BEHAVIORAL EFFECTS OF ASYMPTOMATIC DEVELOPMENTAL PLUMBISM IN RATS Mssertation for the Degree of PM. D. MICHIGAN STATE UNIVERSITY ~ STEPHEN REED OVERMANN 1976 rH E53§ - ‘ This is torcertify that the thesis entitled l I Behavioral Effects of Asymptomatic Plumbism in Rats presented by Stephen R . Overmann has been accepted towards fulfillment of the requirements for Ph . D . degree in Psychology yaw/m fl? fie fl Major pfiessor Date / ‘2 —/5‘ 75/ . , TING bib" ' IIIIIIK BINDERY III ,3 uamnv amp I . ABSTRACT BEHAVIORAL EFFECTS OF ASYMPTOMATIC DEVELOPMENTAL PLUMBISM IN RATS BY Stephen Reed Overmann Lead intoxication is a serious pediatric problem, overtly affecting thousands of children yearly. Moreover, these children may represent only a fraction of the number affected by excess exposure to lead. The population of undetected, asymptomatically poisoned children has been estimated to exceed one quarter million. Overt plumbism results in a constellation of sensory, motor, social, and intellectual deficits. However, the extent of impairment of children in the asymptomatic population is largely unknown. The current study was an attempt to develop an animal behavior model of asymptomatic plumbism. Long-Evans rats were intubated daily from three to twenty one days of age with a O, 10, 30, or 90 mg/kg dose of a lead acetate solution. Following weaning, all subjects began a series of behavioral tests which reflected consideration of the behavioral deficits reported to result from childhood plumbism. The following tests were used: visual acuity (optokinetic drum method); activity level (activity chambers); aversive conditioning (passive and active avoidance, acquisition and extinction); motor coordination (rotarod method); Stephen Reed Overmann response-inhibition (discrete-trial DRL bar-pressing); simple instru- mental learning (turning response in E-maze); complex learning with tactile cues (conditional discrimination in E—maze); and complex learning with visual cues (conditional discrimination in E-maze). Following behavioral testing all subjects were sacrificed and the wet weight of their adrenals and kidneys determined. Blood samples at twenty-one and thirty-five days of age were analyzed for lead content and hematocrit. The lead treatment had no significant effect on simple learning, complex visually-cued learning, and visual acuity. Neonatal lead exposure did result in increased activity, decreased motor coordination, and an impairment in response inhibition. Neither acquisition nor extinction of passive avoidance yielded a significant effect from the lead, but both the acquisition and extinction of active avoidance did. Lead poisoned rats acquired the avoidance response more slowly and extinguished more slowly than controls. Reversal learning of the tactually-cued conditional discrimination was also impaired by the lead treatment. The three levels of lead exposure had no significant effect on growth and all animals were overtly free of poisoning symptoms. Blood samples at twenty-one days of age showed high blood lead levels and decreased hematocrit values among exposed subjects. These indices of poisoning were quite transient, with only a small effect apparent when blood samples were taken on Day 35. The lead treatment resulted in increased adrenal size, but did not affect kidney size among sub- jects given behavioral tests. Additional subjects, treated as the O Stephen Reed Overmann or 90 mg/kg lead poisoning groups, showed that the highest level of lead exposure increased adrenal and kidney weights at twenty-one and thirty-five days of age. The results demonstrate that lasting behavioral impairments may be induced by transient, asymptomatic lead poisoning during early post- natal development. The study also indicates the feasibility of using an animal model in the further study of sub-clinical plumbism. The constellation of behavioral sequelae of developmental plumbism parallel those seen in minimal brain dysfunction (MBD) children. Estimates of a large, undetected poisoned population suggest that plumbism may be significant in the etiology of many cases of MBD. Demonstrations that asymptomatic lead poisoning results in behavioral impairments similar to those of MBD emphasize the urgency of removing lead from children's environment. BEHAVIORAL EFFECTS OF ASYMPTOMATIC DEVELOPMENTAL PLUMBISM IN RATS BY Stephen Reed Overmann A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Psychology 1976 to Kathy ii ACKNOWLEDGMENTS I would like to thank my committee members for their advice throughout this study. I would like to express my particular gratitude to Ray Denny, Glenn Hatton, and Stan Ratner for their scholarly gui- dance throughout my graduate career. Jack King kindly loaned the opto- kinetic drum equipment and Jack Freeman was responsible for the design and construction of the activity chamber sensors. A number of students contributed through assistance in data collection. Their time and effort was greatly appreciated: Julie Canham, Katherine Cartwright, Sandra Cifor, Nancy Hallo, Michael Kamp, Vaughn Rickert, Gary Rutledge, and Vera Sekulvski. iii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . . . . . METHOD . . . . . . . . . . . . . . . . . . . Subjects . . . . . . . . . . . . . . . . Apparatus and Procedure . . . . . . . . . Visual Acuity Measurement--Group I-Test Activity Measurement--Group I-Test II I Measurement of Aversive Conditioning--Group Motor Coordination Measurement--Group I-Test IV . Measurement of Response Inhibition-~Group II-Test I E Maze Testing . . . . . . . . . . . Measurement of Simple Learning--Group II-Test II Measurement of Complex Learning with Tactile Cues-— Group II-Test III . . . . . . . . . Measurement of Complex Learning with Visual Group II-Test IV . . . . . . . . . Physiological Measures . . . . . . . RESULTS . . . . . . . . . . . . . . . . . . . Effect of Poisoning on Growth . . . . . . Visual Acuity Measurement--Group I-Test I Activity Measurement—-Group I-Test II . . Measurement of Aversive Conditioning--Group I-Test III I-Test III Cues-- Motor Coordination Measurement--Group I—Test IV . Measurement of Response Inhibition--Group II-Test I . Measurement of Simple Learning-~Group II-Test II iv Page vi ix 11 11 12 13 14 '15 16 17 18 19 19 20 21 23 23 26 26 26 34 39 39 page Measurement of Complex Learning with Tactile Cues-- Group II-Test III 0 O O O I O O I O O O O O O O O O O O O O 44 Measurement of Complex Learning with Visual Cues-- Group II-Test IV . . . . . . . . . . . . . . . . . . . . . 47 Blood Lead and Blood Hematocrit Values . . . . . . . . . . . 47 Adrenal and Kidney Weights . . . . . . . . . . . . . . . . . 53 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 APPENDIX A . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 APPENDIX B O O O O O O C O C O C O O O O O O O O O O O O O O O O 69 APPENDIX C . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 Table BI. 82. B3. B4. BS. B6. B7. LIST OF TABLES Mean preweaning body weight of all subjects given behavioral tests . . . . . . . . . . . . . Mean postweaning body weight of all subjects given behavioral tests . . . . . . . . . . . . . Number of rats responding to three visual stimuli in optokinetic drum . . . . . . . . . . Mean number of shocks prior to acquisition-- criterion of three trials without a shock . . . Mean blood lead (ug/lOO ml) and blood hematocrit (% RBC) values at 21 and 35 days of age . . . . Mean body weight, adrenal weights, and kidney weights of all subjects given behavioral tests . Mean body weight, adrenal weights, and kidney weights at twenty-one and thirty five days of age of subjects not given behavioral tests . . . Preweaning body weight (g) of subjects given behavioral tests . . . . . . . . . . . . . . . . Postweaning body weight (g) of subjects given behavioral tests . . . . . . . . . . . . . . . . Response of subjects to 28" stimulus in optokinetic drum . . . . . . . . . . . . . . . . Mean activity totals for four days and four nights in activity chambers . . . . . . . . . . Number of shocks prior to acquisition criterion of three trials without a shock . . . Number of active avoidances per block of four trials during active avoidance acquisition . . . Number of active avoidances per block of eight trials during active avoidance extinction . . . vi Page 24 25 27 31 52 54 55 69 72 75 76 78 79 82 Table BB. 89. 810. 811. 312. 813. 314. B15. B16. B17. B18. B19. B20. Number of passive avoidances in the first four trials during passive avoidance acquisition . . Number of passive avoidances per block of eight trials during passive avoidance extinction . . . Mean duration of three trials on six drum x speed combinations of the rotarod . . . . . . . Number of rewarded bar-presentations for ten days of training . . . . . . . . . . . . . . . . Number of correct responses per block of ten trials in the acquisition of a simple turning- response in an E maze . . . . . . . . . . . . . Number of correct responses per block of ten trials in the reversal of a simple turning- response in an E maze . . . . . . . . . . . . Number of correct responses per block of twenty trials in the acquisition of a tactually-cued conditional discrimination . . . . . . . . . . . Number of correct responses per block of twenty trials in the reversal of a tactually—cued conditional discrimination . . . . . . . . . . . Number of correct responses per block of twenty trials in the acquisition of a visually-cued conditional discrimination . . . . . . . . . . . Blood lead values (micro ug/lOO ml) for a sample of rats at four levels of lead exposure . . . . Packed red blood cell volume (%) for a sample of rats at four levels of lead exposure . . . Combined adrenal weights (sum of left and right as a percent of body weight) of subjects given behavioral tests . . . . . . . . . . . . . . . . Combined kidney weights (sum of left and right as a percent of body weight) of subjects given behavioral tests . . . . . . . . . . . . . . . . vii Page 85 86 89 93 97 99 101 103 105 109 110 111 112 Table 321. 322. Page Combined adrenal and combined kidney weights (sum of left and right as a percent of body weight) of rats sacrificed at twenty-one days of age . . . . . . . 113 Combined adrenal and combined kidney weights (sum of left and right as a percent of body weight) of rats sacrificed at thirty—five days of age . . . . . . 114 viii LIST OF FIGURES Mean total activity under light and dark conditions for rats exposed to four levels of lead poisoning . Mean percent successful active avoidances during acquisition and extinction . . . . . . . . . . . . Mean percent successful passive avoidances during acquisition and extinction . . . . . . . . . . . . Mean duration (sec.) on six size x speed rotarod drum combinations . . . . . . . . . . . . . . . . . Mean percent rewarded bar-presentations over ten days of training in the response—inhibition test . Mean percent correct trials in the acquisition and reversal of an E maze turning response . . . . Mean percent correct trials in the acquisition and reversal of a tactually-cued conditional discrimination . . . I . . . . . . . . . . . . O 0 Mean percent correct trials in the acquisition of a visually-cued conditional discrimination . . . Mean percent correct trials in the acquisition of a visually-cued conditional discrimination by male and female rats . . . . . . . . . . . . . . ix Page 29 33 36 38 41 43 46 49 51 I NTRODUCT ION Lead is a powerful, cumulative toxin to biological systems. Poisoning through inhalation or ingestion of lead may result in severe physiological, neurological, and behavioral aberrations. The mobili- zation of vast amounts of lead ore for usage in modern industry (Ziegfeld, 1964) has resulted in a widespread distribution of the metal; contaminating the air, water, soil, and food of man's environ- ment. The ambient level of lead in the environment has been increasing at nearly an exponential rate over the past forty years (Bryce-Smith, 1971). This level is, as of yet, below that which would endanger the health of the general population. Currently, the adverse effects of lead poisoning are largely restricted to children and animals that ingest lead-containing materials. Among these populations, one of the most heavily affected is migratory waterfowl. The birds often ingest spent lead pellets via their water-bottom feeding habits. Once ingested, the lead shot is subjected to prolonged abrasion and grinding by gizzard action. While a single shotgun shell may contain several hundred pellets, only five or six shot constitute a fatal dose for mallards (Karstad, 1971). The loss of waterfowl to lead poisoning is a major concern to wildlife managers. Cases of over 5,000 birds dying at one time are not uncommon and it has been estimated that four percent of the waterfowl population is lost annually to this cause (Belrose, 1964, 1959). Losses among other wildlife populations are not as marked, but concentrations of lead in food-chain animals and plants suggests the need for concern (Gish & Christensen, 1973; Hirao & Patterson, 1974). Lead toxicosis among domesticated animals is also known to occur sporadically. Plumbism is a well recognized veterinarian malady observed with particular frequency in urban areas (Zook, 1973; Zook, Carpenter, & Roberts, 1972). The sources of lead in the poisoning of dogs and zoo animals is often unknown, though lead-based paint is frequently suspected (Berry, 1966; Zook, Eisenberg, & McLanahan, 1973; Zook, Sauer, & Garner, 1972a, b). Similarly, poisoning among cattle and horses is most often attributed to ingestion of such non-food objects as used crankcase oil and storage batteries (Aronson, 1972; Donawick, 1966). In the vicinity of lead smelters these grazing animals may obtain toxic amounts of lead from their forage alone (Hammond & Aronson, 1964; Schmitt, Brown, Devlin, Larsen, McCausland, & Saville, 1971; Stewart & Alcroft, 1956). Although plumbism represents a health problem for the area of animal husbandry, the overwhelming area of concern must be the effects of lead ingestion on children. The danger of lead to children is particularly great because of the greater absorption and susceptibility to damage of developing rather than mature organisms (Barltrop, 1969; Kostial, Simonovic, & Pisonic, 1971). Nationally, two hundred fatalities and twenty thousand cases of overt intoxication from lead poisoning of children are reported yearly (Novick, 1971). Moreover, these children may represent only a fraction of the number affected by excess exposure to lead. The total number of children lead poisoned annually has been estimated to be 225,000 (Oberle, 1969). While lead exposure is known to have latent sequelae (Byers & Lord, 1943; Chisolm & Harrison, 1956; de la Burde & Choate, 1972; Pueschel, Kopito, & Schwachman, 1972; Thurston, Middlekamp, & Mason, 1955; Wiener, 1970) the extent of structural damage or functional impairment of children in this asymptomatic population is largely unknown. There are many sources of lead exposure for children, including atmospheric pollution, house dust, and many commercial products (Barman & McKiel, 1972; Bogden & Singh, 1974; Hankin, Heichel, & Botsford, 1973; Sayre, Charney, Vostal, & Bless, 1974; Shea, 1973). However, there is general agreement that pica, a perveted appetite for non-food objects is primarily responsible for the increased exposure of children to lead (Leonard, 1971; Lin-Fu, 1973; Smith, Baehner, Carney, & Majors, 1963; Wiener, 1970). Numerous studies of lead poisoned children have reported that the majority of affected children had a history of pica (de la Burde & Shapiro, 1975; de la Burde & Choate, 1972; Christian, Celewycz, & Andelman, 1964; Griggs, Sunshine, Newill, Newton, Buchanan, & Rasch, 1964; Jacobziner, 1966). Pica represents a serious health hazard when the child's environment con- tains materials with dangerous amounts of lead, such as particles of paint, plaster, putty, and perhaps newsprint (Bogden, Joselow, & Singh, 1975; Hankin, Heichel, & Botsford, 1973; Joselow & Bogden, 1974). Although current federal legislation restricts the manufacture and utilization of lead-based paints, an estimated 30,000,000 existing dwellings, constructed prior to World War II, likely contain potentially dangerous amounts of lead—based paint (Chisolm, 1973). An estimated 7,000,000 of these residences are in a dilapidated condition such that peeling paint and cracked and falling plaster are common (Chisolm, 1973). The incidence of childhood plumbism is closely associated with areas of older, deteriorating housing. Certain inner-city areas have, in fact, been dubbed "lead belts" due to the prevalence of poisoning (Griggs, Sunshine, Newill, Newton, Buchanan, & Rasch, 1964). Children in these zones are also exposed to greater ambient lead levels resulting from the greater traffic density in metropolitan areas (Cohen, Bowers, & Lepow, 1973; Mouw, Kalitis, Anver, Schwartz, Constan, Hartung, Cohen, & Ringler, 1975). The detrimental effects of lead on health have been extensively documented through clinical observations and experimental investigations. This large body of literature may be broadly divided into three areas: physiological, neurological, and behavioral effects. One of the most commonly reported and most thoroughly investi- gated areas of physiological damage are the hematological changes induced by lead poisoning. Primary among these effects is an inhibition of enzymes associated with heme synthesis, resulting in decreased hemoglobin and erthrocyte values (de Bruin, 1971; Cardona & Lessler, 1974; Chisolm, 1964; Davis & Andelman, 1967; Kao & Forbes, 1973). Additional hematologic changes noted have included: shortened life span and basophilic stippling of erthrocytes; reticulocytosis; and a stimulation of erythropoiesis in the bone marrow (de Bruin, 1971; Hass, Brown, Eisenstein, & Hemmens, 1964; Hernber, Nuriminen, & Hasan, 1967). The long bones, the primary site of erythropoiesis, are also the primary site for lead deposition and storage in the body. Osteopathic changes in bone formation and bone growth have also been found (Hass, Brown, Eisenstein, & Hemmens, 1964). Lead-induced physiopathological alterations in the liver, kid- neys, and gonads are also well documented. These changes include alterations in renal and hepatic metabolism, renal tubular dysfunction, and the formation of intranuclear inclusion bodies (Chisolm, 1962; Goyer, 1971; Goyer, Leonard, Moore, Rhyne, & Krigman, 1970; Singhal, Kacew, Sutherland, & Telli, 1973). The reproductive performance of laboratory animals has commonly been found to be decreased by lead poisoning due to: damage to the seminiferous tubules; decreased sperm motility; irregularity of estrus cycles; development of ovarian follicular cysts; and reduced viability of offspring (Hilderbrand, Der, Griffin, & Fahim, 1973; Lach & Srebro, 1972; Schroeder & Mitchner, 1971; Stowe & Goyer, 1971). Additionally, lead exposure may result in corneal opacification, increased intraocular pressure, and visual system degeneration which includes the eye muscles, the retina, and the optic tract (Grant, 1962; Grant & Kern, 1956; Kerstein, 1971). Neurological damage from lead is not confined to the optic nerve, but occurs throughout the central and peripheral nervous system. Encephalopathy is, in fact, one of the most frequent and most crippling effects of lead poisoning, often resulting in cerebral palsy, epi- lepsy, convulsive disorders, and mental retardation (Barltrop, 1973; Chisolm & Harrison, 1956; Perlstein & Attala, 1966). Systematic investigations of lead encephalopathy have uncovered a protean array of neurotoxic effects of lead. Among these effects are decreased axon size and interference with myelin and Schwann cell formation, resulting in decreased nerve conduction velocity (Feldman, Haddow, Kopito, & Schwachman, 1973; Krigman, Druse, Traylor, Wilson, Newell, & Hogan, 1974; Lampert & Schochet, 1968). Demyelination and degeneration of nerve fibers have also been implicated in the increased muscle contraction thresholds and extensor weakness reported in lead poisoning (Millichap, Llewellin, & Roxburgh, 1952; Seto & Freeman, 1964). Experimental studies of the central nervous system have reported increased cere- brospinal fluid and intracranial pressure, vascular lesions, cerebellar hemorrhages, and changes in brain biochemistry and metabolism (Kostial & Vouk, 1959; Krigman & Hogan, 1974; Michaelson & Sauerhoff, 1973, 1974). The behavioral effects of symptomatic childhood lead poisoning have been well documented through clinical observations. These effects may be loosely organized into three areas of damage: motor, social, and mental impairment. The effects of lead on the motor behavior of children are two- fold. The first of these is the development of hyperactivity or a general increase in motor behavior, resulting in children with plumbism frequently being described as restless, agitated, impulsive, and hyperexcitable (David, 1974; David, Clark, & Voeller, 1972; Thurston, Middlekamp, & Mason, 1955). The second manner in which lead affects motor behavior is to decrease coordination resulting in fine motor dysfunction, clumsiness, and ataxia (Jenkins & Mellins, 1957; Pueschel, 1974; Pueschel, Kopito, & Schwachman, 1972). The effects of lead poisoning on social behavior are also two- fold, both of which result in a failure to establish adequate social relationships. The first of these effects is a tendency for lead poisoned children to be socially withdrawn and listless, while the second effect is an increase in aggressive, hostile, and destructive behavior (Chisolm, 1970; Fulwiler & Wright, 1972; National Academy of Sciences, 1972; White & Fowler, 1960). The most serious and salient behavioral effect of lead on children is an impairment of intellectual functioning. Severe lead poisoning may result in permanent and profound mental retardation. Less severe childhood plumbism also has detrimental effects on intel- lectual performance. These children frequently show abnormally low performance on standardized tests designed to measure intelligence, memory, and learning ability (Byers & Lord, 1943; Chisolm, 1970; Perlstein & Attala, 1966; Wiener, 1970). Specific areas of handicap include poor visual-motor performance, poor form discrimination, short attention spans, and high distractability (Barocas & Weiss, 1974; Bradley & Baumgartner, 1958; de la Burde & Choate, 1972; Mellins & Jenkins, 1955; Thurston, Middlekamp & Mason, 1955). Despite the persistence of childhood lead poisoning as a grave national health problem, experimental analysis of the behavioral effects of lead poisoning has been relatively neglected. Behavioral aberration is manifest in those children severely lead poisoned, but their numbers have been claimed to represent only the "tip of the iceberg" of the total population of children affected by plumbism. Future experimental investigations must focus on the subtle behavioral sequelae of asymptomatic lead poisoning during development. Only a portion of the small number of existing animal experi- mental studies on the behavioral effects of lead poisoning are adequate models of asymptomatic childhood lead poisoning. A number of abstracts of behavioral studies from Iron Curtain countries are available which are informative, but insufficiently detailed for thorough analysis (Boyadzhiev, 1960, 1963; Gorschelva, 1951, 1957; Ungher, Lillis, Moscovici, & Pompilian, 1957; Ungher, Nestiano, & Lillis, 1957). American experimental investigations of the behavioral effects of lead poisoning are relatively recent. These studies have focused primarily on behavioral measures of learning and activity, but have also reported incidental observations on social and motor behaviors. Studies of acute or chronic lead poisoning of adult animals have failed to demonstrate behavioral effects (Brown, Dragann, & Vogel, 1971; Bullock, Wey, Zaia, Zarembok, & Schroeder, 1966; Snowdon, 1973) or have demonstrated disruption of learning—task performance following high levels of lead exposure (Avery, Cross, & Schroeder, 1974; Shapiro, Tritschler, & Ulm, 1973; Snowdon, 1973; Van Gelder, Carson, Smith, & Buck, 1973; Van Gelder, Carson, Smith, Buck, & Karas, 1973; Weir & Hine, 1971). Methodological faults common to many of these studies include: administration of fatal or near—fatal doses of lead, the use of adult rather than developing animals, and a failure to obtain physiological indices of lead exposure. Several studies of low-level lead exposure during prenatal and early postnatal development have all reported significant behavioral disturbance, often in the absence of overt, clinical symptoms of poisoning. These studies can properly be considered appropriate animal behavioral models of asymptomatic childhood lead poisoning. Carson, Van Gelder, Karas, & Buck (1974a, b) fed female sheep lead for five weeks prior to breeding and throughout gestation. Measurement of blood lead concentrations showed a mean level of 34 microg/lOO ml, only slightly above levels currently considered safe for pregnant women. The prenatally lead exposed lambs, tested on an operant visual discrimination task at one year of age, showed signifi- cant learning deficits. Postnatal lead exposure via the dam's milk has been found to result in encephalopathy in suckling rat pups (Pentschew & Garro, 1966; Rosenblum & Johnson, 1968). A similar method of exposure has also been demonstrated to result in post-weaning hyperactivity in mice, rats, and rhesus monkeys (Allen, McWey, & Suomi, 1974; Silbergeld & Goldberg, 1973, 1974; Sauerhoff & Michaelson, 1973). Post-weaning learning deficits have also been reported in rats suckled by lead poisoned dams (Brown, 1975, 1973; Snowdon, 1974). A somewhat different method of preweaning lead exposure was used by Sobotka and Cook (1974). Rat pups were intubated with lead acetate solution from three to twenty-one days of age and tested post-weaning on a two-way shuttle avoidance task. The mean blood lead concentration (23 microg/lOO ml) though considerably below that currently accepted as safe for young children, was sufficient to produce significant learning deficits. Two of these studies of early postnatal exposure also examined the responses of lead poisoned animals to psychoactive drugs. These 10 studies reported decreased motor activity of poisoned animals following injections of amphetamines, and increased motor activity following injections of phenobarbital (Silbergeld & Goldberg, 1974; Sobotka & Cook, 1974). The paradoxical behavioral responses to these medications by lead poisoned animals parallels the effects of these drugs on children with minimal brain dysfunction hyperactivity. Additionally, several of these studies have noted impaired motor behavior in poisoned animals (Silbergeld & Goldberg, 1974), abnormal social behavior and an increase in grooming and aggression (Allen, McWey, & Suomi, 1974; Sauerhoff & Michaelson, 1973; Silbergeld & Goldberg, 1973, 1974). The current research extended the experimental analysis of the behavioral effects of asymptomatic lead poisoning. Briefly stated, the purposes of the research were fourfold: (l) to sub- stantiate further that behavioral impairments may occur in the absence of overt, clinical symptoms of plumbism, (2) to identify additional behavioral tests that are sensitive to the effects of asymptomatic lead poisoning in rats, (3) to examine a possible dose- response relationship between lead exposure and behavioral impairment, and (4) to concomitantly obtain physiological indices of lead exposure. METHOD Subjects The experiment was performed in two replications. For the first replication, seven timed pregnant Long-Evans hooded rats were ordered (Charles Rivers Breeding Labs). Four of these females littered within a two-day period, and pups from these litters were used for the four experimental treatments. Two days following the birth of the last litter, cross-fostering of pups to the four experi- mental dams was performed to minimize any bias introduced through genetic differences in susceptibility to the effects of lead ingestion. After cross-fostering, each litter of ten pups was composed of two or three pups from each dam. For the second replication, seven Long- Evans females were mated in the laboratory. Cross-fostering pro- cedures were again followed. Because of the high pup mortality experi- enced in the first replication, litter sizes were increased to fifteen pups per dam for the second replication. Throughout the entire experiment all animals were maintained on a 12:12 light:dark cycle and given ad libitum access to water. Standard lab chow was provided ad libitum until the onset of 21 hour food restriction required for the later behavioral tests. All animals were weighed daily prior to weaning and weighed on alternate days following weaning. ll 12 Lead poisoning was induced through daily intubation of the rats with a lead acetate solution. This method of exposure allowed delivery of precise amounts of lead to the digestive system. The dosages used, 0, 10, 30, and 90 mg/kg were administered from three through twenty-one days of age. Each animal received its lead acetate in a volume of distilled water equivalent to 0.01 ml/g of body weight. Behavioral testing began the day following the last day of poisoning. Little experimental attention has been given to possible sex differences in the effects of lead poisoning. For this reason both male and female rats were tested. The composition of the groups were: 0 mg/kg--ll males, 9 females; 10 mg/kg--7 males, 10 females; 30 mg/kg—- 9 males, 7 females; and 90 mg/kg—-5 males, 10 females. Apparatus and Procedure The series of eight behavioral tests used reflects consideration of behavioral deficits commonly reported to result from childhood plumbism (e.g., hyperactivity, poor motor coordination, and deficits in visually and non-visually cued learning). Although childhood lead poisoning results in a constellation of behavioral deficits, previous studies have examined lead poisoned animals' performance on only one or two behavioral measures. The current research examined the per- formance of each animal on a series of tests, more adequately investi- gating the entire behavioral syndrome of developmental plumbism. The tests were divided into two groups on the basis of the requirement of food restriction to induce the necessary motivation for performance of several of the tasks. Group I tests preceded Group II 13 tests for all animals, and all subjects proceeded through the tests in the same order. Following the completion of Group I tests, animals were placed on 21 hour food restriction for the duration of testing. The three hours of food access immediately followed completion of each day's behavioral testing. Group II tests began after a minimum of seven days of food restriction. Visual Acuity Measurement-- Group I-Test I To date, no experimental study of the visual acuity of lead poisoned animals has been reported. The current research examined the effectiveness of an optokinetic drum technique for detecting visual acuity deficits in lead poisoned hooded rats. The Optokinetic method utilizes the reflexive nystagmus response to visual pursuit of movement in the visual field. This method has been extensively used with a number of species and has been shown to be the most sensitive measure of the visual acuity of rodents (King & Vestal, 1974). The optokinetic device consisted of a rotatable drum with interchangeable linings of vertical black and white stripes. The equipment used has been previously described by King and Vestal (1974). The animals were individually suspended in a restraining device such that their eyes were approximately 20 cm from the visual stimuli. Testing consisted of eight one minute trials. For each trial, the drum was rotated (3-6 rpm) for four fifteen sec periods in alternating clockwise and counter clockwise directions. Four visual stimuli were used. Three consisted of vertical black and white stripes subtending visual angles of 218, 28, and 14 minutes of arc. The fourth drum l4 lining was solid gray and was used as a control. For all subjects the order of stimulus presentation was 218", gray, 28", l4", 14", 28", gray, 218". Because judgement of the eye movements of the animals was difficult and subjective, three observers independently rated the response of the subjects on each trial. The ratings were: l--the response definitely did not occur; 2--the response probably did not occur; 3--the response probably did occur; and 4--the response definitely did occur. The criterion for recording a positive response on any trial was that the sum of the three observers scores be equal to or greater than nine. Activity Measurement—-Group I—Test II To obtain a measure of overall activity the rats were individ- ually housed in activity boxes (see Appendix A) for four days. The activity scores, accumulated on digital counters, were recorded twice daily at the time of transition of the 12:12 light:dark cycle. Despite efforts to equate their sensitivity, some differences may have existed between these laboratory fabricated activity chambers. To control for these possible differences, each animal spent one day in each of the chambers. The order of housing in the boxes was counterbalanced in a Latin Square design. For this test, and all sub- sequent measures, the animals were tested in squads, with each squad composed of one animal from each poisoning condition. 15 Measurement of Aversive Conditioning-- Group I-Test III To assess the generality of the learning deficits incurred through asymptomatic plumbism, it was of interest to examine the per- formance of lead poisoned on both positively and negatively motivated learning tasks. The current research examined the performance of lead poisoned rats on a test that combined both active and passive avoidance tasks. The procedure and apparatus used was similar to that of Bagne (1971). A typical avoidance chamber (90 x 10 x 32 cm), divided into two compartments by a moveable guillotine door, was used. 0n active avoidance trials, the rat was placed in the black (shock) side of the box and the guillotine door was raised. The raising of the door served as the CS. The CS-US interval, the time between the raising of the guillotine door and the onset of footshock (.8 ma), was five seconds. A successful active avoidance was defined as movement of the rat to the safe area prior to the onset of shock. If the animal did not avoid, the footshock remained on until an escape to the safe chamber was made. Following an avoidance or an escape, the rat was confined in the safe chamber for thirty seconds. After this safe area confinement the subject was manually placed in a holding bucket for twenty seconds prior to the start of the next trial. 0n passive avoidance trials, the rat was placed in the white (safe) side of the box and the guillotine door was raised. A success- ful passive avoidance was scored if the subject remained in the safe area for five seconds. Following a successful passive avoidance the door was lowered and the subject confined in the safe area for thirty 16 seconds. A failure to passively avoid was recorded if the animal moved into the shock (black) chamber. Once in the shock chamber, the con- tingencies became identical to those of an active avoidance trial. That is, the subject had five seconds to leave the chamber before the onset of shock. Following a failure to passively avoid the animal was confined in the safe area for thirty seconds. After the confinement, the subject was placed in a holding bucket for the twenty second ITI. Acquisition was completed in one day and extinction was con- ducted the following day. For acquisition, all subjects received 16 active and 16 passive avoidance trials in a predetermined order. Additionally, the shock chamber was rotated during acquisition and extinction to render direction cues irrelevant. For acquisition, two repetitions of the following sequence were made: A - P - AA - PP - A - PA - A — PP - AP - P - A, where "A" indicates an active avoidance trial, "P" indicates a passive avoidance trial, and “-" indicates a 180° rotation of the apparatus. For extinction, six repetitions of the sequence were completed. Throughout extinction, the shock- generator was turned off, and animals that failed to avoid were manually placed in the safe area for thirty seconds of confinement. Motor Coordination Measurement-- Group I-Test IV No previous testing of the motor coordination of lead poisoned animals has been reported. The current research evaluated the rotarod technique as a measure of lead-induced motor impairment. The rotarod, actually a motor-powered, rotating drum, has frequently been used by 17 pharmacologists as an index of the effects of drugs on the motor per- formance of rats. The apparatus consisted of a sand-paper covered drum mounted on a rod that was attached, via a series of gears, to a small elec- trical motor. The drum was located 130 cm above a burlap catching net. The behavioral test consisted simply of placing the animal on the turning drum and measuring the duration of time that it was able to stay on the drum without falling off. A stop clock was started when the rat's feet left the experimenter's hand and stopped when the rat landed in the burlap net or after ninety seconds. Since different drum size--rotation speed combinations may be differentially sensitive to lead's effects, two drum sizes (2 and 4 inches in diameter) were used at three rotation speeds (12, 20, and 30 rpm). Prior to testing, each animal was given three practice trials at the 4" - 12 rpm condition. Three trials at each of the six drum by speed combinations were then given each animal on a single day of testing. Following rotarod testing the rats were placed on 21 hr food restriction for a minimum of seven days prior to initiation of Group II behavioral tests. Measurement of Response Inhibition-- Group II-Test I A clinically observed symptom of childhood plumbism is an inability to inhibit inappropriate behavioral responding. The current research examined a discrete trial operant discrimination task for effectiveness in detecting impaired response inhibition ability in lead poisoned rats. 18 A standard rat operant chamber (24 x 22 x 21 cm) equipped with a retractable lever was used. All contingencies and recording were programmed with standard electromechanical equipment. The testing procedure required the subjects to inhibit bar-pressing for at least six seconds after the insertion of the retractable lever into the operant chamber. Responses prior to six seconds went unrewarded and resulted in bar-retraction for fifteen seconds. Responses after six seconds were reinforced with one 97 mg food pellet and also resulted in bar-retraction for fifteen seconds. The rats were shaped on one day and the following day were given fifty trials with the retractable lever operative, but without the six second delay contingency. Testing began on the third day of training. Fifty bar—presentations, with the delay contingency, were made on each of ten days of testing and the daily number of rewarded bar-presentations was recorded. E-Maze Testing The following three measures of the learning behaviors of lead poisoned rats all utilized a simple wooden E-maze. The start alley (10 x 14 x 60 cm) and goal boxes (10 x 14 x 24 cm) were attached at right angles to the running alley (10 x 14 x 150 cm). The first of these tests was relatively simple and the second and third relatively difficult. The rationale for varying difficulty of a single learning procedure was to determine if asymptomatic plumbism might impair the acquisition of complex tasks without impairing simple learning tasks. Additionally, these tests were designed to determine if the degree of learning impairment was related to the sensory stimuli involved. A single learning procedure that varied in difficulty and that varied 19 the relevant sensory stimuli, was thought to answer these questions. Such a procedure also minimized the problems of inter-measure com- parability which would have arisen if three distinctly different learning measures were used. Measurement of Simple Learning-- Group II-Test II The learning task was a simple left-right turning response in the E-maze. The rats were placed in the maze, with both goal boxes rewarded (two 97 mg food pellets) and allowed an initial period of habituation and exploration.' After the subjects had found and eaten the pellets in both goal boxes, they were removed from the maze. Each rat was then given a single trial, with both goal boxes again baited. The right or left turn of the rats on this trial was taken as the subject's initial turn preference. Following this trial each rat was trained to the direction opposite the initial turn preference. All subjects were given twenty trials per day for two days and the daily number of correct choices was recorded. 0n the third and fourth days of training, each rat received twenty trials of reversal training. Throughout acquisition and reversal, a variable ITI of approximately four minutes was maintained. Measurement of Complex Learning with Tactile Cues--Group II-Test III No deficits in tactile sensitivity or tactile discrimination have been reported in lead poisoned children. The absence of such reports suggests that either plumbism has relatively little effect on 20 this sensory dimension or investigations of this area have not been conducted. The learning task in this test was the acquisition and reversal of a conditional discrimination of substrate texture. The purpose of this measure was to examine the performance of lead poisoned rats on a complex task requiring utilization of cues from a sensory dimension not ordinarily impaired by plumbism. The E-maze used in the previous test was again used, as well as similar rein- forcement and ITI. The two substrate testures were defined by simi- larly colored interchangeable coarse sandpaper and smooth posterboard linings on the floor of the entire maze. The correct cue-response contingencies (e.g., right or left turn in the presence of coarse or smooth floors) were counterbalanced across animals. All rats received twenty trials per day for six days of acquisition and six days of reversal training. Each day of training consisted of two repetitions of the following sequence: SSRRRSRSSR, where "S" indicates the smooth floor linings and "R" indicates the rough floor linings. Throughout testing the daily number of correct choices was recorded. Measurement of Complex Learninggwith Visual Cues-~Group II-Test IV A common sequelae of developmental plumbism is impairment of learning involving visual cues. The learning task in this test was the acquisition of a conditional discrimination of visual stimuli lining the walls and floor of the E-maze. The purpose of this measure was to examine the performance of lead poisoned rats on a complex task 21 requiring the utilization of cues from a sensory dimension commonly impaired by plumbism. The E-maze, reinforcements, and ITI were similar to those of the previous two tests. The discriminative stimuli were two sets of posterboard linings with black and white horizontal or vertical stripes (1.3 cm in width). The correct cue-response contingencies (e.g., right or left turn in the presence of horizontal or vertical stripes) were counterbalanced across animals. The rats were given twenty trials per day for ten days and the number of correct choices was recorded. Each day of training consisted of two repetitions of the following sequence: HHVVVHVHHV, where "H" indicates the horizontal stripe linings and "V" indicates the vertical stripe linings. Physiological Measures Very few studies of the behavioral effects of lead poisoning in animals have reported ancillary physiological indices of lead exposure. Four physiological measures accompanied the behavioral tests of the current research. Blood lead values were determined by atomic absorption spectrophotometry (analyses performed by Environ- mental Health Laboratories, Farmington, Michigan). Samples of blood obtained by heart puncture on the last day of poisoning and two weeks after the end of poisoning were used to determine blood lead and hematocrit values. Following the completion of behavioral testing all subjects were sacrificed to obtain measures of the wet weight of their kidneys and adrenals. Additionally, several litters of rats treated similarly to the 0 or 90 mg/kg treatment groups, were sacrificed at twenty—one 22 and thirty-five days of age to obtain their kidneys and adrenals. All animals were given an overdose of ether, their kidneys and adrenals were surgically removed and immediately weighed to the nearest tenth of a milligram. RESULTS The results of the various measures were statistically analyzed using a two-way (Treatments x Sex) or a three-way (Treatments x Sex x Trials) analysis of variance (Winer, 1971), unless otherwise noted. Because of disproportional cell frequencies, unweighted means analysis of variance was used. The raw data used in these statistical tests are presented in Appendix B. Effect of Poisoning on Growth Body weight measurements for all subjects given behavioral tests are presented for the preweaning period in Table l and for the postweaning period in Table 2. The tables present the number of sub- jects (N), the mean (R) body weight, and the standard error of the mean (Sm). There were no statistically significant effects on body weight attributable to the treatment conditions, though, as expected, there were significant postweaning sex differences (F = 34.4, df = l, 60, p < .001) in body weight. Additionally, the postweaning effects of days (F = 724.0, df = 7, 420, p < .001) and the sex by days inter— action (f = 30.3, df = 7, 420, p < .001) were significant. There was an obvious treatment difference in the pre-weaning mortality of sub- jects. The control group lost twenty percent of its subjects, while the highest lead exposure group lost forty percent of its subjects. However, of those subjects surviving through behavioral testing, no 23 24 mu—u 2%“ Z 3 3 2 as 3 E In Nam en es s: 2 m. s z 2 z a a z 9.358 3 3 am 3 3 2 E 3; EN ”.2 as we as M 2 s 3 8 a a z 3358 3 3 S as no as E Ex is an as es 3 .x. 2 s s 8 m a 2 £22 3 3 3 S 2 as E ea SN as 3.2 a: S m. ON ON 8 a n a 2 9.350 mm. m um 1m. d .m: “2 “5 was 33 52>ch :35 £8.33 __m B Ems; >3; 5:83.21. :82 H 53: 25 9: 23 To 3e :3 mam as Tom 90 TR A: Emu me 9.2 9m To Em mmm mmm New :m mum EN EN EN 3H «mm «2 ”fl 3: EH mg as M. . gases «.2 92 T: 0.3 Q: 92 MS 9: Z: a; «.2 v.3 as A: m.m we Em omm Sm «mm mom 3N NR. mom «mm m: SN on: ma 9: NS NS 02 M 9:38 g 92 9.0 mi Ag 4.: To 92 3 ma Rm 9: 9m ~.m NB Em Em SN mum NmN mum 2N 8m 3m owm 0% new 0: 8m 2: 02 2: Rd M 9:32 0.2 92 ms 02 ms as :3 «.2 v.2 NA. 9: Z. 3. mm 3 3 Em EN 3m SN 3m mNN SN com New 2; «mu M: 8m m3 5 m2 N: V mxameo mbwuomuomuowkomuomowomuo .3. 3. was: Slat ID: No. mm m. M91 “5 8:5 was 33322 5% 9.8.33 =m co Ema; >3: 55:81.58 :82 N 5m <.F 26 detectable differences in general health or demeanor were evident. Only one subject, a male from the 90 mg/kg treatment group exhibited any overt morphological abnormality: an abnormal growth of the incisors. Visual Acuity Measurement--Group I-Test I The results of optokinetic testing of the visual acuity of all subjects are presented in Table 3. Virtually all of the subjects responded to the 218" stimulus on at least one trial, while none of the subjects showed a nystagmus response to the gray, control stimulus. A statistical test of responding to the 28" stimulus showed that posi- tive or negative responses were unrelated to the treatment conditions (X2 = 9.07, df = 9, N.S.). Only one subject, a female from the 10 mg/kg lead treatment condition, responded to the 14" visual stimulus. Activity Measurement--Group I-Test II Figure 1 shows the mean activity levels for light and dark conditions for all treatment groups. The results showed an effect of the lead exposure on activity, with poisoned subjects being signifi- cantly more active (F = 10.5, df - 3, 60, p < .001). Also, there was a significant effect of illumination conditions, with all treatment groups showing a greater mean activity level during the hours of darkness (F = 7.6, df = 1, 60, p < .01). No significant sex differ- ences or interactions were obtained. Measurement of Aversive Conditioning--Group I-Test III Because of a procedural error the data for 12 subjects for passive avoidance acquisition and passive and active avoidance extinction had to be discarded and are not included in the results presented. 27 TABLE 3 Number of rats responding to three visual stimuli in optokinetic drum lEAD DOSAGE LEVEL Omglkg lOmg/kg 30mg/kg 90mg/kg 218" stimulus 0” 11 7 9 5 O 9 10 7 10 28" stimulus o” 7 3 7 2 4O 5 9 5 6 Gray stimulus +03 28 .mcflcOmHom coma mo mHo>oH snow on oomomxo moon wow mcofluflocoo xumo pom unmfla hopes >ufi>wuom Homo» cmozir.a .oflm 29 "IV/101 All/\IIOV NVBW 8 8 8 8 8 8 8 8 8 e F 5.3.5 o L 9,? m V 31.6 o l 99 '00 x B» 3‘ F §§ E ‘5: l .09 9 0 FY 66 o E 66 l 95—8 F o ,_ r 8—»? 5 \ 3 0’ 5+3” r.l'%'§:o 6 o o 5 o 8 ° 8 8 8 l0 8 m N — 111101 Ail/\IIOV NVEW LEAD DOSAGE LEVEL 30 One criterion of the acquisition of avoidance conditioning is the number of shocks prior to three trials without a shock (Table 4). When this measure was applied to the current results, the effects of the poisoning treatments approached significance (F = 2.45, df = 3.60, p < .10). Examination of the data showed that subjects in the control group (0 mg/kg) tended to receive fewer shocks, while the subjects in the high lead exposure group (90 mg/kg) tended to receive a greater number of shocks prior to reaching this acquisition criterion. When the data were analyzed for number of correct avoidances over trials, significant treatment differences became evident. The treatment groups did show a difference in the acquisition of active avoidance (F = 4.6, df = 3, 60, p < .01) with the learning of the 90 mg/kg treatment subjects being most obviously impaired (Figure 2). The active avoidance data also showed a significant trials effect (F = 61.8, df = 3, 180, p < .001) and a significant treatment by trials interaction (F = 2.52, df = 9, 180, p < .01). This interaction resulted from a lag in the attainment of asymptotic performance of the 90 mg/kg treatment subjects. The results of the extinction of active avoidance (Figure 2) showed a significant treatments effect (F = 3.48, df = 3, 40, p < .05), as control subjects (0 mg/kg of lead) extinguished most quickly. The effects of trials (F = 20.6, df = 5, 240, p < .001) as well as the trials by treatments interaction (F = 2.97, df = 15, 240, p < .01) were significant. While all treatment groups showed essentially equivalent initial extinction performance, subjects in the high lead condition (90 mg/kg) were markedly more persistent in their avoidance 31 TABLE 4 Mean number of shocks prior to acquisition - criterion of three trials without a shock Omglkg OZ 9 N 11 9 X 3.0 4. 9 Sm 0.7 0.7 lOmg/kg N 7 10 Y 4.3 5.3 Sm 1.6 0.9 30mglkg N 9 7 7 5. 4 5. 4 Sm 0. 6 1.5 90mglkg N 5 10 7 6.4 7.7 Sm 1. 2 1.5 32 .cofiuocfiuxo pom coHufimHsvom mowuso moocmoflo>m o>fluom Howmmoooom ucoouom coozrr.m .mflm 33 EONVGIOAV .LNBOHBd NVBW O O O O O Q m a) N (D T I I I r I V?“ Z 9 E g I- t: / I 1 /' " ../ SEES sees 0938 Z 2 I: ”J D O 2 a! 8: 1 Q: I § I 81 BONVCIIOAV .LNBOHBd NVEW' IS 24 32 40 TRIALS l6 l2 34 responses than were animals of other groups. Subjects in the 0, 10, and 30 mg/kg groups showed a sharp decrease in avoidance responses, while the 90 mg/kg subjects showed only a slight decrease in avoidance during extinction. A correlated t-test comparing the first and last block of extinction trials showed that the 90 mg/kg subjects did show a significant extinction of avoidance (t = 2.84, df = 11, p < .05). Because of the rapid attainment of high level performance on passive avoidance acquisition by all groups (Figure 3), only the data for the first four trials were statistically analyzed. These results failed to show any significant differences due to the treatment con- dition or sex of the subjects. The data from the extinction of passive avoidance showed similar results (Figure 3). That is, the effects of trials was sig- nificant (F = 15.7, df = 5, 240, p < .001), while the effects of the experimental treatments only approached significance (P = 2.56, df = 3, 48, p < .10). Examination of the data showed that control subjects tended toward more rapid extinction of passive avoidance. Motor Coordination Measurement--Group I-Test IV Use of the rotarod technique to evaluate motor performance revealed a clear deficit in the coordination of lead poisoned rats (Figure 4). For statistical analysis, the mean of each rat's three trials on each drum by speed combination was used. A significant effect of the poisoning treatments was obtained (F = 5.12, df = 3, 60, p < .005), along with a significant difference in the effect of drum size by speed combinations (F = 41.3, df = 5, 300, p < .001). Subjects in the 90 mg/kg lead exposure group were consistently unable 35 .cofluocwuxo pom cofluflmfiswom mcwudo moocmoflo>m o>flmmom Hdwmmoooom ucoouom cmo271.m .mflm 36 BONVCIIOAV .LNEIOHBd NVHW O O O O O Q m on h o I T I I I T I fi‘ 'I z 9 I'- o .2. I- x m 8888 \\\\ GOOD E E E E 0 098m 2 Q . t 92 3 o 0 <2 .—_F I x o BONVOIOAV 1N3083d NVBW IS 24 32 40 48 TRIALS l6 l2 37 .mcoHuocHnEoo Educ ooumuou ooomm x ouflm xwm co A.oomv COADMHDU cooz|r.v .mflm (395) W080 N0 38 NOLWHI'IG NVBW 8 P 8 ‘3 .9. '2 8 “.2 8 8 8 e I I o o o o' I >17 w 06 >17 w 06 _ l____ S I tin/6w o: E fix/bu: 02 5 5 I—___ :01 I bag/bu: 0| 3’ LBX/bw 0| L Eng/Our o I bx/bw o I tin/Ow 06 I tin/5w 06 L 6w WI 02 I fwbw 02 L bx/fiw on I 69/5!“ or I tin/bu: o L bwfiw o I tin/5w 06 L fan/bu: 06 L ful/bu: 02 I bxxbw 02 L fix/bu: or tin/bu: on I tin/6w o I fix/Dw o 6 6 o 6 c3 «3 6 «3 o «3 o «'3 O} F 0 V’ '0 - O) h (D V” '0 — (093) WTIHCI NO NOIlVHnCI NV3W 20 RPM 30 RPM DRUM ROTATION SPEED l2 RPM 39 to remain on the rotating drum as long as rats in the other groups. Also, while few animals had difficulty staying on the drums at 12 rpm, almost all subjects were unable to maintain themselves on the drum at 30 rpm for the full duration of the trial. Measurement of Response Inhibition-- Group II-Test I Significant effects of the poisoning treatments were also obtained on the test of response inhibition ability (F = 14.2, df = 3, 60, p < .001). All lead exposed groups showed lower mean performance levels than non-exposed controls (Figure 5). These differences became evident with the first test session and persisted throughout the ten days of training. Subjects in the 90 and 30 mg/kg treatment groups were most impaired and the two group's mean performance was similar. The mean performance of the 10 mg/kg treatment subjects was better than that of the other lead-poisoning groups, though still consistently more poor than the performance of control rats. The only other sta— tistically significant effect obtained was that due to trials (F = 7.5, df = 9, 540, p < .001). All groups showed the same trend in perform- ance; approximately a ten percent decrease in the mean number of rewarded bar-presentations. Measurement of Simple Learning--Group II-Test II The simple E-maze task required the animals only to learn to go consistently right or left for reward. Neither the acquisition nor the reversal of this learning task revealed significant effects from the lead poisoning manipulation (Figure 6). The performance of sub- jects in all conditions was very similar and the mean performance of 4O .umou cofluflnHLCAIomcommou on» cw mcHCHmuu mo mwmo co» uo>o mcoflumucomoumruon oooum3ou ucoouom cmoZur.m .mflm 41 9 o——0 l0 mg/kg /k 0—0 30 mg/kg b—A 90mglkg 9 HOm L n 1 1 1 l \— O I0 0 to in ‘ CD 00 ID V 8 r0 SNOIlViNElSBHd 8V8 GBOHVMBU 1N3083d NVBW l0 DAYS 42 .omcommou mcficusu oumelm cm mo Hmmuo>ou ocm cofluflmwsvom on» CH mamauu uoouuoo unmouom cmozir.o .mam 43 1038800 1N30838 NVBW O) Q N o It) I I I I I .J < (I) 0: Lu > m a: 3333 \\\\ GOOD EEEE 0 098m 2 Q (L) D o o < l l l l 1 1 l 1 l \. o o o o o P as no b o n 1038800 1N308Bd NVBIN M 4b 3b 20 4b 30 20 b b TRIALS 44 all groups revealed rapid acquisition and reversal of this simple learning task. The failure of the groups to reach a higher mean per- formance level during acquisition was due to a few subjects in all conditions that showed a marked persistence in their initial turning preference. The only significant effect obtained in both acquisition (F = 8.1, df = 3, 180, p < .001) and reversal (F = 59.9, df = 3, 180, p < .001) were attributable to the increase in rewarded performance over trials. Measurement of Complex Learning with Tactile Cues-- Group II-Test III When the subjects were tested on the E-maze conditional dis- crimination of substrate texture, no treatment effects were noted in acquisition. In general, the groups performed similarly, though the asymptotic mean performance of the 0 and 90 mg/kg groups were somewhat higher than that of the other groups (Figure 7). As with the acqui- sition of the simple E-maze task, the only significant effect in the acquisition of the tactile E-maze task was attributable to trials (F = 9.5, df = 5, 300, p < .001). When the cue-response contingencies were reversed, however, a significant effect of the lead exposure was observed (F = 5.8, df = 3, 60, p < .005). The level of early postnatal lead exposure was inversely related to the initial mean performance of the rats on reversal. That is, those animals given the highest lead dosage tended to retain the previously acquired response the most (Figure 7). As with acquisition, a significant effect of trials was obtained in reversal (F = 20.2, df = 5, 300, p < .001). 45 .cofluocflefluomflo Hmcofluwocoo ooool>aamouomu 8 Mo Hmmuo>ou oco cofluflmfisvom ocu CH maowuu uoouuoo ucoouom coo2r|.n .mflm 46 1038800 .LN3083d NV3W to O ID ‘0 CD If) T I \- 8 e 8 :8 60 80 I00 40 |20 IOO 60 80 40 \‘ i (I) \ug'o (I m x > I.” I: 3333 \\\\ 0000 E EE E 00 2 09mm 9 L". (L) D O 0 4 I l l \I no 0 to o noA (D (D ID ID V 1038800 1N3083ci Nvaw I20 20 20 TRIALS 47 Measurement of Complex Learning with Visual Cues-- Groupill-Test IV The final behavioral test measured the acquisition of a visual conditional discrimination. Ten days of training failed to reveal a significant effect of lead exposure (Figure 8). The mean performance by all groups was consistently similar throughout the training period. A significant trials effect (F = 36.7, df = 9, 540, p < .001), as well as, two significant interactions were obtained. The significant sex by trials interaction (F = 3.3, df = 9, 540, p < .005) resulted from male subjects performing better than females on Day 1 and Day 10, while the opposite was true on the intervening days of training (Figure 9). The three way interaction between trials, sex, and treat- ments was also significant (F = 2.5, df = 27, 540, p < .01). This effect was due to the fact that in the later stages of training, males in the two lowest dosage conditions (0 and 10 mg/kg) performed better than their female counterparts, while the performance of males in the high dosage conditions (30 and 90 mg/kg) did not surpass the per- formance of those groups' females in the last days of training. Blood Lead and Blood Hematocrit Values Samples of blood taken on the last day of poisoning, Day 21, showed that subjects in the 30 mg/kg and 90 mg/kg treatment conditions had sharply elevated blood lead values and lowered hematocrit values (Table 5). Two weeks after the cessation of lead exposure (Day 35 samples) the blood lead values had decreased considerably. Statistical analysis showed a significant effect of the poisoning conditions (F = 104.9, df = 3, 40, p < .001) and a significant treatment by 48 .coflumcflefluomflo Hocofluflocoo bosorwaamomfl> o mo cofluflmwswom on» ca maofluu uoouuoo ucoouom cmoznn.m .mHm I m.._<_m_._. OON 0m. OO. O! ON. 00. 0m 00 Og ON 9.on om arlh 9.on on ulna ox\oE O. I 9.on o I .Om -OO .9. .ON 1038800 .LN3083d NV3W 50 .muou onEom pom mama >2 coflumcflefluomwo Hmcowuflocoo oozonxaamsmfl> o No cofluflmwovom onu cw mamanu uoouuoo ucoouom cmozul.m .mflm 51 l l O O IN (0 1038800 .LN3083d NV3IN so - so - ’{ 40 60 80 |00 |20 I40 I60 I 80 200 20 TRIALS TABLE 5 9 E: 05 5| 2 a: 0+ 38 a: I él o: g) .3 si e 'b 58 2"— “5o: gmg >s_.l Egg 388:2 0+ “U805 Efisfl Hgg’ game-”SING mm 18 V: 13.. $8 3 0+ C: 5'3 8 it E EI c’"o Blood Lead 9.97.2.1. 3 3 3 3 4 l4. 3 15. 7 34. 0 32. 3 159. 5 N x 3.4 2.7 3.1 0.3 26.6 Sm 52 12. O 15. 8 14. 8 23. 5 23. 3 56. 4 55. 6 12.3 8.0 5.7 1.4 2.6 0.9 1.7 2.1 1.8 Hematoc ri 3 4 4 4 4 4 4 33.7 32.5 32.5 26.0 25.8 26.0 25.8 3 33.0 0.9 1.4 1.0 1.4 1.0 1.3 0.7 1.5 3 4 4 4 4 5 36.0 36.5 36.5 35.8 36.3 34.4 3 37.7 0.5 0.9 0.8 1.0 0.7 0.6 0.3 53 sample day interaction (F = 15.75, df = 3, 40, p < .001). This interaction resulted from a small decrease in blood lead values for the low dosage conditions (0 and 10 mg/kg) but a large decrease in the high dosage groups (30 and 90 mg/kg). The results for the hematocrit measurements were similar. There was a significant effect of the poisoning conditions (F = 23.99, df = 3, 44, p < .001), a significant effect of the two week period between samples (F = 184.1, df = l, 44, p < .001), and a significant treatment by sample days interaction (F = 10.88, df = 3, 44, p < .005). Adrenal and Kidney Weights Following the last behavioral test all subjects were sacri- ficed at approximately 133 days of age. Table 6 presents the mean combined (sum of left and right) adrenal and kidney weights of all subjects given behavioral tests. Statistical analysis was performed on the mean percent of body weight data. No significant differences in kidney weights were found. However, a significant lead treatment effect on adrenal size was noted (F = 2.9, df = 3, 60, p < .05). The subjects given behavioral tests were sacrificed as mature adults after a long maturation period following poisoning. This interval may have partially masked treatment differences in adrenal and kidney sizes induced by the neonatal lead exposure. To assess the effect of poisoning at an age closer to the lead exposure period additional subjects were poisoned and sacrificed at 21 and 35 days of age. In order to examine the two extremes of poisoning, only the O and 90 mg/kg lead exposure levels were used. The mean combined kidney and adrenal weights of these subjects are presented in Table 7. 54 sod sad 8... so. so. so. so. as. E was as... mes was sad :3 was :3 .E as s a. x 3 3 we 3 3 em 3 3 E as es s .3 53 we. 24 98 o .3 as. 2%; 329.3. 8. s. s. e. 3. 3. S. s. E s .o e .o s .o e .o 2 .o e .o e .o e .o .E as s .4. m 3.: 2m 9: es 2: $2 3.8 we E s: as as ea :2 as 3: as as. some x $655. o .2 2m 4 .2 m .e 3 , o .2 s .2 o .s E mm we am a E N: am an a. m s m a a 2 a a z z W Aw m . 2%: as. no .6 No a .0 Gamma mamafi gels 32a ._m_>u._ mu25: Em £225 ace 8 .2295 >8: :82 o 5m 5. 55 m8. NS. so. :5. m8. NS. NS. H8. Em ms. 48. me. so. as. 3.. N8. N8. 2%; as s .4 m 3 3 Z Z 3 E 3 as Em 3m in em New a: <2 3: 4.3 as. 5%; m 22.8.. u< 8. s. s. No. 3. e. e. s. E a .o 8 .o 8 .o a .o a; as 2 .H 8 .H 2%; as s .4. x 9% 2m 3m Em MEN 0 .e s .E 2: E E as EU m3 :0 es em as as. 2%; m @355. H...“ em 2 3 Z 3 as 3 E was $2 :2 32 33 33 3% use a. m 2 s 2 : NN : a a 2 0 0 so 0 Mo Mo 2%; as. as $8913 98383 as seam as as 0 “ea 2,2 :2: 6&8 as: was .8223: :33 8: $833 3 man to £8 3:1»: :3 can 08.35%. E 3:92, .65: new 52%; .29. 8 .523, 33 :82 N 5m 5 56 As with the data for the subjects given behavioral tests, the mean percent of body weight data were used for statistical analysis. Com- parisons of adrenal size at both twenty-one (F = 3.9, df = l, 87, p < .05) and thirty-five (F = 9.8, df = l, 65, p < .005) days of age revealed a significant increase in wet weight due to lead poisoning. Similarly, the wet weight of the kidneys was significantly increased by lead exposure at twenty-one (F = 18.8, df = l, 87, p < .001) and thirty-five (F = 4.1, df = 1, 65, p < .05) days of age. No other significant differences were found. DISCUSSION With regard to the primary purpose of this study, the results strongly demonstrate that lasting behavioral impairments may be induced by transient, asymptomatic lead poisoning during development. None of the subjects exhibited the typical symptoms of lead toxicosis in rats, such as anorexia, impaired growth, rough pelage, or tendencies toward ataxia (Michaelson & Sauerhoff, 1974). However, significant behavioral effects attributable to the early postnatal lead exposure were obtained on five of the eight behavioral measures studied. The fact that several of these tests revealed clear performance deficits in the lead poisoned rats also addresses the second purpose of the study: the identification of appropriate behavioral tests of asymptomatic plumbism in rats. These measures add to the array of behavioral tests useful in the further experimental analysis of developmental plumbism's behavioral sequelae. The third purpose of this research was to examine a possible dose-response relationship between lead exposure and behavioral impairment. The results were inconsistent in this regard. Three tests, visual acuity, simple E-maze, and visual E-maze measures, revealed no treatment effects. Examination of the figures depicting the results of the other five measures, shows that relative to con- 'trods, the 10 mg/kg treatment group exhibited performance decrements 57 58 only in the response inhibition and tactile E-maze reversal tasks. On the other measures the performance of the lowest lead exposure level was essentially identical to that of the non-poisoned controls. On the tactile E-maze reversal and the rotarod tests the degree of behavioral impairment did tend to reflect the lead exposure level. As the level of poisoning increased, the degree of behavioral impairment increased. On two of the behavioral tests, the activity and response inhibition measures, the performance of the 30 mg/kg and 90 mg/kg groups were nearly equal, though clearly different from that of con- trol subjects. An overview of the results of the entire experiment does lead to a conclusion of a gross dose-response relationship. That is, animals subjected to the higher levels of poisoning were more likely to show a greater degree of behavioral disruption. The fourth purpose of the study, to obtain physiological indices of lead exposure, was also accomplished. Perhaps the most significant feature of these results was the rapidity with which diagnostic symptoms of plumbism decreased in the blood measures. This may be taken to indicate that while the behavioral effects of lead poisoning are relatively persistent, the typical clinical indices of lead exposure necessary for accurate post-hoc diagnosis of exposure level are quickly transient. Despite the failure of the optokinetic drum technique to reveal visual acuity deficits following lead exposure in this study, its use should be encouraged. Disturbances in visual ability are a well known after-effect of lead poisoning, and the induced structural aberrations have been well described in man (Grant, 1962; Kerstein, 59 1971). However, because of the retrospective nature of these studies very little is known regarding the exposure parameters required to induce visual pathology. Animal experimental studies of lead's visual effects are limited and have often relied on topical rather than systemic exposure (Grant & Kern, 1956). Though these animal models may induce similar pathological changes in structure, the optokinetic task seems ideally suited as an amotivational test unabiguously revealing functional deficits in visual acuity. The increase in overall activity found in the current study confirms previous animal experimental demonstrations of lead induced hyperactivity in asymptomatic mice and rats (Sauerhoff & Michaelson, 1973; Silbergeld & Goldberg, 1973, 1974; Sobotka & Cook, 1974). These prior studies measured activity for shorter periods and only under illuminated conditions. The current research demonstrated increased activity during both the light and the dark phases of the photoperiod. While lead poisoned animals exhibited a greater absolute level of overall activity, the relative ratio of activity under day or night conditions was unaffected. Hyperactivity is a well-documented sequelae of childhood plumbism (Thurston, Middlekamp, & Mason, 1955). Childhood hyper— activity actually describes a syndrome of behaviors partially characterized by high levels of motor behavior, short attention Spans, and impulsivity (Stewart, 1970; Wherry, 1968). In a large portion of the cases the exact etiology of developmental hyperactivity is uncer- tain, but it is known to affect approximately five percent of United States children (David, 1974). The estimates of a large, undetected 60 population of asymptomatically lead poisoned children (Oberle, 1969), animal experimental demonstrations of hyperactivity following asympto- matic plumbism, and demonstrations of increased body lead levels in hyperactive child populations (David, 1974; David, Clark, & Voeller, 1972) combine to raise the alarming suggestion that asymptomatic lead exposure may be an important causative factor in many cases of develop- mental hyperactivity. The results of the aversive conditioning measure were con- sistent with previous reports that lead poisoning disrupts active avoidance acquisition in rats and goldfish (Avery, Cross, & Schroeder, 1974; Sobotka & Cook, 1974; Weir & Hine, 1970). Observations of the subjects during training suggested that the impairment in acquisition was related to the persistence of inappropriate responses by lead poisoned animals. Rather than making the appropriate response of running into the safe chamber, these subjects tended to freeze or make vertical jumping responses both during the CS—US interval and after shock onset. The current study is the first to examine active avoidance extinction in lead poisoned animals. Once the avoidance response was acquired, the high lead exposure subjects showed a greater resistance to extinction. The extinction of avoidance behavior has been inter- preted as resulting from the competing response of relaxation with the previously acquired fearful emotional responses (Denny, 1971). This higher resistance to extinction, then, as well as the emotional responses seen in acquisition may be interpreted as consistent with 61 reports of hyper-excitability seen in hyperkinetic and minimal brain dysfunction children (Paine, 1968). The failure of passive avoidance testing to reveal any treat- ment differences may be due in large measure to the relatively high level of shock used. In passive avoidance acquisition, high levels of shock elicit emotional freezing responses compatible with the required response. The data for passive avoidance extinction approached significance, however. Control subjects tended toward a more rapid extinction of passive avoidance, possibly indicating a failure of lead poisoned animals to exhibit relaxation responses as quickly as controls. The rotarod portion of this study was the first explicit attempt at the experimental analysis of the motor coordination of asymptomatically lead poisoned animals. Despite the lack of obvious motor impairment, animals in the two highest lead exposure treatments showed a pronounced deficit in the ability to maintain themselves on the revolving drum. Fine motor incoordination and clumsiness have been reported following frank lead intoxication (Jenkins & Mellins, 1957). The current animal study is confirmatory evidence of recent human studies demonstrating motor impairment at sub-clinical levels of lead exposure (Pueschel, 1974; Pueschel, Kopito, & Schwachman, 1972). The results of the response inhibition test clearly revealed a deficit in the ability of lead poisoned rats to withhold inappro- priate responding. Since none of the subjects showed acquisition of the delayed response this suggests that this test either represents a 62 poor measure of learning or an insufficient period of training was given. Observations of the subjects during testing showed that con- trols tended to engage in grooming, exploration, or food-cup investi- gation during the bar-retraction and response-delay periods. The behavior of animals in the lead poisoned groups was distinctly dif- ferent and more varied. The entire pace of behavior was noticeably more agitated and frenetic among lead exposed animals. For example, the exploratory behavior of controls was replaced in the poisoned rats by rapid dashes from place to place within the test chamber. Escape responses were most evident in the lead poisoned subjects, with bar— presses sometimes being made by a hind foot or other body part during jumps toward the chamber ceiling. Additionally, aggressive-like behavior was more evident in the lead treated rats. A common observation was a biting attack on the lever as it emerged into the chamber. For the lead poisoned animals, a rewarded response would often follow a protracted and highly agitated period in the food cup. During these periods, the animals were commonly observed lying on their back, biting the wire mesh covering of the food cup. The overall subjective and qualitative impression of the lead poisoned subjects was one of a higher level of agitated activity than shown by controls. The simple E-maze task of the current study failed to reveal a learning deficit as a result of the lead treatment. Previous studies of lead poisoned rats' learning of simple mazes have similarly failed to show an effect (Brown, Dragann, & Vogel, 1971; Bullock, Wey, Zaia, Zarembok, & Schroeder, 1966). However, these two earlier studies are only minimally comparable to the present study for they 63 used adult animals administered overtly toxic doses of lead. The most plausible explanation of the current results is that asymptomatic plumbism has a negligible effect on very simple learning tasks. The data from the tactile E-maze test failed to show a treat- ment effect on acquisition, but did show an effect on reversal learning. Preweaning lead exposure resulted in a marked lag in the acquisition of the reversed cue-response contingencies. This may be taken to indicate a decrease in the ability to inhibit inappropriate, pre— viously acquired responses. If this interpretation is accepted, then, a relationship with the response inhibition test becomes evident. That is, both measures revealed a lead induced deficit in inhibition abilities of the exposed subjects. The results of the visually-cued E-maze test were unexpected. Reports of lead's interference with visually-cued learning are common in the clinical literature (Bradley & Baumgartner, 1958; Mellins & Jenkins, 1955; Thurston, Middlekamp, & Mason, 1955). Additionally, a recent experimental study of early postnatal lead exposure did show disruption of a conditional light-dark discrimination in a T-maze (Brown, 1975). Pilot work preceding the current study did reveal a lead induced impairment in the acquisition of the visually-cued E-maze test. This test was originally considered to be one of the more powerful behavioral measures of the entire study and the lack of significant treatment effects is puzzling. In some instances, behav— ioral disabilities incurred through childhood neurological trauma seem to dissipate with further maturation. This phenomenon has been 64 termed "maturing-out." A tentative hypothesis accounting for the results of the visually-cued E-maze test is that a process similar to the "maturing-out" seen in some lead poisoned and brain damaged children may have occurred (Paine, 1968; Pueschel, Kopito, & Schwach- man, 1972; Thurston, Middlekamp, & Mason, 1955). Since the visually- cued E-maze test was the last behavioral measure, allowing the rats to reach maturity, such an explanation is plausible. The design of the current study confounded order and matura- tional effects making it impossible to adequately assess the role of these variable in the current study.) That is, it cannot be determined if similar results would have been obtained if the behavioral tests were administered in a different order or at different ages. The important question of the persistence of plumbism's behavioral deficits can only be answered through careful longitudinal studies. The physiological data provided informative indices of lead exposure. The samples of blood taken at twenty—one days of age showed very high blood lead levels among animals of the two highest lead exposure groups. Interpretation of these levels is difficult because of the dearth of previous behavioral studies that.have obtained blood lead measures, and also because of the rat's reputed resistance to lead intoxication (Scharding & Oehme, 1973). Sobotka and Cook (1974) used a poisoning procedure nearly identical to the current study. They reported significant behavioral effects as well as the lowered lead content in the blood of thirty-five day old animals. Although the blood lead levels of the thirty-five day samples in the current study were somewhat higher than those of Sobotka and Cook, they 65 generally agree in showing a rapid decline in blood lead content following the cessation of lead exposure. The adrenal and kidney weight data also lack suitable studies for comparison. The present data do however, show that the higher lead levels were sufficiently stressful and toxic to produce increased adrenal and kidney weights (Goyer, 1971; Selye, 1956). The need for more sophisticated ancillary physiological measures of lead exposure became evident in the current study. Measurement of adrenal and kidney weights is only a gross measure of the effects of lead poisoning and these values are subject to inaccuracy due to dessication. The current study investigated the behavioral syndrome of asymptomatic plumbism. The results of animal behavioral studies, as well as, clinical observations increasingly indicate the similarities between the effects of plumbism and the syndrome of minimal brain dysfunction (MBD). The MBD classification is a categorical name for a constellation of behavioral deficits resulting from neurological damage. Children in this classification show such impairments as hyperactivity, poor motor coordination, poor impulse or inhibitory control, and a variety of learning difficulties (Paine, 1968). This symptomology is parallel to that seen in the current study and in developmentally lead poisoned children. In many instances the etiology of MBD is unknown. There is likely no single causitive agent in MBD. However, the realization of the widespread nature of asymptomatic plumbism (Needleman, 1973) raises the question: is lead poisoning an important contributor to this behaviorally crippling childhood dis- order? 66 The current research demonstrated that satisfactory animal behavioral models of asymptomatic plumbism can be developed. Such animal studies are essential to investigation of the functional and structural effects of lead poisoning, and promise to provide a bridge toward empirical investigation of minimal brain dysfunction. The question an experimentalist should ask upon completion of a study is, "what comes next?" Given a problem as complex and rela- tively uninvestigated as the behavioral effects of asymptomatic plumbism, the answer is difficult and multifaceted. There are at least four areas that merit further investigation. First, the attempt to elucidate the behavioral effects of asymptomatic plumbism must continue. This area of investigation would take the form of further exploration of appropriate and powerful animal behavioral preparations, including an emphasis on longitudinal studies. Secondly, a much greater use of physiological and neurological assays must accompany behavioral studies. Only through attempts to correlate structural aberration with functional impairment can an adequate understanding of plumbism's effects be understood and managed. Third, a critical area must be seen as further examination of lead exposure periods. Empirical work in this area would take the form of tests of prenatal exposure, as well as studies of inter- generational transfer and accumulation of body lead burdens. Fourth, factors modifying lead's toxicity deserve further attention. Outstanding among these factors are the effects of clinical 67 treatment procedures and nutritional status in modifying lead poison- ing's behavioral effects. APPENDIX A Appendix A The activity boxes (30 x 30 x 30 cm) were inexpensively produced and were sufficiently sensitive to measure locomotion, rearing, and vigorous grooming as activity. The floor of the boxes, designed to allow slight vertical displacement, were rigidly connected to the vibration sensitive crystal of a phono cartridge. This connection results in an electrical signal in response to floor vibration. A high gain op-amp brought the signal to a useable level and this output controlled a gating circuit. The gating circuit controlled the output of an astable multivibrator calibrated at ten pulses per second. Any activity by the rat which produced a signal allowed the output of the multivibrator-driver circuit to step a digital counter. Discrete motor movements, such as a head wipe, by the rat resulted in two to five counts. Prolonged activity, such as locomotion, resulted in ten counts per second for as long as the rat remained active. 68 APPENDIX B Table Bl Preweaning Body Weight (g) of Subjects Given Behavioral Tests ‘Q_Mg/Kg Treatment _>'_Da ages: 2 .6. .9. .12 .1_5 is. 3.1. 13.8 20.6 19.7 16.7 41.4 52.4 58.9 13.7 19.8 18.1 19.6 31.8 36.3 60.6 9.3 12.7 9.4 21.5 30.3 19.0 50.3 8.2 13.8 18.0 20.1 32.7 36.0 37.1 8.5 13.0 16.3 21.0 18.5 36.9 40.7 8.1 12.6 25.2 23.2 22.8 54.5 44.0 8.1 13.9 17.7 21.7 13.6 39.8 46.9 8.4 9.4 14.3 30.7 27.0 29.0 35.6 7.9 10.2 28.1 24.0 31.2 25.7 35.6 6.4 7.2 14.0 32.7 35.1 25.7 47.0 5.8 9.5 19.6 24.7 26.8 39.6 36.0 7.1 9.6 13.9 20.6 40.6 29.8 39.7 6.4 7.3 10.7 15.4 22.1 31.5 48.3 5.6 7.2 16.9 17.6 27.3 35.8 44.3 6.9 7.3 12.7 19.7 23.6 33.8 41.1 6.5 8.7 8.7 20.4 31.3 37.6 51.5 8.4 8.9 16.8 23.0 25.0 35.6 39.1 9.6 10.4 10.6 25.8 29.8 36.4 43.2 6.6 10.4 17.6 24.8 31.3 27.6 34.5 9.0 11.9 11.8 27.1 32.4 37.8 47.5 8.9 11.7 17.4 9.1 13.0 8.3 13.4 6.8 8.7 19_Mg/Kg Treatment 1 11.0 8.0 14.0 14.2 21.3 41.4 43.2 8.3 9.7 15.0 18.7 27.2 29.6 44.8 9.5 10.5 17.0 22.1 26.6 29.9 52.6 8.8 10.2 11.5 22.1 28.8 38.6 42.8 9.5 10.0 21.6 22.6 32.4 33.2 40.0 8.5 11.2 14.6 23.9 29.6 35.6 49.1 8.1 12.7 18.3 26.7 30.7 38.3 33.3 7.0 10.3 13.6 30.7 31.2 41.5 50.6 69 70 Table Bl (Cont'd) 19.Mg/Kg Treatment _3'_Da 823589. 9. .6. .2 32 32 a a 6.3 14.2 10.7 30.1 34.5 25.3 56.1 6.9 15.7 24.1 30.4 36.7 46.4 65.7 14.5 5.7 19.4 23.9 18.6 42.3 54.6 12.6 20.6 18.6 24.3 30.0 43.8 54.6 8.6 17.9 24.1 11.3 29.2 46.0 50.3 8.7 15.4 20.4 29.0 19.2 27.0 47.6 9.4 13.6 15.2 23.4 39.4 42.4 57.6 9.3 15.1 18.0 24.3 35.1 55.0 32.9 9.8 14.1 18.7 25.6 41.3 43.7 56.7 9.7 13.4 19.1 24.3 40.4 5.7 12.0 9.2 26.9 34.4 6.9 10.0 4.1 7.1 9.3 6.7 6.8 6.6 8.7 5.1 6.2 39_Mg/Kg Treatment 14.8 22.6 17.8 18.6 32.3 51.3 59.6 15.9 22.5 15.6 28.0 17.5 32.8 53.2 13.1 14.6 31.3 40.7 20.5 34.2 54.3 9.1 13.5 19.7 13.0 17.8 26.7 69.0 8.6 13.7 12.6 27.2 18.0 23.2 48.7 9.0 10.7 11.9 13.9 25.9 27.7 43.6 8.6 8.1 10.4 33.6 41.7 38.0 39.9 9.1 7.3 29.5 15.1 39.9 32.3 54.6 6.1 6.5 14.1 22.1 30.9 51.3 49.8 7.8 7.6 16.2 18.6 25.7 37.5 62.3 6.5 7.8 9.0 19.0 18.4 29.4 41.3 6.3 5.2 9.1 21.0 20.5 34.7 38.0 5.9 5.8 11.6 15.6 23.8 32.6 46.1 6.4 11.7 12.4 14.4 21.9 34.7 36.2 7.0 8.0 19.2 19.6 23.1 34.6 40.4 7.0 8.7 15.4 17.0 24.8 31.8 40.6 7.8 7.5 8.9 17.1 25.2 30.4 9.9 9.5 13.4 21.1 27.7 8.1 11.1 10.8 24.3 28.4 6.4 9.1 11.7 7.3 13.0 9.2 11.2 8.2 13.5 8.3 8.13 71 Table Bl (Cont'd) 29_Mg/Kg_Treatment P‘P‘P‘h‘h‘h‘h‘ 2212.25.582 2. 9. .2 .12 .12 .12 .21 6.2 22.7 19.1 18.7 34.5 46.6 40.6 6.0 18.6 31.2 28.6 47.4 41.3 54.7 6.9 12.7 16.1 13.1 36.8 55.2 43.1 5.9 6.0 17.1 39.7 38.3 46.3 57.6 6.6 9.3 9.8 21.3 31.9 40.3 50.0 6.6 9.2 18.7 21.7 18.2 44.7 53.9 7.7 14.1 21.8 21.8 24.1 41.9 64.2 7.1 14.8 16.1 23.9 28.4 24.6 50.7 7.4 11.2 11.7 30.7 33.7 35.0 48.1 8.6 14.6 12.7 23.1 31.6 35.6 50.9 8.4 6.8 14.9 11.5 17.4 36.1 43.7 9.1 10.6 9.8 15.7 24.7 36.1 42.6 11.5 6.0 14.7 19.3 27.7 37.6 52.4 15.3 7.0 16.1 17.7 29.4 40.7 43.9 16.5 8.5 9.0 17.6 27.2 24.7 45.2 6.9 0.6 14.6 17.7 27.7 7.4 0.0 6.6 19.1 34.8 8.5 0.6 11.4 23.6 10.6 2 8 12.7 14.5 9 4 4 2 8 2 4 5 8 7 4 8 5 8 7 8 9 8 72 Table 82 Postweaning Body weight (g) of Subjects Given Behavioral Tests Q_Mg/Kg Treatment _z_Da 8 2i 1.955. s_u_1_b eat. 2 Q 93. a 9_1 192 119. .133. MALES 111 128 153 191 202 241 251 274 265 112 118 165 228 221 273 290 323 346 113 131 177 223 235 290 291 325 312 115 131 208 234 222 291 290 323 324 116 106 187 244 264 315 337 388 387 117 94 140 192 182 242 291 380 372 212 124 186 214 241 268 282 310 331 213 103 180 206 244 264 278 307 321 216 115 184 207 247 260 290 312 348 217 123 192 215 240 249 271 294 301 314 63 114 131 162 185 216 228 258 FEMALES 114 132 190 194 226 255 241 265 279 118 115 172 227 206 241 240 250 248 211 131 159 163 190 210 221 238 248 214 118 158 199 225 216 247 289 308 215 109 138 150 170 182 202 240 263 218 102 177 181 200 188 212 246 268 311 82 168 194 228 206 228 229 226 312 80 116 131 152 187 231 286 327 313 61 103 120 148 170 188 197 207 19_Mg/Kg Treatment MALES 121 123 173 261 263 322 327 358 350 222 128 169 190 270 303 318 321 336 223 128 171 207 265 291 319 351 398 224 120 186 217 248 290 302 318 322 225 132 168 193 224 260 304 341 368 226 120 176 190 231 271 290 334 355 322 89 141 163 202 220 237 249 263 Table B2 (Cont'd) 73 10 13813.8. Treatment Days g£_Age Subject 3_5_ 4_9. 9.3. 17. 9.1 3e 119 1.3_3 FEMALES 122 121 168 211 206 230 231 250 247 123 96 148 182 170 214 205 208 203 124 128 174 179 200 242 241 265 256 125 128 178 211 190 237 ' 238 252 253 r 126 122 141 175 194 173 216 227 272 221 120 150 176 196 226 251 256 268 227 97 134 142 150 168 170 207 215 228 88 130 136 148 171 206 230 260 321 73 137 149 187 196 212 210 211 323 90 134 140 162 186 194 218 222 F 39 Lig/_I(g_ Treatment MALES 133 115 160 206 202 247 256 288 275 134 109 180 194 226 253 285 316 339 135 123 198 211 221 291 305 368 365 136 117 184 263 255 293 287 328 320 233 108 186 195 209 229 261 290 284 234 105 178 192 258 286 314 367 338 236 85 156 168 192 218 237 257 300 332 87 137 155 188 214 231 251 266 FEMALES 131 112 148 209 213 252 241 254 245 132 94 160 147 158 184 186 209 225 231 122 159 181 204 212 234 236 245 232 117 131 140 159 184 204 218 220 235 92 140 163 197 241 266 290 294 237 90 126 131 142 171 193 221 235 331 90 140 150 155 176 188 207 210 74 Table B2 (Cont'd) 29_Mg/Kg Treatment Days pf Age 9. 91>. 1e.ct 15. 52 13. 11 2.1. 39.5. .119. 119. MALES 144 71 143 107 143 189 187 206 233 145 131 237 260 271 341 365 427 441 242 91 159 191 229 258 281 303 322 244 106 178 214 291 327 361 392 416 341 93 151 167 186 217 240 257 279 FEMALES 141 122 128 188 204 230 221 237 229 142 112 167 191 187 221 224 233 257 143 120 155 180 179 218 212 231 226 241 110 151 190 208 230 256 280 287 243 117 156 171 201 219 255 271 280 245 95 136 140 168 220 279 297 302 246 89 136 151 164 191 215 256 269 247 85 131 139 141 181 207 221 223 248 87 127 143 184 217 239 257 266 342 81 131 147 161 181 170 191 190 Response of Subjects to 28" Stumulus 75 Table B3 in Optokinetic Drum 2 H8158. .12 3.18158. 2", f 2 B. a E 2 3 111 +§ 114 + 121 o 122 + 112 0 118 o 222 + 123 + 113 + 211 + 223 0 124 + 115 0 214 + 224 + 125 + 116 + 215 0 225 o 126 0 117 + 218 0 226 0 221 + 212 0 311 + 322 + 227 + 213 + 312 0 228 + 216 0 313 + 321 + 217 + 323 + 314 o 2.9. 118/.158. 2.9. 581$ 2' 3 1 3 d' R 9» R 133 + 131 + 144 0 141 + 134 + 132 + 145 0 142 + 135 + 231 + 242 0 143 + 136 + 232 0 244 + 241 + 233 + 235 0 341 + 243 0 234 0 237 + 245 0 236 + 331 + 246 o 238 + 247 o 332 0 248 + 342 + 1. R - Response 2. +4- Response was observed 3. 0 - Response was not observed 76 wx\wz OH we}: 0 HqNN mcma mNm «mnm NomH HNm oaoN moo mam mmom Nmoa mNN onN HmHH Nam moom mmqa NNN bmmm mNHH Ham come aNOH HNN mqu mmw mHN Hmom whom oNH qun HNNH mHN cmqq sham mNH mHNN ANNH «AN meme NbON «NH maaN mam HHN cmmq mmoa mNH mmwN Nboa mHH mama HQHH NNH Homm mme «Ha mmq<2mm mmqfiuo< cw muswaz “pom can when noon you mamuoa hua>wuo< com: 77 we\mz ca we\mz on onmm mmna qu mNNo quN ch Name mmmN NcN NNom mesa ocN «Hue moNH Hmm Noon wmom qu «mom anN mmN oomm quN qu woom «mHN mmN nmom HHON HQN mon oNNN NmN Hmooa Nonm qu omoc quH HmN mmwm Hood Ned omnq oHON NmH oomm mmma Hod Hume waa HmH mmq¢ m>ammmm magnum manage uaom umuam ms» ca mmoamvao>< m>fimmmm mo uwnaaz mm UNANH 86 Table B9 Number of Passive Avoidances Per Block of Eight Trials During Passive Avoidance Extinction 0 Mg/Kg Blocks g£_Tria1s l 2 2 5*. 2 9 MALES 115 8 8 8 8 7 5 116 3 6 3 2 5 2 117 8 8 7 4 5 3 212 8 8 6 7 6 7 213 8 8 8 8 7 8 216 6 5 8 3 6 4 217 8 8 8 8 8 8 314 8 8 5 6 3 3 FEMALES 114 6 5 7 S 7 7 118 8 7 7 7 6 4 211 8 7 8 7 5 2 214 8 8 8 8 5 5 215 5 4 6 3 S 3 218 5 5 4 6 2 3 311 5 5 l 3 2 2 312 6 1 0 0 4 8 313 6 5 4 3 3 3 lQ_Mg/Kg MALES 222 8 8 7 6 4 6 223 8 8 8 8 8 7 224 8 8 8 8 8 8 225 8 8 6 4 5 4 226 1 2 l 4 6 6 322 8 8 8 7 6 5 87 Table B9 (Cont'd) Blocks of Trials 6: a: A; 1: 2. 1_ FEMALES 67/458453 44878486 [45568576 78758476 78788488 88887678 124 125 126 221 227 228 321 323 MALES 68288466 58888375 78788666 88888867 87888288 88888678 134 135 136 233 234 236 238 332 FEMALES 2686/... 52855 56775 78885 88884 88885 231 232 235 237 331 MALES 66782 67885 88885 88787 88888 88887 144 145 242 244 341 Table B9 (Cont'd) 88 29_Mg/Kg Blocks g§_Trials .1. 3 .3. fl .5. 2 FEMALES 241 8 8 8 5 5 5 243 8 8 8 8 8 8 245 8 8 8 8 8 7 246 8 8 8 8 8 7 247 8 7 7 7 7 8 248 5 5 2 1 2 2 342 8 8 8 8 8 8 Table 810 Mean Duration of Three Trials on Six Drum X Speed Combinations of the Rotarod 89 23 CH 42:12_529_ 4"-20 rEm 4"-30 rpm 2"-12 rpm 2"-20 rEm 2"-30 rEm MALES 90.0 90.0 90.0 81.1 90.0 90.0 111 112 113 115 116 117 90.0 90.0 90.0 90.0 90.0 90.0 20.7 30.5 53.4 25.6 35.0 61.8 90.0 72.6 90.0 74.9 82.8 90.0 83.1 57.8 90.0 90.0 11.0 90.0 90.0 90.0 90.0 56.0 90.0 90.0 90.0 4.0 212 2.1 90.0 90.0 90.0 60.1 90.0 90.0 213 90.0 90.0 90.0 90.0 90.0 216 217 314 53.3 50.2 77.2 3.3 18.3 90.0 51.4 65.5 90.0 3.0 90.0 90.0 FEMALES 9.1 90.0 90.0 75.8 24.6 37.9 90.0 114 90.0 90.0 90.0 90.0 90.0 118 89.8 90.0 90.0 36.7 90.0 90.0 211 90.0 214 215 90.0 7.4 90.0 90.0 57.1 90.0 69.5 90.0 218 311 312 90.0 90.0 90.0 90.0 90.0 90.0 53.0 89.4 90.0 31.4 78.3 90.0 90.0 90.0 90.0 90.0 90.0 313 Table 310 (Cont'd) 90 IR 10 4"-30 rEm 2"-12 rRm 2"-20 rEm 2"-30 rgm 4"-12 rEm 4"-20 rEm MALES 43.9 90.0 90.0 4.9 61.5 72.1 121 222 223 3.4 90.0 72.9 37.5 31.0 90.0 60.9 90.0 90.0 64.5 90.0 90.0 1.3 90.0 90.0 63.6 90.0 60.7 90.0 224 225 226 322 90.0 90.0 90.0 90.0 61.0 12.3 90.0 90.0 20.0 90.0 90.0 90.0 90.0 90.0 90.0 79.2 90.0 FEMALES 90.0 90.0 90.0 69.1 90.0 62.9 122 123 57.6 62.9 90.0 14.3 62.1 90.0 90.0 90.0 90.0 73.5 90.0 90.0 124 125 50.3 90.0 90.0 61.0 62.0 90.0 62.5 82.0 83.3 8.5 56.6 34.6 126 2.6 90.0 90.0 90.0 83.8 32.6 47.1 221 227 71.5 90.0 90.0 90.0 90.0 3.2 90.0 50.5 37.6 90.0 79.5 61.2 228 90.0 79.3 90.0 90.0 73.7 321 323 90.0 90.0 90.0 90.0 90.0 90.0 Table 310 (Cont'd) 91 30M/K 4"-30 er 2"-12 rem 2"-20 rEm 2"-30 rgm 4"-12 rem 4"-20 rpm MALES 90.0 90.0 90.0 90.0 90.0 90.0 133 134 135 136 27.8 90.0 90.0 7.0 7.8 2.6 60.7 90.0 90.0 14.7 31.6 50.1 18.6 29.1 16.3 90.0 90.0 10.6 75.9 90.0 90.0 90.0 90.0 90.0 233 234 4.3 1.9 90.0 90.0 90.0 90.0 90.0 62.4 90.0 90.0 90.0 60.7 236 238 3.8 59.1 23.3 90.0 10.6 55.0 90.0 48.3 90.0 2.2 6.0 60.6 332 FEMALES 41.7 90.0 90.0 47.0 90.0 90.0 131 132 2.7 90.0 63.9 33.2 90.0 33.6 36.5 70.6 90.0 90.0 90.0 61.0 231 12.5 90.0 90.0 5.0 90.0 90.0 90.0 232 90.0 90.0 90.0 90.0 90.0 235 237 31.9 36.7 90.0 15.5 60.7 90.0 90.0 61.9 90.0 17.6 71.1 90.0 331 Table 310 (Cont'd) 92 4"-12 rpm 4"-20 rpm 4"-30 rpm 2"-12 rpm 2"-20 rpm 2"-30 rpm MALES 42.3 40.4 90.0 83.8 4.6 1.9 90.0 144 145 3.9 62.8 5.3 90.0 7.7 90.0 2.1 32.6 2.8 61.0 2.7 242 90.0 90.0 90.0 1.8 32.2 90.0 244 3.6 36.9 66.2 5.2 17.3 29.0 341 FEMALES 63.7 90.0 90.0 44.1 90.0 64.8 141 36.2 36.5 90.0 38.0 74.3 64.1 142 46.2 73.8 90.0 33.0 90.0 90.0 143 4.7 2.3 90.0 90.0 32.1 90.0 90.0 31.6 241 243 90.0 90.0 90.0 90.0 90.0 90.0 90.0 31.1 61.0 90.0 245 61.6 90.0 90.0 90.0 63.5 90.0 246 13.8 90.0 66.1 3.6 90.0 90.0 247 40.0 90.0 90.0 3.1 3.7 90.0 90.0 248 342 3.0 8.9 33.6 8.8 14.4 93 NH mm mm om mm mm mm om mm on MHm «N mm mm «N mm mu Hm Hm mm mm «Hm mm «H «H mm om wN on NH nN Hm HHm ON on «H an mN oN «N mm mm «m mHN mH nH «H mm mu on mm «H Nm Nm mHH «N m~ mm mm on mN mm «N «N mN «Hm NN «N mH mm mm Hm mm mN mm ma HHN mm «H m mH NH HH mH NH mm Hm mHa mm HN «N «N «N HN mm «H am an «HH mmHom onu aH «HoHuH coy mo xUOHm umm moadomwmm uumuuoo NO “09852 MHm «Home 100 «H « « « N«« N « N « ««N « « N « N«N « « « « ««N « N N N H«« N « « H ««N « « « « N«N « « « « ««N « N « « ««N « « « « H«N «H « « N N«N « « H « ««H «H N « « H«N « «H « « N«H «H «H « « N«H «H «H « « H«H «H «H «H N H«H ««Hmm «Lu aH mHmHuH mucosa mo JUOHm umm momaonmmm uoouuoo mo umnadz mHm «Hams 104 HH O NH N O O N«O OH «H O N N N O«N O HH HH OH OH O N«N NH HH «H HH m N O«N OH OH OH OH OH O HOO NH HH OH O N « O«N OH OH O OH OH O NON HH NH HH OH OH OH O«N OH NH OH OH NH O OON HH OH OH OH OH O H«N OH O O O O O NON O HH O O OH N O«H NH OH O O OH o HON OH OH O O O O N«H OH NH NH HH O O NOH HH OH HH O « O H«H OH OH N O O « HOH OMH a mo cOHuHmHavo< «nu OHm UHDmH :H «H«HNH «unoae mo JUOHO mom momaoawom uomuuoo mo Nonasz 106 «H NH «H HH «H NH NH « HH « «N« HH NH HH HH «H «H « «H NH « HN« «H NH «H «H NH «H «H NH «H NH «NN «H NH «H «H «H «H «H « « « NNN NH «H NH HH «H HH HH HH « N HNN «H «H .«H «H NH HH HH «H NH HH «NH NH «H «H NH «H « «H « «H « «NH «H «H «H NH «H « NH NH «H « «NH NH «H «H «H «H HH NH HH NH HH «NH «N «N «N «H NH «H «H NH « «H NNH ««HHmIOuanH um OooHMHuuwm mumm mo Auanoz «com mo unmoumm a «m uanm vow ummH mo somv munOHms «maOHM voaHnaoo can Hmamuv< OmaHnaoo NNm mHDMH APPENDIX C APPENDIX C Eguipment Reagent grade lead acetate 1 cc Plasti pak disposable syringe 250 5/8 PE 40 Intramedic Polyethylene Tubing Panheprin Micro-capillary centrifuge, Model MB Red-Tip heparinized capillary tubes Hematocrit Reading Chart Autogram 1000 Scale Triple-beam balance, 2610 g capacity H33 analytical balance Analytical balance, Model 340-D 115 Supplier Mallinckrodt Chemical Works St. Louis, Missouri Becton Dickinson and Company Rutherford, New Jersey Scientific Products Romulus, Michigan Abbott Laboratories North Chicago, Illinois International Equipment Company Boston, Massachusetts Sherwood Medical Industries, Inc. 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