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A phenotypic and neuropathological assessment of the impact of fetal and secondary adult re-exposures… Banjo, Opeyemi Christiana 2009

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A Phenotypic and Neuropathological Assessment of the Impact of Fetal and Secondary Adult Re-exposures to Steryl Glucosides in Mice    by  Opeyemi Christiana Banjo   B.Sc., University of California, Davis, 2005   A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  The Faculty of Graduate Studies (Neuroscience)   THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   June 2009  © Opeyemi Christiana Banjo, 2009 Abstract Cycad consumption remains one of the strongest etiological factors in the epidemiology of amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS-PDC). Two water insoluble steryl glucosides (stigmasterol β-D-glucoside (SG), and β-sitosterol β-D- glucoside (BSSG)) identified in washed cycad have previously been demonstrated to have neurotoxic effects in both in vitro and in vivo studies. The present study aims to address the variations in age of onset and rate of disease progression, by examining the hypothesis that factors contributing to adult onset diseases may first arise during fetal development. We studied the impact of a prenatal and secondary adult re-exposure to a combination of BSSG and SG on disease onset and progression, and also investigated possible sex differences in disease outcomes. Pregnant CD-1 mice were fed a combination of 0.6mg BSSG and 0.4mg SG on embryonic days 10-11, to target the differentiation of the substantia nigra; other pregnant females received the control diet. Pups were weighed within 24hr of birth, and weekly thereafter. Animals were monitored behaviorally using a series of tests starting at 5 weeks for the duration of the study. At 18 weeks, subsets of both litters were exposed to dietary BSSG and SG in the same doses as before for 15 weeks. Afterwards, animals were perfused and CNS tissue was examined for associated neuropathologies. Behavioral and neuropathological assessments revealed significant differences in the response of adult male and female mice to BSSG and SG exposure. While male mice exposed to BSSG and SG during gestation showed significant weight increases and behavioral deficits irrespective of whether or not they received a secondary exposure, female mice showed marked hyperactivity only if they received BSSG and SG as adults. BSSG and SG exposure also induced significantly decreased tyrosine hydroxylase (TH) labeling in the nigro-striatal pathway, increased apoptosis and gliosis, and resulted in widespread lipid accumulation in male mice; these effects were not observed in female mice. This study demonstrates that developmental exposure to cycad neurotoxins triggers an insult that may elicit a dominant phenotype based on the period of exposure, and that the insult is exacerbated by a secondary exposure, depending on animal sex.    ii TABLE OF CONTENTS  Abstract........................................................................................................................................ ii Table of Contents .......................................................................................................................iii List of Figures............................................................................................................................vii Acknowledgements .................................................................................................................... ix Dedication .................................................................................................................................... x 1 Introduction.............................................................................................................................. 1             1.1 Background: Amyotrophic Lateral Sclerosis – Parkinsonism Dementia         C C          Complex (ALS-PDC) ....................................................................................................... 1                      1.1.1 History............................................................................................................. 1                      1.1.2 Disease Phenotype and Associated Neuropathology ...................................... 2                      1.1.3 Other Areas of ALS-PDC Occurrence............................................................ 4             1.2 Disease Etiology ......................................................................................................... 5                     1.2.1 Genetic and Environmental Factors................................................................. 5                    1.2.2 Cycas Micronesica ........................................................................................... 6                    1.2.3 Putative Cycad Toxins ...................................................................................... 6                    1.2.4 An Animal Model of ALS-PDC ....................................................................... 8                    1.2.5 The FeBAD Hypothesis .................................................................................... 9           1.3 Rationale and Hypotheses Tested ............................................................................... 11                   1.3.1 Rationale .......................................................................................................... 11                   1.3.2 Hypotheses ....................................................................................................... 11 2 Materials and Methods.......................................................................................................... 14           2.1 Materials ..................................................................................................................... 14                   2.1.1 Chemical Reagents........................................................................................... 14                   2.1.2 Laboratory Supplies ......................................................................................... 15                   2.1.3 Primary and Secondary Antibodies ................................................................. 16                   2.1.4 Behavioral Testing Equipment ........................................................................ 16           2.2 Methods....................................................................................................................... 16                   2.2.1 Cycad Steryl Glucoside Synthesis ................................................................... 16                   2.2.2 Animals ............................................................................................................ 17  iii                   2.2.3 Steryl Glucoside Feeding ................................................................................. 19                   2.2.4 Animal Behavior Testing ................................................................................. 20                   2.2.5 Animal Sacrifice and Tissue Processing.......................................................... 23                   2.2.6 Nissl – Lower Motor Neuron Quantification................................................... 24                   2.2.7 Fluorescence Antibody Staining ...................................................................... 25                   2.2.8 Immunoperoxidase Antibody Staining in Lumbar Spinal Cord ...................... 27                   2.2.9 Immunoperoxidase Antibody Staining in Brain Sections................................ 30                   2.2.10 Tyrosine Hydroxylase Immunohistochemistry.............................................. 31                   2.2.11 Lipid Accumulation - Oil Red O ................................................................... 32                   2.2.12 Magnetic Resonance Microscopy (MRM)..................................................... 34                   2.2.13 Statistical Analyses ........................................................................................ 34 3 Effects of Prenatal Exposure to BSSG and SG on Mouse Behavior           3.1 Aims ............................................................................................................................ 36           3.2 Results......................................................................................................................... 36                   3.2.1 Fetal Exposure to BSSG and SG Induces Considerable Increases      ----- --------------Body Weight ............................................................................................................ 37                   3.2.2 The Impact of Fetal Exposure to BSSG and SG on Spinal Reflexes and M               Muscle Strength........................................................................................................ 37 3.2.3 Fetal Exposure to BSSG and SG Induces Changes in Mice Gait and Exploratory Patterns.................................................................................................. 37           3.3 Discussion ................................................................................................................... 38           3.4 Conclusions................................................................................................................. 40 4 Effects of Postnatal Secondary Re-exposure to Dietary BSSG and SG on Adult Male Mice .................................................................................................................................. 58            4.1 Aims ........................................................................................................................... 58            4.2 Results........................................................................................................................ 58                   4.2.1 Male Mice Exposed to BSSG and SG During Development Maintain 000            Larger Body Weights ................................................................................................ 58                   4.2.2 “Prenatal Exposure Only” Male Mice Show Similar Behavioral Trends 000            as Group Exposed Both Pre- and Post-natally .......................................................... 59  iv                   4.2.3 Decrease in Tyrosine Hydroxylase Immunoreactivity in Response to 0000          Dietary BSSG and SG............................................................................................... 60                   4.2.4 Significant Microglial Activation in Response to BSSG and SG 00000000  Exposure ................................................................................................................... 60                   4.2.5 Dietary BSSG and SG Induce Minimal Loss of Lower Motor 00               Neurons .................................................................................................................... 60                   4.2.6 Dietary BSSG and SG Induce Apoptosis in the Lumbar Spinal 000000      Cord........................................................................................................................... 61                   4.2.7 Dietary BSSG and SG Results in No Significant Induction of ATF-3 and 000000      HSP-70 ...................................................................................................................... 61                   4.2.8 Increased Lipid Accumulation in BSSG and SG - Treated Mice .................... 61                   4.2.9 Magnetic Resonance Microscopy (MRM) Analysis........................................ 62           4.3 Discussion ................................................................................................................... 62           4.4 Conclusions................................................................................................................. 65 5 Effects of Postnatal Secondary Re-exposure to Dietary BSSG and SG on Adult Female Mice............................................................................................................................... 95           5.1 Aims ............................................................................................................................ 95           5.2 Results......................................................................................................................... 95                   5.2.1 Body Weights of Female Mice ........................................................................ 95                   5.2.2 Adult Dietary Exposure to BSSG and SG Induces Hyperactivity in 00000        Females ..................................................................................................................... 96                   5.2.3 Female Mice Show No Significant Changes in Tyrosine Hydroxylase 000             Immunoreactivity in Response to Dietary BSSG and SG ....................................... 96                   5.2.4 Female Mice Show No Significant Changes in Glial Activation in 0000000    Response to BSSG and SG Exposure ....................................................................... 96                   5.2.5 No Significant Changes in the Number of Lower Motor Neurons Across 00000         Female Groups ......................................................................................................... 97                   5.2.6 Dietary BSSG and SG Induce Apoptosis in the Lumbar Spinal Cord of 00000        Female Mice.............................................................................................................. 97           5.3 Discussion ................................................................................................................... 98           5.4 Conclusions............................................................................................................... 101  v 6 General Discussion and Future Studies ............................................................................. 111           6.1 General Discussion ................................................................................................... 119           6.2 Future Studies ........................................................................................................... 120 References................................................................................................................................ 122 Appendices............................................................................................................................... 141           Appendix A ..................................................................................................................... 142           Appendix B ..................................................................................................................... 149           Appendix C ..................................................................................................................... 183           Appendix D ..................................................................................................................... 200           Appendix E ..................................................................................................................... 207                       vi List of Figures Figure 1 Cycas Micronesica and Some of its Water Insoluble Components ............................. 13 Figure 2 Schematic Depiction of Experimental Design ............................................................. 35 Figure 3 Body Weights of Animals ............................................................................................ 42 Figure 4 Stepping Patterns of Animals ....................................................................................... 44 Figure 5 Open Field Activity of Animals ................................................................................... 49 Figure 6 Body Weights of Animals ............................................................................................ 67 Figure 7 Open Field Activity ...................................................................................................... 68 Figure 8 Decreased Tyrosine Hydroxylase Immunoreactivity in Nigro-striatal System of BSSG and SG – Treated Mice .................................................................................................... 71 Figure 9 BSSG and SG Induce Microglial Proliferation in Lumbar Spinal Cord and Striatum....................................................................................................................................... 74 Figure 10 Motor Neuron Quantification in the Lumbar Spinal Cord ......................................... 78 Figure 11 Anti-ChAT Motor Neuron Quantification in the Lumbar Spinal Cord...................... 81 Figure 12 Increased Expression of Active-Caspase-3 in the Lumbar Spinal Cord of Treated Mice ............................................................................................................................... 83 Figure 13 Oil Red O (ORO) Staining in Lumbar Spinal Cord Sections Reveals Increased Lipid Deposition/Reduced Clearance in BSSG and SG – Fed Animals..................................... 85 Figure 14 Magnetic Resonance Microscopy (MRM) Analysis Suggests Abnormalities in the Spinal Cord of BSSG and SG - Treated Mice ...................................................................... 90 Figure 15 Body Weights of Animals ........................................................................................ 102 Figure 16 Open Field Activity .................................................................................................. 103 Figure 17 Tyrosine Hydroxylase Immunoreactivity in Nigro-striatal System ......................... 106 Figure 18 Quantification of Activated Astrocytes in Lumbar Spinal Cord and Striatum ........ 109 Figure 19 Quantification of Microglia Proliferation in Lumbar Spinal Cord and Striatum..................................................................................................................................... 112 Figure 20 Motor Neuron Quantification in the Lumbar Spinal Cord ....................................... 115 Figure 21 Increased Expression of Active-Caspase-3 in the Lumbar Spinal Cord of Treated Mice ............................................................................................................................. 118 Figure 22 Open Field Tracking System .................................................................................... 142 Figure 23 Digigait Analysis System ......................................................................................... 145  vii Figure 24 Leg Extension and Wire Hang Data ......................................................................... 149 Figure 25 Additional Gait Analysis Parameters ....................................................................... 152 Figure 26 Additional Open Field Parameters ........................................................................... 176 Figure 27 Leg Extension and Wire Hang Data – Male Mice ................................................... 183 Figure 28 Additional Open Field Parameters – Male Mice ...................................................... 184 Figure 29 Lumbar Spinal Cord Volumes and Motor Neuron Counts Adjusted for Spinal Cord Volumes ........................................................................................................................... 191 Figure 30 ATF-3 Immunoreactivity in the Lumbar Spinal Cord Show No Significant Differences Among the Groups ................................................................................................ 194 Figure 31 HSP-70 Immunoreactivity in the Lumbar Spinal Cord Show No Significant Differences Among the Groups ................................................................................................ 196 Figure 32 Anti-GFAP Astrocyte Quantification in the Lumbar Spinal Cord ........................... 198 Figure 33 Leg Extension and Wire Hang Data – Female Mice ................................................ 200 Figure 34 Additional Open Field Parameters - Female Mice ................................................... 201                    viii  Acknowledgements    I am most grateful to my immediate supervisor, Dr. Christopher Shaw, as well as the other members of my supervisory committee, Dr. Doris Doudet, Dr. Orson Moritz, and Dr. John Steeves for the time they invested and their guidance throughout my program. I also thank Dr. Liisa Galea, my external examiner, for her meticulous edits and suggestions for the final draft of the thesis.  I am also grateful to Dr. Samuel Grant and his team at the Florida State University & National High Magnetic Field Laboratory for the many hours spent scanning my tissue samples.  I thank Grace Lee, Dominica Kwok, and all other members of the Shaw laboratory, past and present, for helping me learn, and for all the memories.  To my parents and siblings, my gratitude to you is beyond words can express. I thank you immensely.  Finally, to all those friends who have become my family, thank you for being a part of my life.     Funding for this work was provided by grants from the ALS Association, the United States Department of Defense, and the National Institutes of Health.    ix          To Mom, for your sacrifice; Olubunmi, for your courage; Emmanuel, for your support; and The Creator, I stand in awe of You.               x  1 1 INTRODUCTION  1.1 Background: Amyotrophic Lateral Sclerosis – Parkinsonism Dementia Complex (ALS-PDC) 1.1.1 History   Amyotrophic Lateral Sclerosis-Parkinsonism Dementia Complex (ALS-PDC) is a progressive and eventually fatal neurodegenerative disease that was first characterized among the Chamorro people of Guam. The initial formal reports of the disease occurred in the late 1940’s when a United States Navy Officer, Zimmerman HM, first reported a high occurrence of a motor neuron disease among the Chamorros (Zimmerman, 1945). The symptoms of this ‘motor neuron disease’ bore striking resemblance to those seen in the classical form of ALS. Further examination revealed that patients affected by this disease often showed several features of parkinsonism, as well as dementia (Kurland and Mulder, 1954; Mulder et al., 1954), hence the disease complex, ALS-PDC. In the decade following the Second World War (WWII), ALS-PDC became a major cause of death in Guam, accounting for about 25% of adult deaths in the small village of Umatac on Guam (Plato et al., 1969; Zhang et al., 1990). At the time, it was reported that the prevalence of the disease in Guam was about 100 times higher than other developed countries around the world (Kurland and Mulder, 1954). Although ALS often occurs later in life, investigators observed ALS features in patients as young as those in their 20’s (Kurland & Mulder, 1954; Plato et al., 2003). The age of onset of Parkinsonism Dementia Complex (PDC) was typically later, with a range of 32-64 years of age (Lessell et al., 1962; Kurland, 1988). Persons afflicted with ALS would usually die between 3-5years following diagnosis (Hirano et al., 1961a; Lessell et al, 1962). Clinical evidence also revealed that the disease onset could either be rapid or slow for reasons that remain unknown (Kurland, 1998). As with many other neurodegenerative diseases, ALS-PDC also shows a sex bias in the rate of occurrence as the disease affects more men at a rate of about 50% more than it does women (Mulder, 1957; Kurland, 1988; Plato et al., 2003). Although ALS-PDC may occur in families, efforts to identify a causative gene have thus far been unsuccessful (Steele and McGeer, 2008). Both the incidence and prevalence of ALS-PDC have since declined steadily, with ALS showing a markedly greater decline compared to PDC. Today, there are hardly any new cases of Guamanian ALS, while there are very few cases of PDC (Garruto et al., 1985; Plato et al., 2003, Galasko et al., 2007), although these figures are often debated, it is generally accepted that the disease on Guam is now almost extinct.  1.1.2 Disease Phenotype and Associated Neuropathology  The clinical phenotype of Guamanian ALS and the classical form of ALS are very similar. In both forms of the disease, patients present with a progressive weakness and atrophy of skeletal muscles due to the loss of lower motor neurons. This is typically accompanied by the degeneration of upper motor neurons as well (Kurland, 1988). Muscle fasciculations occur as a result of neuronal loss, and patients gradually lose the ability to both initiate and control all voluntary movements, except the muscles of the eyes and bowels which are usually, but not always spared (Kurland and Mulder, 1987). Eventually, death occurs as a result of the failure of respiratory muscles, with the patient unable to breathe or swallow (Hirano et al., 1966a; Kurland and Mulder, 1987). Symptoms of PDC present as classical parkinsonism symptoms, associated with dementia. Affected persons generally show involuntary tremors, increased muscle tone and rigidity, akinesia, postural and gait abnormalities, and generalized slowness and poverty of movement accompanied by mental slowness and memory deficits (Hirano et al., 1961a; Hirano et al., 1961b). Patients in late stages often become immobile and mute, and also show several changes in mood and personality including loss of apathy, depression, and sometimes violent behavior (Hirano et al., 1961a; Hirano et al., 1961b; Steele, 2005). The disease ALS-PDC as seen on Guam embodies the spectrum of ALS, Parkinson’s disease (PD) and dementia. Patients afflicted with ALS-PDC usually present with varying levels of the three diseases in the complex (Hirano et al., 1966a; Steele and McGeer, 2008). As with the physical manifestations, the neuropathology of classical ALS and the Guamanian variant are also very similar. Loss of upper and lower motor neurons is the hallmark of both diseases. However, in addition to this, Guamanian ALS patients show widespread neurofibrillary tangles (NFTs) and intracytoplasmic bodies in post-mortem exams, particularly in the hippocampus and other subcortical areas (Kurland, 1988). Histological analyses of Guamanian ALS patients reveal widespread NFTs and neuronal  2 loss in the hippocampus, parahippocampal gyrus, neocortex, amygdaloid nucleus, hypothalamus, and substantia nigra (SN) (Oyanagi et al., 1994), bearing significant overlap with other neurodegenerative diseases such as AD (Rodgers-Johnson et al., 1986) and PD, based on damage to the SN. Although other investigators have found NFTs in Chamorros without any history of neurological disease, the levels were usually lower than in those who were already symptomatic (Anderson et al., 1979: Oyanagi et al., 1994). It remains somewhat unclear whether the tangles seen in these controls were just a mere genetic feature of Chamorros, or whether the tangle intensity was not yet abundant enough to reach a level where it starts to produce clinical signs (Anderson et al., 1979; Hirano and Zimmerman, 1962; Oyanagi et al., 1994). The NFTs found in Guamanian ALS patients are composed of hyperphosphorylated Tau aggregates; a microtubule- associated protein (MAP) which is highly soluble in its non-pathological isoform, and interacts with tubulin, mainly to stabilize axonal microtubles in neuronal cells (Rodgers- Johnson et al., 1986, Hanger et al., 2009). However, when Tau becomes hyperphosphorylated, it often results in the formation of abnormal conformations which in turn form aggregates, such as the self-assembly to form both paired helical filaments (PHF), and straight filaments. These have also been implicated in other neurodegenerative disorders such as Alzheimer’s disease (AD) (Alonso et al., 2001; Hanger et al., 2009), progressive supranuclear palsy (PSP) (Hof et al., 1991; Gaig et al., 2008), frontotemporal lobar degeneration (FTLD) (Gaig et al., 2008), and corticobasal degeneration (Gaig et al, 2008). Collectively, these diseases are termed “Tauopathies.” The pathological features of PDC share significant overlap with both PD and AD. As with PD, loss of neurons in the nigro-striatal pathway is a major pathological feature of the disease (Hirano et al., 1961); however, Lewy body-aggregates mostly composed of the alpha-synuclein protein, another hallmark of classical PD are not often observed in post-mortem analyses of PDC patients (Forman et al., 2002; Winton et al., 2006). PDC patients also show significant loss of cortical neurons, a feature often associated with AD (Hirano et al., 1961). In addition to these features, the NFTs observed in Guamanian ALS patients are also widespread in PDC patients. In both diseases, Tau-immunopositive neurons are observed extensively in the motor cortex, the oculomotor nucleus, and facial nucleus (Oyanagi et al., 1994). PDC pathology also bears considerable similarities with  3 PSP. Both diseases show pathology in the SN, locus coeruleus, periaqueductal gray matter, and several regions of the reticular formation, including marked neuronal loss, as well as gliosis (Steele, 2005). However, each disease presents with a different isoform of hyperphosphorylated Tau. While PDC presents with a Tau triplet, PSP shows a doublet (Steele, 2005). It is also noteworthy that the Tau aggregates in PDC are identical to those observed in Guamanian ALS and AD (Steele, 2005). The clinical and pathological phenotypes of Guamanian ALS and PDC lend much support to the view that these diseases represent a single mixed disease entity, often with a dominant phenotype, occurring in a clinical spectrum. In addition, ALS-PDC shares several features with other neurodegenerative diseases as discussed above. The fact that it has only been found to occur in major clusters makes it all the more remarkable. Consequently, gaining a better understanding of this complex disorder may provide great insight into deciphering other neurodegenerative diseases.  1.1.3 Other Areas of ALS-PDC Occurrence Since ALS-PDC was first identified among the Chamorros on Guam, other areas of an ALS-PDC-like disorder have also been identified. An increase in ALS and PDC incidence and prevalence has been identified in two villages, Hozagawa and Hobara, located in the mountainous southern coast area of the Kii peninsula of Japan (Kimura , 1961; Kurland, 1988; Kuzuhara et al., 2001; Itoh et al., 2003), as well as in the western area of New Guinea (Gajdusek, 1963; Kurland, 1988; Kokubo and Kuzuhara, 2003). Similar to the Guamanian syndrome, ALS and PDC features are sometimes found to co- occur in patients. Although the disease phenotype and associated pathology is quite similar to the ALS-PDC of the Chamorros, several distinctions are observed in the disease etiology and familial occurrence (Itoh et al, 2003), the male/female incidence ratio (Kokubo and Kuzuhara, 2001), and also the age of onset (Kokubo and Kuzuhara, 2003; Kuzuhara, 2007). Unlike the Guamanian variant, the disease prevalence remains relatively high in the Japanese villages, and about 70% of the disease prevalence is familial. The disease also occurs more frequently in women, with symptoms first appearing between ages 53 to 74 (Kokubo and Kuzuhara, 2001). Despite the similarities to the ALS-PDC of Guam, firm conclusions cannot be drawn that these disorders are  4 identical (Steele and McGeer, 2008). However, while the high familial rate of occurrence in the Kii peninsula strongly suggests a genetic cause, a causal vector is yet to be identified, just as in the ALS-PDC of Guam, thereby making the search for environmental causes most imperative.  1.2 Disease Etiology 1.2.1 Genetic and Environmental Factors Over six decades after the disease was first described, the cause(s) of ALS-PDC remain unknown. Given the unique cluster of the disease on the island of Guam (Steele and McGeer, 2008), as well as noted familial aggregation of the disease (Murakami, 1999), a genetic cause was the first inclination of investigators. However, attempts to identify a causative gene have remained unsuccessful to date. Although some investigators suspect the Tau gene because of its prevalence among the Chamorros, detailed analyses of the gene showed no distinct mutation (Steele and McGeer, 2008). The genetic argument has also been further weakened by the gradual decline in the incidence of the disease since the decades following WWII; in fact, some investigators hold that no new pathological verifiable case has occurred in any person born after 1951 (Steele, 2005). Furthermore, some of the Filipino migrants who came to Guam to rebuild the island in the wake of the war also developed ALS-PDC (Steele, 2005) further negating the genetic argument. As a result, the focus shifted to potential environmental toxins, and/or gene-environment interactions. Several candidate toxins such as viruses (Viola et al., 1975; Gibbs and Gajdusek, 1982), abnormal levels of various metals such as calcium, magnesium, and aluminum (Oyanagi, 2005; Oyanagi et al. 2006), abnormal carbohydrate metabolism (Koerner, 1976), consumption of flying foxes (Pteropus mariannus mariannus, Pteropodidae) (Cox and Sacks, 2002; Banack et al., 2006), and consumption of cycad flour (Cycas micronesica) (Whiting, 1963; Whiting, 1964; Kurland, 1972), have all been investigated. Most of these hypotheses have been suspended and/or weakened over the years due to lack of sufficient evidence. However, consumption of cycad, the false sago palm of Guam, remains the most commonly cited hypothesis as a plausible environmental causative factor of the disease (Borenstein et al, 2007).   5 1.2.2 Cycas Micronesica The suspicion of cycad as a possible environmental risk factor for the development of ALS-PDC began in the 1950’s when it was first observed that a unique food staple of the Chamorros may be the causative factor for their disease (Whiting, 1963). Chamorro natives had prepared the starchy endosperm of the false sago palm and used it for both dietary and medicinal purposes for hundreds of years (Fig. 1). Apparently, the natives recognized the potential toxicity of cycad, and therefore employed an extensive method of ‘detoxifying’ the seed prior to consumption (Kurland, 1988). Typically, after harvesting the seeds, the outer husk was removed and the endosperm was cut up into chips and soaked for several days in several changes of water to remove potential toxins. Afterwards, the chips were dried, ground up into flour, and then used to make tortillas, soups, dumplings, and other food items. Prior to consumption however, the Chamorros often tested the bio-safety of the seeds by feeding the last water used in soaking the chips to chickens; if the chickens were still alive when the chips had dried, the chips were assumed to be safe for consumption (Kurland, 1988). The acute toxicity of various species of unwashed cycad consumption in humans, cattle and other animals are well documented (Hall, 1987). During the Japanese occupation of Guam during WWII however, cycad became one of the few sources of food available to the natives, resulting therefore not only in heavy consumption (Kurland, 1994), but also the highly likely consumption of younger seeds, which have been found to be more toxic (Marler et al., 2006). Hence, the cycad hypothesis stems from the observation that the incidence and prevalence of the disease fluctuated around patterns of cycad consumption, with the disease reaching its peak in the decade following the war (Duncan 1992; Zhang et al., 1996), and declining steadily thereafter following the importation of American wheat and corn flour which could be easily purchased from Guamanian stores (Zhang et al., 1990), resulting therefore in a steady decline in cycad consumption.  1.2.3 Putative Cycad Toxins Since the initial identification of cycad as a plausible etiological factor in the development of ALS-PDC, numerous studies have been done to replicate the clinical and  6 pathological features of the disease in animal models. Rats exposed to cycad developed tumors rather than neurological disease (Spatz and Laqueur, 1967). However, there is no evidence of cycad carcinogenicity in humans, neither is cancer endemic to Guam (Kurland, 1972). Closer examination of these earlier studies revealed that most of these studies involved the use of unwashed cycad, which was contrary to normal Chamorro practices. However, in a study done in rhesus monkeys, in which the animals were either fed unwashed, washed, or cooked cycad, it was noted that monkeys fed washed cycad had profound neurological damage compared to those fed unwashed or cooked cycad (Dastur, 1964). Laboratory analysis of cycad for putative toxins revealed several suspects including the amino sugar cycasin, the β-D-glucoside of methylazoxymethanol (MAM) (Laqueur, 1964; Hoffmann and Morgan, 1984), β-N-oxalylamino-L-alanine (BOAA), and β-N-methylamino-L-alanine (BMAA) (Polsky, 1972; Spencer et al., 1987; Weiss et al., 1989). While the hepatotoxicity and carcinogenicity of both cycasin and MAM have been demonstrated (Watanabe et al., 1975a; Watanabe et al., 1975b; Sieber et al., 1980; Laqueur et al., 1981;), these compounds have repeatedly failed to induce a disorder similar to ALS-PDC (Yang et al., 1966; Kurland, 1988). Further examination of these compounds revealed that both are water soluble, and would likely have been eliminated during the extensive washing employed by Chamorros when preparing cycad (Duncan et al., 1990; Khabazian et al., 2002). Monkeys exposed to BOAA developed neurolathyrism (Spencer et al., 1986), but again, this was not a feature common to ALS-PDC. In another study, where monkeys were fed BMAA via gavage, both behavioral and several neuropathological deficits were observed (Spencer et al., 1987); however, the study was challenged and criticized due to the high quantities of BMAA given to the animals in comparison to the low amounts of the toxin found in cycad flour prepared following Chamorro protocols (Garruto et al., 1988). Attempts to reproduce the study also proved unsuccessful (Perry et al., 1989; Cruz-Aguado et al., 2006). The BMAA argument has been further weakened given the knowledge that majority of BMAA in cycad samples were lost, leaving only trace amounts, following traditional methods of processing (Duncan et al., 1990).   7 1.2.4 An Animal Model of ALS-PDC Following years of unsuccessful attempts replicate the ALS-PDC disease phenotype and pathology in animals, our research group began dietary studies in which adult outbred mice were fed cycad flour prepared following traditional Chamorro methods for a period ranging from 30days to 3months. While experimental animals received ground cycad flour made into pellets as 20% of their daily food intake, control animals were given standard white flour pellets in the same amounts. The results of these studies revealed several behavioral disturbances in a range of motor and cognitive tasks. In addition, pathological features similar to those seen in ALS-PDC patients were also observed in mice brain and spinal cord tissue (Wilson et al., 2002; Schulz et al., 2003; Wilson et al., 2003; Wilson et al., 2004). This series of studies led to a re-examination of the cycad hypothesis, with the viewpoint that the potential neurotoxin(s) would have to be water-insoluble, hence not washed off during the mode of preparation employed by the Chamorros, and also be sufficiently lipophilic in order to cross the blood brain barrier (BBB) and exert neurotoxic actions (Khabazian et al., 2002). Following a series of column chromatography extractions, three compounds with the aforementioned properties were isolated from washed cycad chips. The compounds, all β-D-glucosides, were later identified as campesterol β-D-glucoside, stigmasterol β-D-glucoside (SG), and β-sitosterol β-D- glucoside (BSSG) (Khabazian et al., 2002) (Fig. 1). In vitro assays using rat cortical neurons showed marked cell depolarization, apoptosis, glutamate excitotoxicity, and the upregulation of glutamatergic protein kinases, when the neurons were exposed to these identified compounds (Khabazian et al., 2002). BSSG was also found to exert toxic effects on neurons using both organotypic hippocampal and striatal cultures (Khabazian et al., 2002), as well as a motor neuron-derived cell line, NSC-34 cells (Ly and Shaw, 2007). Following these in vitro assays, in vivo studies examining the toxic effects of some of these isolated steryl glucosides have also been performed. Outbred mice fed mouse chow pellets containing 1 mg of synthetic BSSG daily for a period of 15 weeks showed significant neuronal pathology, including loss of lower motor neurons, gliosis, decreased glutamate transporter labelling, activation of cell-stress response proteins,  8 increased caspase-3 labelling in the striatum, as well as decreased tyrosine-hydroxylase labelling in the striatum and substantia nigra (Tabata et al., 2008). Similar experiments using SG also showed significant pathology in the mice CNS tissue. A marked loss of lower motor neurons, increased caspase-3 activation, increased astrocyte and migroglia proliferation, reduced tyrosine hydroxylase labeling in the striatum and SN of treated animals, as well as both Tau and TAR DNA binding protein 43 (TDP-43) aggregation was observed in some SG-treated mice (Tabata, 2008). The outcomes of these studies have not only revived the cycad hypothesis, they have also provided evidence in support of cycad consumption as a causal factor in the development of ALS-PDC. Since the individual effects of these synthesized steryl glucosides found in cycad have been established, we have begun to examine the synergistic effects of both BSSG and SG in an in vivo mouse model.  1.2.5 The FeBAD Hypothesis In recent times, the influence of environmental factors on a variety of disease processes is increasingly being acknowledged. Various data suggest that the developmental exposure to environmental toxins could produce behavioral and neuropathological outcomes later in adult life. The FeBAD (Fetal Basis of Adult Disease) hypothesis was borne based on this emerging shift towards studying the developmental origins of adult diseases, and is based on the premise that environmental toxins encountered early in development (gestational or neonatal) could increase the vulnerability of developing certain diseases related to the toxin in adulthood, if re- exposed as adults (Barlow et al., 2007). Historically, investigators have primarily studied this model as it relates to fetal malnutrition and diseases such as diabetes, heart disease, and cancers (Barker et al., 1989; Valdez et al., 1996; Barker, 2002; Heindel, 2005; Morley, 2006). However, more studies are increasingly being conducted to examine the FeBAD hypothesis in relation to neurological disorders. A study on the developmental origins of schizophrenia shows a strong positive correlation between the disease etiology and factors such as maternal infection with the influenza virus during the first trimester of pregnancy, and gestational exposure to lead, among other causative factors (Opler and Susser, 2005). Other investigators have also examined the long-term neurological effects  9 of developmental exposure to lead (Needleman et al., 1990; Silbergeld, 1992). In another study, prenatal exposure to tobacco and alcohol resulted in reduced body and organ weight, delayed neonatal reflexes, and behavioral and long-term learning deficits (Li and Wang, 2004). In the realm of neurodegenerative disorders, Basha and colleagues recently showed that exposing neonatal rodents to lead triggered an increase in the expression of the amyloid precursor protein (APP) gene which has been associated with AD etiology (Basha et al., 2005). Also, in studies examining the FeBAD hypothesis and environmental toxicants, Thiruchelvam and colleagues showed that prenatal exposure to the common pesticides paraquat and maneb increased the incidence of parkinsonism phenotypes arising in adult mice, following a second exposure to the same pesticides in adulthood (Thiruchelvam et al., 2002; Barlow et al., 2007). In the study, the investigators showed that mice that were previously administered with these compounds neonatally, and then re-exposed as adults suffered the most damage to the nigro-striatal system, suggesting increased vulnerability to developing the disease in these group of animals (Thiruchelvam et al, 2002; Barlow et al., 2007). Several factors have remained mysterious in the studies of ALS-PDC, including observations that the disease onset could occur in young and old alike (Kurland & Mulder, 1954; Lessell et al., 1962; Kurland, 1988; Plato et al., 2003) and that the progression of the disease could be rapid or slow (Kurland, 1998; Kurland et al., 1994). However, an unusual correlate of the disease was the observation that exposure before adulthood to some unknown environmental factor(s) was a predictor of future disease expression (Garruto et al., 1980). Chamorro migrants were reported to have developed the disease phenotype in the United States, Japan, and Germany after periods of absence from the Island ranging from 1-34years (Garruto et al., 1980). Some migrants who had returned to Guam after periods of long absences were also observed to have developed the disease upon return. The common factor among all these migrants was the fact that all of them spent part of their childhood and/or adolescence on Guam (Garruto et al., 1980). Yet, the developmental exposure to plausible causative factors of ALS-PDC remains unexplored. Based on this observations as well as our previous work, the current study examines the effects of BSSG and SG in a mouse model, following a ‘multiple-hit’  10 exposure paradigm, in order to assess the effects of prenatal only, postnatal only, or both pre- and postnatal exposures to these steryl glucosides on disease onset and progression.  1.3 Rationale and Hypotheses Tested 1.3.1 Rationale Our model of the neurological disease ALS-PDC of Guam, is based on neuroepidemiological data linking consumption of the flour of the cycad seed to the disease (Kurland, 1988; Borenstein et al., 2007). We have previously demonstrated that cycad seed flour fed to adult male mice led to similar phenotypes observed in the human disease, including behavioral (motor), olfactory, and cognitive deficits; as well as neuropathologies associated with the respective neuronal populations (Wilson et al., 2002). Later experiments using the purified steryl glucosides found in washed cycad, BSSG and SG also recapitulated many of the same features of the disorder (Tabata et al., 2008, Tabata, 2008). Although strong evidence supports a developmental origin of the disease (Garruto et al., 1980), this area remains uncharted territory in ALS-PDC research. Also, the symbiotic effects of the synthesized steryl glucosides in washed cycad are yet to be examined. The current study therefore aims to advance the knowledge of ALS-PDC research by exploring two novel areas relating to the disease.  1.3.2 Hypotheses  In order to explore the aforementioned themes, the following hypotheses will be tested: - Fetal exposure to BSSG and SG will “sensitize” animals, such that a secondary re-exposure in adulthood will lead to a more rapid disease onset and progression - The period of fetal exposure will selectively impact neurons undergoing differentiation during that time, such that the same neuronal population will be preferentially vulnerable following exposure to the same neurotoxins in adulthood - There will be sex differences, with males showing greater affect, similar to what is observed in human ALS-PDC patients  11 Specifically, the study aims to: 1. Evaluate the effect(s) of prenatal, postnatal, and both prenatal and postnatal exposures to a combination of BSSG and SG on the overall wellness, and motor behaviors of mice; as well as indices of neural pathology in different regions of the CNS; 2. Assess whether or not animals exposed to steryl glucosides prenatally only are able to recover from any neural damage in development, or if any negative impacts are progressive; 3. Assess the effect(s) of targeting specific neural populations during development, by selective temporal exposure of these toxins on the development of a specific adult disease phenotype; 4. Determine if any sex differences occur as a result of the toxic insult. The experiments employed in the investigation of these hypotheses are discussed subsequently.                           12 Figure 1 Cycas Micronesica and Some of its Water Insoluble Components  Cycad pods (A), de-husked cycad (starchy endosperm) (B), molecular structure of β- sitosterol β-D-glucoside (BSSG) (C), and stigmasterol β-D-glucoside (SG) (D). BSSG has a molecular weight of 576.8473 g/mol, molecular formula C35H60O6 and the IUPAC name (2R,3R,4S,5S,6R)-2-[[(3S,8S,10R,13R,17R)-17-(5-ethyl-6-methylheptan-2-yl)- 10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H- cyclopenta[a]phenanthren-3-yl]oxy]-6-(hydroxymethyl)oxane-3,4,5-triol), while SG has a molecular weight of 574.83142 g/mol, molecular formula C35H58O6 and the IUPAC name (2R,3R,4S,5S,6R)-2-[[(10R)-17-[(E)-5-ethyl-6-methylhept-3-en-2-yl]-10,13- dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3- yl]oxy]-6-(hydroxymethyl)oxane-3,4,5-triol)). Both compounds are found in washed cycad flour, and have been chemically synthesized in the laboratory.                                  D C A B  13 2 MATERIALS AND METHODS  2.1 Materials 2.1.1 Chemical reagents Chemicals         Supplier Acetic acid        Sigma Acetic acid, glacial       Fisher Acetone        Fisher Antigen unmasking solution      Vector Bovine serum albumin (BSA)     Sigma 2-Bromo-2-chloro-1,1,1 trifluoroethane (stabilized with 0.01% thymol)     Sigma  Cresyl violet acetate       Sigma Clarion mounting media      Sigma 3,3’-diaminobenzidine tetrahydrochloride (DAB) kit   Vector 4’6 diamidino-2-phenylindole (DAPI) mounting media   Vector Entellen        Merck Ethanol  VGH Ethyl violet (methyl green)      Sigma Gelatin        Fisher Glycerin jelly mounting media     Shaw Lab Glycerol        Fisher Harris modified haematoxylin     Fisher ImmPACT DAB kit       Vector Isofluorane        Sigma Isopropanol        Fisher Methanol        Fisher 2-Methylbutane       Fisher Normal goat serum       Vector Normal rabbit serum       Vector Oil red O        Sigma  14 Paraformaldehyde       Sigma Permount        Fisher Phenol         Fisher Pottasium chloride (KCl)      Fisher Pottasium phosphate, monobasic     Fisher Propylene glycol       Sigma Saponin        Calbiochem Sodium chloride (NaCl)      Fisher Sodium phosphate, dibasic      Sigma β-sitosterol β-D-glucoside    UBC Stigmasterol        Sigma Stigmasterol β-D-glucoside  UBC Sucrose  Fisher Tween-20  Fisher Triton X-100        Fisher Vectastain elite ABC kit      Vector Xylenes        Fisher  2.1.2 Laboratory supplies Supplies        Supplier AccuSpin microcentrifuge      Fisher Analytical balance Denver – Instrument  Cryomold        Tissue-tek Cryostat        Leica Microtome blades      Richard-Allan Optimum cutting temperature (OCT) gel    Tissue-tek Orbit shaker        Lab-Line Perfusion pumps       Cole-Parmer Platinum cured silicon tubing      Cole-Parmer Slide coverslips       VWR  15 Superfrost plus charged glass slides     Fisher Vortex genie 2 Scientific - Industries  2.1.3 Primary and secondary antibodies Antibody       Supplier    Dilution Activated caspase-3      Chemicon 1:250 Activating transcription factor-3 (ATF-3) (H-90)             Santa Cruz 1:100 AlexaFluor 548-goat anti-rabbit IgG conjugate  Invitrogen 1:200 AlexaFluor 568-goat anti-rabbit IgG conjugate  Invitrogen 1:200 Choline acetyl transferase (ChAT)    Chemicon 1:100 Glial acidic fibrillary protein (GFAP)   GeneTex 1:100 Heat shock protein-70 (Hsp-70)    Chemicon  1:100 Ionized calcium binding adaptor molecule-1 (IBA-1) Wako  1:500 Tyrosine hydroxylase (TH)     Affinity 1:500  2.1.4 Behavioral testing equipment Behavioral testing equipment    Supplier Gait analysis runway       Mouse Specifics Open field       Noldus Wirehang       Shaw Laboratory  2.2 Methods 2.2.1 Cycad Steryl Glucoside Synthesis Steryl glucosides were synthesized on a contract basis by the laboratory of Dr. Stephen Withers (Department of Chemistry, University of British Columbia). Stigmasterol (95% purity) was purchased in bulk (Sigma-Aldrich Co., Oakville, ON, Canada) and used as the starting material for the synthesis of BSSG and SG. Stigmasterol was first converted to stigmasterol tosylate, and then treated with methanol and pyridine to yield stigmasterol methyl ether. Hydrogenation of stigmasterol methyl ether was accomplished in ethanol using 5% palladium on carbon as the catalyst.  Finally, β-sitosterol was produced in a reaction with tosic acid in water and dioxane at 80oC.  16 Glycosylation was achieved following a multi-step process to yield the final products, BSSG and SG.  The synthesized compounds were characterized using NMR (1H and 13C) and high-resolution mass spectrometry (HRMS), and a purity of at least 95% was verified by HPLC.  2.2.2 Animals 9 5-month-old CD-1 female mice were purchased from Charles River Laboratories (Wilmington, MA) and allowed one week to acclimatize to the new animal holding facility. CD-1 mice were selected because they are an out-bred strain, and also because our laboratory has previously demonstrated the effects of cycad flour, and cycad steryl glucoside consumption in this strain of mice (Wilson et al., 2002; Shaw and Wilson, 2003; Wilson et al., 2004; Tabata et al., 2008, Tabata, 2008). Following the period of acclimatization, the female mice were group-housed in 3 groups of 3 in cages containing some soiled male bedding to induce and synchronize the estrous cycle in the females (Whitten et al., 1968). On the third day, the females were age-matched with nine CD-1 males at the end of the daily light cycle. Afterwards, the females were checked for an ejaculatory plug every morning. The day a plug was found was recorded as the first day of pregnancy or as embryonic day 1 (E 1). Once a plug was found, the female was separated from the male and returned to her home cage. On E10 and E11, a mouse chow pellet containing a combination of two of the identified cycad steryl glucosides (0.6 mg BSSG and 0.4 mg stigmasterol glucoside (SG) based on the average amount found in 1 g of cycad seed used in previous cycad feeding experiments (Khabazian et al., 2002; Wilson et al., 2002) was fed to five of the pregnant females, while the remaining received the standard mouse chow pellets. The days, E10 and E11 were specifically chosen to target the differentiation of the nigro-striatal pathway in the developing nervous system (Bayer et al., 1995; Rice and Barone, 2000). Pregnant females were monitored daily for any unusual behavior. Parturition typically occurred between E19 and E20, and the litter number ranged between 8-15 pups per litter, with an average of ~10pups per litter. The control dams had an average litter size of 11.6 pups, while the BSSG and SG – fed dams had an average litter size of 9.5 pups. Although the control dams had a slightly higher average litter size, there were no significant differences in either the litter number, or the  17 sex of pups born to the control or BSSG and SG – fed mothers. The mortality rate of the pups was 11%, as one of the control dams lost 6 pups of her litter of 8. Two of the BSSG and SG - fed mothers also lost a pup each out of a litter of 8 and 12 respectively. All surviving pups were weighed within 24hr of birth, and then on a weekly basis. On day 22, mice were weaned, ear-punched for identification, and then group-housed by sex in a 12:12-hour light-dark cycle with food and water supplied ad libitum. Only pups that were siblings were housed in the same cage. Postnatal behavioral analysis commenced at 5 weeks of age, using different behavioral testing methods to be described below. Behavioral tests were performed in a room adjacent to that in which the animals were housed. Animals were housed in a virus-free barrier facility in a temperature-controlled room, and all procedures were performed in strict compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, the Canadian Council of Animal Care guidelines, and the Society for Neuroscience “Guidelines for the Use of Animals in Neuroscience Research” using a protocol approved by the University of British Columbia Animal Care Council (Protocol number: A07-0063). At 4 months (17 weeks old), the mice were separated into single cages in preparation for the secondary exposure to steryl glucosides, in order to ensure that the BSSG and SG – fed mice were consuming the appropriate amount of steryl glucosides. Animals were randomly assigned to separate cohorts of male and female mice in one of 4 groups: - Group 1: Mice from control moms and with no subsequent steryl glucoside exposure. These are the “control” mice. - Group 2: Mice exposed to BSSG and SG during gestation via maternal diet, and with no subsequent steryl glucoside exposure. These mice are called the “prenatal” group - Group 3: Mice from control moms that will receive the steryl glucoside- containing diet. These mice are called the “postnatal” group - Group 4: Mice exposed to BSSG and SG during gestation via maternal diet that will also receive a secondary exposure to steryl glucoside- containing diet. Mice in this group are labeled “both.”  18 There were 8 groups in total with an “n” range of 7-11 per group. More specifically, there were 11 males in the “prenatal” and “both” groups,” 10 males in the “postnatal” group (one animal in this group had to be sacrificed midway through the study due to the development of a skin irritation), and 9 “control” males. In the female cohorts, there were 7 mice each in the “prenatal,” “both,” and “control” groups, while there were 8 female mice in the “postnatal” group. One week after the animals were separated into single cages (18 weeks old), mice in groups 3 and 4 (a total of 36 mice) started to receive mouse chow pellets containing 0.6mg BSSG and 0.4mg SG as a part of their daily diet for a period of 15 weeks, while the remaining 34 mice (groups 1 and 2) received the standard mouse chow. During this period, the mice were weighed weekly and also tested for any associated changes in behavior as before. Daily monitoring of any unusual activity, as well as the general well-being of the animals also continued as usual (Fig. 2).  2.2.3 Steryl Glucoside Feeding The standard mouse chow pellets fed to the mice (Mouse Diet™, Purina) were collected from the animal holding facility weekly and finely ground using a coffee grinder. This “powdered-chow” was stored in an air-tight container at room temperature. Every morning, 36 g of ground chow was weighed out for the 36 mice in groups 3 and 4. A total of 21.6 mg BSSG and 14.4 mg SG was then carefully weighed out using an analytical balance (Denver Instrument, Denver, CO. U.S.A) to ensure a high degree of precision, and added to the chow. The synthetic steryl glucosides were very well mixed in with the chow, using a spatula to ensure a distribution as even as possible. After this was achieved, ~15 ml of ddH20 was added to the mix, and the chow was kneaded into dough. This dough was then weighed, and divided by 36. Each pellet was cut out and weighed, ensuring that the pellets weighed the same to at least one place of decimal. Each mouse in groups 3 and 4 received the steryl-glucoside-containing pellet in their feeding trays daily before the addition of the standard mouse chow, while control animals only received the standard chow. The mice were not starved as they always consumed the experimental pellets in its entirety and even appeared to have a preference for the experimental pellets as they usually ate it as soon as it was placed in their trays, even when there were left  19 over standard chow pellets from the previous day. The mice were fed the experimental BSSG and SG-containing pellet for a period of 15 weeks.  2.2.4 Animal Behavior Testing Aside from the daily observation of the animals for any unusual behavior including stereotypy often associated with singly-housed mice, the mice were tested often using a number of behavioral tests to determine the effects, if any, of the steryl glucoside diet. The behavior tests employed, as well as the methods of testing are briefly described as follows. For all the tests except for the open field test in which the accompanying software required certain parameters such as the definition of the center and outer zones in the arena be set and fixed for the duration of the study, the mice were always randomly selected for testing. Behavior testing was conducted during the day before the start of the dark cycle (18:00 hr), in a room adjacent to that in which the animals were housed in the holding facility. The leg-extension test was used to measure spinal reflexes, and it evaluates the presence (maximum score = 4) or absence (minimum score = 0) of the extension of the hind limbs when mice are suspended from the tail as a measure of lower motor neuron functionality (Barneoud and Curet, 1999). Specifically, the mice were taken from their cages one at a time, and placed on a disinfected table top. The animal’s hind limbs were then suspended by holding up the mouse by the base of the tail while the fore limbs rested on the edge of the table. A score of 4 was assigned to a mouse that completely extended both hind limbs as soon as it was suspended by its tail; such a response was considered the normal response. A mouse received a score of 3 when there were tremors and/or punching observed in either extended hind limb. A score of 2 was assigned when an animal exhibited tremors and/or punching in both limbs, or when an animal fully extended a limb while retracting the other. A score of 1 was given to a mouse with one retracted limb, with tremors and/or punching in the other. Finally, an animal was scored 0 when both legs were fully retracted. The test was designed to show the progressive loss of lower motor neuron functionality that has been observed in previous studies where CD-1 mice have been exposed to cycad flour or steryl glucosides as part of their diet (Wilson et al., 2002; Tabata et al., 2008). All the mice used in this study were tested for  20 this reflex once a week, starting from when the animals were 5 weeks old, and continuing for the duration of the study. Muscular strength was measured using the wire hang test which measures the latency to fall from an inverted grid (Paylor et al., 1999; Wilson et al., 2003; Wilson et al., 2004; Wilson et al., 2005; Tabata et al., 2008). Each mouse was placed on a wire mesh raised with the aid of a box to a height of 50 cm above a soft landing surface. The height was so designed to discourage most mice from refusing to hang on, while the padded surface was designed to prevent injury in animals that fell off before the desired time. Each mouse was allowed a few seconds to latch onto the wire mesh, and then the mesh was inverted 180°. The latency to fall off the grid was measured as soon as the grid was inverted, and the time it took before a mouse fell off was recorded. The maximum time for the wire hang test was set at 120 seconds. If a mouse fell off within 5 seconds of grid- inversion, such an animal was given a second trial as it was assumed that the animal may not have had sufficient time to latch onto the grid. However, if such an animal fell of again within 5 seconds on the second trial, the second time was recorded. All female mice were tested before the males to minimize the possible effects sex pheromones may have on the test, and the mesh was always disinfected with 70% ethanol between each mouse. The wire mesh was also cleaned and disinfected with 1.3% quatricide at the start and end of all the testing sessions. As with the leg extension reflex test, all the mice used in this study were tested on the wire hang on a weekly basis, starting from when the animals were 5 weeks old, and continuing for the entire duration of the study. Exploratory activity was measured in the mice using the open field test. This test is very often used as a measure of assessing locomotor activity in mice (Malleret et al, 1999; Machado et al., 2009) and is considered one of the most standardized measures of motor function (Crawley, 2000). The open field test is also often used to assay anxiety- like behavior in animals (Tonelli et al., 2009; Wultsch et al., 2009). The test was performed under standard light conditions in the animal holding facility using Noldus Ethovision, a video tracking system for detailed recording of animal activity. During the testing sessions, each mouse was placed into an open arena of ~1 m diameter, which enabled the definition of both center and peripheral regions in a single arena. There were no actual physical demarcations in the tub/arena; the regions were defined in the arena  21 using the accompanying software of the Noldus recording system, Ethovision 3.1 (Ethovision® 3.1, Noldus Information Technologies Inc., Leesburg, VA, USA) (Appendix A). Mice were started in the center of the arena one at a time and their activity was measured using an overhead-mounted camera over a five minute period. This timing has been shown adequate by other investigators to assess motor functions and other measures of abnormal behavior (Crawley, 2000; Wultsch et al, 2009). At the end of each five minute trial, animals were returned to their home cages and the arenas were disinfected with 70% ethanol between animals, again to reduce the likelihood of animal scents in the tubs as a possible confounding variable. After all animals had been tested, the arenas were cleaned with 1.3% quatricide and the videos were automatically analyzed using the Ethovision 3.1 software. The mice used in this study were tested in the open field bi-monthly. Mice were first tested in the open field at 8 weeks of age, and the tests continued for the duration of the study. To test for any anomalies in animal gait that may be associated with steryl glucoside feeding, mice were tested on a ventral plane videography device which captured mice stepping patterns in significant detail (Amende et al., 2005). To perform the gait analysis test, mice were run on a colorless (PVC and polycarbonate), treadmill- like motorized belt (Digigait™, treadmill motor: DC, ¼ HP; torque 45 lb-in, Mouse Specifics, Inc., Boston, MA, USA), enclosed in a well-lit, transparent, rectangular box. The mice were placed in the box one at a time and allowed to run on the belt at a speed of 20rpm. A high-speed camera mounted ventrally beneath the colorless belt captured the gait dynamics of each paw at a speed of ~150 frames per second. A total of 350 frames of continuous running were required to analyze each animal gait dynamics, and so the mice were only required to run for ~5 seconds. The gait indices were analyzed using the accompanying digital imaging software (Digigait™, Mouse Specifics, Inc., Boston, MA, USA) version 6.1, strictly following manufacturer’s instructions (Appendix A). To briefly explain, 350 frames of continuous running are selected from the video recording of individual mice. The software then generates a record of mice paw placements from the paw prints in the selected frames and breaks down the paw placements into three main parameters, stride duration, swing duration, and stance duration. The stance duration represents the time when a paw is in contact with the walking surface, while the  22 swing duration represents the time when the paw is not in contact with the walking surface. Both of these parameters make up the third major parameter, the stride duration. The stance time is comprised of the brake time which represents increasing paw contact area with the runway belt over time, and propel time which is the decrease in paw-to-belt contact area over time. The software calculates each of these variables for all four paws per mouse, in addition to several other gait indices such as stride frequency, paw angle, and the number of steps per paw over the selected running time frame. As with the other behavior tests, both the belt and animal holding box were wiped down with 70% ethanol between each mouse trial. The animals were tested on the digigait bi-monthly starting at 8 weeks of age, and the tests continued for the duration of the study. The mice testing schedule in this study was done such that all behavior tests were not performed on the same day, rather the tests were grouped into two sets; the leg extension reflex test, and the wire hang tests were done on the same day right after the mice weights were taken, while the open field and gait analysis tests were performed together following an interval of at least 3 days in an effort to minimize any potential stress the animal may suffer as a result of the tests.  2.2.5 Animal Sacrifice and Tissue Processing Immediately following the 15-week period of secondary exposure to steryl glucosides in the second part of the study, all the animals were sacrificed for histological assays at 33 weeks of age. Mice were deeply anesthetized in a closed chamber littered with halothane (2-Bromo-2-chloro-1,1,1 trifluoroethane; Sigma-Aldrich Co., Oakville, ON, Canada ) - soaked cotton balls, followed by an intracardial perfusion with phosphate buffered saline (PBS). After the animal blood was completely eluted, the tissues were fixed by an intracardial perfusion with cold 4% paraformaldehyde (PFA) in PBS. Both reagents were injected into the animals using separate sterile platinum-cured silicon tubing (Cole-Parmer Canada Inc., Montreal, Canada) attached to peristaltic pumps (MasterFlex peristaltic pump, Cole-Parmer Canada Inc., Montreal, Canada) at a rate of 20 mls per minute. Mice brains, spinal cords, and gastrocnemius muscle tissue were dissected out and post-fixed overnight in 4% PFA. Afterwards the tissues were equilibrated in increasing concentrations of sucrose in PBS (10, 20, and 25%) over a 24-  23 hour period. The tissues were assumed to have completely equilibrated when they sank to the bottom of the scintillation vials in 25% sucrose.  Afterwards, the tissues were set in labeled cryomolds (Tissue-tek, Sakura Finetek, USA, Torrance, CA) covered in an optimum cutting temperature (OCT) gel (Tissue-tek, Sakura Finetek, USA, Torrance, CA) and then frozen over a 2-methylbutane (Fisher Scientific, Pittsburgh, PA) bath on dry ice. Frozen tissues were then transferred and stored at -80oC until they were ready to be processed.  Frozen tissue were cut coronally, using a motorized Leica cryostat, model CM3050S (Leica Microsystems, Wetzler, Germany) maintained at -20oC. Brain sections were cut at 30μm, while spinal cord sections were cut at 20μm. Tissue sections were serially direct-mounted on plus-charged glass slides (Superfrost; Fisher Scientific, Pittsburgh, PA) such that adjacent spinal cord sections were at least 100μm apart, while adjacent brain sections were 150μm apart. Ten spinal cord sections were mounted per slide, while eight sections of brain tissue were mounted per slide. The slides were then stored at -80oC until ready for histological processing.  2.2.6 Nissl - Lower Motor Neuron Quantification Lumbar spinal cord sections in the L1 region (Sidman, Angevine et al, 1971) from each animal were stained for the Nissl substance. To do this, slide-mounted tissue sections were washed in a decreasing gradient of ethanol (95%: 15 min; 70%: 1 min; 50%: 1 min) on an orbit shaker (Lab-Line, Barnstead International, Iowa, USA) at room temperature (RT) in order to hydrate and remove lipids and fixation chemicals from the tissue. The tissue sections were then washed for 1 min each in two changes of distilled water. Afterwards, the sections were stained with twice-filtered (Whatman, Kent, United Kingdom) 0.5% cresyl violet for 5 min, and then rinsed again in two changes of distilled water, 1 min each. Tissue dehydration followed by placing the slides in 50% ethanol for 1 min, 70% ethanol with 1% glacial acetic acid for 30 sec, 95% ethanol for 1 min and lastly in 100% ethanol for another minute. Tissue sections were then cleared in xylene for 1 min, mounted in Entellen mountant (Merck, Darmstadt, Germany), and then cover- slipped (VWR, Mississauga ON, Canada).  24 “Healthy” motor neurons in the Nissl-stained tissue sections were quantified in the lateral ventral horn of the lumbar region of the spinal cord. “Healthy” motor neurons were defined as neurons with a visible round, pale nucleus, and with visible, well defined processes extending from the cell body. Neurons with atrophic cell bodies, and shrunken, pyknotic nuclei were excluded from the counts as these were considered degenerating neurons (Yamazaki et al., 2005). In the same way, chromatolytic and vacuolized neurons were also excluded from the count as these were also deemed to be advanced in the cell death pathway (Chang and Martin, 2009; Kuru et al., 2009). The ventral horn boundaries were determined by the margins of the gray and white matter, as well as with the aid of an invisible line drawn laterally through the central canal (Fig. 9). All the “healthy” motor neurons in the field of view at 40x magnification were counted in both the left and right lateral ventral horns of 10 adjacent sections in each animal, and the counts from all BSSG and SG – fed groups were compared to those of the control mice.  2.2.7 Fluorescence Antibody Staining Immunofluorescence staining for antibodies was performed on tissue sections in the lumbar spinal cord as follows: tissue-mounted slides were heat-fixed using very low heat for a few seconds on a hot plate, and then washed for a total of 15 min in 3 changes of PBS or PBS with 0.5% Triton X-100 (PBST) at RT on the shaker. Slides were then incubated at RT for 1-3 hr in a permeabilizing blocking solution made up of 10% normal goat serum (NGS), or 5% normal rabbit serum (NRS), with 1% bovine serum albumin (BSA) in PBST. Following the blocking step, slides were washed in 2 changes of PBS or PBST for a total of 4 min. Excess solution was suctioned off the slides without allowing the tissue sections to dry, and slides were incubated overnight in a humidified chamber at 4oC in a base solution made of PBST with 1% NGS or 1% NRS, 1% BSA, and one of the primary antibodies diluted as follows: - Anti-activated caspase-3 IgG, rabbit host (monoclonal, 1:250; Chemicon International, Temecula, CA); - Anti-human/mouse choline acetyl transferase (ChAT) IgG, goat host (polyclonal, 1:100; Chemicon International, Temecula, CA);  25 - Anti-human/mouse ionized calcium binding adaptor molecule 1 (IBA-1) IgG, rabbit host (polyclonal, 1:500; Wako Pure Chemical Industries, Ltd, Richmond, VA); - Anti-mouse glial fibrillary acidic protein (GFAP) IgG, rabbit host (polyclonal, 1:100; GeneTex Inc., San Antonio, TX). For both the IBA-1 and GFAP experiments, additional steps were included to ensure proper antibody penetration and antigen labeling. Following the initial washes in PBS, and prior to the blocking step, slides were placed in a PBS solution containing 1% saponin (Calbiochem, EMD Chemicals, Darmstadt, Germany) on the shaker for 1 hr. Afterwards the slides were washed in two changes of PBS at 5 min each, and then boiled for 7 min in a low pH antigen unmasking solution (Vector Laboratories, Burlingame, CA, U.S.A). Slides were allowed a few minutes to cool and were then washed in another 2 changes of PBS at 5 min each, before continuing on to the blocking step. In all the experiments, an additional lumbar spinal cord slide was randomly selected and included as a negative control slide by omitting the primary antibodies in order to test for non- specific staining. Following the overnight primary antibody incubation, slides were rinsed for 15 min at RT in 3 changes of PBS on the shaker. Again, excess solution was suctioned off to prevent the over-dilution of antibodies, and then the slides were incubated in dark chambers for 2 hr at RT with the secondary antibody solutions containing fluorophore conjugated antibodies: AlexaFluor 568 goat anti-rabbit, or rabbit anti-goat IgG (monoclonal, 1:200, absorption: 578 nm, emission 603 nm; Molecular Probes, Eugene, OR), AlexaFluor 546 goat anti-rabbit or rabbit anti-goat IgG (monoclonal; 1:200, absorption 556 nm, emission 573 nm; Molecular Probes, Eugene, OR) diluted in PBS of pH 7.4. After the secondary antibody incubation, slides were washed for a total of 15 min in 3 changes of PBS. Excess PBS was then suctioned off and the tissue sections were cover-slipped with a drop (~15 µl) of 4’6 diamidino-2-phenylindole (DAPI) fluorescent mounting media (Vectashield, absorption: 360 nm, emission: 460 nm, Vector Laboratories, Burlington, ON, Canada) in order to label the nuclei and prevent rapid loss of fluorescence during microscopic examination of the tissue sections. Slides were stored in dark slide boxes at 4oC to hinder photo-bleaching, and were typically observed and  26 quantified under a fluorescent microscope, Zeiss Axiovert 200M (Carl Zeiss Canada Limited, Toronto, ON) within 2 to 3 days of staining. DAPI (blue fluorescence) visualization required a 359/461 nm excitation/emission filter, Alexa Fluor 546TM (red fluorescence) required a 556/573 nm excitation/emission filter, and Alexa Fluor 568TM (green fluorescence) required a 578/603 nm excitation/emission filter. As with the Nissl stain, quantification of all labeled cells was done in the lateral ventral horn of the lumbar spinal cord at 20x magnification, except for GFAP astrocyte labeling that was done at 40x magnification. Tissue sections used for the ChAT, GFAP, IBA-1, and Caspase-3 antibodies were carefully selected to ensure uniformity of the sections across all animals, and the stains were performed on tissue sections in the L1 and L2 levels of the mouse lumbar spinal cord. Only those cells showing distinct labeling were counted in both horns of the spinal cord, and again, 10 sections were counted per animal. Images were captured using the Zeiss Axiovert Zoom Axiovision 3.1 with AxioCam HRM camera attached to the microscope, and the images were processed using the accompanying AxioVision 4.3 software.  2.2.8 Immunoperoxidase Antibody Staining in Lumbar Spinal Cord Lumbar spinal cord sections were also examined for antibody immunoreactivity as follows: slide-mounted lumbar spinal cord sections were heat-fixed to the slides using very low heat for a few seconds on a hot plate. Tissue sections were subsequently washed in 2 changes of PBS for a total of 10 min on the shaker. Sections were then incubated with 0.3% H2O2 in methanol for 30 min to quench any endogenous peroxidase activity. This was followed by another wash in 2 changes of PBS for a total of 4 min. Tissue sections were then incubated in the blocking solution made up of 10% NGS and 1% BSA in PBST for 1 hr at RT. Following the block step, slides were incubated overnight in a humidified chamber at 4oC in a base solution made of PBST with 1% NGS, 1% BSA, and one of the following primary antibodies diluted as indicated: - Anti-human/mouse activating transcription factor 3 (ATF-3) (H-90) IgG, rabbit host (polyclonal, 1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); - Anti-activated caspase-3 IgG, rabbit host (monoclonal, 1:250; Chemicon International, Temecula, CA);  27 - Anti-mouse glial fibrillary acidic protein (GFAP) IgG, rabbit host (polyclonal, 1:100; GeneTex Inc., San Antonio, TX); - Anti human/mouse heat shock protein 70 (HSP-70) IgG, rabbit host (polyclonal, 1:100; Chemicon International, Temecula, CA); - Anti-human/mouse ionized calcium binding adaptor molecule 1 (IBA-1) IgG, rabbit host (polyclonal, 1:500; Wako Pure Chemical Industries, Ltd, Richmond, VA).  On the following day, the slides were washed again for a total of 4 min in 2 changes of PBS, and then incubated with the secondary antibody (biotinylated goat-anti- rabbit IgG) using the Vectastain ABC elite kit (Vector Laboratories, Burlingame, CA, USA) prepared by following the accompanying product insert. Briefly, 3 drops (~150 µl) of the NGS stock solution provided in the Vectastain kit was added to 10 ml of PBST and briefly mixed on the vortex (Scientific Industries, Bohemia, NY, USA). A drop (~50 µl) of the biotinylated goat-anti-rabbit IgG was then combined with the solution and further mixed. Although the product manufacturer’s suggested a 30 min secondary antibody incubation time, secondary antibody incubation was actually done for 1 hr at RT as it was noted to give better results in this tissue. About 35 min into the secondary antibody incubation, the Avidin/Biotin complex (ABC) solution was prepared by adding 1 drop (~50 µl) of reagent A (Avidin DH) and 1 drop (~50 µl) of reagent B (Biotinylated Horseradish Peroxidase H) (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA, USA) to every 2.5 ml of PBST. Following the 1 hr of secondary antibody incubation, slides were again washed for a total of 4 min in 2 changes of PBS and then incubated for 30 min with the ABC solution. Sections were washed afterwards for 4 min in 2 changes of PBS, and then incubated with the peroxidase substrate (DAB) solution for color development. The DAB working solution was prepared using the DAB kit (Vector Laboratories, Burlingame, CA, USA) by combining 2 drops of buffer stock solution, 4 drops of DAB stock solution, and 2 drops of hydrogen peroxide solution for every 5 ml of ddH2O added, per manufacturer’s instructions. Color typically developed within 1 to 2 min of incubation with DAB, and the slides were washed afterwards for 5 min in ddH2O to terminate the enzyme-substrate reaction. Counterstaining of the nuclei was achieved by placing the slides in filtered 0.5% methyl green for 10 min. The methyl green solution  28 was prepared by adding 1.5 g of ethyl violet free - methyl green (Sigma-Aldrich, Oakville, ON, Canada) to 300 ml of 0.1 M sodium acetate buffer (pH 4.2), which was prepared by adding 4.08 g of sodium acetate trihydrate to 300 ml of distilled water. The final pH was adjusted to 4.2 using concentrated glacial acetic acid (Fisher Scientific, Pittsburgh, PA, USA). Following the nuclei counterstaining, slides were washed in several changes of ddH2O to wash of any excess methyl green. Tissue dehydration followed by dipping the slides in 2 changes of 95% ethanol for a total of 20 sec and 2 changes of 100% ethanol for a total of another 20 sec. Sections were cleared in xylene under a fumehood for 1 min and subsequently permanently mounted in clarion media (Sigma-Aldrich, Oakville, ON, Canada), and then cover-slipped. As with the fluorescent protocol, additional steps were included for Iba-1 and GFAP immuno-staining in the lumbar cord to ensure proper antibody penetration and antigen labeling. Following the initial washes in PBS, and prior to the blocking step, slides were placed in a PBS solution containing 1% saponin on the shaker for 1 hr. Afterwards the slides were washed in two changes of PBS at 5 min each, and then boiled for 7 min in a low pH antigen unmasking solution. After allowing the slides a few minutes to cool down, slides were washed in another 2 changes of PBS at 5 min each, before continuing on to the blocking step. Following the DAB color development step, nuclei counterstaining was omitted as it was deemed unnecessary. Lumbar spinal cord sections used for the ATF-3 and HSP-70 stains were selected from spinal cords collected from male mice in the L2/L3 levels of the cord and as with the Nissl stain, only “healthy” motor neurons that expressed both DAB (brown) and nuclei counterstaining (green) in the lateral ventral horn were counted. Such neurons in the field of view as observed under 40x magnification were counted in both horns of the spinal cord sections across all treated and control groups. Sections used for the caspase-3, Iba-1, and GFAP stains were selected from spinal cords collected from female mice in the L2 level of the cord, and were quantified in a similar manner as the male tissue. A total of 10 sections were quantified per animal and the average number of positively labeled neurons across both horns in all sections quantified was taken for each animal.    29 2.2.9 Immunoperoxidase Antibody Staining in Brain Sections In order to examine activated glia in mouse brain striatum sections without necessarily compromising the ability to clearly identify different brain areas, slide mounted striatum sections were stained with anti-Iba-1 (anti-human/mouse Iba-1 IgG, rabbit host, polyclonal, 1:500; Wako Pure Chemical Industries, Ltd, Richmond, VA) and anti-GFAP (anti-mouse GFAP IgG, rabbit host, polyclonal, 1:100; GeneTex Inc., San Antonio, TX) antibodies using a similar immunoperoxidase antibody staining protocol as described for the lumbar spinal cord in the preceding section. As before, sections were heat-fixed and washed in PBS as previously described. Slides were then placed in a PBS solution containing 1% saponin to ensure membrane permeability. Slides were subsequently washed in PBS, followed by the quenching of endogenous peroxidase activity by a 30 min incubation with 0.3% H2O2 in methanol. Sections were washed again in PBS and then boiled in a low pH antigen unmasking solution for 7 min. Following another wash in PBST, sections were incubated with the blocking solution made up of 10% NGS and 1% BSA in PBST for 1 hr at RT, and then incubated with the primary antibodies (anti Iba-1 or anti-GFAP) for 2 hr at RT. Slides were briefly washed in PBS, and then incubated with the secondary antibody using the Vectastain kit as described above. After 1 hr of secondary antibody incubation, slides were incubated with the ABC solution, and following another quick wash in PBS, color development was achieved, this time using the ImmPACT DAB kit, an enhanced and higher-intensity peroxidase detection system (Vector Laboratories, Burlingame, CA, USA). The ImmPACT DAB working solution was prepared by adding a drop of the provided concentrated stock solution of high intensity DAB chromogen for every 1 ml of diluent, also provided in the kit. Following color development, slides were washed to terminate the color development reaction as described above. However, rather than counterstain the nuclei using methyl green, this step was completely skipped in order to be able to clearly assess glial cell morphology and instead, slides were dehydrated in 95% and 100% ethanol as previously described, allowed to air dry, then mounted in clarion media and cover-slipped. Both astrocyte and microglia quantification in the striatum were done at 40x magnification, using the external capsule as a point of reference and counting all positively-labeled cells in a 6.67 x 5.33 in window. Glial cells were quantified in both the right and left  30 hemispheres in a total of 8 sections per animal. Anatomic boundaries were identified using a stereotaxic mouse brain atlas (Franklin and Paxinos, 1997), and special care was taken to ensure homology in brain sections quantified across all animals.  2.2.10 Tyrosine Hydroxylase Immunohistochemistry Tyrosine hyroxylase (TH) – immunoreactive neurons in both the substantia nigra pars compacta (SNpc) and striatum of animal brain tissue were detected using an immunoperoxidase antigen labeling protocol very similar to those already described. Slide-mounted tissue sections were heat fixed, washed in PBST, and incubated with 0.3% H2O2 in methanol to quench endogenous peroxidase activity. Following a quick wash in PBST, and then a 1 hr block in 10% NGS in a base solution of 1% BSA in PBST, tissue sections were incubated overnight in a humidified chamber at 4oC with the primary antibody, anti-tyrosine hydroxylase, rabbit host (Affinity Bioreagents, Golden, CO, USA), using a 1:500 dilution in PBST containing 1% NGS and 1% BSA. Slides were briefly washed the following day in PBST and then incubated with the secondary antibody, biotinylated goat anti-rabbit IgG, using the Vectastain method previously described. This was followed by the Avidin/Biotin complex formation, and finally the DAB substrate color visualization step. The ImmPACT enhanced DAB kit was again used to visualize TH-immunoreactive cells, and each slide was precisely timed for 75 sec during DAB incubation. This was done to prevent any bias in enzyme-substrate reaction time, hence color density, as TH-positive cells were to be quantified using an image analysis software. Slides were washed in ddH2O to terminate DAB reaction, and were subsequently dehydrated in 95% and 100% ethanol. Afterwards slides were allowed to air dry, mounted in a permanent media (Permount; Fisher Scientific, Pittsburgh, PA, USA), and cover-slipped. Images of both the striatum and SN were captured using a light microscope while keeping both the light and exposure rate constant. Other parameters such as the reflection coefficient (gamma), and saturation were also kept constant, again to have an accurate representation of the actual differences in the optical densities of the images. The optical luminosity of the striatum and SN were analyzed across all animals using the average of both right and left hemispheres (no unilateral effects were observed) in 8 sections of each region per animal. As with glial cell quantification, special care was  31 taken to strictly identify anatomic boundaries in all brain sections analyzed to avoid any major bias in measurements.  2.2.11 Lipid Accumulation - Oil Red O The distribution of lipids in the lumbar spinal cord was assayed using the oil red o (ORO) dye. 0.7% ORO was prepared by dissolving 1.4 g of ORO dye (Sigma-Aldrich, Oakville, ON, Canada) in 200 ml of propylene glycol (PG) (Sigma-Aldrich, Oakville, ON, Canada) while heating and stirring. Care was taken not to exceed 110oC. PG-dissolved ORO was then filtered while the solution was still warm to remove any undissolved residues, and then re-filtered again when the mixture had cooled. To stain, tissue- mounted slides were washed for a total of 10 min in 2 changes of PG. ORO solution was heated up to, and maintained at 60oC, and the slides were placed in the solution for 20 min. Following this, slides were washed in 85% PG diluted in distilled water for 5 min, followed by another wash in dH2O for 5 min. Nuclei counterstaining was achieved by placing the slides in haematoxylin (Harris modified haematoxylin; Fisher Scientific, Pittsburgh, PA, USA) diluted 1:10 in dH2O for 1 min. Slides were then rinsed in running tap water for 1 min, followed by another quick rinse in dH2O. To avoid clearing the lipids to be visualized, clearing solvents were skipped; rather, slides were mounted in glycerin jelly, prepared by dissolving 10 mg of gelatin (Fisher Scientific, Pittsburgh, PA, USA) in 60 ml dH2O while heating. 70 ml of glycerol (Fisher Scientific, Pittsburgh, PA, USA), and 0.25 g of phenol (Fisher Scientific, Pittsburgh, PA, USA) were then added and properly mixed in. Glycerin jelly media was generously applied to the slides while still fluid, and the slides were cover-slipped. Slides assayed for oil red o histochemistry were selected from sections in the L2 level of the cord. Lipid distribution was examined across both the ventral and dorsal regions, as well as in the white and grey matters of the cord. Due to the array of lipid deposits observed in treated and control animals, a quantitative assessment was not done, rather, sections were assayed qualitatively to show the type of deposits observed in different areas of the cord. Sections were evaluated using the Motic B5 Professional, Motic Images Advanced 3.0 software (Motic Instruments Inc., Richmond, ON). It should also be noted that tissue sections processed using non- fluorescent protocols were all visualized using the Motic B5 Professional Series 3.0  32 microscope (Motic Instruments Inc., Richmond, ON), while the images were analyzed using the accompanying software.  2.2.12 Magnetic Resonance Microscopy (MRM) Fixed mice lumbar spinal cord tissue was analyzed using magnetic resonance microscopy (MRM) imaging. High resolution MRM images were acquired by Dr. Samuel Grant and his team, with the aid of a 21.1 Tesla (21.1T), 900 MHz vertical magnet built at the National High Magnetic Field Laboratory (NHMFL), Tallahassee, FL, USA. The Ultra wide bore magnet has an inner diameter of 105 mm and the NMR/MRI magnet is equipped with a Bruker Advanced Console with micro 2.5 gradients. A 5 mm- diameter Alderman Grant coil was used to image mouse lumbar spinal cords. To assess volumetric changes in the spinal cord, high resolution 3D-Gradient Recall Echo (GRE) sequence were acquired with TE = 7.5 and TR = 150 ms. Band width (BW) and matrix was accommodated to match the field of view (FOV) in order to get an isotropic resolution of 5 μm. Whole and regional volumetric analysis of spinal cord images were achieved using the image segmentation editor of the Amira Advanced Visualization software (3.1) for data analysis and geometry reconstruction (Amira, Visage Imaging Inc. San Diego, CA, U.S.A). To determine lumbar spinal cord volumes, 100 adjacent slices in the L1 and L2 levels of the cord were analyzed per animal. The averages per animal were used to determine group averages.  2.2.13 Statistical Analyses As mentioned previously, 10 spinal cord and 8 brain sections were analyzed per mouse for every histological assay performed. Both right and left hemispheres were examined to determine both a hemispherical average (in order to examine differences in affect by hemisphere), and whole tissue average per mouse. Individual mouse data were then used to generate group averages. In total, 7 sets each of mouse brain and spinal cord tissue were randomly selected for immunohistochemical analysis from animals in both the “prenatal” and “both” male groups, while 5 sets each of mice brain and spinal cord tissue were also randomly selected for immunohistochemical analysis for animals in both the “control” and “postnatal” male groups. The remaining 4 sets of brains and spinal  33 cords in each of these groups were shipped out to a collaborating group (Dr. Samuel Grant, Florida State University, Tallahassee, Florida, U.S.A) for Magnetic Resonance Microscopy (MRM) analysis. All brain and spinal cord tissue excised from female mice were used for immunohistological assays as there were not enough animals per group for both immunohistochemistry and MRM analyses. Data from the three steryl glucoside - treated groups were always compared to the control groups, and statistical significance was attained when p<0.05, using a student’s t-test. Where necessary, repeated ANOVAs were also performed followed by a Tukey’s post-hoc test. Data analysis was performed using the GraphPad Prism 3.0 software (GraphPad Software Inc., San Diego, CA, U.S.A) and the Statistica for Windows 6.1 statistical software package (Statsoft, Tulsa, OK, U.S.A).                                34 Figure 2 Schematic Depiction of Experimental Design           Timed Pregnant Females (n=4) + Control Diet  Timed Pregnant Females (n=5) + 0.4mg SG and 0.6mg BSSG (to reflect fractions seen in washed cycad) on E10-11 to target SN differentiation Experiment 1         ¾ Pups were weighed within 24 hr of birth, and then weekly ¾ Starting at 5 wks, tests  for muscular strength and reflexes were performed weekly ¾ Starting at 8 wks, tests for exploratory behavior and gait abnormalities were performed bi-monthly Experiment 2     Postnatal (m = 10; f = 8) Control (m = 9; f = 7) Prenatal (m = 11; f = 7) Both (m = 11; f = 7)       ¾ Mice were fed BSSG + SG in the same doses used prenatally for 15 wks ¾ Behavior tests conducted in experiment 1 continued as before     35 3 Effects of Prenatal Exposure to BSSG and SG on Mouse Behavior 3.1 Aims: - To examine the impact of fetal exposure to cycad steryl glucosides on the behavior and general well-being of mouse pups - To determine whether the targeted period of exposure impacts any associated phenotype - To evaluate whether mice are able to recover from any associated negative change(s) in behavior and function - To determine if any associated deficits are progressive  3.2 Results  To examine the effects of prenatal exposure to cycad primary steryl glucosides on the postnatal behavior of mice, pregnant females were fed BSSG and SG – containing mouse chow pellets on days E10 and E11. These days were chosen to specifically target the differentiation of the nigro-striatal pathway, in an effort to determine if the prenatal exposure induces a dominant PD-like phenotype. Pups were weighed within 24 hr of birth, and weekly thereafter. Behavioral tests for reflexes, and muscular strength were performed once weekly starting at 5 weeks of age until the mice were 18 weeks old, while tests for gait analyses and exploratory behavior were performed bi-monthly starting at 8 weeks of age again till the mice were 18 weeks old. Significant changes observed in experimental animals in comparison to controls are discussed below.  3.2.1 Fetal Exposure to BSSG and SG Induces Considerable Increases in Body Weight  Significant weight differences (student’s t-test, p<0.0005) were observed between mice exposed to BSSG and SG during gestation (n = 36) compared to mice from control litters (n = 34).  Mice exposed to BSSG and SG during gestation gained weight at a rate about 76% higher than mice from control litters. The weight differences persisted post- weaning on day 22, with mice from the treated litters consistently maintaining higher body weights for the duration of the study. However, it should be noted that the weight difference between groups was more apparent in the male mice compared to the females. Upon separation of the mice into single cages in preparation for the second part of the  36 study, noticeable weight losses were observed across all groups, except the treated males (control females, -7.76%; control males, -7.51%; treated females, -2.51%; treated males, - 0.38%) (Fig. 3).  3.2.2 The Impact of Fetal Exposure to BSSG and SG on Spinal Reflexes and Muscle Strength  Mice were tested for spinal reflexes to determine lower motor neuron functionality using the leg-extension test and muscular strength using the wire hang test as previously described. Although some of the mice exposed to BSSG and SG prenatally initially showed some punching/tremors in their hind limbs in the leg extension reflex test, this did not attain significance between groups. In addition, starting from the 6th week of testing (~2.5months old) fewer and fewer animals exhibited these tremors, and by the 10th week of testing, the tremors were no longer apparent. Also, there were no noticeable differences between the sexes in their performances in the leg extension reflex test. Treated mice of both sexes initially showed some punching in their hind limbs that progressively disappeared over time. In the wire hang test, the performances of the mice were comparable on a weekly basis and as such there was no consistent trend. However, the females consistently outperformed the males by hanging on to the grid longer at every time point measured (Appendix B).  3.2.3 Fetal Exposure to BSSG and SG Induces Changes in Mice Gait and Exploratory Patterns In the tests for gait analysis and exploratory activity of the mice, significant differences were observed between experimental groups in several parameters measured. In the gait analysis tests, over twenty indices of gait dynamics were measured, including stance, swing, stride, braking, paw-propulsion, and number of steps taken per paw (Appendix B). However, of all these parameters measured, the stride, brake time, stance time, and numbers of steps taken, showed the most consistency at every time-point measured. Treated mice continuously showed increased braking times, increased stance durations, increased stride durations and stride lengths, while both stride frequency and total number of steps taken were decreased when compared to controls (Fig. 4). The open  37 field analysis test corroborated the results seen in the gait analysis tests as treated mice showed significantly reduced activity compared to controls. Most notably, mice exposed to BSSG and SG via maternal diet spent significantly less time moving around the arena (student’s t-test, p<0.05). When they did move, they tended to do so in a winding/circling fashion, as shown by a significant increase in meandering (student’s t-test, p<0.05), angular velocity (student’s t-test, p<0.05), as well as increased turn angles (student’s t- test, p<0.05), in comparison with control mice. These animals also displayed a preference for the periphery of the open field. Although all mice were placed in the center of the arena prior to the start of the open field analysis, treated mice often showed a significant increase in the time it took to cross into the center of the arena for the first time (student’s t-test, p<0.05), suggesting the animals often moved into the periphery soon after they were placed into the arenas, and before the start of the automated tracking. This notion is further strengthened by the observation that treated mice often displayed a shorter distance to the border of the arena, spent significantly decreased time overall in the center of the arena (student’s t-test, p<0.05), and had markedly reduced crossings into the center of the arena (student’s t-test, p<0.05) (Fig. 5). Some changes in behavior were observed across all groups following the change in housing conditions, as reflected in the graphs. The deficits observed in both the gait analysis and open field tests were more evident in the treated male mice; changes in female mice were more subtle and often were not significant (Appendix B). As such, only those parameters that were consistently significantly different between groups are presented.  3.3 Discussion Although the neurotoxic effects of cycad and some of its constituent water- insoluble steryl glucosides on adult mice have previously been demonstrated (Wilson et al., 2002; Schulz et al., 2003; Wilson et al., 2003; Wilson et al., 2004; Tabata et al., 2008, Tabata, 2008), the present experiment investigates the effects of a brief prenatal exposure to cycad steryl glucosides on postnatal behaviors of mice, and provides novel evidence that prenatal exposure to steryl glucosides can induce behavioral deficits and may also impact metabolism and/or food-intake. Furthermore, the data also suggest that targeting specific neuronal populations during development may selectively heighten the toxic  38 impact on these neurons, and lead to the development of an associated dominant phenotype. In the present study, mice were exposed to a combination of BSSG and SG via maternal diet on days specifically chosen to target the differentiation of the nigro-striatal system.  The results showed that animals with developmental BSSG and SG exposure exhibited significantly increased body weights that persisted for the duration of the study compared to their control counterparts. Although anomalies in the functions of the nigro- striatal system are typically not associated with body weight, the differentiation of this area in the developing nervous system of mice significantly overlaps with the development of the hypothalamus (Rice and Barone, 2000), an area known to regulate metabolism and food intake (Yadav et al., 2009; Kalra et al., 2009). Lesions to the ventromedial hypothalamus (VMH) have been shown to result in weight gain due to metabolic irregularities (Powley, 1977; Rabin, 1974; Cox and Powley, 1981; King, 2006), while lesions to the arcuate nucleus (ARC) often result in increased food-intake (Wisse and Schwartz, 2003; Xu et al., 2003; King, 2006). Thus, even though the intent was to target the differentiating nigro-striatal system, some hypothalamic nuclei may have also been impacted. Prenatally-treated mice also displayed several deficits in many of the behavior tests employed. Mice with in utero exposure to BSSG and SG exhibited disturbances in some of the gait indices measured; most notably, these animals showed increased brake time, as well as increased stance time as measured by ventral plane videography, indicating an overall increase in paw-to-belt contact time. Gait disturbances are symptomatic of Parkinson’s disease, and are widely reported in animal models of the disease (Nieuwboer et al., 2001; Salarian et al., 2004; Amende et al., 2005; Bartolic et al., 2005).  The open field test results showed a decrease in the spontaneous activity of treated mice. These mice spent significantly less time moving around the arena, and also showed an increased tendency to meander. Such locomotor behaviors have previously been associated with a PD-phenotype (Crenna et al., 2007; Pothakos et al., 2009). In addition, treated mice showed a preference for the periphery of the arena, measured by the consistent significantly increased time spent in the periphery of the arena rather than the center region. This behavior is often associated with anxiety-like behaviors in mice  39 (Litteljohn et al., 2008; Wultsch et al., 2009). Although PD is largely characterized as a movement disorder, about 40% of patients are reported to suffer from anxiety (Menza et al., 1993; Shiba et al., 2000). Mice exposed to BSSG and SG during gestation did not show significant differences in their performances in the leg extension reflex test as well as the wire hang test for muscle strength compared with their control counterparts. Although some of the treated mice showed some initial punching/tremors in the leg extension test, these effects seemed to disappear over time until they were no longer apparent, suggesting the possibility that some sort of neuronal recovery may have occurred in these animals. The finding that both groups of mice performed similarly on the wire hang test also implies no discernable impairments in lower motor neuron functionality, suggesting that the targeted prenatal exposure to the nigro-striatal system may have an overall increased impact on these neurons, resulting in a dominant PD-like phenotype. As mentioned previously, most of the changes observed in treated mice were more apparent in male mice than the females, supporting the hypothesis that male mice would be more impacted than female mice. Whether the prenatal exposure to BSSG and SG results in a permanent damage to the nigro-striatal system in mice, or the mice are able to progressively recover over time will be assessed in subsequent chapters. The impact of a secondary adult re-exposure to these toxins will also be discussed.  3.4 Conclusions Overall, the data from this experiment provides evidence for the first time in support of the notion that developmental exposure to BSSG and SG can impact the postnatal behaviors of mice and may even impact metabolism. This supports the observation made by earlier investigators into ALS-PDC on Guam that exposure to some environmental factor prior to adulthood was a predictor of future disease expression (Garruto et al., 1980). The present data also suggests that the period of exposure can result in a heightened impact on the neurons differentiating during this period, leading to in an increased susceptibility to later toxic insults in these neuronal populations. Whether the prenatal exposure results in increased vulnerability to a secondary dietary BSSG and  40 SG exposure in adulthood is the subject of the second part of this experiment, the results of which are discussed in later chapters.                               41 Figure 3 Body Weights of Animals Pup weights prior to weaning (A), male mice body weights (B), female mice body weights (C). Highly significant (student’s t-test, p<0.0005) differences were observed between the weights of mice exposed to a combination of BSSG and SG via maternal diet and the mice that were not. Treated mice were bigger at birth, maintained higher body weights, and gained notably more weight through the course of the study, with the treated males gaining ~54% of their starting body weights, while the treated females had gained ~53% of their starting body weights by 4 months of age, compared to ~48% weight gain of the control males, and 48% weight gain of the control females.  B o d y  W e ig h t 1.0 4.0 10.0 16.0 22.0 0 5 1 0 1 5 2 0 C o n tro l T re a te d * * * * * * * * * *** *** %  w e ig h t  g a in  =  8 1 0 . 2 2 % %  w e ig h t  g a in  =  8 8 5 . 8 4 % * * *  = >  p < 0 . 0 0 0 5 P u p  Ag e  in  D a ys B od y w ei gh t ( g)  A   42 SBody Weight of Male Mice 5 12 19 0 20 40 60 Control Treated* *** *** *** ** ** * * * * ** * *** * => p<0.05 ** => p<0.005 *** => p<0.0005 "S" denotes period when animals were separated into single cages % weight gain = 53.7% % weight gain = 48.3% Age in Weeks B od y W ei gh t ( g)  B   S Body Weight of Female Mice 5 12 19 0 15 30 45 Control Treated * * ** * => p<0.05 ** => p<0.005 "S" denotes period when animals were separated into single cages Age in Weeks B od y W ei gh t ( g) % weight gain = 53.4% % weight gain = 47.8%  C      43 Figure 4 Stepping Patterns of Animals  In the test for gait analysis, the most consistent and significant differences were found in the brake time (D, E, F, G) and the stance duration (H, I, J, K) parameters. The data indicates that mice exposed to BSSG and SG during development showed increased braking times, and increased stance time, which implies an overall increase in paw-to-belt contact time in these mice. This further shows that treated mice took longer periods to complete their steps, suggesting some movement deficits in these animals. This notion is supported by the data on the total number of steps taken per animal paw, which shows that control mice took increased number of steps per unit time, although not significantly so (Appendix B). The gap at week 12 indicates the period when the gait analysis equipment was being maintained, as such, the animals could not be tested that week.   44 SLeft Fore Paw Brake Time - Male Mice 8 12 16 20 0.08 0.09 0.10 0.11 0.12 Control Treated* * * => p<0.05 "S" denotes period w hen animals w ere separated into single cages Age in Weeks Ti m e (s ) D S Right Fore Paw Brake Time - Male Mice 8 12 16 20 0.075 0.100 0.125 0.150 Control Treated* * * => p<0.05 "S" denotes period w hen animals w ere separated into single cages Age in Weeks Ti m e (s ) E             45 SLeft Rear Paw Brake Time - Male Mice 8 12 16 20 0.04 0.05 0.06 0.07 0.08 Control M Treated M "S" denotes period w hen animals w ere separated into single cages F Age  in We eks Ti m e (s ) S Right Rear Paw Brake Time - Male Mice 8 12 16 20 0.04 0.05 0.06 0.07 0.08 Control Treated "S" denotes period w hen animals w ere separated into single cages G Age in Weeks Ti m e (s )      46 SLeft Fore Paw Stance Duration - Male Mice 8 12 16 20 0.18 0.19 0.20 0.21 0.22 0.23 Control Treated * * H "S" denotes period w hen animals w ere separated into single cages * => p<0.05 Age  in We e ks Ti m e (s ) S Right Fore Paw Stance Duration - Male Mice 8 12 16 20 0.18 0.19 0.20 0.21 0.22 Control Treated "S" denotes period w hen animals w ere separated into single cages I Age  in We e ks Ti m e (s )   47 SLeft Rear Paw Stance Duration - Male Mice 8 12 16 20 0.175 0.200 0.225 0.250 Control Treated * ** * "S" denotes period w hen animals w ere separated into s ingle cages J * = >  p<0.05 ** = >  p< 0.005 Age  in  We e ks Ti m e (s ) S Right Rear Paw Stance Duration - Male Mice 8 12 16 20 0.175 0.200 0.225 0.250 Control Treated * * ** ** "S" denotes period w hen animals w ere separated into s ingle cages K * =>  p<0.05 ** =>  p<0.005 Age  in  We e ks Ti m e (s )               48 Figure 5 Open Field Activity of Animals  Spontaneous exploratory behavior was assessed in the mice using the open field test. Mice treated prenatally with BSSG and SG showed significant (student’s t-test, p<0.05) behavioral impairments in the open field compared with the control mice. Treated mice moved slower (L), spent less time moving around the arena (M, N), showed a tendency to circle/rotate (O, P, Q, R), and showed a preference for the periphery of the arena, even though all mice were started in the center (S, T, U, V, W, X, Y, Z). Open field activity was measured over a period of 5 min.                        49 SVelocity - Male Mice 8 12 16 20 0.0 2.5 5.0 7.5 Control Treated * * * L * => p<0.05 "S" denotes period when animals were separated into single cages Age in Weeks Ve lo ci ty  (c m /s )     S Total Movement - Male Mice 8 12 16 20 0 100 200 300 Control Treated ** ** * * * * => p<0.05 ** => p<0.005 "S" denotes period when animals were separated into single cages Age in Weeks D ur at io n (s ) M    50 STotal Movement - Female Mice 8 12 16 20 150 200 250 300 Control Treated * N * => p<0.05 "S" denotes period when animals were separated into single cages Age in Weeks D ur at io n (s )    S Mean Turn Angle - Male Mice 8 12 16 20 50 60 70 80 90 Control Treated *** *** * * ** O * => p<0.05 ** => p<0.005 *** => p<0.0005 "S" denotes period when animals were separated into single cages Age in Weeks Tu rn  A ng le  ( de g. )   51 SMean Turn Angle - Female Mice 8 12 16 20 50 60 70 80 Control Treated * * ** * Age in Weeks Tu rn  A ng le  ( de g. ) * => p<0.05 ** => p<0.005 "S" denotes period when animals were separated into single cages P    S Meander - Male Mice 8 12 16 20 -150 -100 -50 0 Control Treated* ** * ** Q * => p<0.05 ** => p<0.005 "S" denotes period when animals were separated into single cages Age in Weeks M ea nd er  ( de g/ cm )    52 SAngular Velocity - Male Mice 8 12 16 20 -150 -125 -100 -75 -50 -25 Control Treated ** * ** * Age in Weeks Ve lo ci ty  (d eg ./s ) * => p<0.05 ** => p<0.005 "S" denotes period when animals were separated into single cages R     S In center Frequency - Male Mice 8 12 16 20 0 10 20 30 Control Treated*** * ** * * S * => p<0.05 ** => p<0.005 *** => p<0.0005 "S" denotes period when animals were separated into single cages Age in Weeks Fr eq ue nc y    53 SIn center Frequency - Female Mice 8 12 16 20 0 10 20 30 Control Treated* ** * => p<0.05 ** => p<0.005 "S" denotes period when animals were separated into single cages T Age in Weeks Fr eq ue nc y     S In Center Duration - Male Mice 8 12 16 20 0 10 20 30 40 Control Treated ** ** U ** => p<0.005 "S" denotes period when animals were separated into single cages Age in Weeks D ur at io n (s )      54 SIn Center Duration - Female Mice 8 12 16 20 0 10 20 30 40 50 Control Treated * * V * => p<0.05 "S" denotes period when animals were separated into single cages Age in Weeks D ur at io n (s )     S Latency of 1st Center - Male Mice 8 12 16 20 0 50 100 150 Control Treated * * W * => p<0.05 "S" denotes period when animals were separated into single cages Age in Weeks Ti m e (s )   55 SLatency of 1st Center - Female Mice 8 12 16 20 0 40 80 120 Control Treated * X * => p<0.05 "S" denotes period when animals were separated into single cages Age in Weeks Ti m e (s )     S Mean Distance to Zone Border - Male Mice 8 12 16 20 0 2 4 6 8 Control Treated *** *** *** *** Y Age in Weeks D is ta nc e (c m ) *** => p<0.0005 "S" denotes period when animals were separated into single cages    56 SMean Distance to Zone Border - Female Mice 8 12 16 20 0 2 4 6 8 Control Treated * * Z * => p<0.05 "S" denotes period when animals were separated into single cages Age in Weeks D is ta nc e (c m )                            57 4 Effects of Postnatal Secondary Re-exposure to Dietary BSSG and SG on Adult Male Mice 4.1 Aims: - To determine if the behavioral deficits already observed in prenatally-treated male mice is exacerbated by a secondary re-exposure to dietary BSSG and SG - To determine whether or not the deficits observed in male mice exposed to BSSG and SG via maternal diet only are progressive - To evaluate the effects of BSSG and SG on adult male mice not previously exposed to the steryl glucosides in utero  4.2 Results In order to investigate the effects of re-exposure to BSSG and SG on adult male mice, male mice from treated and control litters were further divided into sub-groups at 4 months of age. Animals were randomly assigned into one of four groups; prenatal exposure only (prenatal, n=11), both pre- and post-natal exposures (both, n=11), postnatal exposure only (postnatal, n=10), and the control (n=9) group. Mice in the “both” and “postnatal” groups received BSSG and SG – containing mouse chow in the same doses used for the developmental exposure for a period of 15 weeks. Behavioral monitoring continued as previously described. Immediately following the 15-week steryl glucoside feeding, all mice were sacrificed and tissue samples were collected for histological assays.  4.2.1 Male Mice Exposed to BSSG and SG During Development Maintain Larger Body Weights Both groups of mice that received BSSG and SG via maternal diet were found to maintain higher body weights than the mice that did not receive the prenatal treatment. However, the group of mice that received the secondary exposure after first treated prenatally showed significantly higher body weights (student’s t-test, p<0.05) at every time point measured compared to the control mice. A repeated measures ANOVA, as well as a Tukey’s multiple comparison test measuring the variations between groups also showed significance (repeated measures ANOVA, p<0.0001; pairing, p<0.0001; Tukey’s, control vs. postnatal, p<0.001; control vs. both, p<0.001; control vs. prenatal, p<0.001;  58 postnatal vs. both, p<0.001; postnatal vs. prenatal, p<0.001; both vs. prenatal, p<0.001) (Fig. 6).  4.2.2 “Prenatal Exposure Only” Male Mice Show Similar Behavioral Trends as Group Exposed Both Pre- and Post-natally Behavioral differences among the groups were similar to what was observed between mice from treated and control litters in the first part of the study. In both the leg extension reflex test as well as the wire hang test for muscular strength, there were no significant differences observed among groups (Appendix C). However, the exploratory patterns of the mice in the open field test again showed significant differences between the groups. As before, the groups of mice that had previously received BSSG and SG via maternal diet continued to show reduced activity in the open field, regardless of whether or not they received a secondary treatment. Mice in the “prenatal” and “both” groups showed a preference for the periphery of the open field rather than the center, as they not only spent less time in the center of the arena, but also often stayed close to the zone border (Appendix C). These groups of mice also moved slower around the arena, spent significantly less time moving (student’s t-test, p<0.05; repeated measures ANOVA, p=0.0063; pairing, p=0.0104; Tukey’s, control vs. both, p<0.05; postnatal vs. both, p<0.01) (Fig. 7), and covered less distances when compared to both the “postnatal” and “control” groups (Appendix C). Although mice in the “prenatal” and “both” groups were much bigger than those in the other two groups, no significant correlation was found between animal weights and open field activity, suggesting the reduced activity seen in these mice was not necessarily due to their size (Fig. 7). In addition to these observations, mice in the “prenatal” and “both” groups continued to show an increased ‘circling’ tendency in their pattern of movement as evidenced by their larger angular velocity, increased meandering (Appendix C), and significantly larger turn angles (student’s t-test, p<0.05; repeated measures ANOVA, p=0.0007; pairing, p=0.0178; Tukey’s, control vs. both, p<0.01; postnatal vs. both, p<0.001; postnatal vs. prenatal, p<0.05) (Fig. 7). No significant deficits were observed in mice treated only as adults compared to the control group. Gait patterns were not tested as very few mice ran on the treadmill.   59 4.2.3 Decrease in Tyrosine Hydroxylase Immunoreactivity in Response to Dietary BSSG and SG Immunoperoxidase staining for tyrosine hydroxylase (TH) – positive cells in both the striatum and substantia nigra pars compacta (SNpc) showed decreased labeling in all groups treated with BSSG and SG, with the group treated both prenatally and as adults showing the greatest decline in comparison with controls (Fig. 8).  4.2.4 Significant Microglial Activation in Response to BSSG and SG Exposure The glial response in the animals was measured by immunohistochemical staining for GFAP and Iba-1 – positive cells. GFAP - positive cells with star-shaped cell bodies were quantified in the lateral ventral horn of the lumbar spinal cord; the results revealed no significant differences among the groups, although the “prenatal” group expressed very few reactive astrocytes (-39%; student’s t-test p = 0.0630) compared with controls (Appendix C). Iba-1 – positive cells were also quantified in the same lumbar spinal cord region as the GFAP stain, as well as in the striatum. Results showed significant increases in microglia expression in the lumbar spinal cord (student’s t-test, p<0.05, one-way ANOVA, p=0.0028; Tukey’s, prenatal vs. control, p<0.05; both vs. control, p<0.01), as well as in the striatum (student’s t-test, p<0.05) of BSSG and SG treated mice (Fig. 9).  4.2.5 Dietary BSSG and SG Induce Minimal Loss of Lower Motor Neurons Lumbar spinal cord tissue sections in all four groups of male mice were assessed for lower motor neuron numbers. Nissl body staining revealed loss of motor neurons in the lateral ventral horn of mice that received BSSG and SG during both fetal development and as adults, and mice that received the steryl glucosides as adults only, compared with control mice tissue. However, the counts showed a greater decrease in motor neuron numbers in the “postnatal” only group (-25%; student’s t-test p<0.05), than in the group that received both the pre- and post-natal exposures (-14.1%) (Fig. 10). Counts in the “prenatal” group were comparable to the control group. In addition to neuronal loss, some of the surviving neurons in the treated mice showed some irregularities in their morphology, with many neurons showing marked chromatolysis, and formation of vacuoles in the cell bodies (Fig. 10). Shrunken, pyknotic neurons were  60 also observed in some of the tissue sections. Overall, results from the Nissl body staining show that dietary BSSG and SG induces the loss of lower motor neurons, and results in abnormal morphological changes in some surviving motor neurons. Motor neurons were also assayed similarly as the Nissls using anti-ChAT immunofluorescent antibody staining. ChAT-positive cell counts showed a similar trend to the Nissl cell counts, although no group showed a significant difference when compared with controls (Fig. 11). Based on the weight differences, as well as spinal cord volume differences (see MRM analysis below), observed among the groups, motor neuron counts were also normalized for spinal cord volume (Appendix C).  4.2.6 Dietary BSSG and SG Induce Apoptosis in the Lumbar Spinal Cord Apoptotic cells in the lumbar spinal cord were assayed using anti-active caspase-3 immunofluorescent antibody staining. All cells positively labeled with the active capse-3 in the lateral ventral horn of the cord were counted. Results showed an increased expression of caspase-3 in all 3 groups treated with BSSG and SG compared to controls, with mice in the “both” group showing the most increase in active caspase-3 expression (+60%; student’s t-test p<0.05) (Fig. 12).  4.2.7 Dietary BSSG and SG Results in No Significant Induction of ATF-3 and HSP-70 In order to assess some other mechanisms that could be involved in neuronal degeneration, the lateral ventral horn region in lumbar spinal cord tissue sections were examined for the upregulation of the gene transcription factor, ATF-3, as well as the stress-inducible protein, HSP-70. Immunoperoxidase staining for these proteins revealed only subtle differences between groups (Appendix C).  4.2.8 Increased Lipid Accumulation in BSSG and SG - Treated Mice Lumbar spinal cord sections were stained with the oil red o (ORO) dye to assess lipid deposition and clearance. A qualitative assessment of lipid deposits across the white and gray matter showed more robust staining in mice exposed to BSSG and SG in comparison with controls. The deposits observed in BSSG and SG – fed mice were larger in area, and appeared to have more lipids surrounding the macrophages. Oil red O  61 staining was found to be most intense in the groups exposed to BSSG and SG during development, with a secondary exposure further exacerbating lipid accumulation (Fig. 13).  4.2.9 Magnetic Resonance Microscopy (MRM) Analysis To further assess pathology, 4 lumbar spinal cord tissues were randomly selected per group and scanned using high resolution MRM. A 3-D volumetric analysis of the spinal cord images showed significant volume reductions in both the ventral and dorsal regions of mice treated with BSSG and SG both in utero and as adults in comparison with controls. These mice also showed volume reductions in the overall white and gray matter in the lumbar spinal cord (-11% and -17% respectively) compared to control mice. There were no significant volume differences observed in the lumbar spinal cord of all other groups of mice treated with BSSG and SG compared to controls. Diffusion tensor imaging (DTI) results also showed reduced fiber densities, as well as increased apparent diffusion coefficient (ADC) in BSSG and SG – fed mice (Fig. 14).  4.3 Discussion As mentioned previously, ALS-PDC encompasses symptoms of ALS, PD, and dementia, with patients often expressing a predominant phenotype. Previous in vivo studies involving dietary exposure of mice to washed cycad flour or one of its constituent water-insoluble steryl glucosides, BSSG or SG, have recapitulated many of the phenotypic and neuropathological aspects of the disease (Wilson et al., 2002; Schulz et al., 2003; Wilson et al., 2003; Wilson et al., 2004; Tabata et al., 2008, Tabata, 2008). Still, these experiments did not address the variations observed in the disease onset and rate of progression. In addition, even though developmental origins of the disease have long been speculated about, crucial experiments to this effect remain undone. With the aim of addressing some of the variations observed in disease onset as well as rate of disease progression, the present study tested the hypothesis that prenatal exposure to BSSG and SG that selectively targets the differentiation of a specific neuronal subset will result not only in a more rapid onset and progression of the disease if the mice were again re- challenged with the same toxicants as adults, but also lead to the development of a  62 dominant phenotype, associated with the neurons differentiating at the time of the prenatal insult. Both the behavioral and histological findings of the present study corroborate these assertions, and provide new evidence in support of the hypothesis. As with the first part of the study, mice with prenatal BSSG and SG exposure continued to weigh more than mice that were not exposed to these steryl glucosides in utero; with the mice that received both prenatal and adult exposures consistently weighing significantly more than the control mice at every time-point measured. Mice exposed to dietary BSSG and SG only as adults maintained body weights similar to control mice. This mimics observations in previous studies of dietary exposures of adult mice to washed cycad flour, BSSG, or SG (Wilson et al., 2002; Wilson, 2005; Tabata et al., 2008, Tabata, 2008), and lends more support to the prenatal exposure being the confounding variable. In addition, mice treated prenatally continued to show anomalies in locomotor functions, evidenced by the progressive decline in exploratory activity in the open field as well as the increasing tendency to make turns at wider angles, suggesting these animals take more steps to complete a turn (Crenna et al., 2007). Both of these variables had reached significance by the last time-point measured in mice treated both developmentally and re-exposed as adults compared to control mice. The gradual decline in locomotor functions correlates well with what is often observed in studies of neurodegenerative disorders (Luo and Roth, 2000) and also substantiates the observations in previous cycad and BSSG or SG studies (Wilson et al., 2002; Wilson, 2005; Tabata et al., 2008, Tabata, 2008). Also, mice exposed to BSSG and SG via maternal diet only continued to show similar behavioral trends as the mice that received both the pre- and post-natal exposures, indicating that the prenatal exposure may have resulted in permanent injury in these animals. No significant differences were observed in both the leg extension and wire hang tests; although some BSSG and SG – fed mice were starting to show some tremors/punching in their hind limbs by the 13th week of feeding. Immunohistochemical analysis of the nigro-striatal system of the mice showed reduced tyrosine hydroxylase immunoreactivity in both the striatum and the SNpc of mice exposed to dietary BSSG and SG. In both groups of mice treated prenatally however, the loss was more profound, with the group of mice treated both developmentally and as adults showing significantly reduced TH labeling. Tyrosine  63 hydroxylase catalyzes the conversion of the amino acid tyrosine to dihydroxyphenylalanine (or DOPA), which is a precursor for the neurotransmitter, dopamine. Since the loss of dopaminergic neurons in the nigro-striatal pathway is the hallmark of PD, TH-immunoreactivity is often used to assay dopaminergic neurons, and reduced TH-immunoreactivity in the nigro-striatal system of mice is often found to correspond with a PD-like phenotype, including different measures of motor and non- motor dysfunctions associated with the disease (Le Pen et al., 2008; Meredith et al., 2008; Shi et al., 2009; Yuan et al., 2009). In addition to reduced TH-immunoreactivity, significant microglial activation was also observed in both the striatum and the lumbar spinal cord of BSSG and SG – fed mice. Microglia are immune response cells in the CNS, and their activation is widely associated with the neuroinflammatory response in the CNS, which in turn is associated with many neurodegenerative disorders, including ALS, AD, and PD (McGeer and McGeer, 2004; Block and Hong, 2005; Tansey et al., 2007, O’Callaghan et al., 2008). The role of neuroinflammation as a cause or consequence of neurodegeneration remains unclear, and highly debated (Sargysyan et al., 2005; Dheen et al., 2007; O’Callaghan et al., 2008). Nonetheless, microglial activation in neurodegenerative disease states is widely reported. This finding has also been reported in previous BSSG or SG studies by our group (Tabata et al., 2008, Tabata, 2008). However, a comparison of microglia activation across the all groups of mice in this study reveal that mice exposed to BSSG and SG both prenatally and as adults again show the greatest impact, in support of the behavioral data, and strongly suggesting a faster rate of disease onset and progression in these mice. After demonstrating toxicity to the nigro-striatal system, the primary area of target, the lumbar spinal cord was also examined for pathology as this has previously been shown in prior BSSG or SG studies (Tabata et al., 2008, Tabata, 2008). Examination of lower motor neurons in the lumbar spinal cord revealed some motor neuron loss in the groups of mice fed for 15 weeks (“both” and “postnatal”) during the second part of the experiment. It is not clear why the “postnatal” group lost more neurons than the “both” group compared to controls, it’s also not clear why the “prenatal” group showed increases in motor neuron counts compared to controls, but this suggests that there may be some toxin-induced neurogenesis occurring in these mice (Danilov et al., 2006), perhaps due to  64 the prenatal BSSG and SG exposure. Work is in progress to assess markers of neurogenesis in the mice tissue. A marker for activated caspase-3 was used to assay apoptosis in the lower motor neurons. The results showed increased cell death in all groups of BSSG and SG – fed mice, with the “both” group showing significantly increased apoptotic activity compared to controls. To further assess neurodegeneration in the spinal cord, oil red o, which is specific for degenerating myelin and macrophage clearance (Vallieres et al, 2006), was used to assess myelin degeneration, as well as lipid deposition and clearance as a result of BSSG and SG feeding. The data showed abundant oil red o staining in BSSG and SG – fed mice, with the groups exposed during development showing more intense staining. The lipid deposits observed in these mice were larger in area, and appeared to have more lipids surrounding the macrophages, suggesting considerably delayed/reduced clearance of lipid deposits in these groups of mice (Vallieres et al, 2006; Hoehn et al, 2008). To further support lower spinal cord pathology, mice treated during development and as adults showed significant volume losses in both the ventral (student’s t-test, p<0.05) and dorsal (student’s t-test, p<0.05) horns of the lumbar spinal cord as measured by MRM volumetric analysis. Diffusion tensor imaging (DTI) data in the lumbar spinal cord also showed BSSG and SG – induced reductions in fiber densities (one-way ANOVA, p=0.0413; Tukey’s, both vs. control, p<0.05), as well as increased apparent diffusion coefficient (ADC). This indicates less restricted diffusion in the tissues of these mice, and therefore suggests axonal disruption, possibly from reduced myelination (Pavuluri et al., 2009).  4.4 Conclusions Overall, both the behavioral and histological data presented here demonstrates the neurotoxicity of BSSG and SG, further supporting prior work (Tabata et al., 2008, Tabata, 2008). The cumulative data also reveals that the secondary adult re-exposure in mice previously exposed to BSSG and SG during development further worsens the initial impact. However, the finding that the prenatal exposure alone may result in permanent damage in mice is indeed remarkable. This finding supports epidemiological data on some Guamanian Chamorro migrants, who had developed ALS-PDC in places outside of  65 Guam, such as the United States, Germany, and other countries, several years after migrating, having spent parts of their childhood on Guam (Garruto et al., 1980). A similar study that examined developmental origins of PD, using the common pesticides, paraquat and maneb arrived at a similar conclusion (Thiruchelvam et al., 2002; Barlow et al., 2007). Whether or not such findings are limited to prenatal toxic insults to the nigro- striatal system are worth exploring. However, a recent hypothesis “ALS starts in the perinatal period” recently put forth by a prominent ALS researcher (Eisen, 2009) may be an indication that more studies exploring the developmental origins of adult onset neurodegenerative disorders need to be done.                         66 Figure 6 Body Weights of Animals  The data on animal body weights (A) showed that the two groups exposed to BSSG and SG via maternal diet continued to maintain higher body weights (student’s t-test, p<0.05; repeated measures ANOVA, p<0.0001; pairing, p<0.0001; Tukey’s, control vs. postnatal, p<0.001; control vs. both, p<0.001; control vs. prenatal, p<0.001; postnatal vs. both, p<0.001; postnatal vs. prenatal, p<0.001; both vs. prenatal, p<0.001) for the entire duration of the study. However, the mice that received the secondary treatment in adulthood had the largest body weights, consistently maintaining significantly higher weights at every time point measured when compared with controls. (The “dip” at week 26 signifies when the scale in the animal behavior room was temporarily changed by the staff of the animal holding facility).    Weight 18 20 22 24 26 28 30 32 34 35 50 65 Control Postnatal Both Prenatal *=p<0.05 **=p<0.005 Error bars represent SEM **** ** **** **** ** * **** **** **** ** Age in Weeks B od y W ei gh t ( g) A                   67 Figure 7 Open Field Activity  The open field activity of the mice showed some initial inconsistencies in the behavior of the mice following the solitary housing of the animals at the start of the second part of the study. However, by the 5th week of experiment 2, the behavioral trends became more obvious, with both groups of mice exposed to BSSG and SG in the gestational environment continuing to spend less time moving around the arena (B), and turning at wider angles (D). By the last time point measured prior to sacrificing the animals, the differences in both of these parameters had reached significance (student’s t-test p<0.05). Repeated measures ANOVA on both movement duration (repeated measures ANOVA, p=0.0063; pairing, p=0.0104; Tukey’s, control vs. both, p<0.05; postnatal vs. both, p<0.01),  and mean turn angle also showed significance (repeated measures ANOVA, p=0.0007; pairing, p=0.0178; Tukey’s, control vs. both, p<0.01; postnatal vs. both, p<0.001; postnatal vs. prenatal, p<0.05). The slower movement in the open field observed in mice treated prenatally seemed to be independent of their higher body weights (C), as statistical tests for correlation (Spearman) failed to show significance between body weights and movement duration (control p = 0.948, Spearman r = 0.033; prenatal p = 0.082, Spearman r = -0.554; both p = 0.071, Spearman r = -0.513; postnatal p = 0.665, Spearman r = 0.192).               68 Total Movement Duration 18 20 22 24 26 28 30 32 34 100 150 200 250 Control Postnatal Both Prenatal Error bars represent SEM* *=p<0.05 B Age in Weeks D ur at io n (s )    Weight vs Movement Duration 0 25 50 75 100 0 100 200 300 Both Prenatal Control Postnatal Weights M ov em en t D ur at io n (s ) C      69 Mean Turn Angle 18 20 22 24 26 28 30 32 34 40 60 80 100 Control Postnatal Both Prenatal Error bars represent SEM * *=p<0.05 D Age  in Wee ks Tu rn  A ng le  ( de g. )                                70 Figure 8 Decreased Tyrosine Hydroxylase Immunoreactivity in Nigro-striatal System of BSSG and SG – Treated Mice  TH immunoreactivity was assessed in both the striatum and substantia nigra pars compacta (SNpc) of the animals. The optical density of the images showed decreases in TH labeling in both the striatum (E) and SNpc (F) of groups treated with BSSG and SG. Mice treated with BSSG and SG via maternal diet and as adults showed the greatest loss of TH+ cells in the nigro-striatal system. The attached micrographs show sample images of striatum and substantia nigra across all groups. Images were taken with a light microscope using the 4x objective lens for the striatum, and the 10x objective lens for the SNpc. Scale bar = 50µm.                       71 TH+ Labeling - Striatum Prenatal Both Control Postnatal 0 10 20 30 40 Prenatal Both Control Postnatal -11.6% -19.1% -3.4% E Groups St ai n In te ns ity     Prenatal Both   Control Postnatal   72 TH+ Labeling - SNpc Prenatal Both Control Postnatal 0 10 20 30 40 Prenatal Both Control Postnatal -12.5% -18.9%  * -10.4% *=p<0.05 F Groups St ai n In te ns ity    Both Prenatal                         SNpc     SNr                   SNpc      SNr                                SNpc      SNr Control Postnatal                       SNpc     SNr       73 Figure 9 BSSG and SG Induce Microglia Proliferation in Lumbar Spinal Cord and Striatum  Microglia expression was assayed in both the lumbar spinal cord and the striatum using an antibody against the ionized calcium binding adaptor molecule 1 (Iba-1). Quantification of positively labeled cells in both the lateral ventral horn of the spinal cord (G; Iba-1 – positive cells, red; blue label is DAPI to counter-stain the nuclei) and the striatum (H; Iba-1 – positive cells labeled brown using DAB) showed significant increases (lumbar spinal cord, student’s t-test, p<0.05, one-way ANOVA, p=0.0028; Tukey’s, prenatal vs. control, p<0.05; both vs. control, p<0.01; striatum, student’s t-test, p<0.05) in microglia proliferation in BSSG and SG treated mice. Due to the abundance of microglia in the striatum, counts were restricted to the specified region (I; boxed area), using the external capsule (EC) as the point of reference for consistency. Quantification was done under a light microscope under the 20x (lumbar spinal cord) or 40x (striatum) objective lens. The accompanying micrographs show representative data across the groups; an enlarged micrograph from a BSSG and SG – treated mouse striatum is shown (J) to depict microglia morphology. Scale bars = 50µm; 10µm in (J).                74 Iba-1 Positive Glia Cells - Spinal Cord Pre-natal Both Control Post-natal 0 10 20 30 40 Pre-natal Both Control Post-natal +78%  ** + 95%  ** + 28% ** p<0.005 G Groups # of  M ic ro gl ia     Prenatal Both Control Postnatal  75 Iba-1 Positive Glial Cells - Striatum Prenatal Both Control Postnatal 0 9 18 27 Prenatal Both Control Postnatal +5.0% + 35.8%  * + 13.8% H Groups # of  M ic ro gl ia * = >  p< 0.05    Prenatal Both  Control  Postnatal    76   J I EC                                     77 Figure 10 Motor Neuron Quantification in the Lumbar Spinal Cord Cresyl violet staining for Nissl bodies showed a decrease in the number of motor neurons in the lateral ventral horn of the lumbar spinal cord of mice fed BSSG and SG – containing mouse chow pellets for a period of 15 weeks, in comparison with controls (K). Mice in the prenatal group showed a slight increase in the number of healthy motor neurons compared with controls (K). Although the presence of some neurons with abnormal morphology (chromatolytic, vacuolytic, and/or pyknotic) was observed in many of the mice tissue, the frequency of occurrence of such neurons in mice that received BSSG and SG for a prolonged period was remarkable, as shown by the arrows. A representative micrograph of the level of spinal cord, as well as the area quantified is depicted above (L). Counts were done under a light microscope in the field of view under the 40x objective lens. Scale bars = 10µm, 200µm (L).                            78 Lower Motor Neuron Counts Prenatal Both Control Postnatal 0 5 10 15 20 Prenatal Both Control Postnatal *p=0.0294 + 2.7% -14.1% -25%  * K Groups # of  M ot or  N eu ro ns     L   79  Prenatal Both  Control Postnatal                 80 Figure 11 Anti-ChAT Motor Neuron Quantification in the Lumbar Spinal Cord Cholinergic neurons in the lumbar spinal cord were detected using an antibody against choline acetyl transferase (ChAT) in all four groups of mice. ChAT-positive cells (green; blue label is DAPI to counter-stain the nuclei) were quantified in the lateral ventral horn of the spinal cord. Results showed a similar trend as the cresyl violet stain, with both groups that received BSSG and SG treatment for 15 weeks showing moderate reductions in motor neuron number, while the group that received BSSG and SG during gestation only, showed an increased number of positive cells (M). The counts for cholinergic neurons showed no significance between groups. Quantification was performed under a fluorescence microscope in the field of view under the 20x objective lens. Scale bars = 20µm.              81 Cholinergic Neurons in the Lumbar Spinal Cord Prenatal Both Control Postnatal 0 5 10 15 20 Prenatal Both Control Postnatal +17.5% -6.6% -8.4% Groups # of  M ot or  N eu ro ns M    Prenatal Both  Control Postnatal   82 Figure 12 Increased Expression of Active-Caspase-3 in the Lumbar Spinal Cord of Treated Mice  Apoptotic cells in the lumbar spinal cord were detected using an antibody against the active form of caspase-3. Caspase-3 positive cells (red; blue label is DAPI to counter- stain the nuclei) were quantified in the lateral ventral horn of the spinal cord. Results showed an increase in active caspase-3 expression in all 3 groups of mice exposed to BSSG and SG, with the group with both pre- and post-natal exposures to the toxins showing significantly higher expression of the protein in comparison with controls (N). The accompanying micrographs show representative data from all groups. Quantification was performed under a fluorescence microscope in the field of view under the 20x objective lens. Scale bars = 20µm.                                 83 Active Caspase-3 Positive Cells Prenatal Both Control Postnatal 0 4 8 12 16 Prenatal Both Control Postnatal +41% +60%  * *p<0.05 +32% N Groups # of  c el ls  la be le d w / ac tiv at ed  c as pa se -3    Prenatal Both  Postnatal Control   84 Figure 13 Oil Red O (ORO) Staining in Lumbar Spinal Cord Sections Reveals Increased Lipid Deposition/Reduced Clearance in BSSG and SG – Fed Animals  Lipid accumulation was assessed in the lumbar spinal cord of mice using the oil red o (ORO) dye (red, lipid deposits; blue, light hematoxylin nuclei counterstaining). A qualitative assessment of the sections showed more robust staining in BSSG and SG – fed animals, suggesting increased lipid deposition and/or delayed clearance in these mice. Sample cross-sections showing the distributions across the white and gray matter in all 4 groups are shown (O); images from 2 animals per group at a larger magnification are also shown to further show lipid deposition across the groups (P); larger micrographs from the same 2 animals per group are shown to depict morphological and staining differences (Q). Images were taken with a light microscope. Scale bars = 50µm (O), 20µm (P,Q).                                    85  O1 - Prenatal O2 - Both   O3 - Control O4 - Postnatal   P2 - Prenatal P1 - Prenatal    86  P3 – Both  P4 - Both   P5 – Control  P6 – Control   P8 - Postnatal P7 - Postnatal   87  Q1 - Prenatal Q2 – Prenatal   Q4 – Both  Q3 - Both   Q6 – Control  Q5 – Control  88  Q7 – Postnatal  Q8 – Postnatal                                  89 Figure 14 Magnetic Resonance Microscopy (MRM) Analysis Suggests Abnormalities in the Spinal Cord of BSSG and SG - Treated Mice  MRM analysis of the lumbar spinal cord of mice revealed volume reductions in both the white and grey matters, as well as the ventral and dorsal horn regions of mice treated with BSSG both developmentally and as adults (R, S, T, U). Diffusion tensor imaging (DTI) also showed reductions in fiber densities of all groups treated with BSSG and SG, reaching significance in the group treated both pre- and post-natally (student’s t-test, p = 0.02; one-way ANOVA, p=0.0413; Tukey’s, both vs. control, p<0.05) (V). In addition, the apparent diffusion coefficient (ADC) also showed increases in groups treated prenatally, trending towards significance in the group treated both pre- and post-nally (p = 0.08),  while the group treated only as adults maintained values similar to controls (W). The attached pictures depict a sample spinal cord (X), as well as a coronal section of the lumbar spinal cord, with the areas analyzed labeled (Y; WM = white matter, GM = gray matter, VH = ventral horn, DH = dorsal horn).                  90 White Matter Prenatal Both Control Postnatal 0.0 0.4 0.8 1.2 Prenatal Both Control Postnatal +5% -11% +10% Groups Vo lu m e ex pr es se d as  %  o f an im al  w ei gh t R     Gray Matter Prenatal Both Control Postnatal 0 .0 0.4 0.8 1.2 Prenatal Both Control Postnatal Groups Vo lu m e ex pr es se d as  %  o f an im al  w ei gh t -4% -17% +2% S             91 Ventral Horn Prenatal Both Control Postnatal 0 .0 0.4 0.8 1.2 Prenatal Both Control Postnatal -11% -28% * -6% *=p<0.05 Groups Vo lu m e ex pr es se d as  %  o f an im al  w ei gh t T     Dorsal Horn Prenatal Both Control Postnatal 0 .0 0.4 0.8 1.2 Prenatal Both Control Postnatal +1% -23% * -4% Groups Vo lu m e ex pr es se d as  %  o f an im al  w ei gh t *=p<0.05 U     92 Fiber Density Prenatal Both Control Postnatal 0 50 100 150 200 250 Prenatal Both C ontrol Pos tnatal G roups Fi be r D en si ty  # /m m 3 -18.7% -29.8%  * -3.6% *=p<0.05 V      Apparent D iffusion C oefficient Prenatal Both Control Postnatal 0 .0 0 .8 1 .6 2 .4 Prenatal Both C ontrol Pos tnatal G roups AD C s/ m m 2 + 19.7% + 18.6% -1.9% W    93   DH            WM    GM  VH X  Y                                 94 5 Effects of Postnatal Secondary Re-exposure to Dietary BSSG and SG on Adult Female Mice 5.1 Aims: - To determine if the behavioral deficits already observed in prenatally-treated female mice is exacerbated by a secondary re-exposure to dietary BSSG and SG - To determine if any deficits observed in female mice exposed to BSSG and SG via maternal diet only are progressive - To evaluate the effects of BSSG and SG on adult female mice not previously exposed to the steryl glucosides in utero - To assess any sex differences between the male and female BSSG and SG – fed mice  5.2 Results In order to investigate the effects of re-exposure to BSSG and SG on adult female mice, female mice from treated and control litters were further divided into sub-groups at 4 months of age. Animals were randomly assigned into one of four groups; prenatal exposure only (prenatal, n=7), both pre- and post-natal exposures (both, n=7), postnatal exposure only (postnatal, n=8), and the control (n=7) group. Mice in the “both” and “postnatal” groups received BSSG and SG – containing mouse chow in the same doses used for the developmental exposure for a period of 15 weeks. Behavioral monitoring continued as previously described. Immediately following the 15-week steryl glucoside feeding, all mice were sacrificed and CNS tissue samples were collected for histological assays.  5.2.1 Body Weights of Female Mice There were no significant differences observed in the body weights of female mice across all groups at every time point measured. However, all female groups showed some weight loss (control, -2.14%; postnatal, -6.89%; prenatal, -8.04%; both, -9.09%) following the change in housing conditions, with the control mice losing the least percentage of weight, while the mice treated both in utero and as adults showed the  95 highest percentage of weight loss. A repeated measures ANOVA, as well as a Tukey’s multiple comparison test showed significance (repeated measures ANOVA, p<0.0001; pairing, p<0.0001; Tukey’s, control vs. postnatal, p<0.001; control vs. both, p<0.01; postnatal vs. both, p<0.001; postnatal vs. prenatal, p<0.001; both vs. prenatal, p<0.05) (Fig. 15).  5.2.2 Adult Dietary Exposure to BSSG and SG Induces Hyperactivity in Females The behavioral response of adult female mice to dietary BSSG and SG revealed some similarities as well as remarkable differences between mice of both sexes. As with the male mice, there were no significant differences observed in either the leg extension reflex test or the wire hang test for muscular strength among female groups. Nonetheless, some animals in the two groups of females treated as adults began to show some punching/tremors in their hind limbs starting around the 8th week of BSSG and SG feeding (Appendix D). As before, female mice continued to out-perform the males on the wire hang grid as they hung on for longer periods. Although the wire hang test results showed no significant differences among the female groups exposed to BSSG and SG when compared to controls, the females treated both developmentally and as adults consistently showed the lowest latency to fall off the grid (Appendix D). The open field test revealed marked hyperactivity in the female groups fed BSSG and SG as adults. Like the male groups, female groups exposed to BSSG and SG during gestation spent less time exploring the arena (repeated measures ANOVA, p<0.0005, pairing, not significant (ns); Tukey’s, control vs. prenatal, p<0.05; postnatal vs. both, p<0.01; postnatal vs. prenatal, p<0.01) and showed larger turn angles  (repeated measures ANOVA, p<0.0001, pairing, ns; Tukey’s, control vs. both, p<0.01; control vs. prenatal, p<0.001; postnatal vs. both, p<0.01; postnatal vs. prenatal, p<0.001). These parameters progressively changed over time, reaching significance by the last time-point measured when compared with controls (student’s t-test, p<0.05) (Fig. 16). Again, like the males, female groups exposed to BSSG and SG during gestation also showed a preference for the periphery of the arena (Appendix D). However, unlike the observations in the males, females exposed to dietary BSSG and SG as adults showed remarkable hyperactivity, covering about three times the distance, and moving about three times as fast as both the  96 male mice as well as their female contemporaries. A repeated measures ANOVA, as well as a Tukey’s multiple comparison test showed significance among the groups in both variables (total distance covered and velocity) (repeated measures ANOVA, p<0.0001, pairing, ns; Tukey’s, control vs. postnatal, p<0.001; control vs. both, p<0.001; postnatal vs. prenatal, p<0.001; both vs. prenatal, p<0.001) (Fig. 16). Gait patterns were not tested as very few mice ran on the treadmill.  5.2.3 Female Mice Show No Significant Changes in Tyrosine Hydroxylase Immunoreactivity in Response to Dietary BSSG and SG Immunoperoxidase staining of tyrosine hydroxylase (TH) – positive cells in both the striatum and substantia nigra pars compacta (SNpc) showed no significant differences between BSSG and SG – fed groups and controls (Fig. 17).  5.2.4 Female Mice Show No Significant Changes in Glial Activation in Response to BSSG and SG Exposure The glial response in the animals was measured by immunohistochemical staining for GFAP and Iba-1 – positive cells. GFAP - positive cells with star-shaped cell bodies were quantified in the lateral ventral horn of the lumbar spinal cord, as well as in the striatum. Quantification of GFAP-labeled cells in the lumbar spinal cord and striatum showed no significant differences between BSSG and SG – fed mice and controls (Fig. 18). Quantification of Iba-1 positive cells in the same regions as the GFAP stain also showed no significant microglial activation in BSSG and SG – fed mice (Fig. 19).  5.2.5 No Significant Changes in the Number of Lower Motor Neurons Across Female Groups Lumbar spinal cord tissue sections in all four groups of female mice were assessed for lower motor neuron numbers. Nissl body staining revealed no significant differences between all groups of mice exposed to BSSG and SG compared to control mice. Mice exposed to dietary BSSG and SG during development as well as adults showed subtle losses of lower motor neurons (-8.4%), while mice exposed to dietary BSSG and SG only as adults showed a slight increase (+6.7%) in the number of motor  97 neurons present. Female mice exposed to BSSG and SG via maternal diet only had comparable values as controls (Fig. 20). Cholinergic neurons were also assayed similarly as the Nissls using anti-ChAT immunofluorescent antibody staining. As with the Nissl lower motor neuron quantification, ChAT-positive cell counts showed no significant differences between BSSG and SG – treated mice compared with control mice, although the “postnatal” group again showed increases in number of ChAT-positive neurons compared to controls (Fig. 20).  5.2.6 Dietary BSSG and SG Induce Apoptosis in the Lumbar Spinal Cord of Female Mice Apoptosis was assayed in the lateral ventral horn of the lumbar spinal cord of female mice using anti-active caspase-3 immunofluorescent antibody staining. All cells positively labeled with the active capse-3 in the lateral ventral horn of the cord were counted. Results showed an increased expression of caspase-3 in all 3 groups treated with BSSG and SG in comparison with controls. Mice in the group treated both pre- and post- natally showed the most increase in active caspase-3 expression, and showed a trend towards significance when compared to controls (+58%; student’s t-test p = 0.0543) (Fig. 21).  5.3 Discussion The aim of this experiment was to observe any behavioral and neuropathological differences in female mice subjected to the same regimen of dietary steryl glucoside exposure as the male mice previously discussed, in order to determine if the sex differences observed in the incidence of ALS-PDC in human populations are reflected in our model of the disease. The results demonstrate for the first time differential outcomes in male and female mice exposed to BSSG and SG during development and as adults, and suggest differences in the mechanisms employed by mice of both sexes in response to dietary steryl glucoside – exposure. In contrast to the males, there were no significant weight differences observed across all the female groups. In the leg extension reflex test as well as the wire hang test,  98 there were also no significant differences observed across all female groups. However, some female mice in the groups receiving BSSG and SG as adults started to show some tremors/punching in the leg extension reflex test around the 8th week of feeding, and female mice exposed to BSSG and SG both developmentally and as adults started to show the lowest latency to fall off the wire hang starting around the 9th week of feeding. The open field test showed some parallels, but also revealed striking contrasts in the behavioral response of male and female mice following dietary steryl glucoside exposure. As observed in the males, female mice exposed to BSSG and SG via maternal diet continued to exhibit larger turn angles and spent less time moving in the arena, regardless of whether or not they received a secondary adult re-exposure. However, in sharp contrast to male mice, steryl glucoside exposure in adult mice elicited marked hyperactivity in the females, evidenced by the increased distance covered in the arena, as well as the increased velocity of movement. Although the differences were not significant, they were nonetheless very apparent, as female mice treated as adults moved approximately three times the distance, and at three times the speed of male mice, as well as their female counterparts. In both of these parameters (total distance covered and velocity), female mice exposed only in the gestational environment behaved similarly as controls. The increased open field activity observed in treated female mice reveals some interesting differences in the behaviors of male and female mice in response to dietary steryl glucoside exposure. While it appears that male mice exposed to BSSG and SG via maternal diet only express similar phenotypic outcomes as males exposed both developmentally and as adults, females exposed prenatally alone seem so show a more subtle phenotype, unless re-exposed to the toxins as adults. Again referring back to epidemiological data on a group of Chamorro migrants, the only female recorded to have developed the disease prior to age 46 was a woman who had returned to Guam after spending her childhood there; she developed the disease at age 25, only a year after her return in the early ’60’s (Garruto et al., 1980). The disease onset in other female migrants who developed ALS-PDC outside of Guam did not occur until their peri-menopausal or menopausal years, suggesting some hormonal influences may be involved in the disease etiology. In fact, several studies have investigated the possible neuroprotective effects of  99 female hormones, as the incidence of most neurodegenerative disorders tend to increase in women after menopause (Militello et al., 2002; Dluzen and Horstink, 2003; Plato et al, 2003; Gillies et al., 2004; Haaxma et al., 2007).  In the present study, adult females exposed to BSSG and SG only during gestation start to show significant deviations from control at 32 weeks of age, a period that parallels the onset of menopause in human females (Ison and Allen, 2007; Rajareddy et al., 2007). In regard to the hyperactivity observed in female mice treated as adults, other studies have found that hyperactivity in female mice could be related to dysfunctions in dopamine regulation (Bardullas, 2008; Viggiano, 2008). Although previous studies by our group have shown the neurotoxic effects of both BSSG and SG on dopaminergic pathways, specifically, the nigro-striatal system (Tabata et al., 2008; Tabata, 2008), these effects have never been studied in female mice. Also, as this effect was not observed in male mice that were not only subjected to the same treatment regimen, but also came from the same litters as the female mice, it may represent a sex-specific effect. Sex differences in symptoms and disease progression have been reported in other studies on neurodegenerative disorders (Scott et al., 2000; Veldink et al., 2003; Czlonkowska et al., 2005). Histological assessments of female mouse brains showed no significant differences between all groups of mice in all the pathological markers examined. TH analysis in the nigro-striatal pathway showed some increases in TH labeling in both the striatum and SNpc of the groups treated prenatally, while there were subtle decreases in the “postnatal” group. Although this finding remains unclear, it supports the assertion that a targeted developmental neurotoxin exposure could selectively impact the neurons differentiating at the time of exposure. Microglia activation seen in response to BSSG and SG feeding in male mice was not observed in either the striatum or the lower spinal cord of female mice. Astrocyte activity was also minimal in the striatum of female groups, however, groups treated as adults showed increases in astrocyte activation in the lumbar spinal cord, although this was still not significant when compared to controls. Again, these effects are suggestive of the anti-inflammatory actions of estrogen which have previously been demonstrated (Czlonkowska et al., 2005; Vegeto et al., 2008). Lower motor neuron quantification revealed a minimal loss in the “both” group, an increase in  100 the “postnatal” group, and comparable numbers in the “prenatal” group, when compared with controls. Apoptotic activity was however increased in the same area across all BSSG and SG – fed groups, indicating some toxicity, as seen in treated male groups. These seemingly conflicting findings suggest neurogenesis may also be occurring in adult BSSG and SG – fed mice, likely due to increased activity in these animals. This idea is based on reports from other investigators showing the beneficial effects of exercise in attenuating neurodegeneration and also stimulating neurogenesis in different areas of the CNS, including the spinal cord (Kirkinezos et al., 2003; Ferrer-Alcon et al., 2008).  5.4 Conclusions In sum, this experiment presents novel findings on the differences in both the behavior and the neuropathology of male and female mice in response to dietary BSSG and SG. In male mice, the prenatal exposure to BSSG and SG induces significant behavioral and neuropathological impacts that are worsened by a secondary adult re- exposure, whereas female mice appear to have the ability to largely suppress a behavioral phenotype, unless re-exposed as adults. The phenotypic response to BSSG and SG also appear to differ in mice of both sexes; while male mice show progressive reductions in spontaneous activity measured in the open field, female mice become evidently hyperactive. Neuropathological outcomes also show substantial differences; BSSG and SG –fed male mice show significant gliosis and apoptosis, as well as clear reductions in TH-immunoreactivity in both the striatum and SNpc, but these effects are not seen in the female mice. On the contrary, some of the trends in the treated female are opposite of the observations in the males. It will however be interesting to examine if any changes occur in the female mice that eventually mimic male behaviors with continuous exposure to the compounds. While further studies are needed to further elucidate these sex-specific effects, these results provide additional evidence in support of mechanistic differences in males and females in response to chronic neurotoxicity.      101 Figure 15 Body Weights of Animals The data on animal body weights (A) showed no significant inter-group differences between BSSG and SG – fed groups compared to controls at every time point measured. However, a repeated measures ANOVA, as well as a Tukey’s multiple comparison test showed significance (repeated measures ANOVA, p<0.0001; pairing, p<0.0001; Tukey’s, control vs. postnatal, p<0.001; control vs. both, p<0.01; postnatal vs. both, p<0.001; postnatal vs. prenatal, p<0.001; both vs. prenatal, p<0.05) (The “dip” at week 26 signifies when the scale in the animal behavior room was temporarily changed by the staff of the animal holding facility).    Weight 18 20 22 24 26 28 30 32 34 25 35 45 Control Postnatal Both Prenatal Error bars represent SEM Age in Weeks B od y W ei gh t ( g) A               102 Figure 16 Open Field Activity  The open field activity of the mice showed apparent distinctions between the responses of male and female mice to dietary BSSG and SG. While the two groups of female mice exposed to BSSG and SG during gestation spent less time exploring the arena (repeated measures ANOVA, p<0.0005, pairing, not significant (ns); Tukey’s, control vs. prenatal, p<0.05; postnatal vs. both, p<0.01; postnatal vs. prenatal, p<0.01) (B) and showed larger turn angles (repeated measures ANOVA, p<0.0001, pairing, ns; Tukey’s, control vs. both, p<0.01; control vs. prenatal, p<0.001; postnatal vs. both, p<0.01; postnatal vs. prenatal, p<0.001) (C) as the males, female mice exposed to dietary BSSG and SG as adults show marked hyperactivity. These two groups of females (both and postnatal) covered ~3x the distance of the other two groups (prenatal and control) (D), and also moved at a speed ~3x faster (repeated measures ANOVA, p<0.0001, pairing, ns; Tukey’s, control vs. postnatal, p<0.001; control vs. both, p<0.001; postnatal vs. prenatal, p<0.001; both vs. prenatal, p<0.001) (E).             103 Total Movement Duration - Females 18 20 22 24 26 28 30 32 34 100 200 300 Control Postnatal Both Prenatal Error bars represent SEM * *=p<0.05 B Age in Weeks. D ur at io n (s )     Mean Turn Angle - Female Mice 18 20 22 24 26 28 30 32 34 40 60 80 Control Postnatal Both Prenatal * Age in Weeks. Tu rn  A ng le  ( de g. ) Error bars represent SEM *=p<0.05 C     104 Total Distance Covered - Females 18 20 22 24 26 28 30 32 34 1000 3000 5000 7000 9000 Control Postnatal Both Prenatal Error bars represent SEM D Age in Weeks. D is ta nc e (c m )    Velocity - Females 18 20 22 24 26 28 30 32 34 0 10 20 30 Control Postnatal Both Prenatal Error bars represent SEM E Age in Weeks. Sp ee d (c m /s )            105 Figure 17 Tyrosine Hydroxylase Immunoreactivity in Nigro-striatal System  TH immunoreactivity was assessed in both the striatum and substantia nigra pars compacta (SNpc) of the animals. The optical density of the images showed no significant differences in TH labeling in both the striatum (F) and SNpc (G) in all four groups of females. However, females exposed to BSSG and SG during development show increases in TH labeling while the group treated only as adults showed decreases in TH immunoreactivity. The attached micrographs show sample images of striatum and substantia nigra across all groups. Images were taken with a light microscope. Scale bars = 50µm (striatum); 20µm (SNpc).                                 106 TH+ Labeling - Striatum Prenatal Both Control Postnatal 0 20 40 60 Prenatal Both Control Postnatal + 8.5% + 14.5% -10.0% F Groups St ai n In te ns ity    Prenatal Both  Control Postnatal  107 TH+ Labeling - SN Prenatal Both Control Postnatal 0 4 8 12 Prenatal Both Control Postnatal + 7.8% + 14.3% - 4.2% G Groups St ai n In te ns ity    Prenatal Both                      SNpc    SNr                             SNpc   SNr  Control Postnatal                   SNpc  SNr                            SNpc  SNr    108 Figure 18 Quantification of Activated Astrocytes in Lumbar Spinal Cord and Striatum  Activated astrocyte proliferation was assayed in both the lumbar spinal cord and the striatum using an antibody against the glial fibrillary acidic protein (GFAP). Quantification of positively labeled cells (stained brown using DAB) in both the lateral ventral horn of the spinal cord (H) and the striatum (I) showed no significant differences between all female groups. Astrocyte counts were restricted to the specified region (J; boxed area), using the external capsule (EC) as the point of reference for consistency. Quantification was done under a light microscope under the 40x objective lens. The accompanying micrographs show representative data in the striatum across the groups; an enlarged micrograph from a BSSG and SG – treated mouse striatum is shown (K) to depict astrocyte morphology. Scale bars = 20µm; 50µm (J); 10µm (K).                      109 GFAP - Positive Glial Cells - Female Mice Prenatal Both Control Postnatal 0 5 10 15 Prenatal Both Control Postnatal Groups # of  A st ro cy te s -17.3% + 24% + 59% H     GFAP Positive Glial Cells - Striatum Prenatal Both Control Postnatal 0 1 2 3 4 5 Prenatal Both Control Postnatal G roups # of  A st ro cy te s -4.3% -27.6% -14.9% I   110  Prenatal Both   Control Postnatal   J EC K        111 Figure 19 Quantification of Microglia Proliferation in Lumbar Spinal Cord and Striatum  Microglia expression was assayed in both the lumbar spinal cord and the striatum using an antibody against the ionized calcium binding adaptor molecule 1 (Iba-1). Quantification of positively labeled cells (stained brown using DAB) in both the lateral ventral horn of the spinal cord (L) and the striatum (M) showed no significant differences between all female groups. Microglia counts were restricted to the specified region (N; boxed area), using the external capsule (EC) as the point of reference for consistency. Quantification was done under a light microscope under the 40x objective lens. The accompanying micrographs show representative data in the striatum across the groups; an enlarged micrograph from a BSSG and SG – treated mouse striatum is shown (O) to depict microglia morphology. Scale bars = 20µm; 50µm (N); 10µm (O).                       112 Iba-1 - Postive Glial Cells - Spinal Cord Prenatal Both Control Postnatal 0.0 2.5 5.0 7.5 10.0 Prenatal Both Control Postnatal +12.3% - 18.3% - 18.1% Groups M ic ro gl ia L     Iba-1 - Positive Glial Cells - Striatum Prenatal Both Control Postnatal 0 5 10 15 20 25 Prenatal Both Control Postnatal + 7.0% - 4.0% + 10.9% M Groups M ic ro gl ia     113  Prenatal Both   Control Postnatal   N O EC        114 Figure 20 Motor Neuron Quantification in the Lumbar Spinal Cord Cresyl violet staining for Nissl bodies (P), as well as quantification of cholinergic neurons in the lumbar spinal cord (Q) showed no significant differences across all four female groups. However, female mice fed BSSG and SG only as adults showed increases in motor neuron numbers in both the Nissl and ChAT stains. ChAT-positive neurons were quantified under a fluorescence microscope in the field of view under the 20x objective lens, while cresyl violet – stained neurons were quantified under a light microscope in the field of view under the 40x objective lens. The attached micrographs show representative data of cresyl violet staining across the groups. Scale bars = 20µm.                115 Lower Motor Neuron Counts - Females Prenatal Both Control Postnatal 0 5 10 15 Prenatal Both Control Pos tnatal -1.02% -8.38% + 6.66% P G roups # of  M ot or  N eu ro ns    Cholinergic Neurons in the Lumbar Spinal Cord - Females Prenatal Both Control Postnatal 0 4 8 12 Prenatal Both Control Postnatal + 2.2% -2.3% + 19.8% Q Groups # of  M N s     116  Prenatal Both  Control Postnatal                117 Figure 21 Increased Expression of Active-Caspase-3 in the Lumbar Spinal Cord of Treated Mice  Apoptotic cells in the lumbar spinal cord were detected using an antibody against the active form of caspase-3. Caspase-3 positive cells were quantified in the lateral ventral horn of the spinal cord. Results showed an increase in active caspase-3 expression in all 3 groups of mice exposed to BSSG and SG, with the group with both pre- and post-natal exposures to the toxins showing the highest expression of the protein relative to controls (R). Quantification was performed under a light microscope in the field of view under the 40x objective lens.   Anti-Active Caspase 3 - Positive Cells - Lumbar Spinal Cord Prenatal Both Control Postnatal 0 4 8 12 Prenatal Both Control Postnatal +17.3% +58.0% +43.3% Groups # of  c el ls  la be le d w / ac tiv at ed  c as pa se -3 R              118 6 General Discussion and Future Studies  6.1 General Discussion Amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS-PDC) represents a cluster of the neurodegenerative diseases: ALS, PD, and dementia, occurring on a spectrum, but usually expressing a dominant ALS or PDC phenotype. Neuroepidemiological evidence has linked the consumption of cycad seeds (Cycas micronesica) to ALS-PDC. Two water insoluble steryl glucosides (stigmasterol β-D- glucoside (SG), and β-sitosterol β-D-glucoside (BSSG)) identified in washed cycad have previously been demonstrated to have neurotoxic effects both in vitro and in vivo. The objective of the current study was to examine the impact of a prenatal and secondary adult re-exposure to a combination of these two steryl glucosides, BSSG and SG, on disease onset and progression in male and female mice. In addition, I also examined the impact of targeting specific neuronal populations during development on the dominant phenotype expressed. The rationale was based on epidemiological data that linked the exposure to unknown environmental factor(s) prior to adulthood to future disease expression. Also, other investigators have demonstrated fetal/perinatal origins of some adult onset diseases, such as heart disease, schizophrenia, AD, and PD (Thiruchelvam et al., 2002; Basha et al., 2005; Opler and Susser, 2005; Morley, 2006; Barlow et al., 2007).  The experiments were designed to test the three major hypothesis of the study; that a prenatal exposure will increase the vulnerability to future neurotoxicity, and therefore lead to a more rapid disease onset and progression if mice are re-exposed to the steryl glucosides, that the neurons differentiating at the time of prenatal exposure will be more susceptible to a secondary exposure, and lastly, that there will be sex differences, based on the observations in human populations. The present results indeed demonstrated a faster rate of disease onset and perhaps progression in mice first exposed to BSSG and SG during development, as evidenced by the different markers of behavioral and neuropathological deficits assayed. The results also showed many behavioral changes in the mice that are often associated with nigro-striatal dysfunctions; immunohistochemistry on these neuronal populations supported the behavioral evidence. Lastly, significant differences in outcomes were observed between male and female mice, strongly suggesting differences in the mechanism of response between both sexes. While many  119 questions remain unanswered, and further research is needed to fully understand the many intricacies of this disease complex, this study nonetheless adds to the knowledge of potential risk factors for ALS-PDC and perhaps other neurodegenerative disorders. Future work on developmental origins of adult-onset neurodegenerative diseases will not only provide better insight into understanding the diseases, but may also aid the advancement of intervention strategies.  6.2 Future Studies Future work addressing some of the unanswered questions in this study will be worthwhile for the purpose of gaining a better understanding into the etiology and risk factors of ALS-PDC. To start with, significant weight differences were observed between prenatally-treated and control male mice in the current study; as such, it would be worthwhile to examine areas of the hypothalamus, such as the VMH and the arcuate nucleus for any signs of pathology. Also, although there were no significant differences between litter sizes of control and treated dams, it may be better to cull litter size to the same number of pups per litter to better control for animal weights and completely remove any bias in a future study. CNS tissue from the culled pups could also be assayed for any steryl glucoside-induced anomalies resulting from the prenatal exposure. In addition, the inclusion of additional time-points of animal sacrifice and tissue examination, such as at 4 months prior to the secondary adult re-exposure, will provide better insights into the chronology of neuropathological outcomes. Enabling some mice survive into late adulthood after ceasing dietary steryl glucoside exposure will provide further insights into the neurotoxic effects of these compounds. It will also help clarify if the disease progresses faster in mice exposed developmentally and as adults compared with mice exposed as adults only. As discussed earlier, ALS-PDC has a dementia component, and previous cycad studies have demonstrated cognitive impairments in cycad-fed mice. Yet, the behavioral tests employed in this study did not include a cognitive test component, mainly because the nigro-striatal system was the main area of target. However, it would have been insightful to assess cognition in these animals using appropriate behavioral tests such as the radial arm maze test and the water maze spatial memory tests often used to examine cognitive deficits in mice (Marighetto et al., 2008; Talpos et al., 2008). Nonetheless,  120 pathological markers of cognitive deficits could still be examined in these mice tissue at a future date in order to ascertain if other brain areas were impacted aside from the nigro- striatal neurons, and to what degree they were impacted. Although the deleterious effects of both BSSG and SG are well demonstrated, the target receptors, as well as the mechanism of action of these compounds remain unknown, even as some candidate receptors, such as the liver-X receptor beta (LXRbeta) (Kim et al., 2008) are currently being investigated. Still, examining the integrity of tight-junction proteins in the CNS tissue of mice exposed to steryl glucosides may provide some insight as to how these compounds gain access into the CNS in order to exert their toxic effects. Lastly, a similar study as this, but where other areas of the developing CNS are targeted in order to determine the dominant phenotype expressed would lend more support to the present study.                          121 References Alonso, A., Zaidi, T., Novak, M., Grundke-Iqbal, I., & Iqbal, K. (2001). Hyperphosphorylation induces self-assembly of tau into tangles of paired helical filaments/straight filaments. Proceedings of the National Academy of Sciences of the United States of America, 98(12), 6923-6928. Amende, I., Kale, A., McCue, S., Glazier, S., Morgan, J. P., & Hampton, T. G. (2005). Gait dynamics in mouse models of parkinson's disease and huntington's disease. Journal of Neuroengineering and Rehabilitation, 2, 20. Anderson, F. H., Richardson, E. 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Washington, DC 1945.                           140                    Appendices  A                  141 Appendix A Figure 22 Open Field Tracking System A mouse is depicted in the open field arena (A); the four arenas of the open field with the center and peripheral regions defined (B); mouse tracking in the open field (C).                         142  A   B  143 C                    144 Figure 23 Digigait Analysis System Screenshots of mouse gait analysis in progress (D, E, F).           145  D   146  E   147  F              148 Appendix B Figure 24 Leg Extension and Wire Hang Data  There were no significant differences in the leg extension and wire hang performances of animals treated with BSSG and SG developmentally and control animals.                    149 SLeg Extension - Male Mice 5 12 19 3.50 3.75 4.00 4.25 Control Treated "S" denotes period w hen animals w ere separated into single cages A Age in Weeks Sc or e    S Leg Extension - Female Mice 5 12 19 3.50 3.75 4.00 4.25 Control Treated "S" denotes period w hen animals w ere separated into single cages B Age in Weeks Sc or e     150 SWire Hang - Male Mice 5 12 19 0 16 32 48 64 Control Treated Weeks Ti m e (s ) "S" denotes period w hen animals w ere separated into single cages   S Wire Hang - Female Mice 5 12 19 0 30 60 90 Control Treated "S" denotes period w hen animals w ere separated into single cages Age in Weeks Ti m e (s ) D                151 Figure 25 Additional Gait Analysis Parameters  Additional parameters examining stepping patterns of male and female mice prior to the start of experiment 2.                      152 SRight Fore Paw Swing - Male Mice 8 12 16 20 0.08 0.09 0.10 0.11 Control Treated* * Age in Weeks Ti m e (s ) S Left Rear Paw Swing - Male Mice 8 12 16 20 0.07 0.08 0.09 0.10 Control Treated Age in Weeks Ti m e (s ) S Right Rear Paw Swing - Male Mice 8 12 16 20 0.07 0.08 0.09 0.10 Control Treated* Age in Weeks Ti m e (s ) S Left Fore Paw Swing - Male Mice 8 12 16 20 0.08 0.09 0.10 0.11 Control Treated* Age in Weeks Ti m e (s )  E   153 SRight Fore Paw Swing - Female Mice 8 12 16 20 0.08 0.09 0.10 0.11 Control Treated Age in Weeks Ti m e (s ) S Left Rear Paw Swing - Female Mice 8 12 16 20 0.07 0.08 0.09 0.10 Control Treated Age in Weeks Ti m e (s ) S Right Rear Paw Swing - Female Mice 8 12 16 20 0.07 0.08 0.09 0.10 Control Treated* Age in Weeks Ti m e (s ) S Left Fore Paw Swing - Female Mice 8 12 16 20 0.08 0.09 0.10 0.11 Control Treated Age in Weeks Ti m e (s ) F       154 SLeft Fore Paw Brake - Female Mice 8 12 16 20 0.08 0.09 0.10 0.11 0.12 Control Treated * Age in Weeks Ti m e (s ) S Right Fore Paw Brake - Female Mice 8 12 16 20 0.08 0.09 0.10 0.11 0.12 Control Treated Age in Weeks Ti m e (s ) S Left Rear Paw Brake - Female Mice 8 12 16 20 0.04 0.05 0.06 0.07 0.08 Control Treated Age in Weeks Ti m e (s ) S Right Rear Paw Brake - Female Mice 8 12 16 20 0.04 0.05 0.06 0.07 0.08 Control Treated Age in Weeks Ti m e (s ) G    155 SLeft Fore Paw Propel - Male Mice 8 12 16 20 0.09 0.10 0.11 0.12 0.13 Control Treated Age in Weeks Ti m e (s ) S Right Fore Paw Propel - Male Mice 8 12 16 20 0.08 0.09 0.10 0.11 0.12 0.13 Control Treated * * Age in Weeks Ti m e (s ) S Left Rear Paw Propel - Male Mice 8 12 16 20 0.125 0.150 0.175 0.200 Control Treated Age in Weeks Ti m e (s ) S Right Rear Paw Propel - Male Mice 8 12 16 20 0.125 0.150 0.175 0.200 Control Treated** * Age in Weeks Ti m e (s ) H    156 SLeft Fore Paw Propel - Female Mice 8 12 16 20 0.09 0.10 0.11 0.12 0.13 Control Treated * Age in Weeks Ti m e (s ) S Right Fore Paw Propel - Female Mice 8 12 16 20 0.09 0.10 0.11 0.12 0.13 Control Treated Age in Weeks Ti m e (s ) S Left Rear Paw Propel - Female Mice 8 12 16 20 0.125 0.150 0.175 0.200 Control Treated* Age in Weeks Ti m e (s ) S Right Rear Paw Propel - Female Mice 8 12 16 20 0.125 0.150 0.175 0.200 Control Treated Age in Weeks Ti m e (s ) I    157 SLeft Fore Paw Stance - Female Mice 8 12 16 20 0.18 0.19 0.20 0.21 0.22 0.23 Control Treated Age in Weeks Ti m e (s ) S Right Fore Paw Stance - Female Mice 8 12 16 20 0.175 0.200 0.225 Control Treated Age in Weeks Ti m e (s ) S Left Rear Paw Stance - Female Mice 8 12 16 20 0.175 0.200 0.225 0.250 0.275 Control Treated * ** ** Age in Weeks Ti m e (s ) S Right Rear Paw Stance - Female Mice 8 12 16 20 0.175 0.200 0.225 0.250 0.275 Control Treated* Age in Weeks Ti m e (s ) J    158 SLeft Fore Paw Stride - Male Mice 8 12 16 20 0.250 0.275 0.300 0.325 Control Treated * Age in Weeks Ti m e (s ) S Right Fore Paw Stride - Male Mice 8 12 16 20 0.250 0.275 0.300 0.325 Control Treated * Age in Weeks Ti m e (s ) S Left Rear Paw Stride - Male Mice 8 12 16 20 0.250 0.275 0.300 0.325 Control Treated * Age in Weeks Ti m e (s ) S Right Rear Paw Stride - Male Mice 8 12 16 20 0.250 0.275 0.300 0.325 Control Treated ** Age in Weeks Ti m e (s ) K    159 SLeft Fore Paw Stride - Female Mice 8 12 16 20 0.250 0.275 0.300 0.325 Control Treated Age in Weeks Ti m e (s ) S Right Fore Paw Stride - Female Mice 8 12 16 20 0.250 0.275 0.300 0.325 Control Treated Age in Weeks Ti m e (s ) S Left Rear Paw Stride - Female Mice 8 12 16 20 0.250 0.275 0.300 0.325 0.350 Control Treated * Age in Weeks Ti m e (s ) S Right Rear Paw Stride - Female Mice 8 12 16 20 0.250 0.275 0.300 0.325 Control Treated * Age in Weeks Ti m e (s ) L    160 SLeft Fore Paw Stride Length - Male Mice 8 12 16 20 5.0 5.5 6.0 6.5 Control Treated * Age in Weeks St ri de  L en gt h (c m /s ) S Right Fore Paw Stride Length - Male Mice 8 12 16 20 5.0 5.5 6.0 6.5 Control Treated * Age in Weeks St rid e Le ng th  (c m /s ) S Left Rear Paw Stride Length - Male Mice 8 12 16 20 5.0 5.5 6.0 6.5 Control Treated * Age in Weeks St ri de  L en gt h (c m /s ) S Right Rear Paw Stride Length - Male Mice 8 12 16 20 5.0 5.5 6.0 6.5 Control Treated ** Age in Weeks St rid e Le ng th  (c m /s ) M     161 SLeft Fore Paw Stride Length - Female Mice 8 12 16 20 5.0 5.5 6.0 6.5 Control Treated Age in Weeks St rid e Le ng th  (c m /s ) S Right Fore Paw Stride Length - Female Mice 8 12 16 20 5.0 5.5 6.0 6.5 Control Treated Age in Weeks St rid e Le ng th  (c m /s ) S Left Rear Paw Stride Length - Female Mice 8 12 16 20 5.0 5.5 6.0 6.5 Control Treated * Age in Weeks St rid e Le ng th  (c m /s ) S Right Rear Paw Stride Length - Female Mice 8 12 16 20 5.0 5.5 6.0 6.5 Control Treated * Age in Weeks St rid e Le ng th  (c m /s ) N   162 Left Fore Paw Stride Frequency - Male Mice Right Fore Paw Stride Frequency - Male Mice S8 12 16 20 3.00 3.25 3.50 3.75 4.00 Control Treated* Age in Weeks St ri de  F re q.  ( st ep s/ s) S 4.00 Control 8 12 16 20 3.00 3.25 3.50 3.75 * Age in Weeks St ri de  F re q.  ( st ep s/ s) Treated Right Rear Paw Stride Frequency - Male Mice Left Rear Paw Stride Frequency - Male Mice S8 12 16 20 3.00 3.25 3.50 3.75 4.00 Control Treated * Age in Weeks St rid e Fr eq ue nc y (s te ps /s ) S8 12 16 20 3.00 3.25 3.50 3.75 4.00 Control ** * Age in Weeks St rid e Fr eq . ( st ep s/ s) O Treated  163 SLeft Fore Paw Stride Frequency - Female Mice 8 12 16 20 3.00 3.25 3.50 3.75 4.00 Control Treated Age in Weeks St rid e Fr eq . ( st ep s/ s) S Right Fore Paw Stride Frequency - Female Mice 8 12 16 20 3.00 3.25 3.50 3.75 4.00 Control Treated Age in Weeks St rid e Fr eq . ( st ep s/ s) S Left Right Paw Stride Frequency - Female Mice 8 12 16 20 3.00 3.25 3.50 3.75 Control Treated * Age in Weeks St rid e Fr eq ue nc y (s te ps /s ) S Right Rear Paw Stride Frequency - Female Mice 8 12 16 20 3.00 3.25 3.50 3.75 4.00 Control Treated * Age in Weeks St rid e Fr eq . ( st ep s/ s) P    164 SLeft Fore Paw Angle - Male Mice 8 12 16 20 -7.5 -5.0 -2.5 0.0 2.5 Control Treated ** * * Age in Weeks Pa w  A ng le  (d eg .) S Right Fore Paw Angle - Male Mice 8 12 16 20 -2.5 0.0 2.5 5.0 7.5 Control Treated ** * Age in Weeks Pa w  A ng le  (d eg .) S Left Rear Paw Angle - Male Mice 8 12 16 20 -20 -15 -10 -5 0 Control Treated ** Age in Weeks Pa w  A ng le  (d eg .) S Right Rear Paw Angle - Male Mice 8 12 16 20 0 5 10 15 Control Treated Age in Weeks Pa w  A ng le  (d eg .) Q    165 SLeft Fore Paw Angle - Female Mice 8 12 16 20 -2.5 0.0 2.5 5.0 Control Treated Age in Weeks Pa w  A ng le  (d eg .) S Right Fore Paw Angle - Female Mice 8 12 16 20 -5.0 -2.5 0.0 2.5 5.0 Control Treated Age in WeeksPa w  A ng le  (d eg .) S Left Rear Paw Angle - Female Mice 8 12 16 20 -12.5 -10.0 -7.5 -5.0 -2.5 0.0 Control Treated Age in Weeks Pa w  A ng le  (d eg .) S Right Rear Paw Angle - Female Mice 8 12 16 20 0 5 10 15 Control Treated Age in Weeks Pa w  A ng le  (d eg .) R      166 SLeft Fore Paw Steps - Male Mice 8 12 16 20 9 10 11 12 Control Treated* Age in Weeks # of  S te ps S Right Fore Paw Steps - Male Mice 8 12 16 20 9 10 11 12 Control Treated* Age in Weeks # of  S te ps S  Left Rear Paw Steps - Male Mice 8 12 16 20 9 10 11 12 Control Treated * Age in Weeks # of  S te ps S Right Rear Paw Steps - Male Mice 8 12 16 20 8 9 10 11 12 Control Treated** Age in Weeks # of  S te ps S     167 S Left Fore Paw Steps - Female Mice 8 12 16 20 9 10 11 12 13 Control Treated Age in Weeks # of  S te ps S Right Fore Paw Steps - Female Mice 8 12 16 20 9 10 11 12 13 Control Treated Age in Weeks # of  S te ps S  Left Rear Paw Steps - Female Mice 8 12 16 20 9 10 11 12 Control Treated * Age in Weeks # of  S te ps S  Right Rear Paw Steps - Female Mice 8 12 16 20 9 10 11 12 Control Treated Age in Weeks # of  S te ps T   168 Right Fore Paw Area at Peak Stance - Male Mice Left Fore Paw Area at Peak Stance - Male Mice S8 12 16 20 0.0 0.1 0.2 0.3 0.4 0.5 Control Treated * * Age in Weeks Pa w  A re a (s q.  c m ) S8 12 16 20 0.0 0.1 0.2 0.3 0.4 0.5 Control * * * Age in Weeks Pa w  A re a (s q.  c m ) Treated Right Rear Paw Area at Peak Stance - Male Mice Left Rear Paw Area at Peak Stance - Male Mice S8 12 16 20 0.6 0.7 0.8 0.9 1.0 1.1 Control Treated* * * * Age in Weeks Pa w  A re a (s q.  c m ) S8 12 16 20 0.6 0.7 0.8 0.9 1.0 1.1 Control**** ** Age in Weeks Pa w  A re a (s q.  c m ) U Treated  169 SLeft Fore Paw Area at Peak Stance - Female Mice 8 12 16 20 0.3 0.4 0.5 0.6 Control Treated * * Age in Weeks Pa w  A re a (s q.  c m ) S Right Fore Paw Area at Peak Stance - Female Mice 8 12 16 20 0.00 0.25 0.50 0.75 Control Treated* * * Age in Weeks Pa w  A re a (s q.  c m ) S Left Rear Paw Area at Peak Stance - Female Mice 8 12 16 20 0.6 0.7 0.8 0.9 1.0 1.1 1.2 Control Treated * * * Age in Weeks Pa w  A re a (s q.  c m ) S Right Rear Paw Area at Peak Stance - Female Mice 8 12 16 20 0.6 0.7 0.8 0.9 1.0 1.1 1.2 Control Treated * * Age in Weeks Pa w  A re a (s q.  c m ) V    170 SF r o n t  P a w s  S t a n c e  W id t h  -  M a le  M ic e 8 1 2 1 6 2 0 1 . 5 1 . 6 1 . 7 1 . 8 1 . 9 2 . 0 C o n t r o l T r e a t e d A g e  i n  W e e k s St an ce  W id th  (c m ) S H in d  P a w s  S t a n c e  W id t h  -  M a le  M ic e 8 1 2 1 6 2 0 2 . 2 5 2 . 5 0 2 . 7 5 3 . 0 0 3 . 2 5 C o n t r o l T r e a te d * A g e  i n  W e e k s St an ce  W id th  (c m ) W X    171 SF r o n t  P a w s  S t a n c e  W id t h  -  F e m a le M ic e 8 1 2 1 6 2 0 1 . 2 5 1 . 5 0 1 . 7 5 2 . 0 0 2 . 2 5 C o n t r o l T r e a t e d * * * * A g e  i n  W e e k s St an ce  W id th  (c m ) S H in d  P a w s  S t a n c e  W id t h  -  F e m a le M ic e 8 1 2 1 6 2 0 2 . 2 5 2 . 5 0 2 . 7 5 3 . 0 0 C o n t r o l T r e a t e d A g e  i n  W e e k s St an ce  W id th  (c m ) Y Z    172 SF r o n t  P a w s  S t e p  A n g le  -  M a le  M ic e 8 1 2 1 6 2 0 5 5 6 0 6 5 7 0 C o n t r o l T r e a t e d A g e  i n  W e e k s St ep  A ng le  ( de g. ) S H in d  P a w s  S t e p  A n g le  -  M a le  M ic e 8 1 2 1 6 2 0 4 0 5 0 6 0 7 0 C o n t r o l T r e a t e d A g e  i n  W e e k s St ep  A ng le  ( de g. ) A A B B   173 SF r o n t  P a w s  S t e p  A n g le  -  F e m a le  M ic e 8 1 2 1 6 2 0 5 0 6 0 7 0 C o n t r o l T r e a te d * A g e  in  W e e k s St ep  A ng le  ( de g. ) S H in d  P a w s  S t e p  A n g le  -  F e m a le  M ic e 8 1 2 1 6 2 0 4 0 5 0 6 0 7 0 C o n t r o l F T r e a t e d  F * * A g e  in  W e e k s St ep  A ng le  (d eg .) C C D D     174 SH ind Limb Paw s Shared Stance Time - Male Mice 8 12 16 20 0 .100 0 .125 0 .150 0 .175 0 .200 C ontrol Treated ** * *** *** Ag e  in  W e e ks Ti m e (s ) EE   S Hind Limb Paw s Shared Stance Time - Female Mice 8 12 16 20 0 .100 0 .125 0 .150 0 .175 Con tro l T rea ted* *** ** Ag e  in  W e e ks Ti m e (s ) FF           175 Figure 26 Additional Open Field Parameters  Additional open field parameters examining exploratory activity of male and female mice prior to the start of experiment 2.                             176 SV elo city  -  Fem ale  M ice 8 1 2 1 6 2 0 0 5 1 0 1 5 C o n tro l T re a te d Ag e  in  W e e k s Ve lo ci ty  (c m /s ) G G    S Total D istance C overed -  Male Mice 8 1 2 1 6 2 0 0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 2 5 0 0 C o n tro l T re a te d * * * Ag e  in  W e e k s D is ta nc e (c m ) H H     177 STotal D istance C overed -  Female Mice 8 1 2 1 6 2 0 0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 C o n tro l T re a te d Ag e  in  W e e k s D is ta nc e (c m ) II     S M axim u m  D istan ce M o ved  in  O n e D ir ect io n  -  M ale  M ice 8 1 2 1 6 2 0 0 5 1 0 1 5 C o n tro l T re a te d * * JJ Ag e  in  W e e k s D is ta nc e (c m )   178 SM axim u m  D is tan ce  M o ved  in  O n e D ir ec t io n  -  F em ale  M ic e 8 1 2 1 6 2 0 5 6 7 8 9 C o n tro l T re a te d Ag e  in  W e e k s D is ta nc e (c m ) K K       S Total Turn Angle - Male Mice 8 12 16 20 70000 80000 90000 100000 110000 Contro l T rea ted* Ag e  in  W e e ks To ta l T ur n A ng le  (d eg .) LL     179 STotal Turn Angle - Female Mice 8 12 16 20 70000 80000 90000 100000 Contro l T rea ted * * Ag e  in  W e e ks To ta l T ur n A ng le  (d eg .) MM     S Angular  Velocity -  Female Mice 8 1 2 1 6 2 0 -1 0 0 -7 5 -5 0 -2 5 0 C o n tro l T re a te d Ag e  in  W e e k s Ve lo ci ty  (d eg ./s ) N N   180 SM e ande r - Fe male  M ice 8 1 2 1 6 2 0 -1 0 0 -7 5 -5 0 -2 5 0 C o n tro l T re a te d * Ag e  in  W e e k s M ea nd er  ( de g/ cm ) O O      S R ear in g  -  M ale  M ice 8 1 2 1 6 2 0 0 1 0 2 0 3 0 C o n tro l T re a te d Ag e  in  W e e k s R ea ri ng P P    181 SR ear in g  -  F em ale  M ice 8 1 2 1 6 2 0 0 1 0 2 0 3 0 C o n tro l T re a te d Ag e  in  W e e k s R ea ri ng Q Q                                182 Appendix C  Figure 27 Leg Extension and Wire Hang Data – Male Mice  A student’s t-test comparing all groups treated with BSSG and SG to controls at every time point measured revealed no significant inter-group differences in the leg extension and wire hang performances of the animals.   Leg Extension Reflex - Male Mice 18 20 22 24 26 28 30 32 34 3.6 3.9 4.2 Control Postnatal Both Prenatal A Age in Weeks LE  S co re    W ire Hang - Male Mice 18 20 22 24 26 28 30 32 34 0 30 60 90 Control Postnatal Both Prenatal B Age  in Weeks. Ti m e (s )   183   Figure 28 Additional Open Field Parameters – Male Mice  Additional open field parameters measured revealed male mice treated prenatally showed behavioral deficits compared with controls regardless of whether or not they received a secondary treatment. This is evidenced by the tendency of these mice to cover less distance in the arena (C), move slower (E), walk in a circling/winding manner (F, G), and show a preference for the periphery of the arena rather than the center (J, K, M).                          184 Total Distance Covered - Male Mice 18 20 22 24 26 28 30 32 34 0 500 1000 1500 2000 2500 Control Postnatal Both Prenatal C Age in Weeks. D is ta nc e (c m )    Maximum Distance Moved in Same Direction - Male Mice 18 20 22 24 26 28 30 32 34 3 6 9 Control Postnatal Both Prenatal Age  in We e ks. D is ta nc e (c m ) D    185 Velocity - Male Mice 18 20 22 24 26 28 30 32 34 3 6 9 Control Postnatal Both Prenatal E Age in Weeks. Sp ee d (c m /s )     Angular Velocity - Male Mice 18 20 22 24 26 28 30 32 34 -200 -100 0 Control Postnatal Both Prenatal Age in Weeks. Ve lo ci ty  (d eg /s ) F     186 Meandering - Male Mice 18 20 22 24 26 28 30 32 34 -200 -100 0 Control Postnatal Both Prenatal Age in Weeks. M ea nd er  ( de g/ cm ) G    Total Turn Angle - Male Mice 18 20 22 24 26 28 30 32 34 70000 75000 80000 85000 90000 95000 Control Postnatal Both Prenatal Age in Weeks. Tu rn  A ng le  ( de g. ) H     187 Rearing - Male Mice 18 20 22 24 26 28 30 32 34 0 5 10 15 20 25 Control Postnatal Both Prenatal Age  in Weeks. R ea ri ng I     In Center Frequency - Male Mice 18 20 22 24 26 28 30 32 34 0 20 40 Control Postnatal Both Prenatal J Age in Weeks. Fr eq ue nc y of  e nt er in g ce nt er     188 In Center Duration - Male Mice 18 20 22 24 26 28 30 32 34 0 25 50 75 Control Postnatal Both Prenatal K Age  in We eks. Ti m e in  C en te r ( s)    Latency of Entering the Center for the First Time - Male Mice 18 20 22 24 26 28 30 32 34 0 50 100 150 Control Postnatal Both Prenatal L Age in Weeks. Ti m e (s )     189 Distance to the Zone Border - Male Mice 18 20 22 24 26 28 30 32 34 3 6 9 Control Postnatal Both Prenatal * M Age  in We e ks. D is ta nc e (c m )                               190 Figure 29 Lumbar Spinal Cord Volumes and Motor Neuron Counts Adjusted for Spinal Cord Volumes  Based on the significant size differences between the groups, 4 spinal cords were randomly selected per group for MRM volumetric analysis. The results showed increased lumbar spinal cord volumes in all BSSG and SG – fed groups, with the groups treated prenatally having the highest volumes (N). Motor neuron counts were therefore adjusted for spinal cord volume. The Nissl counts showed that mice in the “both” group showed a significant (-24%; student’s t-test p<0.05) decrease in number of healthy motor neurons compared with controls, while mice in the “postnatal” group also showed a reduction in motor neuron numbers (-8.3%), although not significant compared with controls (O). ChAT-positive cell counts showed motor neuron loss in the “both” group only, although no group showed significant difference compared with controls (P). The discrepancy in the ChAT and Nissl counts may be due to the increased specificity of ChAT for labeling motor neurons, and also the difficulty in ascertaining neuron morphology when using fluorescent probes. Therefore, some motor neurons considered “unhealthy” due to pyknosis, or chromatolysis which are very visible with the Nissl stain may have still been labeled and therefore counted.                191 Total Lumbar Spinal Cord Volume Prenatal Both Control Postnatal 0 200000 400000 600000 Prenatal Both Control Postnatal +16% * +13% +10% Groups Vo lu m e *=>p<0.05 N     Lower Motor Neuron Counts Adjusted for Spinal Cord Volume (Nissl) Prenatal Both Control Postnatal 0.0 0.4 0.8 1.2 Prenatal Both Control Postnatal +0.1% -24.4% * -8.3% * => p<0.05 O Groups N or m al iz ed  m ot or  n eu ro n co un ts  a dj us te d fo r c or d vo lu m e    192 Cholinergic Neurons Adjusted for Spinal Cord Volume Prenatal Both Control Postnatal 0.0 0.5 1.0 1.5 Prenatal Both Control Postnatal P +24% -17.6% +14% Groups N or m al iz ed  C hA T+  c el l co un ts  a dj us te d fo r co rd vo lu m e                                193 Figure 30 ATF-3 Immunoreactivity in the Lumbar Spinal Cord Show No Significant Differences Among the Groups  The activating transcription factor 3 (ATF-3) was assayed in the lateral ventral horn of the lumbar spinal cord of the mice. Quantification of positively labeled motor neurons (ATF-3 – positive cells labeled brown using DAB; green, methyl green staining for nuclei counterstaining) revealed no significant differences among the groups in comparison with controls, although BSSG and SG – fed mice showed increased ATF-3 immunoreactivity (Q). Images were taken with a light microscope. Scale bars = 20µm.                        194 ATF-3 Labeled Motor Neurons - Male Mice Prenatal Both Control Postnatal 0 25 50 75 100 Prenatal Both Control Postnatal +24.4% +20.5% +1.12% Q Groups A TF -3  L ab el le d M N s ex pr es se d as  a  p er ce nt ag e of  t ot al  M N s pr es en t                Prenatal Both               Control Postnatal  195 Figure 31 HSP-70 Immunoreactivity in the Lumbar Spinal Cord Show No Significant Differences Among the Groups  The stress-inducible heat shock protein 70 (HSP-70) was assayed in the lateral ventral horn of the lumbar spinal cord of the mice. Quantification of positively labeled motor neurons (HSP-70 – positive cells labeled brown using DAB; green, methyl green staining for nuclei counterstaining) revealed no significant differences among the groups in comparison with controls (R). Images were taken with a light microscope. Scale bars = 20µm.                             196 HSP-70 Labeled Motor Neurons - Male Mice Prenatal Both Control Postnatal 0 30 60 90 Prenatal Both Control Postnatal -7.7% +11% -0.4% Groups H SP -7 0 La be lle d M N s ex pr es se d as  a  p er ce nt ag e of  t ot al  M N s pr es en t R                 Prenatal Both               Control Postnatal  197 Figure 32 Anti-GFAP Astrocyte Quantification in the Lumbar Spinal Cord Activated astrocytes in the lateral ventral horn of the lumbar spinal cord were detected using an antibody against the glial fibrillary acidic protein (GFAP) in all four groups of mice. GFAP-positive cells (green; blue label is DAPI to counter-stain the nuclei) were quantified in the lateral ventral horn of the spinal cord. Results showed no significant differences among the groups. However, the group of mice treated prenatally had much fewer activated astrocytes as compared with controls (-39%), and showed a trend towards significance (p = 0.0630) (P). Quantification was performed under a fluorescence microscope in the field of view under the 40x objective lens. Scale bars = 20µm                                198 GFAP Positive G lial Cells - Male Mice Prenatal Both Control Postnatal 0 9 18 Prenatal Both Control Pos tnatal -39.0% -3.8% -2.3% G roups # of  A st ro cy te s S       Control Postnatal Prenatal Both  199 Appendix D  Figure 33 Leg Extension and Wire Hang Data – Female Mice  A student’s t-test comparing all female groups treated with BSSG and SG to controls at every time point measured revealed no significant inter-group differences in the leg extension and wire hang performances of the mice.   Leg Extension Reflex - Female Mice 18 20 22 24 26 28 30 32 34 3.6 3.9 4.2 Control Postnatal Both Prenatal Age in Weeks. LE  S co re A    Wire Hang - Female Mice 18 20 22 24 26 28 30 32 34 0 40 80 120 Control Postnatal Both Prenatal Age in Weeks. Ti m e (s ) B   200   Figure 34 Additional Open Field Parameters - Female Mice  Additional open field parameters measured in female mice.                             201 Maximum Distance Moved in Same Direction - Female Mice 18 20 22 24 26 28 30 32 34 4 9 14 Control Postnatal Both Prenatal C Age  in Wee ks. D is ta nc e (c m )   Angular Velocity - Female Mice 18 20 22 24 26 28 30 32 34 -150 -100 -50 0 50 Control Postnatal Both Prenatal Age in Weeks. Ve lo ci ty  (d eg /s ) D     202 Meandering - Female Mice 18 20 22 24 26 28 30 32 34 -100 -50 0 Control Postnatal Both Prenatal Age in Weeks. M ea nd er  ( de g/ cm ) E    Total Turn Angle - Female Mice 18 20 22 24 26 28 30 32 34 80000 90000 100000 Control Postnatal Both Prenatal F Age in Weeks. Tu rn  A ng le  ( de g. )   203 Rearing - Female Mice 18 20 22 24 26 28 30 32 34 0 10 20 30 40 Control Postnatal Both Prenatal Age  in Wee ks. R ea ri ng G     In Center Frequency - Female Mice 18 20 22 24 26 28 30 32 34 0 20 40 60 80 Control Postnatal Both Prenatal H Age in Weeks. Fr eq ue nc y of  e nt er in g ce nt er      204 In Center Duration - Female Mice 18 20 22 24 26 28 30 32 34 0 25 50 75 Control Postnatal Both Prenatal I Age  in Wee ks. Ti m e in  C en te r ( s)    Latency of Entering the Center for the First Time - Female Mice 18 20 22 24 26 28 30 32 34 0 100 200 Control Postnatal Both Prenatal J Age in Weeks. Ti m e (s )     205 Mean Distance to Zone Border - Female Mice 18 20 22 24 26 28 30 32 34 3 6 9 Control Postnatal Both Prenatal K Age  in We e ks. D is ta nc e (c m )                               206 Appendix E    207

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