Neurotoxicants and Synaptic Function: Session VII-B Summary and Research Needs

NeuroToxicology 25 (2004) 515–519

Neurotoxicants and Synaptic Function: Session VII-B Summary and Research Needs William D. Atchison* Department of Pharmacology/Toxicology, Michigan State University, Life Science Bldg., B-331, East Lansing, MI 48824-1317, USA Available online 14 January 2004

Keywords: Session summary; Neurotoxicants; Synaptic functions

SESSION SUMMARY Chemical synaptic transmission is the fundamental process by which information is transferred between cells in the nervous system. It is a cellular process by which an electrical impulse in the sending cell is converted to a chemical signal and then in the receiving cell the chemical signal is reconverted back to an electrical response. Each of these processes, in turn, is remarkably complex, and comprised of numerous distinct steps involving proteins at the sending and receiving side with multiple sites for modulation. Synaptic transmission is critical to learning and memory as well as growth and differentiation in the nervous system. It is also a surprisingly ‘‘plastic’’ function which can be modified in response to changes in activity in the brain. Synaptic transmission is very sensitive to the actions of a number of environmental chemicals which can affect the process on either the sending (presynaptic), or receiving (postsynaptic) ends of the process or at multiple sites. Some of these chemicals such as lead have been proposed to alter learning and memory perhaps by actions on aspects of synaptic function. Others such as methylmercury and ethyl alcohol clearly affect neuronal development by actions which too may involve impaired synaptic transmission. Talks in this session focused on the variety of actions which environmental neurotoxicants including therapeutic drugs, ethyl alcohol and two neurotoxic *

Tel.: þ1-517-353-4947; fax: þ1-517-432-1341. E-mail address: [email protected] (W.D. Atchison). 0161-813X/$ – see front matter # 2003 Published by Elsevier Inc. doi:10.1016/j.neuro.2003.11.002

metals—lead and mercury—have on synaptic function. The talks ranged from the whole animal level of organization down to the single cell, or subcellular processes involving specific ion channel proteins. Presynaptic Disruption of Transmitter Release by Pb—an ‘‘Illegal Substitution’’ Janusz B. Suszkiw Since the pioneering work of Manalis and Cooper (1973), the ability of Pb2þ, and subsequently other polyvalent cations, to block synaptic transmission in the peripheral nervous system has been well known. Low concentrations of inorganic Pb2þ ions have the capacity to disrupt physiological transmitter release by causing aberrant augmentation of spontaneous and suppression of the nerve-evoked release of neurotransmiter. Dr. Jan Suszkiw described recent results from his (Shao and Suszkiw, 1991; Tomsig and Suszkiw, 1993) and other labs directed at examining the ability of Pb2þ to interact with the normally-Ca2þ-dependent secretory process. These studies are beginning to yield new insights into the molecular basis of disruptive effects of Pb2þ on neurotransmitter release. These effects are thought to result from high affinity substitutions of Pb2þ for Ca2þ in the Ca2þ-signaling proteins that subserve the synaptic vesicle mobilization, docking, and exocytosis processes. Based on the potential target proteins identified to date for Pb2þ, it has been suggested that augmentation of spontaneous release of neurotransmitter may involve intracellular actions either to increase

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vesicle mobilization subsequent to Pb2þ activation of Ca2þ-sensitive, calmodulin-dependent protein kinase II (CaMKII)-dependent phosphorylation (Ferguson et al., 2000; Kern et al., 2000), possibly of synapsin I, and/or through direct binding of Pb2þ to, and subsequent activation of synaptotagmin, the putative exocytotic Ca2þ-trigger protein. This latter idea is based on the demonstration in vitro of high affinity interactions of Pb2þ with synaptotagmin I (Bouton et al., 2001; Suszkiw, unpublished observation). Further characterization of Pb2þ-synaptotagmin interactions in relation to transmitter release is an important area for future studies. In addition, synergistic activation of phospholipase C (PLC) and diacylglycerol/Pb2þ-dependent activation of protein kinase C (PKC) described in vitro (Sun et al., 1999; Tian et al., 2000) may enhance the secretagogue effects of Pb2þ by increasing metal sensitivity of exocytosis following phosphorylation of synaptotagmin, and/or phosphorylation and activation of L-type Ca2þ channels. In contrast to the intracellularly-mediated actions of Pb2þ to augment spontaneous release, inhibition of evoked release of transmitter by Pb2þ is principally attributable to extracellular block of the voltagegated Ca2þ channels. Recent expression studies (Peng et al., 2002) of Pb2þ interactions with discrete Ca2þ channel subtypes are now beginning to yield detailed information about interactions of Pb2þ with specific subtypes of voltage-gated Ca2þ channels. Demonstration of intracellular actions of Pb2þ enhancing the activity of L-type Ca2þ channels in adrenal chromaffin cells (Sun and Suszkiw, 1995) implies that in addition to extracellular block, Pb2þ can modulate channel activity by intracellular mechanisms, presumably involving PKC. Further characterization of this effect as well as assessment of intracellular interactions of Pb2þ with other channel types (e.g., N- and P/Q-) that are coupled to the exocytotic machinery in presynaptic terminals remains an important area for future research. However, it is possible that even in the absence of significant inhibition of Ca2þ channel function, evoked release of neurotransmitter may be depressed in Pb2þ-intoxicated cells as a result of failure of the depolarization-evoked Ca2þ currents to activate synaptotagmin once it is bound to Pb2þ. Studies of Pb2þ interactions with other proteins of the exocytotic complex are also needed to characterize fully the molecular targets of Pb2þ in presynaptic nerve terminals. Finally, the role of high affinity interactions of Pb2þ with PKC/PLC and calmodulin (Ferguson et al., 2000; Kern et al., 2000) studied in vitro, should be characterized further with respect to regulation of synaptic vesicle mobilization, docking, and fusion processes in intact cells.

Significance of Neuronal Nicotinic Acetylcholine Receptors in Drug Actions: Alcohol Modulation Toshio Narahashi, Yi Zuo, Gary L. Aistrup, William Marszalec, Keiichi Nagata, Jay Z. Yeh Dr. Narahashi provided an in-depth perspective of how the action of a commonly used chemical, in this case ethyl alcohol (ethanol), can be dissected at the level of single neurotransmitter receptor-channel proteins. In contrast to early studies suggesting that ethanol, acts to disrupt fluidity of the plasma membrane, it is now clear that a major target of alcohol are the neurotransmitter-activated receptor/channel proteins associated with synaptic function. Among various receptors/channels that have been studied, the a4b2 type neuronal nicotinic acetylcholine receptor (nAChR) has been shown to be particularly sensitive to ethanol. At concentrations of 10 mM and above, concentrations easily achieved in modest levels of alcohol intoxication, ethanol greatly potentiates ACh-induced currents in rat cortical neurons in primary culture. Expression studies of cloned nAChRs expressed in human embryonic kidney (HEK) cells were used to compare, alcohols with various carbon chain lengths (Cx). Markedly different effects were seen with shortchain alcohols (C1–C3), and long-chain alcohols (C5–C12). These effects were exerted at different sites on the nAChR and by different mechanisms. Studies of single-channels revealed that fundamental properties of the channel such as open time, burst duration and probability of opening were affected by ethanol, resulting in an increased flow of current through the ion channel activated by the nAChR. Because nAChRs are located on both the presynaptic terminals (sending side) as well as in the postsynaptic membrane (receiving side) and have a relatively high permeability to Ca2þ, activation of nAChRs can cause release of transmitters such as GABA and dopamine. This effect is thought to play a pivotal role in the behavioral changes caused by ethanol intoxication.

Chronic Exposure to NMDA Receptor and Sodium Channel Blockers During Development in Monkeys and Rats: Long-Term Effects on Cognitive Function Merle G. Paule, C. Matthew Fogle, Richard R. Allen, Edwin Pearson, Tim Hammond, E. Jon Popke Dr. Paule’s presentation focused on functional disruption of synaptic transmission using whole animal behavioral assessment. The first part of the talk focused

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on a previous study in rhesus monkeys comparing the effects of chronic inhibition of N–methyl d-aspartate (NMDA)-type glutamatergic receptors using MK-801 or sodium channel block by remacemide on acquisition and subsequent performance of behavioral tasks. Whereas MK-801 is a relatively specific non-competitive antagonist of the NMDA receptor, remacemide has less specific actions. It has the ability to block voltage-gated sodium channels with much weaker NMDA receptor blocking properties. Low and high doses of both drugs were administered orally, daily for 18 months beginning when the animals were about 9 months of age. Whereas low doses of either compound were without effect, high doses of both compounds had differential effects on acquisition of a visual discrimination task. Remacemide, retarded the task for several months beyond the effect of MK-801. Moreover, acquisition of learning task performance was prevented by high doses of remacemide. This effect persisted even months after treatment ceased. Thus, chronic block of either NMDA receptors or voltage-gated sodium channels (perhaps in conjunction with NMDA receptor blockade) had different effects on acquisition of a relatively simple visual discrimination task. On the other hand, only chronic block of sodium channels (perhaps in conjunction with NMDA receptor block) had long-term—perhaps permanent—adverse consequences in the acquisition when compared to learning task performance in nonhuman primates. This rather selective adverse effect on a fairly specific brain function occurred in the absence of any detectable changes in other behaviors, or in clinical chemistry and hematology, among other alternative explanations. Subsequently, studies used a rodent model to attempt to distinguish the relative contributions of block of NMDA receptor and sodium channel to effects of remacemide seen in monkeys. In these studies, low and high doses of MK-801, alone or in combination with phenytoin (which only blocks voltage-gated sodium channels) were administered orally every day for 6 months to female rats beginning at weaning. The doses of MK-801 used were the same as those used in the monkey study. Acquisition and performance of tasks similar to those used in the monkey study were assessed. Acquisition of performance of an audio/ visual discrimination task was inhibited by both the high dose of MK-801 and high dose combination treatments. Acquisition of learning task performance (the same task used in the monkey study) was, however, only inhibited by the high dose of MK-801. In both groups, these noted effects outlasted drug treatment by several months and, thus, appeared permanent.

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The rat data, in which block of NMDA receptors alone had significant and long-lasting adverse consequences, contrasted markedly with primate data, for which MK801 was almost without effect. Effects of MK-801 in rodents resembled those of remacemide in monkeys. Phenytoin was virtually without effect in the rat model and appeared to block the adverse effects of MK-801 on the learning task. This was evidenced by the fact that performance in the group treated with the high dose combination of MK-801 plus phenytoin was not affected by treatment, whereas that in the group treated with only the high dose of MK-801 was severely affected. Thus for some drug classes, the rat may not be a good predictor of adverse drug effects in primates. Whether, this observation holds true for exposures beginning in adulthood is as yet unknown. Future studies of remacemide in the rodent will also allow for a direct comparison of its effects with those noted earlier in the monkey. Disruption of GABAergic Function of Cerebellum by Methylmercury: A Possible Approach to Differential Vulnerability William D. Atchison Dr. Atchison described a new area of research emerging from his lab to identify sites responsible for differential sensitivity of cerebellar neurons to actions of methylmercury (MeHg). Cerebellar granule and Purkinje cells have dramatically different sensitivities to neurotoxicity to MeHg both in vivo and in vitro. Among the major differences between the two cells are differential subunit composition of GABAA receptors. In earlier work from our lab, we found that MeHg caused a paradoxical increase in excitatory synaptic function which was due to block of inhibitory interneuron pathways. Subsequent studies in hippocampal slices showed that GABAA-mediated inhibitory postsynaptic potentials (IPSPs) and inhibitory postsynaptic currents (IPSCs) were very sensitive to block by MeHg. In cerebellar slice, GABAA receptor-mediated inhibition is blocked more readily by MeHg than is glutamergic-mediated excitation. Furthermore, depressant effects of MeHg on granule cell GABAA receptor currents occur earlier than on Purkinje cell GABAA receptor currents. Differences in GABAA subunit subtype can exert profound differences in pharmacological sensitivity of the receptor to agonists and antagonists. Granule cells are the only cell in the brain which expresses the a6 subunit of the GABAA receptor subunit; this is variably expressed alone or in combination with the a1 subunit. Moreover, granule cells frequently

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coexpress the d subunit with a6. Purkinje cells express neither of these subunits, instead expressing only the a1 subunit and coexpressing it with the g subunit. Cerebellar granule cells in culture are extremely sensitive to the blocking action of MeHg. Significant blocking effects occur at concentrations as low as 100 nM. A significant difference between between a1 and a6 subunit-containing GABAA receptors is their sensitivity to block by Zn2þ. a6 Subunit-containing GABAA receptors are readily blocked by Zn2þ whereas a1 subunit-containing GABAA receptors are insensitive to block by Zn2þ. MeHg has been shown to release intracellular Zn2þ. This could contribute to block of GABAA receptor function in granule cells by MeHg. Thus differential sensitivity to Zn2þ-induced block of cerebellar GABAA receptors could contribute to differential sensitivity to MeHg in cerebellum.

RESEARCH NEEDS While there are numerous research needs in furthering the understanding of actions of chemicals on synaptic function, speakers identified several areas in which research needs in the area of chemical-induced modulation of synaptic transmission are especially important. 1. A major area of research need is study of chronic actions of neurotoxicants on synaptic function. This issue is especially vexing for two reasons. The first is the multiplicity of effects which some environmental toxicants are evidently capable of exerting (examples include neurotoxic metals such as lead or mercury, PCBs and solvents), which can include extra- and intracellular actions. This makes it very difficult to sort out ‘‘primary’’ or initiating actions from secondary and perhaps ancillary actions or effects. The second problem is the inherent instability of electrophysiological recordings, which necessarily involve simple and isolated preparations to obviate the many potential confounding physiological processes such as respiration, blood flow, metabolism, etc. As a result of needing to use these simple systems in which the length of the experiment is often severely limited, a number of compromises need to be made. The most obvious of these is often the choice of toxicant concentration (dose ‘‘relevance’’) and, quite likely oversimplification of intracellular actions of toxicant. 2. More studies need to be directed at understanding the complexity of actions of neurotoxicants on

synaptic transmission in the CNS. The original studies of Manalis and Cooper (1973) stimulated a spate of studies on the actions of environmental metals and other possible contaminants on synaptic transmission at the vertebrate neuromuscular junction. While these studies provided a wealth of basic information on susceptibility of synaptic transmission to metals in particular, and have lighted some ‘‘beacons’’ to serve as guides for further mechanistic analyses, these studies were for most part never followed to their logical extension of comparative actions of neurotoxicants on CNS synaptic function. As should be abundantly clear, the brain is not merely a collection of ‘‘neuromuscular junctions’’! There are obvious and subtle differences in synaptic function that can be understood only by studying synaptic transmission in the brain. With the exception of a handful of studies from a couple of labs, this area remains vastly understudied. This is despite the fact that brain slice preparations have been in use for studies of neurophysiology and pharmacology for almost 20 years. Couple this problem with the issue addressed above in point 2, and there are virtually no papers describing effects of neurotoxicants on brain synaptic function following chronic exposure regimens. As pointed out by studies in Dr. Paule’s talk, behavioral assessment indicates that there are clearly actions on synaptic function being induced by chronic exposure of animals to pharmacological agents. Yet dissection of these actions cannot be made realistically at the whole animal level; they are studied best in preparations of relevant brain regions from animals exposed to neurotoxicants to some behavioral endpoint. 3. Effects of toxicants on specific (kinetic) properties of ion channel currents is another area in which studies are generally lacking. Kinetic properties are generally best examined in microscopic studies of single channel currents. With the exception of insecticides, studies of the functional actions of neurotoxicants on kinetic properties of distinct types of ion channels are generally lacking. One important caveat however, is that in some cases studies of kinetic effects have only been done at ‘‘equilibrium’’ effect of neurotoxicant. This may bias results or overlook early actions on the protein. 4. There is a crucial need for better use of contemporary ‘‘molecular’’ techniques to study the action of toxic environmental chemicals on function of specific components of proteins associated with synaptic function. As noted in Dr. Suszkiw’s talk, these types of studies are only now beginning to be

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done with intracellular actions of Pb2þ on the release proteins. With the exception again of the insecticides and ethanol, studies using recombinant proteins of known composition and expressed heterologously under controlled biochemical conditions are lacking. As shown in Dr. Atchison’s talk, application of these techniques may help to understand how neurotoxicants interact preferentially with cells containing distinct isoforms of proteins needed in synaptic function. Thus, as this discussion demonstrates, despite being the fundamental process by which neuronal function is produced, there are immense chasms in our understanding of the processes by which environmental chemicals disrupt synaptic function. A resurgence of interest in this problem similar to that seen in the late 1970s– 1980s following Manalis and Cooper (1973) pioneering work, coupled with contemporary techniques could begin to ‘‘span’’ this chasm.

REFERENCES Bouton CM, Frelin LP, Forde CE, Arnold Godwin H, Pevsner J. Synaptotagmin I is a molecular target for lead. J Neurochem 2001;76(6):1724–35.

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