Use of bullfrog tadpoles (Rana catesbeiana) to examine the mechanisms of lead neurotoxicity

SYNOPSIS. Sublethal exposure to lead elicits changes in behavior, particularly learning. Previously, we had shown that bullfrog tadpoles exposed to 1 mg Pb/liter for 7 days exhibit learning deficits in a discriminate avoidance learning assay. The precise mechanisms involved in these lead-induced learning deficits are not understood, but CNS monoamine neurotransmitter systems have been implicated in the learning process. In the present study, we exposed bullfrog tadpoles to 1.7 +/- 0.2 mg Pb/liter for 7 days and then compared concentrations of neurotransmitters from whole brain samples with those of controls. Serotonin or 5-hydroxytryptamine (5-HT) was significantly decreased in the lead exposed group while the serotonin metabolite, 5-hydroxyindoleacetic acid, (5-HIAA) was similar to controls. No changes were observed in the catecholamines epinephrine and norepinephrine following lead exposure. The ratio of 5-HIAA/5-HT was significantly increased in lead exposed animals as a result of the decrease in 5-HT, suggesting a decrease in S-HT biosynthesis rather than an increase in 5-HT metabolism. These findings are the first to suggest that lead exposure in bullfrog tadpoles affects the monoamine neurotransmitters that are implicated in the learning process. The results of the present study, in conjunction with previous evidence of learning deficits following lead exposure, offer the possibility of correlating lead exposure with learning deficits and alterations in CNS neurotransmitters in bullfrog tadpoles. The use of this tadpole model shows promise as a means to examine and understand the mechanisms involved in lead neurotoxicity.

INTRODUCTION

Despite a decrease in lead emissions in automobile exhaust and a reduction in the use of lead-based paint products, substantial amounts of lead can still be found in the environment (Davis et al., 1993). Leadbased paint found in older homes is considered to be the primary source of lead exposure for children in the United States (U.S. Environmental Protection Agency, 1990). Lead can affect both the renal and hematological systems and specifically targets the central nervous system (Goyer, 1991). For example, exposures to lead that result in blood levels of 80.0 (mu)g Pb/dL or greater have been shown to cause severe encephalopathies and mental retardation in humans (Goyer and Rhine, 1973). Research has shown that low-level lead exposure, which typically does not result in overt clinical symptoms, can also cause negative behavioral effects, particularly in infants and children (Goyer, 1993). Blood lead levels as low as 10.0-15.0 jig Pb/dL (see Goyer, 1993 for review) can elicit alterations in learning skills, as measured by a variety of behavioral and cognitive tests such as IQ and MDI (Mental Developmental Index) (Bellinger et al., 1987; Bellinger et al., 1991; Stiles and Bellinger, 1993).

Behavioral studies examining the effects of lead on non-human mammalian models such as monkeys and rats have revealed that lead exposure can induce deficits in learning tasks such as DRL (differential reinforcement of low-rate schedule of reinforcement), discrimination reversal, spatial delayed alternation, repeated acquisition and spatial learning (Bushnell and Bowman, 1979; Alfano and Petit, 1981; Taylor et al., 1982; Rice and Gilbert, 1985; Cohn et all., 1993; Rice, 1993). These effects can be detected at blood lead levels as low as 10.0 (mu)g Pb/dL (Cory-Slechta, 1995).

Behavioral effects following lead exposure have also been demonstrated in nonmammalian vertebrates. Goldfish (Carassius auratus) exposed for 24 hr to 1.0 mg Pb/liter showed greater than 10% impairment in a conditioned avoidance response (Weir and Hine, 1970). Following a 4-week exposure to a similar concentration, fathead minnows (Pimephales promelas) exhibited altered feeding behavior (Weber et al., 1991).

In neurotransmitter studies, catfish (Clarias batrachus) exposed to 5.0 mg Pb/L for 150 days exhibited decreased levels of GABA (gamma amino butyric acid) and increased levels of 5-HT (serotonin) and histamine in the whole brain (Katti and Sathyanesan, 1986). Fathead minnows exposed to 1 mg Pb/liter for 4 weeks showed significantly elevated whole brain levels of NE (norepinephrine) and 5-HT (Weber et al., 1991).

Our laboratory uses a discriminate avoidance learning assay to examine the behavioral effects of lead on ranid tadpoles, specifically bullfrog (Rana catesbeiana) and green frog (Rana clamitans) tadpoles. In our assay, we condition tadpoles to associate a change in illumination with an electric shock and tadpoles subsequently learn to discriminately avoid the shock by moving in response to the illumination change. In these behavioral studies, we have found that both bullfrog and green frog tadpoles exhibit impaired learning following exposure to 1.0 mg Pb/liter for 7 days (StricklerShaw and Taylor, 1990; Strickler-Shaw and Taylor, 1991). In a subsequent morphological study, neurons in the medial pallium, a brain region homologous to the mammalian hippocampus (Hoffman, 1967; Kicliter and Ebbesson, 1976) appeared hypertrophied following a similar exposure (StricklerShaw and Taylor, 1991). The mammalian hippocampus is believed to be a primary site of learning (Andersen and Trommald, 1995; Sprick, 1995); thus it is possible that the homologous medial pallium in tadpoles could be involved in amphibian learning and memory.