Effects of lead exposure on the physiology of neurons

Progress in Neurobiology Vol. 24, pp. 199 to 231, 1985 Printed in Great Britain.All fights reserved

0301-0082/85 $0.00+ .50 Copyright © 1985PergamonPress Ltd

EFFECTS OF LEAD EXPOSURE ON THE PHYSIOLOGY OF NEURONS GERALD AUDE$IRK Biology Department, University o f Colorado at Denver, 1100 14th Street, Denver, CO 80202, U.S.A.

(Received 15 March 1985)

Contents 1. Introduction 1.1. The search for structural and physiological mechanisms 1.2. Experimental methodologies 1.2.1. In vivo vs/n vitro exposure 1.2.2. Organic vs inorganic lead 2. Neuronal structure 2.1. Effects on the central nervous system 2.1.1. Lead encephalopathy 2.1.2. Effects of low-level exposure 2.2. Axonal morphology and myelination 3. Effects on cellular physiology 3.1. Conduction velocity 3.1.1. Peripheral nerves 3.1.2. Central nerves 3.2. Neuronal excitability 3.2.1. Spontaneous activity 3.2.2. Excitability in response to stimulation 3.3. Synaptic transmission 3.3.1. Chemical synapses 3.3.1.1. Stimulus-evoked presynaptic release of transmitter 3.3.1.2. Spontaneous release of transmitter 3.3.1.3. Lead effects on synaptic receptors and receptor actions 3.3.1.4. Termination of transmitter action 3.3.2. Electrical synapses 4. Subcellular mechanisms 4.1. Lead and calcium interactions 4. I. 1. Lead effects on calcium channels 4.1.2. Lead effects on calcium homeostasis 4.1.2.1. Calcium in organelles 4.1.2.2. Intracellular free calcium ion concentration 4.1.3. Direct effects of lead ions on calcium-mediated processes 4.2. Lead-protein interactions 4.2.1. Neurotransmitter receptors 4.2.2. Lead effects on enzyme activity 5. Perspective and future directions 5.1. In vitro vs/n vivo exposure 5.1.1. Standardization of in vivo exposure in mammals 5.1.2. In vitro experiments 5.2. Cellular physiological techniques 5.2.1. Model systems 5.3. Inorganic vs organic lead and other heavy metals Acknowledgements References

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1. Introduction The fact that lead has toxic effects o n b e h a v i o r a n d h u m a n health has been k n o w n for t h o u s a n d s o f years. I n the second c e n t u r y B.C., the G r e e k physician N i c a n d e r accurately described the s y m p t o m s o f massive lead p o i s o n i n g . T h e b e h a v i o r a l effects o f lead p o i s o n i n g , i n c l u d i n g p o o r n e u r o m u s c u l a r c o o r d i n a t i o n , "wrist d r o p " , paralysis o f the extremities a n d i n s a n i t y are, o f course, caused b y d a m a g e to the n e u r o m u s c u l a r system. 199

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Such overt and devastating symptoms are usually due to rather massive lead intake, such as from lead-lined vessels for drinking liquids, poor glazing of pottery or from occupational exposure. Lead poisoning from household items is now very infrequent, and modern health, safety and antipoilution laws have greatly reduced occupational exposure. As a result, classical plumbism is rare today. Nevertheless, physicians, public health officials and neurobiologists are still concerned that lead poses a hazard to the normal functioning of the nervous systems of many people: subtle dysfunctions brought about by very low levels. The exact nature of the behavioral deficits induced by low levels of lead and the precise level of lead exposure required to elicit the deficits is still a matter of controversy. However, there is widespread agreement that lead levels, which only a few decades ago were considered low enough to be completely safe, probably produce neural deficits, especially in children (for reviews see Baker et al., 1983; Needleman, 1983; Needleman and Landrigan, 1981). Further, the nervous systems of all animals, humans included, evolved in a practically lead-free environment. As a result of human activities, lead is now so ubiquitous that even the most remote human populations (Hecker et al., 1974; Piomelli et al., 1980) have body burdens of lead that are probably orders of magnitude greater than those of our prehistoric ancestors (Settle and Patterson, 1980). It is possible that all human nervous systems function suboptimally due to chronic exposure to low levels of lead. Indeed, at least one study (Benignus et al., 1981) suggests that if there is a threshold to lead's effects on the human brain, it may well be below that found in any person dwelling in an industrial society. Against this background, for the past two decades physicians, psychologists and neurobiologists have sought the answers to three related questions: (1) What behaviors are affected by low levels of lead? (2) What structures in the human brain are affected by lead, and in what ways? (3) What morphological, biochemical and physiological effects does lead have on the functioning of the individual cells and cell assemblies that make up the nervous system? This review will discuss experiments that have attempted to answer the third question, and will be further restricted almost entirely to data specifically relating to the effects of lead exposure on the morphology and physiology of individual neurons. Therefore, I will not discuss the extensive behavioral literature on lead. These experiments are important in attempting to define exactly what behaviors are affected by lead, and to a lesser extent what regions of the brain might be most affected. However, they do not as yet provide very much information as to how the underlying neural mechanisms are altered, nor is that their goal. For discussions of lead effects on the behavior of humans, the reader is referred to reviews by Baker et al. (1983), Needleman (1983) and Needleman and Landrigan (1981). The effects in animals have been reviewed by Silbergeld (1984). I will also omit a discussion of the equally extensive neurochemical literature, for several reasons. First, there are several excellent recent reviews available to the interested reader (Shellenberger, 1984; Silbergeld and Hruska, 1980; Winder and Kitchen, 1984). Second, in many respects the various neurochemical investigators have designed and carried out their experiments so differently that the results are often confusing and contradictory. Sheilenberger (1984) recently evaluated the neurochemical literature and concluded that, although the data are suggestive, "low level lead exposure has not yet been shown to produce a selective or specific action on any of the transmitter systems considered". Third, neurochemical effects may not be the same in all neurons of a given brain region, even if they all use the same transmitter. A finding of, say, lowered norepinephrine levels does not tell us what happens to the norepinephrine content of specific adrenergic neurons, which may differ among subtypes. Fourth, the types of neurochemical effects found so far usually do not predict a clear physiological effect. 1.1. THE SEARCH FOR STRUCTURAL AND PHYSIOLOGICAL MECHANISMS In seeking mechanisms of action whereby lead affects the functioning of nerve cells, and consequently behavior, we must consider both the similarities and differences between

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neurons in structure and physiology. Some of these structures and physiological properties are shared by virtually all neurons, while others vary widely among neuronal types. Depending upon which, if any, of these properties are affected by lead exposure, we might expect some lead effects to be common to all neurons, while other effects might be specific to particular neuronal types. For example, the stimulation-evoked release of neurotransmitters has been found to be calcium-dependent at a wide variety of synapses, both in the central and peripheral nervous systems, and in many different phyla of animals. In vitro lead exposure has been found to inhibit transmitter release at neuromuscular junctions of both frogs and rats, presumably by blocking inward calcium currents at the presynaptic terminal (Atchison and Narahashi, 1984; Manalis and Cooper, 1973; Manalis et al., 1984; Pickett and Bornstein, 1984). Although the exact susceptibility of a given synapse might differ among organisms and among synaptic sites within a single organism, we would expect inhibition of transmission at chemical synapses to be a common feature of in vitro lead exposure in all animals. In contrast, the ability of lead to inhibit adenylate cyclase (Lemay and Jarrett, 1975; Nathanson and Bloom, 1976; Wilson, 1982) would only affect transmission at synapses in which the postsynaptic action is coupled to a cyclic nucleotide cascade. Thus, how, or even if, lead affects a particular neuron will depend upon what neuronal properties are altered by lead and what properties are possessed by that neuron. Further, a neuron's susceptibility to insult from lead exposure may also depend on the neuron's location in the brain. For example, in mammals it appears that chronic exposure to low levels of lead, particularly during development, results in excess accumulation of lead in certain areas of the hippocampus and amygdala (Collins et al., 1982; Danscher et al., 1975, 1976; Fjerdingstad et al., 1974; Grandjean, 1978; Kishi et al., 1982; Scheuhammer and Cherian, 1982). Behavioral evidence also suggests that limbic system functions are more disrupted than those of many other areas of the brain (Alfano and Petit, 1981; Bushnell and Bowman, 1979; Kostas et al., 1976; Petit and Alfano, 1979; Tilson et al., 1982; Walsh and Tilson, 1984). This may be due to the particular properties of limbic system neurons, to preferential access of lead to limbic structures, or both. We do not as yet know enough about lead metabolism in the brain to distinguish between these possibilities. 1.2. EXPERIMENTAL METHODOLOGIES Experimental approaches to the elucidation of the structural and physiological bases of lead effects on neurons have been quite varied. In general, however, the experiments can be grouped into two categories, chronic in vivo exposure and acute in vitro exposure. Each of these methods has unique advantages and disadvantages. Likewise, two general forms of lead are used in experiments, inorganic (lead salts, usually acetate, carbonate, chloride or nitrate) and organic (tetraethyl lead, triethyl lead, tributyl lead, trimethyl lead, etc.). 1.2.1. In vivo vs in vitro exposure In most experiments, the ultimate aim is to understand the mechanisms whereby chronic exposure to small amounts of lead in the workplace or general environment might cause alterations in neuronal physiology of human beings. Obviously, studies of human physiology almost always involve in vivo exposure to relatively uncontrolled quantities of lead. This causes potentially severe difficulties in defining what constitutes undue lead exposure, since usually the rate of lead exposure for any given individual is unknown and the time of exposure often varies by an order of magnitude or more between members of the lead-exposed population. In some experiments, a relatively uniform population is simply split into "high-lead" vs "low-lead" based on blood lead measurements. Not surprisingly, this may result in confusing and conflicting results. Animal models of in vivo exposure attempt to reproduce the human situation, not in exact quantities or routes of exposure, but in probable specific neuronal effects. There are, however, three potential difficulties in developing good animal models: levels of exposure

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compared to humans, non-specific effects and multiple sites of action. First, species vary widely in their susceptibility to the deleterious effects of lead. Therefore, neuronal effects are not necessarily equivalent between humans and an animal model simply because the two develop the same blood lead concentration. For example, it is now well established that rats are much more tolerant to lead than are humans; equivalent neuronal effects probably occur at blood concentrations three or four times higher in rats than in humans (Petit and Alfano, 1983). Second, in vivo animal models must take non-specific effects into account when designing exposure routines. Behavioral deficits probably occur in humans at lead concentrations that produce no overt clinical symptoms (Baker et al., 1983; Needleman, 1983; Needleman and Landrigan, 1981), and the most comparable animal studies would also involve exposures which do not cause overt neurological symptoms (e.g. paralysis, epileptogenic episodes) or malnutrition. Finally, in vivo lead exposure is likely to have multiple sites of action, both within the nervous system and within the rest of the body. For example, a reduction in neuronal dendritic fields following lead exposure could be produced, at a minimum, by effects on glial cells, postsynaptic cells or any of several types of presynaptic cells. This greatly increases the uncertainties in determining the mechanism(s) which cause any defects, especially structural or biochemical defects. For these reasons, and for ease of quantitation of exposure, many investigators have turned to in vitro exposure to study precise sites and mechanisms of lead action. The chief difficulty with this approach, of course, is that in vitro exposure for a few minutes may not be comparable to in vivo exposure for months or years. A few studies have compared the effects of in vivo and in vitro exposure on the same neuronal parameters (e.g. Govoni et al., 1984; Silbergeld et al., 1980). This approach, which will hopefully become more common, can attempt to find out if the mechanism of in vitro lead exposure, usually much easier to study, is similar to that of in vivo exposure. At present, however, most experiments use either in vivo or in vitro exposure, but not both. 1.2.2. Organic vs inorganic lead The vast majority of the experiments to be described in this review have studied the effects of inorganic lead, and when the word "lead" is used without modifiers, I will be referring to inorganic lead. In general, inorganic lead salts are insoluble in lipids (and only a few, such as acetate and nitrate, are even very soluble in water), while organic lead compounds are lipid soluble. With in vitro exposures, one would expect organic lead to penetrate into the interiors of cells more readily than inorganic lead, and thus to have somewhat different effects, at least in the short term. For in vivo exposures, there might be differences in rate and route of uptake from the environment, and perhaps in ultimate effects, although thus far the behavioral data seem fairly comparable for the two forms (Walsh and Tilson, 1984). 2. Neuronal Structure

2.1. EFFECTSON THE CENTRALNERVOUS SYSTEM 2.1.1. L e a d encephalopathy

Relatively acute exposure to massive amounts of lead (sufficient to cause death in a significant proportion of the experimental animals within a few days) causes massive accompanying changes in brain and neuronal structure, including necrosis o f neurons, especially in the cerebellum of young animals. In neonatal rats, lead encephalopathy is well described by Press (1977), Holtzman et al. (1980, 1982) and Lefauconnier et al. (1983). Exposure of older animals to similar doses of lead produces much less neuronal damage (Holtzman et al., 1982, 1984). Holtzman et al. (1982, 1984) suggest that interference with cellular respiration by mitochondria is the key event which triggers the brain damage seen in younger animals, and that older brains are protected by glial uptake and storage of lead in non-mitochondrial sites (lysosomes and nucleus).

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2.1.2. Effects o f low-level exposure Chronic exposure to lower levels of lead can also affect neuronal structure. Such studies of structural effects have utilized rodents, primarily rats, almost exclusively. Several different protocols have been used in exposing young rodents to lead. Most of these have involved feeding lead (usually in drinking water) to the dams during their nursing period (sometimes during pregnancy as well), so that the pups receive lead in the mother's milk. In some studies the pups were dosed by gastric intubation or injection. In longer-term experiments, pups may also be given lead in their drinking water or food after weaning. When evaluating individual studies, one must be careful to note that in some experiments lead feeding caused overall reductions in body and/or brain growth, thus compounding specific lead-induced effects with non-specific changes due to malnutrition. Several investigators, however, have designed their experiments so that there is no difference in growth between lead-exposed and control animals, for example by reducing the amount of lead or by restricting access of control animals to food. While the relative contribution of malnutrition varies among studies, in most cases the structural effects seen in lead-exposed animals are at least quantitatively and often qualitatively different from those found in malnourished animals. For this reason, the extent of malnutrition will not be specified in the following discussion. Exposure of fetal and neonatal rodents to relatively low levels of inorganic lead results in changes in gross morphology of brain regions and in fine structure of neurons. Because of the accumulation of lead in limbic structures and the susceptibility of limbic-controlled behaviors to be affected by lead exposure (see Section 1.1), several laboratories have studied the development of the hippocampus in lead-exposed neonatal rodents (Alfano and Petit, 1982; Alfano et al., 1982; Campbell et al., 1982; Kawamoto et al., 1984; Kiraly and Jones, 1982; Louis-Ferdinand et al., 1978). Various dimensions of hippocampal structures were reduced in lead-exposed animals, including maximum width of the hippocampus, length of the dentate gyrus and overall length and width of the mossy fiber pathway (Alfano et al., 1982) and thickness of the hippocampal cell layer (Louis-Ferdinand et al., 1978). In rats examined 35 days after lead exposure was stopped, Alfano et al. (1982) found some recovery in hippocampal dimensions, but there was still a significant reduction in size compared to controls never exposed to lead. Similarly, Kawamoto et al. (1984) found that the areas of the strata pyramidale, granulosum and moleculare, the dentate hilus and the CA3 apical dendritic region were reduced at 15 days, but compensatory growth restored most regions to normal size by 90 days. In hippocampal fine structure, Kiraly and Jones (1982) measured spine densities on the apical dendrites of CA 1 pyramidal cells, and found about 38~ fewer spines in lead-exposed animals. Campbell et al. (1982) found that lead reduced or delayed synaptogenesis in the suprapyramidal mossy fiber area. In the development of the dendritic fields of the granule cells of the dentate gyrus, lead exposure results in an increase in dendritic branching close to the cell body, but a decrease in the length of the dendritic field and the number of branches far from the cell body (Alfano and Petit, 1982). Both the timing of development of the dendritic tree and the source of the major synaptic inputs to the proximal and distal portions of the granule cell dendritic tree differ (Loy et al., 1977). Therefore, the differences in dendritic field size could be due to differential susceptibility to lead at various times during development, or differential susceptibility of the synapses formed, or both. The proximal inputs arise primarily in the medial septum and contralateral hippocampus and develop early, while the distal inputs from the entorhinal cortex, develop later. These data may therefore imply an overall slowdown in the development of the dendritic field, whereby the early (proximal) dendrites proliferate for a longer period of time and thus produce more branches, while the later (distal) branches are delayed in development and thus do not produce as many branches as usual before proliferation stops. Alternatively, there may be opposite actions of lead in modifying synapse formation in the two specific pathways involved. Similar morphological studies have been made of the rat cerebral cortex. The number of dendritic branches on the apical dendrites of pyramidal cells of the rat somatosensory LP.N. 24/~B

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cortex is unaffected close to the cell body, but is significantly reduced in more distal regions (Petit and Leboutillier, 1979), which is similar to the situation in the dentate gyrus (Alfano and Petit, 1982). In studies designed to count synapses in various cortical regions, lead exposure has been found to retard synaptogenesis (Averill and Needleman, 1980; Bull et al., 1983; McCauley et al., 1982) and, in some cases at least, result in a presumably permanent reduction in the number of synapses per unit volume (Averill and Needleman, 1980; Petit and LeBoutillier, 1979) or per neuron (Krigman et al., 1974). In other brain regions, McConnell and Berry (1979) found a 35~o reduction in the overall length of the dendritic field of cerebellar Purkinje cells as a result of lead exposure, although spine density was unaffected. A reduction in the density of synapses per neuron has also been found in the caudate nucleus of lead-exposed rats (Krigman et al., 1978). If we assume that reductions in dendrite length, dendritic branching and dendritic spine density all correspond to a reduction in the number of synapses likely to be found on a given neuron, then these studies agree that chronic inorganic lead exposure generally (1) delays the formation of synaptic connections during development and (2) often results in a permanent deficit in the number of synapses. Unfortunately, these data do not allow a direct evaluation of the efficacy of synaptic transmission. Electrophysiological measurements of the strength of individual synapses, although technically feasible in brain slice preparations, have not yet been done. The effects of chronic exposure to organic lead have been much less thoroughly studied. In general, organic lead (triethyl or tetramethyl) causes a decrease in the brain/body weight ratio (Ferris and Cragg, 1984). No significant changes occur in the fine structure of the cerebellum, dentate gyrus or Ammon's horn of rats chronically exposed to tetramethyl lead (Ferris and Cragg, 1984). However, chick embryo brain cells in culture show reduced outgrowth of neurites when exposed to low concentrations of triethyl lead (Ammitzboll et al., 1978). High (lethal) doses of tetraethyl lead (which is decomposed in the body to triethyl lead) also cause marked changes in neuronal ultrastructure in rabbit brains (Niklowitz, 1974). Comparisons both in vivo and in vitro of the effects of tetramethyl vs triethyl lead would be most instructive, particularly in the light of the significant differences in brain neurochemistry which are elicited by chronic neonatal exposure to trimethyl vs triethyl tin (Hanin et al., 1984). 2.2. AXONAL MORPHOLOGY AND MYELINATION Relatively high-level lead exposure in humans produces striking effects on the peripheral nervous system, resulting in loss of coordination and weakness of the extremities. Lower level exposure seems to cause a reduction in conduction velocity (see Section 3.1.1). Numerous morphological studies have sought a physical basis for these physiological deficits. Most of the experimental studies have used rodents of various sorts. In adult rodents, chronic exposure to inorganic lead causes segmental demyelination of individual axons (Coria et al., 1984; Dyck et al., 1980; Fullerton, 1966; Myers et al., 1980; Ohnishi and Dyck, 1981; Ohnishi et al., 1977; Powell et al., 1982; Windebank et al., 1980). Reductions in myelination of optic axons (Tennekon et al., 1979; Toews et al., 1980) and forebrain (Toews et al., 1980, 1983) may also occur, showing that myelin deficits are not limited to the peripheral nervous system. However, the effects of lead exposure on myelination appear to be greatly influenced by both location (central vs peripheral nervous system) and age of exposure (neonatal vs adult). In the adult rodent, the evidence indicates that demyelination of peripheral axons results from direct injury to Schwann cells by lead following long-term exposures usually for several months (Myers et al., 1980; Ohnishi and Dyck, 1981; Powell et al., 1982)~ Lead inclusions are found within Schwann cells, especially inside the nuclei (Myers et al., 1980; Powell et al., 1982). Further, both Schwann cell death and proliferation occur in lead-exposed animals (Powell et al., 1982). Finally, the number of lameUae of myelin per unit area of axon is reduced in lead-exposed animals (Ohnishi et al., 1977). These observations suggest, but do not yet prove, that lead enters Schwann cells and causes cell

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death, resulting in demyelination. Lead exposure may also promote proliferation of immature Schwann cells (PoweU et al., 1982), either because of the toxic effects of lead itself, or because proliferation is stimulated by the presence of demyelinated axons following the death of the mature Schwann cells. Thus a continual process of demyelination and remyelination may occur. As mature Schwann cells die, new ones migrate into the vacated region and begin to remyelinate the nerve. As these "replacement" Schwann cells mature, they too die, once again demyelinating the axon, setting the stage for the cycle to repeat. In neonatal rodents, the picture is quite different. Brashear et al. (1978) exposed rats to lead from days 2 to 20 of postnatal life, resulting in extremely high blood lead levels. Although some initial changes in Schwann cell morphology and in rate of myelin compaction occurred, these changes waned during the 20 days of exposure, and segmental demyelination of peripheral axons was not observed. Similarly, Toews et al. (1980) found no reduction in myelin content of the sciatic nerves of neonatally lead-exposed rats. In the central nervous system of neonatal lead-exposed rats, on the other hand, the total amount of myelin is greatly decreased (Tennekoon et al., 1979; Toews et al., 1980, 1983), indicating a diminished rate of myelination. However, the number of myelin lamellae per unit diameter of axon is not changed (Krigrnan et al., 1974; Tennekoon et al., 1979), suggesting that the glial cells are not adversely affected. In the CNS, lead exposure reduces the average axon diameter (Tennekoon et al., 1979). Since, in general, smaller axons have fewer myelin lamellae, a reduction in average axon diameter therefore leads to a lower overall myelin content. What causes the differences between neonatal vs adult and central vs peripheral nervous systems? Unfortunately, differences in methodology among the studies might contribute to the differences in results. For example, in the various studies animals were exposed to lead by gastric intubation, lead in the diet and lead in mothers' milk, lead levels varied and methods of assessing myelination ranged from biochemical measurements to counting lamellae around axons. These caveats aside, Brashear et al. (1978) suggest that the short duration of exposure in their study of neonatal rats (20 days), compared to the usually much longer durations of exposure in studies of adult rats (often several months, e.g. Coria et al., 1984; Ohnishi and Dyck, 1981; Ohnishi et al., 1977) might account for the failure of segmental demyelination to appear in the neonatal peripheral nervous system. However, comparably short exposures of adult rodents have been reported to result in both destruction of Schwann cells (about four weeks: Powell et al., 1982) and segmental demyelination (20 to 35 days: Windebank et al., 1980), indicating that duration of exposure is probably not the only factor involved. For the case of central vs peripheral nervous system effects, Toews et al. (1980) found no reduction in myelination in the sciatic nerve of neonatal rats that showed a 42~ reduction in myelin content of the forebrain. Further, although decreases in axon diameter have been noted in the central nervous system of neonatally lead-exposed rats (Tennekoon et al., 1979), no reduction in diameter occurs in peripheral axons (Brashear et al., 1978). This suggests that the central and peripheral nervous systems of neonatal animals respond very differently to chronic lead exposure. The growth of axons in the CNS is markedly affected, but the myelin-producing glia are not, while in the PNS both axonal growth and myelinating Schwann cells are relatively unaffected. With long-term exposure of adult rats, of course, the Schwann cells do become damaged. In rapidly myelinating cultures derived from mouse dorsal root ganglia, long-term exposure to 10 ]AMinorganic lead in vitro also leads to segmental demyelination (Whetsell, 1984). Axons remain intact during demyelination, suggesting, as with the in vivo experiments described above, that in peripheral nerves lead interferes with Schwann cells rather than the neurons themselves. Co-incubation with lead and high concentrations of heme prevents demyelination, indicating that the toxicity of lead on Schwann cells may be mediated through effects on heine biosynthesis (Sassa et al., 1979). Organic lead also affects myelination of axons, at least in the CNS. Konat and Clausen (1974) found that chronic exposure to triethyl lead causes a 429/o reduction in myelin

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content of the brains of neonatal rats, although the myelin composition appears normal (Konat and Clausen, 1978). Both in vivo and in vitro exposure to triethyl lead causes a reduction in synthesis of myelin proteins in forebrain (Konat et al., 1979) and in incorporation of these proteins into myelin (Konat and Clausen, 1980; Konat and Offner, 1982). Counting lamellae and measuring axon diameters, which suggest that inorganic lead acts to decrease myelination in the CNS by decreasing axon development, have not yet been carried out with organic lead. 3. Effects on Cellular Physiology

Lead may alter the cellular physiology of neurons in a wide variety of ways, including interference with, for example, ion movement through membrane channels, activation or inactivation of voltage-dependent ion channels, release or reception of neurotransmitters, or postsynaptic responses, whether mediated through ion channels or metabolic changes. Unfortunately, actual data are very scanty for virtually all of these processes, with the major exception of the release of neurotransmitters. The best-defined (though not always best-understood) effects are those on conduction velocity in axons, neuronal excitability and synaptic transmission. 3.1. CONDUCTIONVELOCITY 3.1.1. Peripheral nerves

In humans, it has long been known that high doses of lead damage the peripheral nervous system producing, in extreme cases, symptoms such as wrist drop and weakness in the extremities. Over the last two decades, numerous studies have been conducted to determine if lower level lead exposure, which does not produce any overt clinical symptoms of lead poisoning, nevertheless might impair the functioning of peripheral nerves (Araki and Honma, 1976; Ashby, 1980; Baloh et al., 1979; Buchthal and Behse, 1979; Catton et al., 1970; Feldman et al., 1977; Repko et al., 1978; Sepp~l~inen, 1984; Sepp~l/iinen and Hernberg, t972; Sepp~l~inen et al., 1975, 1979, 1983; Singer et al., 1983; Spivey et al., 1980; Triebig et aL, 1984; Verbek, 1976). Typically, conduction velocity measurements are taken from nerves of the arms (usually ulnar or median nerves) or legs (usually the peroneal nerves). The various studies attempted to measure maximal motor nerve conduction velocity, conduction velocity of the slower motor fibers (CVSF) and/or conduction velocity of sensory fibers. As is common with human toxicological studies of this type, it is difficult to compare the results of the different studies. First, the various research groups often measured conduction velocities in slightly different ways and on different nerves. Second, and potentially extremely serious, the "control" groups differ from study to study. Third, sample sizes (from 6 to 94 lead-exposed subjects), lead concentrations in the blood of lead-exposed subjects (means of about 40 to 80/~g/dl) and durations of exposure (49 days to over 30 years) vary widely. Finally, the statistical analyses used are often inappropriate. Most of the studies measured velocities, latencies and amplitudes of compound action potentials in several nerves. Specifics of statistical analyses are usually not given in the literature, but appear most often to be simple univariate comparisons, with significance for each comparison set at the 0.05 level. Multiple univariate comparisons, however, increase the probability of false positives occurring purely by chance (Harper, 1984; Muller et aL, 1984). When multiple comparisons are used, and only certain measurements are found to differ significantly between lead-exposed individuals vs unexposed controls, the interpretation of the results becomes difficult. To illustrate these points, we might compare the studies of Bordo et aL (1982) and Batoh et al. (1979). Both evaluated workers at secondary lead smelters. However, as controls, Bordo et al. (1982) used maintenance workers at a hospital, while Baloh et al. (1979; Spivey et al., 1980) used men working at an aluminum processing plant. Interestingly, Bordo et al. (1982) found that nerve conduction velocity was slower in lead workers than in hospital

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LEAD EFFECTSON NEURONALPHYSIOLOGY TABLE 1. EFFECTSOF CHRONICLEAD EXPOSUREON NERVECONDUCTIONVELOCITY IN HUMANS* Ref. Araki and Honma, 1976 Ashby, 1980 Baloh et al., 1979 Bordo et al., 1982 Buchthal and Behse, 1979 Feldman et al., 1977 Repko et al., 1978t Sepp~iliiinen and Hernberg, 1972 Seppiiliiinen et al., 1975, 1979, 1983 Singer et al., 1983 Spivey et al., 1980 Triebig et al., 1984

Median

Ulnar

Peroneal

Tibial

slow slow NS slow slow NS slow slow slow slow NS no

NS slow no NS NS NS slow slow slow NS no slow

NS slow no no slow slow slow slow slow no no NS

slow NS NS NS NS NS slow NS slow NS NS NS

* Sensory, motor or mixed nerve conduction velocities grouped together in the table. In studies which examined several parameters of conduction velocity in a single nerve, the designation "slow" was given if any of the parameters showed statistically significant slowing. Statistical significance at the 0.05 level, without regard for the appropriateness of the statistical procedures used. NS, not studied; no, no significant slowing. t From secondary sources (Purser et al., 1983; Singer et al., 1983).

workers, while Baloh et al. (1979) found no difference in conduction velocity between lead and aluminum workers. At least two strikingly different hypotheses might be proposed to explain these results. First, perhaps lead does not diminish conduction velocity, but certain other characteristics of the workplace do. If this were true, this might explain why Baloh et al. (1979) found no differences between aluminum workers and lead workers, who work in fairly similar environments (except for the metal involved), while the lead workers studied by Bordo et al. (1982) showed reduced conduction velocity compared to hospital workers, who perhaps work in a more benign environment. The second hypothesis would be that lead does indeed reduce conduction velocity, but so might other substances, such as aluminum. The different conclusions of the two studies might also be due to measurement of different nerves. Bordo et al. (1982) measured maximal motor conduction velocities in the median and peroneal nerves; lead-correlated slowing was found in the median but not peroneal nerve. Baloh et al. (1979) measured maximal motor conduction velocities in ulnar and peroneal nerves, and found slowing in neither nerve. In peroneal nerve velocity, both studies agree. Perhaps, had both groups measured the same nerves, their other results would also agree. Both studies also made multiple measurements and appear to have used multiple univariate comparisons to analyze the statistical significance of the results. If we ignore such differences in procedure, in examining a large number of studies we find widespread, but not universal, agreement that lead exposure is correlated with slower conduction velocity, at least in certain nerves (Table 1). The degree of slowing is small, with velocities usually only a few ~o below those of the control population. Interestingly, changes in conduction velocity of smaller motor fibers (CVSF) vary widely among studies. Seppfilfiinen et al. (1975, 1979) found a very significant slowing of CVSF, while Repko et al. (1978) and Verbek (1976) actually found a (statistically insignificant) increase in CVSF (see below). Few comparable experimental studies in animals have been done. Perhaps the most relevant are those of Hopkins (1970) and Purser et al. (1983). Hopkins repeatedly injected baboons with lead carbonate and followed the animals for up to a year. The dose was high enough that most animals showed severe weight loss and epileptic episodes. Most died during the study. Despite this evidence of massive lead poisoning, neither motor nor sensory conduction velocity was significantly diminished, even in baboons exposed to lead for nearly a year. Purser et al. (1983) fed cynomolgus monkeys oral doses of lead acetate daily for nine months, and found only minor, statistically insignificant decreases in certain measures of maximal motor nerve conduction velocity and a substantial, significant

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increase in CVSF (see below). These primate studies agree that lead-induced changes in nerve conduction velocity, if they occur at all, are small. In comparing non-human primates with human workers, one must conclude that (1) there are large species differences, (2) very long exposures to lead are needed to produce notable effects, and/or (3) lead causes only minimal changes in conduction velocity, at least of larger axons. An interesting situation exists with regard to conduction velocity of slower (and smaller) fibers (CVSF). Of necessity, CVSF must be measured in a rather indirect way, and it is difficult to interpret the CVSF data both in humans and monkeys. Essentially, measurements of CVSF record the conduction velocity of whatever fibers in the nerve are conducting the most slowly. In the case of lead-exposed monkeys, Purser et al. (1983) concluded that their CVSF measurements prior to lead exposure were taken from a population of very small fibers that stopped functioning entirely as lead exposure progressed. After several weeks, the slowest remaining axons would be measured for CVSF. These fibers were probably larger and faster-conducting than those initially measured, and would yield an apparent increase in CVSF. A similar situation might explain the discrepant results in humans. Some studies might be measuring impaired but not yet nonfunctional conduction in the very smallest fibers in the nerves, and thus record a reduced CVSF, while in other studies these smallest axons might have become nonfunctional, leaving the CVSF measurements to be taken from other, larger axons, yielding an increased CVSF. Thus far, there are no firm physiological or morphological defects known which would account for a slower conduction velocity in the peripheral nerves of primates. In rodent studies, lead exposure has often been found to induce demyelination of peripheral nerves (see Section 2.2). Histological studies of nerves in humans exposed to relatively low levels of lead seem to be rare. In one analysis, Buchthal and Behse (1979) found no overt segmental demyelination. A slight increase in fibers with paranodal remyelination was noted, as were rare instances of reduced axonal diameter. They concluded that the reduction in conduction velocity was probably due to changes in the properties of the axonal membrane itself, rather than to myelin defects. Purser et al. (1983) performed histological examinations of the nerves of their lead-exposed monkeys. Segmental demyelination was absent, but some minor axon and myelin degeneration was observed. Two explanations for the combined physiological and histological data may be offered. First, as Buchthal and Behse (1979) suggest, low level lead exposure may cause changes in the axon membrane itself, thereby reducing conduction velocity. Direct proof of this hypothesis would be quite difficult to obtain in mammals, in which the cable properties of myelinated axons are a combination of the properties of both the axon membrane itself and the myelin sheath. Although the phylogenetic distances are great, such experiments would be quite simple to perform on, for example, the giant (unmyelinated) axons of crayfish. Voltage clamp analysis of the voltage dependence and kinetics of electrically excitable ion channels might also offer insights into possible membrane alterations that could decrease conduction velocity. If, for example, the threshold voltage for opening sodium channels was increased (made more positive), then the conduction velocity would be expected to decrease even if the cable properties of the axon remained unchanged. Clamp analysis of ion channels is possible using vertebrate axons, usually using sucrose gap techniques (for example, Arhem (1980) analyzed the effects of acute in vitro application of several heavy metals, but not lead, on sodium and potassium currents in Xenopus myelinated axons), vertebrate neuron cell bodies in slice preparations or in culture (one- or two-electrode conventional clamps), or one of several convenient invertebrate preparations. The second explanation for decreased conduction velocity, suggested by Purser et al. (1983), is that the minor changes in myelin and axon structure which they found in lead-exposed monkeys might be exactly the sort of subtle change expected in instances in which the physiological effects on conduction velocity are themselves small. This also would be difficult to prove experimentally. While there are chemical agents which can be used to induce demyelination, it probably would be essentially impossible to ascertain that

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the extent and nature of the myelin damage caused by an experimental drug would mimic that seen in chronically lead-exposed animals. 3.1.2. Central nerves Few studies of conduction velocity in central nerves have been performed. However, morphological data (Tennekoon et al., 1979; Toews et al., 1980, 1983) show that lead exposure during development impairs myelination in the central nervous system of rats, including the optic nerve, probably due to lead-induced decreases in the diameters of the axons. Impelman et al. (1982) found that chronic lead exposure of neonatal rats via mother's milk (the same route of administration but only 40~ the concentration used in the morphological study of Tennekoon et al., 1979) causes decreases in the conduction velocity of the optic nerve. The mechanism is likely to be the decreased diameter and consequently decreased myelination of axons in the nerve. 3.2. NEURONAL EXCITABILITY Strictly speaking, "neuronal excitability" refers to (1) the incremental voltage to reach threshold for excitation of action potentials when a standard stimulus, preferably intracellularly injected electrical current, is applied to a neuron (a reflection of the absolute value of the resting potential and the voltage dependence of inward current-carrying channels, principally sodium and/or calcium), (2) the amount of depolarization evoked by a standard stimulus (a reflection of the input resistance of the neuron) or (3) the spike frequency elicited by a standard stimulus (a reflection of all these processes and usually others as well, such as sodium or calcium channel inactivation, potassium "A" currents or adaptation currents). However, the literature on the neuronal effects of lead exposure uses the term "excitability" rather more broadly (e.g. Bjrrklund et al., 1983b; Fox et al., 1979; Otto and Reiter, 1984). Therefore, for the purposes of this review, I will also consider "excitability" to refer to (1) the level of "spontaneous activity" of a neuron (i.e. activity not evoked by an experimenter-controlled stimulus) and (2) the magnitude of response of neurons or populations of neurons, often recorded extracellularly from entire regions of the brain, which have been stimulated via polysynaptic pathways (e.g. firing of cortical neurons in response to a flash of light). 3.2.1. Spontaneous activity Palmer et al. (1981, 1984; see also Bjrrklund et al., 1983b) have analyzed the spontaneous activity of Purkinje cells of rat cerebella which were chronically exposed to low levels of lead. Pieces of developing cerebellum were removed from fetal rats and transplanted to the anterior chamber of the eye of adult rats. Some of the host rats were given lead in their drinking water, starting one week before the transplant and continuing for two months. Recording from Purkinje cells from both host and transplanted cerebella occurred four to five months after cessation of lead treatment. In animals never exposed to lead, both host and transplant Purkinje cells fire spontaneously, at an average rate of about 25 to 35 spikes/sec. In lead-exposed animals, the host Purkinje cells fire normally, but Purkinje cells in the transplant usually fail to fire at all unless stimulated. Similar, although less dramatic, results were obtained from Purkinje cells of neonatal rats injected with 8 mg/kg of inorganic lead for the first 20 days of postnatal life (Bjrrklund et al., 1983a). While relatively few cells become completely silent after lead exposure, the proportion of cells with fast firing rates is greatly decreased. Injection with 1 mg/kg of lead produced no effect on Purkinje cell firing rate. Unfortunately, at present there are no data demonstrating a physiological mechanism for the reduced spontaneous activity. Obvious candidate mechanisms are (1) reduced intrinsic excitability (e.g. more negative resting potential, increased threshold for voltagedependent sodium and/or calcium channels or decreased input resistance); (2) decreased

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"spontaneous" excitation (e.g. decreased presynaptic release of excitatory transmitters or decreased postsynaptic reception of, or response to, the transmitter) or (3) increased "spontaneous" inhibition (e.g. increased release, reception or response to inhibitory transmitters). The latter two mechanisms, of course, need not even occur at the neuron in question, but might occur one or more synapses "upstream". Spontaneous activity in cerebellar Purkinje neurons occurs prior to synaptogenesis (Woodward et al., 1969), and bathing cerebellar tissue cultures with high magnesium to block chemical synapses does not block spontaneous activity. These findings indicate that spontaneous activity is an intrinsic property of the Purkinje cells. However, the discharge rate can be slowed by noradrenergic input (Hoffer et al., 1971). Acute lead exposure has been shown to block the inhibition of Purkinje cells by iontophoretically applied norepinephrine (Taylor et al., 1978), while in the iris, chronic lead exposure induces a hyperinnervation of the catecholaminergic ground plexus (Bj6rklund et al., 1981). Palmer et al. (1984) combine these findings to generate the following hypothesis: Chronic lead exposure, like acute lead exposure, interferes with Purkinje cells' reception of, or response to, norepinephrine. This effect is temporary, but in the developing cerebellum it induces hyperinnervation of the noradrenergic fibers innervating the Purkinje cells. As a result, after prolonged exposure to lead, Purkinje cells suffer from excessive inhibitory noradrenergic input, thereby reducing spontaneous firing. Attractive as this hypothesis is, there is no direct evidence in its support, and other changes, particularly in intrinsic membrane properties of the Purkinje cells themselves, remain equally viable hypotheses. Audesirk and Audesirk (1983) exposed pond snails, Lymnaea stagnalis, to low levels of lead in vivo for 6 to 12 weeks, and recorded intracellularly from six distinct, wellcharacterized identifiable neurons or neuron types in vitro in isolated brains bathed in lead-free Ringer's. They found that chronic lead exposure during development decreases spontaneous activity in all six neuron types tested. Again, these experiments could not distinguish between the three possibilities of decreased intrinsic activity or excitability, decreased synaptic excitation and increased synaptic inhibition. However, direct stimulation with electrical current via an intracellular electrode showed that some, but not all, cell types are intrinsically less excitable. In experiments using acute exposure to lead via iontophoresis from a micropipette, Rozear et al. (1971) found that lead depresses spontaneous activity in neurons of both the cerebral cortex and brainstem of cats. Of a series of heavy metals tested, lead had the weakest depressant effect, although severe problems with clogging of lead nitrate-filled electrodes made quantitative comparisons uncertain. No mechanism for the suppression of spontaneous activity was demonstrated for any metal. 3.2.2. Excitability in response to stimulation A variety of studies in several different organisms indicate that lead exposure, both chronic and acute, may reduce the responsiveness of neurons to external stimulation. I will discuss these in sequence from the simplest and most unambiguous experiments to the more complex procedures which are open to several interpretations. Electrical stimulation of the optic nerve of neonatal rats chronically exposed to inorganic lead reveals that the chronaxie is about doubled, suggesting a great decrease in intrinsic excitability (Impelman et al., 1982). Audesirk and Audesirk (1983) assayed excitability in six neuron types of chronically lead-exposed pond snails, L. stagnalis. Excitability was measured as the frequency of spiking in response to intracellular injection of depolarizing current, with spontaneous activity subtracted out. Certain neuronal types show markedly reduced excitability following lead exposure, while other types show no effect at all. In general, resting potentials are more negative in lead-exposed neurons and input resistances are decreased, both of which would tend to reduce excitability. However, all neuron types from lead-exposed animals have hyperpolarized resting potentials and decreased input resistance, even those which show no reduction in excitability, and neither resting potential

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nor input resistance correlate with excitability. The mechanism for reduced excitability therefore remains unknown, although voltage clamp studies should be able to determine if lead exposure alters any of a number of membrane channels. Loop and Cooper (1974) reported that excitability of cat spinal motoneurons is decreased by acute injection of lead into the spinal cord. Using stimulation of the dorsal roots to evoke the monosynaptic spinal reflex, they recorded intracellularly from the motoneurons and found that the amplitude of the excitatory postsynaptic potential does not change, while the threshold for spike firing increases. Concomitantly, the motoneuron antidromic spike amplitude decreases, input resistance increases and the resting potential becomes more negative. They suggest that lead decreases the sodium conductance of the motoneuron, but provide no concrete evidence for this proposal. Voltage clamp studies could be profitably pursued in this preparation. Parenthetically, it is curious that the synaptic potential was unaltered by lead, since lead blocks transmitter release from presynaptic terminals (see Section 3.3.1). Perhaps the increased input resistance in lead enhanced the voltage response to the postsynaptic current. The current would probably be decreased because of diminished output of transmitter from the presynaptic cell, and the two effects may have been nearly balanced, giving no net effect on the size of the postsynaptic potential. In the bullfrog retina acutely perfused with 5 to 50 #M lead chloride, Fox and Sillman (1979) found that the rod ERG amplitude is suppressed, while the cone ERG amplitude is unaffected. Threshold sensitivity to light is decreased by 0.7 log units in lead-exposed rods. However, it is not known at what stage lead interferes in photoreception and generation of the rod receptor potential. In rats, although chronic lead exposure diminishes the response of both rods and cones, rods are more severely affected (Fox and Farber, 1983). Cyclic GMP levels in rat retina are increased by chronic lead exposure, presumably due to a simultaneous decrease in V ~ for cGMP phosphodiesterase and increase in guanylate cyclase activity. Since cGMP is primarily associated with rods rather than cones, these biochemical data support the electrophysiological evidence for a preferential effect of lead on rods. Nevertheless, it is not known if these biochemical changes cause the electrophysiological effects. In the mammalian central nervous system, most studies of neuronal excitability have measured extracellularly recorded responses of various parts of the brain to either electrical or psychophysical stimulation. These studies, of necessity, provide only indirect evidence about intrinsic neuronal excitability, since the magnitude and sign of synaptic interactions and conduction velocities of pathways, to mention only two possibilities, can greatly influence the speed, duration and amplitude of such responses. Nevertheless, several studies report results that could be caused by altered excitability of neurons. McCarren and Eccles (1983) found that chronic lead exposure causes increases in the duration of electrically elicited afterdischarge in the rat hippocampus. However, control rats show an increase in afterdischarge following kindling (repeated stimulation), while lead-exposed rats do not (McCarren et al., 1984), Fox and co-workers observed that lead exposure in neonatal rats causes permanent increases in the latency of the visual evoked response (Fox et al., 1977) and increased excitability of the visual cortex as measured by the amplitude of the evoked response to the second of a pair of closely timed light flashes (Fox et al., 1979). In humans, the amplitude and latencies of both visual and auditory evoked responses have been reported to be negatively correlated with the concentration of lead in hair (Thatcher et al., 1984). An age dependence of lead effects on cortical slow wave potentials has been suggested by Otto and colleagues (Otto et al., 1981; Otto and Reiter, 1984). Younger children (below the age of five) with elevated blood lead levels tend to have more negative slow wave potentials than low-lead controls, while older lead-exposed children have less negative slow waves. Otto and Reiter (1984) speculate that these data indicate different effects of lead on excitability depending on age, so that lead increases CNS excitability in younger children, but decreases excitability later on. As in the other studies described above, many causes might be invoked to explain the observed lead effects,

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including alterations in intrinsic excitability, synaptic excitation, synaptic inhibition and conduction velocity in central pathways, each of which might change with age and vary among cell types.

3.3. SYNAPTIC TRANSMISSION 3.3.1. Chemical synapses Transmission at chemical synapses normally depends upon the successful execution of a number of steps, including synthesis of neurotransmitter; packaging of transmitter into synaptic vesicles; depolarization of the presynaptic terminal, usually via action potentials; depolarization-evoked opening of calcium channels in the presynaptic membrane; influx of calcium ions into the terminal; interaction between calcium and subcellular molecules to promote fusion of vesicles with presynaptic membrane, resulting in release of transmitter into the synaptic cleft; binding of transmitter to receptor molecules on the postsynaptic membrane, resulting in opening of ion channels and subsequent flow of ions across the postsynaptic membrane and/or activation of specific postsynaptic metabolic processes (e.g. adenylate cyclase). In principle, lead exposure might interfere with any or all of these functions. Scores of studies have focussed on effects of lead on neurotransmitter synthesis, turnover and uptake. Since these have been extensively reviewed (Shellenberger, 1984; Silbergeld and Hruska, 1980; Winder and Kitchen, 1984) and, as of yet, provide no conclusive physiological mechanisms for alteration of neuronal function, they will not be discussed here. I have already outlined some of the evidence suggesting that lead alters the excitability of neurons and the conduction of action potentials along axons, both of which might block the initial steps in transmission. In this section, I will concentrate on the opening of calcium channels and the movement of calcium ions across the presynaptic membrane, changes in intracellular calcium concentrations, possible direct effects of lead on transmitter release and the effects of lead on transmitter binding to receptors and associated receptor-mediated functions. 3.3.1.1. Stimulus-evoked presynaptic release of transmitter From a physiological point of view, probably the most thoroughly studied and best-understood effect of lead on neurons is the alteration of transmitter release caused by acute exposure to low levels of inorganic lead in vitro, especially at neuromuscular junctions. These experiments have been reviewed by Cooper and his colleagues in recent years (Cooper and Manalis, 1983; Cooper et al., 1984a, b) and the reader should consult one or more of these excellent reviews for further details. In 1957, Kostial and Vouk measured the amplitude of the nictitating membrane contraction in the cat in response to stimulation of the preganglionic nerve of the superior cervical ganglion. They found that acute perfusion with as little as 4.8 #M lead diminished the force of contraction. Collection of acetylcholine from the perfused ganglion revealed a decreased release of transmitter caused by lead at concentrations as low as 2.4#M. Finally, addition of extra calcium to the lead-containing perfusion medium largely restored both the nictitating membrane response and the release of acetylcholine. Control experiments indicated that lead did not block muscle contraction, axonal conduction or the response of the muscle to exogenous acetylcholine. These results suggest that lead blocks the influx of calcium which is a prerequisite to release of transmitter from presynaptic terminals. Subsequently, Cooper and colleagues have confirmed and extended these findings at the cholinergic synapses of the frog neuromuscular junction (Cooper et al., 1984a, b; Manalis and Cooper, 1973; Manalis et al., 1984), the bullfrog sympathetic ganglion (Kober and Cooper, 1976) and the adrenergic synapse of the rabbit saphenous artery (Cooper and Steinberg, 1977). Essentially identical results have been obtained at mammalian neuromuscular junctions by Atchison and Narahashi (1984) and Pickett and Bornstein (I984). The following discussion will be based largely on findings at neuromuscular junctions.

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Acute exposure of either frog or rat neuromuscular junctions to inorganic lead, in concentrations as low as 0.5 #M (frog) to 1 #M (rat), results in a reduced amplitude of the evoked end plate potential (EPP) recorded in the muscle. The direct response of the muscle to exogenously applied acetylcholine is only minimally affected, even by much higher concentrations of lead, indicating that the action of lead is presynaptic, reducing the amount of transmitter released per action potential in the presynaptic axon. Under the vesicular hypothesis of chemical transmission, the amount of transmitter released by each action potential is a function of both the number of transmitter molecules per vesicle and the number of vesicles released. At least during the initial stages of lead perfusion, and at low concentrations, lead does not affect the amplitude of spontaneously occurring miniature EPPs (e.g. Atchison and Narahashi, 1984; Manalis et al., 1984; Pickett and Bornstein, 1984). Therefore, lead probably does not decrease the amount of transmitter stored in and released from each vesicle. Quantal analysis in both frog and rat preparations agree that lead reduces the mean quantal content (m). The lead-induced transmission block can be relieved by elevating the calcium concentration in the bathing medium, with kinetics suggesting competitive inhibition between lead and calcium (Kober and Cooper, 1976; Manalis et al., 1984; Pickett and Bornstein, 1984). Finally, lead exposure reduces the influx of radioactive calcium in electrically stimulated frog sympathetic ganglia (Kober and Cooper, 1976) and potassium-stimulated rat brain synaptosomes (Nachsen, 1984; Suszkiw et al., 1984). The most likely interpretation of these data is as follows. Action potentials invading the presynaptic nerve terminal open voltage-dependent calcium channels, which then allow an influx of calcium into the terminal down its concentration gradient. The increased intracellular free calcium ion concentration, [Ca 2÷]i, promotes binding of synaptic vesicles to the active zone of the presynaptic membrane, where they fuse with the membrane and release their transmitter molecules into the synaptic cleft. Acute lead exposure probably exerts its main action by blocking the voltage-dependent calcium channels, reducing calcium entry during the presynaptic depolarization, thereby diminishing the increase in [Ca2÷]i, which in turn restricts vesicle fusion and transmitter release. It is worth noting that although most of these data were obtained at peripheral synapses, generally supportive results have been found at the frog sympathetic ganglion (Kober and Cooper, 1976) and in rat brain synaptosomes (Nachsen, 1984; Suszkiw et al., 1984). Acute lead exposure also blocks inward calcium currents in snail neurons (personal observation). Many other heavy metals, including cadmium, cobalt and lanthanum, are also known to inhibit calcium channels in a variety of preparations, ranging from rat brain synaptosomes to snail neurons (for reviews of calcium channels in neurons, see Edwards, 1982; Hagiwara and Byerly, 1981). Therefore it seems likely that acute lead exposure blocks the influx of calcium through all calcium channels. Since transmitter release at both peripheral and central synapses seems to be calcium-dependent (Salzberg et al., 1983), extracellular lead probably blocks synaptic transmission in fundamentally similar ways at all synapses. Comparable data are generally not available on the effects of chronic lead exposure on transmitter release in vivo. Using slices or minces of brain tissue from chronically exposed rats, investigators have reported decreased potassium-stimulated release of acetylcholine (Carroll et al., 1977) and gamma-butyric acid (Silbergeld et al., 1979, 1980); no change in release of norepinephrine (Kant et al., 1984); and increased (Silbergeld, 1977; Kant et al., 1984), decreased (Wince et al., 1980) and unaltered (Kant et al., 1984) release of dopamine, depending, among other things, on the brain region sampled. These experiments, of course, measure at least two possible lead effects, namely alterations in transmitter available for release (e.g. by changes in uptake, synthesis, breakdown or packaging of transmitter) and alterations in (presumably calcium-mediated) exocytosis in response to depolarization. A recent study by Govoni et al. (1984) supports the hypothesis that chronic lead exposure in vivo blocks calcium channels in presynaptic terminals. These investigators assayed the binding of nitrendipine to calcium channels of synaptosomes prepared from the brains of rats never exposed to lead or exposed chronically in vivo, and synaptosomes exposed acutely in vitro. Both acute and chronic lead exposure enhanced nitrendipine

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binding, an effect that was lost when the synaptosomes were washed with an EDTA/EGTA mixture. These data suggest that both chronic and acute exposure to lead results in lead binding to the outer surface of presynaptic membranes, at or near the calcium channels. At this site, lead presumably acts as a calcium agonist in some respects (promoting the binding of nitrendipine) and an antagonist in other respects (blocking calcium influx through the channels). More direct studies on the effects of lead exposure in vivo will be necessary to determine if this hypothesis is correct. These data do not, of course, rule out other sites of action of lead on transmitter release from nerve terminals. For example, Atchison and Narahashi (1984) found that acute lead exposure appears to reduce the pool of readily available transmitter at the rat diaphragm and suggest that lead may act at intracellular sites to bring about this effect. 3.3.1.2. Spontaneous release o f transmitter Presynaptic terminals sporadically release transmitters even in the absence of stimulation, resulting in miniature post-synaptic potentials. Although lead blocks stimulationevoked transmitter release, it increases the spontaneous release of transmitter, as measured by an increased frequency of miniature potentials (Atchison and Narahashi, 1984; Kolton and Yaari, 1982; Manalis and Cooper, 1973; Manalis et al., 1984; Pickett and Bornstein, 1984). This enhancement of spontaneous release by lead is inhibited by the simultaneous presence of cadmium, another calcium channel blocker (Cooper and Manalis, 1984). These findings have been interpreted to mean that, during acute exposure to lead, lead ions enter the terminal by passing through the voltage-dependent calcium channels. Once inside the terminal, lead increases the frequency of miniature postsynaptic potentials. Addition of cadmium blocks the calcium channels, reducing the influx of lead through the channels, which thereby reduces the buildup of lead inside the terminal and prevents lead-induced enhancement of spontaneous release (Cooper and Manalis, 1984). It should be empha, sized, however, that lead influx into presynaptic terminals, through calcium channels or otherwise, is probably small and has not been directly observed. Spontaneous transmitter release, under normal conditions, is thought to be determined by the concentration of free calcium ions within the presynaptic terminal: high concentrations increase the frequency of miniatures, while low concentrations reduce the frequency. Among several possibilities, two likely hypotheses which might explain the effectiveness of lead in increasing spontaneous release are (1) lead substitutes for calcium in promoting vesicle--plasma membrane fusion and (2) lead interferes with calcium regulation within the terminal, resulting in an increased concentration of calcium. There is no direct evidence supporting the first hypothesis. However, lead is known to be able to substitute for calcium in activating calmodulin (Chao et al., 1984; Goldstein and Ar, 1983; Habermann et al., 1983; but see also Cox and Harrison, 1983) and there is suggestive evidence that calmodulin may play a role in the release of neurotransmitters (DeLorenzo, 1981; DeLorenzo et al., 1979; Fujisawa et al., 1984). Thus, lead entering the presynaptic terminal might increase the spontaneous release of neurotransmitters by activating calmodulin. Somewhat more information is available to support the second hypothesis. Ionized calcium within nerve cells is maintained at a very low level through uptake and sequestering by various organelles, including mitochondria, synaptic vesicles and endoplasmic reticulum (see Section 4.1.2). Interruption of calcium uptake by mitochondria with inhibitors either of mitochondrial respiration or calcium transport, presumably causing an increase in intraterminal free calcium ions, results in an increase in spontaneous transmitter release (Alnaes and Rahamimoff, 1975). Although the data are somewhat ambiguous, lead may impair the ability of mitochondria to take up and store calcium. This would probably cause [Ca2+]i to increase, which would result in increased spontaneous release of transmitter. There are at least three potential difficulties with this hypothesis, however. First, the evidence that lead impairs net calcium uptake by organelles (uptake minus release) is inconclusive, suggesting that a lead-induced increase in [Ca 2+ ]i may not occur. Second, in the frog neuromuscular junction, Manalis et al. (1984) found that 50 #M lead

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caused an order of magnitude increase in the frequency of miniature potentials within a minute, even though a complete solution change in their experimental chamber took about the same length of time. A similar time course was found by Kolton and Yaari (1982) for 20 # i lead. This would mean that within less than 60 see lead entered the terminal, reached a high enough concentration to inhibit calcium uptake by organelles, and prevented enough calcium sequestration to raise [Ca 2+ ]i to a level sufficient to cause a 10-fold increase in spontaneous release. Kinetic studies are not available to support or disprove this scenario. Third, lead may well have other effects which would promote decreases in calcium concentration. Intracellular calcium is regulated not only by uptake, storage and release by organelles, but also by extrusion from the cell by Na+-Ca 2+ exchange pumps and a calcium-specific ATPase. The calcium-specific ATPase is activated by calmodulin (Carafoli, 1984; Vincenzi and Hinds, 1982). Although this has not been directly tested, lead activation of calmodulin may well activate this calcium pump, which would lower [Ca 2+]i. Obviously, it would be advantageous to make direct tests of these hypotheses, particularly (1) does lead enter the presynaptic terminal, and with what time course? (2) does [Ca 2+ ]i increase or decrease after lead exposure, by how much, and with what time course? (3) do similar changes in calcium concentrations occur if lead is injected directly into the presynaptic terminal through a microelectrode? For a variety of reasons, these experiments are technically very difficult. Radioactive lead tracer experiments, exposing neurons to lead for precisely controlled times during stimulation, might be able to answer the first question. The second might be approached using calcium-sensitive dyes (e.g. aequorin or arsenazo) or microelectrodes in particularly advantageous preparations, such as the squid giant synapse or neuronal cell bodies with calcium currents, such as most gastropod and some mammalian neurons. These experiments might be compromised by lead interference with the dyes or electrodes. Although lead-filled microelectrodes have a tendency to clog (Rozear et al., 1971), the third experiment might also be attempted in the squid giant synapse or in neuronal cell bodies. 3.3.1.3. Lead effects on synaptic receptors and receptor actions Postsynaptic responses to neurotransmitters consist of several components. Minimally, (1) the neurotransmitter binds to specific receptors on the postsynaptic membrane, (2) it either changes the conductance state of associated membrane ion channels or activates intracellular metabolic processes, or both and (3) it is inactivated in some way, usually enzymatically, by uptake into pre- and/or postsynaptic terminals, or by diffusion. There seem to be no studies in which the influence of lead exposure on all three events has been studied simultaneously. At the neuromuscular junction, relatively low levels of lead (less than 100 # u ) appear to have only minimal effects on the amplitude and time course of the potential developed in the muscle in response to iontophoretic application of acetylcholine in the immediate vicinity of the end plate (Atchison and Narahashi, 1984; Manalis and Cooper, 1973; Manalis et al., 1984). This implies that there has been little or no change in the binding of acetylcholine to its receptors, opening of postsynaptic ion channels, the flux of ions through the channels or hydrolysis of acetylcholine. However, none of these events has been directly examined. It is possible, although probably not very likely, that lead in fact has multiple actions that are mutually compensatory. A few somewhat more indirect experiments also found that acute lead exposure causes no significant changes in postsynaptic responses. In the bullfrog sympathetic ganglion, the amplitude of the postganglionic compound action potential in response to bath application of acetylcholine is essentially unaffected by lead (Kober and Cooper, 1976). The muscle contractions evoked by acetylcholine applied to the cat superior cervical ganglion (Kostial and Vouk, 1957) and norepinephrine applied to the rabbit saphenous artery (Cooper and Steinberg, 1977) are also unchanged by lead exposure. The spontaneous firing rate of Purkinje neurons in rat cerebellum is inhibited by iontophoretic application of norepinephrine (Hoffer et al., 1971), an action that may be mediated through activation of adenylate cyclase (Nathanson et al., 1976). This inhibition

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is blocked by acute application of inorganic lead at low concentrations (Taylor et al., 1978). In the same preparation, excitation by acetylcholine is unaffected by lead. Since inorganic lead has been shown to block adenylate cyclase activity (Lemay and Jarrett, 1975; Nathanson and Bloom, 1976), it is possible that the suppression of the inhibitory effect of norepinephrine is due to inhibition of adenylate cyclase. However, the ultimate electrophysiological response to norepinephrine involves a complex cAMP-initiated cascade, and lead may exert its inhibitory effect at one or more further steps in the cascade (Taylor et al., 1978). Wince et al. (1980) also reported that chronic lead exposure in vivo suppresses dopamine receptor-mediated adenylate cyclase in homogenates of neostriatum, further suggesting that lead exerts postsynaptic effects in cAMP-mediated systems. Acute in vitro exposure to several organic lead compounds, in concentrations ranging from about 5 to 50 # i , inhibits both basal and dopamine-sensitive adenylate cyclase in homogenates of rat caudate nucleus (Wilson, 1982). Kinetic analysis indicates that, for the dopamine-sensitive adenylate cyclase, organic lead is a competitive inhibitor of dopamine, with a K~ value of less than 10 # i for two different organolead compounds. This in turn suggests that organic lead is a dopamine antagonist at D1 dopamine receptors. There have been several studies of the effects of acute lead exposure on binding of transmitter agonists or antagonists to receptors in various brain tissues. Bondy and Agrawal (1980) examined high-affinity binding of various agents to membranes prepared from rat brain. In concentrations of 5 to 10/~i, tributyl lead in vitro strongly inhibits binding of spiroperidol (a specific binding agent for dopamine receptors) and quinuclidinyl benzilate (muscarinic acetylcholine receptors). At these concentrations, inorganic lead has only minimal inhibitory effects. Binding of muscimol (GABA receptors), strychnine (glycine receptors) and diazepam (benzodiazepine receptors) is only minimally affected by either organic or inorganic lead. Similarly, Aronstam et al. (1978) found that inorganic lead inhibits quinuclidinyl benzilate binding to muscarinic receptors only at very high concentrations (>1 mi), and Costa and Fox (1983) found no inhibition of quinuclidinyl benzilate binding by inorganic lead in concentrations ranging from 1 nM to 100 ~M. Lead exposure in vivo has been reported to alter the density of muscarinic receptors from rat cortex. Moingeon et al. (1984) injected rats with lead acetate and observed a transitory increase in the density of muscarinic receptors in both cortex and striatum, without any change in receptor affinity. Receptor density returned to baseline within 24 hr, even though lead concentrations in brain tissue remained high and constant. Costa and Fox (1983; Fox et al., 1982) examined the effects of long-term lead exposure of neonatal rats, and observed a significant decrease in muscarinic receptor density in the visual cortex but not in retina, superior colliculus, striatum, hippocampus, frontal cortex or lateral geniculus. No effect was found on receptor affinity. It is difficult to reach any general conclusions concerning postsynaptic lead effects, since there is a general paucity of studies and the procedures of exposure and analysis are so different. The data suggest, but do not prove, the following hypotheses. Acute exposure to low concentrations of inorganic lead in vitro has only minimal effects on the binding of neurotransmitters to their postsynaptic receptors (Aronstam et al., 1978; Atchison and Narahashi, 1984; Bondy and Agrawal, 1980; Cooper and Steinberg, 1977; Costa and Fox, 1983; Kober and Cooper, 1976; Kostial and Vouk, 1957; Manalis and Cooper, 1973; Manalis et al., 1984). Acute exposure to organic lead compounds, on the other hand, alters binding to some, but not all, types of postsynaptic receptors. Similarly, acute in vitro exposure to inorganic lead probably does not affect the opening of postsynaptic ion channels or the flow of ions through these channels (Atchison and Narahashi, 1984; Cooper and Sternberg, 1977; Kober and Cooper, 1976; Kostial and Vouk, 1957; Manalis and Cooper, 1973; Manalis et al., 1984). In contrast, both organic and inorganic lead, in vitro and in vivo, inhibit adenylate cyclase (Lemay and Jarrett, 1975; Nathanson and Bloom, 1976; Wilson, 1982; Wince et al., 1980), at least in some preparations, and so lead should inhibit postsynaptic responses based on cAMP cascades (Taylor et al., 1978; Wince et al., 1980). In vivo lead exposure may alter the concentrations of some receptors (Costa and Fox, 1983; Moingeon et al., 1984), but much more data are needed regarding which

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types of receptors are affected, which brain regions are susceptible and the time course of the changes. 3.3.1.4. Termination o f transmitter action A number of studies have assayed the effect of acute in vitro and chronic in vivo lead exposure on acetylcholinesterase concentrations and on the uptake of various transmitters, particularly dopamine. These studies have been extensively reviewed recently (Shellenberger, 1984; Silbergeld and Hruska, 1980; Winder, 1982; Winder and Kitchen, 1984) and the reader should consult these reviews for details. Briefly, several studies have found that chronic lead exposure in neonatal rodents appears to cause decreased acetylcholinesterase activity in some, but not all, brain regions (Carroll et al., 1977; Costa and Fox, 1983; Louis-Ferdinand et al., 1978), although other studies have reported no change (Hrdina et al., 1976). Acute exposure to inorganic lead in vitro has no effect (Costa and Fox, 1983), suggesting that the chronic effect must not be due to lead interference with enzyme activity per se, but rather with enzyme synthesis or breakdown. Although the precise cellular localization of acetylcholinesterase activity assayed is not clear, the simplest interpretation would suggest that the action of acetylcholine would be prolonged at selected central synapses, due to a lack of acetylcholinesterase. The data on high-affinity uptake of neurotransmitters including, presumably, re-uptake mechanisms which help to terminate synaptic action, are quite conflicting and confusing. The interested reader is referred to the comprehensive reviews by Shellenberger (1984) and Winder and Kitchen (1984) which compare and contrast the findings of numerous studies. At present, it is probably fair to say that no conclusion can be confidently drawn about the effects of either acute or chronic lead exposure on transmitter uptake, at reasonably low lead concentrations. 3.3.2. Electrical synapses Various types of neurons in the mammalian brain are known to be electrically coupled, presumably through gap junctions (see, for example, Andrew et al., 1981; MacVicar and Dudek, 1981, 1982). Electrical synapses in the brains of mammals are, however, difficult to study electrophysiologically. Audesirk and Audesirk (1984), using identifiable neurons in the snail L y m n a e a stagnalis, found that chronic lead exposure greatly decreases junctional resistance. The two most likely hypotheses to explain this finding are that lead exposure leads to (1) synthesis of additional gap junctional channels or (2) opening of pre-existing, but normally blocked, channels. Although no data are available on the long-term regulation of gap junctional channels in neurons, it has been recently demonstrated in cell culture that cAMP can initiate the development of electrical coupling between sympathetic neurons isolated from the superior cervical gangion of neonatal rats (Kessler et al., 1984). Acute in vitro experiments have suggested that gap junctional permeability is regulated by intracellular levels of various substances, including H ÷ (deCarvalho et al., 1984; DeMello, 1980; Giaume et al., 1980; Reber and Weingart, 1982; Spray et al., 1981, 1982; Turin and Warner, 1980), Ca 2÷ (Dahl and Isenberg, 1980; DeMello, 1975, 1984; Jacob, 1983; Peracchia, 1978; Rose and Loewenstein, 1976; Rose and Rick, 1978), cAMP (Duffey et al., 1981; Flagg-Newton et al., 1981) and calmodulin (Peracchia, 1984; Peracchia and Bernardini, 1984; Peracchia et al., 1983; Welsh et al., 1981, 1982). Lead has been shown to interact with various systems regulating calcium concentration, adenyl cyclase activity and calmodulin activity, but effects on intraceUular pH have not been investigated. Possible lead-induced changes in any or all of these intracellular agents might underlie the effect of chronic lead exposure on electrical coupling. 4. Subcellular Mechanisms

The effects of lead exposure on neuronal structure and physiology discussed in the previous sections must, of course, be secondary to effects on subcellular processes, several

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of which have been mentioned in passing. In this section, I will consider several subcellular processes which might be affected by lead exposure, and the corresponding aspects of neuronal physiology which they are most likely to influence. At the molecular level, most of the effects attributed to lead exposure can probably be explained by one of two general mechanisms. First, lead might interact with any process which is normally influenced by calcium ions. Depending on the system, lead may either act as a calcium antagonist (probably most common) or as an agonist. Second, lead has the potential to interfere with protein function, probably by interacting with sulfhydryl groups. This might cause interference both with specifically neuronal proteins (e.g. neurotransmitter receptors, enzymes involved in neurotransmitter synthesis) and with more prevalent enzymes which are also crucial to neuronal functioning (e.g. Na P-K ÷ ATPase, mitochondrial respiratory enzymes). 4.1. LEAD AND CALCIUM INTERACTIONS The functioning of neurons, and indeed of many cells in the animal body, is critically dependent upon the intracellular concentration of free calcium ions, [Ca2÷]i. This is particularly true of local concentrations in presynaptic terminals and near the membranes of neurons with voltage-dependent calcium channels and/or calcium-activated potassium channels. Lead exposure can exert two different direct effects on [Ca2+]i: (1) interference with transient calcium fluxes through calcium channels in the plasma membrane, and (2) interference with "steady-state" calcium homeostasis, thereby altering the "resting" [C a2+ ]i. Besides changing [Ca 2÷ ]i, lead may also (3) directly substitute for, or antagonize, processes which are normally activated by calcium. 4.1.1. L e a d effects on calcium channels

Extracellular application of many, perhaps most, heavy metals inhibits the movement of calcium ions through voltage-dependent calcium channels, perhaps by interfering with an active recognition site on the outside of the membrane (for reviews see Edwards, 1982; Hagiwara and Byerly, 1981). In this regard, lead is probably one of the most potent of the heavy metals. However, it should be noted that interference with calcium currents by lead has thus far been inferred from indirect experiments, either by a reduction in postsynaptic potentials (Atchison and Narahashi, 1984; Manalis and Cooper, 1973; Manalis et al., 1984; Pickett and Bornstein, 1984) at neuromuscular junctions, or by a reduction in radioactive calcium uptake by synaptosomes depolarized with high potassium solutions (Nachsen, 1984; Suszkiw et al., 1984). Direct quantification of calcium fluxes, whether by measurement of inward calcium currents under voltage damp, with calciumsensitive microelectrodes or with calcium-sensitive dyes, has not been undertaken in lead-exposed neurons. Nachsen (1984), measuring uptake of radioactive calcium into rat brain synaptosomes depolarized with high potassium concentrations, found that the half-inhibition concentration for extracellular inorganic lead is about 1 #M. Assuming that this experiment accurately reflects reductions in calcium currents, then non-trivial interference with these currents might be expected to occur in animals with even extremely low levels of lead exposure. Such inhibition of inward calcium currents would reduce the transient increases in [Ca 2+ ]i that are the proximate causes of calcium-dependent action potentials (Edwards, 1982; Hagiwara and Byerly, 1981), calcium-activated potassium currents (Adams et al., 1982; Alger and Nicoll, 1980; Eckert and Tillotson, 1978; Meech, 1972; Meech and Standen, 1975), calcium-dependent chloride currents (Owen et al.. 1984) and neurotransmitter release. Govoni et al. (1984) measured binding of nitrendipine to calcium channels of synaptosomes prepared from rat brains and found that binding was inhibited both by chronic in vivo and acute in vitro lead exposure. Interestingly, washing the synaptosomes with a mixture of EGTA and EDTA removed the inhibition in both cases, suggesting that the

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chronic lead effect is similar to the acute effect, namely interference with an active site on the outside of the cell membrane. 4.1.2. Lead effects on calcium homeostasis

To a first approximation, animal cells contain two pools of calcium. Probably by far the major portion of intracellular calcium is sequestered within organelles, including mitochondria. Although the calcium in this pool may have major consequences for cellular respiration and other functions it does not immediately affect neuronal processes, such as activation of calmodulin, activation of calcium-dependent potassium and chloride channels, inactivation of voltage-dependent calcium channels or release of neurotransmitter, either spontaneous or evoked. The second, smaller, intracellular calcium pool, which does affect these neuronal processes, consists of free ionized calcium. 4.1.2.1. Calcium in organelles

In all animal cells, [Ca2+ ]i is kept extremely low (usually less than 1/AM) by a variety of mechanisms. Mitochondria, endoplasmic reticulum and synaptic vesicles can all acquire calcium by active transport (see, for example, Blaustein et al., 1978; Chan et al., 1984; Israel et al., 1980; Nicholls and Crompton, 1980; Nicholls and Scott, 1980; Trotta and DeMeis, 1975; for reviews of mitochondrial calcium regulation, see Nicholls, 1981; Fiskum and Lehninger, 1982). These organelles probably provide a buffer against changes in [Ca2+]i. Mitochondria, at least, also have calcium release mechanisms (Nicholls, 1981; Fiskum and Lehninger, 1982). In addition, calcium is extruded from cells via at least two transport systems, Na÷-Ca 2÷ exchange (Baker, 1972) and a calcium-specific ATPase (Carafoli, 1984; Vincenzi and Hinds, 1982). Lead has the potential to interfere with all of these processes. In vitro exposure to inorganic lead, in concentrations at least as low as 5 #M, inhibits uptake of radioactive calcium both by isolated mitochondria and mitochondria within cells in tissue slices (Goldstein, 1977; Kapoor and van Rossum, 1984; Parr and Harris, 1976). Studies of mitochondrial release of calcium have been contradictory. In measurements of release of radioactive calcium from preloaded, isolated mitochondria, one study found decreased or unchanged calcium efflux, depending on lead concentration (Silbergeld and Adler, 1978), while another found increased calcium release (Kapoor and van Rossum, 1984). Unfortunately, these uptake and release studies do not necessarily reflect the true net fluxes of calcium across the mitochondrial membrane, because movements of non-radioactive calcium cannot be assessed. Calcium concentrations have been measured in mitochondria of isolated synaptosomes (Silbergeld and Adler, 1978) and of whole hepatocytes (Pounds et al., 1982). In both cases, the mitochondrial calcium content increases following exposure to lead in vitro. These results can only occur if calcium uptake exceeds release during lead exposure, a finding that would not be predicted by the uptake and release studies reported above. Further, the calcium concentration of whole cells apparently increases when the cells are exposed to lead in vitro (Piccinini et al., 1977; Rosen, 1983). (Note that these experiments measured total calcium content, not [Ca2+]i. ) Since mitochondria and other organelles probably contain most of the calcium in a cell, decreased mitochondrial calcium uptake, especially if coupled with increased release, would be expected to result in a decreased whole-cell calcium concentration as well, unless there were a tremendous increase in [Ca 2÷ ]i. Thus these experiments lend further support to the conclusion that in vitro lead exposure increases net calcium uptake by mitochondria. For a thorough discussion of the possible explanations underlying the seemingly contradictory results of the uptake/release studies and calcium content studies, the reader is referred to a recent review by Pounds (1984). To my knowledge, the effects of lead exposure on calcium extrusion by the plasma membrane of nerve cells, either in vivo or in vitro, have not been specifically studied. There are, however, four suggestive findings in this regard. First, in kidney cells, lead has been found to be only an extremely weak inhibitor of calcium-activated ATPase, even at 100/AM J.P.N 24/~-C

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concentration (Thompson and Nechay, 1981). Second, Goldstein et al. (1977), studying capillaries isolated from rat brain, found that exposure to 100/~M lead tripled the uptake of radioactive calcium by endothelial cells. ATP-dependent uptake (transport) of radioactive calcium by membranes prepared from endothelial cells was abolished by 10 ~ ~ lead. Lead did not affect uptake of radioactive rubidium (a potassium mimic) by intact endothelial cells. The authors hypothesized that lead probably does not alter general membrane permeability and that the enhanced net uptake of calcium by intact cells is probably the result of the elimination of an ATP-dependent calcium efflux mechanism. Interference with a Na+-Ca 2+ exchange cannot, however, be ruled out. Third, brain slices from rats exposed to lead in vivo show no change in uptake, but increased retention, of radioactive calcium (Kim et al., 1980). The authors concluded that in vivo lead exposure probably inhibits calcium efflux. Finally, the calcium-specific ATPase has been shown to be stimulated by calmodulin, at least in red blood cells (Carafoli, 1984). Although the effects of lead on this particular calmodulin-sensitive process have not been studied, lead has been shown to substitute for calcium in activating calmodulin in, for example, the stimulation of phosphodiesterase (Chao et al., 1983; Goldstein and Ar, 1983; Habermann et al., 1983). If lead-activated calmodulin stimulates the calcium-specific ATPase, then this would be expected to increase the efflux of calcium. In summary, the total calcium content of cells or mitochondria, which represents the large, relatively inactive pool of calcium, is probably increased by lead exposure. The mechanism for this increase is not yet clear. 4.1.2.2. Intracellular f r e e calcium ion concentration There are, unfortunately, no direct data on the effects of lead exposure on [Ca2+]i. Of some bearing on this question, however, is the finding that lead exposure in vitro increases the spontaneous release of transmitter at neuromuscular junctions (Atchison and Narahashi, 1984; Manalis and Cooper, 1973; Manalis et al., 1984; Pickett and Bornstein, 1984). Treatments that should increase [Ca 2+ ]i, such as inhibition of mitochondrial respiration or calcium uptake (Alnaes and Rahamimoff, 1975), increase spontaneous transmitter release and therefore one explanation for the lead-induced increase in release would be that lead increases [Ca 2+ ]~. However, the usual reasoning given for this conclusion is that lead enters the presynaptic terminal and interferes with calcium uptake and/or sequestration by organelles, particularly mitochondria, a hypothesis that must be considered at least debatable (see above). If we assume that lead does enter the neuron, then another hypothesis that could explain the increase in spontaneous release would be that lead exerts a direct effect on release processes. Several lines of evidence suggest that calmodulin might mediate the role of calcium in regulating transmitter release (for a review see DeLorenzo, 1981). Since lead can activate calmodulin, at least as measured by stimulation of phosphodiesterase activity and a few other processes (Cheung, 1984), intracellular lead might directly enhance spontaneous release, independent of any effects on [Ca 2+ ]~. [Ca2+]~ can be measured using calcium-sensitive microelectrodes or calcium-sensitive dyes (see Blinks et al., 1982). In theory, one should be able to use these technologies to see if lead exposure in vitro or in vivo alters [Ca 2+ ]i. Pitfalls in the process, especially as far as nerve cells are concerned, include the large size of most ion-sensitive microelectrodes and possible interference with the measurement techniques by intracellular lead. Nevertheless, the attempt should be made to measure [Ca 2+]i, at least in favorable preparations such as some invertebrate neurons, to resolve this question. 4.1.3. Direct effects o f lead ions on calcium-mediated processes

Research on calcium-lead interactions has thus far tended to focus on the first two categories listed above. However, it has been recently reported that lead can substitute for calcium in activation of calmodulin, and, in fact, lead is slightly more effective than calcium (Habermann et al., 1983; Cheung, 1984). Calmodulin is involved in many cellular activities, including synthesis and hydrolysis of cAMP and cGMP, calcium pumps and both

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phosphorylation and dephosphorylation of many proteins (Cheung, 1984). This raises the possibility that lead exposure could permanently "turn on" many calmodulin-dependent activities, freeing them of their normal regulation by [Ca 2÷]i, ultimately to the detriment of the cell. This hypothesis might explain many of lead's toxic effects, for example the increase in spontaneous transmitter release mentioned above, and clearly merits extensive further study. Lead might also act as a calcium agonist in cellular activities that are not modulated by calmodulin. To present just two possibilities, let us consider the inactivation of voltage-dependent calcium channels and the activation of calcium-dependent potassium channels. Both of these processes are normally controlled by [Ca2÷]i (Adams et al., 1982; Alger and Nicoll, 1980; Eckert and Ewald, 1981, 1982, 1983; Eckert and Tillotson, 1978; Meech, 1972; Meech and Standen, 1975). There have, to date been no experiments to test the effects of chronic or acute lead exposure on these activities, either excitatory (agonist for calcium) or inhibitory (antagonist). If lead acted as a calcium agonist in these cases, then chronic lead exposure, at least, would be expected to activate the calcium-dependent potassium channels, perhaps permanently (but see Eckert and Ewald, 1982), resulting in a maintained hyperpolarization of the cells. Such an increased negativity of the resting potential has, in fact, been observed in chronically lead-exposed snail neurons (Audesirk and Audesirk, 1983) and acutely lead-exposed cat spinal motor neurons (Loop and Cooper, 1974). The causes of the hyperpolarizations, however, remain unknown. Simultaneously, an agonist action of lead would inactivate voltage-dependent calcium channels, causing a sustained reduction in calcium currents during depolarization. This has not yet been tested. In both cases, of course, similar effects might occur if lead increases [Ca2÷]i.

4.2. LEAD--PROTEININTERACTIONS 4.2.1. Neurotransmitter receptors The possible effects of lead on postsynaptic receptors have been summarized above (see Section 3.3.1.3). Briefly, in vitro experiments have tended to find significant effects on agonist or antagonist binding to receptors only with organic lead compounds (e.g. tributyl lead) or with inorganic lead at very high concentrations. It is not clear how relevant these studies might be to the effects of lead in vivo. It is possible that chronic in vivo exposure may alter the synthesis or breakdown of certain receptors, particularly muscarinic acetylcholine receptors (Costa and Fox, 1983), leading to changes in receptor density in at least some brain areas, but much more data are needed about chronic lead effects on all types of receptors. 4.2.2. Lead effects on enzyme activity Lead and several other heavy metals are thought to inhibit the activity of many enzymes by binding to sulfhydryl groups. Although this mechanism has not been conclusively proven, various studies have found enzyme inhibition by lead. Lead exposure both in vitro (Nathanson and Bloom, 1976; Wilson, 1982) and in vivo (Wince et al., 1980) has been reported to inhibit catecholamine-activated adenylate cyclase activity. However, calcium-activated calmodulin can potentiate adenylate cyclase activity, including dopamine-stimulated adenylate cyclase, at least in some preparations (Gnegy and Treisman, 1981; Gnegy et al., 1984). The adenylate cyclase assays which showed inhibition by lead did not specifically control for calmodulin effects. Since lead can substitute for calcium in activation of calmodulin, it is possible that quite a different result would have been obtained had the reaction allowed for calmodulin activity. In vitro lead also simulates phosphodiesterase, probably through activation of calmodulin (Nathanson and Bloom, 1976; Chao et al., 1983; Cheung, 1984; Goldstein and Ar, 1983; Habermann et al., 1983). Thus the net effect of lead on cAMP metabolism in any given neuronal system cannot be predicted a priori.

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In t,itro lead also inhibits Na+-K " ATPase in microsomes or tissue homogenales (Nechay and Saunders, 1978; Siegel and Fogt, 1977) from several tissues in a variety of vertebrates. Such an inhibition would, if sustained, result in a breakdown of the sodium and potassium concentration gradients across the neural membrane. Chronic exposure in lead workers causes reduced Na+-K + ATPase activity in membranes from red blood cells (Hasan et al., 1967b; Raghavan et al., 1981) and enhanced efflux of potassium (Hasan et al., 1967a). Interestingly, no such effect of chronic or acute lead exposure has been reported in neurons of humans or experimental animals; in fact, when measured, the resting potential seems to be more, not less, negative, and the action potential amplitude seems to be relatively unaffected (Audesirk and Audesirk, 1983; Loop and Cooper, 1974). Lead is a potent inhibitor of the enzymes involved in energy metabolism, including glucose breakdown (Sterling et al., 1982) and mitochondrial respiration (Holtzman et al., 1978a, b, 1980). This would not only be expected to reduce energy-requiring calcium transport into or out of mitochondria but would also affect any energy-requiring process within the cell, including active transport across the plasma membrane, synthesis of many molecules and growth. Indeed, Holtzman et al. (1984) suggest that inhibition of mitochondrial respiration is important in the development of lead encephalopathy in neonatal rats. Finally, lead may well inhibit enzymes involved in the synthesis and breakdown of neurotransmitters. Even if we were to assume that in all cases lead either inhibits such enzymes or has no effect, the effects of chronic lead exposure in vivo may be very complex. Enzyme-regulating interactions among substrates, transmitters and metabolites might well translate into either increases or decreases in enzyme synthesis and steady-state levels, and hence in overall enzyme activity, for each enzyme in any given pathway. Relatively few studies have examined enzyme activity following chronic lead exposure. These results are summarized in the recent review by Schellenberger (1984).

5. Perspective and Future Directions It is clear from the above discussion that, despite fairly intensive investigation over the past 20 years, we know remarkably little about the many possible effects of lead on neuronal physiology, and still less about the mechanisms of action. The major exception to this generalization is the effect of acute lead exposure on chemical synaptic transmission, in which the elegant studies of Cooper and his associates, and more recently Atchison and Narahashi (1984) and Pickett and Bornstein (1984), have given us a fairly clear picture. The following section will present some ideas about future directions which research into the neuronal effects of lead, or indeed any heavy metal and many organic neurotoxins as well, might profitably take. The reader should note that this is written from the admittedly biased perspective of the cellular ncurophysiologist. 5. I. IN VITRO VS IN VIVO EXPOSURE

From a toxicological viewpoint, the important mechanisms to discover are those that operate in vivo during chronic lead exposure. In particular, the challenge at hand is to determine the mechanisms of action of low levels of lead, from the relatively low levels found in workers in lead-related industries or in inner-city children exposed to lead-based paint, to the very low levels in the general population, which nevertheless probably exceed preindustrial levels by orders of magnitude (Settle and Patterson, 1980). 5.1.1. Standardization o f in vivo exposure in m a m m a l s

For obvious reasons, the organisms of choice in studying chronic in vivo exposure have thus far been the laboratory rat and, to a lesser extent, the mouse. However, differences in mode of exposure (maternal milk, water, food, injection, intubation), durations of exposure and developmental stage have no doubt contributed greatly to the conflicting

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data which have been generated, particularly with respect to biochemical changes (Shellenberger, 1984). Given the difficulties in finding out exactly what lead does to the brain, it would be most desirable to standardize exposure parameters, thereby removing an aggravating source of variance among laboratories. Departures from a standard protocol probably should only be done in conjunction with, and compared to, simultaneous experiments on animals run through the standard procedure. Lead levels should be chosen so that they do not cause malnutrition, which is a confounding factor in many in vivo experiments. This does not necessarily mean using lead exposures which would produce blood levels in the rat which are similar to those in humans, because rats seem to be much less sensitive to lead effects than are humans (Petit and Alfano, 1983). Nevertheless, appropriate doses would be those that minimize non-specific effects. 5.1.2. In vitro experiments Finding out the mechanisms of action of lead on neurons in some cases requires experiments involving in vitro exposure. Many in vitro experiments have been regarded, and rightly so, as pilot studies to see if lead, often in relatively massive quantities, has any effect at all on a given neuronal function. However, this time is probably past. In vitro concentrations exceeding 20 #M, and indeed probably 5 tol0/~M, are unlikely to have much bearing on in vivo lead effects--the animals would be dead long before these levels were attained either within cells or in the extracellular fluid. Parallel experiments run on in vivo and in vitro exposed tissue, such as those of Govoni et al. (1984), are particularly valuable, since they are most likely to directly illuminate the mechanisms of in vivo lead actions and to assess the validity of in vitro models. 5.2. CELLULARPHYSIOLOGICALTECHNIQUES For some reason, few cellular electrophysiologists seem to have become interested in questions of neurotoxicology. This is unfortunate since the mechanisms of action of lead, both in vitro and in vivo, can in many instances be most profitably studied with the techniques of the electrophysiologist. To take one example, a number of exquisite experiments have sought to test the effectiveness of lead and other heavy metals in blocking voltage-dependent calcium channels. These studies, however, have been almost entirely indirect, either measuring amplitudes of postsynaptic potentials at neuromuscular junctions (Atchison and Narahashi, 1984; Manalis and Cooper, 1973; Manalis et al., 1984; Pickett and Bornstein, 1984), measuring uptake of radioactive calcium in synaptosomes (Nachsen, 1984; Suszkiw et al., 1984) or measuring binding of calcium channel antagonists to synaptosomes (Govoni et al., 1984). All of these methods have their drawbacks, particularly in that several processes may intervene between calcium influx and measured parameter, or may occur simultaneously and influence the measurement. The direct measurement of calcium currents through several different types of voltage-dependent calcium channels under voltage clamp, or the estimation of changes in [Ca2+]i by using calcium-sensitive dyes such as aequorin or arsenazo have been standard features of experiments on gastropod neurons for several years (see for example, Ahmed and Connor, 1979; Byerly and Moody, 1984; Byerly et al., 1984; Eckert and Ewald, 1981, 1983; Gorman and Thomas, 1980; Gorman et al., 1984) and more recently in certain vertebrate neurons as well. With the suitable choice of experimental preparations, these measurements could be made on neurons exposed to lead both in vivo and in vitro. Application of these techniques could rapidly advance our knowledge of the mechanisms whereby lead affects neuronal physiology. 5.2.1. Model systems Favorable preparations for the study of lead effects, both in vivo and in vitro, should be used. The classic example of this is the neuromuscular junction of frogs and rats as a model for central synapses. For in vivo exposure suitable models, in addition to the rodent,

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might include (1) crayfish, which have giant axons eminently suited to the study of membrane biophysical parameters under voltage clamp, of conduction velocity and changes that might affect conduction velocity, and electrical synapses and (2) various freshwater snails, which have giant neurons suitable for biophysical studies and biochemical analysis of neurotransmitters within single neurons, electrical synapses between identifiable neurons and known monosynaptic (chemical) connections between identifiable neurons. Slice preparations from rat brains, especially hippocampus, have been extensively used in recent years to investigate cellular neurophysiological events in single neurons and these should find widespread use in the neurotoxicological community. For in vitro experiments both rat brain slices and certain invertebrate preparations, including the crayfish and freshwater snail, could prove particularly advantageous. If the problems of lead solubility could be worked out through the use of suitable anions, preparations from marine animals would be even more favorable, including the squid giant axon and giant synapse (the only preparation in which events in both the presynaptic and postsynaptic terminals can be recorded) and the huge, well-characterized neurons of several opisthobranchs, especially Aplysia californ&a. 5.3. INORGANICVS ORGANIC LEAD AND OTHER HEAVY METALS By far the majority of studies on lead, both in vitro and in vivo, have used inorganic lead, usually as the acetate, chloride, nitrate or carbonate salts. Organic lead compounds have been tested in a relative handful of experiments. For in vitro studies, the effects of organic and inorganic lead can be very different (see for example, Bondy and Agrawal, 1980; Bondy et al., 1979a, b). In particular, organic lead is lipid-soluble and can easily carry lead into the interior of cells, whereas inorganic lead is much less permeable, with only small amounts entering the cell, probably through calcium channels. (It is worth noting here that these conclusions, although reasonable, seem to be untested. To my knowledge, the concentrations of neither the intracellular free lead ion nor organic lead compounds have actually been measured following in vitro exposure.) The effects of in vivo exposure to inorganic and organic lead compounds have recently been reviewed by Walsh and Tilson (1984) who suggest that, in mammals, both have the limbic system as their primary, though not exclusive, site of action. Exactly parallel studies of in vivo exposure to organic vs inorganic lead, in the same laboratory, are rare. Not surprisingly, it is therefore impossible to precisely compare their relative toxicities and specific effects. However, at least some behavioral effects, as such as impaired learning, seem similar. A major difficulty in evaluating the similarities and differences between organic and inorganic lead following in vivo exposure is that, in general, no one knows in what molecular forms lead ultimately resides in cells of the nervous system or other tissues, or even in extracellular fluid. Inside cells, some inorganic lead is bound in precipitates of various sorts, including phosphates, within organelles; some is bound to membranes, including the plasma membrane; some, probably a very small amount, is likely to be in ionized form within the cytoplasm and some is soluble but bound to proteins or other soluble cytoplasmic constituents (Mittelstaedt and Pounds, 1984; Ong and Lee, 1980; Sabbioni and Marafante, 1976; Walton, 1973). The soluble lead may very well resemble "organic lead" in being mostly bound to organic molecules. If so, then organic lead might be the most appropriate form to use for in vitro studies which are attempting to duplicate the effects of in vivo lead exposure. Until the molecular form of lead within cells and extracellular fluid is determined, in vitro studies might, in general, profit from running parallel experiments with organic and inorganic lead, as Bondy and coworkers have done (Bondy and Agrawal, 1980; Bondy et al., 1979a, b).

Acknowledgements I would like to thank Dr. Teresa Audesirk for a careful reading of this manuscript and Drs G. P. Cooper, D. A. Fox, M. R. Krigrnan and J. G. Pounds for their generosity in

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