Changes in the Murine Nigrostriatal Pathway Following Pyrethroid and Organophosphate

Insecticide Exposure: An Immunohistochemical Study

By

Julian Thomas Pittman

Thesis submitted to the Faculty of the

Virginia Polytechnic Institute and State University

in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

in

Veterinary Medical Sciences

___________________________________

Dr. Bradley Klein, Chair

___________________________________ Dr. Jeffrey Bloomquist

___________________________________ Dr. Bernard Jortner

Defense Date: August 22, 2002

Blacksburg, VA

Keywords: Neurotoxicity, Parkinson’s Disease, Immunohistochemistry, Permethrin,

Chlorpyrifos

ii

Abstract

Changes in the Murine Nigrostriatal Pathway Following Pyrethroid and Organophosphate

Insecticide Exposure: An Immunohistochemical Study

Julian Thomas Pittman

Committee Chairman: Dr. Bradley G. Klein

Veterinary Medical Sciences

Parkinson’s disease (PD) is a debilitating motor disorder that primarily afflicts older individuals

(> 50yrs). Although its cause is unknown, many factors are thought to contribute to the disease.

There is growing epidemiological evidence supporting a link between pesticide exposure and

PD. The present immunohistochemical study was undertaken to characterize the role of

insecticide exposure in the etiology of idiopathic PD. The insecticides selected for study were

the pyrethroid permethrin (PE) and the organophosphate chlorpyrifos (CP), both of which

possess properties that could damage or disrupt the nigrostriatal pathway, which is the principal

neurodegenerative target in PD. The present study examined possible alteration of the amount of

dopamine re-uptake transporter protein (DAT), within the striatum of the C57BL/6 mouse, using

DAT antibodies, following low (0.8, 1.5 & 3.0 mg/kg) and high (200 mg/kg) doses of PE,

respectively. Possible nigrostriatal terminal degeneration was examined using antibodies to

tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, following treatment

with 50 mg/kg of CP alone or in combination with the high dose of PE. For both the high dose

of PE alone and for the combined PE/CP treatment, glial fibrillary acidic protein (GFAP)

antibodies were used to examine the possibility of non-degenerative tissue injury. Groups of

matched treated/vehicle-control mice received three IP injections of the insecticide/dose of

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interest over a 2-week period. Counts of immunoreactive (IR) neuropil in the dorsolateral

striatum were made from four pre-selected fields per striatal tissue section. Counts were

compared between matched sections, processed on the same slide, from a treated mouse and its

vehicle control. A mean difference score, across slides, for each treated/vehicle control pair was

determined. All low dose PE groups showed a trend of decreased DAT IR neuropil, but only the

3.0mg/kg group showed a statistically significant reduction (p<.0078). The 200 mg/kg PE group

showed a trend toward reduced TH IR neuropil that was not statistically significant, but a

significant increase in GFAP IR (p = .048) was observed. No significant change in TH IR

neuropil was observed for CP (50mg/kg) alone. A significant increase was observed for GFAP

IR neuropil for the PE/CP (200/50 mg/kg) combination dose (p = .033). The combined

insecticide treatment failed, however, to produce a significant change in TH IR within the

striatum, compared to vehicle controls. These data suggest that the significant increases in

GFAP IR neuropil, in the striatum, reflect some form of tissue insult, following exposure to a

high dose of PE, or PE/CP in combination, that is insufficient to induce degeneration of

dopaminergic terminals within the temporal interval investigated. Although such damage may be

sufficient to account for previously reported decreases in maximal dopamine uptake observed

with high doses of these compounds, the DAT IR data appear to indicate that this damage is

unlikely to be a change in the amount of DAT in these high dose conditions. The decreases in

striatal DAT IR neuropil observed for low doses of PE suggest an alteration in the normal

integrity of the nigrostriatal pathway and in the route by which environmental toxins may enter

dopaminergic neurons.

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Acknowledgments

Many individuals contributed to the successful completion of this thesis. I wish to extend

my sincerest appreciation to each of them.

I thank my advisor, Dr. Bradley G. Klein, for providing me with the opportunity to work

on this research project and for his invaluable mentorship and humor. I also wish to thank my

committee members, Drs. Jeffrey Bloomquist, Bernard Jortner, and Neels van der Schyf (former

committee member) for their support.

I am also grateful to Rebecca Barlow for providing instruction on dosing techniques and

Celia Dodd for providing technical assistance with a portion of the camera lucida drawings. I

also thank Dan Ward for statistical consultation and the Laboratory Animal Resources personnel

at Virginia Tech for animal care and maintenance.

Lastly, a special thanks to my mom and grandparents for their unwavering support of my

goals in life. I would not be where I am today without your love and guidance.

Supported by: U.S. Army, contract DAMD17-98-1-8633

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Table of Contents

Abstract………………………………………………………………………p. ii

Acknowledgments……………………………………………………………p. iv

List of Figures……………………………………………………..………….p. vi

List of Abbreviations………………………………………………..………..p. vii

Chapter 1: Introduction………………..……………………………….…….p. 1

Chapter 2: Materials and Methods…………………………………….…..…p. 28

Chapter 3: Results…………………………………………………………….p. 36

Chapter 4: Discussion………………………………………………………...p. 38

Chapter 5: Conclusion…………………………………………………..…….p 47

References…..……………………………………………….…………………p.49

Vita…………………………….……………………………………………….p. 70

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List of Figures

Figure 1. Basal Ganglia Circuitry and Significant Neurotransmitters……….……...p. 58

Figure 2. Synaptic Relationships of Importance………………………………….…p. 59

Figure 3. Measured Loci……………………………………………….……….……p. 60

Figure 4. Dosing Regimen …………………………………………………………..p. 61

Figure 5. Tissue Sectioning and Antibody Exposure……………..………………….p. 62

Figure 6. Striatal Section/Measured Loci…………………………………………..…p. 63

Figure 7. Illustration of Photoshop® Histogram……………………….……………...p.64

Figure 8. Examples of Staining for DAT, GFAP, TH……………...…………………p. 65

Figure 9. Changes in DAT Immunoreactivity / Low Doses PE………….……….…...p. 66

Figure 10. Changes in DAT, GFAP, TH Immunoreactivity / High Dose PE…......…...p. 67

Figure 11. Changes in TH Immunoreactivity / CP………………………………..…….p. 68

Figure 12. Changes in TH, GFAP Immunoreactivity / PE & CP in combination….……p.69

vii

Abbreviations

AChE = Acetylcholinesterase

CP = Chlorpyrifos

DA = Dopamine

DAT = Dopamine transporter

GFAP = Glial fibrillary acidic protein

IP = Intraperitoneal

IR = Immunoreactivity

PE = Permethrin

PD = Parkinson’s Disease

TH = Tyrosine hydroxylase

Introduction

The present investigation examined the potential role of insecticide exposure in the development

of idiopathic Parkinson’s Disease (PD). The insecticides selected for study were the pyrethroid

permethrin (Ambush ®, Pounce ®) and the organophosporus compound chlorpyrifos (Dursban

®, Lorsban ®). These compounds possess properties that could damage or disrupt the

nigrostriatal pathway, which is the principal target in PD. Reports of parkinsonism following

pesticide exposure (Sechi et al., 1992; Sanchez-Ramos et al., 1987; Bocchetta et al., 1986) make

pesticide-induced parkinsonism biologically plausible. The importance for investigating both

permethrin and chlorpyrifos is highlighted by the fact that both are widely used in military

environments, with particular emphasis on U.S. military personnel who served during the Gulf

War (President’s Advisory Committee Final Report, 1996). Employing immunohistochemistry

techniques, the present study looked at whether chlorpyrifos and permethrin alone, or in

combination, induce neuropathological hallmarks of PD.

General Hypothesis

Exposure to chlorpyrifos and permethrin, alone or in combination, produces anatomical changes

in the nigrostriatal pathway.

Review of Literature

Characterization of Parkinson’s Disease

2

PD was first described as a “shaking palsy” of unknown origin by James Parkinson in 1817. PD

is characterized by brady- and hypokinesia, resting tremor, rigidity, gait disorder and postural

reflex impairment (Marsden, 1994). The cardinal symptoms of PD result from the loss of

dopamine-containing cells of the substantia nigra that project to the striatum. The most

characteristic histological findings are loss of substantia nigra neurons, with Lewy bodies in the

surviving neurons (Gibb & Lees, 1988). Lewy bodies are small spherical inclusions, which

consist of a dense granular core surrounded by a halo of radiating filaments. It is suspected that

these deposits are involved in inducing the death of dopaminergic neurons. In general, it is

believed that the majority of pigmented nigral cells must be lost before clinical signs appear

(Morrish et al., 1996). PD is exceptionally rare before age 40, although there are juvenile forms

that may be inherited (Matsumine et al., 1997). PD incidence and prevalence rises sharply

beginning at age 50 and increases throughout life (Mayeux et al., 1995). In the U.S., PD

prevalence and mortality are slightly higher among Caucasians than African Americans (Mayeux

et al., 1995). Society pays an enormous price for Parkinson’s disease. According to the National

Parkinson Foundation, each patient spends an average of $2,500 a year for medications. After

factoring in office visits, Social Security payments, nursing home expenditures, and lost income,

the total cost to the nation is estimated to exceed $5.6 billion annually.

Etiology of Parkinson’s Disease

1. Age

3

The causes of PD have been debated for 150 years, with no resolution. As noted above, PD is

primarily associated with populations of middle aged or older individuals. In non-PD brains, the

number of nigral cells is reduced by 4.7-6.0% per decade from the fifth to the ninth decade of

life (Gibb & Lees, 1991), but this cell loss is not sufficient to produce Parkinsonian features,

which requires 80-90% loss (Thiessen et al., 1990). Moreover, the pattern of striatal dopamine

loss (Kish et al., 1992) and nigral cell loss (Fearnley & Lees, 1991), in old age, are significantly

different from that in PD. These and other studies (Wolters & Calne, 1989; Scherman et al.,

1989) make it readily apparent that although increasing age is the single most unequivocal risk

factor for development of PD, PD is apparently not caused by the aging process alone.

2. Genetics

The likelihood of a genetic cause for PD has been diminished somewhat by the publication of a

study of 20,000 twins (Caroline et al., 1999). The study demonstrated that identical twins do not

develop PD any more often than two unrelated individuals. In addition, analysis of familial PD

provides no evidence for mitochondrial, X-linked, or polygenic inheritance (Maraganore et al.,

1991). More recently, several mutations have been found that may lead to familial PD (Dunnett,

2000). Two of these mutations, in the genes encoding parkin and ubiquitin carboxy-terminal

hydrolase-L1 (UCH-L1), lead to diseases that are fairly distinct, both clinically and

pathologically, from idiopathic PD and how they relate to classical PD remains ambiguous. A

third category of mutations has been more informative however. These mutations affect the gene

for alpha-synuclein, a synaptic protein which is a major component of the filaments associated

with Lewy bodies. In mice, over-expression of human alpha-synuclein results in impaired motor

4

function, neuronal degeneration, and neuritic and nuclear alpha-synuclein aggregates (Masliah,

2000). Finally, a genetic defect might generate endogenous neurotoxins, impair the ability to

deal with toxic substances produced in the brain through normal metabolism, or impair the

ability to deal with exogenous toxins. At present, the consensus is that an inherited factor may

play a role in some cases of PD, but heredity cannot by itself account for the totality of the

disease.

3. Metabolic Defects

Various metabolic defects in PD have been reported (Steventon et al., 1989; Tanner, 1991)

implying that PD patients have less effective detoxification systems. This raises the

possibility that PD is the result of increased susceptibility to endogenous or exogenous

toxins (Koller et al., 1990). For example, defects in enzyme detoxification systems

including debrisoquine hydroxylation, sulfur conjugation, oxidation, and

thiolmethylransferase activity could lead to potentiation of relatively low-level neurotoxic

exposures in PD patients (Steventon et al., 1989).

Pharmaceutical therapies for PD target MAO-B, an enzyme that metabolizes dopamine in the

brain, by inhibiting the activity of this enzyme, thus delaying the breakdown of naturally

occurring dopamine and dopamine formed by such drugs as levodopa. There is also evidence of

oxidative damage associated with PD. Elevated levels of lipid peroxidation products

malondialdyde and lipid hydroperoxide have been found in the substantia nigra of PD patients

(Dexter et al., 1994). Furthermore, increased levels of protein carbonyls and 8-hydroxy-2-

deoxyguanosine, reflecting oxidative damage to proteins and DNA, respectively, have been

5

found in the substantia nigra as well as in various other brain regions of PD patients (Alam et al.,

1997).

Increased iron has been found in several areas of the brain including the globus pallidus and

substantia nigra of PD patients (Morris et al., 1992). The increased Fe3+ content, especially in

the substantia nigra, and the production of H202 during dopamine metabolism in dopaminergic

neurons, points to the possibility that these neurons are damaged by the highly reactive hydroxyl

radicals produced by the Haber-Weiss reaction. The iron that is present, with no increased

content of ferritin in the substantia nigra, leaves free, accessible iron. Therefore, Fe3+, H2O2 and

O2 are all present and available for the Haber-Weiss reaction, leading to oxygen damage by the

hydroxyl radical (Sofic et al., 1988). How iron accumulates within the globus pallidus and

substania nigra remains a mystery to date. It is unclear if iron accumulation in PD is primary or

secondary. Moreover, it remains to be determined if oxidative damage in PD is a primary event

or occurs secondary to some other etiology, postmortem event, or drugs. For instance, iron

accumulation in PD has been detected following treatment with the toxin 1-methyl-4-phenyl-

1,2,3,6-tetrahydropyridine (MPTP) (Temlett et al., 1994).

Lastly, a possible relationship between metabolic defects and environmental factors, such as

MPTP, may play a role in the development of PD. The hepatic cytochrome P450 enzyme,

debrisoquine hydroxylase (CYP2D6), which is also expressed in substantia nigra dopaminergic

neurons, may detoxify MPTP (Gilham et al., 1997). Several hepatic enzymes have been

implicated in PD including CYP2D6, and cystine dioxygenase. In fact, variant alleles coding for

specific cytochrome P-450 isoenzymes have been identified with increased frequency in

6

individuals with PD, especially those with “early onset” PD (Steventon et al., 1989). Thus,

defective detoxification systems may render the organism unable to deal with toxins of internal

or external origin. One should note that it is difficult to determine whether metabolic defects are

precursors or results of PD.

4a. Environmental Factors: MPTP

The discovery that the toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces

parkinsonism in humans has provided further support for the role of exogenous toxins in PD. In

addition, MPTP has provided valuable insight into the biochemical processes involved in

PD-like nigral cell loss (Langston et al., 1983). MPTP leads to marked decreases in the levels of

dopamine and its metabolites, and in the number of dopaminergic terminals in the striatum

(Zuddas et al., 1992) as well as a severe loss of the tyrosine hydroxylase (TH)-immunoreactive

cell bodies in the substantia nigra, pars compacta (Ricaurte et al., 1987). The mouse is the most

responsive rodent to MPTP in its neurochemical aspects (Markey et al., 1986), i.e. a decrease in

tyrosine hydroxylase activity (Hallman et al., 1985) and a depletion of DA in the striatum

(Bradbury et al., 1986). MPTP injections have been shown to reduce DA levels acutely in the

mouse striatum by as much as 90% (Hallman et al., 1984).

MPTP is converted to MPP+ by MAO-B-containing cells, which in turn causes a selective

destruction of nigral dopaminergic neurons. MPP+ inhibits complex I, which results in

mitochondrial respiratory chain dysfunction and consequently diminishes the production of

energy. Complex I inhibition is sufficient on its own to cause nigral cell death (Tipton & Singer,

7

1993). Inhibition of complex I also leads to increased shunting of electrons through complex II,

which may generate 5-7 times as many reactive oxygen species compared with passing electrons

through complex I (Dykens, 1997). Complex I is a major site of proton pumping and it is

possible that a complex I defect in PD may contribute to neuronal vulnerability and lead to

apoptosis. Nigrostriatal MPTP neurotoxicity is dependent on nitric oxide synthase, which forms

nitric oxide, a potential precursor to the toxic peroxynitrite anion and hydroxyl free radical

(Beckman et al., 1993). These oxidative stresses likely contribute to the death of vulnerable

neurons. Similar processes may occur in PD, as mitochondria from multiple PD tissues,

including platelets, fibroblast, muscle, substantia nigra and non-nigral brain regions, exhibit

reduced complex I activity or protein levels, suggesting a systemic mitochondrial defect

(Nieminen et al., 1995; Parker et al., 1989).

Although MPTP has been shown to cause a profound depression of striatal dopamine in most

studies, the non-homogenous pattern of striatal dopamine depletion that occurs in Parkinson’s

disease, greater loss in putamen as compared to caudate nucleus, has not been clearly

demonstrated (Hornykiewicz et al., 1988). Furthermore, cell degeneration in Parkinson’s disease

is more widespread than that produced by MPTP (Forno, 1982). Perhaps even more importantly,

the characteristic inclusion bodies (Lewy bodies) seen in Parkinson’s disease have yet to be

convincingly demonstrated in MPTP-treated animals. Despite such differences, MPTP-induced

neurotoxicity provides the most widely used current model for examining the mechanisms of

Parkinson’s-like neurodegeneration.

MPTP-induced neurotoxicity suggests that exogenous natural or synthetic compounds,

8

resembling MPTP in actions, may be responsible for PD. MPP+ bears structural similarities to

the herbicide paraquat (Barbeau et al., 1986), suggesting a possible role for this and other

pesticides in PD etiology. In fact, MPP+ was earlier proposed as a herbicide (Cyperquat) but was

never registered (Markey et al., 1984).

4b. Environmental Factors: Pesticides

Pesticides have benefited our society by improving our food supply, controlling harmful pests

and improving our overall quality of living. However, the widespread use of pesticides is not

without risk for our environment and our health. In 1991 the United States used 2.7 billion

pounds of pesticides, approximately 10 pounds of pesticide for every man, woman and child

living in the US (EPA, 1991). It is virtually impossible for anyone to avoid exposure to low

levels of numerous pesticides in our environment (Morgan, 1992).

Several studies suggest that environmental factors, such as pesticides, may be related to the

cause of PD. Hertzman et al. (1994) found a significant association between PD and an

occupation of handling pesticides in British Columbia. PD is most prevalent in industrialized

countries (Tanner, 1989). In China for example, the ubiquity of PD is much lower as compared

to the more industrialized USA (Li et al., 1985). However, even in China, PD appears to be

associated with exposure to industrialized chemicals (Tanner et al., 1989). For example, Ho et al.

(1989) found that subjects who had previously used herbicides/pesticides had a 3.6-fold

increased risk of developing PD in Hong Kong.

9

Many factors associated with a rural environment have been studied, the most frequent being

well-water use and pesticides. Rajput et al. (1984, 1987) reported for the first time the

relationship between living in a rural environment and the risk of PD. Low nigral neuronal cell

counts have also been detected in rural provincial residents (Thiessen et al., 1990). In Young

Onset Parkinsonism (YOPD), a greater exposure to rural living and well-water consumption has

been found, suggesting a dose-response relationship between environmental risk factors and age

of PD-onset (Koller et al., 1990). It is paradoxical that industrialization has been proposed to

explain the differences in geographical prevalence rates of PD between countries, yet within

countries, residing in a rural area has been noted to increase risk as well. However, this may be

related to the fact that agricultural pesticide use is often associated with rural areas, and water

supplies in these areas are not as diligently checked for such chemicals compared with urban,

public water supplies.

Two recent studies, one from Germany (Seider et al., 1996) and another from Taiwan (Liou et

al., 1997), suggest increasing risk gradients with years of application of organophosphates and

various herbicides. There is some clinical data specifically linking pesticide exposure to PD.

Acute exposure to the herbicide diquat has been associated with the development of a

parkinsonian syndrome (Sechi et al., 1992). In addition, case history studies have identified

individuals exposed to mixtures of pesticides who suffered from young-onset PD (Bochetta &

Corsini, 1986). Furthermore, post mortem brain tissue from PD patients has been analyzed for

the presence of organochlorine pesticides (Fleming et al., 1994), with the incidence of PD

significantly correlated with the presence of brain residues of the insecticide dieldrin.

10

Kirby et al. (2001), has shown that heptachlor, an organochlorine, exerts selective effects on

striatal dopaminergic neurons and thus may play a role in the etiology of PD. Walters et al.

(1999) has demonstrated that diethyldithiocarbamate (DDC) enhances MPTP-induced nigral cell

loss. Finally, Betarbet et al. (2000) has shown that chronic intravenous administration of the

mitochondrial toxin rotenone, in rats, produces effects that closely resemble human PD. The rats

exhibited several cardinal behavioral features of the disease. Histological examination indicated

that rotenone treatment lead to progressive degeneration of the nigrostriatal pathway. Most

importantly, the surviving nigral neurons developed intracytoplasmic alpha-synuclein inclusions

that appeared similar to Lewy bodies. Furthermore, the author’s results suggested that striatal

nerve terminals were affected earlier and more severely by rotenone than nigral cell bodies. The

fact that the dompaminergic nerve terminals degenerate first may be a reason why the effects of

PD are not detected when looking at substantia nigra instead of the striatum and possibly missing

this more subtle effect. Correspondingly, in the brains of individuals with PD, there is a more

profound loss of dopamine in the striatum as compared to the substantia nigra. These findings

could have implications for theories of the cause of PD.

It is puzzling that, since the introduction of pesticides, the prevalence of PD has not drastically

increased (Koller et al., 1990; Wermuth et al., 1997; Rajput et al., 1990). Also, the type of

Parkinsonism associated with pesticide exposure is often pathologically and morphologically

different from PD (Koller et al., 1990; Tanner, 1989; Rajput et al., 1984). However, the studies

of the relationship between pesticides and parkinsonism are confined in scope and supported by

limited experimental investigation.

11

There is some evidence of increasing incidences of PD among persons aged 75 years and older

in the United States, many Western European countries and Japan (Kurtzke et al., 1990). Thus,

the effects of pesticides may accumulate over time, resulting in a long latency period of

neurotoxic effects. In addition, pesticides may still act as a risk factor when used in combination

with each other. Furthermore, these compounds may induce sub-clinical changes in the

nigrostriatal pathway. Such changes could lower the threshold for effects of other endogenous

or exogenous factors that may be contributing to PD.

Since there is very little experimental data available on pesticides and their relationship to PD,

the present investigation looked at this experimentally. The study with rotenone by Betarbet et

al. (2000), discussed above, supports a mitochondrial role in the pesticide/PD link. The current

investigation looked at pesticides that may exert their effects through a different mechanism. In

order to clarify these mechanisms, it is essential to have a general working knowledge of the

organization of the basal ganglia as it relates to PD.

Basal Ganglia Anatomy

1. Direct and Indirect Pathways

The basal ganglia have been implicated in a wide variety of motor functions, including the

planning, initiation and execution of movements, the performance of learned movements and

sequencing of movements (Martin et al., 1994). As alluded to above, PD interferes with the

integrated action of the basal ganglia. The basal ganglia consist of four main structures: the

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striatum (caudate nucleus, putamen and nucleus accumbens), the pallidum (external and internal

segments of the globus pallidus and ventral pallidum), the subthalamic nucleus and the substania

nigra (pars compacta and pars reticulata). The striatum is the input structure of the basal ganglia,

receiving afferents, in a topographical fashion, from the entire cerebral cortex, the midline and

intralaminar thalamic nuclei, and midbrain serotonergic and dopaminergic neuron groups. The

output structure of the basal ganglia consists of the internal segment of the globus pallidus and

the pars reticulata of the substantia nigra, which project in a topographical manner as well to

different medial and ventral thalamic nuclei, the deep layers of the superior colliculus and the

reticular formation of the mesencephalon. The various thalamic nuclei that are innervated by

these output structures of the basal ganglia project to different cortical areas of the frontal lobe,

including motor, premotor and prefrontal cortical areas.

The input (striatum) and output structures (internal segments of the globus pallidus and pars

reticulata of the substantia nigra) of the basal ganglia are connected to each other by means of

two pathways, a “direct” and “indirect” pathway (Martin et al., 1994). Activation of the direct

pathway is thought to be involved with the facilitation of “desired” motor behaviors, with

activation of the indirect pathway being involved in “suppression of undesirable” motor

components (Mink et al., 1993). The direct pathway consists of co-localized GABA/substance

P/dynorphin-containing striatopallidal (striatum to internal segment of the globus pallidus) and

striatonigral (striatum to pars reticulata of the substantia nigra) projections, marked by “D” in

Figure 1. The indirect pathway is comprised of the sequence of the GABA/enkephalin-

containing striatopallidal (striatum to external segment of the globus pallidus), the GABA-ergic

pallido-subthalamic (external segment of globus pallidus to subthalamic nucleus), and the

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glutamatergic subthalamo-pallidal (sub thalamic nucleus to internal segment of the globus

pallidus) projections, denoted by “I” in Figure 1. At the level of the output structures of the

basal ganglia, these direct and indirect pathways have opposite effects on the GABA-ergic

neurons that project to the thalamic nuclei, the superior colliculus and the reticular formation. A

“balance” between these two striatal output pathways appears to be essential for the normal

regulation of movement. In PD, loss of dopamine leads to enhanced activation of the indirect

pathway, with increased excitation of the internal segment of the globus pallidus/substania nigra

pars reticulata neurons. This results in decreased facilitation of cortical motor areas and

subsequent development of akinesia and bradykinesia that is exhibited in PD (Wichmann et al.,

1993).

2. Dopamine Components

The dopaminergic projection from the substania nigra to the striatum, denoted by “DA” in Figure

2, modulates the flow of cortical information through the basal ganglia. The dopaminergic

terminals form symmetric synaptic contacts primarily with the necks of dendritic spines of spiny

projection neurons in the striatum (Smith et al., 1994). The head of these spines receive input

from terminals of glutamatergic neurons originating in the cortex, which form asymmetric

synapses. This anatomical arrangement is well suited for the dopamine released from the

nigrostriatal terminal, which acts on dopamine receptors localized both within the synapse and at

extrasynaptic sites, to selectively modulate the response to the excitatory cortical input at the

head of the spine (Yung et al., 1995).

14

Dopamine has opposing effects on the direct and indirect pathways (Gerfen, 1992): via

dopamine D1 receptors a stimulatory effect on the direct pathway, whereas via D2 receptors an

inhibitory effect on the indirect pathway (see Figure 1). Normally, the overall effect of

dopamine release within the striatum is to reduce inhibitory basal ganglia output to the thalamus,

leading to increased activity of the thalamocortical projection neurons. In PD, a compromised

dopaminergic pathway leaves an unantagonized, excitatory cortical input to influence the

striatum (Wichmann et al., 1994) (see Figure 1). The repercussion of the loss of dopaminergic

input to the striatum, as occurs in PD, is thought to be that the indirect pathway is stimulated,

resulting in a greater than normal excitation of the globus pallidus internal segment and substania

nigra pars reticulata. This increases the GABAergic output from the internal segment of the

globus pallidus and the pars reticulata of the substania nigra, to the thalamus (DeLong, 1990).

This ultimately results in a reduction of cortical activation, which accounts for most of the

symptoms of PD. Furthermore, any alteration of the normal dopaminergic input to the striatum

should have serious consequences for normal motor behavior, potentially producing Parkinson’s-

related signs.

3. Acetylcholine Components

The role of acetylcholine in the striatum is also of importance. Acetylcholine is contained in

many of the large spiny inter-neurons that make up about 5 percent of the striatal neurons. The

remaining 95% of striatal neurons are spiny GABA projection neurons (Marin et al., 1997). The

cholinergic interneurons have relatively short axons that are confined to the striatum and synapse

predominantly with medium spiny neurons that project to the globus pallidus internal segment

15

/substania nigra pars reticulata (Izzo et al., 1988) (see Figure 2B). The striatum is rich in the

enzyme acetylcholinesterase (Lehmann et al., 1983), which is normally present at cholinergic

synapses and terminates the synaptic action of acetylcholine. Thus, a cholinesterase-inhibiting

compound, such as the insecticide chlorpyrifos, could disrupt the normal synaptic interaction

between cholinergic interneurons and spiny projection neurons within the striatum. This also has

the potential to produce PD-like motor signs.

4. Dopaminergic-Cholinergic Interactions

Cholinergic interneurons appear to have an excitatory effect on striatal projection neurons, which

can be counteracted by dopamine (see Figure 2). The interaction between cholinergic and

dopaminergic systems, at the medium spiny projection neurons, is therefore central to the

pharmacology of the basal ganglia as the balance between acetylcholine and dopamine levels

appears necessary for normal striatal function (Lehmann et al., 1983). Furthermore, the synaptic

effects of dopaminergic and cholinergic neurons, on each other, could also profoundly affect

normal basal ganglia function. Increases in striatal acetylcholine levels, as a result of dopamine

depletion, is associated with the symptoms of PD. This can be explained by the interactions

between dopamine and acetylcholine at the striatal level since dopamine is known to tonically

inhibit the release of acetylcholine in the striatum under normal conditions (Lehmann et al.,

1993). In addition, nicotinic acetylcholine receptors exist on the terminals of dopaminergic

nigrostriatal neurons. Stimulation of these receptors can modulate the release of dopamine from

terminals. It therefore appears that the dopaminergic and cholinergic systems, within the

striatum, can potentially alter the normal function of the basal ganglia through their individual

16

actions or through synaptic interactions between cholinergic and dopaminergic neurons.

As will be discussed below, the present investigation examined the effects of insecticides that

have been shown to interfere with cholinergic and dopaminergic systems, upon the integrity of

the nigrostriatal dopaminergic projection. Again, this projection is a major regulator of normal

basal ganglia function and the major target of pathological changes in PD.

Pesticides Used In The Present Investigation

1. Chlorpyrifos

Chlorpyrifos is a broad-spectrum organophosphate insecticide. It is used as an insecticide on

grain, cotton, fruit, nut and vegetable crops as well as lawns and ornamental plants. In addition,

it is also registered for direct use on sheep and turkeys, for horse site treatment, dog kennels,

domestic dwellings, farm buildings, and commercial establishments (EPA, 1996). Chlorpyrifos

is moderately toxic to humans. While some organophosphates are readily absorbed through the

skin, studies of chlorpyrifos in humans suggest that skin absorption is limited (Gallo et al.,

1991). Symptoms of acute exposure to organophosphate compounds may include the following:

numbness, tingling sensations, incoordination, headache, dizziness, and tremor. The oral LD50

for chlorpyrifos is 60mg/kg in mice (Gallo et al., 1991).

The insecticidal mode of action, as well as the acute toxicity of organophosphorous insecticides

in non-target species, arise from inhibition of acetylcholinesterase (AChE) in cholinergic

17

synapses by the parent compound or an active metabolite. This results in subsequent

accumulation of acetylcholine at synaptic terminals (Murphy, 1986). Accumulation of ACh

within synapses, following extensive AChE inhibition, leads to overstimulation of postsynaptic

muscarinic or nicotinic cholinergic receptors, which in turn elicits signs of cholinergic

overstimulation such as autonomic dysfunction and involuntary movement.

Chlorpyrifos has been shown to cause persistent inhibition of cortical and striatal cholinesterase

and a reduction of total muscarinic binding sites (Pope et al., 1992). This has the potential to

disrupt cholinergic neurotransmission in the striatum mediated by the cholinergic interneurons

(Bowman and Rand, 1980). Given the existence of acetylcholine receptors on the terminals of

nigrostriatal dopaminergic neurons, acetylcholine inhibition could disrupt the normal integrity of

this dopaminergic striatal input. Li et al. (2000) has shown that chlorpyrifos can affect dopamine

turnover in the striatum, as indicated by significant increases in the dopamine metabolite 3, 4-

dihydroxyphenylacetic acid (DOPAC). Therefore, it is conceivable that inhibition of AChE at

the synaptic terminals of cholinergic interneurons, from chlorpyrifos exposure, could produce

PD-related alterations of normal motor behavior. However, very little clinical evidence exists of

a possible OP-induced parkinsonism. Only two authors, Davis (1978) and Bhatt (1999), report of

patients who developed acute and reversible parkinsonism following OP exposure. However,

this does not preclude the existence of sub-clinical effects of chlorpyrifos on basal ganglia

function or a synergistic contribution when combined with other insecticides.

Inhibition of a critical fraction of neurotoxic esterase (neuropathy target esterase, NTE) in target

neural tissue by pentacovalent organophosphate compounds (chlorpyrifos) of the phosphate,

18

phosphonate, or phosphoramidate classes triggers a symmetrical degeneration of sensory and

motor axons in distal regions of peripheral nerves and/or spinal cord tracts. This condition is

called organophosphate compound-induced delayed neurotoxicity (OPIDN) (Johnson, 1982).

Although very high doses of chlorpyrifos are necessary for OPIDN, little is know about the

possibility that a similar type of degeneration may occur in nigrostriatal axons. It should also be

noted that chlorpyrifos is capable of inducing an increase in cell stress, as measured by decreased

mitochondrial integrity (Karen et al., 2001). As mentioned previously, the mitochondrial toxin

rotenone is capable of inducing PD-like degeneration of the nigrostriatal pathway. Thus,

chlorpyrifos-induced effects on mitochondria might result in nigrostriatal cell degeneration as

well.

2. Permethrin

The synthetic pyrethroids are potent and widely used insecticides derived from natural pyrethrins

found in the flowers of the genus Chrysanthemum. Synthetic pyrethroids are increasingly being

used in veterinary applications on farm and pet animals, for the protection of foodstuffs, for the

control of endemics and parasites in public health programs as well as for household applications

in kitchens and bedrooms. Particularly with indoor applications, a sustained contamination can

result from the absorption of pyrethroids to small dust particles and various other surfaces

(Schwabe et al., 1994). Chronic exposure of unaware individuals to low doses of synthetic

pyrethroids consequently results. Synthetic pyrethroid insecticides may therefore represent an

important indoor and environmental toxin. Based on a clinical analysis, a causative link between

the exposure to synthetic pyrethroids and the development of motor and sensory disorders,

19

including Parkinson’s-like syndromes, has been postulated (Mulle-Mohnssen and Hahn, 1995).

Pyrethroids are lipophilic molecules that generally undergo rapid absorption and distribution

following ingestion by birds and mammals. Unless isolated in lipid deposits, they are quickly

metabolized and eliminated from the body (Gaughan et al., 1977). They have a low oral toxicity

to mammals and in general, their insect (topical) to mammal (oral) toxicity ratio is much higher

than that of the other major classes of insecticides (Elliott et al., 1978). For example, the acute

oral toxicity of permethrin is 490mg/kg in the mouse (Casida, 1983). Although pyrethroids elicit

nerve toxicity at high doses, low levels have not been observed to cause any deleterious

histological changes in the mammalian peripheral nerves. Doses of 100mg/kg, however, can

cause some pathological alterations in peripheral nerves of mice and rats (Parker et al., 1985).

Pyrethroid insecticides prolong the open-state of voltage-sensitive sodium channels, therefore

enhancing the depolarization and activity of affected neurons (Vijverb & Van den Berken, 1990;

Tatebayashi & Narahasi, 1994). Other toxic mechanisms associated with synthetic pyrethroids

include inhibition of Na+/Ca++ ATPase, Ca++/Mg++ ATPase, and calmodulin (Calck &

Matsumura, 1982). Under voltage-clamp conditions pyrethroids produce a slowly-decaying

sodium tail current following a depolarizing pulse and, in some preparations, also delays sodium

current inactivation during depolarization or alters the voltage dependence of sodium channel

activation or inactivation. Patch clamp studies of pyrethroid action confirm that pyrethroids

retard channel activation and inactivation at the single channel level (Jacques et al., 1980).

Based on the symptomology after acute intoxication of insects and mammals, pyrethroids fall

20

into two classes: Type I pyrethroids, such as permethrin, are non-cyano containing and cause

repetitive discharges in nerve fibers as a result of prolongation of sodium currents. This effect

does not result in large membrane depolarization, thus impulse conduction block is not observed.

Type II pyrethroids do cause a large depolarization of nerve membranes and impulse conduction

block due to an extremely prolonged sodium current (Leahey, 1985). In mammals, Type I

pyrethroid effects include aggressive sparring behavior, increased sensitivity to external stimuli,

fine tremors, coarse body tremor, and an increased body temperature. Effects of pyrethroids on

the ligand-binding characteristics of the acetylcholine (ACh) receptor-ionic channel complex

have led to the suggestion that these insecticides delay the closing or desensitization of the ionic

channels coupled to the pre-synaptic nicotinic ACh receptor (Sherby et al., 1986; Abbassy et al.,

1983). Such receptors exist on the terminals of dopaminergic nigrostriatal neurons.

There is some evidence that components of the nigrostriatal system appear to be the origin of

pyrethroid intoxication (Ray, 1980). There is evidence that pyrethroids can affect dopamine

turnover in the striatum (Husain et al., 1991; Karen et al., 2001). Type I & Type II pyrethroids

have been shown to cause 23-37% increases in striatal content of the dopamine metabolite 3, 4-

dihydroxyphenylacetic acid (DOPAC). Toxic symptoms and increases in DOPAC were

associated with higher brain concentrations for type I than type II pyrethroids (Doherty et al.,

1988). In addition, high doses of permethrin have been shown to interfere with the

mitochondrial respiratory chain (complex I) (Wassner et al., 1997) which, in the case of some

compounds, such as MPP+, has been shown to induce nigrostriatal degeneration. Some

pyrethroids may modulate dopamine transporter (DAT) function in striatal synaptosomes. For

example, increased dopamine uptake has been found with low doses and decreased uptake at

21

higher doses of permethrin, as well as the related pyrethroid deltamethrin (Kirby et al., 1998;

Karen et al., 2001). Finally, Harp et al., (2000) has shown that permethrin can alter synaptosomal

dopamine uptake at doses orders of magnitude below the LD50 of the compound.

Given the synaptic organization of the basal ganglia and a limited body of experimental

evidence, it appears that both the anti-cholinesterase organophosphate insecticides and the

pyrethroids are capable of altering the functional integrity of dopaminergic nigrostriatal neurons.

The current study will attempt to expand the limited body of experimental literature on the

relationship between insecticides and PD by examining putative anatomical changes in the

dopaminergic nigrostriatal pathway of the mouse following exposure to permethrin, chlorpyrifos

or both compounds together. The markers that will be used to indicate anatomical changes are

described below.

Dependent Variables (indicators of PD-like damage)

1. DAT

The present investigation examined changes in the amount of DAT protein in dopamine striatal

afferents through staining, using DAT immunohistochemistry. Such a change can occur in the

presence or absence of neural degeneration and could indicate a functional alteration of

dopaminergic neurons.

Dopamine neurotransmission is terminated largely by reuptake through a specific,

22

sodium-dependent dopamine transporter (DAT) (Amara et al., 1993). The synaptic

concentration of dopamine, and therefore, the level of dopamine receptor stimulation, is

regulated by DAT activity. Through this mechanism, DAT may be critically involved in certain

pharmacological or pathological conditions. DAT is absent in parkinsonian putamen and is

reduced in the caudate nucleus (Niznik et al., 1991). DAT is an excellent marker for

dopaminergic neurons because it is more selective than tyrosine hydroxylase, which is also

expressed in other types of catecholaminergic neurons. DAT plays a role in the toxicity of MPTP

(Kopin et al., 1988) by transporting the toxic metabolite, MPP+, into the vulnerable neuron,

leading to dopaminergic neuronal death (Niznik et al., 1991; Chinaglia et al., 1992). In addition,

DAT is the site through which amphetamine stimulates nonvesicular release of dopamine (Giros

et al., 1996). PD is an example where the observed reduction in DAT is due, at least in part, to

the loss of nerve terminals carrying these transporters (Uhl & Kitayama, 1993).

In vivo DAT imaging studies have demonstrated a reduction in DAT density in PD patients as

compared to healthy controls. The reduction in DAT density is both region specific, (putamen

being greater than caudate), and asymmetric, which is consistent with both pathologic

assessment of DAT loss and clinical presentation of PD (Fischman et al., 1998). Studies using

light microscopic immunocytochemistry for DAT (Ciliax et al. 1995; Freed et al., 1995) have

demonstrated that DAT is expressed at the highest levels in the patch compartments of the

striatum, which are known to receive dense inputs from dopaminergic perikarya in the ventral

tier of the substantia nigra (Gerfen et al., 1987). Because DAT can transport dopamine

neurotoxins, the expression of high levels of DAT in nigrostriatal dopaminergic neurons

represents an attractive model to explain the selective vulnerability of these neurons (Edwards,

23

1993).

It therefore appears that the amount of DAT protein, within the striatum, represents a sensitive

anatomical substrate indicative of an alteration in the integrity of the nigrostriatal pathway. Such

changes could occur in the absence of neuronal death or degeneration. In this study, DAT

immunocytochemistry was used to evaluate the effects of low doses of permethrin on the amount

of DAT in the striatum, since there is pharmacological evidence that such doses of permethrin

can increase dopamine uptake in mouse striatal synaptosomes (Karen et al., 2001).

2. TH

The present study examined changes in the number of dopaminergic striatal afferents through

immunocytochemical staining for the enzyme TH (tyrosine hydroxylase). TH is the first enzyme

(rate-limiting) in the pathway for dopamine synthesis and therefore was used as a marker to

examine the effects on the survival of dopamine-containing terminals. In the striatum,

TH-positive structures represent the axons and axon terminals of dopamine neurons (Anden et

al., 1964). TH is a mixed-function oxidase that uses molecular oxygen and tyrosine as its

substrates and biopterin as its cofactor. TH is a homotetramer, each of whose subunits has a

molecular weight of 60 kDa. TH catalyzes the addition of a hydroxyl group to the meta position

of tyrosine, thus forming, 3, 4-dihydroxy-L-phenyalanine (L-dopa). It is primarily a soluble

enzyme, localized in the cytosol of catecholamine-containing neuronal processes. Tyrosine

hydroxylase levels are 25% higher in the substantia nigra, compared to that in the striatum

(MacKay et al., 1978). Immunocytochemical techniques which use antibodies against tyrosine

24

hydroxylase TH have permitted the mapping of catecholaminergic cell bodies and fiber networks

in the CNS at the light microscopic level (Hokfelt et al., 1976; Pickel et al., 1975). Decreased TH

immunoreactivity has been widely used as an indicator of dopaminergic neuronal degeneration

(Haycock et al., 1991; Freund et al., 1984; Pickel et al., 1981). The paucity of norepinephrine

and epinephrine input into the striatum makes TH immunoreactivity an appropriate marker for

dopamine neuronal degeneration, which could be induced by chlorpyrifos, an organophosphate

that is capable of producing OPIDN and cell stress. Furthermore, TH immunocytochemistry was

used to determine whether previously reported decreases in dopamine uptake, induced both by

high doses of permethrin, as well as chlorpyrifos, are attributable to degeneration of

dopaminergic nigrostriatal neurons.

3. GFAP

MPP+ and structurally related compounds have been shown to modulate the TH system in tissue

slices of rat striatum. Therefore, it is conceivable that an upregulation of TH in surviving

dopamine terminals could mask any nigrostriatal terminal degeneration (Tatton et al., 1991).

Given this possibility, an additional marker, GFAP, was used to detect pesticide-induced tissue

insult within the striatum. Such damage could be more subtle than frank degeneration.

GFAP immunohistochemistry is the most commonly used method to examine the distribution of

astrocytes in response to neural degeneration or injury (Dahl et al., 1986; Kobayashi et al.,

1986). Assays of GFAP reveal dose-, time-, and region-dependent patterns of neurotoxicity at

toxic doses below those that cause light microscopic evidence of cell loss or damage. GFAP

25

immunostaining has been shown to be a sensitive marker of MPTP neurotoxicity (Francis et al.,

1995). MPTP treatment in mice causes astrocyte hypertrophy and increases in GFAP

immunoreactivity in the striatum (Stromberg et al., 1986). The highest concentrations of GFAP

staining in the mouse have been found in the brainstem, and the lowest concentrations found in

the striatum and cortex (Martin & O’Callaghan, 1995).

GFAP, a 50 kDa protein, represents the major intermediate filament of mature, differentiated

astrocytes (Norenberg, 1994). Its function is unknown, but may play a role in the maintenance of

cell structure. Increases in GFAP immunoreactivity may occur within several hours of an insult

(Schmidt-Kastner et al., 1993). However, this is likely not due to an actual increase in the GFAP

protein level, but may be associated with edema resulting in a dispersion of intermediate

filaments and the exposure of additional epitopes as visualized by immunohistochemistry.

Therefore, GFAP content does not necessarily correspond with the immunohistochemistry (Eng

et al., 1989). When direct measurement of GFAP protein is performed, the increase is usually

seen after 1 to 3 days, reaching peak 5 to 8 days, and depending on the nature of the injury, may

thereafter subside or remain elevated. In addition, corresponding rises in GFAP mRNA occur

(Condorelli et al., 1990; Burtrum et al., 1994). While elevation in GFAP is characteristic of CNS

injury, immunostaining will not occur if astrocytes are destroyed in the area of injury (Schmidt-

Kastner et al., 1993). The increase will be observed in the surrounding viable tissue.

Justification and Hypotheses

Karen et al., (2001) have demonstrated that low IP doses of the pyrethroid insecticide

26

permethrin can increase maximal dopamine uptake in striatal synaptosomes to a peak of 134% of

control at doses of 1.5 – 6 mg/kg. Such changes could reflect a change in transporter kinetics or

a change in the amount of transporter protein employed by dopamine nigrostriatal neurons. To

investigate the latter possibility, the current investigation used immunohistochemistry staining

for DAT in the striatum of mice treated with low doses of permethrin and in matched vehicle

controls.

Hypothesis 1

Low doses of the pyrethroid insecticide permethrin will significantly increase the amount of

DAT immunoreactive protein within the striatum of the C57BL/6 mouse.

As noted above, at low IP doses, permethrin increases maximal dopamine uptake in striatal

synaptosomes. However, at high doses of 200 mg/kg, dopamine uptake is significantly less than

that of controls, (about 50%) (Karen et al., 2001). This could reflect a change in transporter

kinetics or a loss of dopamine nigrostriatal terminals from the sampled region. Karen et al.,

(2001) have shown that 200 mg/kg doses of permethrin do not alter the efficiency of dopamine

transport. However, as noted above, there is some evidence that permethrin can produce some

histopathologies in peripheral nerves of rodents and can also induce potentially cytotoxic effects

on striatal mitochondria. To investigate whether such neuro-pathology occurs centrally, TH

immunohistochemistry was used to assess whether there was a loss of the TH-synthesizing axon

terminals within the striatum. GFAP immunohistochemistry was employed to indicate whether

histopathological insult occurs in the absence of a loss of TH-immunopositive terminals.

27

Hypothesis 2

High doses of the pyrethroid insecticide permethrin will produce significant degeneration of

nigrostriatal axon terminals as indicated by a decrease in the amount of TH immunoreactive

protein within the striatum, while increasing the amount of GFAP immunoreactive protein.

With regard to the effects of chlorpyrifos upon synaptosomal dopamine transport, Karen et al.,

(2001) has reported no significant effects for sub-cutaneous doses of 25 or 50 mg/kg. However,

at the highest dose used, 100 mg/kg, there was a small, but significant 10% reduction in

dopamine uptake. In addition, the 100 mg/kg dose of chlorpyrifos has been reported to increase

dopamine turnover in mouse striatum, although it does not change dopamine concentration. As

will be reported below, the high dose of permethrin tested in hypothesis 2, failed to show

evidence of nigrostriatal terminal death, but did indicate some form of striatal tissue damage as

indicated by an increase in GFAP immunoreactivity. Therefore, in order in investigate potential

synergism between permethrin and chlorpyrifos, a dose of chlorpyrifos was used that produces

no observable effect upon dopamine uptake and which is below that which affects dopamine

turnover. The present study explored whether this dose of chlorpyrifos can render a dose of

permethrin, that produces some form of tissue insult in the striatum, deadly to dopaminergic

nigrostriatal terminals. TH immunohistochemistry was used to assess any loss of TH-

synthesizing terminals within the striatum and GFAP immunohistochemistry was used to

indicate histopathological damage in the case where no loss of TH immunopositive terminals is

indicated.

28

Hypothesis 3A

A dose of chlorpyrifos that does not affect dopamine uptake or turnover will not produce

significant degeneration of nigrostriatal axon terminals.

Hypothesis 3B

A high dose of permethrin, that does not induce degeneration of nigrostriatal terminals when

given alone, will act synergistically with chlorpyrifos to produce such degeneration.

Materials and Methods

1. Animals and Treatments

Retired breeder C57BL/6 male mice, 28-35g live weight, 7-9 months of age, were obtained from

Harlan Sprague-Dawley, Dublin, VA, USA. Mice were housed with free access to food and

water and kept under environmentally controlled conditions. The mice were assigned to groups

by weight, such that every mouse designated for insecticide treatment had a paired, weight-

matched vehicle control. These weight-matched pairs were randomly assigned to different dose

concentration groups. Mice were treated with 0.8, 1.5, or 3.0 mg/kg of permethrin (95.4% pure,

Sigma-Aldrich Chemical Co., St. Louis, MO, USA), the “low dose” condition; 200 mg/kg of

permethrin, the “high dose” condition; 50 mg/kg of chlorpyrifos (99.5% pure, ChemService, Inc.

29

West Chester, PA, USA) alone; or both 200 mg/kg of permethrin and 50 mg/kg of chlorpyrifos.

Permethrin, dissolved in methoxytriglycol (MTG), was injected intraperitoneally (i.p.).

Chlorpyrifos, dissolved in corn oil, was injected subcutaneously (s.c.). The preceding treatments

were administered 3 times over the course of two weeks; day 1, 8, 15 and sacrificed 24hours

after the last injection (see Figure 4). Control animals received only the appropriate vehicle

injections on the same schedule. For every insecticide-treated mouse that was injected or

sacrificed on a given day, its weight-matched, vehicle control partner was also injected or

sacrificed. A “case” in this investigation consisted of one insecticide-treated mouse and its

matching vehicle control animal, with 6-10 cases per dose concentration group (e.g. 3.0 mg/kg

group).

2. Tissue Preparation for Immunohistochemistry

Animals were deeply anesthetized with Nembutal (0.1 ml of sodium pentobarbital (50mg/ml) in

0.5 ml of sterile saline) i.p. and perfused transcardially using a ministaltic Monostat pump to

ensure a constant flow rate. The blood was washed out with 30ml of phosphate buffered saline

(PBS), (0.05M, pH 7.4, room temp) followed immediately with 80ml of low pH fixative (pH 7.0,

4% paraformaldehyde in 0.1M phosphate buffer). The pump was then stopped for 5 minutes

before beginning fixation with 80 ml of high pH fixative (pH 10.5, 4% paraformaldehyde in

0.1M phosphate buffer). Following the perfusion, the brain was removed from the skull and

postfixed in a vial of 10 ml of high pH fixative for 4 hours. The vial was then rinsed with PBS

followed by 3 X 30 min washes of the brain in PBS. The brain was then cryoprotected with 10ml

of 10% sucrose (in PBS) at 4oC for 18hrs.

30

3. Immunohistochemical Staining for DAT, TH, and GFAP

A tissue block containing the striatum was prepared by making coronal slices, with a razor blade,

at the caudal-most region of the olfactory bulbs and the rostral most portion of the cerebellum.

The right side of the brain was then “notched”, adjacent to the midline, rostro-caudally, to assist

with tissue orientation. The brain was then rapidly frozen on a cryostat stage submerged in a dry

ice and 95% isopropol alcohol mixture, then placed in a cryostat at -15 oC. The brain was then

prepared for cutting with the application of OTC (Tissue-Tek, Miles Inc., Elkhart, IN, USA)

over the outer surface of the brain. After the OTC solidified, 16uM coronal sections were cut.

The first appearance of the lateral ventricles was used as a marker for commencement of tissue

acquisition. The entire striatum was cut with the cryostat using a systematic procedure in which

two consecutive 16um sections were each placed on two separate sets of 0.5% gelatin-coated

slides (12 slides per stain for each case), one for TH immunohistochemistry, or DAT

immunohistochemistry, or GFAP immunohistochemistry, and one for cresyl violet staining. This

process was repeated every 320 um until tissue was acquired for all 24 slides (see Figure 5).

For each case, the matched vehicle brain and treated brain were placed adjacent to one another

on each of the slides. This insured that sections from the treated and matched vehicle control

brains were always exposed to identical tissue processing conditions. Sections were counter-

balanced between cases to prevent possible position effects. In addition, each set of slides from a

given case included an additional slide used as an omission control for non-specific staining. The

remaining portion of the brain was frozen and kept for future use.

31

After cutting the tissue, a circle was then made around each section using a “PAP” pen,

hydrophobic marker (Research Products, Mount Prospect, IL, USA), to facilitate the

containment of incubation solutions placed on the sections. Tissue was processed by the avidin-

biotin-peroxidase complex (ABC) method originally described by Hsu (1991). Slides were

gently rinsed with PBS and placed in Coplin jars filled with PBS on a shaker table (~0.5 RPM)

for 10 minutes. Slides were then dried using Kimwipes (being careful not to touch the tissue

sections) and incubated for 20 minutes in a 10% blocking serum (10% normal goat serum

(Vector Labs, Burlingame, CA, USA) in PBS with .15% Triton-X-100) in a humidity chamber.

Slides were then dried again and incubated in well characterized, commercially available

primary antibody to TH (Protos Biotech Corp., New York, NY, USA, #CA-101), or DAT

(Chemicon Int., Temecula, CA, USA, #AB1591P), or GFAP (DAKO Corp., Glostrup, Denmark,

#Z0334). Primary antibodies were diluted with a solution of 1% goat serum in PBS with 0.15%

Triton-X-100 to 600:1 for anti-DAT, 600:1 for anti-TH and 6400:1 for anti GFAP for 18 hours in

the humidity chamber. Omission control sections for the case were processed in identical

solutions but without primary antibody exposure. At the end of the primary antibody incubation,

slides were gently rinsed and placed in Coplin jars for 3 X 5 minute wash baths (PBS). The

tissue was then incubated for 45 minutes in a biotinylated secondary antibody solution

(biotinylated goat anti-rabbit secondary antibody solution (Vector Labs, Burlingame, CA, USA)

in 1% diluent with 0.15% Trition-X-100) in the incubation chamber. Following the biotinylated

antibody incubation, the slides were rinsed (3 X 5 minute wash baths (PBS)), then incubated in

3% hydrogen peroxide in PBS for 10 minutes (to quench endogenous peroxidase activity), then

rinsed (3 X 5 minute wash baths (PBS)). The sections were then incubated for 45 minutes in an

32

avidin-biotin peroxidase complex solution (ABC elite kit, Vector laboratories). At the end of the

ABC incubation slides were rinsed again (3 X 5 minute wash baths (PBS)). Immunoreactivity

was revealed by incubation in diaminobenzidine (DAB) solution (0.05% diaminobenzidine and

66 ul of 30% hydrogen peroxide in PBS). TH sections were incubated in DAB for 10 minutes,

DAT and omission control sections for 5 minutes. Following DAB incubation, slides were rinsed

(3 X 5 minute wash baths (PBS)). Sections were then dehydrated through a series of ethanol

baths (70%, 80%, 95%, 100%), cleared in xylene, and coverslipped with Permount.

4. Data Analysis

An individual data point in this study was defined as the difference in the amount of

immunoreactive neuropil (DAT, GFAP, or TH) between a brain section from an insecticide-

treated mouse and the corresponding section from its matched vehicle control located on the

same slide. The amount of immunoreactive neuropil in a given brain section was a mean,

calculated from four 3070 um2 fields, distributed within the dorsolateral portion of the striatum

(see Figure 6B). This region has been reported to contain the highest density of dopaminergic

nigrostriatal afferents. In order to insure consistent sampling between sections from treated and

matched control sections, a morphometric procedure was used to define the location of the four

fields within each brain section. Consistent identification of the caudate-putamen, across

sections, was aided by the fact that throughout most of its rostrocaudal course, the caudate-

putamen is bordered dorsally, ventrally and laterally by the external capsule, a well defined band

of white matter (see Figure 6). Second, the dorsomedial border of the nucleus is adjacent to and

can be easily distinguished from the cavity of the lateral ventricle. Finally, the ventromedial

33

border of the nucleus can be distinguished from the less densely staining globus pallidus in both

immunostained and cresyl violet stained sections. In the rostral third of the nucleus, where the

external capsule does not extend to the ventral border, the lateral striatal stripe and the anterior

commissure were used to locate the ventral border of the caudate-putamen and distinguish it

from the nucleus accumbens. The identity of anatomical structures was confirmed using a

stereotaxic atlas of the mouse brain (Franklin and Paxinos, 1996).

The four loci measured in each coronal section of the striatum were located as follows: Camera

lucida drawings were made from the immunohistochemistry sections through the rostrocaudal

length of the striatum. The lateral border of the striatum was traced, noting the lateral ventricle

and external capsule, respectively, as anatomical references for the medial and lateral borders,

respectively. Along the lateral border a mark was made at 1/3 and 2/3 of the distance between

the ventral-most point and the dorsal ventricular border of the striatum (see Figure 3). The center

of the striatum was then determined by measuring the distance from the dorsal-most point of the

nucleus to the ventral-most point, calculating the mid-point and, at this point, drawing a

horizontal line across the nucleus, from the medial border to the lateral border. The midpoint of

this line was considered the center of the nucleus. A radius was then drawn from the 1/3 and 2/3

markers of the lateral border of the nucleus to the center mark of the nucleus. A mark was then

made 1/4 and 1/2 the distance from the lateral border of the striatum to the center marker of the

striatum along each of the two radii. This procedure produced 4 sampling regions from the

dorsolateral portion of the striatum, which is the principal area of nigral dopaminergic afference

to the striatum (see Figure 3). Around these sampling regions, numerous tissue landmarks were

traced, such as unstained fiber bundles or imperfections, to aid in relocating these fields for

34

photography. A digital image of each of the four sampling regions for each section was then

captured using a Vanox microscope (100X objective, Flashbus image capture software, Integral

Tech Inc.) (see Figure 7).

5. Image Analysis

The digital images captured from each section were then analyzed using the “Histogram” tool of

Adobe Photoshop© 5.0 software. For any selected area of pixels from a digital photograph, this

function can determine the mean gray scale value (0-255, where 0 is black and 255 is white) and

the number of pixels that are darker than a specified grayscale value (see Figure 7). All images

were converted to grayscale and a mean minimum grayscale value was determined for

immunolabeled varicosities. This grayscale value was determined from all vehicle control

brains. All pixels darker than this minimum value would subsequently be counted as positive

immunostaining. The mean minimum grayscale value was determined by finding the mean

grayscale value of two immunolabled varicosities per field (loci), judged as the minimum

acceptable for measurement. The grand section mean across the 4 fields of each section was then

determined. Then a grand mean for the brain was determined across each of the grand section

means. The minimum acceptable grayscale value, calculated from all the vehicle control brains,

was then used to make pixel counts on both the vehicle control and treated brains. The number of

pixels that had a value less than, i.e. darker than, the minimum acceptable grayscale value was

recorded for each field within a brain section. The count for each field was used to calculate a

mean striatal pixel count of immunopositive labeling for each section. The mean pixel count for

each insecticide treated section on a single slide was compared with its adjacent, matched

35

vehicle-treated section. As noted earlier, this difference comprised a single data point of the

study. The mean of these differences was then calculated across slides for a given case (case =

one treated brain and matched vehicle control brain). These mean differences in pixel counts,

across cases within a dose, were tested for their difference from zero.

6. Statistical Analyses

A comparison of the difference in immunopositive labeling between the paired treated and

vehicle control tissue on each slide was made. As mentioned before, the paired mice were

injected on the same days, sacrificed on the same day and sections were taken from similar

rostrocaudal positions within the striatum on the same day. In addition, the paired sections on a

slide shared identical tissue processing conditions. The difference found for each slide was

averaged across slides for each pair of matched treated and vehicle animals.

Scatterplots were employed to assess the effects of microscope slide section position and date of

tissue processing on differences in immunostained neuropil between paired sections. Based on

this analysis, an ANOVA model, fitted using the GLM procedure of SAS (SAS Institute Inc.,

Cary, NC, USA), was used to correct for the effect of processing date on each data point. The

corrected differences in immunostaining were then consistent with the assumptions for analysis

by a paired t-test. For a pair of insecticide-treated and matched vehicle control brains, the

difference in immunostained neuropil was averaged across microscope slides. Then, for each

dose concentration group, the grand mean of these corrected mean differences was tested for its

difference from zero using an alpha level of 0.05. Inflation of the type I error rate was avoided

36

by treating each dose concentration group as a separate experiment.

Results

Figure 8 shows three separate coronal sections through the striatum as an example of staining for

the three antibodies used in the study (the “x” in the figure is the same spot, in higher power, as

in Figure 6A). Again, Figure 7, taken from a video monitor screen, shows an example of a

varicosity considered to be positively immunostained (see “box” in lower left of field,

surrounding a varicosity). As noted earlier a population of such varicosities was used to

calculate a minimum acceptable grayscale value for immunolabeling. The image frame in Figure

7 also shows the extent of one of the four loci used for measuring immuno-positive staining in a

given striatal section. This area is enclosed within the dashed box, a portion of which is covered

by the histogram information. Figures 9-12 show box and whisker plots of the distribution of

mean differences in immunolabeling, between matched pairs of vehicle and insecticide-treated

mice. Unless an extreme value is present, indicated by an open circle, the tips of the whiskers

represent the respective minimum and maximum mean differences, while the length of the box

represents the inter-quartile range of the mean differences. The line and the solid square within

the box, respectively, represent the median and mean of the distribution of difference scores. The

number of matched pairs of mice examined at each dose is also shown in each box. An open

circle is used to represent a value that is more than 1.5 inter-quartile ranges above the mean. A

grand mean of zero represents no change in immunostaining between matched pairs of treated

and vehicle control mice, while an asterisk indicates a non-zero significant difference between

matched pairs.

37

Permethrin Treatment Alone

As can be seen in Figure 9, the mean difference scores between vehicle and matched treated

mice were negative for all low doses of permethrin, indicating an overall trend toward a decrease

in DAT immunolabeling in the treated mice. As indicated by the asterisk, only the 3.0 mg/kg

dose of permethrin produced a significant decrease in DAT immunolabeling within the caudate-

putamen, compared to vehicle control mice (df = 7, p = .007).

As can be see in the box and whisker plots of Figure 10, the high dose of permethrin did not

produce a significant amount of change in the amount of TH or DAT immunostaining within the

striatum as compared to matched vehicle controls. However, a high dose of permethrin did

produce a significant increase in GFAP immunostaining within the striatum (df = 15, p = .048).

Chlorpyrifos Treatment Alone

Figure 11 shows a single box and whisker plot illustrating the mean difference in TH

immunopositive staining between mice treated with 50 mg/kg of chlorpyrifos and matched

vehicle controls. At this dose, the mean change was not significantly different from zero.

Permethrin and Chlorpyrifos Treatments Combined

Figure 12 shows similarly constructed box and whisker plots of the distribution of mean

38

differences in TH immunolabeling between matched pairs of vehicle and insecticide-treated mice

with both a high dose of 200 mg/kg permethrin and 50 mg/kg dose of chlorpyrifos. As shown in

the figure, the combined insecticide treatment failed to produce a significant change in TH

immunostaining within the striatum, compared to matched vehicle controls. However, the total

amount of GFAP immunostaining was significantly increased (df = 7, p = .033) compared to

matched vehicle controls, suggesting an increase in reactive astrocytes.

Discussion

The present investigation examined the premise that pyrethroid and organophosphate

insecticides, either alone or in combination, are capable of producing anatomical changes in the

nigrostriatal pathway of the C57BL/6 mouse. Low doses of the pyrethroid insecticide

permethrin were predicted to significantly increase the amount of DAT protein within the

striatum of the C57BL/6 mouse. High doses of the pyrethroid insecticide permethrin were

predicted to produce degeneration of nigrostriatal terminals as indicated by a significant decrease

in the amount of TH immunoreactive protein within the striatum, while increasing the amount of

GFAP immunoreactive protein, indicative of an increase in astrocytic response to tissue damage.

Furthermore, a dose of chlorpyrifos, that does not affect dopamine uptake or turnover, was

predicted to not produce degeneration of nigrostriatal terminals. Finally, chlorpyrifos (50mg/kg)

was predicted to potentiate the effects of permethrin (200mg/kg), resulting in nigrostriatal

terminal death, as indicated by decreased TH immunoreactivity and increased GFAP

immunoreactivity in the striatum.

39

1. Low Dose PE Effects

Previous studies by Bloomquist and colleagues (Karen et al.) have demonstrated that low IP

doses (1.5-6mg/kg ) of the pyrethroid insecticide permethrin can increase maximal dopamine

uptake in striatal synaptosomes to a peak of 134% of control. Such changes could reflect a

change in transporter kinetics or a change in the amount of transporter protein employed by

dopamine nigrostriatal neurons. To investigate the latter possibility, the present study used

immunohistochemical staining for DAT in mice treated with low doses of permethrin.

It is evident that low doses of permethrin can alter not only the function of DAT, as

demonstrated by Karen et al. (2001), but also the amount of DAT protein within the caudate-

putamen as demonstrated by the present study.

Although low doses of permethrin can affect both dopamine uptake and the amount of DAT

immunoreactive protein in components of the dopaminergic nigrostriatal pathway, the effects are

in different directions. A 1.5 mg/kg dose of permethrin has been reported to produce an increase

in maximal dopamine uptake in striatal synaptosomes (Karen et al., 2001), while in the present

investigation 3.0 mg/kg produced a decrease in DAT immunoreactive neuropil within the

caudate-putamen. These two findings would be concordant if an increase in the efficiency of

DAT transport overcompensated for a decrease in the amount of DAT protein. However, Karen

et al. (2001) failed to a find a change in the Km for synaptosomal dopamine uptake, arguing

against increased DAT efficiency.

Alternative explanations for the difference in the direction of the two findings noted above relate

40

to differences in the nature of the tissue samples analyzed. For example, in the present

immunohistochemcial study, anatomical landmarks within transverse forebrain sections were

used to select analysis fields within the caudate-putamen, the region of the striatum containing

the principal dopaminergic afference from the substantia nigra (Heimer et al., 1995). Moreover,

each of these analysis fields was only on the order of 3070um2. The kinetic data of Karen et al.

(2001) employed a synaptosomal preparation made from the entire dissected striatum. This

sample may have included varying amounts of tissue from striatal regions outside the caudate-

putamen, such as the nucleus accumbens or non-striatal regions such as the globus pallidus, both

of which are physically contiguous with the caudate-putamen (Franklin & Paxinos, 1996).

Therefore, the present investigation analyzed changes in a smaller sample of tissue, from a more

topographically restricted region of the striatum, the region that is most closely associated with

degenerative changes in PD (Kish et al., 1988; Graybiel et al., 1990). This suggests the

possibility that different topographic sub-regions within the striatum may be differentially

affected by permethrin exposure. In fact, it has been shown that portions of the dopaminergic

afference to the ventral striatum are more resistant to the neurodegenerative effects of MPTP

compared with the dopaminergic input to the more dorsolaterally located caudate-putamen

region of the striatum (Gerhardt et al., 1985; Mizukawa et al., 1990; Betarbet et al., 2000).

Although the sampling within the present investigation was more restricted with regard to striatal

topography, the immunohistochemical analysis most likely surveyed a more extensive portion of

individual nigrostriatal neuronal morphology compared with analysis of synaptosomes.

Synaptosomal preparations are designed to isolate the synaptic bouton and sometimes contain a

portion of the immediate post-synaptic membrane (Webster., 2001). Alternatively, any DAT-

41

containing portion of a neuron that lies near the surface of the striatal tissue section is available

for labeling by the immunohistochemical procedure. Electron microscopic

immunohistochemical studies of the dorsolateral striatum and substania nigra pars compacta

have reported that DAT immunoreactive protein can be found not only at the plasma membranes

of synaptic boutons, but at the membranes of axonal segments lying between the boutons, as well

as at somal and dendritic membranes of dopaminergic neurons (Nirenberg et al., 1996; Hersch et

al., 1997). In fact, it has been reported that there is a higher density of DAT at inter-bouton

axonal membranes than at the transmitter release sites of dopaminergic neurons. Since these

inter-bouton axonal segments course through the caudate-putamen, immunohistochemical

procedures should label bouton-associated DAT, as well as the more plentiful axon-associated

DAT. Synaptosomal preparations would be expected to primarily sample bouton-associated

DAT, since these structures do not contain axonal membrane and any portion derived from the

membrane of a post-synaptic striatal neuron would be almost exclusively non-dopaminergic.

Therefore, the regional intra-neuronal population of DATs examined in the present study may

have differed from that examined by Karen et al. (2001). In some dopaminergic neurons, there is

a functional differentiation between DATs located in different regions of the cell. For example,

dendritic DATs in the substantia nigra are capable of releasing dopamine through reverse

transport. This potential for functional differentiation between DATs in different parts of the

neuron, combined with a probable difference in regional populations of DATs examined, could

account for the difference in direction of the kinetic data of Karen et al. (2001) and the present

immunohistochemical study.

2. High Dose PE Effects

42

As noted above, at low IP doses, permethrin increases dopamine uptake in striatal synaptosomes

and decreases the amount of DAT immunoreactive protein. However, at high doses (200mg/kg),

dopamine uptake is significantly less than that of controls (about 50%) (Karen et al., 2001). This

could reflect a change in transporter kinetics or a loss of dopamine nigrostriatal terminals from

the sampled region. Bloomquist and colleagues (Karen et al., 2001) have shown that 200mg/kg

doses of permethrin do not alter the Km of DAT transport. The absence of a significant change in

DAT immunoreactive protein in the high dose permethrin group, as demonstrated in the present

study, indicates that a change in the amount of DAT protein does not underlie the decrease in

dopamine uptake that was previously demonstrated for this dose by Karen et al (2001), pointing

to the possibility that the terminals may be leakier.

There is some evidence that permethrin can produce some lesions in peripheral nerves of rodents

(Wright et al., 1988). To investigate whether such pathology occurs centrally, we used TH

immunohistochemistry to assess whether there is a loss of the TH-synthesizing axon terminals

within the striatum (Gerhardt et al., 1985; Nirenberg et al., 1996; Ho and Blum, 1998; Brooks et

al., 1999; Betarbet et al., 2000). The dopamine synthesizing enzyme TH is commonly used as an

immunohistochemcial marker to identify the presence of dopaminergic neuropil within the

striatum. Although this enzyme is also critical for synthesis of norepinephrine and epinephrine,

there is an insignificant presence of neurons that contain these transmitters within the striatum

(Aston-Jones et al., 1995; Saper, 2000). Therefore, TH immunoreactivity was used as an

additional marker for dopaminergic terminal death within the striatum since it is possible that the

DAT protein could be downregulated without accompanying degeneration of dopaminergic

43

striatal afferents. The absence of a significant change in TH immunoreactive neuropil in the

high dose permethrin group, as indicated in the present study, suggests that the decrease in

dopamine uptake that has previously been demonstrated at this dose is not a function of the

degeneration of dopaminergic terminals within the striatum. However, our data cannot preclude

the possibility that the lack of change in TH immunolabel is attributable to an upregulation of

TH in striatal terminals that survive a wave of degeneration, or other compensatory mechanisms

such as growth or sprouting of new terminals from surviving DA neurons.

GFAP immunohistochemistry was used to indicate whether histopathological insult occurred in

the absence of a loss of TH-immunopositive terminals. Increased glial fibrillary acidic protein

(GFAP), an intermediate filament protein of astrocytes, has been shown to be a marker of the

onset, degree and locus of neuropathology (Norten, 1992; O’Callaghan et al., 1995; Eng et al.,

2000). Such increases correspond to sites where there is histological evidence of tissue damage

that is more subtle than frank degeneration (Miller et al., 1991). Although the lack of change in

the TH and DAT immunoreactivities within the caudate-putamen, demonstrated in the present

study, argue against the death of dopaminergic nigrostriatal terminals in the “high dose”

condition, the corresponding increase in GFAP immunoreactivity suggests axon terminal damage

that has not advanced to frank degeneration. Such damage may be sufficient to render the DAT

inoperative, which would be consistent with the decrease in maximal dopamine uptake reported

by Karen et al. (2001). Again, such comparisons with the aforementioned synaptosomal study

should be tempered by the possibility of a topographic difference in the striatal tissue samples

examined. It should also be noted that the observed increases in GFAP immunoreactivity could

also represent damage to glial cells, since such increases have been reported following damage

44

to oligodendrocytes and in astrocytes that survive direct astrocytic insult (Smith et al., 1983;

Takada et al., 1990).

3. CP Effects

As noted earlier, previous work (Karen et al., 2001) has shown that a 100 mg/kg dose of

chlorpyrifos can decrease open field behavior, mitochondrial function, and produce a small

decrease in dopamine uptake. It can also increase dopamine turnover, but fails to decrease

dopamine concentration in the striatum. A 50 mg/kg dose fails to produce a change in open field

behavior or dopamine uptake, but actually increases mitochondrial function. Therefore, the

absence of a significant change in TH immunoreactive neuropil in the striatum of the

chlorpyrifos-treated group, as demonstrated in the present study, is not surprising given the

previous finding that dopamine concentration does not change following exposure to 100 mg/kg

of chlorpyrifos (Karen et al., 2001). It suggest that 50 mg/kg of chlorpyrifos fails to produce

nigrostriatal terminal degeneration and provides further support for the notion that this is a

physiologically sub-clinical dose of chlorpyrifos. It should be noted, however, that due to time

constraints, GFAP immunohistochemistry could not be performed for this condition. Therefore,

it is possible that non-degenerative tissue insult could have been produced that could not be

detected by TH immunohistochemmistry alone.

4. Combined PE & CP Effects

In order to investigate potential synergistic effects of permethrin and chlorpyrifos, a dose of

45

chlorpyrifos (50 mg/kg) was used that produces no observable effect upon dopamine uptake,

which is below that which affects dopamine turnover and which fails to provide evidence of

nigrostriatal terminal degeneration; in conjunction with a dose of permethrin that also fails to

produce terminal degeneration, but which appears to induce some form of striatal tissue insult.

The present investigation explored whether this 50 mg/kg dose of chlorpyrifos, that appears non-

toxic for dopaminergic nigrostriatal neurons, can exacerbate the tissue insult produced by the

high dose of permethrin into terminal degeneration. TH immunohistochemistry was employed to

assess any loss of TH-synthesizing terminals within the striatum and GFAP

immunohistochemistry was used to indicate histopathological damage in the case where no loss

of TH immunopositive terminals is indicated.

The finding that a combined dose of 200mg/kg of permethrin and 50mg/kg of chlorpyrifos fails

to produce a change in striatal TH immunostaining in the present study, suggests that a sub-

threshold dose of chlorpyrifos fails to act synergistically with permethrin to exacerbate the

permethrin-induced tissue insult into full blown terminal degeneration. The tissue insult seen

with the high dose of permethrin alone, as indicated by a significant increase in striatal GFAP

immunostaining, persists in the combined treatment. However, the contribution of chlorpyrifos

to this effect is not clear since an analysis of GFAP was not performed with a dose of 50 mg/kg

of chlorpyrifos alone.

5. Improvements

The exposure of the animal to permethrin and chlorpyrifos in this study was intermittent and

46

brief in nature, unlike “real-life”, sustained, low doses one is likely to encounter at the work

place, home, or through food and drinking water. The non-exposure periods between dosings

may have enabled elimination and repair processes to minimize neurotoxicity relative to a

continuous exposure. Therefore, a continuous exposure study may provide quite different results

than the present study. The route by which a chemical encounters the body can alter substantially

its effects and the quantity required to cause the effect (Casarett and Doull, 1996). With IP

administrations, compounds are absorbed through the hepatic portal circulation and rapidly

metabolized by the liver so that its toxic effects are greatly diminished. Therefore, different

routes of administration should be explored in the future, such as inhalation and dermal, two

routes by which military service personnel were exposed during the Gulf War and routes of

exposure for the general public as well. In addition, animals could have been sacrificed at each

of the three dosing time points to investigate possible recovery effects, particularly with regard

to GFAP immunostaining. As noted above, GFAP immunohistochemistry was not performed for

the 50mg/kg CP group alone, due to time restrictions. This data would have been valuable given

the effects observed for the combination treatment. Furthermore, given the discrepancy between

the TH and GFAP data, the use of immunohistochemistry for other neurotransmitter systems,

namely GABAergic and cholinergic systems, would have been advantageous to elucidate the

potential contribution of these pathways to the increase in GFAP noted. Finally, a greater

number of striatal samples within each section could have been taken.

Conclusions

The findings of the present investigation suggest that exposure to permethrin and chlorpyrifos,

47

individually and in combination, at the doses, exposure intervals and conditions tested, have a

marginal capacity to alter the normal status of the nigrostriatal pathway, even when administered

together. The tissue damage suggested by increased GFAP within the caudate-putamen has

implications for the putative connection between pesticide exposure and Parkinson’s disease,

specifically with regard to the accumulation of subtle tissue damage over time. Alterations of

DAT protein within this region may also represent an important substrate modulating the onset

and severity of Parkinson’s disease since this transporter is the means by which Parkinson’s

disease-like inducing chemicals, such as MPP+, can enter dopaminergic neurons. Such changes

in the integrity of DAT may be important for future investigations of the role of synergistic

interactions between environmental chemicals in the development of Parkinson’s disease.

It is conceivable that an upregulation of TH in surviving dopamine terminals could mask

degeneration, and the upregulation of GFAP points to this possibility. Given the fact that the

lowest concentrations of GFAP immunoreactivity in the mouse have been found in the striatum

(Martin & O’Callaghan, 1995), the significant upregulation observed in the present investigation

is of great interest if one assumes that it would be easier to induce a statistically significant

upregulation of GFAP in brain regions with a greater concentration such as the hippocampus or

olfactory bulbs. Furthermore, the discrepancy between the TH and GFAP data reveal an

interesting aspect of pesticide induced metabolic change in dopamine neurons. Other

neurotransmitter systems could be involved, namely the GABAergic and cholinergic striatal

neurons. If permethrin and chlorpyrifos do damage or alter these other striatal neurochemical

systems, then the possibility of targeting multiple sites of the nigrostriatal pathway may

compromise homeostatic regulation, potentially resulting in toxicity and ultimately cell death.

48

Numerous agrochemicals share patterns of geographical use with permethrin and chlorpyrifos

(Gorell et al., 1998). Thus, there is clearly a concern that “supramixtures” of agrochemical

exposures are likely. The findings of the present investigation represent an attempt to dissect the

respective and combined role of two insecticides as risk factors for the development of PD. The

results of this study merit further consideration, to ascertain whether with chronic exposure,

chlorpyrifos and permethrin might act as parkinsonism-provoking, dopaminergic toxins. It is

important to note that it is unlikely that such exposures, per se, produce PD. Most studies suggest

that no single factor is solely responsible for PD. Evidence to date seems to suggest that PD is a

multifactorial disorder, most likely caused by a combination of age, genetics, and environmental

factors.

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58

Effects of PD on Neural Function

GABA

Cerebral Cortex

D1

Glutamate

+

-

EP(Gpi)/SNr

GP

STN

ThalamusGABA

SPD2

-+

--

+

-

Normal function

DA

Cerebral Cortex

D1

Glutamate

+

-

EP(Gpi)/SNr

GP

STN

ThalamusGABA

SPD2

-+

--

+

-

Dopamine deficiency (PD)

GABA

DAGABA

GABA

GlutGlut

DI

Figure 1. Schematic diagram of the basal ganglia-thalamocortical circuitry under normal conditions and

parkinsonism. Inhibitory connections are depicted as ( - ); excitatory connections as ( + ). Thickness of the lines

indicates changes in the disease state. D = direct pathway; I = indirect pathway, denoted by the black and blue lines

respectively ; D1 and D2 = dopamine receptors. STN = Subthalamic nucleus, GP = Globus pallidus, SNr =

Substantia nigra pars reticulata.

59

Figure2. Schematic representation of striatal synaptic circuitry, relevant neurotransmitters, and

their actions. Inhibitory connections denoted by - ; excitatory connections by +. ACh =

acetylcholine; DA = dopamine; GLU = glutamate.

Spiny Projection

Neuron

DA

GLU

Substantia Nigra

A. B.

Spiny Projection

Neuron

ACh

DA

(+)

(-)

(+) or (-)

Cortex

60

Figure 3. Diagrammatic representation of the four loci measured in each coronal section of the

striatum.

61

Figure 4. Dosing and sacrifice regimens.

Dose

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 6

Dose Dose S a c r i f i c e

Day

T R E A T E D M O U SE A N D M A T C H E D V E H I CLE C O N T R O L

62

Figure 5. Tissue sectioning and staining strategy. The entire striatum was cut with a cryostat using a systematic procedure in which two consecutive 16 um sections were placed on two separate sets of slides (12 slides per stain for each case) one for TH, or DAT, or GFAP immunohistochemistry, and one for cresyl violet. This process was repeated every 320 um until tissue was acquired for all 24 slides. GFAP stained sections are not schematically represented.

63

Figure 6. Left, is a typical low magnification image of a coronal striatal section, stained for

DAT, showing some of the prominent anatomical landmarks in the region. Right, is a schematic

of the four 3070 um2 analysis fields, distributed within the dorsal lateral portion of the striatum.

CPu = caudate-putamen, ec = external capsule, LV = lateral ventricle, ac = anterior commissure,

LGP = lateral globus pallidus, DAT = dopamine transporter.

64

\Figure 7. Illustration of Adobe Photoshop® histogram tool and an example of one of the four loci (within dashed square) used to

measure immunopositive pixels in each striatal section. The small solid black square shows an example of the minimum intensity

of staining that was defined as immunopositive. The shaded area in the histogram contains the pixels that are darker than the

minimum acceptable grayscale value for measurement. The number of these pixels is indicated by “count”.

65

Figure 8. Typical high magnification images of coronal sections through the striatum, showing

an example of staining for the three antibodies used in the study. The “x” indicates the same

tissue mark as in Figure 6. EC = external capsule.

66

Figure 9. Changes in DAT immunoreactivity for low doses of PE. Box and whisker plots

represent distribution of differences in immunopositive pixels between matched pairs of treated

and vehicle control mice. The line crossing each box is the median and the solid square is the

mean. The asterisk indicates a significant change from zero. See text for further details.

-80,000

-60,000

-40,000

-20,000

0

20,000

0.8 1.5 3.0

*

Dose of permethrin (mg/kg)

Diff

eren

ces

in im

mun

opos

itive

pix

els�

(trea

ted

min

us m

atch

ed v

ehic

le)

67

Figure 10. Box and Whisker plot of changes in DAT, TH and GFAP immunoreactivity for a

high dose (200 mg/kg) of PE. The open circle represents a value more than 1.5 inter-quartile

ranges above the mean.

-120,000

-80,000

-40,000

0

40,000

80,000

120,000

DAT TH

-12,000

-8,000

-4,000

0

4,000

8,000

12,000

GFAP

*

Primary antibody

Diff

eren

ces

in im

mun

opos

itive

pix

els�

(trea

ted

min

us m

atch

ed v

ehic

le)

68

Figure 11. Changes in TH immunoreactivity for a dose of 50 mg/kg of CP.

-40,000

-30,000

-20,000

-10,000

0

10,000

20,000

30,000

40,000

TH

Diff

eren

ces

in im

mun

opos

itive

pix

els�

(trea

ted

min

us m

atch

ed v

ehic

le)

Primary antibody

Dose: CPF (50 mg/kg)

n = 6

69

Figure 12. Changes in TH and GFAP immunoreactivity for a combined dose of PE (200 mg/kg) and CP

(50 mg/kg).

-40,000

-30,000

-20,000

-10,000

0

10,000

20,000

30,000

40,000

TH GFAP-3,000

-2,000

-1,000

0

1,000

2,000

3,000

*

Diff

eren

ces

in im

mun

opos

itive

pix

els�

(trea

ted

min

us m

atch

ed v

ehic

le)

Primary antibody

n=10 n=10

Dose: CPF (50 mg/kg) + PM (200 mg/kg)

70

Vita

Julian Thomas Pittman

A native of Williamsburg, VA. Graduated from the College of William & Mary (98) B.S. with

high honors. Presently pursuing a Ph.D. in Toxicology at Mississippi State University, College

of Veterinary Medicine.