971). Further. Snowdon ( 1973) found that weanling and adult rats could be given 5. 8. and 12 mg of Pb/kg for 37 days without effects on Hebb-Williams maze performance. Adult sheepgiven 100 mg of Pb/kg/day for 9 weeks performed lesswell on an auditory signal detection task. but the effect was associated with toxicosis (Van Gelder et al.. 1973). Our data and those of Driscoll and Stegner (1976) suggestthat effects of lead on schedule-controlled
282
BARTHALMUS
ET AL.
behavior can be seen at doses that do not produce gross toxicity. Driscoll and Stegner (1976) chronically exposed rats to lead acetate in drinking water and found effects on acquisition of a visual discrimination reversal problem and on shuttle avoidance performance, although it is difficult to determine the actual lead intake of their animals. Effects of lead on locomotor activity have been produced in animals exposed during prenatal or preweaning periods. Mice chronically exposed to lead acetate in drinking water before and after weaning, or through the milk of lead-exposed mothers, developed high levels of spontaneous motor activity (Silbergeld and Goldberg, 1973, 1974, 1975). Hyperactivity, apparently resulting from exposure during critical periods of development, has also been observed in rats (Sauerhoff and Michaelson, 1973; Golter and Michaelson, 1975) and infant rhesus monkeys (Allen et al., 1974). Increased rates of responding were seen in some of our pigeons under both schedule components, although rate-decreasing effects were observed more frequently. Whether or not these increases in rates of responding are related to the increases in rates of locomotor activity reported in other species is not clear. The blood-lead concentrations reported in our pigeons were IO-100 timer higher than those reported in the above studies where lead was lethal to mice, rats, sheep, and monkeys. The pigeon’s capacity to survive these high blood-lead concentrations may be associated with the nuclear inclusion bodies which we identified in the erythrocytes. The erythrocyte inclusions were apparent only at the ultrastructural level and were distinctly different from the nuclear hemoglobin crystals described by Fawcett and Witebsky (1964) and from the cytoplasmic hemoglobin observed in the lead-intoxicated mud puppy (Necturus) by Dawson (1930). Goyer et al. (1970) have described these bodies as lead-protein complexes consisting of 50 pugof lead/mg of protein. Others have suggestedthat they serve as a cellular detoxifying mechanismwhich isolates the lead from lead-sensitive cytoplasmic organelles such as mitochondria (Moore et al., 1973). thereby providing a protective mechanismduring transcellular transport of lead (Goyer and Rhyne, 1975). Horn (1970) has reported that lead-inclusion bodies contain 70- 100 times more than that found within the whole kidney homogenate. Consequently, nucleated erythrocytes of pigeonsmay serve as an extensive storage site, in addition to the liver and kidneys, for large quantities of partitioned lead. Our observation of these lead-inclusion bodies in the nucleated erythrocytes of pigeons may account in part for survival without overt signs of toxicosis at high blood concentrations and the low correlation between blood lead concentrations and behavior in some pigeons. although more detailed analyses of the uptake and tissue distribution of lead in the pigeon are being determined to evaluate the role of these processes.
ACKNOWLEDGMENTS We wish to thank John Gatzy for helpful criticism typing the manuscript.
of the manuscript
and Barbie Scott for
REFERENCES J.. R.. MCWEY. P. .I.. AND SUOMI. S. J. (1974). Pathological and behavioral lead intoxication in the infant rhesus monkey. Etzt,irotr. Henifh Persped. 7.239-246.
ALLEN,
etfects of
LEAD
AND
PIGEON
BEHAVIOR
283
T. W., AND KRIGMAN. M. R. (1975). Acute lead encephalopathy in the guinea pig. Acta Neuropathol. 33, 185-190. BROWN, D. R. (1975). Neonatal lead exposure in the rat: Decreased learning as a function of age and blood lead concentrations. Toxicol. Appl. Pharrnacol. 32.628-637. BROWN, S.. DRAGANN, N., AND VOGEL, W. H. (1971). Effects of lead acetate on learning and memory in rats. Arch. Environ. Health 22, 370-312. BULLOCK. J. D., WEY. R. J., ZAIA. J. A.. ZAREMBOK, I., AND SCHROEDER. H. A. (1966). Effects of tetraethyllead on learning and memory in the rat. Arch Environ. Health 13. 2 l-22. CARSON. T. L., VAN GELDER. G. A., KARAS. G. C.. ANU Buck, W. B. (1974). Slowed learning in lambs prenatally exposed to lead. Arch. Environ. Health 29, 154-156. DAWSON. A. B. (1930). Changes in the erythrocytes of Necturus associated with intracellular crystallization of hemoglobin. A nut. Rec. 46. 16 l- 17 1. D~:L.vEs. H. T. A. (1970). Micro-sampling method for the rapid determination of lead in blood and urine by atomic absorption spectrometry. Analyst (London) 95, 431. DRISCOLL, J. W., AND STEGNER, S. E. (1976). Behavioral effects of chronic lead ingestion on laboratory rats. Pharmacol. Biochem. Behav. 4.411-417. EIIIGEK. R. D.. AND COLEMAN. R. S. (1972). Modified Delves cup atomic absorption procedure for the determination of lead in blood. At. Absorption Newslett. 11, 33. FAWCETT, D. E., AND WITEBSKY, F. (1964). Observations on the ultrastructure on nucleated erythrocytes and thrombocytes with particular reference to the structural basis of their discordal shape. Z. Zellforsch. 62, 785-806. FEKSTER. C. B., AND SKINNER. B. F. (1957). Schedules of Reinforcement. Appleton-CenturyCrofts, New York. GOLLUB. L. R. (1964). The relations among measures of performance on fixed-interval schedules. J. Exp. Anal. Behav. 7, 337-343. GOLT~.K. M.. AND MICHAELSON. I. A. (1975). Growth, behavior and brain catecholamines in lead-exposed neonatal rats: A reappraisal. Science 187, 359-361. GOYER. R. A.. MAY. P., CATES. M.. AND KRIGMAN, M. R. (1970). Lead and protein content of isolated intranuclear inclusion bodies from kidneys of lead-poisoned rats. Lab. Invest. 22, 245-25 1. Goue~. R. A.. AND RHYNE. B. C. (1975). Pathological effects of lead. Int. Rev. Exp. Pathol. 12. I--77. GO~EK, R. A., AND WILSON, M. H. ( 1975). Lead-induced inclusion bodies. Results of ethylenediaminetetraacetic acid treatment. Lab. Incest. 32, 149-156. HORN. J. (1970). Isolation and examination of inclusion bodies of the rat kidney after chronic lead poisoning. Virchows Arch. B. 6,3 13-3 17. KRIGMAN, M. R.. Burrs. S. A., HOGAN. E. L., AND SHIN~~MAN. M. (1972). Morphological, neurochemical, and behavioral correlates of lead intoxication and undernourishment in developing rats (abstract). Fed. Proc. Fed. Amer. Sot. E-up. Biol. 137,665. KKI~IMAN. M. R., DRUSE. M. J.. T~YLOK. T. D.. WILSON. M. H.. NEWELL.. L. R.. ANI) HOGAN. E. L. (1974). Lead encephalopathy in the developing rat: Effect on cortical ontogenesis. J. .Veuropathol. E.Y~. Neurol. 33, 67 l-686. M,\HAFF~Y. K. R.. GOYEK, R. A.. AND HASEMAN. J. K. (1973). Dose-response to lead ingestion in rats fed low dietary calcium. J. Lab. Clin. Med. 82. 92-100. MOORI:. J. F.. GOYER. R. A., ANII WILSON. M. (1973). Solubility, amino acid content. and relationship to residual acidic nuclear proteins. Lab. Invest. 29.488-494. SAI~I-XHOFF. M. W.. AND MICIIAEL.SON. I. A. (1973). Hyperactivity and brain catecholamines in Icad-exposed developing rats. Science 182. 1022-1024. SII.HEKGEI.D. E. K.. .4x11 GOLDHERC;. A. M. (1973). Lead-induced behavioral disorder. L@ Sci. 13.1275-1283. SILBERGELD. E. K.. AND GOLDBERG, A. M.. (1974). Lead-induced behavioral dysfunction: An animal model of hyperactivity. Exp. Neural. 42. 146-157. SII-I%ERC;ELD. E. K.. ,\ND GOLDI~ERG. A. M. (1975). Pharmacological and neurochemical investigations of lead-induced hyperactivity. Nercropharmaeology 14,43 l-444. SNO\CIIOU. C. T. ( 1973). Learning deficits in lead-injected rats. PhamacoL Biochm. Behar. 1. 5’99-603. BOULDIN.
284
RART~~.~~.MUS
V \I\; GI I I)t K. G. A.. C,\KSOK. T. L.. Sn-ilitl. toxicologic assessment of the neurologic effect WI IR. P. A.. .\UII Hlct.. C. H. (1070). Effects goldfish. .-1 rch. Eurirorr. Flea//h 20. 45-5 I
tc7‘ .dL.
R. M.. ;IND Bi.(‘h. W. B. (11173). Behavior-a of lead in sheep. C/i/l. Tosicol. 6. 405-418. of various metals on behavior of conditioncc
Effects
of Lead
on Schedule-Controlled Behavior’
Pigeon
GEOKGE T. BAKTHAL.ML:S.~ J. DAVII) Lt-ANI)I-I<.’ DONAL.I)E. Mc.MII.I..\N.’ P \I I ML;SHAK.~ANL)MAIWN R. KKIC;MAN~
Chronic Effects of Lead on Schedule-Controlled Pigeon Beha\lor. B \KIII \I XII \. G. T.. Lt .\uI),:K. .I. D.. McM11.1 A\. D. E.. Mu >,I \K. P.. \NI) KICK,\! \N. M. R. ( lY77). Toxicol. ./lppI. Phnr~nacol. 42, 271-284. Pigeons were trained to peck a response key under a multiple tixed ratio 30 response (FR 30) fixed-interval 5-min (Fl 5) schedule of food presentation. When rate\ of responding stabilized, lead acetate (6.25. 12.5. and 25 mg/kg body weight) or sodium acetate solutions were given daily by gastric intubation. Blood-lead concentrations were measure? weekly by atomic absorption spcctrometry. and responding under the multiple schedule wab measured Monday through Friday. Control lntubations of sodium acetate did not atl’ect responding under either component of the schedule. The 25-mg/kg dose of lead decreased rate\ of responding after 3 to IO days of administration and usually was lethal between 18 and 35 Jays. Post mortem examination of the birds ahowed obvious esophageal dilatation. damage to the crop. discontinued but rates
weight loss. in some of responding
and aubarachnoid hemorrhages. Admimstration of the birds after responding had ceased. Responding gradually remained low and variable under both components
25-mg/kg dose was began to recover. of the schedule for
many days. Daily administration of the l2.5-mg/kg dose of lead caused one death. but no obvious signs of intoxication occurred in the other birds. However, three of the four bird, yurbiving daily l2.5-mg/kg doses of lead showed decreased rates of responding under both schedule components within 30 days of the initial intubation. The fourth bird showed a high11 1 ariable rate under the FI component of the schedule during lead administration: however. when lead administration was discontinued. dramatic increases in ra!es of responding above lead pretreatment levels occurred. particularly under the FI component. These rate increase\ persisted for 75 days thereafter. The 6.25-mgikg dose of lead produced only small changes in rates of responding during 70 days of intubation. Blood-lead concentrations were very high ( 150-3470 pgldl) during treatment with all doses of lead. Increasing doses produced increasing blood concentrations which were correlated with increasing behavioral effects. although correlations individual
between birds.
blood-lead
concentrations
’ Supported by Grant No. ES01 104 from ’ Department of Zoology. North Carolina ’ Department of Pharmacology, School Carolina 275 14. To whom reprint requests ’ Department of Pharmacology. School Carolina 275 14. ’ Department of Pathology. School of Carolina 27514.
and
behavioral
the NIEHS. State University. Raleigh, North of Medicine. University of North should be addressed. of Medicine. University of North Medicine.
University
of North
changes
were
low
within
Carolina Carolina,
27607. Chappel
I Hill.
North
Carolina.
Chappel
Hill.
North
Hill.
North
Carolma.
Chappel
272
BARTHALMUS
ET AL
Considerable research has been focused on the pathological, physiological, and neurological consequences of both acute and chronic exposure to lead (Bouldin and Krigman, 1975; Goyer and Rhyne, 1975; Goyer and Wilson, 1975; Krigman et al., 1972, 1974; Mahaffey et al., 1973). However, there are few controlled experiments on the behavioral effects of lead ingestion. Lead-induced hyperactivity in mice has been suggested as a model for studying drug responses in children with minimal brain dysfunction hyperactivity (Silbergeld and Goldberg, 1974, 1975). Deficits in the learned behavior of animals including visual discrimination (Carson et al., 1974). conditioned avoidance responding (Weir and Hine, 1970), and maze learning (Brown, 1975: Bullock ef al., 1966; Snowdon, 1973) have been reported, but there have been few attempts to use schedule-controlled behavior to assess the effects of chronic exposure to lead. The present experiments were performed to examine the effects of chronic oral lead administration on responding maintained by schedules of food presentation to the pigeon.
METHODS
Animals Adult male White Carneaux pigeons (Palmetto Pigeon Plant, Sumter, South Carolina) were maintained at 80% of their free-feeding weights throughout all experiments. Their 80% body weights ranged from 410 and 480 g. Birds were fed mixed grain (Ralston-Purina pigeon diet), and crushed oyster shell was freely available in home cages as a source of gizzard grit. Water was freely available both in home cages and test chambers. Materials and Dosing Lead solutions were prepared weekly for administration as 6.25, 12.5, and 25.0 mg of lead (as lead acetate)/kg body weight. Solutions of sodium acetate served as a control intubation solution. Birds were given lead or sodium acetate 7 days/week by oral intubation into the proventriculus by passing a vinyl catheter down the esophagus and past the crop. Intubations followed the test sessions on weekdays.’ Determination
of Blood-Lead
Concentrations
Blood samples from the radial vein were obtained each Wednesday after the test sessions. Syringes and acid-washed, Teflon-sealed storage vials were precoated with anticoagulant consisting of 5% w/v sodium citrate in 0.9% saline. Samples were frozen immediately and stored until lead determinations were made by atomic absorption spectrometry employing a Perkin-Elmer Model 306 double-beam instrument. The method of Delves (1970) as modified by Ediger and Coleman (1972) was used. Ten- to twenty-microliter samples of titrated whole blood. with or without prior dilution, were delivered in lead-free nickel crucibles, preignited in the cool portion of the flame and then directly inserted into the flame for lead atomization. An aligned deuterium arc background correction accessory minimized signal errors. Levels of total lead in the oyster shell and pigeon gram also were determined.
LEAD
AND
PIGEON
BEHAVIOR
273
Histology Tissues removed at necropsy were fixed in formalin for light microscopy and some tissues, peripheral nerve, kidney, liver, and lung, were selected for electron microscopy. Tissues for electron microscopy were initially fixed in 10% formalin, postfixed in 1% osmium tetraoxide in 0.1 M phosphate buffer, dehydrated in alcohol and propylene oxide, and embedded in an Epon (D.E.R. 732) plastic. Thin sections were stained with many1 acetate. Apparatus for Behavioral
Testing
Behavioral testing was conducted in sound- and light-attentuated chambers similar to those described by Ferster and Skinner (1957). The chamber was illuminated by a 7.5. W light (ac) and contained a translucent plastic response key (2.0 cm in diameter) which was centrally mounted in the chamber wall 22 cm above a wire-mesh floor. The response key could be transilluminated by red or blue lights. The minimal force required to operate the key was about 0.15 N. Centrally below the key and 6 cm above the floor was a rectangular opening through which the pigeon could be given access to mixed grain. Relay programming and recording apparatus housed in the adjacent room controlled the delivery of grain and recorded the pattern of key pecking. A masking noise was supplied by a speaker mounted inside the chamber. Procedure Pigeons performed Monday through Friday under a multiple fixed-ratio, fixedinterval schedule (mult FR FI) of food presentation. In the presence of a blue light, the 30th key peck (FR 30) produced 4-set access to grain. In the presence of the red light, the first response after 5 min (FI 5) produced 4-set access to grain. The FR and FI components alternated after each food presentation. If a bird failed to respond within 40 set after 5 min had elapsed during the FI component, the schedule changed to the FR component. If the bird failed to make 30 responses within 40 set during the FR component, the schedule changes to the FI component. Sessions were started in the FR component 10 min after the pigeon was placed in the chamber and were terminated the first time the schedule component changed after an hour of testing had elapsed. Birds were weighed before each session and were weighed and dosed immediately following each session except Wednesday when dosing followed the blood sampling. Evaluation Average rates of responding for the I-hr sessions were computed separately for the FR and FI components from data recorded on digital counters and elapsed-time meters. The distribution of responses within the FI component was obtained by dividing the interval into 10 30.set segments and recording the number of responses in each segment on counters. These data were used to calculate the quarter-life value, a statistic which is independent of response rate and is used to describe quantitatively the positively accelerated pattern of responding that occurs under the FI schedule component. The quarterlife is defined as the percentage of the FI taken to emit 25% of all responses in the FI (Gollub, 1964). All pigeons were tested for 40-50 nontreatment sessions to establish a stable control baseline of behavior. Then they began the experiment, with some birds remaining as
214
BARTHALMUS
ET AL
untreated controls, whereas others were treated with either sodium acetate or lead. Data points on the figures which fell outside the range of the untreated controls and/or the sodium acetate control ranges were considered to be behavioral effects of lead. RESULTS Figures l-5 show the response rate data of 14 birds. The quarter-life no trends and, therefore, are not shown in the figures.
values showed
0
A FR-30
BIRD
5610
FR-30
BIRD
1469
FI-5
SUCCESSIVE
DAYS
SUCCESSIVE
C BIRD
FR- 30 3r
25
ACET.
;
DAYS
4756
25 LEAD
t OL
1
5
’
IO I5
8’
SUCCESSIVE
6
5
’
IO
’
15
’
20
sL
25
’
30
35
DAYS
FIG. I Effects of 3-5 mg/kg of lead on the average rate of responding FR 30 FI 5 schedule of food presentation. Bird 1469 (B) was given times the volume of water given to bird 56 10 (A) and bird 4758 (C). lead, Abscissa. successive ahsociatcd with the FR micrograms per deciliter.
days: ordinate. 30 rates of
mean responding
rate
under each component of the mult on 2S-mg/kg lead intubation in t&o All pigeons died from treatment with
of responding during indicate Wednesday
a complete blood-lead
session. Numbers concentrations in
Figure 1 shows the response rates under the FR 30 and FI 5 schedule of food presentation fcr three pigeons that died following 17-33 days of 25-mg/kg intubations. Rates of responding decreased for all birds within 7 days of exposure to lead. Gross effects. observed 8-20 days following the response-rate decreases, included the copious
LEAD
AND
PIGEON
275
BEHAVIOR
accumulation of crop fluid, esophageal dilatation, and a large weight loss. At mortem examination, the crop of each bird was distended with grain; however, the had not entered the gizzard. The brains of all three birds were covered with subarachnoid hemorrhage. Gross effects were seen first in birds 5610 and 4758, and since these effects may resulted from the high concentration of lead acetate dissolved in distilled water,
FIG. 2. Effects of 25 mg/kg of lead on the average FR 30 Fl 5 xhedule of food presentation. Bird 110 Icad. Other details as in Fig. 1.
rate (A),
of responding under 3933 (B). and 4619
post grain thin have bird
each component of the mult (C) survived treatment with
1469 received the 25.mg/kg dose in twice the volume of water. Again, response-rate decreases under both the FR and FI components were soon followed by gross signs and death. Figure 2 shows the data for three pigeons which did not die from the repeated administration of 25 mg of Pb/kg. Birds 110, 3933, and 4619 were dosed for 52, 43, and 13 days, respectively. Within 2 days, birds 110 and 3933 showed obvious changes in the rates of responding under both the FR and FI components. Throughout the period of lead dosage, bird 110 exhibited a moderate decrease in rates under the FR component. while rates under the Fl tended to increase slightly, but with wide daily
276
BARTHALMUS ET.4l..
variation. When lead intubations were discontinued, the rates of responding under the FR component continued to fall and then partially recovered. However, the FR rates remained below the baseline rates for 75 days after lead administration was discontinued. In contrast, the rates under the FI tended to remain within the control range. The rates of responding under the FR and FI components of bird 3933 declined during lead exposure until responding was abolishedafter about 3.5days of exposure. A
FIG. 3. Effects of 12.5 mg,/kg of lead on the average rate of responding under each component of the mult FR 30 and FI 5 schedule of food presentation. Bird 5775 (A) died during treatment with lead; birds 4522 (B) and 3594 (C) survived treatment with lead. Other details as in Fig. I.
few days later, lead intubations were discontinued. After 31 days without any appreciable responding, key pecking began to recover under both FR and Fl components. Rates under the FR returned to and remained at baselinefor 37 days but then dropped to a consistent subbaselinelevel for the remaining 48 days. However. when responding under the FI returned, it was highly variable for about 25 days after which the rates fell below baseline for the remaining 60 days. Excessive crop fluid was observed in bird 3933 during the period when responding had ceased;however, bird 110 and 46 19 showedno grosssigns. Bird 46 19. who received control intubations for 55 days, showed no change in either
ILAD
AND
PIGEON
BEHAVIOK
277
schedule component during this time. nor during the following 13 days of lead intubations and the 36.day period without intubations. Three pigeons treated with 12.5 mg/kg of lead are shown in Fig. 3. Bird 5715. who died after 42 days, showed decreased rates of responding under both schedules after 15 days of exposure. The autopsy showed evidence of subarachnoid hemorrhage but excessive crop fluid. esophageal dilatation, and weight loss did not occur.
Y r
FIG. 4. Effects of 12.5 mg/kg of lead on the average rate of responding under each component of the mult FR 30 FI 5 schedule of food presentation. Bird 7773 (A) and bird 5397 (B) were given lead: bird 2923 (C)was given only sodium acetate. All birds survived the treatment. Other details as in Fig. I.
After 27 days of lead intubation, the rate of responding under the FI component of bird 4522 increased, whereas the response rate under the FR component was un changed. Continued lead administration led to rate decreasesunder both schedule components, although rates were near normal during some sessionsduring the last 30 days of lead administration. Rates of responding by bird 4522 gradually recovered under both schedulecomponentsduring the 42 days following lead intubation. The rates of responding by bird 3594 decreasedunder both the FR and FI schedule components after 2 1 days of lead intubations. The rates of responding decreasedabout 30% over a 19-day span and then remained relatively constant for the remaining 41 days of treatment. When lead administration was discontinued, 25 days elapsedwithout signsof a return to baselineperformance.
278
t.7’ -II..
HAKTH.4LMUS
Figure 4 shows the data from two additional birds given 12.5 mg of Pb/kg. A third bird. bird 2923, received sodium acetate as a control for 90 days without behavioral disruptions. Bird 7773 showed a temporary decrease in the rates under the FR schedule between 7 and IO days of exposure, a return to baseline rates between Days 11 and 34. and then the rates of responding decreased or were abolished for the remaining 24 days. When lead was discontinued, the rates of responding under the FR schedule recovered quickly, and most of the subsequent sessions were characterized by rates above the baseline rate.
FIG. mult
5. Effects
of 6.25
FR 30 FI 5 schedule
mg/kg
of Lead
of food
presentation.
on
the average Both
birds
rate
of responding
survived
under
the treatment.
each Other
component details
of the
as in Fig.
I.
The rates of responding under the FI component by bird 7773 were less affected than the FR rates, although the FI rates did decrease after 40 and 48 days of lead intubation. The discontinuation of lead produced no further changes in rates of responding under the FI component over 55 days of testing. Pigeon 5397 showed no consistent changes in rates under the FR after 71 days exposure to 12.5 mg of Pb/kg, whereas the rates under the FI became more variable and were often higher than control rates. After 22 days without lead intubations. rates of responding under the FR component exceeded control levels and remained elevated for 73 days of testing. After discontinuation of lead administration, rates under the FI exceeded baseline values almost immediately. maintaining levels nearly double those of the control sessions. These high rates were maintained for more than 90 days. Figure 5 shows two pigeons treated with 6.25 mg of Pb/kg for 72 days. In bird 855 the rates under the FI component were decreased immediately after lead intubation started. but the rates were only slightly below baseline. The rate under the FR component was unaffected. In bird 1764, there was a slight increase in the rates under the FR component. whereas the rate under the FI was largely unaffected.
LEAD
AND
PIGEON
279
BEHAVIOK
Table 1 summarizes the effects of lead on the schedule-controlled behavior of the pigeon. Both rate increases and rate decreases were observed under both schedule components, although rate decreases were the predominant effect in 80% of the cases where rate changes were observed regularly. There was no evidence that behavior controlled by one schedule component was more sensitive to the effects of lead than the other. The mean number of intubation days elapsed to produce a behavioral effect at the 25. 12.5. and 6.25 mg of Pb/kg doses were 3.8. 15.8 and 17.0 days, respectively, suggesting that the behavioral effects of lead were dose-dependent. TABLE
1
SUMMAKY OF EFFECTS OF LEAD ON RATI’ OF RI~SPONDING OF PIGEONS
Initial effect observed Dose Bird
hdkd
Day effect established
FR
FI
Predominanteffect over days -~
FR
FI
” Eventually died. ” +. Rate increases: I rate decreases. ’ No effect. ” Given sodium acetate only.
The weekly blood-lead concentrations are shown in Figs. l-5 above the response data for the FR component. Three of the birds receiving 25 mg of Pb/kg in Figs. 1 and 2 exceeded 100 Ergof Pb/dl of whole blood, with bird 110 reaching a peak blood level of 3400 pugof Pb/dl. The blood-lead concentrations appeared to be dose-related since lower levels were found in the birds receiving 12.5 and 6.25 mg of Pb/kg. Nevertheless. the two birds receiving 6.25 mg of Pb/kg still attained peak blood-lead levelsof 285 and 4 I2 ,ugof Pb/dl. Table 2 shows that there was not a strong correlation between blood concentration and behavioral effects within individual birds, despite the fact that both the blood concentration and the behavioral effect were dose-relatedacrossbirds. Neither the initial behavioral effect nor the maximum behavioral effect occurred at the time of peak blood lead concentrations for most birds. Histologic examination revealed no specific organ changesexcept in the kidney. The sciatic nerves were within normal limits. The brains showed no changes except for
280
BARTHALMUS
TABLE COMPARISON
OF BLOOD-LEAD
ET .4L.
2
CONCENTRATIONS AND RESPONDING IN PIGEONS
Blood-lead
Bird 5610” 1469” 4758” 110
3933 1113 5397 5715” 4522 3594 855 I164 2923”
Dose (w/kg)
At time of initial behavioral effect
25 25 25 25 25 12.5 12.5 12.5 12.5 12.5 6.25 6.25 0
” Eventually died. h Given sodium acetate only. C Range of observations (no behavioral
503 491 IO1 408 339 427 429 414 353 280 285 311 -
CHANGES
IN RATES
concentrations
At time of maximum behaviorial effect .--~ 923 380 918 608 269 175 71 513 409 145 285 193
OF
@g/dl) Highest concentration observed 1235 624 918 3415 1147 441 848 569 409 398 285 412 17-64k’
change).
FIG. 6. Electron photomicrograph depicts two red ceils in a pulmonary capillary, one of which containb a nuclear lead-inclusion body (arrow). Formalin-fixed necropsy tissue. postfixed with osmium tetraoxide. and the ultrathin sections were stained with uranyl acetate. Magnification Y 1.98 x IO’.
LEAD
AND
PIGEON
BEHAVIOR
281
congestion and recent subarachnoid hemorrhage in the three pigeons that died on the 25-mg/kg dose schedule. Light microscopy examination revealed typical lead-inclusion bodies in the proximal convoluted tubules of the kidney, and by electron microscopy, inclusion bodies were also identified in erythrocyte nuclei. The erythrocytic nuclear inclusions (Fig. 6) were circular to oval, electron-dense bodies, which were distinctly separate and different from the heterochromatin, nucleolus, and nuclear hemoglobin. Occasionally a red-cell nucleus contained two contiguous inclusion bodies. Further details of the histological changes will be reported elsewhere. Analysis of two samples from different sacks of pigeon grain yielded values of 0.3 1 and 0.38 pg of lead/g of grain, and analysis of two samples of oyster shell yielded values of 0.19 and 0.16 pg of lead/g of shell. Since the daily grain intake of these birds was about 20 g/day, the birds would be consuming less than 8 ,uglday, or about 0.02 mg/kg/day. This dose from the diet is a very small fraction of the dose delivered by intubation. Although exact values on the ingestion of oyster shell are not available, smaller amounts of lead would be available from this source, since 25 g of shell lasts for several weeks, and the concentration of lead in the shell is lower than in the grain. DISCUSSION
This study shows that schedule-controlled responding of the adult male White Carneaux pigeon is sensitiveto the effects of chronic low-level exposure to lead. A dose of 6.25 mg/kg produced marginal behavioral effects. A dose of 12.5 mg/kg usually disrupted rates of schedule-controlledresponding in four of five birds without producing either ataxia or other grosssignsof lead toxicosis. A doseof 25 mg/kg of lead produced both gross toxicity (marked dilatation of the esophagusand marked weight loss) and effects on schedule-controlledbehavior. It was difficult to characterize precisely the behavioral effects of lead on the schedulecontrolled behavior of the pigeon, since both rate increases and rate decreaseswere observed. although the latter predominated. The increasesand decreasesusually were observed under both schedulecomponents, although one bird (56 10 in Table 1) showed effects during only the FI component, and two birds (4522 and 855 in Table I) showed effects only in the FR component. On the basisof these data no case can be made that one schedulecomponent was more sentitive to the effects of lead than the other. Several studieshave failed to find behavioral changes after exposure to lead or lead compounds. in some casesat doseswhich produce grosstoxicity. For example. 15-20 mg/kg of tetraethyl lead (5-mg/day cumulative doses)produced toxicosis and death but did not affect the ability of 150-g rats to learn a water T-maze task before the gross effects occurred (Bullock et al., 1966). and neonatal and young adult rats given 100 m&g of lead acetate (ip) initiated at 8. 21. or 35 days of age showed no deficits in learning or memory in a water escape maze (Brown et al., 1971). Further. Snowdon ( 1973) found that weanling and adult rats could be given 5. 8. and 12 mg of Pb/kg for 37 days without effects on Hebb-Williams maze performance. Adult sheepgiven 100 mg of Pb/kg/day for 9 weeks performed lesswell on an auditory signal detection task. but the effect was associated with toxicosis (Van Gelder et al.. 1973). Our data and those of Driscoll and Stegner (1976) suggestthat effects of lead on schedule-controlled
282
BARTHALMUS
ET AL.
behavior can be seen at doses that do not produce gross toxicity. Driscoll and Stegner (1976) chronically exposed rats to lead acetate in drinking water and found effects on acquisition of a visual discrimination reversal problem and on shuttle avoidance performance, although it is difficult to determine the actual lead intake of their animals. Effects of lead on locomotor activity have been produced in animals exposed during prenatal or preweaning periods. Mice chronically exposed to lead acetate in drinking water before and after weaning, or through the milk of lead-exposed mothers, developed high levels of spontaneous motor activity (Silbergeld and Goldberg, 1973, 1974, 1975). Hyperactivity, apparently resulting from exposure during critical periods of development, has also been observed in rats (Sauerhoff and Michaelson, 1973; Golter and Michaelson, 1975) and infant rhesus monkeys (Allen et al., 1974). Increased rates of responding were seen in some of our pigeons under both schedule components, although rate-decreasing effects were observed more frequently. Whether or not these increases in rates of responding are related to the increases in rates of locomotor activity reported in other species is not clear. The blood-lead concentrations reported in our pigeons were IO-100 timer higher than those reported in the above studies where lead was lethal to mice, rats, sheep, and monkeys. The pigeon’s capacity to survive these high blood-lead concentrations may be associated with the nuclear inclusion bodies which we identified in the erythrocytes. The erythrocyte inclusions were apparent only at the ultrastructural level and were distinctly different from the nuclear hemoglobin crystals described by Fawcett and Witebsky (1964) and from the cytoplasmic hemoglobin observed in the lead-intoxicated mud puppy (Necturus) by Dawson (1930). Goyer et al. (1970) have described these bodies as lead-protein complexes consisting of 50 pugof lead/mg of protein. Others have suggestedthat they serve as a cellular detoxifying mechanismwhich isolates the lead from lead-sensitive cytoplasmic organelles such as mitochondria (Moore et al., 1973). thereby providing a protective mechanismduring transcellular transport of lead (Goyer and Rhyne, 1975). Horn (1970) has reported that lead-inclusion bodies contain 70- 100 times more than that found within the whole kidney homogenate. Consequently, nucleated erythrocytes of pigeonsmay serve as an extensive storage site, in addition to the liver and kidneys, for large quantities of partitioned lead. Our observation of these lead-inclusion bodies in the nucleated erythrocytes of pigeons may account in part for survival without overt signs of toxicosis at high blood concentrations and the low correlation between blood lead concentrations and behavior in some pigeons. although more detailed analyses of the uptake and tissue distribution of lead in the pigeon are being determined to evaluate the role of these processes.
ACKNOWLEDGMENTS We wish to thank John Gatzy for helpful criticism typing the manuscript.
of the manuscript
and Barbie Scott for
REFERENCES J.. R.. MCWEY. P. .I.. AND SUOMI. S. J. (1974). Pathological and behavioral lead intoxication in the infant rhesus monkey. Etzt,irotr. Henifh Persped. 7.239-246.
ALLEN,
etfects of
LEAD
AND
PIGEON
BEHAVIOR
283
T. W., AND KRIGMAN. M. R. (1975). Acute lead encephalopathy in the guinea pig. Acta Neuropathol. 33, 185-190. BROWN, D. R. (1975). Neonatal lead exposure in the rat: Decreased learning as a function of age and blood lead concentrations. Toxicol. Appl. Pharrnacol. 32.628-637. BROWN, S.. DRAGANN, N., AND VOGEL, W. H. (1971). Effects of lead acetate on learning and memory in rats. Arch. Environ. Health 22, 370-312. BULLOCK. J. D., WEY. R. J., ZAIA. J. A.. ZAREMBOK, I., AND SCHROEDER. H. A. (1966). Effects of tetraethyllead on learning and memory in the rat. Arch Environ. Health 13. 2 l-22. CARSON. T. L., VAN GELDER. G. A., KARAS. G. C.. ANU Buck, W. B. (1974). Slowed learning in lambs prenatally exposed to lead. Arch. Environ. Health 29, 154-156. DAWSON. A. B. (1930). Changes in the erythrocytes of Necturus associated with intracellular crystallization of hemoglobin. A nut. Rec. 46. 16 l- 17 1. D~:L.vEs. H. T. A. (1970). Micro-sampling method for the rapid determination of lead in blood and urine by atomic absorption spectrometry. Analyst (London) 95, 431. DRISCOLL, J. W., AND STEGNER, S. E. (1976). Behavioral effects of chronic lead ingestion on laboratory rats. Pharmacol. Biochem. Behav. 4.411-417. EIIIGEK. R. D.. AND COLEMAN. R. S. (1972). Modified Delves cup atomic absorption procedure for the determination of lead in blood. At. Absorption Newslett. 11, 33. FAWCETT, D. E., AND WITEBSKY, F. (1964). Observations on the ultrastructure on nucleated erythrocytes and thrombocytes with particular reference to the structural basis of their discordal shape. Z. Zellforsch. 62, 785-806. FEKSTER. C. B., AND SKINNER. B. F. (1957). Schedules of Reinforcement. Appleton-CenturyCrofts, New York. GOLLUB. L. R. (1964). The relations among measures of performance on fixed-interval schedules. J. Exp. Anal. Behav. 7, 337-343. GOLT~.K. M.. AND MICHAELSON. I. A. (1975). Growth, behavior and brain catecholamines in lead-exposed neonatal rats: A reappraisal. Science 187, 359-361. GOYER. R. A.. MAY. P., CATES. M.. AND KRIGMAN, M. R. (1970). Lead and protein content of isolated intranuclear inclusion bodies from kidneys of lead-poisoned rats. Lab. Invest. 22, 245-25 1. Goue~. R. A.. AND RHYNE. B. C. (1975). Pathological effects of lead. Int. Rev. Exp. Pathol. 12. I--77. GO~EK, R. A., AND WILSON, M. H. ( 1975). Lead-induced inclusion bodies. Results of ethylenediaminetetraacetic acid treatment. Lab. Incest. 32, 149-156. HORN. J. (1970). Isolation and examination of inclusion bodies of the rat kidney after chronic lead poisoning. Virchows Arch. B. 6,3 13-3 17. KRIGMAN, M. R.. Burrs. S. A., HOGAN. E. L., AND SHIN~~MAN. M. (1972). Morphological, neurochemical, and behavioral correlates of lead intoxication and undernourishment in developing rats (abstract). Fed. Proc. Fed. Amer. Sot. E-up. Biol. 137,665. KKI~IMAN. M. R., DRUSE. M. J.. T~YLOK. T. D.. WILSON. M. H.. NEWELL.. L. R.. ANI) HOGAN. E. L. (1974). Lead encephalopathy in the developing rat: Effect on cortical ontogenesis. J. .Veuropathol. E.Y~. Neurol. 33, 67 l-686. M,\HAFF~Y. K. R.. GOYEK, R. A.. AND HASEMAN. J. K. (1973). Dose-response to lead ingestion in rats fed low dietary calcium. J. Lab. Clin. Med. 82. 92-100. MOORI:. J. F.. GOYER. R. A., ANII WILSON. M. (1973). Solubility, amino acid content. and relationship to residual acidic nuclear proteins. Lab. Invest. 29.488-494. SAI~I-XHOFF. M. W.. AND MICIIAEL.SON. I. A. (1973). Hyperactivity and brain catecholamines in Icad-exposed developing rats. Science 182. 1022-1024. SII.HEKGEI.D. E. K.. .4x11 GOLDHERC;. A. M. (1973). Lead-induced behavioral disorder. L@ Sci. 13.1275-1283. SILBERGELD. E. K.. AND GOLDBERG, A. M.. (1974). Lead-induced behavioral dysfunction: An animal model of hyperactivity. Exp. Neural. 42. 146-157. SII-I%ERC;ELD. E. K.. ,\ND GOLDI~ERG. A. M. (1975). Pharmacological and neurochemical investigations of lead-induced hyperactivity. Nercropharmaeology 14,43 l-444. SNO\CIIOU. C. T. ( 1973). Learning deficits in lead-injected rats. PhamacoL Biochm. Behar. 1. 5’99-603. BOULDIN.
284
RART~~.~~.MUS
V \I\; GI I I)t K. G. A.. C,\KSOK. T. L.. Sn-ilitl. toxicologic assessment of the neurologic effect WI IR. P. A.. .\UII Hlct.. C. H. (1070). Effects goldfish. .-1 rch. Eurirorr. Flea//h 20. 45-5 I
tc7‘ .dL.
R. M.. ;IND Bi.(‘h. W. B. (11173). Behavior-a of lead in sheep. C/i/l. Tosicol. 6. 405-418. of various metals on behavior of conditioncc