ORGANIC POLLUTANTS: An Ecotoxicological Perspective - Chapter 16 doc

quanti-In the first place, there are a number of different sites of action for toxic chemicals within the central and peripheral nervous system of both vertebrates and inverte-brates.. W

so Examples include the organomercury fungicides and tetraethyl lead, which has been used as an antiknock in petrol (both in Chapter 8) It would appear, therefore, that the nervous system represents an “Achilles heel” within both vertebrates and invertebrates when it comes to the toxic action of chemicals When pesticide manu-facturers have screened for insecticidal activity across a wide diversity of organic chemicals, many of the substances that have proved successful in subsequent com-mercial development have been neurotoxic.

This line of argument can be extended to natural toxins as well (Chapter 1) Thus, many plant toxins such as the pyrethrins, physostigmine, strychnine, veratridine, aconitine, etc., all act upon the nervous system As discussed earlier, the presence

of such compounds in plants is taken as evidence for a coevolutionary arms race between higher plants and the animals that graze upon them The production of these compounds may protect the plants against grazing by vertebrates and invertebrates Apart from plants, animals and microorganisms also produce neurotoxins that have deadly effects upon vertebrates or invertebrates or both in the living environment For example, snakes, spiders, and scorpions all produce neurotoxins, which they inject into their prey to immobilize them (see Chapter 1, Section 1.3.1) Also, tetrado-toxin is stored within the puffer fish, and ergot alkaloids are produced by the fungus

Claviceps purpurea Indeed, these natural toxins have given many useful leads in the

design of new pesticides, biocides, or drugs

In earlier chapters, many examples were given of lethal effects and associated rotoxic or behavioral effects or both caused by pesticides in the field These included effects of organomercury fungicides upon birds (Chapter 8, Section 8.2.5), organo-chlorine insecticides on birds, and both organophosphorous and carbamate insecti-cides upon birds (Chapter 10, Section 10.2.4) Also, a retrospective analysis of field data on dieldrin residues in predatory birds in the U.K suggested that sublethal neu-rotoxic effects were once widespread and may have contributed to population declines observed at that time (Chapter 5, Section 5.3.5.1) Lethal and sublethal effects of neu-rotoxic insecticides upon bees is a long-standing problem (see Chapter 10, Section10.2.5) Speaking generally, it has been difficult to clearly identify and quantify neuro-toxic and behavioral effects caused by pesticides to wild populations, especially where the compounds in question have been nonpersistent (e.g., OP, carbamate, or pyrethroid insecticides), and where any sublethal effects would have been only transitory

neu-It is very clear, therefore, that there have been many examples of neurotoxic effects, both lethal and sublethal, caused by pesticides in the field over a long period of time Far less clear, despite certain well-documented cases, is to what extent these effects, especially sublethal ones, have had consequent effects at the population level and above Interest in this question remains because neurotoxic pesticides such as pyre-throids, neonicotinoids, OPs, and carbamates continue to be used, and questions con-tinue to be asked about their side effects, for example, on fish (Sandahl et al 2005), and on bees and other beneficial insects (see, for example, Barnett et al 2007).The present account will consider, in a structured way, how neurotoxic com-pounds may have effects upon animals, and how these effects can progress through different organizational levels, culminating in behavioral and other effects at the

“whole animal” level Emphasis will be placed upon the identification and fication of these effects using biomarker assays, and upon attempts to relate these biomarker responses to consequent effects at the population level and above, refer-ring to appropriate examples The concluding discussion will focus on the use of this approach to identify and quantify existing pollution problems and on its potential in environmental risk assessment

quanti-In the first place, there are a number of different sites of action for toxic chemicals within the central and peripheral nervous system of both vertebrates and inverte-brates When studying the effects of neurotoxic compounds, it is desirable to monitor the different stages in response to them using appropriate biomarker assays, begin-ning with initial interaction at the target site (site of action), progressing through consequent disturbances in neurotransmission, and culminating in effects at the level

of the whole organism, including effects upon behavior Thus, in concept, a suite of biomarker assays can be used to measure the time-dependent sequence of changes that follows initial exposure to a neurotoxic compound—changes that constitute the process of toxicity From integrated studies of this kind should come principles and techniques that can be employed to develop and validate new approaches and assays for the purpose of environmental monitoring and environmental risk assessment In reality, however, only a very limited range of biomarker assays are available at the time of writing, and much work still needs to be done to realize this objective

An overview will first be given of the interaction of neurotoxic compounds with target sites within the nervous system before moving on to discuss disturbances caused in neurons and, finally, effects at the whole-organism level; prominent among the latter will be behavioral effects Throughout, consideration will be given to bio-marker assays that may be used to monitor the toxic process Examples will be given

of the successful use of biomarker assays, where, by judicious use of such assays, effects observed in the field have been attributed to neurotoxic chemicals In conclu-sion, there will be a discussion of attempts to relate biomarker responses to conse-quent effects upon populations and above

16.2 NEUROTOXICITY AND BEHAVIORAL EFFECTS

Animal behavior has been defined by Odum (1971) as “the overt action an ism takes to adjust to its environment so as to ensure its survival.” A simpler def-inition is “the dynamic interaction of an animal with its environment” (D’Mello 1992) Another, more elaborate, one is, “the outward expression of the net interac-tion between the sensory, motor arousal, and integrative components of the central and peripheral nervous systems” (Norton 1977) The last definition spells out the important point that behavior represents the integrated function of the nervous sys-tem Accordingly, disruption of the nervous system by neurotoxic chemicals may be expected to cause changes in behavior (see Klaasen 1996, pp 466–467)

organ-Throughout the present text, toxicity is described as a sequence of changes ated by the interaction of a chemical with its site (or sites) of action, progressing through consequent localized effects and culminating in adverse changes seen at the level of the whole organism Thus, in what follows, the description of the bio-chemical mode of action of neurotoxic compounds will be followed by an account of localized effects before concluding with effects seen at the level of the whole animal, particularly behavioral effects

initi-By approaching neurotoxicity in this way, it should be possible, in the longer term, to develop biomarker assays that can monitor the different stages in toxicity and to produce combinations of biomarker assays that will give a quantitative in-depth picture of the sequence of changes that occurs when an organism is exposed

to a neurotoxic compound or a mixture of neurotoxic compounds In following this

progression, one moves from biochemical interactions, which are particular for a certain type of compound, to behavioral effects that are far less specific However, by following this integrated approach, it should be possible to distinguish the contribu-tion of individual members of a mixture to a common effect at a higher level of bio-logical organization, for example, an alteration in the conduction of nervous impulse

or a change in behavior Later in this account, examples will be given describing experiments that have successfully linked mechanistic biomarker assays to behav-ioral changes despite the complexity of the nervous system

Following from the above, behavioral assays, which can be relatively simple and cost-effective, can be very useful as primary screens when testing chemicals for their neurotoxicity in the context of medical toxicology (see Dewar 1983, Atterwill et al

1991, and Tilson 1993) Where disturbances of behavior are identified, subsequent more specific tests, including in vitro assays, may then be performed to establish

where and how damage is being caused to the nervous system It should be added that behavioral effects of chemicals may be very important in ecotoxicology They may be critical in determining adverse changes at the population level (Walker 2003, Thompson 2003).

Some authors have drawn attention to evidence for the greater sensitivity of early developmental stages of mammals to neurotoxins in comparison to adults (Colborn

et al 1998, Eriksson and Talts 2000) It has been claimed that neurotoxic and crine-disrupting chemicals are most damaging if there is exposure during embryonic, fetal, or postnatal life stages This is a point to be borne in mind when investigating the long-term effects of neurotoxins using biomarker strategies

NEUROTOXIC COMPOUNDS

The principal, known mechanisms of action of some neurotoxic environmental chemicals are summarized in Table 16.1 In considering these, it needs to be borne in mind that the interactions between chemicals and the nervous system in vivo can be very complex, and there is a danger of oversimplification when arguing from mecha-nisms of action shown to occur in vitro It is very important to relate results obtained

in vitro to interactions that occur in vivo, taking into account toxicokinetic factors The distribution of chemicals over the entire nervous system and the concentrations reached at different sites within it are critical in determining the consequent interac-tions and toxic responses Further, any given neurotoxic compound may interact not just with one well-defined target but with contrasting target sites in different parts of the nervous system Thus, one chemical may interact with two or more quite differ-ent receptor sites (e.g., Na+ channel and GABA receptor) at the same time, albeit in different parts of the nerve network Also, there may be different forms of the same type of active site—with contrasting affinities for neurotoxic compounds That said, this account will attempt to focus on the principal modes of action that particular chemicals have shown to particular species of animals in vivo

Taking first the voltage-sensitive Na+ channels (Chapter 5, Figure 5.4) that are found in the plasma membranes of nerve and muscle cells of both vertebrates and invertebrates, it is seen that these are regulated by two separate processes: (1) activa-tion, which controls the rate and voltage-dependence of the opening of this hydro-phobic channel, and (2) inactivation, which controls the rate and voltage-dependence

of the closure of the channel These channels are known to exist in many different forms despite the fact that they all have the same common function, that is, the regulation of sodium currents across the plasma membrane Three different types are recognized in rat brain, and strongly contrasting forms are recognized in differ-ent strains of the same species Resistant strains of houseflies and other insects have different forms from susceptible strains of the same species For example, kdr and super kdr strains have forms of the proteins constituting Na+ channels which are dif-ferent from those found in susceptible strains (see Chapter 5, Section 5.2.5.2), and the forms present in these resistant strains are insensitive to both DDT and pyrethroid insecticides; that is, they provide the basis for resistance to the insecticides

Neurotoxic Action of Some Environmental Chemicals

Veratridine appears to act at a different part of pore channel from DDT or pyrethroids Nicotinic acetylcholine

receptors

Neonicotinoids Similar action to Nicotine Nicotine Act as agonists causing

desensitization of receptor Gamma aminobutyric acid

Picrotoxinin Inhibitor of GABA receptors

Acetylcholinesterase OP and carbamate insecticides Inhibitors of enzyme causing

buildup of acetylcholine in synapses

Physostigmine Inhibitor of acetylcholinesterase

Neuropathy target esterase Certain OP compounds including

DFP, mipafox, and leptophos

Aging of inhibited enzyme leads to degeneration of peripheral nerves Cause damage to CNS of

The Na+ channel is the target for certain naturally occurring toxins (see Chapter 5, Figure 5.4) The lipid-soluble alkaloid veratridine can activate the channel by binding

to it and stabilizing it in a permanently open conformation (Eldefrawi and Eldefrawi 1990) This causes a prolongation of the sodium current and disruption of the action potential—typically, repetitive firing of the action potential The marine toxins tetro-dotoxin and saxitoxin have the opposite effect They are organic ions bearing a posi-tive charge that can bind to the channel near its extracellular opening and thereby block the movement of sodium ions Of the insecticides, the principal mode of action

of both DDT and the pyrethroid insecticides is thought to be upon Na+ channels Rather like veratridine, they bind to the channel causing a prolongation of the Na+current, although they appear to bind to a different part of the protein than does this alkaloid (Chapter 5, Figure 5.4) Nerves poisoned by DDT typically produce multiple rather than single action potentials when they are electrically stimulated (Figure 16.1)

B Current Generated on Postsynaptic Membrane of Inhibitory Synapse

following Stimulation with Gab

The nicotinic receptor for acetylcholine is located on postsynaptic membranes of

nerve and muscle cells It is found in both the central and peripheral nervous system of vertebrates, but only in the central nervous system of insects (Eldefrawi and Eldefrawi 1990) A hydrophobic cationic channel is an integral part of this transmembrane pro-tein With normal synaptic transmission, acetylcholine released from nerve endings interacts with its binding site on the receptor protein, and this leads to an opening of the pore channel and an influx of cations The consequent depolarization of the mem-brane triggers the generation of an action potential by neighboring sodium channels, and so the message is passed on The natural insecticide nicotine acts as an agonist for acetylcholine and can cause desensitization of the receptor Neonicotinoid insecticides such as imidacloprid act in a similar way to nicotine They are more lipophilic than the natural compound and are more effective as insecticides

Gamma aminobutyric acid (GABA) receptors are located on the postsynaptic

membranes of inhibitory synapses of both vertebrates and insects and contain within their membrane-spanning structure a chloride ion channel They are found in both vertebrate brains and invertebrate cerebral ganglia (sometimes referred to as brains)

as well as in insect muscles Particular attention has been given to one form of this receptor—the GABA-A receptor—as a target for novel insecticides (Eldefrawi and Eldefrawi 1990) It is found both in insect muscle and vertebrate brain The remain-der of this description will be restricted to this form

GABA-A possesses a variety of binding sites (Chapter 5, Figure 5.4) One of them is for the natural transmitter GABA, an interaction that leads to the opening

of the pore channel and the influx of chloride ions (Figure 16.1) Another, close

to or in the chloride ion channel, binds the naturally occurring convulsant toxinin, the cyclodiene insecticides (e.g., dieldrin, endrin), gamma HCH (lindane), and toxaphene Convulsions accompany severe poisoning by these insecticides The GABA-A receptor of mammalian brain is believed to be the primary target for cyclo-diene insecticides in that organ Binding of picrotoxinin and cyclodiene insecticides

picro-to the receppicro-tor retards the influx of chloride ions through the pore channel following stimulation with GABA; that is, they inhibit the normal functioning of the receptor.Acetylcholinesterase is a component of the postsynaptic membrane of cholinergic synapses of the nervous system in both vertebrates and invertebrates Its structure and function has been described in Chapter 10, Section 10.2.4 Its essential role in the postsynaptic membrane is hydrolysis of the neurotransmitter acetylcholine in order

to terminate the stimulation of nicotinic and muscarinic receptors (Figure 16.2) Thus, inhibitors of the enzyme cause a buildup of acetylcholine in the synaptic cleft and consequent overstimulation of the receptors, leading to depolarization of the postsynaptic membrane and synaptic block

The carbamate and OP insecticides and the organophosphorous “nerve gases” soman, sarin, and tabun all act as anticholinesterases, and most of their toxicity is attributed to this property The naturally occurring carbamate physostigmine, which has been used in medicine, is also an anticholinesterase Some OP compounds can cause relatively long-lasting inhibition of the enzyme because of the phenomenon of

“aging”; the inhibited enzyme undergoes chemical modification, and inhibition then becomes effectively irreversible.

A few OP compounds cause delayed neuropathy in vertebrates because they

inhibit another esterase located in the nervous system, which has been termed

neu-ropathy target esterase (NTE) This enzyme is described in Chapter 10, Section 10.2.4 OPs that cause delayed neuropathy include diisopropyl phosphofluoridate (DFP), mipafox, leptophos, methamidophos, and triorthocresol phosphate The delay

in the appearance of neurotoxic symptoms following exposure is associated with the aging process In most cases, nerve degeneration is not seen with initial inhibition of the esterase but appears some 2–3 weeks after commencement of exposure, as the inhibited enzyme undergoes aging (see Section 16.4.1) The condition is described as OP-induced delayed neuropathy (OPIDN)

Organometallic compounds such as alkylmercury fungicides, and tetraethyl lead, used as an antiknock in petrol, are neurotoxic, especially to the central nervous system

of vertebrates (Wolfe et al 1998, Environmental Health Criteria 101, and Chapter 8,

BOX 16.1 TECHNIQUES FOR MEASURING THE

INTERACTION OF NEUROTOXIC CHEMICALS

WITH THEIR SITES OF ACTION

A central theme of this text is the development of biomarker assays to measure the extent of toxic effects caused by chemicals both in the field studies and for the purposes of environmental risk assessment

Considering the examples given in Table 16.1, a number of possibilities present themselves In the first place, competitive binding studies may reveal the extent to which a toxic compound is attached to a critical binding site For example, the convulsant TBPS binds to the same site on GABA-A receptors of rat brain as do cyclodiene insecticides such as dieldrin In samples preexposed

to dieldrin, the binding of radiolabeled TBPS will be less than in controls not exposed to the cyclodiene (Abalis et al 1985) The difference in binding of the radioactive ligand to the treated sample in comparison to binding to the control sample provides a measure of the extent of binding of dieldrin to this target Similarly, the competitive binding of tetrodotoxin and saxitoxin to the

Na+ channel may be exploited to develop an assay procedure

In cases where the mode of action is the strong or irreversible inhibition

of an enzyme system, the assay may measure the extent of inhibition of this enzyme This may be accomplished by first measuring the activity of the inhibited enzyme and then making comparison with the uninhibited enzyme This practice is followed when studying acetylcholinesterase inhibition by organophosphates (OP) Acetylcholinesterase activity is measured in a sample

of tissue of brain from an animal that has been exposed to an OP Activity is measured in the same way in tissue samples from untreated controls of the same species, sex, age, etc Comparison is then made between the two activity measurements, and the percentage inhibition is estimated

Section 8.2.4 and Section 8.2.5 in this book) Neurotoxic effects in adult mammals include ataxia, difficulty in locomotion, neurasthenia, tremor, impairment of vision and, finally, loss of consciousness and death Necrosis, lysis, and phagocytosis of neu-rons are effects coinciding with these symptoms of toxicity As described earlier, sub-lethal neurotoxic effects on humans and wild vertebrates have occurred and still occur

as the result of environmental contamination by methylmercury The mechanism of neurotoxic action is complex and is not well understood There is strong evidence that methylmercury compounds can have adverse effects upon a number of proteins, including enzymes and membrane-spanning proteins involved in ion transport (ETAC 101) It seems probable that the strong tendency of these compounds to bind with—and thereby render ineffective—functional –SH groups of the proteins is the main reason for this (see, for example, Jacobs et al 1977, who studied the inhibition of protein syn-thesis by methylmercury compounds) There is also evidence that exposure to sublethal levels of methyl mercury can cause changes in the concentration of neurochemical receptors in the brains of mammals and birds (Basu et al 2006, Scheuhammer et al 2008) Thus, an increase in concentration of brain muscarinic receptors for acetylcho-line and a decrease in the concentration of brain receptors for glutamate was observed

Post-Synaptic Membrane

Vesicles with Neurotransmitter

Cholinergic (Nicotinic) Synapse

ACh Receptor

Na Channel

Direction of Transmission Synaptic Cleft

following exposure to environmentally realistic levels of methylmercury This vation was made both in mink and common loons.

obser-In summary, the toxic effects of methylmercury on vertebrates are complex and wide ranging, and with the present state of knowledge it is not possible to ascribe this neurotoxicity to one clearly defined mode of action

16.4 EFFECTS ON THE FUNCTIONING OF THE NERVOUS SYSTEM

Following combination with their sites of action, the main consequent effects of the neurotoxic compounds described here are upon synaptic transmission or propagation

of action potential In some cases (e.g., methylmercury and some OPs) there are signs

of physical damage such as demyelination, phagocytosis of neurons, etc The ing account will be mainly concerned with effects of the first kind—that is, electro-physiological effects—which may provide the basis for assays that can monitor the progression of toxicity from an early stage and thus provide a measure of sublethal effects caused by differing levels of exposure Effects on the peripheral nervous sys-tem and the central nervous system will now be considered separately

follow-16.4.1 E FFECTS ON THE P ERIPHERAL N ERVOUS S YSTEM

Electrical impulses are passed along nerves as a consequence of the rapid sion of a depolarization of the axonal membrane In the resting state, a transmem-brane potential is maintained on account of the impermeability of the nerve to ions such as Na+ and K+ Were the membrane freely permeable, these ionic gradients could not be sustained Active transport processes maintain ionic gradients in excess

progres-of those that could be achieved purely by passive diffusion However, when Na+channels open in the axonal membrane, a very brief inwardly flowing Na+ current causes a transient depolarization This is rapidly corrected by a subsequent outward flow of K+ions The Na+ current is terminated when the pore channel closes, and the succeeding K+ current flows briefly until the transmembrane potential returns to its resting state (Figure 16.1)

The passage of action potentials along a nerve can be recorded by inserting microelectrodes across the neuronal membrane and using them to record changes in the transmembrane potential in relation to time This has been done in a variety of ways Microelectrodes can be inserted into nerves of living animals, or into isolated nerves, or cellular preparations of nerve cells (see Box 16.2) An important refine-ment of the technique involves “voltage clamping.” This permits the “fixing” of the transmembrane potential, which restricts the movement of ions across the mem-brane Thus, it is possible to measure just the Na+ current or the K+ current in control and in “poisoned” nerves, thereby producing a clearer picture of the mechanism of action of neurotoxic compounds that affect the conduction of action potentials along nerves Measurements of this kind may be just of spontaneous action potentials or of potentials that are elicited by electrical or chemical stimulation Chemical stimula-tion may be accomplished using natural neurotransmitters such as acetylcholine.The effects of neurotoxic chemicals upon nerve action potential have been mea-sured both in vertebrates and insects Of particular interest has been the comparison

of the responses of different species and strains of insects to insecticides Returning

to the examples given in Table 16.1, both DDT and pyrethroid insecticides interact with the Na+ channel of the axonal membrane of insects With repeated use of DDT, insects such as houseflies came to develop kdr and super kdr resistance against the insecticide Both types of resistance are due to the appearance of forms of the Na+channel that are insensitive to the insecticide (see Chapter 4, Section 4.5, and Chapter

12, Section 12.6) The fact that these strains also show marked cross-resistance to pyrethroids is compelling evidence that this pore channel represents the principal site of action for both types of insecticide in insects

The effects of DDT on nerve action potential are illustrated in Figure 16.1 In nerves poisoned by the insecticide, there is a prolongation of the sodium current and a consequent delay in returning to the resting potential This can result in the

There has long been an interest in the development of in vitro assays for ing neurotoxic effects of chemicals from the point of view of both human risk assessment and environmental risk assessment The effects of neurotoxic chemicals on laboratory animals is a major concern of animal welfare organi-zations An outstanding problem is that, because of the complexity of the ner-vous system, some neurotoxic effects can only be detected in vivo—in whole animal systems (Dewar 1983, Atterwill et al 1991) Thus, it is difficult to fore-see the total banning of in vivo tests However, in vitro assays can still make an important contribution to testing protocols for chemicals These protocols can include a combination of in vivo and in vitro tests, with a consequent reduction

detect-in the use of animals for testdetect-ing procedures (Atterwill et al 1991)

Atterwill et al (1991) list six categories of nervous system culture that have been used in in vivo testing procedures These are dispersed cell cultures, explant cultures, whole organ cultures, reaggregate cultures, whole embryo models, and cell lines It is possible in cultures such as these to measure the cellular response to neurotoxic chemicals Electrophysiological measurements can be made even on single cells, revealing effects of chemicals upon ion cur-rents and transmembrane potential Also, there is the possibility of following effects on the release of chemical messengers such as cyclic AMP from post-synaptic membranes, when neurotransmitters interact with their receptors

In one example (Lawrence and Casida 1984, Abalis et al 1985) rat brain microsacs were used to test the action of cyclodiene insecticides such as dieldrin and endrin on the GABA receptors contained therein The influx of radiolabeled Cl− into the microsacs via the pore channel of the receptor was inhibited by these chemicals A similar assay was developed using microsacs from cockroach nerve Assays with this preparation showed again the inhibi-tory effect of a cyclodiene (this time heptachlor epoxide) on Cl− influx Also, that microsacs from cyclodiene resistant cockroaches were insensitive to the inhibitory effect of picrotoxinin, which binds to the same site on the GABA receptor (Kadous et al 1983)

generation of further spontaneous action potentials, that is, there can be repetitive action potentials following a single stimulus.

As described earlier, the chloride channels, which are associated with GABA

receptors, are affected by the action of cyclodienes and certain other chlorinated insecticides These chemicals can inhibit the action of the neurotransmitter GABA

by binding to a site in or near the pore channel, with consequent reduction in the inward flow of Cl−(see Figure 16.1) Electrophysiological studies have been car-ried out that involve the stimulation of GABA receptors of insect muscle (e.g., of the locust) Treatment with GABA causes hyperpolarization of the membrane, an effect that is retarded when the receptors are preexposed to cyclodienes, or to the natural product, picrotoxinin The action of picrotoxinin on GABA receptors of the

locust Calliphora erythrocephala and the resulting neurophysiological effects are

described by Von Keyserlingk and Willis (1992) So, again, the interaction of a rotoxic compound with a receptor can be related to consequent electrophysiological effects (see also Box 16.2)

neu-The neurophysiological effects of anticholinesterases have been studied in the peripheral nervous system of experimental animals and humans In some cases of human poisoning, effects on motor conduction were measured using electromyogra-phy (EMG), which involves the insertion of a needle-recording electrode into muscle (Misra 1992) In cases of OP poisoning, there was evidence of several types of neu-rophysiological effects, including repetitive activity Poisoning in vertebrates leads to

a buildup of the neurotransmitter on cholinergic junctions, which, if severe enough, will cause a depolarization of the synaptic membrane and loss of synaptic transmis-sion Thus, the later stages of poisoning should be evident from measurement of the postsynaptic signal by EMG Effects of anticholinesterases on the sensory system of the mammalian PNS have also been monitored using electrophysiological methods.The neurophysiological effects of nicotine have been widely reported in the pharmacological literature, and the neonicotinoid insecticides are known to act in a similar way Initially, these compounds act as agonists of nicotinic receptors of ace-tylcholine, but this interaction leads to desensitization of the receptor, resulting in a loss of synaptic transmission Thus, their effects can be monitored by recording the signals from cholinergic synapses such as the neuromuscular junction of vertebrates and testing responsiveness to acetylcholine stimulation by EMG measurements This can be done, for example, with denervated muscle of the rat

The delayed neuropathy caused by certain OPs that inhibit neuropathy target esterase is characterized by a number of pathological changes in the peripheral nervous system of vertebrates (Johnson 1992, Veronesi 1992) Electrophysiological measurements on the sciatic nerve of hens have shown a significant increase in excit-ability 24 hours after dosing with one of these compounds The hen is used as a test organism on account of its high susceptibility to this type of poisoning In the longer term (2–3 weeks), degenerative changes appear in peripheral nerves that are characteristic of this type of poisoning, changes that affect the distal extremities and are associated with a sensory–motor deficit These later effects have been observed

in mammals, including humans