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The effects of cypermethrin on neurite development and intracellular calcium concentrations of cultured embryonic rat hippocampal neurons

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Title:
The effects of cypermethrin on neurite development and intracellular calcium concentrations of cultured embryonic rat hippocampal neurons
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Keller, Lisa Gwyn
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English
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xiii, 129 leaves : illustrations ; 28 cm

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Pyrethroids -- Toxicology ( lcsh )
Rats -- Experiments ( lcsh )
Pyrethroids -- Toxicology ( fast )
Rats -- Experiments ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Includes bibliographical references (leaves 112-129).
General Note:
Submitted in partial fulfillment of the requirements for the degree, Master of Arts, Biology.
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by Lisa Gwyn Keller.

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University of Colorado Denver
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Auraria Library
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32714224 ( OCLC )
ocm32714224
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LD1190.L45 1994m .K45 ( lcc )

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THE EFFECTS OF CYPERMETHRIN ON NEURITE DEVELOPMENT AND INTRACELLULAR CALCIUM CONCEN1RATIONS OF CULTURED EMBRYONIC RAT HIPPOCAMPAL NEURONS by Lisa Gwyn Keller B.A., University of Colorado, 1989 A thesis submitted to the Faculty of the Graduate ,School of the University of Colorado at Denver in partial fulfillment of the requirements for the degree of Master of Arts Biology 1994

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This thesis for the Master of Arts degree by Lisa Gwyn Keller has been approved for the Graduate School by Gerald Audesirk Teresa Audesirk I 9 Y Date.

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Keller, Lisa Gwyn (M.A., Biology) The Effects of Cypermethrin on Neurite Development and Intracellular Calcium Concentrations of Cultured Embryonic Rat Hippocampal Neurons Thesis directed by Associate Professor Gerald Audesirk ABSTRACT The severe neurological effectS of the pyrethroid insecticides are due to their. modification of nerve membrane sodium channel activity. The pyrethroids may have effects on other cellular processes, however, such as nerve cell differentiation and outgrowth, which may or may not be linked to their action on the sodium _channel. The present study was designed to determine the effects of cypermethrin, a Type II pyrethroid, on various parameters of neurite outgrowth in fetal rat hippocampal neurons. In addition, the possibility that the effects of both cypermethrin and permethrin, a Type I pyrethroid, might be mediated by the pyrethroid actions on the sodium channel was also explored by use of tetrodotoxin, a potent sodium channel blocker. Finally, since calcium has been implicated in many neurite outgrowth processes, the effects of cypermethrin and permethrin on intracellular calcium concentration at one, two, and three hour time points .were also determined. The effects of cypermethrin on neurite development were inhibitory and concentration dependent with a concentration of 100 11M causing an inhibition of

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almost every parameter. Tetrodotoxin caused a partial reversal of the effects of cypennethrin and permethrin on the initiation of neurites, but had no reversing effect on any other parameter of neurite outgrowth. Cypermethrin had no effect on intracellular calcium concentration at any timepoint, while permethrin caused a significant decrease. These results indicate that pyrethroid-induced effects on neurite outgrowth are not mediated by alterations of sodium channel activity. This abstract accurately represents the content of the candidate's thesis. I recommend its publication. Gerald Audesirk

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To Steven v

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CONTENTS Figures ..................................................... x Tables . . . . . .-. . . . . . . xii CHAPTER 1. INTRODUCTION ............................ ............ 1 General Overview ................. : ........................ 1 The Pyrethroids . . . . . . . . . . 3 Background ............................. .... ........... 3 Two Classes of Symptoms of Pyrethroid Intoxication . . . . . . . . .4 Structure . . . . : . . . . . . . 6 Pyrethroid Effects . . . . . . . . . . 10 Pyrethroids and the Hippocampus ............................. 27 Neurite Development ........................................ 29 Neurite Structure and Growth . . . . . . . .29 Calcium Regulation by the Ner\re Cell.-......................... .33 Calcium-Mediated Effects on Neurite Growth ...................... 35 Second Messenger Systems that Mediate Neuronal Growth Processes .......... ....................... 40

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Tetrodotoxin Effects on Neurite Development .................... 43 A Model of Neurite Development ....................... .... 46 Hypothesis . . . .... . . . . . . . 50 2. METHODS ............................................... 52 Cell Culture Experiments ..................................... 52 Isolation and Dissociation of Hippocampal Cells ...................................... 52 Cryopreservation_ of Cells ................................... 53 Culturing Techniques ...................................... 53 Assessment of Survival and Differentiation .... ; .................. 55 Measurements of Intracellular Calcium ............................ 57 Background of Fura-2 .................................... 57 Imaging System Setup ..................................... 59 Calibration . . . . .. . . . ; . . . 60 [Ca2+]iri in Cells Exposed to Permethrin or Cypermethrin .................................. 61 Statistical Analysis ............ ............................ 62 3. RESULTS ................................................ 63 Cell Culture Experiments . . . . . ; . . . 63 Cypermethrin Effects on Neurite Outgrowth ...................... 63 vii

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Tetrodotoxin Effects on Cultures Exposed to Pennethrin or Cypennethrin ...................... : ........ 70 Measurements of [Ca2+]in .................................. 84 Cypennethrin Effects on [Ca2+]in ....... ........................ 84 Pennethrin Effects on [Ca2+lm . .. . . . . . . 86 4. DISCUSSION ............................................. 88 General Overview ......................................... 88 Cypennethrin Effects on Neurite Outgrowth ........................ 89. Survival versus Initiation ............ ....................... 91 Axon versus Dendrite Elongation .............................. 91 Comparisons Among Initiation, Dendrite Production, and Axonal Branching .... ....................... 92 Perrnethrin Effects on Neurite Outgrowth : . . . . . 94 Pennethrin' s versus Cypennethrin' s Effects on Neurite Outgrowth ................................ 95 The Feasibility of a Sodium Channel Response .................... ................... 96 Tetrodotoxin Effects on Cultures Exposed to Pennethrin or Cypennethrin . . . . . . . . 98 Initiation . . . . . . . . . . . 99 [Ca2+lm MEASUREMENTS .................................. 101 Overall Conclusion . . . . . . . . . 105 Vlll

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Calcium Regulation by Rat Hippocampal Neurons .................. 1 06 Pyrethroid Effects on Cell Survival . . . . . . 106 Pyrethroid Effects. on lntiation ............................... 107 A Potential Mechanism for Type II Pyrethroid Effects on Neurite Development . . . . . 108 Final Summary . . . . . . . . . . 111 LITERATURE CITED ........................................... 113 IX

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FIGURES Figure Page 1.1 Structural Base of the Pyrethroids ............................. 7 1.2. Structures of Various Pyrethroids . . . . . . . ... 9 1.3. Effect of Pyrethroid on Action Potential ...... .. . . . . 11 1.4. Voltage-Sensitive Na+ Channel ............................... 12 .. 1.5. Sites of Action of the Pyrethroids ............................. 15 1.6. Structure of a Neurite ............... ....................... 30 1.7. [Ca2+]in within an Active Growth Cone ....................... 36 1.8. Effect of Tetrodotoxin on the Na+ Current ....................... 45 1.9. A Model of Neurite Development ... ... : .................... 48 2.1 Fura-2 Excitation Spectrum .................................. 59 3.1. Percent Cell Survival ........................ . . . 64 3.2. Percent Cells Initiating Neurites ............................... 65 3.3. Percent Axonal Cells per Growing Cell ......................... 67 3.4. Mean Axon Length ........................ .............. 68 3.5. Number of Branches per Axon .............................. 69 3.6. Number of Dendrites per Cell ................................ 71 X

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3.7. Percent Cell Survival (Tetrodotoxin Experiments) .................. 74 3.8. Percent Cells Initiating Neurites ............................... 75 3.9. Percent Axonal Cells per Growing Cell ............ ." ............ 77 3.10. Mean Axon Length ....................................... 78 3.11. Number of Branches per Axon ............................... 79 3.12. Axon Branch Length .. ; ................................... 81 3.13. Number of Dendrites per Cell ................................ 82 3.14. Mean Dendrite Length ........................ ; ............ 83 3.15. Effect of Cypermethrin on [Ca2+Jm ............................. 85 3.16. Effect of Permethrin on [Ca2+]in ............................ 87 xi

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TABLES Table Page 4.1. Cypermethrin Effects on Neurite Development .................... 90 4.2. Reversal of Pyrethroid Effects on Neurite Development by Tetrodotoxin ......................... 100 xu

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ACKNOWLEDGEMENTS I would like to offer a special thanks to Drs. Gerald Audesirk and Teresa Audesirk for their invaluable instruction, help, and encouragement over the past several years. I would also like to thank Charles Ferguson and Dr. Alan Brockway for their valuable counsel. Finally, I would like to offer my sincerest gratitude to Marcey Kern, Leigh Cabell, and John Dohanyos for their untiring support and assistance. This work was supported by.a grant from the National Institutes of Environmental Health Sciences. Xlll

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CHAPTER I INTRODUCTION General Overview Pyrethroid insecticides currently constitute more than 30% of the world market, and are replacing many of the traditional insecticides (Eriksson, 1992; Eells et al., 1993). There is increasing concern about the health impact that these highly potent neurotoxicant:S will have on the surrounding environment and non-target pyrethroid sensitive animals. One important environmental concern is the fact that the severe neurotoxic effects of the pyrethroids are not just' restricted to insects; pyrethroids are also severely neurotoxic to fish, crustaceans, and frogs (Glickman and Casida, 1982). There is concern about whether aquatic populations of .porids and streams will be exposed to significant levels of pyrethroids by way of drift from spraying or from run-off from exposed fields (Hill, 1985). Another important consideration is the potential effect of pyrethroids on humans and mammals. Recurrent reports of respiratory irritation and various peripheral sensory phenomena have been made by humans occupationally exposed to pyrethroids (Vijverberg and van den Bercken, 1990). In addition, even though pyrethroids are rapidly metabolized and eliminated in mammals and are therefore considered relatively nontoxic, they are highly soluble

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in fat and can be transferred from a lactating mother to her offspring (Gaughan et al., 1978; Kavlok et al., 1979). If the pyrethroids were absorbed by the offspring in sufficient quantities, they could induce detrimental neurotoxic effects (Eriksson and Norberg, 1990; Eriksson and Fredriksson, 1991). Studies indicate that neonatal rats are more sensitive to pyrethroid toxicity than adult rats, and that exposure to pyrethroids during a critical time period of neonatal brain development causes a decline in adult brain function (Eriksson, 1992; Cantalamessa, 1993). Relatively little information has been acquired on the effects that pyrethroids have on prenatal brain development. This thesis will attempt to investigate the effects that two different types of pyrethroids, permethrin and cypermethrin, can have on several different aspects of neurite development in neurons from the hippocampus of prenatal rats. It will also investigate the possibility that pyrethroid effects on neurite development may be mediated in part by calcium, an important intracellular messenger. The introduction will be divided into three sections. The first part will continue to discuss the pyrethroids by examining the background of their development, followed by their two classes of symptoms of pyrethroid intoxication, their structural make-up, their cellular effects, and finally their effects on the hippocampus. The second part will examine aspects of neurite development, and the role that calcium and several other second messengers might play in this development. It will discuss neurite 2

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structure and growth, followed by regulation of calcium by the cell, calcium-mediated effects on neurite growth, and other (non-calcium) second messenger systems that mediate neurite growth. Following this will be a brief discussion of the etiects of tetrodotoxin, a potent sodium channel blocker and antagonist of pyrethroid effects on the sodium channel (Narahashi, 1982b,1986b; Ghiasuddin and Soderland, 1985), on neuronal growth. It will end with a possible model for neurite development. The last part will conclude the introduction with a hypothesis about the effects that the two types of pyrethroids will have on both rat hippocampal cell neurite development and on intracellular calcium concentration. The Pyrethroids Background Pyrethroid insecticides are synthetic analogues of the naturally occurring pyrethrin compounds found in pyrethrum (Chrysanthemum cinerariaefolium) tlowers. Although the pyrethrins are potent insecticides, with a high insect-to-mammal lethality ratio, they are highly photodegradable, and are costly to extract (Wouters and van den Bercken, 1978; Williamson et al., 1989). These two factors, along with the increasing concern about environmental impact from the organophosphate, carbamate, and organochlorine insecticides, prompted the development of the pyrethroids (Elliot and Janes, 1978; Narahashi, 1982b). The pyrethroids are similar to the pyrethrins 3

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regarding their high selectivity towards insects, and relatively low mammalian and avian oral toxicity. Indeed, the pyrethroids have the highest average ratio for rat oral LD5ofinsect topical LD50 (mglkg) of all the highly potent insecticides (Casida et al., 1983). In addition, they maintain limited soil persistence, yet they have enhanced potency and are more photostable compared to the naturally occurring pyrethrins (Elliot, 1976; Wouters and van den Bercken, 1978). Finally, the pyrethroids are more practical economically, due to a low dosage requirement and greater duration of protection (Glickman and Casida, 1982). These important characteristics imply an increasing and more widespread use in field pest control as well as in various other situations, including household insecticides and "knockdown" agents (agents which prevent insects from continuing in flight), mosquito and fly repellents, the protection of stored foodstuffs,and veterinary and human delousing agents (Williamson et al., 1989). Pennethrin and cypennethrin, the pyrethroids studied in this thesis, are used as agricultural insecticides, a tick repellant and killer (permethrin), a protector of wheat stocks (cypennethrin), and as veterinary fly repellents (Herve, 1985). Two Classes of Symotoms of Pyrethroid Intoxication In general, pyrethroids have a strong excitatory action on the nervous system. The pyrethroids have been divided into two distinct classes, based on their symptomology of poisoning in mammals and insects and on their chemical structure. Verschoyle and 4

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Aldridge (1980) classified the T-Syndrome (Tremor) as occurring in rats injected with pyrethroids lac-.dng an a-cyano group, and the CS-Syndrome (Choreoathetosis and Salivation) occurring in rats injected with pyrethroids containing an a-cyano group. The T-Syndrome is characterized in rats and mice by a rapid onset of aggressive sparring, hyperresponsiveness to external stimuli, hyperthermia, and hyperactivity. Subsequently, fine tremors in the extremities develop, which then gradually progress to prostration with whole-body tremors, to tonic-clonic seizures and ultimately death (Verschoyle and Aldridge, 1980; Lawrence and Casida, 1982; Hudson et al., 1986; McDaniel and Moser, 1993). CS-Syndrome toxicity consists of pawing and burrowing behavior, profuse salivation, hypothermia, and a decreased response to external stimuli. This is followed by a coarse whole body tremor, which proceeds to a splayed gait of the hind limbs, and ultimately choreoathetosis, clonic seizures and death (Barnes and Verschoyle, 1974; Verschoyle and Aldridge, 1980; Lawrence and Casida, 1982; McDaniel and Moser, 1993). Analogous syndromes occur in the cockroach, which Gammon et al., (1981) classified as Type I and Type II effects. Type I effects correspond to T-Syndrome and are similarly caused by pyrethroids lacking an a-cyano group. Its symptoms include: restlessness, incoordination, prostration, and paralysis. Type IT symptoms are cased by pyrethroids containing the a-cyano moiety, and consist of ataxia and incoordination accompanied by periods of intense hyperactivity and seizures, followed 5

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by prostration and death. Structure The natural pyrethrin insecticides are esters of cyclopropane carboxylic acids with alkenylmethyl cyclopentenolone alcohols. The first pyrethroids were also esters, with derivations of the acid and alcohol moieties (Fig. 1.1). Activity of the pyrethroids is dependent upon the presence of the ester, as derivatives of the acid and alcohol constituents themselves contain no insectiCidal activity (Elliot, 1976). The ester probably adopts a trans configuration, meaning that the cyclopropane ring and the alcohol are on opposite sides of the molecule. High activity is also related to the ability of the molecule to adopt a particular conformation at the site of action, and is therefore influenced by the configurations of the asymmetric carbons at C-1 of the cyclopropane ring and C-4 of the cyclopentenolone ring (Fig. 1.1 ). All pyrethroids with high insecticidal activity have an R-configuration at C-1 of the cyclopropane ring, and an S-configuration at C-4 of the cyclopentenolone. A dimethyl group on C2 of the cyclopropane ring, and an unsaturated butyl side chain on C-3 are also requisite for high insecticidal activity (Wouters and van den Bercken, 1978). 6

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Gem-dimethy I group I Pyrethrln I CH3' CH3 lrR ',(o ... 'l .l 4. I 1 0 "" .. Cyclopropane rlnQ : Acid I Alcohol Fig. 1.1. Structural base of the pyrethrolds. (Reprinted from Gen. Pharm., Vol. 9, Wouters, W. & van den Bercken, J., Acti9n of pyrethroids, 1978, pp. 387-398, with kind permission from Elsevier Science Ltd., The Boulevard, Langford Lane, Kidlington OXS 1GB, UK). The first pyrethroid, allethrin, was by shortening the side chain of the cyclopentenolone (see Fig. 1.2 for various pyrethroid structures) (Schechter et al., 1949). Allethrin and its isomers bioallethrin and S-bioallethrin showed greater thennal stability than the natural pyrethrins, in addition to high pote.ncy as knockdown agents (Elliot, 1976; Casida et al., 1983). Subsequently, it was discovered that 5-henzyl-3furyl-methyl alcohol could substitute for the cyclopentenolone, yielding bioresmethrin, which maintained the low mammalian toxicity of the pyrethrins, but which showed greatly augmented insect toxicity. Indeed, has the highest LD50 oral rat oral/insect topical (mglkg) ratio of all pesticides (Elliot, 1971). Further enhancement 7

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of insect toxicity was made by replacement of the furylmethyl group with 3-phenoxy benzyl alcohol. This, along with a dihalovinyl substitution at C-3 of the cyclopropane ring, resulted in the first highly effective, photostable pyrethrin, permethrin (Elliot et al., 1973). The most active pyrethroids, however, have the addition of a cyano group in an S-configuration on the alpha (a) carbon of 3-phenoxybenzyl group, along with a dihalovinyl group off the cyclopropane ring. NDRC 161, or deltamethrin, has the highest insecticidal activity of all insecticides,. with an u:>;0 in the range of 0.03 mglkg insect (Elliot et al., 1974). Further alterations of the pyrethroid structure involved the use of an aromatic ring in lieu of the cyclopropane ring (i.e., fen valerate), which has increased the potential applicability for pyrethroid insecticides (Ohno et al., _1974). As in fen valerate, modification of the side components has become so great that some compounds classified as pyrethroids now possess nothing in common with the natural pyrethrins except the ester group. Nevertheless, these compounds are still classified as pyrethroids, due to similarities _with the naturalpyrethrins regarding their overall molecular configuration and insecticidal actions. 8

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Acid Moi_ety )=J{ ''''C!Ol-) pyrethrin I allethrin tetramethrin X resmethrin (X= CH3l phenothrin (X = CH3l permethrin (X c Cl) eypermethrin (X= Cl l v ti 1111C(Ol-s kadethrin Br)=. '' ( s ''C(Ol-) Br deltamethrin \! $ 111'C(Ol-s fenprapathrin fenvalerate Moiety R 0 pyrethrin I (R = CH=CH2l allethrin (R = H) 0 tetromethrin resmethrin tiodethrin phenothrin permethrin s-o ,,tt;J 0' eypermelhrin deltamethrin fenpropalhrin fen valerate. Fig. 1.2. Structures of various pyrethroid.s. (Re printed from Neurobehavioral Toxicology and Teratology, Vol. 4, Glickman, A.H. & Casida. J.E., Species and Structural Variations Mfecting Pyretbroid Neurotoxicity, pp. 793-799, 1982, with kind permission from Elsevier Science Ltd., The Boulevard, Langford Lane, Kidlington O:XS 1GB, UK) 9

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. Pyrethroid Effects It is well documented that the acute neurological symptoms of the pyrethroids are due to their effects on sodium channel activit}' (Narahashi, 1992). However, the pyrethroids may also have other effects on biological activities. Both sodium channel effects and the well-established non-sodium channel effects will be discussed in this section. Sodium Channel Effects. Electrophysiological studies have revealed that the voltage-sensitive sodium channel is a major target site of pyrethroid action in both vertebrates and invertebrates. Both Type I and Type II pyrethroids prolong the mean open time of the sodium channel during patch clamp recordings of single ion channels (Yamamoto et al., 1983) .. Because of this prolongation, the duration of the sodium current is extended as shown under conventional voltage clamp conditions (Narahashi, 1982b; Kiss, 1988b). This protraction of sodium current, in tum, invokes a long lasting depolarization of the membrane following .a spike (depolarizing after-potential) as revealed under current clamp conditions (Narahashi, 1982a; Yamamoto et al., 1983)(Fig. 1.3). However, since the pyrethroids demonstrate structure-related differential actions on the sodium channel, they therefore yield differing overall effects on the nerve membrane, as will be discussed later. 10

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50 + pyrethroid mV 0 -50 -100 t---1 100 ms 0 mA cm-2 -2 -4 t----1 10 ms Fig. 1.3. Effect of pyrethroid on action potentia: (a), Na current (b) and single Na channel current (c). openings of single Na channels C(!.use prolongation of N;; ctrrrent recorded from a b c a whole cell, resulting in an elevation of :ne depolarizing afterpotential, which eventually reaches th"
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The prolonged opening of voltage-sensitive sodium channels by pyrethroids is due to alterations, in effect, a slowing down, of their gating kinetics (Narahashi 1982h). It is thought that the pyrethroids induce a stabilization of the open state and the other states of the sodium channel by reducing the transition rates among them (Chinn and Narahashi, 1989; de Weille and Leinders, 1989; Brown and Narahashi, 1992). However, the stabilization is thought to primarily when the channel is in a closed or resting state (de Weille and Leinders, 1989; Brown and Narahashi, 1992)(see Fig. 1.4 for Na+ channel structure). Inactivation gate AT REST CLOSED Activation gate DEPOLARIZED OPEN 1-----t 2nm DEPOLARIZED INACTIVATED TTX Fig. 1.4. Schematic diagram of the voltage-sensitive Na+ channel. (Reprinted from Kuffler, S.W., Nicholls, J.G, & Martin, R.A., From Neuron to Brain (2nd edition), Sinauer Associates, Inc., 1984). 12

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Although both Type I and Type II pyrethroids slow down voltage-sensitive sodium channel gating kinetics, they differ in their degree of kinetic modification. This is revealed during voltage clamp studies that step up the membrane potential to a depolarized level, and then bring it back down again to a hyperpolarized level. The resulting membrane current that remains upon repolarization is called a tail current. In voltage clamped frog myelinated nerve fibers, pyrethroids produce a characteristic time constant of tail current decay that is dependent upon their particular structure. The Type II pyrethroids, in general, produce much greater time constants than the Type I pyrethroids (Vijverberg et al;, 1986). The greater enhancement of the tail current durations by the Type II pyrethroids haS been correlated with the greater Type II pyrethroid insecticidal potency (Nishimura et al., 1991). What this means during normal, non-voltage clamped activity is that the Type II pyrethroids will delay of the sodium current more than the Type I pyrethroids. Pyrethroids are thought to modify voltage-sensitive sodium channel kinetics by binding to the sodium channel protein after penetrating the lipid phase of the plasma membrane (Narahashi, 1986b). This binding site appears to be separate from those of other known sodium channel toxins, as shown by binding studies using tritiated toxins on mammalian brain membranes, and by voltage clamp studies on tail currents. Pyrethroids applied to rat brain membranes do not block the binding of tetrodotoxin 13

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(ITX), which binds to the external mouth of the channel and blocks voltage-sensitive sodium channel conductance (Lombet et al., 1988). Moreover, TTX counteracts tetrarnethrin's effect on tail current duration in a non-competitive manner (Lund and Narahashi, 1982). Thus the pyrethroid binding site is distinct from the TIX binding side. Also excluded is the batrachotoxin (BTX) binding site, which has been demonstrated to be located on the inactivation gate receptor site found on the internal mouth of the channel (Tanguy et al., 1984). Binding of BTX makes inactivation of sodium channels inoperative, and greatly prolongs the sodium current. Pyrethroids actually enhance the binding of BTX in rat brain membranes (Lombet et al., 1988), and application of tetrarnethrin to BTX-poisoned squid axons still invokes a prolonged tail current characteristic of tetramethrin action (Narahashi, 1986b). Finally, the binding site cannot be located on the interior of the channel, as the permeability ratios of various permeant cations remained unchanged following exposure to tetramethrin (Yamamoto et al., 1986). Normally, the sodium channels of neuroblastoma cells remain open for several milliseconds following a depolarizing stimulus. However, upon application of pyrethroids, the remain open up to hundreds of milliseconds, or even seconds (Narahashi et al., 1992). Only a very small fraction of the total population of sodium channels requires modification by the pyrethroid in order to prolong the sodium current and thereby invoke a depolarizing after-potential to the level of threshold for 14

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action potentials. Type I pyrethroids provoke a depolarizing after-potential that has been found to be directly responsible for eliciting repetitive activity in squid giant axons at concentrations of 1 to 100 JJM (Narahashi, 1986b), in the neuromuscular junction of the clawed frog at concentrations of 1 to 50 J.I.M (Ruigt and van den Bercken, 1986), and in the cockroach cereal sensory nerve at concentrations in the femtomolar range (Gammon et al., 1983). These repetitive discharges are generated in a diverse group of neurons in the CNS and the peripheral nervous system (PNS), including nerve terminals, intemeurons, sensory nerve fibers, and motor nerve fibers (Narahashi, 1986a) (Fig 1.5). They drastically enhance neurotransmitter release and thereby cause a severe disruption of synaptic transmission. This occurs especially in areas with a dense population of synapses, such as the CNS and the peripheral ganglia, thus explaining the symptoms of hyperexcitation, ataxia, tremors and convulsions that occur l.n Type I pyrethroid-poisoned animals. Fig. 1.5. Sites of action of Type I and Type n pyrethroids on the nervous system.+, degree of generation of repetitive activity (Reprinted from TIPSReviews, Vol. 13, Narahashi, T., Nerve membrane sodium channels as targets of insecticides, pp. 236-241, 1992, with kind sensory neuron permission from Elsevier Sciences Ltd., The CNS Boulevard, Langford Lane, Kidlington OX5 1GB, UK). muscle 15 type I type II -+ ++ -+ -++ ++ -+ -++ ++

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Type II pyrethroids cause the sodium channels to remain open for a much longer duration, and therefore they produce a much greater protraction of sodium current than the Type I pyrethroids (Narahashi, 1986b) .. This produces a substantial and sustained membrane depolarization, which will, in tum, lead to a blockade of action potential generation in many large nerve fibers such as the crayfish and giant squid axon at a concentration of around 1 j.1M (Narah,ashi, 1985) or a frequency dependent depression of action potentials in clawed frog peripheral nerves a:t concentrations greater than 1 J.1M (Glickman and Casida, 1982). Both of these effects are probably due to the sustained membrane depolarization causing a reduction in the driving force pushing sodium into the cell, in addition to sodium channel inactivation (Matsuda et al.; 1991). In other neurons, such as sensory neurons, the depolarizing action on the membrane potential yields repetitive discharges (Fig. 1.5). As sensory neurons are particularly sensitive to even slight membrane depolarizations, the sensory nervous system is especially prone to severe effects (Narahashi, 1986a), and this explains the abnormal facial sensations experienced by humans who have been exposed to Type II pyrethroids (He et al., 1988). The depolarizing action on the membrane potential will also affect presynaptic nerve terminals (Narahashi, 1985). Large amounts of neurotransmitter are released, causing a severe disruption in synaptic transmission. In addition, depletion of neurotransmitter could occur in certain regions of the CNS, thus inducing seizures (Clark and Brooks, 1989). Therefore, the various poisoning 16

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symptoms of Type II pyrethroids as represented by salivation, choreathetosis, and tremors can be explained by their action on the nerve membrane. Non-Sodium Voltage-Sensitive Ion Channel Effects. Pyrethroids have been found to have various effects on other ion channels. High concentrations of pyrethroids (100 uM) cause blockage of potassium channels (Omatsu et al., 1988) and suppression of the potassium current (Nishimura et al., 1989) in crayfish giant axons. In addition, the transient outward potassium current in molluscan neurons has also been found to be attenuated (Kiss, 1988a). In contrast to these results, however, is the finding that 10 fenvalerate demonstrated no effects on the potassium current in voltage clamped crayfish giant axons, but did cause a great prolongation of the sodium current at this concentration (Salgado et al., 1989). Further studies are needed to determine the toxicological significance of these findings. Several investigations have suggested that calcium channels may also be affected by pyrethroids. Tetramethrin, a Type I pyrethroid, greatly attenuates one of two types of calcium current in whole-cell patch clamping of neuroblastoma cells (Narahashi, 1986a). Moreover, at concentrations of 50 both deltamethrin and allethrin partially block two types of calcium curren.ts in rat hippocampal neurons (Frey and Narahashi, 1990). Interestingly, at a concentration of 10 both Type I and Type II pyrethroids inhibit the binding of nimodipine, an L-type calcium channel blocker, to rat brain synaptosomes (Ramadan et al., 1988). Finally, These data suggest that. 17

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a Ca2+ current resulting from the depolarizing effect of the pyrethroids could be partially counteracted by a calcium channel blocking effect. Interestingly, Clark and Brooks (1989) have remarked on the structural and chemical similarity between the Type IT pyrethroids and phenethylamine-type Ca2+ channel blockers such as verapamil and 0595. Neurotransmitter-Operated Ion Channels. In addition to voltage-sensitive ion channel effects, several lines of evidence have suggest thatpyrethroids may also affect neurotransmitter-gated ion channels. In rat brain membranes, Typen pyrethroids (at concentrations .ranging from 1 to 5 partially .Inhibit the. binding of radiolabelled ligands which bind to a specific site on the gamma-aminobutyric acid (GABAA) receptor channel complex (Lawrence and Casida, 1983; Casida and Lawrence, 1985). Facilitation of this Type IT-induced inhibition occurs in the presence of GABA in piscine brain membranes (Eshleman and Murray, 1990). In addition, symptoms of type n toxicity in cockroaches can be postponed by diazepam, an allosteric modulator of the GABAA receptor which increases the GABA;.. channel's opening frequency (Gammon et al., 1982). It has been suggested that these modulatory effects are GABA-antagonistic. Chloride uptake is inhibited in GABA stimulated mouse brain vesicles upon addition of Type IT pyrethroids (Abalis et al., 1986). However, other studies have failed to yield GABA-antagonistic effects of the pyrethroids. Deltamethrin, at a concentration of 10 J.IM, has no effect on the GABA-18

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activated inward chloride current in rat dorsal root ganglion neurons (Ogata et al., 1988). Moreover, deltamethrin and fenvalerate actually enhance GABA-ergic recurrent inhibition in the hippocampus of conscious rats (Gilbert et al., 1989). Similarly, deltamethrin evokes an exaggeration of inhibition in rat dentate gyrus granule cells (Joy and Albertson, 1991). Pyrethroids have also been "found to interact with the nicotinic acetylcholine (ACh) gated ion channel. In Torpedo electric organs, a 100 J.LM concentration of various pyrethroids inhibits the binding of the non-competitive antagonist, perhydrohistrionicotoxin (HTX) to ACh receptors, with the Type I pyrethroids being more potent than the Type II pyrethroids (Eldefrawi et al., 1985). However, the study also found that pyrethroids did not alter receptor-activated sodium influx, suggesting that they do not inhibit receptor activation. In contrasno this finding, both 10 J.LM allethrin and 10 J.LM cyphenothrin reduce the peak amplitude of the nicotinic membrane dei>Oliuization by half in cultured mouse neuroblastoma cells. (Oortgiesen, et al., 1989); Similarly, 10 J.LM deltamethrin reduces the Ach-induced current in snail neurons (Kiss and Osipenko, 1991). The somewhat contradictory results. on pyrethroid effects on the GABA receptor channel and on the Ach receptor channel indicate that further experiments are needed to help determine any possible interactions between pyrethroids and neurotransmitter operated ion channels. However the results thus far indicate that the effects are 19

PAGE 33

mainly indirect and may not contribute greatly to the cellular effects of pyrethroids. ATPase Activity. High concentrations of pyrethroids (around 100 J.IM) inhibit ATPase activity in both vertebrates and invertebrates (Clark and Matsumura, 1982, 1987; Sahib et al., 1987; Al-Rajhi, 1990). Ca2+/Mg2+ ATPase enzyme activity in microsomal fractions isolated from rat brain has been shown to be very sensitive to pyrethroids, with the Type II pyrethroids causing greater inhibition than the Type I pyrethroids (Al-Rajhi, 1990). Congruent results regarding the differential sensitivity of brain Ca2+/Mg2+ ATPase enzyme activity to pyrethroid type have been obtained in both the cockroach and the squid (Clark and Matsumura, 1982; Clark and Matsumura, 1987; Clark, 1986). In contrast, the hydrolysis of ATP by the Na+/Ca2+ exchange is inhibited to a greater extent by Type I _pyrethroids than by the Type II pyrethroids, and it appears to be less sensitive to pyrethroids in general than the Ca2+/Mg2+ ATPase enzyme (Clark and Matsumura, 1987; Clark, 1986). Since both ATPase and the Na+/Ca2+ exchange are involved in calcium regulation in the cell, inhibition of either of these two. enzymes could result in a disruption of calcium homeostasis and thereby modify neuronal growth (as discussed in Section II). Calmodulin. Pyrethroids have been found to inhibit of calmodulin-stimulated Ca2+ ATPase activity, but not basal Ca2+ ATPase activity at concentrations of around 100 J.1M (Sahib et al., 1987). Therefore a possible interaction may exist between pyrethroids and calmodulin. Both permethrin and cypermethrin inhibit calmodulin 20

PAGE 34

from bovine heart at relatively high concentrations (100 1JM) (Rashatwar and Matsumura, 1985). The anti-calmodulin agent W-7, however, does not demonstrate similar neurophysiological effects on crayfish giant axons as the pyrethroid meta methylbenzyl pyrethrate (Matsuda et al., 1993). Moreover, calmodulins from pyrethroid resistant strains of the German cockroach and housetly exhibit properties which have no significant differences from the calmodulins of susceptible strains, indicating that calmodulin inhibition is probably not an avenue of pyrethroid toxicity in these insects (Charalambous and Matsumura, 1992). Further studies are needed to help clarify whether or not pyrethroids interact with calmodulin. Since calmodulin is an important regulator. of [Ca2+]in, and performs an important role in many biological reactions, many Ca2+/calmodulin-related systems could potentially be affected. Protein, Phosphorylation. Deltamethrin augments the protein phosphorylation brought on by depolarization in synaptosomes of rat brain (Enan and Matsumura, 1991, 1993) and squid optic lobe (Matsumura et al., 1989). The investigators observed an increase in phosphorylation of synapsin I, calcium-calmodulin dependent protein kinase II (CaM-Kinase II), neuromodulin, and an unidentified 38 KD protein upon exposure to rtanomolar concentrations of deltamethrin. Charalambous and Matsumura (1992) observed similar effects in synaptosomes from the German cockroach and the housefly. In the presence of deltamethrin, a significant increase 21

PAGE 35

in the total level of endogenous protein phosphorylation occurs in susceptible strains, but not in knockdown resistant strains (KDR). The deltamethrin-induced effects on protein phosphorylation activities were assumed to be mediated, in part, by calcium. For one, deltamethrin only invokes an increase in protein phosphorylation after depolarization of the synaptosomal membrane. Moreover, fura-2 measurements have revealed that the increase in protein phosphorylation is accompanied by an increase in intrasynaptosomal free calcium ion concentration beyond that induced by depolarization alone (Enan and Matsumura, 1991). Dunkley et al. (1986) have shown that increases in 32P labelling of phosphopeptides in rat cortical synaptosomes occurs in a Ca2+-dependent manner. Enan and Matsumura (1991) found that the increase in protein phosphorylation is mediated in part by an increase in intracellular Ca2+ that is released from intracellular stores. This rise is may not be entirely attributable to the interaction of pyrethroids with sodium or calcium channels. The addition of tetrodotoxin, a sodium channel blocker, and verapamil, a blocker of certain types of calcium channels, does not completely eliminate the deltamethrin-induced increase in protein phosphorylation in depolarized synaptosomes. In addition, the increase still occurs when extracellular Ca2+ is replaced with barium, which although able to pass through the calcium channel upon depolarization, can not bind to calmodulin and does not activate CaM-Kinase II. Thus the increase in calcium may be partially ascribable to a release of calcium from intracellular stores. 22

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Enan and Matsumura speculate (1991) that the release of calcium from intracellular stores is caused by a deltamethrin-induced activation of the phosphoinositide pathway. Activation of the pathway would cause an increase in inositol trisphosphate (IP3 ) and also the activation of protein kinase C. The increase in IP3 in turn, would cause release of calcium from the endoplasmic reticulum. The raised intracellular calcium would then induce activation of calcium dependent protein kinase C, CaM Kinase II and calcineurin, a calcium and calmodulin-dependent phosphatase. Since neuromodulin is a preferred substrate for protein kinase C, the enhancement of its phosphorylation by deltamethrin upon depolarization of the synaptosome could corroborate the involvement of the IP3 pathway. Enan and Matsumura (1993) have indeed found that there is activation of the phosphoinositide/protein kinase C pathway in rat brain synaptosomes by deltamethrin, although the exact mechanism of such is unclear, since the membrane bound receptor portion of the pathway appears not to be involved in the increase in IP3 In addition, stimulation of phosphoinositide formation by Type II pyrethroids is largely inhibited by dibucaine, which inhibits sodium channel activation, and by tetrodotoxin, suggesting that it involves the sodium channel to some extent (Gusovsky et al., 1989). In addition, picomolar concentrations of deltamethrin have been found to directly stimulate protein kinase C (Enan and Matsumura, 1993). In addition to protein kinase C, picomolar concentrations of deltamethrin also have 23

PAGE 37

the capacity to directly stimulate cAMP-dependent protein kinase. Such direct stimulation of protein kinases by deltamethrin could explain the late increases in protein phosphorylation that occur even after 3 to 5 minutes, by which time increased calcium levels should have activated phosphatases (Matsumura et al., 1989). In addition, nanomolar concentrations of Type II pyrethroids have been shown to specifically inhibit calcineurin, the predominant calcium-calmodulin stimulated phosphatase (Enan and Matsumura, 1992). Thus phosphoproteins such as neuromodulin and Synapsin I may not be returned to their dephosphorylated state. Some of these protein phosphorylation effects might also be explained by the pyrethroid-mediated effects on the sodium channel. Enan and Matsumura (1991) suggest that the protein phosphorylation increase is in part due to an IP3-mediated release of calcium from intracellular stores since Na+ influx-mediated depolarization was blocked. by TIX and Ca2+-influx was blocked by veraparnil. However, TTX insensitive (Ginsburg and Narahashi, 1991) and veraparnil-insensitive channels have been found. Thus some of the enhanced phosphorylation could still be caused by depolarization of the membrane causing Ca2+ influx. Calcium intlux through voltage sensitive calcium channels would then activate phospholipase C and thus induce activation of protein kinase C (Kendall and Nahorski, 1985). In addition, cAMP dependent protein kinase activity could be stimulated by way of Ca2+/calmodulin activation of adenylate cyclase. Thus the pyrethroid Na+ channel effects could explain 24

PAGE 38

an increase in phosphorylation in addition to or instead of any possible direct stimulation of PKC or cAMP. Neurotransmitter Release. Nanomolar concentrations of pyrethroids promote neurotransmitter release in both mammalian and non-mammalian nerve terminal preparations (Salgado et al., 1983; Schouest et al., 1986; Eells and Dubocovich, 1988). Both Type I pyrethroids and Type II pyrethroids evoke a concentration-dependent increase in spontaneous release of [3H]acetylcholine from rat brain synaptosomes (Eells et al., 1992). A concentration-dependent release of both dopamine and acetylcholine by fenvalerate is also observed in rabbit striatal slices (Eells and Dubocovich, 1988). In insects, deltamethrin induces massive amounts of neurotransmitter release and subsequent depletion of synaptic vesicles in motor nerve terminals (Salgado et al., 1983; Schouest et al., 1986) .. In contrast to the previous experimenters, Brooks and Clark (1987) found that pyrethroids had no effect on neurotransmitter release in rat brain synaptosomes that had not been previously depolarized, but enhanced release in potassium depolarized synaptosomes. Type II pyrethroids appear to have greater efficacy at enhancing neurotransmitter release than the Type I pyrethroids (Eells et al., 1992; Brooks and Clark, 1987). At concentrations ranging between 10 to 100 Type II pyrethroids effect a much greater release of neurotransmitter than the Type I pyrethroids. In accordance with this, plasma levels of catecholamines iii rats with deltamethrin-induced choreoathetosis 25

PAGE 39

are much higher than those of rats with cismethrin-induced tremors (Cremer and Seville, 1982). Interestingly, Brooks and Clark (1987) discerned that the enhancement of norepinephrine release induced by deltamethrin, cypermethrin and fenvalenite corresponds well with the pyrethroids' respective efficacies in prolongation of the sodium currents. Depolarization-induced increases in protein phosphorylation have been correlated with release of neurotransmitter (Dunkley et al., 1986). Due to the depolarizing effect of pyrethroids on nerve membrane, it is therefore probable that there is a corresponding relationship between the augmentation of neurotransmitter release and of protein phosphorylation l>y delta.rnethrin in depolarized synaptosomes. Synapsin I inhibits mobilization of neurotransmitter-containing vesicles in its dephosphorylated. form. A deltamethrin-enhanced increase. in cytosolic ccilcium concentration upon depolarization will iricrease activation of CaM Kinase, and therefore the phosphorylation ofsynapsin I. Upon phosphorylation by CaM Kinase, release is no longer inhibited (Schwartz, 1991). Therefore it seems plausible to assume that an increase in intracellular calcium which occurs upon deltamethrin addition will yield greater neurotransmitter release due to aniricrease in CaM activation._ Indeed, the rate of occurrence of miniature EPSPs was found to be greatly enhanced in insect neuromuscular preparations in the presence of Type IT pyrethroids, but not in the presence of Type I pyrethroids (Salgado et al., 1983). Inhibition of calcineurin by 26

PAGE 40

Type II pyrethroids (Enan and Matsumura, 1991) could prolong the phosphorylated state of Synapsin I. In addition, since phosphorylation of neuromodulin is correlated with neurotransmitter release (Dekker et al., 1989), and since it is dephosphorylated by calcineurin, prolonged neurotransmitter release could be evoked by way of this pathway also. Pyrethroids and the Hippocampus Verschoyle and Aldridge (1980), who were the original classifiers of the T and CS syndromes, suggest that the T syndrome is due to pyrethroid actions on the PNS, whereas the CS syndrome has more of a central component. However, the distinct motor symptoms of both syndromes were later discovered to originate in the CNS at the level of the spinal cord (Bradbury et al., 1983; Gray and Rickard, 1982), and are associated with the levels of unmetabolized pyrethroid in the brain (Rickard and Brodie, 1985; Anadon et al., 1991). In addition, injection of pyrethroids into the CNS of mice invokes much greater toxicity than does injection in to the PNS (Staatz et al., 1982). Permethrin has been found to accumulate in concentrations which exceed plasma level in various regions of the brain, with main targets being the hypothalamus, hippocampus, and frontal cortex (Anadon et al., 1991). Interestingly, the elimination half-life for the hippocampus and the frontal cortex is also much greater than that of the plasma. 27

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Although it has been suggested that pyrethroid toxicity attenuates CNS inhibition, in part due to the similarity between behavioral syndromes following administration of deltamethrin and picrotoxin, a neurotoxin which blocks the A-type gamma-amino butyric acid (GABAA) receptor (Gammon et al., 1982), this assumption has been proven to be false. CNS inhibition remains and is even augmented throughout the course of either Type I or Type IT severe pyrethroid intoxication (Joy et al., 1990). In the hippocampus, stimulation of the primary (excitatory) synaptic input to the granule cells of the dentate gyrus (the perforant path) invokes activation of collaterals which mediate GABA-ergic inhibition. Both Type I and Type II pyrethroids prolong the inhibition of granule cell excitability, with the Type II pyrethroids causing a much greater prolongation than the Type I pyrethroids (Joy et al., 1990; Gilbert et al., 1989). It has been demonstrated that deltamethrin-induced actions on the perforant path axons and their respective receptors on the granule cells do not cause the observed effects. This suggests that it is the basket cell intemeurons of the hippocampus that mediate the pyrethroid-induced enhancement of granule cell inhibition (Joy et al., 1989). Supporting this is the finding that mephenesin, an agent which selectively depresses activity of the polysynaptic intemeurons of the spinal cord (Bradbury et al., 1981), antagonizes the effects of deltamethrin on granule cells (Joy et al., 1990). As mephenesin appears to have selectivity for intemeurons (Klee and Wagner, 1967), it could be that mephenesin's antagonizing effects are due to its actions on the basket 28

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cells. Neurite Development The pyrethroid-induced effects on various cellular activities may invoke changes in neuronal development. Normal neurite structure and growth processes will be described in this section in order to further elucidate potential avenues of pyrethroid action. The section will include a description of calcium homeostasis in addition to an examination of calcium-mediated effects on .neurite growth. Second messenger systems that mediate neuronal growth will also be examined. Neurite Structure and Growth An actively growing neurite consists of a neurite. shaft extending from the soma of the neuron, with a specialized motile structure at its tip called the growth cone (Fig. 1.6). The growth cone is an enlargement of the shaft of the neurite and has a flattened appearance. Finger-like projections of the plasma membrane called filopodia repeatedly extend and retract from the leading edge of the growth cone. Lamellipodia, which are also motile, consist of ruffled edges of thin membrane located between the filopodia. 29

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Soma Neurite Shaft Low [ca2+Jin enhances I I I I I I I I Growth Cone High [ca2+Jin enhances actin-based motility and exocytosis Filopodia (pull) Fig. 1.6 .. Structure of a neurite showing some of the Iirocesses involved in neurite growth, which are favored by differeni levels of calciUm. A high density" of .actin filaments is found in both filopodia and lamellipodia (Forscher and 19S8; Miichison and Kirschner, 1988), and their motility is regulated by actin-dependent processes (Letourneau, 1983; Mitchison and Kirschner, 1988). Indeed, addition of actin.,depolymerizing drugs such as cytochalasin cause a decrease in or cessation of growth cone motility (Marsh and Letourneau, 1984; Forscher and Smith, 1988). Lamellipodial and filopodia! protrusions of a growth cone result from a net polymerization of actin filaments at their leading edges (Mitchison .and Kirschner, 1988). 30

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In contrast, the neurite shaft is mainly composed of microtubules which extend in parallel array along its length and into the growth cone lamella (Letourneau, 1983). Elongation of the neurite shaft involves both the collapse of the dense network of actin fihunents and associated proteins just beneath the plasma membrane (cell cortex) and the elongation and bundling of microtubules behind the advancing growth cone (Tanaka and Kirschner, 1991). Bamburg .et al. (1986) have found that the microtubules in the growth cone are much more sensitive to depolymerizing drugs than microtubules in the shaft of the neurite. Since the primary target of these depolymerizing drugs is where assembly of microtUbules is occurring, this suggests that it is in the growth cone where microtubule elongation is occurring. In addition, labelled tubulin subunits, are preferentially incorporated at the tip of the neurite (Okabe and IJirokawa, 1988; Lim et al., 1990.; Tanaka and Kirschner, 1991). Two widely debated theories, termed the "ptill" and "push" theories, have been offered to explain the process of neurite elongation. Bray (19&4. 1987) proposes that actin-based growth cone motility.creates mechanical tension in the neurite, and that this is used to "pull" the neurite along. Supporting this are experiments performed by Lamoureux. et al. ( 1989) that show a strong terriporai correlation between neurite tension increases and growth cone advance. This pulling mechanism may be due to an interaction of the actin cortex withmyosin, which has been found to be localized in lamellipodial and filopodia! regions. (Brigman and Dailey, 1989). 31

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In contrast, the "push" theory contends that neurite elongation is driven by microtubule based transport of cytoplasm down the neurite shaft and into the growth cone. Goldberg and Burmeister (1986) have used video-enhanced contrast-differential interference contrast microscopy on Aplysia axons to discern that membrane-bound vesicles are added to the main body of a growth cone, and that there is a progressive filling of lamellipodia with Aletta and Greene (1988) also have used high resolution video microscopy, and visualized the entrance of'cytoplasm into newly extended lamellipodia. Neither group has observed either fiiopodial contraction or forward movement of the main body of the growth cone, which would be consistent with the "pull" mechanism of neurite elongation. In addition; cytochalasins cause a disappearance of lamellipodia and filopodia and consequent cessation in growth cone activity, but they do not affect microtubule polymerization and neurite elongation (Marsh and Letourneau, 1984; Forscher Smith, 1988). Finally, as suggested by the "pull" theory, quiescence of the actin rich cortex should cause a release of constriction on the neurite microtubules, and thus instigate the microtubule bundling that occurs during neurite elongation. Tanaka and Kirschner. (1991) have found, however, that microtubule bundling precedes collapse of the cortex. These data suggest that neurite elongation is not due to the tension exerted on the neurite by growth cone motility, but rather that elongation results from the entrance of cytoplasm into the growth cone. 32

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In summary, a hypothesis for the sequence of events during neurite growth is as follows: Just prior to the initial appearance of a neurite, called initiation, tuhulin and actin filaments accumulate next to the area where a growth cone will emerge from the cell body (Lefcort and Bentley, 1989). Mter emergence of the growth cone the filapodia randomly extend and explore the environment Their directionality is determined by interactions of the actin cortex of the leading edge with the extracellular matrix and with diffusible molecules which bind to surface receptors and transmit signals across the plasma membrane (Jessen, 1991). The growth cone attaches to the substrate underneath its central portion, and then elongation begins by the spreading of the lamellipodia. Subsequently, in a process called consolidation, new membrane is inserted into the growth cone aild the lamellipodia progressively fills in with cytoplasm and organelleS. Consolidation continues as the thickening growth cone loses motility and microtubules in the growth cone become stabilized (Aletta and Greene, 1988). Finally the cylindrical neurite shaft is formed as the microtubules form bundles and the actin cortex of the broadened growth cone collapses (Tanaka and Kirschner, 1991). Calcium Regulation by the Nerve Cell A cell maintains a very low intracellular calcium concentration (about lOOnm) compared to the concentration in the extracellular fluid (about 1mM). Since a 33

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transient rise or fall in [Ca2+]in modulates many second messenger pathways, maintenance of Ca2+ homeostasis is crucial for control of neuronal processes. The first of three major control pathways is the plasma membrane, which utilizes both Ca2+ ion channels and membrane-bound pumps for Ca2+ regulation (Verity, 1992). Ion channels are proteins surrounding pores in the plasma membrane. They can he either ligandor voltage-gated and influx of calcium through these channels can effect transient rises in intracellular calcium. The plasma membrane also contains the ATP/Mg2+ -dependent Ca2+ extrusion pump whiCh utilizes energy derived hydrolysis of the high-energy phosphate bonds of adenosine triphosphate (ATP) in order to pump calcium against its electrochemicai gradient into the extracellular fluid. The Na+/Ca2+ exchange system also pumps Ca2+, but couples:the influx of Na+ with the efflux of Ca2+. The exchanger has a relatively low affinity for Ca2+ and therefore does not efficiently operate until [Ca2+]in reaches 10 times its normal level. The second major control pathway is the Ca2+ sequestration and release organelles such as the smooth endoplasmic reticulum and mitochondria, which like the plasma membrane, contains an ATP-dependent Ca2+ pump to pump Ca2+ against its concentration gradient into the organelle. Lastly, the high-affinity Ca2+ binding proteins calmodulin and parvalbumin also regulate Ca2+ homeostasis by acting as Ca2+ buffers. 34

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Calcium-Mediated Effects on Neurite Outgrowth There is accumulating evidence that calcium plays an integral role in the regulation of the events involved in neurite growth, including initiation, growth cone motility and elongation in both vertebrate and invertebrate neurons (Anglister et al., 1982; Lipton, 1987; Mattson and Kater, 1987; Mattson et al., 1988b; Silver et al., 1989; Audesirk et al., 1990). Studies have found that these stages may require different optimal levels of Ca2+ concentrations during their time of occurrence (Cohan et al., 1987; Mattson et al., 1988b,d) (Fig. 1.6). Moreover, Ca2+ may instigate transformation from one process. to the next (Goldberg, 1988). Therefore it seems reasonable to assume that cells might maintain their [Ca2+]in within a compromise range which is an overlap for all three events. Additionally or alternatively, the neuron could utilize localized Ca2+ influx across the plasma membrane which would allow for differing levels of Ca2+ in different areas of the neuron 1. 7). Initiation of Neurites; There is coQflicting evidence on the roles that intracellular calcium concentration ([Ca2+]in) and calcium influx play on neurite in_itiation. Mattson et al. (1988b), have found that addition of low concentrations of the Ca2+ channel blockers La3+, Co2+, and Cd2+ to the media enhanced neurite outgrowth in Helisoma neurons, while the calcium ionophore A21387 reduced it. In contrast, inhibition of calcium influx by La3+, Mn2+, Co2+ and nitrendipine in chick retinal ganglion cells decreases neurite initiation (Suarez-Isla et al., 1984). Similarly, the specific L-type 35

PAGE 49

Ca2+ channel inhibitors nifedipine and Cd2+ decrease initiation in chick embryo brain neurons, embryonic rat hippocampal cells, and NIE-115 neuroblastoma cells (J. Dohariyos, unpublished observations). These results could indicate that there are differences between vertebrates and invertebrates in regard to their optimal [Ca2+lw levels. Fig. 1.7. A pseudocolor representation of Ca2+ concentration within an active growth cone as derived from a digital analysis of paired fura-2 images. (Reprinted from TINS, Vol. 11, Kater, S.B., M_attson, M.P.; Cohan, C. & Connor, J., Calcium regulation of the neuronal growth cone, pp. 315-321, 1988, with kind permission from Eisevier Science Ltd., The Boulevard, Langford Lane, Kidlington OX5 1GB, UK). Growth Cone Motility. The .viewpoints are also disparate in regard the level of [Ca2+]in in growth cones arid the effect of influx on growth cone motility. Several experiments provide evidence that a higher calcium concentration and/or calcium influx is required in the growth cone in order for motility to occur. Addition of EGT A to the cell media of chick dorsal root ganglion cells causes a cessation of both 36

PAGE 50

lamellipodial and filopodia! motility (Lankford and Letourneau, 1989). In addition, the addition of the calcium channel blocker, Co2+, to the media of Aplysia neurons decrease the formation of new lamellipodia (Goldberg, 1988). Moreover, the calcium ionophore A23187 induces the formation of new lamellipodia in Aplysia neurons and the expansion of growth cones in neuroblastoma cells. Finally, Helisoma neurons show reduced growth cone motility in the presence of various calcium channel blockers (Mattson and Kater, 1987; Mattson et al., 1988b). In accord with all of these experiments is the finding. by Silver et al. (1990), that calcium channel clustering occurs in the growth cones of neuri.tes of NIE-neuroblastoma cells, and that these clusters cause an elevation of calcium levels within the growth cone. In addition, motile growth cones of Helisoma neurons a higher free [Ca2+]in than growth cones that have ceaSed movement (C()haD. et al., 1987). In seeming contrast to these .findings are several experimentS suggesting that a rise in [Ca2+lm decreases .growth cone motility. The presence (5-HT), which causes a depolarization"dependent increase in [Ca2+lm in growth cones (Cohan et al., 1987), induces retraction of lamellipodia and the loss of tilopodia in Helisoma neurons et al., 1991). In addition, Ca2+ ionophore-treated cultures of chick dorsal root ganglia neurons show changes in.growth cone morphology, presumably by disruption of the actin filament network in the peripheral areas of the growth cone (Lankford and 1989). Finally, [Ca2+lm measurements using fura-2 37

PAGE 51

demonstrated that there were no differences between [Ca2+lm in motile growth cones as compared to quiescent growth cones of neuroblastoma cells (Silver et al., 1989). These results indicating that an increase in [Ca2+lm inhibits growth cone motility, however, are not necessarily contradictory to the results showing that a decrease in [Ca2+]in causes growth cone motility inhibition .. A plausible explanation is that there might be an optimal [Ca2+]in for growth cone motility, and concentration levels. that are either too high or too low will inhibit motility. Elongation. Studies ort elongation have been relatively consistent, with the overall conclusion being that influx of calcium decreases neurite elongation. Increased calcium influx caused by ionophores or by activation of voltage-sensitive calcium channels from neurotransmitter-induced depolarization of nerve membrane inhibits or eliminates elongation or_ causes neurite retraction in Helisoma -neurons, rat hippocampal neurons, chick dorsal root neurons (Mattson and Kater, 1987; Mattson et al., 1988a,b,c; Polak at al., 1991; Lankford and Letourneau, 1989). Consistent with these fmdings are data from studies on LA-N-5 neuroblastoma cells, Helisoma neurons, and rat hippocampal neurons, showing that calcium channel blockers augment elongation rate (Mattson and Kater, 1987; Mattson et al., 1988a,b; Sidell et al., 1990). Furthermore, reduced Ca2+ media enhances elongation rate in Helisoma neurons (Mattson and Kater, 1987). Audesirk et al. (1990), however, have found no effect of voltage-sensitive calcium channel blockers on mean neurite length 38

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of either chick embryo brain neurons or N1E-115 neuroblastoma cells. Axonversus Dendrite Sensitivity to Calcium. Guthrie et al. (1988) have found that, in actively growing cultured rat neurons, the intracellular calcium levels are lower in the axon than in the dendrites. Consistent with this are data from Mattson et al. (1990) that a gradient of Ca2+ concentration might ciffect axon initiation Focal application of K+ or the Ca2+ ionophore A23l87 to an initial axon in embryonic rat hippocampal neurons causes the_ formation of an additional axon at a site on the soma that was distant to the site of the initial axon. They also observed a Ca2+ gradient spread between the point of application and the soma. Finally, hippocampal pyramidal neuron dendrites have greater to excitatory amino acid-induced Ca2+ increases and to A23187 than does the axon (Mattson et al., 1988a,c). Axons -continue to extend in the presence of 50 -glutamate whereas dendrites retract at a steady rate. The greater excitatory amino acid sensitivity of the dendrites to calcium could have a substantial affect on the differential neuroarchitectural forms of the axon and dendrites. In such an instance, axons would be able to achieve a greater elongation rate and grow the long distances needed to reach their targets,while dendrites would not be able to elongate as much and would be contined to the local area. Furthermore, since axons and dendrites contain some differences in their cytoskeletal proteins (Matus, 1988) it is plausible that these proteins mediate their differential sensitivity to calcium. 39

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Branching. Branching of neurites also seems to be correlated with calcium influx. Helisoma neurons grown in the presence of La3+ show a large decrease in the amount of neurite branching. In addition, these neurons display a decrease in the number of filopodia per growth cone (Mattson et al., 1988b). Since growth cone motility and' neurite branching appear to be directly related and similar conditions favor both, it is reasonable to assume that filopodia and lamellipodia play an active role in generating neurite branching and that this process is influenced by calcium (Mattson and Kater, 1987; Mattson et al., 1988b). Second Messenger Systems that Mediate Neuron Growth Processes Several second messenger pathways have been implicated.in processes involved with neurite outgrowth. Activation of the pathways occurs in respo-nse to a primary signal, such as neurotransmitters, growth factors, certain substrate molecules, and electrical activity. These pathways presumably act by altering the structure or function of cytoskeletal proteins or other neuronal proteins (Jessen, 1991). Protein Kinase C. The phosphorylation of key cell membrane and transport proteins by protein kinase C (PKC) implicates PKC as having a significant role in neurite outgrowth (Nishizuka, 1986). A study using antibodies to PKC in PC12 cells simulated by nerve growth factor (NGF) revealed that activation of PKC promotes neurite initiation (Altin et al., 1992). Consistent with these results are findings that 40

PAGE 54

the PKC inhibitors sphingosine and calphostin C suppress neurite initiation in NGF stimulated PC12 cells (Hallet al., 1988) and in rat hippocampal neurons (Cabell and Audesirk, 1993). The results are less consistent however, regarding phorbol esters, which activate PKC. Initiation is induced by tetradecanoyl phorbol acetate (TPA) in chick sensory ganglia explants '(Hsu et al., 1989) and in PC12 cell cultures (Hall et al., 1988), whereas phorbol12-myristate 13-acetate (PMA) has no affect on initiation in cultures of rat hippocampal cells (Cabell and Audesirk, 1993). Elongation is probably not significantly affected by PKC, since both inhibitors and activators have little affect on axonal and dendritic elongation in rat hippocampal neurons (Cabell and Audesirk, 1993). Interestingly, PMA causes a reduction in growth cone tilopodial activity upon focal application (Mattson et al., 1988c ), but also increases neurite branching at concentrations greater than 10 nM (Cabell and Audesirk, 1993). These data indicate that PKC does have soine influence on neurite growth; however, its exact roles remain to be elucidated. Cyclic Adenosine Monophosphate. Cabell and Audesirk (1993) discovered that inhibition of cyclic adenosine monophosphate (cAMP) -dependent protein kinase appears to have little or no effect on neurite initiation in rat hippocampal cells (Cabell and Audesirk, 1993). Activation of the cAMP second messenger system with forskolin causes enhancement of neurite elongation in rat hippocampal neurons, especially in dendrites (Mattson et al., 1988c). These effects could have been 41-

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mediated locally through the growth cone, since focal application of forskolin to the growth cones causes enhanced outgrowth also. The cAMP-dependent protein kinase inhibitor, KT5720, however, does not have any effect on either axon or dendrite length in rat hippocampal cultures, but does decrease branching (Cabell and Audesirk, ). The effects of cAMP on neurite development are inconclusive, but could be governed in part by cAMP-dependent protein kinase phosphorylation of microtubule associated proteins, causing a decrease in actin-microtubule association (Seldon and Pollard, 1983). Calmodulin. Calmodulin probably mediates its effects on neurite growth by way of interaction with calcium-calmodulin dependent enzymes and other proteins. For instance, calcium-activated calmodulin can alter ihe of microtubules with microtubule associated_ proteins (MAPs) __ and thereby affect the stability of microtubules (Matus, 1988). In addition, calmodulin has also been shown, in the presence of nanomolar concentrations of Ca2+, to inhibit binding of spectrin, a major neuronal structural protein,to membrane proteins in nit brain synaptosomes (Steiner et al., 1989). Calmodulin has also_ been found to bind to and be localized by neuromodulin, a major constituent of neuronal growth cone membranes, and to stimulate calcineurin, a phosphatase which dephosphorylates neuromodulin (Liu and Storm, 1989). High concentrations of calmidazolium, a calmodulin antagonist, inhibit initiation in 42

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rat hippocampal neurons (Cabell and Audesirk; 1993). These effects are probably not mediated through calcium-calmodulin dependent protein kinase IT (CaM kinase II), since the Ca2+/calmodulin-dependent protein kinase II inhibitor KN62 has no effect on neurite initiation (Cabell and Audesirk, 1993). Calmidazolium also causes an inhibition of growth cone motility in Aplysia neurons (Goldberg, 1988). Consistent with this is the finding that the calmodulin antagonist CGS 9343B mitigates the 5-HT induced calcium suppression of neurite growth and growth cone motility in Helisom.a buccal ganglion neurons (Polak et al.,. 1991). These data all demonstrate the importance .of calmodulin. in regulating neurite growth processes. Tetrodotoxin EffectS on Neurite Development Tetrodotoxin ('ITX) is a potent neurotoxin which acts by selectively blocking voltage-sensitive sodium channels and thereby eliniinating the sodium current. The action of TIX on sodium channels antagonizes pyrethroid-induced effects on the sodium current (Narahashi, 1982b, 1986a;. Ghiasuddin and Soderland, 1985). Therefore TTX was utilized as an investigative tool in some of the experiments undertaken for this thesis. By blocking the sodium current by addition of ITX to the cell media, any significant morphological effects produced by the pyrethroids could be assumed to be non-sodium channel effects on neuronal growth. Although 'ITX resiStant channels have been found (Ginsburg and Narahashi, 1991; Narahashi et al., 43

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1992), our laboratory demonstrated that the sodium current in rat hippocampal neurons is completely eliminated by 500 nM TfX at membrane potentials greater than 0 m V (see Fig. 1.8). Data on the effects of TfX on neuronal growth mainly come from studies about chronic blockade of synaptically-transmitted, spontaneous action potentials (spontaneous bioelectric activity). Neonatal rat cortex explants demonstrate a decrease in cell survival between one and two weeks when their electric activity is chronically --. blocked by exposure to 100 nM ITX (Ruijter et al., 1991). -Process formation initially drops during the first two weeks of exposure to .ITX, but then is actually enhanced during weeks 3 and 4. In two-day-old cultures of cerebral cortex fetal rat neurons, 1 jlM TTX enhances initiation, the.number of processes per cell, and neurite.elon:gation and subsequent (Van Huizen and Romijn, 1987). These data are in seeming contrasno an earlier study whiCh showed a retardation of growth cone and synapse formati9il by TTX in cerebral cortex during the _tirst three weeks in culture (Van Huiren et al., 1985). However, these differential effects might be explained by the different concentrations of' ITX used or by different ages of the cultures. Since the cultures do not exhibit regular spontaneous bioelectric activity until day 5 in vitro (Van Huizen and Romijn, 1987), ITX enhancement of neurite outgrowth in 2-day-old culture are may not be due to supression of bioelectric activity. Rather, 44

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they are probably induced by a lowered intracellular sodium concentration or more likely by a maintainance of a hyperpolarized membrane potential. A hyperpolarized membrane potential would not allow voltage-sensitive calcium channels to open up and therefore a lack of Ca2+ influx and/or a continuous lower [Ca2+lm could affect neurite growth. pre-TTX 500 nM TTX, 2d at 0 37 c 10 50.0 0 0 ___l 10 -500 10 -rooo 10 -..:1500 10 -2000 10 -2500 -20 0 20 40 60 80 100 120 -20. 0 20 40 60 80 1 00 1 20 Fig. 1.8. Effect of tetrodotoxin on the Na.+ current. Abscissa is time in msec. Ordinate is current in picoamps 45

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Other studies suggest that there is no effect at all of TIX on neurite outgrowth. One group of experimenters found that 100 nM 1TX added to 6-day-old primary cultures of rat cerebral cortex did not alter either growth cone morphology or axonal elongation rate (Ramakers et al., 1991). Another group found that in vivo TTX treatment of regenerating goldfish retinal axons did not alter their rate of regeneration in comparison with the control (Hartlieb and Stoermer, 1989). Finally, when developing retinal ganglion cells from a species of salamander that is sensitive to TIX are chronically blocked by transplantation into another species that produces the toxin, they still can grow to form synaptic connections with their proper targets (Harris, 1980). A Model of Neurite Development Regulation of neuronal growth is mediated by intracellular proteins that function as substrates for the binding of second messengers and protein kinases (Jessell, 1991). These proteins affect neurite growth by modifying the structure and function of the cytoskeleton and its associated proteins. One such protein is the neuron-specific calmodulin-binding protein neuromodulin, which is thought to he involved in neurite initiation (Skene and Willard, 1981), axonal elongation (Zuber et al., 1989; Ramakers et al., 1991), synaptic plasticity (Chan et al., 1986), and neurotransmitter release (Dekker et al., 1989). Neuromodulin is found in great abundance in the brain, 46

PAGE 60

especially during development, with the fetai brain containing 15 to 20 times more neuromodulin than the adult brain. During development, the highest levels of neuromodulin expression occur in the growth cone. It is primarily found in the filapodial regions of axonal growth cones (Goslin et al., 1991), where it associated with the plasma membrane (Van Lookeren et al., 1989). Neuromodulin probably affects neurite outgrowth by modifying the rate that new membrane is added to the growth cone (Meiri et al., 1991; Zuber et al., 1989) An large increa.Se in neuromodulin expression occurs concurrently with the inital appearance of a neurite (Shea et al.,. 198?). Present in such great amounts, neuromodulin is then able to bind up and localize all the calmodulin within a specific region at low Ca2+ levels. Phosphorylation of neuromodulin causes neuromodulin to lower itS affinity for calmodulin and thereby release it in high concentrations. It appears that protein kinase C is the mediator of neuromodulin phosphorylation, since the PKC activators phosphatidylserine and diolein stimulate neuromodulin phosphorylation (Lui and Storm, 1990). Activation of PKC occurs after activation of phospholipase C. Activation of phospholipase C itself, is triggered in response to extracellular cues such as neurotransmitters and neuromodulators or by the influx of Ca2+ through voltage-sensitive Ca2+ channelS (Kendall and Nahorski; 1985). 47

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anchor proteins + CaM-sensitive PIP-+-PIP2 enzymes Fig. 1.9. Model for neurite development showing regulation of free calmodulin levels in neurons by protein kihase C and calcinemin. Extracellular signals coupled to activation of phospholipase C .. may stimulate phosphorylation of neuromodulin (NM) by protein kinase C (PKC) and release of free calmodulin (CaM). Free calmodulin could then stimulate a number of different calmodulin-sensitive enzymes and/or: affect cytoskeleton-membrane interactions. Depolymerization or uncrcisslinking of cytoskeleton J}olymers and release from membrane anchoring sites would result in a local membrane "softening" as a primary event in filapodial extension from growth cones .. Activation of calcineurin (CN) by calmodulin would lead to dephosphorylation of neuromodulin and of calmodulin bound to neuromod!llin. (Reprinted from TIPS, Vol. 11, Lui, Y. & Storm, D.R., Regulation of free calmOdulin levels by neuromodulin: neuron growth and regeneration, pp. 107n 1, 1990, with kind permission from Elsevier Science Ltd., The Boulevard, Langford Lane, Kldlington OX5.1GB, UK). 48

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Local release of calmodulin upon phosphorylation of neuromodulin by PKC would allow for the triggering of many calmodulin-dependent processes which can alter cytoskeletal structure and cytoskeleton-membrane interactions (Fig. 1.9). For example, calmodulin can bind to Ca2+ and form a complex (Ca2+/calmodulin) which inhibits the binding of brain spectrin, a major structural protein in neural tissues, to membrane proteins (Steiner et al., 1989). Moreover, Ca2+/calmodulin dependent protein kinase phosphorylation can regulate the stability of microtubules (Job et al., 1981) and the binding of some microtubule-associated proteins (Sobue et al., 1981). Thus the polymerization and the cross-linking of the cytoskeleton can be temporarily altered to allow for the addition of plasma mem.brane and the formation of filopodia. Cessation of this membrane alteration would occur upon the eventual activation of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase (Lui and Storm, 1989). Dephosphoi-ylated neuromoduJ,in would then be able to bind up calmodulin again: An increase or decrease in Ca2+ could have a major impact on the neuromodulinlcalcineurin regulation of the neurite outgrowth. A [Ca2+]in that was too high could cause a prolonged release of calmodulin by neuromodulin and cause severe disruptions in cytoskeletal dynamics. Alternatively, Ca2+/calmodulin could prematurely activate calcineurin, and not allow time for cytoskeletal restructuring to occur. Finally, activation of cal pain might occur, causing degradation of neurofilaments in the developing axon. Too low [Ca2+]in would not allow for the 49

PAGE 63

release of calmodulin by neuromodulin, nor would it activate calmodulin anyway, and thus no outgrowth would occur at all. Hypothesis The effects of two pyrethroids, pennethrin and cypennethrin, on rat hippocampal cell neurite outgrowth and intracellular calciUm. concentrations were investigated in the group of experiments presented here. The pyrethroid-induced effects of prolonging. the sodium current should cause a depolarization of the rat hippocamp;U cell plasma membrane. This depolarization should then cause an opening of voltage-sensitive calcium channels and thus an influx of caldum. Thus the intracellular calcium cnncentration a5 measured by fura-2 should be expected to rise. Since the Type II pyrethroids generally prolong the opening of sodium channels to a much greater degree than the Type I pyrethroids (Narahashi, 1992), cypennethrin should cause a greater increase in intracellular calcium concentration. than pennethrin. The increase .in intracellular calcium should causes changes in neurite ou.tgrowth. The morphological effectS based on previous experiments with rat hippocampal cells using agents which elevate intracellular catcium should be as follows: 1) Cell survival should be decreased .Glutamate-induced calcium increases cause cell death .in rat hippocampal neurons (Mattson et al., 1988c). 50

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2) The initiation of neurites should be decreased. The calcium ionophore A23187 decreases the outgrowth of neurites (Mattson et al., 1988a). 3) Elongation of neurites should be reduced. Neurotransmitter-induced calcium increases cause suppression of neurite elongation (Mattson et al., 1988a). 4) Mean axon length should not be reduced as greatly as mean dendrite length. Axons are less sensitive to excitatory amino acid and A23187-induced calcium increases than dendrites are. 5) Branching of neurites should be reduced. Branching appears to be directly related to growth cone motility (Mattson et al., 1988b), and since growth cone motility probably requires an optimal range of calcium concentration, then a disturbance of this optimal range (by the pyrethroids) will inhibit branching. Finally, since tetrodotoxin antagonizes pyrethroid-induced etJects on the sodium channel, and since our laboratory demonstrated complete inhibition of the rat 1/ hippocampal cell sodium current by. TfX, then TIX should mitigate the morphological effects of the pyrethroids. 51

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CHAPTER 2 MATERIALS AND METHODS Cell Culture Experiments Isolation and DissoCiation of Hippocampal Cells Embryonic rat hippocampal cells were acquired from timed-pregnant Sprague Dawley (Harlan Sprague-Dawley) rats. The pregnant rats were sacrificed at El8 (embryonic day 18) by cervical dislocation following C02 anesthesia. The brains of the fetal rats were removed and then the hippocampi were dissected and dissociated according to a modification of a protocol described by Banker and Cowan (1977). The hippocampi were placed in a Ca2+ -, Mg2+ -free Hank's balanced salt solution (HBSS, Sigma) containing 10 mM HEPES buffer and 1% antibiotic/antimycotic solution (Sigma). After collection, the hippocampi were dissociated by incubation at 37C in HBSS containing 2 mg/ml trypsin. After 15 min, The trypsin solution was removed and the brain tissue was rinsed with Ca2+-, Mg2+ -free HBSS. The -brain tissue was then incubated in HBSS containing (2 mg/ml) trypsin inhibitor at 37C for 5 minutes, and then rinsed again in HBSS. Finally, the cells were dissociated by trituration with a 52

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sterile fire-polished glass pipette. Cryopreservation of Cells Cryopreservation techniques were perfonned in a slightly modified manner from those of Mattson and Kater (1988). Cells were suspended at a density of 4 million cells/ml in a solution consisting of Eagle's Minimum Essential Medium (MEM) buffered with 10 mM NaHC03 and.25 mM HEPES, and supplemented with 2 mM 1-glutamine, 2% glucose, 1 mM sodium pyruvate, 15 mM KCl (tinal concentration 20 mM) and 10% heat-inactivated fetal bovine serum (FBS, Sigma) and containing 8% dimethyl sulfoxide (DMSO). The cells were counted in a hemocytometer, and then dispensed in 0.25 ml quantities (2 million cells) into 2 ml freezing tubes. The cells were then frozen at -70C in 1" thick styrofoam containers which slow the freezing process. Culturing Techniques For culturing, hippocampal neurons were rapidly thawed by adding 1 ml of 37C media to the cryopreservation vials, and then agitating the cells for 1 to 2 minutes in a 37C water bath. The neurons were then plated in poly-D-lysine (M.W. approx. 100,000 Daltons) coated, gridded plastic culture dishes containing 2 ml of medium, at a density of 200,000 cells per dish. This density was used since the cells will not 53

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normally come in contact with one another, thus allowing measurements to be made of neurites on individual cells. The culture media was the same as the media used for freezing the cells, except that it contained 0.2% glucose, no added DMSO, and 2% FBS. Permethrin and cyperniethrin were dissolved in DMSO in stock solution concentrations so that the total percentage of DMSO dissolved in the culture media was always 0.1% DMSO. The control dishes also contained 0.1% DMSO. Previous experiments have revealed that this concentration 'will not affect any parameters of neurite growth. In experiments on cypermethrin' s effects on neurite outgrowth, the experimental dishes contained 1 10 or 100 cypermethrin. In experiments on the possible counteracting effects of tetrodotoxin (TTX) on permethrin or cypermethrin effects on neurite outgrowth, 100 penriethrin or cypermethrin was used in the experimental dishes, in combination with 500 nM TIX (2% of a stock solution of 25 TfX in deionized water). Three separate controls were used in these experiments and they consisted of 0.1% DMSO and 2% deionized water, 0.1% DMSO and 2% TTX, and 100 pennethrin or cypermethrin (0.1% DMSO) and 2% deionized water. The cells were incubated at 37C in a humidified, 5% C02 atmosphere for 2-4 hours to allow for settling and attachment. At this time, the media were replaced with fresh culture media, so that residual DMSO from freezing was removed. Also at this time, 54

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attached and living neurons were counted in four 250X microscope fields. Subsequently, the cells were incubated for an additional 42-46 hours for a total incubation time of 48 hours. At 48 hours, living neurons (as detined by trypan blue exclusion) were round, phase-bright, and firmly attached to the substrate (cells that did not initiate neurites), or flattened, variably bright or dark, with no apparent vacuolization of the cytoplasm, and usually bearing processes. Very few non-neuronal cells existed at 48 hours, and those that did could be easily ditierentiated from neurons by morphology and immunostaining (Caceres et al., 1986 and Kern, unpublished observations).. The neuronal cells were probably predominantly pyramidal cells, since these are the majority of the types of hippocampal cells taken from E18 rats (Goslin and Banker, 1991). Assessment ofSurvival and Differentiation Survival and differentiation measurements were performed at 48 hours using a digitizing tablet and morphometric software (Sigmascan: Jandel Corp.). Rat hippocampal neurons produce one axon which is morphologically and immunocytochemically distinct from the dendrites in culture (Goslin and Banker, 1991). Axons have been defmed as processes that are at least twice as long as the next longest neurite (Mattson and Kater, 1988). All dishes in every set of experiments were coded in order to avoid any experimenter bias. Decoding occurred 55

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only after completion of parameter measurements at 48 hours. The following parameters were measured and then calculated: (a) Percent survival is defmed as the percentage of cells that remain alive in the same four 250x microscope fields that were examined at 4 hours. (b) Percent neurite initiation is the percentage of living neurons that have produced at least one process that is one soma diameter. This was determined by counting fields until there were at least 30 cells that were alive and at least 10 cells that were initiating neurites. Counting was continued until up. to 50 cells, if 10 growing cells had not yet been fourid. (c) Percent of axonal cells per growing cell was calculated by comparing the number of neurite-bearing cells that had produced an axon to those who had not. (d) Mean axon length was calculated by dividing the sum total of all the axon lengths by the number of axonal cells. (e) The mean number of branches per axon was calculated. (f) The mean axon branch length was calculated. (g) The mean number of dendrites pet pyramidal-like cell was calculated. (h) The mean dendrite length was calculated. (i) The mean number of branches per dendrite was calculated. Each dish was considered a single data point, and a total of 10 or more dishes was used for each concentration in the pennethrin and cypennethrin experiments or each mixture of 56

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pennethrin and cypennethrin with TfX, in the TfX experiments. Control dishes were run with cells from the same cryopreserved vial from which the cells of the experimental dishes were taken. Measurements of [Ca2+l" Background of Fura-2 Measurements of intracellular calcium ([Ca2+lm> were perfonned on rat hippocampal cells using the Ca2+ -specific fluorescent probe, fura-2. Fura2 contains a Ca2+selective binding site, which is similar in structure to that of the Ca2+ chelator EGTA (Tsien, 1988), and binds to Ca2+ with a high affinity (Grynkiewicz et al., 1985). The binding of fura-2 to Ca2+ is also highly selective, preferring Ca2+ over the most seriously competing ion, Mg2+, by about five orders of magnitude (Tsien, 1989). As [Ca2+]m is relatively low (10.7 -106 M) compared to cytosolic magnesium (10"3 M), this selectivity is critical (Tsien, 1988). Fura-2, a polycarboxylate, is able to. be loaded into the cytoplasm of the cells in the lipophilic fonn of fura-2-acetoxymethyl ester (AM). Fura-2 AM contains ester groups which allow for easy penetration of the molecule across the plasma membrane, without causing damage or disruption to the lipid bilayer (Tsien, 1994). Once inside the cytoplasm, the ester group is hydrolyzed by cytoplasmic esterases, and thus the polycarboxylate anion is regenerated and trapped iri the cytosol (Tsien,l988). 57

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Upon excitation by ultraviolet (UV) light, fura-2 emits a longer wavelength of light (around 510 nm) by fluorescing (Tsien, 1988). When fura-2 is bo.und to Ca2+, its excitation spectrum shifts about 30 nm to shorter wavelengths of light (Grynkiewicz et al., 1985) (Fig. 2.1). Thus the excitation efficiency that occurs upon exposure to 340 nm UV light is increased, whereas the excitation efficiency that occurs at 380 nm UV light is depressed. Therefore, an increase in Ca2+ will cause an increase in fluorescing at 340 nm, and a decrease in fluorescing at 380 nm. The emission spectrum itself, however, changes very little when Ca2+ binds. The shift in fura-2 's excitation wavelengths upon the binding of Ca2+ allows for [Ca2+]in to be deduced from a ratio of the fluorescence intensity amplitudes. Because of this ratioing, a measurement of [Ca2+]m can be obtained that is independent of dye concentration, cell thickness, optical path length, and illumination intensity. The ratio of the brightness intensity of the fluorescence obtained at 340 nm compared to the that obtained at 380 nm can be used to calculate absolute [Ca2+]in, according to the formula [Ca2+lm = Kd[(R-1\nm)/(1\nax-R)] (F jF5), where Kd is the effective dissociation constant for binding of fura-2 to calcium (224 nM); R = the intensity of the ratioed image (340/380) obtained from an experimental solution; Rmax = 340/380 intensity ratio in a Ca2+ -saturated solution; = 340/380 intensity ratio in a Ca2+ -free (EGTA) solution; and FjF5 =emission intensity stimulated by excitation at 380 nm in a Ca2+-free solution/intensity in a Ca2+-saturated solution (Grynkiewicz et al., 1985). 58

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>--iii c: Q) -.5 2 300 Wavelength l nm Fig. 2.1. The excitation of fura-2 shifts upon binding of calcium ions (Reprinted from TIBS, Vol. 11, Tsien, R.Y. & Poenie, M., Flourescence ratio imaging:a new window into intracellular ionic sig1;1alling; pp. 450-455, 1986, with kind pennission from Elsevier Sciences Ltd., The Boulevard, Langford Lane, Kidlington OX5 1GB, UK). Imaging System Setup A Quantex QX-7 imaging system with an inverted Nikon microscope was used to perform the [Ca2+1m measurements. The set up inCludes a xenon light source for emission of ultraviolet light and a computer-controlled filter wheel containing filters which eliminate all but the desired wavelength (340 nm or 380 nm). The cells are 59

PAGE 73

imaged by the use of an intensified charge-coupled device camera (CCD) that is connected to the microscope. A neutral density filter is used to reduce the saturation of the camera and to diminish bleaching of the fura-2. The camera acquires sixteen frames each at 340 nm and 380 nm, and then relays them to the Quantex QX-7 image processing system. The system converts the fluorescence intensities to numbers, and the computer stores and then calculates [Ca2+]in according to the aforementioned formula. Calibration Calibration of the imaging system was performed in vitro, using solutions with known Ca2+ concentrations (Molecular Probes). In vivo calibrations were not performed due to the sensitivity of the cells to the Ca2+ ionophores normally used for this purpose, and due to the uncertainty that the cells were clamped to a known [Ca2+]in. The dishes used for the calibrations were fabricated by cutting a hole out of the bottom of a 35 mm culture dish, and then applying a #1 glass coverslip over the hole with Sylgard 184 (Dow Coming). Two hundred microliters of Hank's balanced salt solution (HBSS) (a Ca2+ -saturated solution) or of 60 mM EGTA (a Ca2+ -free solution) were combined with 10 Ill of [lmM] fura-2 pentapotassium salt. Images were then acquired for both mixtures, and grey scale pixel intensities were measured for determination of and F fFs 60

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[Ca2+L Measurements in Cells Exposed to Pennethrin or Cypennethrin Culture dishes for Ca2+ imaging were fabricated in the same manner as the calibration dishes. Just prior to culturing cells, the dishes were sterilized by exposure to UV radiation for 30 minutes, coated with poly-D-lysine (MW >500,000) and then rinsed with sterile deionized water. Three hundred microliters of culture media was added to the glass well in the center of the dish, and then 200,000 rat hippocampal neurons were gently pipetted into this media. The dishes were incubated at 37C in 5% C02 for 2-4 hours to allow for settling and attachment of the cells. An additional 2 ml of culture medium was then added and the dishes were incubated for 48 hours. Mter 48 hours, dishes were checked to make sure they were uncontaminated and contained actively growing neurites. Two microliters of .fura-2 AM (1 Jlg/Jll) were pipetted into the dish wells of all of the dishes and the dishes were incubated at 37C. Mter 30 minutes of loading time for the fura-2 AM, the .dishes had all the media, except for a small amount of remaining in the cell wells, gently pipetted away, and replaced with fresh media. The cells were then incubated for an additional 30 minutes to allow for the intracellular de-esterification of the fura-2 AM into fura-2 acid. After this second incubation period, culture medium containing 100 11M pennethrin or 100 JlM cyperrnethrin was added to 6 randomly selected dishes (two dishes per time point), and culture medium containing 0.1% DMSO was added to six additional dishes. The cells were then allowed to incubate in the media for one, two, or three 61

PAGE 75

hours. Mter one, two, or three hours of incubation, the dishes were placed on a heated microscope stage kept at 37C. Every cell was only imaged one time to represent a particular time point, and at least 30 cells were imaged from each dish. Control dishes were only considered valid if the means of their [Ca2+]in levels fell between 20-150 nM. Two control dishes and two permethrin or cypermethrin dishes were imaged at each time point during a particular experiment, with a sum total of 8 to 20 dishes imaged for each condition. Statistical Analysis Data from both the morphology experiments (those that did not involve TTX), and the intracellular calcium imaging experiments were statistically analyzed by performing a one-way analysis of variance (ANOV A). This was followed by a two tailed Dunnett's test for comparing between each experimental condition and its control (with a significance level of p < 0.05). Means were obtained by replicating each data point at least 8 times. A one-way ANOV A was also performed for the morphology experiments that involved TIX in combination with cypermethrin or permethrin. This was followed by a Student-Newman-Keuls test for multiple comparisons among experimental means. Each data point was the mean of at least 8 replicates. 62

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CHAPTER 3 RESULTS Cell Culture Experiments Previous experiments conducted in our laboratory indicated that pennethrin, a Type I pyrethroid, produces effects on several different parameters of rat hippocampal cell neurite outgrowth (C. Ferguson, unpublished observations). Cypennethrin, whose structure differs from pennethrin only by the addition of .a cyano group, is over 100 times as potent at prolonging the opening of sodium channels (Vijverberg et al., 1986). Thus it was of interest to discern whether cypennethrin had any effects on various parameters of neurite outgrowth, and to what degree they were manifested. Cypennethrin Effects on Neurite Outgrowth Percent Cell Survival. Exposure to 100 J.1M cypennethrin caused a significant decrease in cell survival (p < .006; Fig. 3.1). A significant difference was not observed, however, between 1 10 the control. Percent Cells Initiating Neurites. The percent cells that initiated neurites was significantly decreased by exposure to cypennethrin over the range of concentrations 63

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100 90 80 -' 70 > 60 0::: :::> (f) .1-50 z w 40 u 0::: w 0... 30 20 10 0 1 10 100 CYPERMETHRIN Fig. 3.1. The effect of cypennethrin on cell survival. Asterisk indicates statistical significance (p < .006) The dashed line represents the control level. Data points are means s.e.m. 64

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100 90 80 --------..... -. ... --. -.----------------------------------.... 70 z 0 I-.L <( 60 Iz 50 Iz w 40 u 0::: w 0... 30 20 10 0 10 100 CYPERMETHRIN (.uM) Fig. 3.2. The effect of on initiation. Asterisks indicate.statistical signifcance (p < .0001). The dashed line represents the control level. Data points are means s.e.m. 65

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tested (p < .0001; Fig. 3.2). The effect appears to be concentration dependent, with 100 J.1M cypermethrin producing the greatest decrease in initiation in comparison to the control (31% vs. 81% ). Percent Axonal Cells per Growing Cell. The percent axonal cells per growing cell was found to be significantly decreased by 100 J.1M cypermethrin (p < .0001; Fig. 3.3). A significant effect was not displayed by either 1 JJM or 10 J.!M, however, the trend was down at 10 J.1M also. Mean Axon Length. Mean axon length was significantly decreased by both 10 J.!M and 100 J.1M (p < .0001; Fig. 3.4), with 100 J.1M producing the greatest decrease in comparison to the control (57 J.1lil down from 130 J.1111). One micromolar cypermethrin had no significant effect on mean axon length. Number of Branches per Axon. The number of branches per axon was significantly decreased by all the concentrations of cypermethrin that were tested (p < .0001; Fig. 3.5). The decrease occurred in a dose-dependent manner, with 100 J.1M cypermethrin producing the greatest reduction in branch number compared to the control {0 vs. 1.4). Mean Axon Branch Length. Cypermethrin did not significandy affect axon branch length at concentrations of either 1 J.1M or 10 J.IM. No branches were observed at 100 J.1M cypermethrin, therefore axon branch length was not measurable. 66

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Fig. 3.3. The effect of cypermethrin on the percent axonal cells per growing cell. Asterisk indicates statistical significance (p < .QOOl). The dashed line represents the control level. Data points are means s.e.m. 67

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,...... E ::t .......... I I-C) z w ...J z 0 X <: z <: w 140 120 100 80 60 40 20 OL..L-------..l...---..,...--------1 1 10 CYPERMETHRIN (,uM) 100 Fig. 3.4. The effect of cypennethrin on mean axon length. Asterisks indicate statistical significance (p < .0001. The dashed line represents the control level. Diua points are means s.e.m. 68

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z 0 X <( 0::: w a.. l/) w :::r:: u z <( 0::: co l.J.... 0 0::: w co ::::> z 2.0 1.5 1.0 0.5 1 10 CYPERMETHRIN (J.LM) 100 Fig. 3.5. The effect of cypermethrin on number of branches per axon. Asterisks indicate statistical significance (p < .0001). The dashed line designates the control level. Data points are means s.e.m. 69

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Number of Dendrites per Cell. Exposure to cypermethrin significantly reduced the mean number of dendrites per pyramidal-like cell in a dose-dependent manner (p < .0001; Fig. 3.6). One hundred micromolar cypermethrin reduced the number of dendrites to the greatest degree (1.0 down from 3.4). Mean Dendrite Length. Mean dendrite length was not significantly affected by cypennethrin over the range of concentrations that was tested. Number of Branches per Dendrite. Meaningful interpretations can not be made about the number of branches per dendrite, due to the extreme rarity of dendrite branches (0.16 branches/dendrite). Tetrodotoxin Effects on Cultures Exposed to Permethrin or Cypermethrin Since sodium channels are a major target site of action for the pyrethroids (Narahashi, 1992), it was of interest to determine what role, if any, that sodium int1ux and the resultant depolarization of the nerve membrane have on neurite development. Since tetrodotoxin has been found to completely block pyrethroid enhancement of the sodium current (Ghiasuddin and Soderland, ), TTX can be used as an 70

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....J ....J w u a::: w a_ (/) w !::: a::: 4 3 0 2 z w 0 l.J.... 0 a::: w CD ::::> z 0 10 CYPERMETHRIN (,uM) 100 Fig. 3.6 The effect of cypennethrin on number of dendrites per cell. Asterisks designate statistical significance (p < .0001). The dashed line represents the control level. Data points are means s.e.m. 71

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investigative tool in determining sodium channel effects. Tetrodotoxin-resistant sodium channels have been found in certain types. of cell; however, a voltage clamp study performed in our laboratory clearly demonstrated that the sodium current is completely abolished by 500 nM tetrodotoxin at membrane potentials greater than 0 m V (Fig. 1.8). Thus application of TTX in conjunction with the pyrethroids was assumed to eliminate any sodium channel-related effects on neurite development. In addition, preliminary experiments (data not presented here) revealed that 500 nM TIX added to culture media does not signifiCantly affect neurite growth in two day old cultures. Therefore any observed effects on neurite growth were assumed to be induced by the. pyrethroids. Three controls were used in the TfX experiments: one containing 500 nM TfX, one containing 100 pyrethroid, and one which contained neither TTX nor pyrethroid (the overall control). All three of these controls were requisite in order to reaffirm that the pyrethroid by itself affected neurite growth in various parameters, and that TIX by itself did not. In both the cypermethrin with TIX and the permethrin with TIX sets of experiments, the TfX control was not significantly different from the overall control in any parameter. Unless otherwise mentioned, in the cypermethrin with TTX set of experiments, the cypermethrin control was signiticantly different from the overall control in the same parameters that the 100 J.LM expetimental concentration was different from its control in the cypermethrin set of experiments. 72

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In the permethrin TTX set of experiments, the permethrin control was signiticantly different from overall control in several neurite growth parameters that were not found to be significantly different at the same concentration in a set of experiments previously performed in our laboratory (C. Ferguson, unpublished observations). These parameters will not be pointed out in the results, since they were performed by a different experimenter and several years prior; however, a possible explanation for the discrepancies will be discussed in the conclusion section of this thesis. Percent Cell Survival. Tetrodotoxin did not significantly alter the effect of 100 cypennethrin on percent cell survival (Fig. 3.7a). However, percent cell survival was no longer significantly reduced from the TTX control. Tetrodotoxin also did not significantly alter the effect of 100 J.1M permethrin on percent cell survival (Fig. 3.7b). In this case however, both the experimental dishes and the cypermethrin control were significantly decreased from the overall and TTX controls. Percent Cells Initiating Neurites. A significant mitigating effect was produced by TTX on 100 J.1M cypermethrin's effect on neurite initiation (52% up from 34%, p < .0001). The experimental dishes, however, still significantly differed from the overall and TTX controls (82% and 79%, respectively; Fig. 3.8a). A similar signiticant mitigating influence of TTX was also observed on 100 permethrin' s decrease in initiation (27% up from 10%, p < .0001). Again, the experimental dishes still were significantly different from the overall and TTX controls (Fig. 3.8b ). 73

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so 70 60 -1 50 > a:: :;) a) 1/) 40 1z w u a:: w 30 Q. 20 10 0 o" ... CJO 70 60 50 -1 > 40 a:: b) :;) 1/) .... z 30 w u a:: w c.. 20 10 0 o" .,_.,.+ ... )( Ci q q Fig. 3.7. Reversal of effect of cypemrethrin (a) or permethrin (b) on cell survival by TIX. Data points are means s.e.m. 74

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90 80 70 z. 60 0 i= < E 50 a) z ,_ z 40 w u t a: w 30 a. 20 10 0 c" .... CJ ,J.Q. c.. 80 z 0 i= 60 < b) ;:: z ,_ z ILl 40 u a:: ILl a. 20 Fig. 3.8. Reversal of effect of cypermethrin (a) or permethrin (b) on initiation by TTX. Dagger indicates statistical significance (p < .0001). Data points means s.e.m. 75

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Percent Axonal Cells per Growing Cell. Cypermethrin' s intluence on the percent of axonal cells per growing cell was not significantly changed by TTX; there was still a significant decrease in percent axonal cells per growing cell (Fig. 3.9a). Similarly, TIX did not alter the decrease in percent axonal cells per growing cell caused by 100 J.1M pennethrin (Fig. 3.9b). Mean Axon Length. The reduction in mean axon length that was induced by exposure to 100 J,1M cypermethrin was not significantly altered by TTX (Fig. 3.10a). Tetrodotoxin also produced no significant moderating influence on the reduction in mean axon length that is induced by exposure to 100 J.1M pennethrin (Fig. 3.10b). Number of Branches per Axon. Tetrodotoxin had no significant mitigating effect on the decrease in the number of axon branches that is caused by exposure to 100 !JM cypennethrin (Fig. 3.11a). One hundred micromolar pennethrin (the permethrin control) did not induce a significant change in the number of branches per axon compared to the other two controls (Fig. 3.11b). The experimental dishes also did not significantly vary from either the permethrin or the other two controls. It must be noted, however, that both the permethrin control and the experimental dishes contained only five and six data points, Thus whether these comparisons are meaningful or not is questionable. 76

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70 _, _, .... 60 u 0 z 3: so 0 a: (,!) a: .... 40 Q. til _, a) _, .... u 30 _, < z 0 X 20 < ,_ z .... u 10 a: w Q. 0 o"' ...._ ..... + -<:-+ ..... X c; ,.J..q ,.J..q v v 60 _, _, w u 50 0 z 3: 0 a: 40" 0 a: w Q. b) t/) 30 _, _, w u _, < % 20 0 X < 1-z w 10 u a: w Q. 0 o"' ...._,+ X c; q<.; q Fig. 3.9 Reversal of effect of cypermethrin (a) or permethrin (b) on percent axonal cells per growing cell by TIX. Data points are means s.e.m. 77

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140 120 -100 E ::t ..... J: ... 80 \,) a) z w ...J z 60 0 X < 40 20 0 o" ,_-<' c.; r..,q;. ,l,.q c:., 160 140 120 E 100 ..3 J: b) I-<.:! 80 z w ...J z 60 0 X < 40 20 0 o" ..._ .... :+ <:--+ )( c.; q<,; q Fig. 3.10. Reversal of effect of cypermethrin (a) or pennethrin (b) on mean axon length by TIX. Data points are means s.e.m. 78

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1.2 z 1.0 0 X < c:: w 0.8 Q.. Ul w :I: u z 0.6 < c:: CD ...... 0 0.4 c:: w CD :::i! :::> 0.2 z t 0.0 .<:-+ .<:-+ ov X Cj c? v Fig. 3.11. Reversal of effect of cypennethrin on number of branches per axon. Dagger denotes that no branches were present at this concentration. Data points are means s.e.m. 79

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Mean Axon Branch Length. No axon branches were present iri the cypermethrin control, therefore axon branch length was not measurable. Thus rio significant quantitative comparisons were able to be made. In the permethrin in conjunction with TIX set of experiments, the permethrin control for axon branch length did not significantly vary from the other two controls. Interestingly, the experimental dishes contained a significantly lower mean than all three of the controls (p < .011; Fig. 3.12). However, since the experimental dishes contained very few axon branches, this finding may not be reliable. Number of Dendrites per Cell. The decrease in the number of dendrites per axonal cell upon exposure to 100 j.1M cypermethrin wa.S not significantly changed by addition of TfX (Fig. 3.13a). Similarly, TTX had no effect on the permethrin-induced reduction in the number of dendrites per cell (Fig. 3.13b). Mean Dendrite Length. Tetrodotoxin caused a significant counteracting effect on the reduction in mean dendrite length caused by 100 j.1M cypermethrin (15 11m up from 12 j.liD.; p < .0041). The reversal was complete, since the value of the experimental dishes did not vary from either the overall or the TIX control (Fig. 3.14a). It should be noted however, that there was a not a significant reduction between the control and 100 j.1M cypermethrin in the cypermethrin set of cell culture experiments. One hundred micromolar pennethrin did not produce any significant effect on mean dendrite length (Fig. 3.14b). Correspondingly, there was no significant 80

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25 ......... E :::3.. ...__, I 20 t-0 z w _J I u z <{ cr:::: OJ 15 .z 0 X t <{ Fig : 3.12. Reversal of effect of pemiethrin on mean axon bran!=h.Iength by TTX Dagger designates statistical significance (p < .011). Data points are means: s.e m 81

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3.0 _, 2 5 _, LJ.I (..) 0:: w 2.0 a.. Vl w 1-cr 0 1.5 a) z w 0 LL 0 0:: 1.0 w Ql :::; ::::l z 0 5 0 0 o" )( c; q_<.; c: c. 3.0 _, 2.5 --' w (..) 0:: w 2.0 a.. Vl w 1b) cr 0 1.5 z w 0 LL 0 0:: 1.0 w Ql :::; :::> z 0 5 0.0 o" -0;.. .(:-;.. )( c; q_<.; q_ Fig. 3.13. Reversal of effect of cypermethrin (a) or permethrin (b) on number of dendrites per cell. Data points are means s.e.m. 82

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18 16 14 ,..., E 12 3 J: t-10 C> z LoJ -' LoJ 8 ta) 0 6 z LoJ 0 4 2 0 o" -(j ,J..q_ c. 24 20 ,..... E _16 :t ,_. :I: t-C> z LoJ 12 -' b) LoJ-t-0 8 z LoJ 0 4 0 o" ..._,+ ..._,..,+ x (j
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difference between the mean dendrite length of the experimental dishes and those of the controls. Measurements of [Ca2+l" Regulation of intracellular calcium has been shown to play an integral role in neurite growth. Thus one avenue for the pyrethroids to affect neurite growth could be through alterations in intracellular calcium levels. These alterations in intracellular calcium levels could occur by way of inteiference. with calcium regulating systems (such as Ca2+1Mi+ATPase activity) or by way.of an increase in calcium int1ux due to the pyrethroid-induced depolarization of the membrane. Whether there is an alteration in [Ca2+]in or not must first be Cvoermethrin Effects on JCa2+l" In a set of static imaging no significant effect on the [Ca2+]iu of two day old rat hippocampal neurons was found after exposure to 100 1-1M cypermethrin for one hour, two hour or three hour time periods (Fig. 3.15). 84

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.......... :::E 1:: .......... z 0 t-< ll:: tz w (.) z 0 u :::E :::> () -l <(" u ll:: < ..J :::> ..J ..J w u <( ll:: tz 100 90 80 70 60 50 40 30 20 10 0 .. 2 HOURS OF EXPOSURE TO CYPERMETHRIN e-e -cypermethrin 0 = control Fig. 3.15. Effect of cypennethrin on [Ca2 ... Data points are means s.e.m. 85 3

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Permethrin Effects on [Ca2+l" In a set of static imaging experiments, 100 J.IM permethrin caused a significant reduction in the [Ca2+]in of two day old rat hippocampal neurons upon exposure for one, two and three hour time periods (p < .004; Fig. 3.16). 86

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-100 ::iE c .._., 90 z 0 I-80 < 0::: 1-70 z w (.) z 60 0 (.) ::iE 50 ::::> u _J 40 < (.) 0::: .3.0 5 :::J _J 20 ._I w (j 10 < 0::: 1z 0 l_________________ A A .1. 2 HOURS OF EXPOSURE TO PERMETHRIN ..... = permethrin 0-0 = control .-! 3 3.16. Effect of permethrin on [Ca2+]in. Asterisks indicate statistical significance (p < .004). Data points are means :1:: s.e.m. 87

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CHAPTER4 DISCUSSION General Overview The primary intent of this thesis was to determine the effects that cypermethrin, a Type IT pyrethroid, had on neurite outgrowth. Another set of experiments examining the effects of permethrin, a Type I pyrethroid, on neurite outgrowth, had been conducted previously in our laboratory. Thus, the second intent was to investigate two potential avenues by which both types of pyrethroids might have exerted their effects on neurite outgrowth. These two potential mechanisms are: 1) Pyrethroid interaction with the voltage-sensitive sodium channel and 2) Pyrethroid-induced alterations in intracellular calcium concentration (which may or may not be affected by pyrethroid interaction with the voltage-sensitive sodium channel). So, for the purpose of reading ease the effects of cypermethrin on neurite outgrowth parameters will be discussed first, followed by a brief discussion of permethrin' s versus cypermethrin's effects on neurite outgrowth. The feasibility of a sodium channel response and the extent of the role that the sodium channel plays in pyrethroid induced effects will be discussed next. This will in turn be followed by a discussion 88

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regarding the effects that cypennethrin and pennethrin had on intracellular calcium concentration. A final conclusion and summary will integrate the three sets of experiments by attempting to correlate, to some degree, the findings about the extent of the role that the sodium channel plays and the findings about intracellular calcium concentration with the observed morphological effects of cypennethrin. Finally, a potential avenue for how cypermethrin might exert its effects on neurite outgrowth will also be explored. Cypermethrin Effects on Neurite Outgrowth Table 1 summarizes the effects that different concentrations of cypermethrin had on various parameters of neurite outgrowth in rat hippocampal neurons. In combination with Figures 3.1 through 3.6, several interesting conclusions can be drawn about both cypermethrin's mechanism of action and also about aspects of neurite outgrowth itself. Perhaps the most important finding is that 100 cypermethrin causes an inhibition of almost every parameter of neurite outgrowth. Such pervasive inhibition of outgrowth did not occur at 1 or 10 indicating that concentrations in the vicinity of 100 J.!M are the level where severe neurodevelopmental toxic effects occur. This is of comfort to know since such high concentrations of pyrethroids are not encountered in the environment (Hill, 1985). 89

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\0 0 Cypermethrin Effects on Neurite Development Concentration Survival Initiation %Axonal Mean Axon Branches per Mean Axon Dendrites per Mean Cells Length Axon Branch Length Cell Dendrite Length -.!. ---.!. -.!. --.!. .!. .!. .!. 100 JIM .!. .!. .!. .!. .!. n.m. .1. --Table 4.1. A".!." denotes a significant decrease (p < .01). A "--"denotes that there was no effect. A "n.m." denotes that there were no branches at this concentration.

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Survival versus Initiation The percent of neuron survival appears to be one of the least sensitive growth parameters to cypermethrin's toxicity, whereas initiation appears to be one of the most sensitive. Survival was only affected by cypermethrin at the concentration (1 00 11M) which inhibited almost every other growth parameter. This corresponds to some degree previous observations from our laboratory that there is an inverse relationship between neuron survival and initiation (Audesirk et al., 1991; Kern et al., 1993). These observations suggested that differentiation of a neuron may predispose it to death. However, the present finding that survival was not enhanced suggests that cypennethrin is interfering with a mediator of neurite outgrowth that is involved the production of neurites but which has little or no affect on cell viability. Axon versus Dendrite Elongation. The only aspect of neurite outgrowth that was not affected by 100 11M cypermethrin was mean dendrite length. This finding is quite interesting in that it suggests that axons and dendrites have a different underlying mechanism(s) mediating elongation since mean axon length was significantly decreased even at 10 !JM. One mediator that may have a role in axon elongation is Ca2+ -calmodulin-dependent protein kinase II (CaM-KIT), a protein kinase which phosphorylates serine and threonine residues. Ten micromolar KN62, an inhibitor of CaM-KIT, also causes a reduction in mean axon 91

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length, but not of mean dendrite length in rat hippocampal neurons (Cabell and Audesirk, 1993). Notably, cypennethrin has been found to inhibit calmodulin at concentrations of around 1 o3 to 104 M. One could surmise that 100 J.!M cypermethrin is inhibiting CaM-KIT phosphorylation of axonal cytoskeletal proteins by inhibiting calmodulin's interaction with the kinase and thereby causing a decrease in axon elongation. The observation that dendrite elongation is unaffected would not necessarily mean that CaM-Kll has no role in dendrite elongation; other serine/threonine kinases might make up for a decrease in phosphorylation by CaM-KIT of dendritic cytoskeletal proteins. Conceivably axonal cytoskeletal proteins could have amino acid residues that are more favorably phosphorylated by CaM-KIT. Comparisons among Initiation, Dendrite Production, arid Axonal Branching Initiation, the number Of dendrites per cell and the number of branches per axon were all reduced in a dose-dependent manner by cypermethtin. This observation might lead to the hypothesis that the processes of initiation of neurites, the production of dendrites and the branching of axons have similar underlying mechanisms. Nevertheless, previous experiments pei:formed in our laboratory have demonstrated differential effects of various neurotoxins and neurotoxicants on these processes in rat hippocampal neurons (Audesirk et al., 1991; Cabell and Audesirk, 1993; Kern et al., 1993). These results suggested that initiation, dendrite production and axon branching 92

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may all be regulated by different developmental mechanisms, perhaps by different protein kinases. That is, different protein kinases would preferentially phosphorylate different target proteins involved in the individual processes and" therefore might act as triggering devices for these processes (Klee et al., 1990). However, although these parameters may be primarily regulated by d,ifferential mechanisms, there may be some overlap. Some target proteins may be phosphorylated by several different kinases. One set of experiments using the protein kinase c inhibitor calphostin c demonstrated a partial inhibition of both initiation and axon branching (Cabell and Audesirk, 1993). As calphostin C is a highly selective for PKC over inhibitor, this suggest that PKC may influence, to some degree both of these processes. Protein kinase C inhibition might also seem to be a mechanism for cypermethrin's decrease in both initiation and axon branching, however, the Type II pyrethroid deltamethrin has been found to directly stimulate protein kinase C activity. One mediator that both dendrite production and axonal branching might have in common. iSCa2+-calmodulin-protein kinase II (CaM-KIT). The CaM-KIT inhibitor KN62 inhibits both dendrite production and axonal As suggested previously, cypermethrin may interfere with the interaction of calmodulin with the kinase. This could cause a decrease in phosphorylation of crucial target proteins involved in the production of dendrites or in axonal branching. .. Additionally or alternatively, cypermethrin could interfere with several different 93

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moderators of neurite outgrowth, or perhaps with an overall moderator which influences many different processes. This could easily explain cypermethrin' s multimodal inhibition of neurite outgrowth. Permethrin Effects on Neurite Outgrowth It was expected that neurite outgrowth in the (100 JJM) permethrin control of the set of experiments investigating tetrodotoxin effects on cultures exposed to permethrin would be congruent with the 100 J.1M concentration from a previous performed set of experiments investigating various concentrations of permethrin on neurite outgrowth of rat hippocampal neurons (C. Ferguson, unpublished results). However, in several different parameters of neurite outgrowth, the lQO permethrin control in the set of experiments presented here differed from the effects observed at 100 J.LM permethrin in the previously performed set of experiments. Namely, the present set caused significant reductions in survival, mean axon length, and the number of processes per cell, all of which did not occur in the other set Not disregarding the fact that the sets of experiments were performed by two separate experimenters, one potential explanation for the greater potency of permethrin in the present set of experiments is that a higher purity of permethrin was used. Other possible reasons that may have caused the discrepancies to occur are the use of a slightly different cell media or the implementation of slightly different cell cryopreservation and/or culturing 94

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protocols. Due to the disparate findings, the observations of the previously conducted experiment will not be discussed here. Rather, a brief comparison between 100 JJM cypermethrin' s effects on neurite outgrowth and 100 JlM permethrin' s effects on neurite outgrowth will be presented. Permethrin's versus Cvnermethrin's Effects on Neurite Outgrowth Cypermethrin is over one hundred times as potent in prolonging the sodium current than permethrin (Vijverberg et al., 1986), therefore it was expected that cypermethrin would have more potent effects on neurite outgrowth. Surprisingly, however, 100 JJM permethrin inhibited outgrowth in almost all the same parameters as did 100 JlM cypermethrin. In addition, when comparing percent of the controls for these 100 JlM permethrin varied very little from 100 JJM cypermethrin except in initiation. In fact, permethrin actually had a greater effect on initiation than did cypermethrin (11% and 42% of their controls, respectively). These results are interesting in that they suggest either that sodium channel effects do not have a major influence onpyrethroid-exposed neuron morphology, or alternatively, that they are not magnified by cypermethrin' s greater niean open time. The only outgrowth parameter which 100 JJM permethrin appeared to have no effect on, in contrast to 100 JJM cyperinethrin, was the number of branches per axon. The significance of this is unknown, however it is congruent with previous findings from 95

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our laboratory that suggest that neurite branching has different underlying mechanisms than do initiation or dendrite production (Audesirk et al., 1991; Kern et al., 1993). The Feasibility of a Sodium Channel Response One question that is fundamental to address when investigating potential sodium channel-related pyrethroid effects, is whether or not the rat hippocampal cells are exhibiting spontaneous bioelectric activity (SBA) in culture. Van Huizen et al. (1985) have found that SBA is not extracellulatly measurable until day 5 in in vitro reaggregates of embryonic day 19 (E19) neocortex cells. Electrophysiological studies demonstrate a repetitive activity response or a depolarization block response of a nerve fiber to Type I and Type II pyrethroids, respectively ,following a single stimulus (Narahashi, 1982b, 1986a). Therefore the question arises as to whether a sodium channel response to the pyrethroids can even be generated in cultures of widely dispersed embryonic day 18 (E18) hippocampal cells that may not yet be demonstrating SBA. This author believes that the pyrethroids can have sodium channel -related effects, even without SBA. First of all, even though most voltage-sensitive sodium channels open at relatively depolarized membrane potentials, there are a small number of open sodium channels at any given time, even at non-depolarized membrane potentials. That is, individual sodium channels vary in the level of membrane potential at which 96

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they open. The current flowing through these channels could be prolonged by the pyrethroid and therefore could cause a slight membrane depolarization, provoking the opening of more channels (and therefore more current) for the pyrethroids to act upon (Eells et al., 1992). Secondly, the media that the hippocampal cells are cultured in contains a high concentration of potassium (20 Mm K+) and is therefore slightly depolarizing, resulting in a resting potential that is 15 to 20 mV less negative than the in vivo resting potential. Therefore there is a comparatively greater number of voltage sensitive sodium channels open upon which the pyrethroids can produce a positive feedback effect. Thirdly, in addition to being slightly depolarizing because of a high concentration of K+, the media also is depolarizing due to the fact that it contains the neurotransmitter glutamate. Rat hippocampal cells contain N-methyl-D-aspartate (NMDA), a-amino3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate, and metabotropic receptors, all of which bind the excitatory neurotransmitter glutamate. Experiments performed in our laboratory have demonstrated inhibiting effects on neurite outgrowth in cultures of El8 rat hippocampal cells by antagonists to these three receptor types (M. Kern and L. Cabell, unpublished observations). This suggests that E18 hippocampal cells do indeed contain glutamate receptors. The binding of glutamate to the NJviDA, AMPA; and kainate receptors would cause a depolarization of the cell 97

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also. Indeed, NMDA evokes whole cell currents in E18-E20 rat hippocampal neurons after one day in cultUre (Ujihara and Albuquerque, 1992). The binding of glutamate to metabotropic receptors would trigger the release of Ca2+ from intracellular stores by way of activation of the phosphoinositide-linked second messenger system and thereby further depolarize the cell. In conclusion, it is believed that their was ample opportunity for the hippocampal cells to become depolarized and for the pyrethroids to have sodium-channel related effects. Tetrodotoxin Effects on Cultures Exposed to Permethrin or Cypermethrin It was hypothesized that tetrodotoxin would mitigate many of the effects that pennethrin and cypermethrin have on various parameters of neurite outgrowth. Since the sodium channel is the major target site for pyrethroid-induced effects on the excitability of nerve membrane, it might be expected that it would also be the root of their morphological effects. That is, the morphological effects would be indirectly related to the depolarization of the nerve membrane caused by pyrethroid-induced sodium influx. Tetrodotoxin, a sodium channel antagonist which also antagonizes pyrethroid-induced depolarization of the nerve membrane, should therefore counteract, to some extent, pyrethroid .effects on neurite outgrowth. A summary of TTX's effects on rat hippocampal_ cultures that have been exposed to permethrin or cypermethrin is provided by Table II. The table, in combination with Figures 3.17 through 3.14 98

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reveals the extent of the role that the sodium channels plays in mediating pyrethroid effects on various parameters of neurite outgrowth. Surprisingly, sodium channel effects appear to play almost no role in mediating permethrin' s and cypermethrin' s effects on neurite outgrowth. The only parameter on which TIX had a mitigating effect is discussed here. Initiation Tetrodotoxin demonstrated only a partial mitigation of both permethrin' s and cypermethrin's decrease iii the. percent cells that initiated neurites. Therefore it appears that a pyrethroid-induced depolarization of the nerve membrane is only partially responsible for a decrease in initiation. It is interesting that TIX had a significant mitigating affect on the decrease in initiation caused by permethrin, in addition to its mitigation of the decrease caused by cypermethrin (up 18% of the control and up 22% of the control, respectively). Since cypermethrin is extremely more potent at prolonging the sodium current than permethrin, it would be expected that TIX would have a greater mitigating effect. This suggests that the depolarization of the nerve membrane by permethrin is sufficient enough or long enough to produce an effect on initiation that is qualitatively comparable to the effect produced by cypermethrin. 99

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-8 Reversal of Pyrethroid Effects on Neurite Development by 1TX -Pyrethroid Survival Initiation %Axonal Mean Axon Branches per Mean Axon Dendrites per Mean Cells Length Axon Branch Length Cell Dendrite Length Cypermethrin 0 + 0 0 0 ++ 0 Permethrin 0 + 0 0 ---0 Table 4.2. A"+" denotes a partial reversal (p < .05). A "++"denotes a complete reversal. A "0" denotes no reversal at all. A "--" denotes that there was no significant effect of cypermethrin on this parameter. )I. n.m. denotes that no branches were present in the cypermethrin control, therefore measurement of axon branch length was not possible ++ -I

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[Ca2+L Measurements Several lines of evidence led to the hypothesis tha:t both permethrin and cypermethrin should cause an increase in the intracelhilar calcium level in hippocampal cells. Firstly, pyrethroids cause a prolonged opening of the sodium channel, resulting in a significant depolarization of the nerve membrane. The depolarization of the nerve membrane, in tum, invokes the opening of voltage sensitive calcium channels and thus there is an influx of calcium. Therefore it was expected that there would be a resultant increase in [Ca2+]in upon exposure of rat hippocampal cells to either pyrethroid. Cypermethrin was proposed to effect a greater increase in [Ca2+]in because of the greater prolongation by the Type II pyrethroids of the mean open time of the sodium channel. In accord with the theory that the two types of pyrethroids would cause a depolarization-induced increase in [Ca2+]in, is a study that measured [Ca2+]in in rat brain synaptosomes using fura-2. Enan and Matsumura (1991) demonstrated that deltamethrin invokes an increase in intrasynaptosomal [Ca2+] which is, in part, due to deltamethrin's actions on the sodium channel. Moreover, they suggest that the enhancement of protein phosphorylation that occurs even five minutes after addition of deltamethrin to the synaptosomes is caused by an increase in [Ca2+lm Finally, and also along the same line of reasoning, an increase in [Ca2+]in could also explain pyrethroid-induced increases neurotransmitter release (Eells and Dubocovich, 1988), 101

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since neurotransmitter release is dependent upon Ca2+ influx. In addition to a potential augmentation in [Ca2+lm due to pyrethroids-induced depolarization of the nerve membrane, studies of pyrethroid effects on A TPase activity and on the Na+/Ca2+ exchange system also suggest that [Ca2+ln would increase. Permethrin and cypermethrin have both been found to inhibit Ca2+-ATPase and Ca2+ + Mg2+-ATPase activity in rat brain synaptosomal, microsomal, and nuclear fractions (Sahib et al., 1987; Al-Rajhi, 1990; Kodavanti et al., 1993). Since Ca2+-ATPase and Ca2+ + Mg2+-A TPase function as Ca2+ sequestering and eft1ux mechanisms, respectively, the inhibition of these enzymes would elevate [Ca2+lw Moreover, ATP hydrolysis that is increased in the presence of Na+ and Ca2+, is inhibited by both permethrin and cypermethrin, with permethrin being the more potent inhibitor (Clark and Matsumura, 1987). This suggests that the pyrethroids may have an atJect on the Na+/Ca2+ exchange system. Inhibition of this Ca2+ efflux mechanism could also potentially effect a rise in [Ca2+lm For these reasons, the finding that permethrin and cypermethrin did not cause an increase in [Ca2+lm in the static Ca2+ imaging experiments was quite surprising. Even more unexpected was the fact that permethrin caused a significant decrease in Ca2+ at the one, two and three hour timepoints. These results would seem to indicate that if there are any increases in [Ca2+]in by pyrethroid-induced depolarization of the membrane and/or by pyrethroid inhibition of ATPases, then they are being 102

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counteracted by mechanisms that decrease [Ca2+lm In saying this however, several things must be examined with greater scrutiny. First of all, the time aspect of this study must be taken into account. The [Ca2+liu measurements that were performed here were obtained at one, two, and three hours after the addition of permethrin or cypermethrin to the dishes. Thus it can not be said that there was no increase in [Ca2+]in at any time. The experiments performed by Enan and Matsumura (1991) clearly demonstrated that some of the increase in [Ca2+liu that they found with their fura-2 measurements was attributable to a deltamethrin induced interaction with the sodium channel. Their measurements were performed on the preparation following about ten minutes of incubation time. After one hour of time, however, as in these experiments, Ca2+ regulatory mechanisms could have kicked in and brought the [Ca2+]in back down to around normal levels. Alternatively or additionally, the sustained opening of sodium channels induced by the pyrethroids would yield a prolonged depolarization of the membrane. This could, in tum, effect an eventual inactivation of Ca2+ channels. Even the L-type calcium channel, which has an extremely slow rate of inactivation (Tsien et al., 1988) would be inactivated after an hour of membrane depolarization. Therefore [Ca2+lw would no longer increase by way of voltage-sensitive Ca2+ channel influx, and Ca2+ regulatory mechanisms could bind up or pump out extra Ca2+ that could have intluxed immediately following pyrethroid-induced depolarization of the nerve membrane. 103

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Secondly, the previously mentioned studies on Ca2+ and Ca2+ + Mg2+ATPases and on the Na+/Ca2+ exchange revealed an inhibition of ATP hydrolysis shown by an decrease in radiolabelled inorganic phosphate. This type of assay does not demonstrate a direct effect on a specific enzyme system or a protein. Thus it is uncertain whether permethrin and cypermethrin affect these Ca2+ regulatory mechanisms directly, or if they are just indirectly affecting ATPase hydrolysis. Thus even though it might seem as though cypermethrinor permethrin-inhibition of these enzyme systems would cause an increase in [Ca2+]in, it may not be the case. For example, Doherty et al. (1987) examined the effect of pyrethroids on Ca2+ regulation. They demonstrated that most pyrethroids at a concentration of 5 11M cause an inhibition of 45Ca2+ uptake into rat brain synaptosomes. This "uptake" by the synaptosomes presumably represents the normal Ca2+-ATPase extrusion process in non-synaptosomal preparations, since control samples readily take up 45Ca2+ in the presence of ATP, but not in its absence. Interestingly, they found that cis-permethrin and cis-cypennethrin at a concentration of 5 !1M. in contrast to most of the other pyrethroids, actually cause increases in the amounts of 45Ca2+ incorporated by the synaptosomes. This would therefore reflect that these two pyrethroids would actually cause an increase in Ca2+ extrusion. Thus this would provide yet another possible explanation for why [Ca2+lm was not. found to have been increased by exposure to permethrin or cypermethrin. 104

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A third potential explanation for the lack of increase in [Ca2+1in could be an inhibition of Ca2+ channels by the pyrethroids. Fifty micromolar tetramethrin greatly reduces one of two types of calcium current in neuroblastoma cells (Narahashi, 1986a). In addition, at a concentration of allethrin and deltamethrin block the peak amplitudes of both the non-inactivating and inactivating Ca2+ currents by around 30% and 50%, respectively, in rat hippocampal neurons (Frey and Narahashi, 1990). Whether or not permethrin and cypermethrin would have similar affects on Ca2+ currents, and whether this would result in a decrease in [Ca2+]in, is of interest to discover. Further analysis for the lack of Ca2+ increase will be examined in the next section. Overall Conclusion It was hypothesized that the Type I pyrethroid permethrin and the Type II pyrethroid cypermethrin would both exert their effects on rat hippocampal cell neurite outgrowth by way of their actions on the sodium channel. Specifically, the delay in inactivation of the. sodium channels by the pyrethroids would cause a depolarization-induced increase in intracellular calcium. Since Ca2+ has a major role in regulating neurite outgrowth, the increase in [Ca2+]in would then go on to interfere with various parameters of outgrowth. Therefore, the findings from the tetrodotoxin experiments that neither of these pyrethroids have no sodium-channel related effects on neurite 105

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outgrowth, except for a slight effect on initiation, was very surprising. Equally as surprising was the finding that the hippocampal cells did not demonstrate a rise in [Ca2+]in after exposure for one, two or three hours. Calcium Regulation by Rat Hippocampal Neurons Since other investigators demonstrated a rise in rat brain intrasynaptosomal [Ca2+] upon exposure to pyrethroids (Enan and Matsumura, 1991; Kodavanti et al., 1993), it seems almost inconceivable that there was never any increase in [Ca2+]in at alL It is well known that depolarization causes an influx of Ca2+; The pyrethroids must be invoking at least an initial increase in [Ca2+lmHence in less than one hour's time the cell has pumped out the excess Ca2+ and returned [Ca2+]in back down to normal levels. Eventual closure of Ca2+ channels from sustained depolarization would aid in this process. Alternatively, the pyrethroids could initially inhibit Ca2+ influx by partially blocking voltage-sensitive Ca2+ channel current (Narahashi, 1986a; Frey and Narahashi, 1990); however it seems as though this would still be greatly outweighed from the initial depolarization-induced influx of Ca2+. Thus the cell must have a remarkable ability to regulate Ca2+. Pyrethroid Effects on Cell Survival The ability of the cell to regulate Ca2+ after exposure to pyrethroids, may explain why there was not a huge decrease in cell survival upon exposure to permethrin or 106

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cypennethrin. Interestingly, the cytotoxicity of pyrethroids to rat cerebellar granule cells was found to be much less than the cytotoxicity of certain other agents that interfere with Ca2+-transport systems (Kodavanti et al., 1993). Accordingly, the Ca2+ATPase inhibitor chlorpromazine (CPZ) inhibits Ca2+-ATPase activity and 45Ca2+ uptake by mitochondria and microsomes to a much greater degree than does permethrin or deltamethrin. Thus less of a decrease in survival appears to be associated with a better ability to regulate Ca2+ homeostasis. This is congruent with in vivo fmdings have shown a lack of neuropathic action of the pyrethroids. Pvrethroid Effects on Initiation Initiation is the first process to occur of any aspect of neurite outgrowth. The new little stubs .of neurite processes can first be viewed under the microscope one to two hours after plating rat hippocampal cells. Presumably the underlying mechanisms that invoke initiation have already occurred by the time the stubs are visible. As permethrin and cypermethrin both appear to have a (partial) sodium channel related action on initiation, it may be that initiation is occurring early enough to coincide with a Ca2+ influx from a pyrethroid-induced depolarization of the nerve membrane. Thus before Ca2+ homeostasis is restored by the cell, a high level of intracellular Ca2+ would be able to provoke an inhibition of the events involved with initiation. 107

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A Potential Mechanism for Type II Pyrethroid Effects on Neurite Development The question arises that if permethrin and cypermethrin are not affecting neurite outgrowth by their actions on the sodium channel, then what is their mechanism of action? Recently, two separate groups of investigators have found that Type II pyrethroids inhibit calcineurin, the predominant Ca2+-and calmodulin-dependent protein phosphatase in the mammalian nervous system, at nanomolar and sub nanomolar concentrations (Enan and Matsumura, 1992; B. Martin and D. Brautigan, unpublished results). As mentioned earlier, one explanation for the fact that permethrin and cypermethrin inhibit many aspects of neurite outgrowth could be that they are interfering with an overall modulator that influences many of these processes. In order for protein kinases to act as triggering devices for neuronal growth processes, the target phosphoproteins that they act upon must be in their dephosphorylated state (IGee et al., 1990). Calcineurin, which is constitutively expressed, could act as a negative control and keep the target phosphoproteins in a dephosphorylated state that is primed for phosphorylation by protein kinases. For example, calcineurin dephosphorylates microtubule-associated protein 2 (MAP2), an important protein involved in dendritic architecture. Calcineurin inhibition would also affect the regulation of neuromodulin, a neuron specific calmodulin-binding protein found in great abundance in the brain and which has been linked to several different processes of neurite outgrowth. Specifically 108

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neuromodulin has been implicated in neurite initiation (Skene and Willard, 1981), axon elongation (Zuber et al., 1989; Ramakers et al., 1991), and synaptic plasticity (Chan et al., 1986). Its influence on these processes is probably due to its capacity to bind calmodulin. Although neuromodulin binds calmodulin with low affinity, it can conceivably bind up great amounts of the molecule due to its great abundance in certain areas of the neuron (Skene, 1990). The phosphorylation of neuromodulin causes it to lose its affinity for calmodulin and hence release it in great quantities. The calmodulin can then go on to stimulate calmodulin-dependent enzymes. Activation of these enzymes can then cause alterations in cytoskeletal structure and cytoskeletal-membrane interactions which could then allow for a "step" to take place in processes such as initiation and axon elongation. Cessation of a "step" would occur upon dephosphorylation of neuromodulin by calcineurin (Lui and Storm, 1989). Inhibition of calcineurin by cypermethrin, however, would not allow for individual "steps" in neurite outgrowth to occur, and render phosphoproteins unable to respond to the next stimulus (Enim and Matsumura, 1992). Inhibition of calcineurin would also account for several non-morphological affects of the pyrethroids on neurons. For example, Enan and Matsumura have found increases in protein phosphorylation to occur for up to five minutes after exposure of synaptosomes to deltamethrin. Inhibition of calcineurin would potentiate the increase phosphorylation of phosphoproteins depolarization by not allowing for 109

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phosphoproteins to return to the dephosphorylated state. This could also explain the increased release of neurotransmitters upon exposure to Type II pyrethroids. Synapsin I, which inhibits mobilization of neurotransmitter-containing vesicles in its dephosphorylated state, has been shown to be phosphorylated upon stimulation by deltamethrin (Enan and Matsumura, 1991). A failure to return synapsin I to its dephosphorylated state would extend the release of neurotransmitters. In addition, the phosphorylated state of neuromodulin has also been associated with neurotransmitter release (Dekker et al., 1989). Failure to return neuromodulin to its dephosphorylated state by calcineurin inhibition would also exaggerate neurotransmitter release. Finally, inhibition of calcineurin could provide an explanation for the inhibition of ATPase activity by (Type II) pyrethroids. If these enzymes are only active in a dephosphorylated state for example, than calcineurin inhibition might potentiate an inactive phosphorylated state. In conclusion, inhibition of calcineurin can explain many oflhe morphological and non-morphological effects of cypermethrin on neurite outgrowth. While it does not explain the effects of permethrin on neurite outgrowth, it does render a general mechanism by which a rteurotoxicant can exert physiological and morphological effects on neurons. That is, inhibition of a predominant modulator such as a protein phosphatase, can affect many different aspects of neurite outgrowth. 110

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Final Summary Since pyrethroids are lipophilic molecules they are rapidly taken up by organisms. Thus the fmding that even the lowest concentration of cypermethrin ( 1 IJM) had a pronounced inhibiting effect on several of the measured parameters of neurite outgrowth is disconcerting. However, such levels of pyrethroids do not occur in the environment even directly following application to fields, ponds, or forests by spraying (Hill, 1985). In addition, workers that have been engaged in packaging pyrethroids excrete urine that contains pyrethroid .concentrations in the range of 1 o-9 to 1 o-s M. Since pyrethroids are rapidly excreted, this would seem to indicate that pyrethroid concentrations are not occurring that are in the range of 1 o-6 M. In laboratory conditions that subject animals to sustained pyrethroid concentrations plateau residue levels of pyrethroid are quickly obtained due to high metabolism and excretion rates (Hill, 1985). Finally, practical observations have suggested that there is no biomagnification of pyrethroid concentrations in the food chain, nor is there any evidence of significant bioaccumulation by animals. All these findings suggest that there is little practical neurodevelopmental hazard to animals or humans exposed to the pyrethroids. However, further experiments are needed to discern whether sub-micromolar concentrations of pyrethroids have an effect on any aspects of neurodevelopment. 111

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