The Behavior and Neurochemistry of the Methylazoxymethanol-Induced Microencephalic Rat

The Behavior and Neurochemistry of the Methylazoxymethanol-Induced Microencephalic Rat PIPPA S. LOUPE AND STEPHEN R. SCHROEDER LIFE SPAN INSTITUTE UNIVERSITY OF KANSAS LAWRENCE, KANSAS 66045

RICHARD E. TESSEL DEPARTMENT OF PHARMACOLOGY A N D TOXICOLOGY UNIVERSITY OF KANSAS LAWRENCE, KANSAS 66045

I. A.

INTRODUCTION

Microcephaly in Humans

There are currently over 255,000 developmentally disabled individuals in the United States living in public residential facilities and over 270,000 who receive community day services (J. Jacobson, 1991). The classification of developmental disabilities includes the presence of a mental or physical impairment with an onset prior to the age of 22 and a continuance throughout the individual's lifetime. Furthermore, this mental or physical impairment results in limitations in self-care, language, learning, mobility, and independent living (J. Jacobson, 1991). Of the developmentally disabled population, there are those with the condition of microcephaly. Microcephaly is defined as a condition in which the circumference of an individual's head is more than two standard deviations below the population mean of head circumferences adjusted for age and sex (Abuelo, 1991). In humans, the presence of microcephaly is an indication of a disruption in brain development as the rate of growth of the skull coincides with the rate of growth of the brain (Cowie, l"ERNATIONAL REVIEW OF RESEARCH IN MMTAL WARDATION. Vol. 21 00747750197 525.00

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1987).Therefore the terms microcephaly, micrencephaly, and microencephaly are used interchangeable in the human population, whereas in animal research, microencephaly is generally the preferred term (Goldstein & Oakley, 1985). Although there are no published estimations on the number of developmentally disabled with microcephaly. there are studies on the number of microcephalic individuals who are developmentally disabled. Estimations of the prevalence of developmental disabilities within the microcephalic population range from 70% (Sassaman & Zartler, 1982) to 90% (Martin, 1970) to 100% (O’Connell,Feldt, & Stickler, 1965). Of the developmentallydisabled with microcephaly,a greater proportion is classified as severely and profoundly disabled as opposed to mild or moderately disabled. However, microcephalic individuals fall into all classifications of developmental disabilities (e.g., mild, moderate, severe, and profound), including those with average IQ scores (Martin, 1970;Sassaman & Zartler, 1982).The reasons for the differences in IQ scores of microcephalic individuals are unclear and may be related to the magnitude of disorganizationof central nervous system (CNS) pathways associated with microcephaly. In general, the learning dysfunctions of the microcephalic developmentally disabled individual are believed to be related to the underlying physiological abnormalities that occur in conjunction with the reduction in brain size, and not due to the size of the head per se (Dolk, 1991). The learning dysfunctionsof the microcephalicdevelopmentallydisabled individual have been typically defined in terms of IQ scores. There are few published behavioral assessments of learning in the microcephalic individual that indicate the nature of the learning disability. In a study cited by Goldstein and Oakley (1985), Sidman used stimulus fading and response shaping in teaching form discrimination and the use of a pencil to a 40-year-old microcephalicman with a developmental age of 18 months. In a report by L. Jacobson, Bernal, and Lopez (1973), a microcephalic 17-year-old with cerebral palsy and classified as uneducable was studied using a two-choice discrimination procedure. Within 50 hr, the individual was able to accurately discriminate stimuli using one-, two-, and threedimensional concepts (color, shape, and number). After the discrimination training, the individual’s developmental score on the Stanford-Binet was 3.2 years of age; prior to the training his IQ was considered unmeasurable.Previous studies by the authors found that disadvantaged preschool children learned the same discriminationswithin 20 hr (Jacobson et al., 1973). The results of these two studies suggest that microcephalic developmentallydisabled show a slower acquisition of accurate performance in discrimination tasks as compared to disadvantaged preschool children. These findings are similar to the results of other studies that indicate that the developmentally disabled population as a whole shows delayed acquisitionin discriminationtasks. Unfortunately, these studies do not indicate the presence or nonpresence of microcephaly in their developmentallydisabled populations (Hale & Borkowski, 1991). In humans, microcephaly is caused by an interruption of the neurophysiologi-

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cal processes involved in cellular formation and migration in the CNS.Microcephaly is believed to be caused by several different factors:chromosome disorders, teratogens, intrauterine infections, perinatal brain damage due to asphyxia, and idiopathic congenital malformations (Abuelo, 1991). The specific physiological abnormalities related to the learning dysfunctions of microcephalic individuals have been difficult to discern, however, because of the variety of possible causes and multiple symptomatology. The development of microcephaly in humans can lead to impairments in several areas of the brain, resulting in varied types of sensory, motor, and cognitive deficits. However, there are relatively few areas in the brain that when affected result only in learning and memory problems. The areas of the brain generally believed to be involved in learning and memory are the cortex, striatum, and hippocampus (Bloom, 1990). For humans, it is currently unclear what effect microcephaly has on these particular brain regions, which may be involved in learning impairments. Until procedures such as position emission tomography (PETScans) and magnetic resonance imaging (MRI), which provide information on regional brain function, are routinely used in the developmentally disabled microcephalic population, it will be difficult to determine in humans the exact physiological mechanisms involved in their learning impairments. One way researchers have looked at the physiological mechanisms underlying behavioral disorders is to develop an animal model of the disorder. Microcephaly in animals can be produced in the same ways it is produced in humans. For instances, teratogens such as alcohol, cocaine, lead, and irradiation produce severe microcephaly in animals with deficits in physical, motor, and cognitive development. Because researchers have been primarily focused on the learning impairments in the microcephalic population, an animal model was developed that confined the disruption of brain development only to areas directly involved in learning and memory (cortex, striatum, and hippocampus). Spatz and Lacquer (1968) developed an animal model that allowed the assessment of the effects of microcephaly on the resulting disorganization of neural pathways and subsequent learning abilities. Spatz and Lacquer (1968) observed that administration of the antimitoticagent methylazoxymethanol (MAM) to pregnant rat dams during a particular point in gestation leads to disruption of cell division in the cortex, striatum, and hippocampus in their offspring. This chapter reviews the research findings on the behavioral deficits and neurochemical abnormalities in an animal model of microcephaly,the MAM-exposedrat. Evaluation of this research should provide an understanding of the neurochemistry associated with the learning deficits of some individuals with microcephaly. B.

The Effect of Prenatal MAM Administration

MAM treatment leads to microencephaly by reducing the number of brain cells that develop during gestation. New brain areas are formed by brain cells undergo-

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ing differentiation, division, and migration to brain regions. MAM, a powerful alkylating agent, passes through the placenta (Nagata & Matsumoto, 1969) and kills the dividing cells of fetal brain tissue by methylating purine and pyrimidine bases in brain nucleic acids (Sanberg, Moran, & Coyle, 1987). Prenatal MAM treatment is believed to suspend cell division for 2 to 24 hr, depending on the dosage (Sanberg et al., 1987). The brain areas that are affected by MAM are reduced in size, thus producing microencephaly. Depending on when MAM is administered,it can also affect the development of specific brain regions, while leaving the pregnant female and the fetuses otherwise healthy. The development of the rat forebrain occurs during the mid- to later-half of gestation (days 14 to 21) and the early postnatal period (days 1 to 21; Sanberg et al., 1987).MAM treatment during early gestation, that is, prior to day 15, affects the development of the brain stem and hindbrain structures (i.e., locus coerulus and cerebellar purkinje cells; Rodier, 1986), whereas MAM treatment at gestation day 15 affects the neocortex, hippocampus, and corpus striatum (Rodier, 1986). Postnatal administration of MAM affects the cerebellar interneurons (Rodier, 1986). C.

The Literature Reviewed

This chapter focuses on the studies of learning and memory in the MAM rat so that informationmay be derived on the type of learning disorders exhibited by microencephalic organisms and the possible neurophysiological mechanisms of these disorders. Additionally, although there are several studies on the effects of MAM on the development of the cerebellum, this review concentrates on the effects of MAM on the development of the cortex, striatum, and hippocampus, which are believed to be important in learning and memory (Balduini et al., 1986). In order to describe these relations, this chapter reviews the following types of studies. First, it reviews the literature on the effects of prenatal MAM treatment on physical and motor development, and on performance in simple and complex mazes and operant chambers. Second, it reviews the effects of MAM on the neurochemistry in the neocortex, hippocampus, and corpus striaturn of the prenatally MAM-treated rats. And third, it suggests what future research might be conducted to improve our understanding of specific types of learning dysfunctions of the MAM rat and how knowledge of the neurophysiology of those dysfunctions may be useful in deriving treatments for microcephalic individuals with lesions in cortical, hippocampal and striatal brain regions. This review is restricted to studies that administered MAM between gestation days 14 to 17, the time at which the cortex,hippocampus,and striatum are believed to be most affected by MAM. Data from studies using MAM doses ranging between 20-35 mgkg administeredon gestation day 15 will be emphasizedbecause doses in this range on gestation day 15 result in the severest abnormalities in the

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brain structure and neurochemical mechanisms believed to be involved in leaming and memory.

11.

A.

THE EFFECTS OF PRENATAL METHYLAZOXYMETHANOL ADMINISTRATION ON GROWTH AND LEARNING IN THE RAT

Physical and Motor Development

For rats, developmental indices that can be assessed for birth defects are (a) gestational and litter parameters, (b) seizures, (c) weight gain, (d) locomotor activity and rearing, and (e) reflex development. As for gestational and litter parameters (e.g., implantation sites, maternal weight gain, and litter size), MAM is not believed to produce any detrimental effects to the mother during pregnancy (Rodier, 1986). As for seizures in the MAM-treated offspring, these have been documented for MAM doses as low as 20 mgkg (Kabat, Buterbaugh, & Eccles, 1985). 0ther research indicates that at a dose of 25 mg/kg, MAM-treated animals have seizures during early adulthood (3 to 5 months of age), but that the seizures disappear by 8 months (R. Tessel, personal communication, April 1992). Sufficient research is availableon the other three indices of maturation that they are discussed separately, as follows. 1. WEIGHT GAIN Several studies have reported on changes in birth and adult weights in rats prenatally exposed to MAM as compared to controls. Some studies have found significant weight loss in adult MAM-exposed rats (Fischer, Welker, & Waisman, 1971;Vorhees, Fernandez, Dumas, & Haddad, 1984). whereas others have found a significant weight gain (Johnston & Coyle, 1979). Still others have found no differences in the weights of adult MAM-treated and control animals (Balduini, Lombardelli, Peruzzi, & Cattabeni, 1991;Dambska, Haddad, Kozlowski, Lee, & Shek, 1982; Tamaru, Hirata, Nagayoshi, & Matsutani, 1988). The reasons for this variability may lie in the difficulty of precisely determining the days of gestation and the apparent rapid change in MAM’s effect on weight depending upon which day it is administered. A recent study by Rodier, Kates, White, and Muhs (1991) compared the growth and weight effects of administering MAM at a dose of 20 mg/kg on gestation days 14 or 16. The dose delivery on day 14 resulted in dwarf rats in 10% of the pups from the MAM-treated groups, that is, rats whose total body weight was less than that of untreated animals. These dwarf rats had a normal weight at birth, but suddenly decreased in weight gain around postnatal day 28. This decrease continued such that the MAM-treated group had a 50% reduction in body size as compared to controls by postnatal day

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40. If the dwarf MAM-treated pups were omitted from these calculations, then the weight and size of the other treated pups did not significantly differ from controls. In contrast, administeringMAM on gestation day 16 resulted in accelerated postnatal weight gain, such that on postnatal day 40 the animals treated with MAM were significantly larger than the untreated animals. In this study, animals from both of the MAM-treated groups (gestation days 14 and 16) had massive brain weight reductions (between 15-35%) whether or not their overall growth was affected. For the dwarfrats, these weight reductions were correlated with reductions in the cell count of growth hormone-releasing factors of the hypothalamus, which are involved in the pituitary regulation of growth hormone secretion. These rats also had a significant increase in growth hormone release inhibitory factors (GHRIFs) and extremely small pituitaries. For the day 16 rats, whose overall weight gain was accelerated,neither of these hormones was affected. These rats did, however, have very large pituitaries and hypertrophy of the somatotropins. Given the pattern of these results, Rodier et al. (1991) concluded that the MAM administrationmay cause neurological birth defects that lead to endocrine abnormalities in adulthood. The variability in body weight produced by administering MAM on gestation day 15 may be due to the rapid change in MAM’s effects on the endocrine system between gestation days 14 and 16 and the difficulty in determining precisely the days of gestation. The Rodier et al. (1991) study provides evidence that the day of administration is an important determinant of the effects of MAh4 on body weight. 2. LOCOMOTOR ACTIVITY AND REARING MAM-treated animals are considered by many investigators to be hyperactive when compared to control animals (Archeret al., 1988;Balduini,Lombardelli,Peruzzi, Cattabeni. & Elsner, 1989; Kiyono, Seo, & Shibagaki, 1980; Sanberg et al., 1987; Vorhees et al.. 1984).This is measured operationally by locomotor activity and rearing in a variety of situations: open field tests with and without a noseboard floor. cages with activity wheels, and mazes equipped to measure spontaneous activity (Sanberg et al., 1987). Compared to untreated animals, MAM-treated animals appear to be hyperactive, at least during their first 30-60 min in an observation chamber (Sanberg et al., 1987). Several studies have measured the daytime activity of MAM animals for short periods of time and found hyperactivity throughout these periods. Vorhees et al. (1984). for instance, reported increased daytime activity in MAMtreated animals (30 mgkg on gestation day 15) when measured for 9 min in an open field and in a figure-eight maze. Cannon-Spoor and Freed (1984) and Archer et al. (1988) have also found increased daytime hyperactivity in the MAM-exposed animals (25 mgkg on gestation day 15) in spontaneous activity chambers when measured for 60-or 30-min periods, respectively. MAM-exposed rats (25

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mgkg on gestation day 15) also exhibited more locomotor activity and rearing between food reinforcements occumng in 30-min sessions of a fixed-time 60-sec schedule of reinforcement U u p e , Stodgell, & Tessel, 1992). Other studies, however, have found that when the MAM-exposed animals are observed for a more extensive period of time (e.g., 24 hr), the differences in daytime activity levels between the MAM-exposed and the control animals disappear (Kiyono et al., 1980; Sanberg, Moran, Kubos, Auterono, & Coyle. 1983; Sanberg et al., 1987). These studies, however, used a lower dose of MAM, (i.e.. 20 mg/kg) than the studies by Vorhees et al. (1984). Cannon-Spoorand Freed (1984),Archer et al. (1988), and Loupe et al. (1992), and thus the results are confounded. In a study that assessed whether there were differences in the hyperactivity of MAM animals in a residential maze depending upon dose, Balduini et al. (1989) found no differences in the daytime activity among the control animals and the experimental animals at either MAM dose (15 and 25 mglkg) when measured during a 23-hr session. The animals treated with 25 mg/kg of MAM, however, displayed significantlymore nocturnal locomotor activity and rearing than the controls. This increase in nocturnal hyperactivity is consistent with the results of other studies (Kiyono et al., 1980; Sanberg et al., 1983, 1987). It appears then, that hyperactivity in MAM animals is not uniform. The research suggests that the MAM-treated animals exhibit more locomotor activity compared to controls during the fust hour of observation in a novel environment, but not after prolonged exposure. Furthermore, their hyperactivity varies with MAM dose size and whether their activity is measured during diurnal or nocturnal periods. In addition to differencesin the amountof nocturnal locomotor activity and rearing between MAM and control animals, there may be differences in the duration of rearing (Hanada, Nakatsuka, Hayasaka, & Fujii, 1982; Rabe & Haddad, 1972; Sanberg et al., 1987). For instance, Sanberg et al. (1987) found that although the MAMs did not differ from the controls in the onset of rearing or in the frequency of rearing when measured for a 24-hr period, the rats keep a rearing position for significantly longer durations by postnatal day 30. Finally, why differences occur in nocturnal. but not daytime locomotor activity and rearing between controls and animals treated with MAM doses of 20 or 25 mgkg is unclear. A few studies though, have found that particular types of brain damage lead to increases in nocturnal but not daytime activity. For example, research on the effects of chronic brain damage resulting from toxic agents such as X-ray irradiation, which results in hippocampal cell deficits, has found increased levels of nocturnal locomotoractivity (Peters & Brunner, 1976).Relatedly, in prenatal MAM administration, varying doses on gestation day 15 can result in differences in the locations of cell damage. A MAM dose of 25 mgkg results in cell damage in both the hippocampus and the striatum, as well as the cortical regions (Balduini et al., 1989).In contrast, a 15-mg/kgdose of MAM results only in cortical cell loss (Balduini et al., 1989).The increased levels of nighttime locomotor

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activity and rearing with MAM administration with doses above 15 mgkg may be related to the specific brain damage caused by MAM to the hippocampus and the striatum. 3. REFLEX DEVELOPMENT

The effects of MAM on reflex development and coordination has been assessed with the following tests: the air-righting reflex, rotarod performance, and the horizontal wire test. a. Air-righting reflex. To measure the air-righting reflex, the animal is held in a supine position and then dropped from a height of 60 cm onto a padded surface and observed for its landing position (Adams, 1986). The righting response includes a sequence of coordinated movements involving head and limb rotation such that the animal lands on its feet. This ability normally develops in the rat between postnatal days 12 and 17.A study by Rodier, Webster, and Langman (1975) reported that rats prenatally exposed to 25 mgkg of MAM do not appear to have any deficits in the air-righting reflex. b. The rotarod task. The rotarod task involves placing the animal on a revolving cylindrical rod (similar to a dowel stick) and measuring at what speed and for how long the animal is able to stay balanced on it (Adams. 1986). The ability to remain balanced on the rod usually develops between 18 and 25 days. Giurgea, Greindl, Preat. and Puidevall (1982) found no difference in rotarod performance after MAM exposure (25 mg/kg) between gestation days 14 and 17. Balduini et al. (1991), however, recently reported that MAM-treated rats showed an impairment in rotarod performance on postnatal day 50 when given MAM (25 mg/kg) on gestation days 14, 15, 16, or 17. In addition, Kabat et al. (1985) found deficits in rotarod performance in MAM-treated animals (25 mgkg) when the MAM was administered on postnatal day 1; these deficits may be related to the detrimental effects of postnatal MAM treatment on the development of the cerebellum. c. The horizontal wire test. The horizontal wire test involves lifting animals by their tails and allowing them to grasp a horizontal wire with their forelimbs. The animals are then released and a measure is taken of how many animals are able to grasp the wire with one or more hindlimbs within a 20-sec period of suspension. Balduini et al. (1991) found that rats treated with MAM (25 mgkg) on gestation days 14, 15, 16, or 17 showed small deficits in the horizontal wire test, but that their performance was not significantly different from controls. The results of these studies on reflex development indicate that exposure to MAM on gestation days 14-17 does not produce severe deficits. This is consistent with the reports of other antimitotic treatments (X-ray irradiation and azacytidine), which also do not adversely affect reflex development when administered between gestation days 14 and 17 (Rodier, 1984). In contrast, administrationof any antimitotic agents, including MAM, earlier in

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the fetal period or postnatally, does cause deficits in reflex development that are believed to be related to their effects on the development of the cerebellum (Haddad, Rabe, Dumas, Shek, & Valsamis, 1977; Kabat et al., 1985; Rodier, 1984).The importance of the time of administration on the effects of MAM in reflex development is thus clearly evident. This may be related to the time of development for particular areas of the brain. The development of the brain stem, the locus coerulus, and the cerebellar purkinje cells, for instance, have been shown to be affected by MAM exposure prior to gestation day 14. Postnatal MAM exposure is believed to afFect the development of the cerebellar interneurons.The deficits in the reflex development of motor coordination in animals treated with MAM during the gestational developmental periods associated with brain stem and cerebellum growth is consistent with findings that both the brain stem and the cerebellum are involved in motor coordination (Carlson, 198 1 ).

B.

Performance in Simple and Complex Mazes

Several studies have assessed the effects of MAM exposure on rats’ performance in a variety of mazes. 1 . WATER-FILLED MAZES

The study by Hanada et al. (1982) assessed the ability of prenatally MAM-exposed (25 mg/kg) rats to escape from a water-filled triple T maze. In their T maze, the escape platform was located at one end of a blind alley for the first three days of testing and then relocated in another blind alley on the opposite end for the fourth and fifth day of testing. Hanada et al. (1982) found that the MAM rats made three times as many errors in learning how to find the escape platform as compared to the controls during the first 3 days of testing. The MAM-treated rats also showed a greater increase in errors as compared to the controls on the first day of a reversal in the location of the escape platform. Both groups showed similar decreases in errors on the second day of testing with the reversal of the escape platform. Using a higher dose of MAM (30 mg/kg) administered on gestation day 14, Vorhees, et al., (1984) found that the MAM-treated rats made 64% more errors in the initial learning and 608%more errors in the reversal learning on how to find the escape platform. The MAM-treated rats spend a significantly shorter time (39%)than the controls on the initial learning of the maze and spent a significantly longer time (297%)on the reversal learning of the maze. Archer et al. (1988) also found that a group of MAM-treated rats had difficulty in learning how to perform in a swim maze task. In their maze, a circular pool was filled with water to a depth of 30 cm, with an escape platform located in a specific position 1 cm below the surface of the water. The MAMs took longer to reach the platform and failed to find the platform as often as the controls.

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In the Archer et al. (1988) study, the MAM-treated rats also showed deficits in performance in a radial arm maze task. The radial arm maze or Olton spatial maze consists of an central area with eight arms extending from it. Exploratory behavior is measured by the total number of arm entries and the number of different arm entries. Here, the MAM-treated rats took longer to reach the goal box (located at the end of one of the arms) and took more visits to different arms of the mazes than the controls. In a study by Shimizu, Tamaru, Katsukara, Matsutani, and Nagata (1991). the MAM-treated rats again displayed significantly more errors than the controls in learning the radial arm maze and in subsequent stimulus retention tests. 3. LASHLEY 111 MAZE

Lee, Haddad, and Rabe (1982) assessed the MAM-treated rats’ performance in a Lashley III maze. This maze consists of a rectangular chamber with four alleys, a start box, and a goal box. The correct path from the start to the goal boxes involves zigzagging through doorways located in the alleys. The rats treated on gestation day 15 with 25 mgkg of MAM had 128 errors in completing the task, which was significantly different from the 73 errors of the control rats (Lee et al., 1982). 4. REVERSAL TASKS

In a study by Mohammed, Jonsson. Soderberg, and Archer (1 986), MAM-treated animals had difficulty performing a successive position reversal in a T-maze. The task included (a) a forced run in which alternately one of two arms was blocked which forced the animals to run in the other arm and (b) a choice run in which the arm blocked in the forced run now contained reinforcement. The animals were therefore required to run alternately between the two arms. The MAMtreated animals had difficulty in learning to alternate in that they persistently ran to the arm that contained reinforcement on the preceding trial. Rabe and Haddad (1972) studied whether immature MAM-treated pups (15 days old) prenatally exposed to 25 mgkg of MAM have the same difficulty as older MAM-treated rats in learning to reverse a previously-learnedresponse. In comparison to saline-treatedcontrol pups, the MAM-treated pups were impaired in the initial learning and the later reversal learning of the position discrimination task. The investigators also tested a group of naive MAM-treated adult rats (50 days of age) and found that the adult MAM-treated rats had no difficulty in learning the position discrimination,but had difficulty in learning to reverse it (Rabe & Haddad, 1972).

C.

Performance in Operant Chamber Tasks

Most of the studies on MAM-treated animals in operant chambers have assessed differences in light or position discriminations, passive avoidance, and differential-reinforcement-of-low-rate-behavior (DRL) tasks.

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1. DISCRIMINATION TASK One of the first studies to look at the possible learning deficits of MAM-treated rats was conducted by Rabe and Haddad in 1972. Their discrimination task involved an acquisition phase in which the rats were reinforced for lever pressing during a 3-min period when the houselight was illuminated (time-on periods). During the acquisition phase, these time-on periods were alternately presented with periods of no reinforcement (time-out) when the house light was not illuminated. After the acquisition phase, the rats were placed in an extinction phase where they were no longer reinforced for pressing in the presence of the houselight. Rabe and Haddad (1972) found no differences in performance during the acquisition phase of a light-discriminationtask, but significant differences in the extinction phase between the severely microcephalic rats (60% forebrain loss), moderately microcephalic rats (30%forebrain loss), and the control rats. The severely microcephalic MAM rats responded similarly to the moderately microcephalic and the controls during the time-out periods, but they made many more responses than the other groups in the previously reinforced time-on periods. A study by Cannon-Spoor and Freed (1984) found that MAM-treated rats outperformed controls in a food-reinforcedtwo-lever discriminationtask. In this task, the rats were required to press one of two levers in order to receive reinforcement depending upon whether the cue light above the lever was illuminated.The MAh4s showed a tendency to make more level presses during the sessions and were significantly more accurate than the controls. 2. PASSIVE AVOIDANCE TASK

Cannon-Spoor and Freed (1984) also assessed the effects of MAM treatment in a passive avoidance task. In these tasks, rats are required to stay in a small corn partment to avoid shock in an accessible larger compartment. Here, MAM-treated rats took significantly longer to reach the criterion of 2 min in the smaller compartment. In the reversal of passive avoidance, however, the rats needed to move from the larger compartment to the smaller compartment. No shock was delivered in either compartment. The MAM-treated rats took longer to reach criterion in the passive avoidancetask,yet were much faster in a reversal of the passive avoidancetask In comparing these last two measures, Cannon-Spoor and Freed (1984) concluded that the MAMs were more hyperactive and that this led to differences in performance. They also concluded that the MAM-treated rats completed more trials than controls in the food-reinforced discrimination task and thus had more opportunity to learn the discrimination. 3. DRLTASK Archer et al., (1988) found that MAM-treated rats did not differ from controls in learning a DRL-72-sec contingency. The MAM-treated rats did not respond as

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frequently as the controls when acquiring the initial FRl response, but showed no significant differences in response rate and in the number of reinforcements when the schedule was gradually changed from the FRl to the DRL-72 sec. D.

Summary on Growth and Learning in the MAM-Exposed Rat

The effects of MAM administrationon the rat’s development and learning abilities have been studied by several methods (e.g., growth, reflex development, mazes, and operant tasks). MAM administration does not appear to affect weight gain and growth if administered on gestation day 15, but it appears to decrease weight gain in some rats if given on gestation day 14 and increase weight gain if given on gestation day 16. In measures of locomotor activity and rearing, the MAM-treated animals appear to be hyperactive as compared to controls in nighttime locomotor activity and during an initial period of exploration in a novel environment. Reflex developmentdoes not appear to be affected by MAM-administration. In learning tasks involving simple mazes, the MAM-treated rats performed as well as the control animals. In tasks involving complex mazes, however, the MAM-treated animals produced more errors. The MAM-treated animals appeared to have difficulty in learning to reverse a learned position response. They also appeared to make more errors in mazes that had multiple arms. In operant chambers, the MAM-treated animals performed various operant tasks (fixed-ratio, a DRL, and a 2-lever discrimination task) adequately, but those classified as severely microencephalic took significantly longer to extinguish response during extinction trials. Understanding the types of learning deficits exhibited by MAM-treated animals provides information on how neurophysiological changes due to microencephaly in specific brain regions may be related to learning deficits. Another area of prenatal MAM exposure research involves assessing the neurochemical changes in the brain that result from MAM administration.The next section reviews the research on the effects of prenatal MAM administration on the neurochemistry in brain regions believed to be involved in learning and memory. 111.

ALTERATIONS IN BRAIN STRUCTURE AND NEUROCHEMISTRY DUE TO PRENATAL METHYLAZOXYMETHANOL EXPOSURE

The cortex, the striaturn, and the hippucampus have been studied extensively for their roles in the processes of learning and memory. The study of their neuronal connections, in turn, is a more recent advance. The next section describes the

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known structure and neurochemistryof the cortex, striatum, and the hippocampus, preceded first with an overview of the general structure of the brain, as well as type and nature of the neural connections and neurotransmitters. A.

Brain Structure and Neural Connections

1. BRAIN STRUCTURE The cortex is composed of 50 billion neurons constituted in layers of tissue that receive sensory information-somatosensory, visual, auditory, olfactory, and motor (Bloom, 1990). These neuronal layers form associations among themselves (corticocortical systems) and with noncortical areas of the brain (subcortical systems) that are believed to enable abstract thought, memory, and consciousness.The striatum and the hippocampus are part of the limbic system, which is believed to integrate emotional state with motor and visceral activities (Bloom, 1990). The striatum is a part of the extrapyramidal motor system, damage to which leads to motor disorders characterized by involuntary motor movements such as Parkinson’s disease or Huntington’s chorea (Bloom, 1990).The hippocampus is believed to be involved in the formation of recent memory because the loss of this function has been found in patients with bilateral damage to the hippocampus and in patients with Alzheimer’s disease, which destroys the hippocampus’s intrinsic structure (Bloom, 1990). 2. NEURAL CONNECTIONS

These three different brain regions interact through neuronal connections of three types: long hierarchial connections, local circuits, and multiple-branched neurons. The long-hierarchial connections relay sensory information through an ascending hierarchical series of neurons to the cortex and output motor information from the cortex to the motorneuron of the spinal cord using a descending hierarchical series of neurons, with acetylcholine as the postsynaptic neurotransmitter to the muscle system. The second type of neuronal system-local circuits-involves neurons whose synaptic connections occur relatively close to their cell bodies. These neurons are believed to regulate information within specific brain regions and to use several different neurotransmitters,including gamma-aminobutyricacid (GABA), glycine, glutamate, and several types of peptides. Multiple-branched neurons, the third type, arise from a single neuronal location and extend multiple branches to several target cells in varying brain regions. These connections are not considered to be organized in a hierarchical fashion because severing a portion of the fibers does not completely disrupt the functions of the target organs. These systems contain catecholamines, such as norepinephrine, dopamine, 5-hydroxytryptamine(5-HT), and several peptide systems.

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3. NEUROTRANSMIlTEiRS

The importanceof each of these types of neuronal connections for learning and memory is being investigated (Bloom, 1990).One method of identifying the functions of neurotransmitters is to study the changes that occur when the neurotransmitter systems are disrupted. MAM treatment during gestation results in structural abnormalities and therefore is also believed to result in changes in the neurochemistry of these structures. Studying the neurochemistry of MAM-exposed animals who show structural abnormalities and deficits in learning and memory should provide further information on the relationships between brain regions and the neurochemistry involved in learning and memory. The neurotransmitters that have been studied in the cortex, striatum, and h i p pocampus of MAM-treated rats are GABA, acetylcholine, norepinephrine, dopamine, serotonin, and glutamate. Other neurotransmittersin these regions are either not affected by MAM administration between gestation days 14 and 17 (when the above brain regions are most affected by MAM), or the present information is inconclusive. The effects of MAM on the neurotransmitters in the cortex, as well as in the striatum and hippocampus, have been assessed in terms of the brain tissue content of synthetic enzyme activity, high-affinity uptake system activity, endogenous concentrations of the neurotransmitter, and degrading enzyme activity. Assessment of these factors provides an indication of how much of the neurotransmitter is produced (synthetic enzyme activity), the capacity of the neurotransmitter-releasing neuron to accumulateneurotransmitterpreviously released by them (highaffinity uptake activity),how much of the neurotransmitteris available for release (endogenous tissue neurotransmitterconcentrations),and the capacity of the neurotransmitter to be eliminated through metabolism to nonneurotransmitter substances (degrading enzyme activity). Taken together, these measures provide an estimation of the functional capacities of individual neurotransmittersystems in a brain region. The capacity of an enzyme to convert one substance (e.g.. the substrate for the enzymatic reaction) into another (e.g., the product of the enzymatic reaction) is quantified as either the enzyme’s specific activity or total activity. Specific activity of an enzyme is typically defined as the amount of product formed (e.g., nmol [nanomole] of product) per unit time per milligram of tissue protein or milligram of tissue weight (e.g., nmol/hr/gm protein), whereas total activity of the enzyme is defined as nmol/hr/tissuesample. Similarly, the specific activity of a neurotransmitter or of receptors for the neurotransmitter(receptors being membrane-bound proteins that bind neurotransmitter, the result of which is some detectable biochemical effect) can be defined as nanomole of neurotransmitter(or receptor) /mg protein or nmoYmg tissue weight, whereas total neurotransmitter or receptor activity can be defined as nmol of neurotransmitter(or receptor) /tissue sample. Spe-

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20 1

cific activity and total activity measures thus differ in that the specific activity is influenced by any changes in tissue protein concentrationsor tissue weight, whereas total activity is not. The relationship between specific and total activity is important in understanding the changes that occur in the neurochemistry of these brain regions after MAM exposure. The specific activity of an enzyme or neurotransmittercan increase in a MAM-treated animal as compared to a control because the amount of tissue sample protein is often decreased by the MAM treatment. Thus, for example, if the total enzyme activities in the MAM-treated hippocampus were the same as the control, but the amount of tissue in the MAM-treated hippocampus was decreased relative to the control, then the specific activity is increased. If the specific activity of an enzyme is increased in the treated animal relative to control, but the total activity between treated and control does not differ, then the tissue lost contained neurons other than neurons in which the above enzyme is located. If however, the total activities of an enzyme were decreased in the MAM-exposed animal, but the specific activities of that enzyme are unchanged as compared to control animals, then similar amounts of tissue and enzymes have been lost. Additionally, MAMtreated tissue has therefore lost neurons that contained that enzyme. Another measure of neurotransmitter action is detection of the binding of agonists and antagonists to the neurotransmitter’s receptors. Using radioactively labeled ligands (agonists or antagonists) to bind to neurotransmitterreceptors, an approximation of the number of receptors present in a given tissue sample as well as the affinities of the agonists and antagonists for the receptors can be obtained. The next section reviews the information on the neurochemical alterations in the MAM-treated animals in the specific brain regions believed to be involved in learning and memory. B.

Effects in the Cortex

Prenatal administration of MAM between gestation days 14 and 17 results in a reduced rate of cell division in the cortex, with the greatest reduction occurring when MAM is administered on gestation day 15 (Matsumoto, Spatz, & Lacquer, 1972). Johnston and Coyle (1979) found a 67% reduction in cortical size in adult rats prenatally treated with 20 mgkg of MAM. In MAM-treated rats, the layer I of the cortex is thicker than controls; layers II-IV are thinner; and layer V is more densely packed with granule cells than normal; layer VI is not affected (Johnson & Coyle, 1979). These differential effects are important because the functions of the layers of the cortex vary. Incoming sensory nerves are believed to terminate in layer IV, whereas the outgoing information to the brain stem and spinal cord leaves through the neurons of layers V and VI (Guyton, 1991). The neurons of the layers I, 11, and

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HI of the cortex perform most of the intracortical association functions (Guyton, 1991). Consequently, the reduction of cortical mass in the layers 11-IV caused by

MAM administration can possibly affect the areas of intracortical transmission (layers II and III) and the receiving of incoming sensory information (layer IV). Likewise, the increased cellular density of layer V may affect the transmission of output signals to the brain stem and spinal cord (Johnston & Coyle, 1979). A subsequent study by Jones, Valentino, and Fleshman (1982) found that although the number of cells per cortex section decreases due to MAM-exposure, the mean surface area of each cell increases. Jones et al. (1982) proposed that the surviving cells were able to establish normal synaptic connections between afferent projection fibers and efferent neurons. That the surviving cells of the cortex are able to maintain synaptic neurotransmission may explain why the severity of the MAM-induced deficits is not as large as would be expected given the amount of tissue reduction in the cortical layers. To see whether the surviving cells of the MAh4-exposed cortex undergo changes in their neurotransmittersystems, GABA, acetylcholine,norepinephrine,and glutamate in the cortex have been investigated. 1. GABA GABA innervates the cortex in a local circuit and is believed to act as an inhibitory neurotransmitterin the interconnections between the sensory (layer IV) and the association layers (I, 11, and m)of the cortex (Guyton, 1991). Based on the results of a study by Johnston and Coyle (1979), GABA neurons are believed to be severely depleted following prenatal MAM treatment between gestation days 14 and 17. This study assessed the effects of MAM on the total and specific activities of GABA's synthesizing enzyme, glutamate decarboxylase (GAD), the specific and total uptake of GABA, and the specific and total content of GABA in the cortical tissue. The specific activity of GAD (nmol/hr/mgprotein) was slightly reduced, whereas the total activity of GAD (nmol/hr/corticalslab) was markedly reduced to 50% of control. Likewise, the specific activity of the high-affhity uptake process of GABA (nmoVmg protein) was unchanged, but the total uptake of [3H] GABA (nmoVcortical slab) was decreased by 63% (Table I). Finally, the concentration of GABA per milligram of corhial tissue was unchanged, but the total content of GABA for each cortex was reduced to 41% of the control subjects (Table II). The specific activity of GAD,the specific uptake of GABA, and the specific content of GABA were essentially identical in the control and MAM-treated animals. In addition, as compared to control subjects, there was a percent reduction in the MAM-treatedanimals in the total activity of GAD, the total uptake of GABA, and the total content of GABA. These results are consistent with a large MAM-induced reduction in the number of cortical GABAergic neurons (neurons releasing GABA). The reduction of GABAergic neurons in the cortex means there is a decrease in the inhibitory ac-

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TABLE I NEUROTRANSMITTER UPTAKEIN THE CORTEX FOLLOWING PRENATAL METHYLAZOXYMETHANOL TREATMENT^ Specific uptake (pmol/min/mg tissue) Neurotransmitter

Control

[3H]GABAC [3H]Glutamate 13H]Norepinephrine

240 575 543

MAM"

Change (%)

+ 16

279 549 1251*

-5 +130

Total uptake (pmol/min/slab) Control

737 1800 1386

MAM"

Change (%)

273** 523** 1244

-63 -71 - 10

"The data in this table are from a study by Johnston and Coyle (1979) "MAM, methylazoxymethanol. 'GABA, gamma-aminobutyric acid. * p < .05. **p c .01.

tivity of interneurons in the cortex, which may explain the transient Occurrences of seizures during early adolescence and adulthood in the MAM-exposed rats. Although the mechanism of actions for seizures is still unclear, the loss of GABAer-

gic neurons in the cortices of patients suffering From epilepsy has previously been reported (Rall & Schleifer, 1990). The specific relationship between the inhibito-

TABLE I1 ENW E N O U S NEUROTRANSMITTER LEVELS IN MAM=-TREATED CORTEX FOLLOWING PRENATAL MAM TREATMENP Total content

Specific content Neurotransmitter GABA' Acetylcholine Glutamate Norepinephrine

Control

MAM

Change (%)

1.2 +9 (nmol /mg tissue) 12 20** +64 (nmol/mg tissue) 18.3 17 -6 440 1026** +I33 (pg/mg tissue) 1.1

" MAM, methylazoxymethanol.

Control

62 764 1082 31

"The data in this table are from a study by Johnston and Coyle (1979). "GABA, gamma-aminobutyricacid. * p < .05. **p < .01.

MAM 26* * (mollslab) 450** (nmol/slab) 383** 24* (ng/slab)

Change(%)

-59 -41

-65 -22

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ry actions of GABAergic neurons and the processes of learning and memory development has not yet been determined. 2. ACETYLCHOLINE Acetylcholine is an excitatory neurotransmitter in the autonomic nervous system, and its role in the CNS is also being studied. For instance, a deficiency in the functioningof cholinergic neurons has been found in Alzheimer’s disease patients (Guyton, 1991). In addition, drugs that inhibit the action of the degrading enzyme acetylcholinesterase have been found to delay the loss of memory in the early stages of this disease (Taylor, 1990). In the cortex, acetylcholine is believed to activate the neuronal processes for storage and retrieval of memories (Guyton, 1991). Johnston and Coyle (1979) looked at the effects of MAM exposure on the acetylcholine-containingneurons (cholinergic neurons), finding that cholinergic neurons are not affected as severely as GABAergic neurons by MAM treatment on gestation day 15. The synthesizing enzyme for acetylcholine, choline acetyltransferase (CAT), showed an increase of 97% in specific activity, while the total activity for CAT occurring in the cortex was reduced by 33% (Table III). Another marker of cholinergic function in the cortex is the activity of the acetylcholine-degradingenzyme, acetylcholinesterase.The amount of specific activity of acetylcholinesterase was increased in the cortex by MAM administration on gestation day 15; however, the total activity of acetylcholinesterasefor the cortex (nmollminkortex slab) was severely reduced (Table LII; Nagata, Nakamura. & Watanabe, 1978). TABLE III OF SYNTHESIZING ENZYMES IN THE CORTEX FOLLOWING ACTIVITY PRENATAL METHYLAZOXYMETHANOL TREATMENT= ~~~

Specific activity Enzyme Glutamate decarboxylase Choline acetyltransferase Acetylcholinesterase Tyrosine hydroxylase

Control

MAMb

Change(%)

- 13 21 (nmol/hr/mg.prot) 32 65** +97 (nmol/hr/mg.pot) 16** 23 +44 (pmol/min/wet weight, g) 14 51** +276 (pmol/hr/mg.prot)

24

Total activity

Control

MAMb

Change(%)

44** -71 (pmol/hr/tissue) 22 I 149** -33 (nmol/hr/tissue) 18 13 -30 (pmol/min/tissue) 96 125 +31 (pmol/hr/tissue) 155

OData in table are from a study by Johnston and Coyle (1979), except for the data on acetylcholinesterase, which are from a study by Nagata, Nakamura, and Watanabe (1978). bMAM, methylazoxymethanol. * p < .05. **p < .01.

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Finally, Johnston and Coyle (1979) found that the specific content of endogenous acetylcholine shows an increase of 64% in the MAM-treated rats in comparison to controls, whereas the total content of acetylcholine is decreased by 41% in the MAM-treated animals (Table II). Together these results-the increase in the specific activities of the synthesizing (CAT) and degrading (acetylcholinesterase) enzymes, and the relative increase in the endogenous content of acetylcholinesuggest hyperinnervation of acetylcholine in MAM-treated animals. However, the decrease in total activity of acetylcholine within the cortex suggests that choline uptake is occurring by noncholinergic neurons.

3. NOREPINEPHRINE Norepinephrine is believed to have an inhibitory action on cortical neuronal function by causing postsynaptic inhibitory electrical potentials (Guyton, 1991). Norepinephrine-containingneurons (noradrenergicfibers)from the locus coeruleus innervate layers I to IV of the cortex (Guyton, 1991). Johnston and Coyle (1979) found that the normal number of noradrenergic axons are present in the MAMtreated cortex. The specific activity for norepinephrine’s synthesizing enzyme, tyrosine hydroxylase, is increased in the tissue of MAM-treated animals, relative to control animals (Table III), such that the total activity of tyrosine hydroxylase in the cortex of the MAM-treated rats is increased. The specific amount of uptake of [3H]norepinephrinewas increased by 130%, but the total uptake of norepinephrine per cortex was not significantly altered (Table I). The specific content of norepinephrine was also increased by 133%, although the total content of norepinephrine per cortical slab was reduced by 22% (Table II). Johnston and Coyle’s (1979) results indicate that the total uptake or total content of norepinephrine is not altered due to MAM treatment. Therefore, the increase in specific uptake and specific content of norepinephrineonly reflects loss of other nonadrenergic neurons. The increase in the total activity of tyrosine hydroxylase indicates that there may be an increase in the synthesis of norepinephrine in the MAM-treated animal secondary to the loss of these other neurons. Johnston and Coyle (1979) found a decrease in total beta-adrenoreceptor densities in MAM-exposed cortices. Beaulieu and Coyle (1982) suggested that this decrease reflected down regulation of beta-adrenoreceptors,an adaptive response to the increased innervation of norepinephrine in the MAM-treated cortex. Additionally, equivalent increases in the total densities of norepinephrine uptake sites were relative to the total amount of norepinephrine in the MAM-treated cortex (Watanabe, Kinuya, Ohtakeno, Watanabe, & Mamiya, 1992). 4. GLUTAMATE

L-glutamate is an excitatory neurotransmitter in the CNS with its receptors divided into three subtypes based on their sensitivity to exogenous compoundsN-methyl-D-aspartate(NMDA), quisquisalate (QA), and kainate (KA) (Cooper, Bloom, & Roth, 1991). Tamaru, Yoneda, Ogita, Shimizu, Matsutani, and Nagata

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(1992) assessed the binding of glutamate agonists and antagonists to the NMDA, KA, and QA receptors in the MAM-exposed rat. Specific bindings of NMDA-sensitive [3H]glutamate and [3H]glycine,another neurotransmitter which binds to the NMDA receptor complex, were slightly but insignificantly elevated (20%) in the cortex. Binding of a noncompetitive NMDA antagonist, MK-801, was found to be unchanged in the MAM-exposed cortex. The specific bindings of [3H]AMPA, a highly specific QA agonist and [3H]KAin the cortex were unchanged in the MAM-exposed cortex. Total bindings of [3H]glutamate,[3H]glycine,[3H]MK-801, [3H]AMPA, and [3H]KAwere reduced to 35% of those of controls. The results of the Tamaru et al. (1992) study are consistent with the earlier findings of Johnson and Coyle (1979) in that there appears to be a reduction in the number of glutamate neurons (neurons releasing glutamate) in the cortex of the MAM-treated rats. Although there was relatively little change in the specific activities of the uptake sites for glutamate (nmoYmg protein) and in the content of glutamate, there were significant reductions in total activity and content for glutamate in the MAM-treated rats (Tables II and m). Among the subtypes, of glutamate receptors, NMDA receptors exist in greater density in the cortex and hippocampus than the QA and KAreceptors, (Monaghan, Bridges, & Cotman, 1985).The NMDAreceptoris thought to be important in longterm potentiation (LTP), which is believed to be responsible for memory formation. LTP occurs when there is a long-lasting membrane potential resulting from the synchronous firing of several high-frequency action potentials within a small population of neurons (Kandel & O'Dell, 1992). In synapses that are capable of LTP, the presynaptic terminals release glutamate. Glutamate binds to a NMDA receptor and several non-NMDA receptors (binding to the non-NMDA receptors enables the depolarization of the postsynaptic cell). When a NMDA receptor channel opens due to the binding of glutamate and the depolarization of the postsynaptic cell, this leads to the enhancement of synaptic transmission. Evidence that LTP is involved in memory processes comes from studies using mutant mice strains that lack the calcium-calmodulin-dependentprotein kinase type II (aCaMKII) in the hippocampus and do not exhibit any LTP (Silva, Stevens, Tonegawa, & Wang, 1992). The a-CaMKII enzyme is believed to enable activation for continual synaptic potentiation without the presence of calcium (Silva. Stevens, Tonegawa, & Wang, 1992). In a parallel study by the same research team, the mutant mice showed deficits in spatial learning in a water T-maze task (Silva, Paylor, Wehner, & Tonegawa, 1992). A recent study on the cortex and hippocampus of MAM-exposed animals found that there is a reduction in the phosphorylation of B-50, a protein substrate for protein kinase C (PKC; Di Luca et d., 1991). PKC is another enzyme believed to be important in LTP in the hippocampus (Akers, Lovinger, Colley, Linden, & Routtenberg, 1986; Hu et al., 1987; Linden & Routtenberg, 1989). This loss of phosphorylation of the B-50enzyme is not due tot he reduction of B-50because there

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is no difference in the specific or total content of B-50in the MAM-treated animals (DiLuca et al., 1991). This loss of phosphorylation of B-50 is believed to be due to reduced PKC activity and supports the theory that the MAM-exposed animals have cognitive deficits that may be related to deficits in LIT (DiLuca et al., 1991).

C.

Effects in the Striatum

The effects of prenatal h4AM treatment on the neurochemistry of the striatum has also been assessed. The corpus striatum (caudate nucleus, putamen), globus pallidus, and the substantianigra comprise the largest componentsof the basal ganglia. The corpus striatum is believed to be involved with somatic motor function. The corpus striatum has been linked with motor dyskinesias and Parkinson’s disease. A study by Beaulieu and Coyle (198 1) assessed the effects of MAM on the neurochemistry of the striatum. MAM administrationon gestation day 15 causes a decrease in striatal mass reduction of 37%from that of control animals; on gestation day 14, a reduction of 49%; and on gestation day 7, a reduction of 20%.No disruptions in cellular organization were found in histological examination. The effects of MAM exposure on the neurotransmitters GABA, acetylcholine, dopamine, and glutamate in the striatum are reviewed in the next sections. 1. GABA

GABA neurons lie in a pathway from the striaturn to the globus pallidus and substantianigra. The axon terminals of these neurons cause inhibition in the globus pallidus and substantia nigra, and thus reduce the firing rate of the neurons of the substantia nigra (i.e., dopamine neurons). It is believed that the abnormal movements of Huntington’s chorea are due to the loss of cell bodies of GABA neurons, which results in the loss of inhibition in the globus pallidus and substantia nigra (Guyton, 1991). Beaulieu and Coyle (1981) assessed the effects of prenatal MAM exposure in the striatum on the total and specific activities of GAD, the specific and total uptake of GABA, and the specific and total content of GABA. MAM administration on gestation day 15 leads to a reduction in the total activity of the synthesizing enzyme, GAD, in the total uptake of GABA, and in the total content of GABA for the striatum (Beaulieu & Coyle, 198 1). This suggests that, as in the cortex, a large reduction of GABAergic neurons occurs in the striatum following prenatal exposure to MAM. The specific and total activity of GAD was assessed as a marker of GABAergic neurons in the striatum by Beaulieu and Coyle (1981). The specific activity of GAD in the striatum of MAM-treated animals remained similar to control animals, whereas the total activity of GAD was significantly reduced in the MAM animals (Table N).The specific uptake of GABA in the striatum of the MAM-treated animals was not significantly different from the controls; however, the total uptake

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TABLE IV ACTIVITY OF SYNTHESIZING ENZYMES IN THE STRIATUM FOLLOWING PRENATAL TREATMENP Specific activity Conuol Glutamate decarboxylase Choline acetyltransferase msine hydroxylase

MAMb

Change(%)

3 +2 (nmolh/mg tissue) 17 23** +31 (nmolkrlmg tissue) .56 .77** +38 (nmolhlmg tissue) 2

Total activity Control 75 532 17

MAMb

Change(%)

43** -43 (nmolh/slab) 414 -22 (nmolh/slab) 14 - 18 (nmolh/slab)

'The data in this table are from a study by Beaulieu and Coyle (1981). Reprinted by permission of the International Society for Neurochemistry. bMAM, methylazoxymethanol. * p < .05. **p < .01.

of GABA was significantly depleted in the MAM-treated striaturn (See Table V). The specific content of GABA was elevated by 20% in the MAM-treated striaturn, but the total content of GABA was decreased by 21% (Table VI). Proportionally similar decreases also occurred in the total activity of GAD, the total uptake of GABA, and the total content of GABA. Generally no differences occurred between MAM and control in the specific activity of GAD, or in the specific uptake

TABLE V NEUROTRANSMITTER UPTAKE IN THE STRIATUM FOLLOWING PRENATAL METHYLAZOXYMETHANOL TREATMENP Specific uptake Neurotransmitter r3HIGABAC [3H]Choline [3~~~pamine

Control

MAMb

Change(%)

20 22 +9 (pmoll2 min/mg tissue) .16 .26** +59 (pmol/4 min/mg tissue) 1.3 1.8** +32 (pmoll4 m i n h g tissue)

Total uptake Control

MAMb

Change(%)

779

516** -34 (pmoll2 minlslab) 7 7 + + 2 (pmoll4 minlslab) 52 39: -25 (pmol/4 minklab)

'The data in this table are from a study by Beaulieu and Coyle (1981). Reprinted by permission of the International Society for Neurochemistry. bMAM, methylazoxymethanol. 'GABA, gamma-aminobutyricacid. * p < .05. **p < .01.

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TABLE VI ENDOGENOUS NEUROTRANSMITTER IN THE STRIATUM FOLLOWING PRENATAL METHYLAZOXYMETHANOL TREATMEN-P Total content

Specific content Neurotransmitter GABA Acetylcholine Dopamine

Control 2 76 14

MAM*

Change(%)

2.4** +20 (nmollmg tissue) 78

+3

(pmol/mg tissue) 18** +31 (ng/mg tissue)

Control

68 2830 493

MAM* 54** (nmol/slab) 1652** (pmol/slab) 331* (ng/slab)

Change(%) -21

-42 -33

'The data in tlus table are from a study by Beaulieu and Coyle (1981). Reprinted by permission of the International Society for Neurochemisuy. *MAM,methylazoxymethanol. * p < .05. **p < .01.

of GABA. The slight elevation of specific content of GABA in the MAM-treated striatum suggests a slower rate of turnover of GABA in the MAM-treated striatum (Beaulieu & Coyle, 1981).

2. ACETnCHOLINE Acetylcholine neurons act as an excitatory pathway from the cortex to the caudate nucleus and putamen of the striatum. The dementia that occurs in Huntington's chorea is believed to be due to the loss of the acetylcholine neurons in the striatum and in the cortex (Guyton, 1991). The effects of MAM on acetylcholine neurons in the corpus striatum were also investigated by Beaulieu and Coyle (1981). The specific activity of CAT increased 31% when MAM was administered on gestation day 15. The total activity of CAT was reduced by 22% (Table IV). The specific activity of the synaptosomalcholine high-aftinity uptake process was increased by 59%. The total activity of choline uptake in MAM-treated striatum was comparable to levels in control animals (Table V). The specific content of acetylcholine in the MAM-treated striatum did not differ significantly from that in the controls (Table VI). The total content of striatal acetylcholine in the MAM-treated animals was reduced by 42% as compared to control animals, which was in proportion to the amount of striatal atrophy in the MAM-treated animal (Beaulieu & Coyle. 1981). These results indicate that significant increases occur in the specific activity of CAT, and in the specific uptake of acetylcholine. Significant decreases occurred in the total activity of CAT, but little change in the total uptake of acetylcholine. No differences were found between the MAM and control striaturn in the specific content of endogenous acetylcholine; however, a large decrease occurred in the

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total content of acetylcholinein the MAM-treated striatum. The disparity between the increased rates of specific uptake of acetylcholine and the significant reduction in the total content of acetylcholine suggests that the neurons affected by the MAM administration are those that are involved in the synthesis of acetylcholine (cholinergic neurons), and not those involved in the uptake of choline (which includes a large proportion of noncholinergic neurons) in the striatum. 3. DOPAMINE The striatal dopaminergic system is of interest because alterations in this system are believed to be involved in motor disorders with symptoms such as tics, stereotyped behaviors, dyskinesias (Breese, Baumeister, Napier, Frye, & Mueller, 1983, and disorders exhibited by some developmentally disabled children (Gualtieri & Schroeder, 1989). In a subset of these children who are also microencephalic, these abnormal movements may relate to the changes in the nigrastriatal dopamine system due to the microencephaly. Beaulieu and Coyle (1981) assessed the effects of prenatal MAM exposure on the activity of the dopamine system in the striatum. The specific activity of tyrosine hydroxylase increased by 38% in MAM-treated animals as compared to that of control animals, whereas its total activity decreased slightly (Table IV).The specific uptake of dopamine was elevated by 32%, whereas its total uptake was decreased by 25% (Table V). The specific content of dopamine within the striatum was significantly elevated by 31% in MAM-treated animals, but the total content of dopamine in the striatum was reduced by 33% (Table VI). These results suggest that a similar increase occurred in the specific activity of tyrosine hydroxylase, specific uptake of dopamine, and the specific content of endogenous dopamine. Similar decreases occurred in the total activity of tyrosine hydroxylase, total uptake of dopamine, and in the total content of dopamine. A later study showed an increase in the density of [3H]GBR12935binding to dopamine uptake sites in the shiatum (Watanabe et al. 1990).The results of the Beaulieu and Coyle (198 1) and Watanabe et al(1990) indicate a relative enrichment of dopamine in the existing striatal tissue (Beaulieu & Coyle, 1981). Recent studies on dopaminergic receptors indicate differences in the effects of MAM on the D, and D, receptors in the striatum. MAM treatment does not appear to affect the binding of a D, dopamine antagonist, [3H] spiperone and a D, dopamine antagonist, SCH23390, to D, and D, dopamine receptors, respectively (Beaulieu & Coyle, 1981; Watanabe et al., 1990). However, a study by Balduini, Abbracchio, Lombardelli,and Cattabeni (1984) found that there was a decrease in D,-stimulated cyclic adenosine monophosphate (CAMP)production. Thus MAM treatment appears to result in a relative hyperinnervation of dopamine neurons in the presence of a normal number of D, and D, receptors available for binding but that the D, receptors may be functioning abnormally.

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4. GLUTAMATE

The glutaminergic neuronal fibers that innervate the striatum are derived from the cortex. For the these neuronal fibers, binding of agonists and antagonists to the NMDA. QA, and KA glutaminergicreceptor subtypes was assessed (Tamaru et al., 1992). Specific bindings of NMDA-sensitive [3H]glutamateand [3H]glycinewere 90% and 60% higher, respectively, in the striatum of MAM-exposed rats than in the control rats. In the MAM-treated striatum, the specific binding of a noncompetitive NMDA antagonist, 13H]MK-801,was 160% and 180%higher than in that of control animals. Likewise, the specific binding of [3H]AMF’A to QA receptors (in the presence of KSCN ions) was 30% higher and the specific binding of [3H]KA was 40% higher in the striatum of MAM-treated rats. In contrast, total bindings of [3H]glutamate, [3H]glycine, [’H]MK-801, [3H]AMPA, and [3H]KA were not significantly different between MAM-treated and control striati. Additionally, the decreased total activity of these agents did not correlate with the decrease in striatal weight. These results suggest that glutaminergic neurons in the smatum are not severely affected by MAM exposure and, similar to the effects of MAM on noradrenergic, dopaminergic, and cholinergic fibers, and “condensation” of glutaminergic neurons occurs in the remaining tissue leading to a relative increase in their density.

D.

Effects in the Hippocampus

1. STRUCTURALABNORMALITIES

The hippocampus is believed to be involved in learning and memory because it is a site of LTP, a possible mechanism of memory formation, as discussed above. To review that discussion briefly, LTP involves the binding of glutamate to NMDA receptor, which leads to a long-lasting membrane action potential that enables memory formation. The NMDA receptor channel opens when the binding of glum a t e and the depolarization of the postsynaptic cell occurs, leading to the enhancement of synaptic transmission. The mutant mouse strain that lacks the aCah4KII enzyme necessary for continued synaptic potentiation, and therefore lacks LTP in the hippocampus, also shows spatial learning deficits (Silva, Paylor, Wehner, & Tonegawa 1992). Additionally, MAM rats showed a decrease in the phosphorylation of B-50 by PKC, another enzyme believed to be important in LTP (Di Luca et al., 1991). These results suggest that the learning deficits seen in MAM-exposed rats may be related to the neurophysiologicalalterations in the cortex and hippocampus. Histological examinations of the hippocampus indicate that MAM exposure leads to changes in the CA1 pyramidal neurons, such as abnormal dendritic configurations and connections, which results in reduced hippocampal commissural

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input and general disorganization(Singh, 1980). Singh (1 980) found that the number of dendritic spines on a segment of a dendrite from a MAM-induced mispositioned cell was reduced by 75%from normal cells in hippocampus of MAM-treated rats and by 84%from the cells of saline controls. The reason for the dendritic abnormalities is unclear. Sanberg et al. (1987) suggested that the changes in the pyramidal cells may be due to abnormal afferent innervationresulting from MAM treatment and the subsequent inappropriate position of these cells. Cheema and Lauder (1983) investigated the organization of the granule cells and the pyramidal cell pathway in the hippocampus and found the abnormal development of infrapyramidal mossy fibers (dentate granule cell mons) in CA3 pyramidal neurons. This also occurs in neonatal hyperthyroidism,prenatal ethanol exposure, and neonatal lesions of the hippocampal pyramidal neurons (Cheema & Lauder, 1983). Why MAM exposure or these other neonatal injuries would cause the development of these infrapyramidal mossy fibers is unclear. Additionally, it is unknown how the development of these infrapyramidal fibers affect the neurotransmitter systems in the hippocampus.The neurotransmittersthat have been investigated are norepinephrine, dopamine, serotonin, and glutamate. 2. NEUROTRANSMITTERS: NOREPINEPHRINE, DOPAMINE, AND SEROTONIN

Noradrenergic fibers arise from the locus coerulus and appear to have two functions in the hippocampus. First, norepinephrinehas an inhibitory action on the inhibitory interneurons, thus leading to a decrease in inhibition to the pyramidal cells. The second function is to decrease the calcium-activatedpotassium current thus enhancing excitation. Serotonergicfibers come from the raphe nucleus to innervate the hippocampus. Both serotonergic and noradrenergic inputs produce slow postsynaptic potentials and are believed to act as modulators of activity in the hippocampus and are involved in brain plasticity. Because the only measures for norepinephrine,dopamine, and serotonin in the hippocampus of MAM-treated animals are the endogenous concentrationsof these neurotransmitters,they will be discussed together. Jonsson and Hallman (1982) took measurements of the effects of prenatal MAM administration on the endogenous-specificand total content of norepinephrine,dopamine, and serotonin (Table VII). The specific content of endogenous norepinephrine, dopamine, and serotonin was increased in the MAM-treated animals to 166%, 197%,and 205%of control, respectively; however, there were no significant differences between the MAM-treated and control animals in the total content of any of these neurotransmitters.These results suggest that despite the changes in the ectopic cells and the abnormal growth of intrapyramidal mossy fibers, no differencesoccurs in the concentrationof these neurotransmittersand the tissue has a relative hyperinnervation of neurons containing these neurotransmitters.

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TABLE VII ENDOGENOUS NEUROTRANSMITTER LEVELSIN M A M a - ~ ~ ~ ~ ~ ~ HIPPOCAMPUS FOLLOWING PRENATAL MAM TREATMENT^ Specific Content Neurotransmitter Norepinephrine Dopamine serotonin

Control

MAM"

Change (a)

422 ? 55 6.8 2 13 331 2 35

701 t 48** 13.4 t 2.5* 690 t 50**

-k 97

+66 +lo5

MAM, methylazoxymethanol. bThe data in this table from a study by Jonsson and Hallman (1982). Reprinted from Developmental Brain Research, 2, by G . Jonnson and H. Hallman. Effects of prenatal methylazoxymethanol treatments on the development of central monamine neurons (pp. 513-530), 1982,with kind permission of Elsevier S c i e n s N L , Sara Burgerhartstraat 25,1055 KV Amsterdam, The Netherlands. *p < .05. **p < .01.

3. GLUTAMATE! L-glutamate is a major excitatory neurotransmitter in the hippocampus. Among the NMDA, KA, and QA receptor subtypes, NMDA receptors-which are believed to be important for LIT-are found in a greater density in the cortex and hippocampus than the other two subtypes (Monaghan, Bridges, & Cotman, 1985). As described earlier in the section on glutamate in the cortex, Di Luca et al. (1991) indicated that a reduction of the phosphorylation of B-50 occurs in the cortex and hippocampus of MAM-treated animals. This reduction in phosphorylation may affect the production of LTP in the hippocampus (Akers et al., 1986; Hu et al., 1987; Linden & Routtenberg, 1989). As mentioned earlier, this loss of phosphorylation of B-50 is thought to be due to reduced PKC activity and may explain the cognitive deficits of the MAM-exposed animals (Di Luca et al., 1991). An assessment of the binding of glutamate agonists and antagonists to the NMDA, KA, QA receptors was conducted (Tamaru et al., 1992). Specific bindings of NMDA-sensitive [3H]glutamateand [3H]glycine were unchanged in the hippocampuses of MAM-treated and control animals. The binding of noncompetitive NMDA antagonist, MK-801. was found to be unchanged in the MAMexposed hippocampus. The specific bindings of [3H]AMPA, a highly specific QA agonist, and [3H]KA in the cortex were unchanged in the MAM-exposed hippocampus. Total bindings of [3H]glutamate, [3H]glycine. [3H]MK-801, [3H]AMpA. and [3H]KAin the hippocampus were reduced to 45% of controls. Tamaru et al.'s (1992) results indicate that glutaminergic neurons are severely affected by the prenatal MAM exposure. The reduction of phosphorylation of the B-50enzyme and the disruption in glutaminergic involvementin LTP may explain

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some of the neurochemical mechanisms of the learning deficits of the MAM-exposed animals.

E.

Summary on the Neurochemical Changes

The studies reviewed here indicate that the neurochemistry of the cortex, striatum,and hippocampus is affected by MAM prenatal administration.A review by Rodier (1986) suggests that the results of Johnston and Coyle (1979) are consistent with the theory that all of the norepinephrine-containingneurons and most of the acetylcholine neurons derive from cell bodies located outside the cortex and that these neurons form before or after gestation day 15 in the rat. These multiplebranched extrinsic neurons, therefore, are not reduced by the MAM administration at this time even though cortical mass is reduced. Both noradrenergic and cholinergic projections into the cortex and dopaminergic projections in the striatum occur as a relative hyperinnervation.A consistent increase in the specific activities of tyrosine hydroxylase, specific content of dopamine, and specific uptake of dopamine and a consistent decrease in the their total levels per striatum suggests an enrichment of dopaminergicneurons in the remaining striatum tissue. Finally, no changes in the total content of the monoamines occur in the hippocampus despite the significant loss of tissue. In contrast, the GABAergic neurons, the intrinsic neurons of the cortex and striatum. are affected by the MAM treatment. The number of GABAergicneurons and their projections are reduced by MAM in proportion to the amount of cortical and striatal mass reduction. The evidence indicating that of GABAergic neurotransmitter activity (total activity of GAD, total uptake of [3H] GABA, and total content of GABA) is reduced in both the cortex and the striatum is consistent with the theory that the GABAergicneurons are in local circuits in the cortex and in the striatum. Finally, glutamate, another local circuit neuron, is reduced in both the cortex and hippocampus, but is relatively unchanged in the striatum. IV.

FUTURE DIRECTIONS

This chapter focused on the learning deficits and neurochemical changes that occur in rats due to prenatal MAM exposure. By looking at the effects of MAM, it is believed that information can be obtained on the effects of microcephaly on the learning impairments of developmentally disabled humans. The studies reviewed emphasized the effectsof microcephaly specifically in the cortex, striatum, and hippocampus-areas important in learning and memory. In learning tasks, MAM-treatedanimalsperform similarly to controlsin simple mazes, but show decreased ability in performing the reversals or the extinctions of learned responses that occur in complex mazes. In operant chamber tasks,MAMs appear to be hyperactive, thereby interfering with performance in a passive avoidance task. In

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contrast, alterations in the cortex, striatum. and hippocampus do not lead to changes in physical and reflex development. Although the results of the behavioral studies on MAM-treated animals indicate that MAM produces learning impairments, how these impairments relate to neurochemical changes in the MAM-induced microencephaly is still being investigated. MAM-treated animals show enrichment of norepinephrine and dopamine relative to the amount of tissue lost in the cortex, hippocampus, and striatum. It appears as though the innervation of noradrenergic and dopaminergic extrinsic fibers are unaffected by prenatal MAM treatment, unlike the GABAergic and glutaminergic intrinsic projections that appear to be severely affected by MAM exposure. The effect of MAM treatment on acetylcholine is confusing: Although a hyperinnervation of acetylcholine occurs similarly to that of dopamine and norepinephrine,there is an overall decrease in the total activity of acetylcholinein the cortex and hippocampus, similar to the effects of MAM on GABA and glutamate. Information on the neurochemistry involved in learning and memory is being investigated in several ways. Most theories suggest that cholinergic and glutaminergic neurotransmission play a role in learning and memory. Drug studies using anti-acetylcholinesteraseshave shown that these agents improve the retention of a learned task if given within 1 week after the training (Fibiger, Damsma, & Day, 1991).Other evidence for the role of acetylcholine in memory comes from studies on Alzheimer’spatients where clinical reports indicate that the loss of the acetylcholine-synthesizingenzyme, choline acetyltransferase, correlated with the loss of cognitive function and the development of senile plaques (Fibiger et al. 1991). Research on the relationships between cholinergic transmission and memory formation can be extended to include the assessment of neurochemistry in microcephalic developmentally disabled. One way to study the importance of this neurotransmitter system in developmental disabilities is to assess its functioning in MAM-treated animals that are believed to have learning difficulties. If the learning deficits assessed in the MAM-treated animal are related to the MAM-induced neurochemical changes in acetylcholine activity, then using drugs that alleviate those alterations might reduce the learning impairments caused by the prenatal MAM exposure. Future research could assess whether giving a cholinomimetic agent, which would increase the levels of acetylcholine activity on the postsynaptic receptor, or an acetylcholinesteraseinhibitor, which would reduce the metabolism of acetylcholine,could improve the learning abilities of the MAM-treated animals. Studies looking at the improvement of memory have been testing the memory-enhancing effects of cholinomimetics in normal rats (Sarter, 1991). Future studies could assess the effects of these drugs on the MAM-treated animal in learning tasks. Another hypothesis of memory development suggests that the glutaminergic system is involved in the strengthening of synaptic connections, LIT, which results in memory formation. The total activity of glutamate is reduced in both the

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cortex and hippocampus of the MAM-treated animal, which could therefore lead to a reduced capacity for LIT. Additionally, MAM-treated animals show the loss of phosphorylation of B-50, an enzyme important in LIT, in both the cortex and hippocampus. This loss of phosphorylation is believed to be due to reduced PKC activity and supports the theory that the MAM-exposed animals have learning impairments that may be related to deficits in LTP(Di Lucaet al., 1991).It would be interestingto see whether glutaminergic agonists would facilitate LTP in the MAM-treated animals and improve their learning abilities. The behavioral research on MAM-treated animals’ learning and memory suggest they have specific learning impairments that disrupt their performancein tasks involving multiple choices and reversals of learned responses. The stimulus manipulations that occur in complex mazes can be adapted to a task that is similar to tasks given to the developmentally disabled-the repeated acquisition task, (see chapter by Williams and Saunders, this volume). For both humans and nonhumans, the repeated acquisition task requires the subjects to learn and then relearn variations of a sequence of responses across different discriminate stimuli in order to receive reinforcement (Thompson, Mastropaolo, Winsauer, & Moerschbaecher, 1986). For instance, the rat learns a response chain across three levers in one order and then is required to learn another response chain using the same levers in a different order. The MAM-treated animals may have difficulty in learning a response chain of three lever presses as shown by their increased errors in multiple arm mazes, or they may have a problem in relearning the order of responses as shown by their problems in tasks that required alternating between arms and reversals of learned positions, or both are as may be problematic. It would be interesting to see whether the learning deficits exhibited by MAM-treated animals in complex mazes occurred in the operant chamber using the repeated acquisition task.

The repeated acquisition task is one of the few operant tasks that shows similar sensitivity to drugs in both humans and nonhumans (see chapter by Williams & Saunders, this volume). The repeated acquisition task could be used to assess whether cholinomimetics or NMDA agonists decrease these particular learning impairments in MAM-treated animals. This assessment can provide information on the effects of these drug agents on the learning disabilities of the microencephalic mentally retarded individual. This in turn may lead to improved drug therapy in these individuals and thus enhance their living situation. In conclusion, this chapter has discussed research on the types of learning deficits and neurochemical changes that are the result of microencephaly in the MAM-treated rat. Future research with the MAM-treated rat should focus on treatments for these learning deficits and neurochemical alternations that may be applicable as treatments for similar dysfunctions in the microcephalic human population.

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