Nosce te ipsum

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Karlsson, O., Berg, C., Brittebo, EB., Lindquist, NG. (2009) Retention of the cyanobacterial neurotoxin β-N-methylamino-L-alanine in melanin and neuromelanin-containg cells – a poss-ible link between Parkinson-dementia complex and pigmentary retinopathy. Pigment Cell Melanoma Res, 22 (1):120–130

II Karlsson, O., Lindquist, NG., Brittebo, EB., Roman, E. (2009) Selective brain uptake and behavioral effects of the cyanobac-terial toxin BMAA (β-N-methylamino-L-alanine) following neonatal administration to rodents. Toxicol Sci, 109(2):286-95

III Karlsson, O., Roman, E., Brittebo, EB. (2009) Long-term cog-nitive impairments in adult rats treated neonatally with β-N-methylamino-L-alanine. Toxicol Sci, 112(1):185-95

IV Karlsson, O., Roman, E., Berg, AL., Brittebo, EB. (2010) Early hippocampal cell death and late learning and memory deficits in rats exposed to the environmental toxin BMAA (β-N-methylamino-L-alanine) during the neonatal period. Submitted

V Karlsson, O., Berg, AL., Arnerup, G., Roman, E., Lindström, AK., Bergquist, J., Lindquist, NG., Andersson, M., Brittebo, EB. (2010) Long-term morphological and protein changes in the brain of adult rats neonatally treated with the environmental toxin BMAA (β-N-methylamino-L-alanine). Manuscript

Reprints were made with permission from the respective publishers.

Contents

Introduction ................................................................................................... 11 Developmental Neurotoxicity .................................................................. 11 

Critical periods of brain development ................................................. 12 Brain growth spurt .......................................................................... 14 

Natural toxins ........................................................................................... 17 Nonprotein amino acids ....................................................................... 17 

BMAA – an environmental neurotoxin ........................................... 18 BMAA – mechanism of action ....................................................... 18 

ALS/PDC and retinal pigment epitheliopathy of Guam ........................... 21 Properties and function of melanin .......................................................... 22 

Neuromelanin ...................................................................................... 23 

Objectives ..................................................................................................... 25 

Material and methods .................................................................................... 26 Comments on methods ............................................................................. 27 

Autoradiography .................................................................................. 27 Whole body tape-section autoradiography ...................................... 27 Light-microscopic autoradiography ................................................ 28 

Matrix-assisted laser desorption ionization imaging mass spectrometry ............................................................................................................. 28 

Behavioral tests ........................................................................................ 30 Test of early neurobehavioral development ......................................... 31 The elevated plus maze test ................................................................. 31 The open field test ............................................................................... 32 The multivariate concentric square field™ test ................................... 32 The object recognition test ................................................................... 33 The water maze test ............................................................................. 33 The radial arm maze test ...................................................................... 35 

Results and Discussion ................................................................................. 36 Interaction of 3H-BMAA with neuromelanin and melanin ...................... 36 Tissue-distribution of 3H-BMAA in mice – possible protein incorporation .................................................................................................................. 40 Localization of ³H-BMAA in the mouse brain ......................................... 41 Effects of BMAA in neonatal rats ............................................................ 43 

Acute and short-term locomotor impairments ..................................... 43 

Long-term cognitive impairments ....................................................... 45 Effects on anxiety-like behavior .......................................................... 49 Early morphological brain changes ..................................................... 50 Glucocorticoid levels in serum ............................................................ 52 Protein expression in the brain ............................................................ 52 

Summary of findings ..................................................................................... 54 

Concluding remarks ...................................................................................... 55 

Acknowledgements ....................................................................................... 58 

References ..................................................................................................... 61 

Abbreviations

AD Alzheimer’s disease ADME absorption, distribution, metabolism, and excretion ALS Amyotrophic lateral sclerosis AMPA Amino-3-hydroxy-5-methylisoxazoleproprionic acid BBB Blood-brain barrier BGS Brain growth spurt BMAA β-N-methylamino-L-alanine BOAA β-N-oxalylamino-L-alanine CA Cornu ammonis CNS Central nervous system CORR Corridor (in the MCSF test) CTRCI Central circle (in the MCSF test) Da Dalton DCR Dark corner room (in the MCSF test) DDT Dichlorodiphenyltrichloroethane DHAP 2’,4’,-dihydroxyacetophenone DNA Deoxyribonucleic acid EDTA Ethylenediaminetetraacetic acid etc Et cetera e.g. Exempli gratia EPM Elevated plus maze HPA Hypothalamic-pituitary-adrenal i.e. Id est IMS Imaging mass spectrometry iv Intravenous KA Kainic acid L-DOPA L-3,4-dihydroxyphenylalanine MALDI Matrix-assisted laser desorption ionization MAM Methylazoxymethanol MCSF Multivariate concentric square field™ min Minutes MPP+ 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ion MPTP 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine MS Mass spectrometry mRNA Messenger ribonucleic acid m/z Mass to charge

N Novel object (in the object recognition test) NE North east (quadrant in the water maze) NFT Neurofibrillary tangles NMDA N-methyl-D-aspartate NW North west (quadrant in the water maze) OF Open field OR Object recognition PCA Principal component analysis PCB Polychlorinated biphenyl PD Parkinson’s disease PDC Parkinsonism-dementia complex PND Postnatal day PNS Peripheral nervous system RAM Radial arm maze ROS Reactive oxygen species S Samples (in the object recognition test) sc Subcutaneous SE South east (quadrant in the water maze) SEM Standard error of the mean SNP single nucleotide polymorphism SW South west (quadrant in the water maze) TEM Transmission electron microscopy TOF Time of flight tRNA Transfer ribonucleic acid TUNEL Terminal deoxynucleotidyl transferase nick end labeling UV Ultraviolet WM Water maze

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Introduction

Developmental Neurotoxicity There are a great number of industrial chemicals, environmental pollutants and toxicants as well as natural toxins in the environment that wildlife and humans can be exposed to throughout their life time. Gestation and early postnatal development are probably the most vulnerable periods of human life, in regard to adverse effects caused by environmental insults. The course of human development from conception to adulthood is extremely complex. Myriads of biochemical, physical, and organizational processes must be exactly coordinated to ensure accurate development, maintain health, and avoid disease.

The brain is the most complex human organ with its sophisticated func-tions and ability to control behavior including learning and memory, perfor-mance of a vast number of skills, interaction with the social environment etc. The human brain develops over a long period of time and the development is also exceptionally intricate compared to other organs. Unique windows of susceptibility to insults from toxic agents occur during development that have no counterpart in the mature brain [1, 2]. If the developmental processes in the immature nervous system are disturbed, the effects are like-ly to be lasting and possibly permanent, which could manifest as subclinical deficits in mental abilities or more severe behavioral disorders [3-7]. Selec-tive vulnerability of the developing organism could be due to a number of factors including differences in metabolizing enzymes, rates of excretion, and differences in the blood-brain barrier (BBB) [8]. In order for a toxic agent to directly affect brain development it first has to pass the placenta and/or the BBB. The placenta provides some protection, but xenobiotics can cross the placenta and sometimes concentrate in the fetal nervous system [9, 10]. The development of the BBB is a gradual process that starts in utero and ends after birth in humans [11, 12]. The characteristics of the early BBB differs from the adult BBB and has for instance a higher transport of some amino acids to meet the demands of the rapid growing brain [13, 14]. Toxic agents that are unlikely to enter the mature brain may pass into the develop-ing brain during this period [9].

Over the last decades evidence has been accumulating that toxicants can damage the developing human brain to produce a variety of neurodevelop-mental disorders ranging from overt toxicity at high levels of exposure down

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to subclinical dysfunction. However, pioneering studies such as the work by Byers and Lord [15] in 1943 demonstrating permanent brain damage in lead-poisoned children had little immediate scientific impact. The paradigm of early origin of human disease did not start to gain recognition until 1979 after reports such as the one by Needleman and coworkers who demonstrat-ed dose-related mental deficits in children using retained lead in teeth as a marker of exposure [16, 17].

Today a growing number of synthetic chemicals are implicated in the causation of neurodevelopmental disabilities. Chemicals identified as human developmental neurotoxicants include lead, methylmercury, polychlorinated biphenyls (PCBs), arsenic, manganese, organophosphate insecticides, dich-lorodiphenyltrichloroethane (DDT) and ethanol [18]. This could, however, be the tip of an iceberg. We are surrounded by thousands of synthetic chemi-cals as well as natural toxins. Less than 20% of high-volume synthetic chem-icals have been tested for neurodevelopmental toxicity and few natural tox-ins [18]. Developmental disabilities such as developmental delays, learning disabilities, and emotional or behavioral problems are common health issues [19]. Moreover, the reported increase in the incidence of neurodevelopmen-tal disabilities such as autism and the empirical data suggesting that envi-ronmental toxicants could play an important part has increased the interest for research on effects of pre- and postnatal exposure to toxicants [18, 20-24]. Recently more attention has been focused on the developmental origin of adult disease. David Barker first proposed the concept that factors during early growth and development may be predictors of adult disease (“the Barker hypothesis”) [25]. Barker studied birth cohorts and found that infants with low birth weight, low ponderal index, and small head circumference at birth had an increased risk for developing coronary heart disease, hyperten-sion, stroke, insulin resistance, and diabetes as adults [26]. The hypothesis has been extended by others to encompass brain development and it has been suggested that exposure during critical windows of vulnerability during brain development may also contribute to neurodegenerative diseases [27, 28].

Critical periods of brain development The architecture and physiology of the brain develops throughout gestation and continues postnatally through adolescence [29]. The brain develops from a strip of cells along the dorsal ectoderm of the embryo into a complex organ consisting of billions of precisely located, interconnected, and specialized cells. Insult at any point during this period may result in aberrant neuronal structure or function with outcomes ranging from structural abnormalities incompatible with survival, to ultrastructural or molecular abnormalities potentially associated with functional deficits.

After fertilization of the egg cell by the sperm a series of rapid cell divi-sions starts which generates the blastula. The cells are then arranged into

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three primary germ layers (endoderm, mesoderm and ectoderm) during the process of gastrulation [30]. The first stage of the central nervous system (CNS) development when the cell precursors of the brain and spinal cord start to develop is called neurulation. At approximately 2 weeks of gestation in humans the notochord induces the formation of the neural plate from the overlying ectodermal tissue [1]. The neural plate then starts to invaginate along its central axis to form the neural groove with neural folds on each side. The neural folds move together both cranially and caudally to fuse and form the neural tube [1, 30]. Interruption of neural tube closure can result in severe abnormalities of the brain and spinal cord.

A large part of the neural tube will give rise to the spinal cord while the rostral end forms the three primary brain vesicles: the prosencephalon (fore-brain), the mesencephalon (midbrain), and the rhombencephalon (hindbrain) [1, 30]. These brain vesicles further subdivide and during the first month of gestation specific areas of the CNS start to form with a region and time de-pendent neurogenesis and migration of cells in the forebrain, midbrain, and hindbrain (Figure 1) [31, 32]. Development continues with further prolifera-tion and migration followed by differentiation, synaptogenesis, apoptosis, gliogenesis, and myelination (Figure 2). All of these events rely upon the interplay of transcription factors, secreted signals, and their receptors, as well as adhesion and recognition molecules for accurate development. Re-cent studies have clearly shown that the brain continues to develop through-out adolescence and into adulthood [33, 34]. Different brain regions, includ-ing the prefrontal cortex and several subcortical structures undergo important structural and functional changes in synaptic plasticity and neuronal connec-tivity during the juvenile and adolescence periods [35, 36].

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Figure 1. Estimated timelines of regional neurogenesis in rats and humans. Gesta-tional day (GD), postnatal day (PND). Adapted from Bayer et al. [31].

Figure 2. Timelines of developmental processes in the nervous system. The scale are estimated time for development in rats. Gestational day (GD), postnatal day (PND). Adapted from Rice and Barone [1].

Brain growth spurt Although the human brain undergoes important changes in terms of its com-position, volume, and function during childhood and adolescence these changes in brain morphology are clearly more subtle than the changes during the first four years in life (Figure 3) [37, 38]. After the initial embryonic development of the brain, when precursors of glia and neurons proliferate and the general shape of the brain forms another phase of brain development known as the brain growth spurt (BGS) takes place. During this period rapid

GD 11 GD 12 GD 13 GD 14 GD 15 GD 16 GD 17 GD 18 GD 19 GD 20 GD 21-22 PND 0-3 PND 4-7 PND 8-11

3.5-4.0 4.1-5.2 5.3-5.7 5.8-6.6 6.7-7.0 7.1-7.4 7.5-7.9 8.0-9.9 10.0-11.9 12.0-14.9 15.0-18.9 19.0-23.9 24.0-27.9 28.0-31.9 32.0-35.9 36.0-40.0

Cerebellum

Spinal cord

Medulla

Pons

Substantia nigra

Pallidum

Striatum

Amygdala

Neocortex & limbic cortex

Entorhinal cortex

Hippocampus (CA1-3)

Hippocampus (dentate granule cells)

Time of development in humans (weeks)

PND 12-15 PND 16-19

Time of development in rats (days)

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neurodevelopmental changes such as axonal and dendritic growth, estab-lishment of neuronal connections, synaptogenesis, apoptosis, and prolifera-tion of glial cells followed by myelination take place in a strictly controlled way [2]. To achieve mature function as transmitters of signals, the neurons must form connections. Newly differentiated nerve cells send out dendrites and axons to form synaptic connections and the basic circuitries of the nerv-ous system. A typical mature neuron is covered with thousands of synapses [39]. The BGS is also characterized by proliferation of oligodendrocytes and rapid myelination. Increased myelination of axons accelerate the communi-cation between neurons and make more stable connections [36, 39]. In many parts of the CNS an excess of neurons is initially produced and the number of neurons is then reduced by periods of programmed cell death. This period of cell death typically coincides with the period of synaptogenesis. Neurons that do not receive the right number/kind of connections or get proper troph-ic signals are removed by apoptosis to render an optimal number of well-connected neurons [39, 40].

0 1 5 12 21 50 6565 80 950.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Age (years)

Bra

in w

eig

ht

(kg

)

Figure 3. Human brain weight as a function of age. The largest increases in brain weight occur during the first year of life, when the weight more than doubles that at birth. The rapid growth of the brain continuous during the first 4 years of life. Fur-ther growth of the brain is considerably slower. The human brain reaches its maxi-mum weight in early adult life and then decreases progressively from about 50 years of age. Adapted from Dekaban and Sadowsky [37].

During nervous system development, neurons often express and release neu-rotransmitters before their axons establish contacts with their target cells needed for synaptic communication. Many lines of evidence show that neu-rotransmitters in the immature nervous system can act as trophic factors that influence different developmental events including cell proliferation and differentiation [41]. The excitatory amino acid glutamate is the major excita-tory neurotransmitter in the mature brain and is important for CNS develop-ment. The glutamatergic system is involved in the modulation of many of the events occurring during the BGS but its complex role is not fully understood. Acting mainly at N-methyl-D-aspartate (NMDA), but also at kainic acid

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(KA), amino-3-hydroxy-5-methylisoxazoleproprionic acid (AMPA) and metabotropic receptors, glutamate provides trophic functions in the develop-ing brain, influencing synaptic plasticity, axonal and dendritic growth as well as growth cone guidance, promoting proliferation and migration of neu-ronal cells [41-45].

The timing of the BGS differs among species. In humans, it starts during the last trimester of pregnancy and continues through the first few years of life (Figure 3), while the corresponding period in rodents is the first 3–4 weeks after birth [37, 38, 46]. The BGS is a critical period regarding expo-sure to toxicants and neurotoxins since disturbance of the developmental processes can manifest as permanent changes in adults [47-49]. Substances that mimic or block the actions of important neurotransmitters/messengers and have transient effects in the adult may have permanent effects during brain development. The BGS is a sensitive period for glutamatergic com-pounds. Several studies in rodents have reported long-term changes in brain and behavior following neonatal exposure to glutamate receptor agon-ists/antagonists, especially those targeting the NMDA receptors [50-57]. The biochemistry and pharmacology of glutamate receptors in the developing CNS differs from those in the adult CNS. The receptors and their subunits have distinct regional and temporal expression profiles, often with transient peak levels during the BGS. For example, glutamate binding sites in the hippocampus peak at PND 9 [58] and most of the NMDA receptor subunits peak during the same period [59]. Neonatal exposure to glutamatergic com-pounds has also been shown to induce more severe neuronal injury than adult exposure [60, 61]. In addition, blockade of NMDA receptors during this period may cause widespread apoptotic neurodegeneration in the brain [62]. Animal studies during the BGS also support the concept that a wide range of industrial chemicals can induce developmental neurotoxicity at low doses that do not have harmful effects in adult animals [63, 64]. The time of exposure is of major importance, since there is regional heterogeneity in the time course of developmental trajectories. This results in area- and system-specific periods of development and maturation [65]. For example, the gra-nule cells in the dentate gyrus of the hippocampus have an exceptionally late time of origin and functional maturation (Figure 1). Approximately 85% of these neurons are generated after birth in rats, mainly during the first post-natal week [31]. Hence, depending on timing, a toxicant with the potential to interfere with a specific developmental processes could possibly affect dif-ferent brain regions and may produce a variety of neurological and behavior deficits [62].

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Natural toxins Natural toxins produced by plants, fungi, marine organisms, or animals play an important role in defense against herbivores and interspecies defense as well as predation. In nature a range of different secondary metabolites that interfere with fundamental biochemical processes shared by virtually all eukaryotes (e.g. inhibition of DNA replication and dysfunction of synaptic transmission) has evolved. Therefore some of these toxins exert potent ef-fects on organisms at virtually all levels of phylogeny, including humans. There are several known classes of natural toxins that are harmful in hu-mans, including carcinogenic aflatoxins and neurotoxic nonprotein amino acids.

Nonprotein amino acids Accurate decoding of the genetic code depends on the presence of an ami-noacyl-tRNA whose anticodon matches the mRNA codon, with the cognate amino acid esterified to the 3’-terminal adenosine of the tRNA. Natural pro-teins are synthesized via the ribosomal pathway using the same complement of 22 amino acids. However, more than 900 other amino acids, produced by plants, fungi, and microorganisms exist in nature. These nonprotein amino acids are normally not incorporated within proteins but serve as nitrogen storage and as an arsenal of antinutrients/antimetabolites aimed to protect its host. Such compounds may be present in commonly eaten foods and they have the ability to interfere with a wide range of fundamental biochemical processes [66, 67]. Some of the nonprotein amino acids are structural mimics of common amino acids and may interfere with intermediary metabolism. For example, canavanine found in several types of legumes can disturb in-termediary metabolism as it is an analogue of arginine. Arginyl-tRNA syn-thases sometimes fail to discriminate between the two amino acids which leads to misincorporation of canavanine into proteins which may disrupt fundamental life processes and cause autoimmunity disorders [67]. There are also several naturally occurring excitatory amino acids, which have the po-tential to disturb neurodevelopment. KA and NMDA are two potent excitato-ry amino acids that have been identified in several algae. These amino acids have named two subclasses of glutamate receptors and are widely used as tools in neurophysiological research. Other excitatory amino acids like the algal toxin domoic acid and the nonprotein amino acid BOAA (β-N-oxalylamino-L-alanine) have been implicated in the cause of neurological disease in humans [66]. The ingestion of mussels containing domoic acid was identified as the cause of an outbreak of gastrointestinal and neurologi-cal illness in eastern Canada in 1987. In this outbreak over 100 persons sus-tained permanent brain damage, memory impairment, and seizure activity [68]. Consumption of chickling peas (Lathyrus sativus) containing BOAA

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has been implicated as a cause of neurolathyrism in humans [66, 69]. Neuro-lathyrism is endemic in certain regions of the world and characterized by a progressive and irreversible spastic paralysis of the legs. Cynomologus monkeys fed with extract of the peas demonstrated a similar syndrome [69]. The nonprotein amino acid β-N-methylamino-L-alanine (BMAA) was first detected in Cycas micronesica (a gymnospermous tree) on the island of Guam in the Pacific Ocean [70, 71]. Dietary exposure to BMAA has been suggested to be involved in the etiology of a particular neurodegenerative disease (Amyotrophic lateral sclerosis/Parkinsonism-dementia complex; ALS/PDC) on the island [70, 71] but the role of BMAA in neurodegenera-tive disease is still controversial [72, 73].

BMAA – an environmental neurotoxin Cyanobacteria (blue-green algae) are extensively distributed and exist as free-living or symbiotic organisms in terrestrial and aquatic environments. These organisms produce numerous organic compounds, including several cyanotoxins (e.g. dermatoxins, hepatoxins, and neurotoxins) associated with harmful effects in animals and humans [74, 75]. Several studies have indi-cated that cyanobacteria can produce the neurotoxic nonprotein amino acid BMAA [76, 77] whereas other studies have not been able to detect the pro-duction of BMAA [78, 79]. Cyanobacteria are associated with surface blooms (massive increases in population) in fresh, brackish, and marine wa-ter all over the world, and recently BMAA was detected in several water systems including temperate aquatic ecosystems such as the Baltic Sea [80-82]. The presence of BMAA in mollusks and fish destined for human con-sumption suggests that BMAA may accumulate in aquatic food chains [83, 84]. Cox and coworkers have reported that BMAA is biomagnified from symbiotic cyanobacteria to cycads to fruit bats (flying foxes) on the island of Guam [70, 85]. This nonprotein amino acid has also been detected in brain tissue from ALS/PDC patients. Moreover, BMAA has been found in the brains of Alzheimer’s disease (AD) patients and ALS patients [85, 86], but so far the analytical data are conflicting and have been challenged [79, 87, 88]. Algal blooming is of increasing concern with respect to cyanobacteria since it is promoted and favored by the current eutrophication of aquatic environments and global warming [89, 90]. Hence, the human and wild-life exposure to this nonprotein amino acid may increase.

BMAA – mechanism of action BMAA is a non-lipophilic amino acid with a secondary amine in the side chain. BMAA is a zwitter ion and has a neutral net charge at physiological pH (Figure 4) [91]. It is known to activate ionotropic NMDA and AM-PA/KA receptors as well as metabotropic glutamate receptors and to induce neuronal degeneration via an excitotoxic mechanism [92-97]. The BMAA-induced activation of glutamate receptors appears to be bicarbonate (HCO3)

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dependent [98, 99]. BMAA reacts with CO2 at physiological pH to form reversible α- and β-carbamate adducts, where the latter are most structurally similar to glutamate and its selective agonist NMDA (Figure 4) [98, 100, 101]. BMAA is also one of the most avid of amino acids in chelating diva-lent metal ions such as Cu2+, Zn2+ and Ni2+ [102]. Relatively high concentra-tions of BMAA (1-3 mM) are required to induce cell death in cortical cell cultures [96, 98, 103]. However, BMAA concentrations as low as 10 μM can enhance neuronal death induced by amyloid-β or MPP+ (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ion) [93]. BMAA has also been shown to induce production of reactive oxygen species (ROS) and selective motor neuron death at 30 μM in mixed spinal cord cultures [94]. In addition, BMAA af-fects the cystine/glutamate antiporter (sytem Xc-) and inhibits cysteine up-take resulting in cellular glutathione depletion. These findings suggest that BMAA also stimulates glutamate release, which indicates transport of BMAA into cells by the sytem Xc- [104]. Moreover, BMAA has been sug-gested to be misincorporated into proteins, as other nonprotein amino acids, which could lead to disruption of protein function as well as a reservoir of BMAA that may be released over time [85, 105-110].

Figure 4. BMAA reacts with CO2 at physiological pH to form reversible α- and β-carbamate adducts that are structurally similar to glutamate. pKa1 2.10; pKa2 6.50; pKa3 9.80 [111]

Bell and co-workers first demonstrated the neurotoxic potential of BMAA in vivo [112]. They found that L-BMAA but not D-BMAA induced motor ab-normalities in young chicks and rats. The symptoms produced in chicks were general unsteadiness in standing, followed by inability to extend legs and stand straight. The birds tended to fall over and were unable to get up. In rats, dragging gait, weakness and convulsions were observed. Later they also reported similar symptoms in adult mice after an extremely high dose (2520-

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3340 mg/kg). The acute symptoms were reversible and no neuropathological changes in the brain were observed [113, 114]. In agreement with Bell and coworkers, intracerebroventriculary injected BMMA induced a unique whole-body shake/wobble in mice, which was attenuated by a NMDA recep-tor antagonist [103]. A similar study resulted in the conclusion that the acute symptoms were mediated via AMPA receptors and not NMDA receptors [115]. Others have suggested that the observed behavioral changes were mediated by several subtypes of glutamate receptors [116]. Additional in vivo studies have confirmed that BMAA is neurotoxic in rodents but only one study in monkeys could produce neurological changes similar to human ALS/PDC [71, 112]. Spencer and colleagues reported that one-year old ma-caques fed large amounts of BMAA (100-315 mg/kg daily for up to 12 weeks) developed clinical, electrophysiological, and neuropathological evi-dence of ALS-like motor dysfunction with parkinsonian features. Degenera-tive changes of motor neurons in cerebral cortex and spinal cord were found. However, administration of BMAA in primates produced an acute/subacute toxicity model rather than simulating the long latency associated with the development of ALS/PDC. Furthermore, no BMAA-treated monkeys had extrapyramidal and AD-type neuropathology with extensive neurofibrillary tangles (NFTs) which is found in ALS/PDC [71]. BMAA is considered to have a relatively low neurotoxic potency in adult rodents. For example, large amounts of BMAA administrated by gavage to adult mice over 11 weeks did not induce behavioral, neurochemical or neuropathological changes [117]. These mice received 500 mg/kg daily for 18 days, 500 mg/kg every other day over 28 days, and 1000 mg/kg every other day for a further 30 days [117].

The oral bioavailability has been shown to be rapid and almost complete in both rats and primates [118, 119]. The metabolism of BMMAA appears to be limited as in vitro studies have not demonstrated any significant decrease in BMAA concentration during incubation with homogenates from liver, muscle, kidney and brain for two hours [118]. However, one recent in vitro study suggests that metabolism may be of importance [120]. The access of BMAA to the adult rodent brain is reported to be low and appears to be re-stricted by the large neutral amino acid transporter in the BBB [118, 121]. However, as the characteristics of the developing BBB differ from those of the adult; the developing BBB can permit xenobiotics and toxins to enter the brain [11, 14]. The permeability of the neonatal BBB to BMAA and the ex-tent of transfer of this neurotoxin to the neonatal brain have not previously been examined, nor has the transplacental transfer of BMAA to the fetal brain been investigated. The knowledge of the effects of developmental ex-posure to BMAA is also limited. A previous report in which BMAA (500 mg/kg) was administered on postnatal day (PND) 2 and PND 5 showed no major short- or long-term behavioral effects on rats [122]. This study fo-cused on neurochemical effects and observed changes that appeared to be

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related to modification of neuronal development rather than to a direct exci-totoxic action [122].

ALS/PDC and retinal pigment epitheliopathy of Guam Neurodegenerative diseases are characterized by selective and progressive loss of specific neuronal populations. It is a heterogeneous group of patholo-gies that includes both complex multifactorial diseases and monogenic dis-orders. There are genetic underpinnings in most of the common neurodege-nerative disorders, but only a small fraction of cases are due to causative mutations in defined genes [123]. Three of the major neurodegenerative diseases AD, Parkinson’s disease (PD) and ALS are known to have complex multifactorial pathologies. A small group has a strict familial etiology while the majority of cases are sporadic with unknown cause [123-125]. The inter-est of the role of environmental factors in the pathogenesis of neurodegener-ative disorders was intensified by the discovery of MPTP (1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine), an illicit drug impurity that was the first environmental chemical that could be causally linked with specific do-paminergic denervation and parkinsonism in humans [126]. Epidemiological studies have shown that exposure to certain pesticides may increase the risk of developing PD [127, 128]. The risk may also increase when pesticide exposure occurs early in life [129]. Studies of monozygotic and dizygotic pairs of twins conclude that environmental factors are a major etiologic component of neurodegenerative diseases [130, 131]. In most sporadic cases of neurodegenerative disorders environmental factors in combination with genetic susceptibility probably contributes to the onset of the disorders.

The taupathy ALS/PDC primarily found on the island of Guam is proba-bly the most enigmatic of all neurological diseases and characterized by extensive formation of NFTs [132]. The clinical picture of ALS/PDC is complex with ALS or PDC or both [133, 134]. Typical onset of PDC is 10 years later than ALS [135]. In the 1950s the ALS/PDC incidence and preva-lence for residents of Guam was much higher than for similar neurodegener-ative disorders in the rest of the world [135]. Since 1955 the ALS/PDC inci-dence has steadily declined and the age of onset increased [136-138]. The cause of the disease is unknown. Genetic factors may be of importance since cases cluster among families to an extent, but no obvious pattern of inherit-ance has been identified [137-139]. Despite detailed screening over many years, no causative gene has been found [73, 140]. Studies of apolipoprotein E (Apo-E) and tau genes important for NFT formation revealed no causative mutations [140-142], but three single nucleotide polymorphisms (SNPs) were detected in tau and suggested to be a mild risk factor [143]. Several migrants from Guam have developed ALS/PDC after long absence from the island. This lead Garruto and coworkers to suggest that the etiological

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process had occurred in utero, infancy, childhood, or adolescence [144, 145]. Environmental factors such as calcium deficiency and infectious agents have been investigated as possible causes for ALS/PDC but they have not been proven to be part of the etiology [73, 144, 146-148]. Epidemiological studies found that traditional food was significantly associated with the incidence of ALS/PDC [149]. The rapid change in incidence and onset of ALS/PDC, suggests that the disease is caused by environmental factors, with dietary factors as one candidate. Studies of the traditional diet identified food de-rived from seeds of the cycad Cycas micronesica as a risk factor [150]. Cy-cads and its components are known historically to induce locomotor disord-ers in livestock. Cycad flour has been shown to induce motor neuron degene-ration and limb weakness when feed to primates while other similar studies did not produce any clinical signs [151]. Chronic dietary exposure to washed cycad flour leads to neurodegeneration in mice [152]. Recent studies have also demonstrated that long-term administration of the flour may induces α-synuclein and ubiquitin aggregates in the striatum as well in the locus coeru-leus and cingulate cortex in one-year old rats [153]. Chemical analyses of cycad seeds have revealed several toxins including BMAA, cycasin, methy-lazoxymethanol (MAM), BOAA, and β-sitosterol glucosids [152]. However no animal model has been able to closely mimic the clinical and pathophysi-ological findings seen in ALS/PDC patients [112, 151]. The cycad hypothe-sis and the role of BMAA in the etiology of ALS/PDC have also been highly debated [73, 154-157].

One important clue in the search for the cause of the disease could be that more than 50% of patients with ALS/PDC also have a very uncommon re-tinal disease, a pigmentary retinopathy that is often bilateral [158, 159]. In patients suffering from PDC, as in patients with idiopathic PD, the neurome-lanin-containing neurons in the substantia nigra are degenerated [160-162]. The extraordinary combination of a rare ocular disease, pigmentary retinopa-thy, and PDC with degeneration of the neuromelanin-containing neurons in substantia nigra could indicate a common link.

Properties and function of melanin Melanin is a naturally occurring pigment that colors the hair, skin and eyes. The biosynthesis of melanin mainly takes place in melanocytes [163]. These cells originate from the neural crest and migrate during embryogenesis, mainly to the basal layer of the epidermis, but also to the choroid, ciliary body, and iris of the eye, inner ear, and leptomeninges of the brain [30, 163]. Also the retinal pigment epithelium of the eye contains melanin. The retinal pigment epithelium has neuroectodermal origin and is derived from the optic vesicle [30, 163]. In general, melanin appears to protect cells and tissue. A major biological function of melanin is to attenuate UV-light penetration of

23

the skin and protect from UV-induced DNA damage. Because of the chemi-cal properties, melanin scavenges ROS, toxic free radicals, redox active met-al ions and various xenobiotics [164-167]. The large number of free carbox-ylic acid residues and semiquinones in the polymer are responsible for cation exchange properties and provide most of the ionic bindings sites in melanin. Hydrogen bonds and van der Waal’s attractions are other mechanisms that are thought to contribute to melanin’s scavenger properties [168]. The bind-ing of toxicants to melanin probably protects the cells initially. However, the binding is normally slowly reversible and melanin may therefore serve as a depot that accumulates the toxicant and slowly releases it into the cytosol [166, 167]. Certain pharmaceuticals (e.g. chloroquine) that bind to melanin has also been suggested to be involved in pigmentary retinopathy [165, 169]

Chemically there are two distinct groups of melanin, brown to black eu-melanin and yellow to reddish pheomelanin. Both types of melanin are de-rived from the precursor dopaquinone formed by oxidation of the amino acid L-tyrosine by tyrosinase [170, 171]. Dopaquinone undergoes a series of spontaneous reactions, leading to the production of eumelanin. In addition to tyrosine, the amino acid cysteine participates in melanin synthesis. Cysteine reacts with melanin precursor dopaquinone and forms the intermediates 5-S- and 2-S-cysteinyldopa which also can be incorporated into the melanin po-lymer [170, 171]. The resulting pheomelanin is significantly less efficient in binding drugs and metal ions than eumelanin where cysteinyldopa is not included in the polymer [172]. In addition eumelanin acts as a photoprotec-tive anti-oxidant while pheomelanin exhibits phototoxic pro-oxidant beha-vior that rather increases the risk of UV-induced skin damage [173]. Most mammalian pigments consist of a mixture of eumelanin and pheomelanin [174, 175]. Pheomelanin appears to be restricted to the core of the pigment granule while the surface may be covered with eumelanin [176]. Melanin is also known to incorporate “false” precursors such as thiourea in its synthesis which render a pigment with a different function and characteristics [172, 177].

Neuromelanin A closely related brown pigment, neuromelanin is mainly produced in the substantia nigra but also in catecholaminergic neurons in other brain areas such as the locus coeruleus. A considerable number of dopaminergic mid-brain cells do not produce the pigment [163, 178]. Neuromelanin shares most properties with melanin produced by melanocytes. The mechanism for neuromelanin synthesis is not fully understood and the role of tyrosinase is debated [179, 180]. The main precursor to neuromelanin is dopamine and cysteine appears to be partially incorporated. Neuromelanin is a complex polymeric multilayer system and each layer consists of a polymer of melanic groups bound to aliphatic and peptide chains [181]. It is located in organelles

24

that often are surrounded by a double membrane [182]. The initiation of pigmentation in human brain starts at approximately 3 years of age after which neuromelanin continues to accumulate in the substantia nigra during aging [183-185]. In the substantia nigra of normal subjects, neuromelanin is proposed to be neuroprotective since its synthesis removes excess of oxi-dized catechols [186] and due to the binding and accumulation of a large amount of various drugs, metals and toxicants [165, 167, 181].

In recent years, research on neuromelanin has attracted much attention because of its possible role in the etiology and pathogenesis of PD. In PD there is a selective loss of dopaminergic neurons containing neuromelanin, whereas the non-pigmented dopaminergic neurons are relatively spared [162, 187]. An early accumulation and overload of redox active iron that can par-ticipate in the formation of ROS is observed in neuromelanin in the substan-tia nigra of PD patients [188-190]. In addition, some toxicants that bind to melanin have been suggested to be involved in PD and parkinsonism [188, 191-193]. The neurotoxin MPTP selectively damages dopaminergic neurons predominantly in the substantia nigra pars compacta causing parkinsonism in humans and nonhuman primates. The active uptake of the metabolite MPP+ in dopaminergic nerve terminals via the dopamine transporter is one impor-tant factor for the selectivity [194]. In addition both MPTP and MPP+ have high affinity for melanin and MPTP-induced degeneration of dopaminergic neurons in monkeys has been reported to be correlated with the melanin content in these cells [191-193, 195]. Accordingly, the nigrostriatal tissue damage of MPTP is less prominent in rodents that lack neuromelanin [196-199]. Pretreatment with chloroquine has been demonstrated to partially pre-vent MPTP-induced parkinsonism in monkeys, possibly by blocking binding sites on the neuromelanin polymer, which supports the importance of neu-romelanin binding in MPTP-induced toxicity [200]. Langston and co-workers observed ongoing nerve cell loss in humans decades after exposure to MPTP and suggest that MPP+ bound to neuromelanin continues to exert toxicity as it is gradually released [201]. Neuromelanin may also induce a neurodegenerative process via an inflammatory mechanism since it has been revealed that extracellular neuromelanin has the ability to activate the CNS immune cells, microglia [202-204]. Extracellular neuromelanin has been observed close to activated migroglia cells in brains from patients suffering from PD as well as MPTP-induced parkinsonism [201, 205].

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Objectives

The overall objective of this thesis is to characterize the tissue distribution and developmental neurotoxicity of the mixed glutamate receptor agonist and non-protein amino acid BMAA in experimental animals. The specific aims are:

To investigate the uptake and tissue distribution of 3H-BMAA in adult pigmented and albino mice (a species lacking neuromelanin) as well as in frogs (species with neuromelanin) [Paper I]

To investigate the interaction of 3H-BMAA with the pigment me-lanin in vitro [Paper I]

To investigate the uptake and distribution of 3H-BMAA in the brain of neonatal and pregnant mice [Paper I and II]

To investigate behavioral changes during the development in pups and young rats following neonatal exposure to BMAA [Paper II]

To investigate long-term behavioral changes in adult rats follow-ing neonatal exposure to BMAA [Paper III and IV]

To investigate morphological changes and differences in protein expression in the brain of adult rats following neonatal exposure to BMAA [Paper IV and V]

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Material and methods

The methods used in this thesis are listed in Table 1. The reader is referred to the Material and method sections in the specific papers for details. The auto-radiographic technique, matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS), and the behavioral studies are further discussed.

Table 1. Methods used

Method Paper(s)

Behavioral studies The nest-finding test IIThe modified open field test IIThe inclined plane test IIThe audiogenic immobility test IIThe elevated plus maze test II, III and IVThe open field test II, III and IVThe multivariate concentric square field™ test IIIThe radial arm maze test III and IV The water maze test IVThe object recognition test IV Histological techniques Whole body tape-section autoradiography I and IILight-microscopic autoradiography IMALDI IMS VImmunohistochemistry IV and VTUNEL-staining IVTransmission electron microscopy (TEM) V Quantification of tissue radioactivity Liquid scintillation counting I Studies of melanin interaction Interaction with melanin synthesis in vitro IMelanin binding in vitro I

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Comments on methods Autoradiography In Papers I and II whole body tape-section autoradiography and light-microscopic autoradiography were used to study tissue distribution and transplacental transfer of 3H-BMAA. In order to study the possible melanin interaction both pigmented and albino mice were used. As mice lack neuro-melanin in the substantia nigra [196, 199] we also examined the uptake and binding of ³H-BMAA in the frog Rana temporaria, a species with neurome-lanin in ventral motor neurons of the brain [206].

Autoradiography is an application of the photographic effect of radioiso-topes first described by Becquerel [207]. Radioactive particles/energy (be-ta/gamma) localized within a solid substance is imaged by close contact to a detection media. The original detection media was X-ray film, or another media that contained a photographic emulsion. Crystals of silver halides function as independent detectors in the photographic emulsion, which re-spond to the radioactivity by the formation of an image that is made perma-nent by the process of development.

Whole body tape-section autoradiography The whole-body autoradiography technique was originally developed by Ullberg in 1954 [208]. The technique makes it possible to detect radiola-beled substances in all organs and tissues of an intact animal body. No prior hypothesis regarding the distribution is required. The isotopes which are widely used for labeling the test substances are 14C, 3H, 35S, 32P, 131I, or 125I [207]. A radiolabeled test substance is administrated to an animal followed by euthanasia and snap freezing of the animal. The animal is embedded in a carboxymethyl cellulose block, and tissue sections are collected onto adhe-sive tape. The tissue sections are dehydrated and mounted on X-ray film. In order to study the distribution of irreversibly tissue bound radioactivity addi-tional tissue sections can be extracted stepwise with trichloroacetic acid and solvents. After exposure for a defined time images are obtained. Whole-body autoradiography is commonly used by pharmaceutical companies in absorp-tion, distribution, metabolism, and excretion (ADME) studies of new drug candidates. The key strength of the technique is that it shows true tissue dis-tribution of radiolabeled test substances in a relatively unaltered sample. The major drawback is that the technique provides data on the concentration of radioactivity only, which may include the parent substance and its metabo-lites and/or degradation products and exclude metabolites without the la-beled atoms.

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Light-microscopic autoradiography In Paper I, microscopic autoradiography was used in order to be able to study the cellular localization of 3H-BMAA in paraffin-embedded samples of frog brain. After treatment with radiolabeled compound, the animal is sacrificed and tissues are dissected and snap frozen or immersed in liquid tissue fixation such as formalin. In Paper I the frog skulls were fixed in for-malin, decalcified in 5% EDTA for several days and then stepwise extracted and dehydrated in an ethanol series before paraffin embedding. This proce-dure is expected to extract unbound labeled substance, leaving the incorpo-rated/bound fraction in the tissue section. After sectioning the tissue is mounted on glass microscope slides, which are dipped in liquid film emul-sion. Following exposure, the slides are developed before being stained us-ing histological staining protocols. Microscopic autoradiography gives qua-litative information at the cellular level but cannot provide good quantitative results as a result of the inability to ensure uniform emulsion thicknesses [209].

Matrix-assisted laser desorption ionization imaging mass spectrometry In Paper V, MALDI IMS was used to investigate differences in protein ex-pression in adult brain after neonatal exposure to BMAA. Caprioli and co-workers introduced a MALDI time of flight (TOF) IMS approach for spatial analysis of large molecular species directly on tissue sections in 1997 [210]. This technology allows analysis and visualization of proteins, peptides and lipids as well as administrated substances and their potential metabolites, provided that they have sufficient ionization efficiency [209]. The major steps leading to the introduction of IMS were the development of suitable mass spectrometry (MS) instrumentation. MALDI MS is a soft ionization technique that gives the opportunity to study intact macromolecules in solid samples and is usually the technology used in IMS studies. It is based on laser irradiation induced desorption and ionization of analytes that are incor-porated into a crystalline matrix. There are several different chemical ma-trices that are suitable for different analytes. In Paper V, a mixture of sina-pinic acid and 2’,4’,-dihydroxyacetophenone (DHAP) optimized for analysis of proteins was used. After laser irradiation energy is transferred to the ana-lyte which makes it vaporize and ionize. The molecular species, typically singly protonated, are separated and individually detected according to their mass to charge ratio (m/z) in the TOF-mass analyzer. The key strengths of MALDI TOF are high resolution, sensitivity and insensitivity to sample im-purities.

A common MALDI IMS experiment starts with the sectioning of sample tissue. The sections are mounted on conductive MALDI glasses and the ma-

29

trix solution is applied on the section by sprayers or robotic spotters. The mass spectra are collected in a raster with distinct spatial resolution and can later be used to visualize the distribution of each detected analyte (Figure 5). The ability of the technique to give spatial localization together with quantit-ative estimates of the analyte is of particular importance in brain research since the brain is highly somatotopically organized. The spatial resolution depends on the diameter of the focused laser spot (typically 20-100 μm) [209]. Robotic matrix spotters further limit the spatial resolution (~300 μm) but have the advantage of high reproducibility [211]. MALDI IMS has the capability to analyze several types of compounds simultaneously, with high molecular specificity and sensitivity, and does not have to rely on known chemistry/protein species as traditional histological techniques such as im-munohistochemistry. Thus, no prior hypothesis regarding mechanism of action, biological function etc is required. However, since MALDI IMS cha-racterizes molecules exclusively on the accurate mass of an analyte, addi-tional validation experiments are often necessary.

Figure 5. Experimental design of MALDI imaging. Frozen tissue samples are cut and mounted on conductive glass slides. MALDI matrix is applied before mass spectra are collected in a quadratic pattern with a defined lateral resolution (e.g. 300 µm). The peak intensity distribution of individual peaks is visualized using user-defined colors.

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Behavioral tests Behavior is the final output of the nervous system at the level of the whole organism. Investigation of discrete animal behavior can be used to test hypo-theses about anatomical, neurochemical, neurophysiological, and genetic substrates of behavior [212, 213]. The results and insights in experimental animals are to a large extent generalizable to humans. The comparative ap-proach that relies on animal models is based on the evolutionary theory and the assumption that fundamental aspects of the behavior of humans have a genetic basis with a common evolutionary trajectory (i.e. are shared with other animals) [214]. Much of the current knowledge on human biology originates from research in experimental animals. However, animal models are often limited and crude approximations of the human condition since brain functions in humans are more highly organized and phylogenetic closeness does not guarantee involvement of the same subsystems in differ-ent processes [214].

Animal models can be used to investigate how substances (e.g. drug can-didates and toxicants) modulate molecular events in the nervous system and produce differences in behavior. Rats are generalists, found in virtually every ecological niche on earth and the primary species used in behavioral neuroscience [213]. Rats are also the animals of choice for performing neu-rotoxicity and developmental neurotoxicity testing. Developmental neuro-toxicity studies in rats are recognized as good predictors for a compounds potential to affect human neurodevelopment [215]. In principle, the devel-opment of the nervous system of all mammals follows the same schedule and is comparable with the brain development in humans. However, there are vast differences in the period of BGS in relation to the time of birth as discussed above. The newborn rat is capable of some specific activities such as suckling and vocalizing. However, the pup is essentially motionless, its tactile sensitivity is not fully developed, and its ear canals and eyes remain closed until approximately two weeks after birth. At present, there is little known of how the correct contacts are formed and how dendrites and axons are organized into circuits that control behaviors. Clearly the development of behavior cannot occur until the functional integrity of neuronal units re-quired to perform the behavior has matured. Thus the BGS is a period essen-tially marked by acquisition of new motor and sensory capabilities in ani-mals [216-218].

The knowledge of the effects of developmental exposure to BMAA is li-mited. In Papers II, III and IV short- and long-term behavioral effects of neonatal BMAA exposure were investigated in rats. In order to study beha-vioral changes, pups were given one daily subcutaneous (sc) injection of BMAA at 50 mg/kg, 200 mg/kg, 600 mg/kg or vehicle, for 2 days (PNDs 9 and 10). Sc injections were used to have a controlled exposure as feeding by gavage in small pups can be uncontrolled.

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Test of early neurobehavioral development In Paper I, developmental effects of BMAA exposure were studied by look-ing at developmental landmarks such as eye opening and by using a series of behavioral tests. Olfactory discrimination, locomotor development and the hardwired behavior to not leave the nest were studied in the nest-finding test. The nest-finding ability increases significantly during the early postnatal development in rodents [219, 220]. Early locomotor ability and activity of the rat pups was also tested using a size adjusted open field test. Muscle performance was tested in an inclined plane test (Figure 6), where the maxi-mum angle at which the pup could maintain itself on the plane was recorded [221, 222]. Furthermore, the audiogenic immobility reaction (freezing) was studied. Freezing is a passive defensive mechanism that first appears around PND 16 in rats and reaches is developmental peak around 3 weeks of age [223, 224].

Figure 6. The inclined plane

The elevated plus maze test The elevated plus maze (EPM) test is one of the most widely used tests for assessment of anxiety-like behavior [225]. The EPM consists of two en-closed and two open arms which represent an aversive environment for the rodent (Figure 7). The intrinsic fear for open and narrow elevated spaces is used for interpretation of anxiety-like behavior. The animal can seek shelter in the enclosed arms or explore the aversive open arms. Elevated open arm activity is generally considered to be a characteristic of low anxiety-like behavior [225, 226] but has also been suggested to be indicative of a flight response [227]. The light intensity is an important variable in the EPM test as low light intensities can attenuate the aversive effect of the open arms [225, 226]. Also the total trial time is of importance as the activity declines over time [228]. Repeated testing in the arena is not suitable for assessing anxiety-like behavior since the animals habituate to the open arm and de-crease their exploration [229].

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Figure 7. The elevated plus maze

The open field test The open field (OF) test is used for studies of general exploration, locomotor activity, and emotional reactivity. The arena is usually square or circular in its form and divided into peripheral and central zones (Figure 8). The origi-nal interpretation of behavior in the OF test was that an animal displaying high anxiety-like behavior would demonstrate low ambulation and high de-fecation [230]. Generally, entrance to and time spent in the central zones indicates increased exploration and reduced anxiety-like behavior, in con-trast to more time spent close to the wall (thigmotaxis) [231, 232]. Light intensity affects the behavior in the same manner as in the EPM test. Thus, dimmed light should be used if the focus is to study locomotion [225]. Re-peated trials in the OF test can be used to study habituation learning.

Figure 8. The open field arena

The multivariate concentric square field™ test The multivariate concentric square field™ (MCSF) provides several areas for the animal to explore including sheltered, open, and elevated areas, a holeboard device, and areas with different illumination (Figure 9). The pur-pose of the multivariate design is to gather parameters into functional cate-gories i.e. general activity, exploration, risk assessment, risk taking, and shelter-seeking behavior, which enables a behavioral profiling of the animals

CENTER

MIDDLE CIRCLE

OUTER CIRCLE

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[233]. The functional interpretation of the MCSF has been described in de-tail elsewhere [234, 235]. The cognitive part of the test can include an aver-sive stimulus (air puff) administered at the BRIDGE ENTRANCE in the MCSF test followed by assessment of the experience in a second trial. This test paradigm has been useful for evaluation of avoidance learning [236]. The strategy is similar to that of passive avoidance [218], with the advantage of collection of additional behavioral parameters.

Figure 9. The multivariate concentric square field™

The object recognition test The object recognition (OR) test is a non-spatial cognitive test based on the natural behavior of animals to spend more time exploring a novel, rather than a familiar, object [237]. The test consists of three different phases. First the animals are familiarized with the test environment (habituation phase). In the following sample phase the animals are presented to two identical objects the samples (S). The discrimination phase usually starts 30 min to 24 hours after the sample phase. In this phase the animals are presented to one of the familiar objects and one novel object (N). The total exploration time and the discrimination ratio, N/(N + S) are common parameters used to evaluate the result in the test [237, 238]. It is important to screen the objects before run-ning the test since the complexity of objects affects how much exploration they evoke [213]. Studies suggest that the hippocampus is not critical for object recognition memory in rodents, whereas the perirhinal cortex plays a more important role [239, 240].

The water maze test The temporal lobe, including the hippocampus, the entorhinal cortex, and the perirhinal cortex, is important for spatial memory [241]. Memory formation

CTRCI

BRIDGE

AIR PUFF

BRIDGEENTRANCE

SLOPE

CORRHURDLE

CORR

DCR

CORR

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consists of distinct phases: acquisition, consolidation and storage, and re-trieval. The consolidation process, to convert new memories into long-term memories, can take several days or weeks after an initial learning expe-rience. Spatial memory can be categorized into working memory and refer-ence memory, with potentially important differences between the substrates supporting the respective types of memory [242, 243]. Spatial working memory can be defined as the information useful during a single session of an experiment, and reference memory as the holding of information of con-tinued value across all subsequent sessions.

The water maze (WM) test is commonly used in studies of spatial learn-ing and memory [244, 245].The animals should locate a hidden platform in a water pool using navigational cues. Generally the arena is a circular tank divided into four equal quadrant zones (Figure 10). During the acquisition period several trials are conducted per day for a number of consecutive days until the latency to find the platform is short. To verify the memory of the precise location of the platform a retention (probe) test can be performed. The platform is then removed and parameters such as accurate swim direc-tion and time spent in the former platform quadrant indicates that the animal has learned the spatial location of the platform relative to the navigational cues [212, 245]. The protocol used in Paper IV with randomized starting positions and fixed platform position is primarily a test for spatial learning and reference memory. Using another protocol with fixed starting position and changed platform position it is possible to evaluate working memory in the WM test [212, 245].

Figure 10. The water maze with platform in south west quadrant

SW SE

NENW

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The radial arm maze test The radial arm maze (RAM) test is another test widely used in studies of spatial learning and memory [246, 247]. The apparatus used is a radial maze typically with eight arms (Figure 11). The animals should in a restricted time locate rewards hidden at the end of the arms without reentering any of the arms using navigational cues. Usually small food pellets are used as rewards and restricted feeding is used to further motivate the animals [246, 247]. During the acquisition period the animals are run for several consecutive days. The long-term memory can then be evaluated in a retention trial days or weeks after the last acquisition trial. The performance in the test can be evaluated using parameters such as number of pellets collected, time to col-lect all pellets and percentage of correct choices made. The protocol used in Paper III and IV, with all 8 arms baited, is mainly sensitive to impairments in spatial learning and working memory. A protocol with four of eight arms baited includes more of reference memory [212].

Both the RAM and the WM are designed to measure place learning and memory, using visuospatial cues, but they differ in several respects, which could be of importance and possibly render different results. The differences include constrained—as opposed to free—search and different types of rein-forcement, i.e. food reward in the RAM test and aversive motivation in the WM test [248].

Figure 11. The radial arm maze

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Results and Discussion

Interaction of 3H-BMAA with neuromelanin and melanin Paper I The in vivo studies revealed a general tissue distribution pattern of 3H-BMAA that was similar in adult non-pigmented NMRI mice and in pig-mented C57BL/6J mice (see below) with the exception of the pigmented tissues. In the pigmented mice there was a distinct localization of radioac-tivity in pigmented tissues such as the eye and hair follicles (Figure 12) fol-lowing a single intravenous (iv) injection of ³H-BMAA. Within the eye, the highest level of radioactivity was detected in the iris and ciliary body (Figure 13A and 13C), known to have active melanin synthesis in adult animals [165, 249]. The levels of radioactivity in the iris and ciliary body increased from 30 min to 3 hours. There was a slight uptake of radioactivity also in the posterior parts of the uvea/retinal pigment epithelium, but this uptake was much lower than in the anterior parts of the eye. The localization of radioac-tivity in the iris and ciliary body was still present 12 days after the adminis-tration of ³H-BMAA. No similar localization of radioactivity was detected in any part of the eye in the adult albino mice (Figure 13B) indicating that the retention in pigmented mice is related to the presence of melanin. As the melanin synthesis, catalyzed by tyrosinase, in the eyes of pigmented mice is very active during the first postnatal weeks [165, 249] the distribution of 3H-BMAA in the eye of 10-day old C57BL/6J mice was also investigated. In these mice there was a high uptake of radioactivity in the entire uveal tract/retinal pigment epithelium (Figure 13D).

As mice do not have neuromelanin in the pigmented dopaminergic neu-rons of the substantia nigra the uptake and distribution of 3H-BMAA was examined in the frog Rana temporaria, a species that has neuromelanin in the corresponding neurons. In adult frogs a high uptake and retention of ra-dioactivity was seen in all pigmented tissues after a single sc injection of ³H-BMAA (Figure 14). The level of radioactivity in melanin-containing tissues, such as the eye and melanocytes surrounding blood vessels and visceral or-gans, were higher than in non-pigmented tissue at all studied survival times (up to 11 days after administration). There was a distinct uptake of radioac-tivity in pigmented neurons and meninges in the brain whereas the level of radioactivity in other brain regions was relatively low (Figure 15). Light-

37

microscopic autoradiography of organic solvent-extracted brain sections demonstrated a selective localization of black silver grains corresponding to high levels of radioactivity in neuromelanin-containing neurons and in mela-nocytes localized in the meninges and adjacent to cerebral blood vessels of the brain up to 11 days after a sc injection of 3H-BMAA. The neuromelanin pigment appeared in the neuronal cytoplasm as golden-brown granules close to the nuclei in the neuronal soma. The melanin granules were co-localized with the black silver grains (Figure 15). The highest levels of radioactivity in the brain were found in neuromelanin-containing neurons in the trochlear chiasma under the optic tectum. The level of radioactivity in neuromelanin-containing neurons did not seem to decrease up to the longest studied sur-vival interval (11 days after administration). In the other melanin-containing tissues the highest uptake of radioactivity was seen at short survival times (up to 24 hours).

Figure 12. Whole-body autoradiogram demonstrating the distribution of 3H-BMAA in an adult pigmented mouse 3h after an iv injection. Eye [E], kidney [K], liver [L], hair follicles [H], pancreas [P], salivary gland [S], bone marrow [Bm], heart blood [Hb], skeletal muscle [M] and brain [B]. Black areas correspond to high levels of radioactivity.

Figure 13. Details of whole-body autoradiograms showing the distribution in the eye 3 (A and B) and 24 (C) hours after iv injection of 3H-BMAA in an adult mouse and 3 hours after a sc injection in a 10-day old mouse (D). There was a high uptake in pigmented parts of eyes in adult pigmented mice, particularly in the iris and ciliary body (A and C). No similar uptake was present in any part of the eye of adult albino mice (B). In the neonatal mice there were high levels of radioactivity in the entire uveal tract/retinal pigment epithelium (D). Black areas correspond to high levels of radioactivity.

DCA B

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The data suggest that retention of 3H-BMAA in melanin- and neuromela-nin-containing cells may result in a reservoir of this neurotoxin. The general tissue levels of 3H-BMAA in adult mice and frogs decreased during the ob-servation period. Significant amounts of 3H-BMAA were, however, still retained in melanin-containing tissues at the longest survival time, indicating a persistent association/incorporation of BMAA with melanin and neurome-lanin. Extraction of tissue-sections with a strong acid and water confirmed this observation and demonstrated that 3H-BMAA was partly associated and or incorporated in the pigmented tissues. Analysis of the binding of 3H-BMAA to melanin granules from the common cuttlefish (Sepia officinalis) confirmed that BMAA is bound to melanin with two apparent binding sites in vitro. Furthermore the in vitro studies revealed a higher association of 3H-BMAA with melanin during synthesis than binding of 3H-BMAA to pre-formed melanin (Figure 16). Together with the increased in vivo uptake and retention of ³H-BMAA in cells and tissues that actively synthesize melanin, this strongly suggests that ³H-BMAA is incorporated into the growing mela-nin polymer. This could involve a reaction between the 1,2-diamino group in BMAA and the 1,2-dicarbonyl group of ortho-quinone intermediates of me-lanin. The latter group may also be present in the preformed melanin, but at lower levels and may have lower reactivity (Shosuke Ito, personal communi-cation).

Melanin has previously been reported to incorporate “false” precursors such as thiourea in its synthesis and render a pigment with different func-tions and characteristics [172]. A comparison between pheomelanin and eumelanin illustrates the importance of the melanin structure as pheomelanin with cysteinyldopa included in the polymer, is significantly less efficient in binding drugs/metal ions and acts as a pro-oxidant rather than anti-oxidant, compared to eumelanin [172, 173]. The suggested incorporation of BMAA into melanin may possibly lead to an altered structure of the melanin poly-mer, which in turn could change the ability of the novel melanin polymer to bind metals and other molecules, and/or render the melanin polymer more susceptible to disintegration. This could result in lowered protective effects, release of high concentrations of metals and other toxins, and also an induc-tion of inflammatory responses via microglia activation.

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Figure 14. Details of tissue sections showing the kidney and testis (A) as well as liver (B) and corresponding details of whole-body autoradiograms (C and D) of a frog 11 days after a sc injection of 3H-BMAA. Arrows show melanin-containing cells and tissue. Kidney [k], testis [t] and blood [b]. Black areas correspond to high levels of radioactivity.

Figure 15. Light-microscopic autoradiograms demonstrating the distribution of non-extractable radioactivity in frog brain 11 days after a sc injection of 3H-BMAA. A selective retention of radioactivity (shown as black silver grains) is present in neu-romelanin-containing motor neurons. The pigment appears as brown granules close to the nuclei in the neuronal soma with black silver grains in close association. A detail of the solvent-extracted, hematoxylin-eosin stained tissue section is shown in higher magnification.

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Figure 16. Association/incorporation of 3H-BMAA with melanin during synthesis from dopamine or L-DOPA and binding of 3H-BMAA to preformed melanin. *p < 0.05, *** p < 0.001 compared with binding to preformed melanin (two tailed un-paired t-test).

Tissue-distribution of 3H-BMAA in mice – possible protein incorporation Paper I The non-protein amino acid BMAA does not belong to the amino acids that proteins normally consist of. In spite of this, reports have indicated that BMAA is associated to, or incorporated into, proteins [85, 105-107]. It has been suggested that association of BMAA with proteins could lead to disrup-tion of protein function as well as a reservoir of BMAA that may be released over time [105]. The whole-body autoradiographic study of 3H-BMAA in adult mice (Paper I) revealed a general tissue distribution pattern that is simi-lar to that of protein-forming amino acids such as phenylalanine [250, 251]. There was a distinct uptake of radioactivity in tissues with high cell turn-over and/or protein synthesis, such as exocrine pancreas, salivary glands, bone marrow, hair follicles, pituitary and the gastro-intestinal mucosa in mice injected with 3H-BMAA (Figure 12). Previous whole-body autoradio-graphic studies of amino acids have demonstrated retention in tissues with high cell turn-over and/or protein synthesis also after extraction of tissue-sections with strong acids that precipitate proteins, suggesting an incorpora-tion of the amino acids into proteins [250]. In analogy, the present studies demonstrated that major parts of the 3H-BMAA radioactivity in tissues with high cell turn-over and/or protein synthesis was retained following acid-extraction of tissue sections from 3H-BMAA-injected mice and frogs. These autoradiographic observations support the idea that BMAA is incorporated into proteins or that it is associated with proteins during their synthesis.

The data also revealed a high level of radioactivity in the liver and the corticomedullary region of the kidney (an area corresponding to the straight segment of the proximal tubules) up to 24 hours after injection (Figure 12). At the longest survival interval, 12 days, there was also a significant reten-

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tion of radioactivity in the kidney, with a tissue-blood ratio of >10, after injection of ³H-BMAA.

Localization of ³H-BMAA in the mouse brain Paper I and II All studied organs except the brain had a higher level of radioactivity than that of the blood (tissue-blood ratio >1) at all survival times in adult mice injected with a trace dose of 3H-BMAA. The level of radioactivity in the brain and spinal cord was lower than that of the blood up to 1 hour after 3H-BMAA injection. The brain distribution in the adult brain was homogenous with no specific uptake and binding in discrete regions. Autoradiography revealed that the level of radioactivity in the pituitary gland, choroid plexus and brain ventricles was higher than that of the brain up to 24 hours after injection. These data confirmed previous studies showing low uptake of BMAA in the rodent brain [118, 121].

In contrast, the transfer of BMAA into the neonatal brain was markedly higher after a single sc injection of 3H-BMAA in 10-day old mice and the level of radioactivity increased from 30 min to 24 hours Selective uptake was demonstrated in the striatum and hippocampus at all survival intervals studied (30 min, 3 and 24 hours). The radioactivity was present in the CA1, CA2, CA3 and dentate gyrus regions of the hippocampus. In addition, there was selective uptake in the brain ventricles, choroid plexus, and the pituitary gland at 30 min and 3 hours. Twenty-four hours after the injection, radioac-tivity was also present in the thalamus, cerebellum, brain stem, and spinal cord, as well as the striatum and hippocampus (Figure 17). The rapid binding of 3H-BMAA in some of the neonatal brain regions is most likely due to binding of BMAA to glutamate receptors in these areas. The glutamate re-ceptors and their subunits are differentially regulated during development, with distinct regional and temporal expression profiles, and often with tran-sient peak levels during the BGS [58, 252-254]. For instance, glutamate binding sites in the hippocampus peaks at PND 9 and gradually decreases to adult levels at PND 23 in rats [58]. In accordance, most of the NMDA recep-tor subunits, are highly expressed in the hippocampus and peaks in the same period [59]. It cannot be excluded that BMAA is associated to, or incorpo-rated into, proteins in the brain.

Furthermore, the autoradiographic study demonstrated transplacental transfer of 3H-BMAA after a single iv injection to pregnant mice on day 14 of gestation (Figure 18). The results revealed a distinct BBB in the dams; the level in maternal brain was lower than the level in blood at the 30 min sur-vival interval. In contrast, the level of radioactivity in the fetal tissues was higher than that in the maternal blood at 30 min and increased gradually at the later survival intervals. The general tissue-distribution in fetuses was

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homogeneous at 30 min except for slightly higher radioactivity in the eyes. At 3 h, there was high, selective uptake by the fetal CNS and the level was even higher at the longest survival interval of 24 hours (Figure 18).

BMAA appears to be transported by the large neutral amino acid trans-porter in the adult BBB [121]. However, the characteristics of the developing BBB differ from those of the adult in that some amino acids need to pass the BBB more easily in order to meet the demands of the rapidly growing brain [13, 14]. The marked uptake of 3H-BMAA in the developing brain may thus be related to facilitated transport through the BBB.

Figure 17. Details of autoradiograms showing the distribution of radioactivity in the neonatal brain 24 hours after a sc injection of 3H-BMAA to a 10-day old mouse. Hippocampus [H], cerebellum [C], striatum [S], brain stem [Bs], thalamus [T], and spinal cord [Sc]. Black areas correspond to high levels of radioactivity.

Figure 18. Autoradiogram demonstrating transplacental transfer of radioactivity to mouse fetuses 24 hours after an iv injection of 3H-BMAA to a pregnant mouse on gestation day 14. The upper figure shows the pregnant dam with fetuses and the lower figures are details from two fetuses. Heart blood [Hb], brain [B], spinal cord [Sc]. Black areas correspond to high levels of radioactivity.

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Effects of BMAA in neonatal rats Acute and short-term locomotor impairments Paper II All rat pups were closely monitored for seizure activity immediately after two BMAA injections on PND 9 and 10. The high dose of BMAA (600 mg/kg) induced delayed motor convulsions with loss of righting reflex in two animals. The pups showed emprosthotonic-like seizures with the neck, spine and limbs curled in a flexed ball-like position [255]. These acute ef-fects confirmed that the BGS is a sensitive period for glutamatergic com-pounds [60, 61]. The emprosthotonic seizures are a unique type of seizure that appears to be induced exclusively by NMDA receptor agonists in ro-dents no older than 25 days [255, 256]. Older mice receiving high doses of BMAA intracerebroventriculary have been shown to develop a transient hyperexcitable state followed by a delayed whole-body shake/wobble phase which could be selectively inhibited by pretreatment with a NMDA receptor antagonist [103]. High levels of BMAA have also been reported to induce ataxia and selective cerebellar damage after an intraperitoneal injection in rats [257]. However, the observed seizures in rat pups treated with BMAA (600 mg/kg) on PND 9-10 do not appear to be similar to the described ef-fects in older animals. The two affected pups, along with two other pups that lost the righting reflex, were sacrificed for ethical reasons and to avoid con-founding effects.

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The nest-finding test (on PND 12) and the modified open field test (on PND 13) demonstrated that the BMAA-treated pups displayed acute effects cha-racterized by hyperactivity in both treatment groups (200 and 600 mg/kg) together with ataxia in the high dose group (Figure 19). No weight loss was observed in the BMAA-treated rats. There was, however, a small, transient

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effect on body weight gain in the group receiving 600 mg/kg which resulted in a significant difference in body weight on PND 10-16. The motor impair-ments probably affected the pups’ ability to feed and may have caused this transient reduction in weight gain. These results are in contrast with the pre-vious study by Dawson et al., in which BMAA treatment on PND 2 and 5 (500 mg/kg) induced no seizures or major motor system impairment [122]. Our findings are most likely related to the BMAA exposure during a more sensitive time of the BGS [48], although it cannot be excluded that the slightly higher dose used in our study may also play a role. The acute effects observed in pups could have been caused by BMAA stimulation of the glu-tamatergic system. The autoradiographic study demonstrated selective up-take and binding of 3H-BMAA in the neonatal striatum, brain stem and cere-bellum, brain areas of importance for motor function. The cerebellum and the basal ganglia play a major role in the control and adjustment of balance and motor coordination [258]. Glutamate receptors are abundant in the basal ganglia, and especially in the striatum, where the interaction between dopa-mine and glutamate plays an important role in motor function [259]. Local injections of glutamate agonists into the striatum have been shown to en-hance locomotor activity [260, 261]. The audiogenic immobility reaction, tested on PND 19, demonstrated a reduced freezing reaction in the high dose group (Figure 20). The reduced freezing response was not caused by lower muscle strength, since there were no differences between the groups in the inclined plane test performed on PND 16. The monoaminergic systems and brain areas such as the amygdala and the periaqueductal gray are thought to be involved in the control of the audiogenic freezing response but the neuro-biological mechanisms are largely unknown [224, 262]. The shorter freezing response observed in the pups treated with BMAA at 600 mg/kg could have been caused by the hyperactivity observed in this group of animals.

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Figure 20. The audiogenic immobility reaction test on PND 19 demonstrated altered freezing behavior in BMAA-treated rats. Bars represent mean ± SEM for time spent immobile (A) and the percentage of animals that remained immobile during the total trial time, 600 s (B). *p < 0.05 compared with control.

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The acute BMAA-induced effects on locomotor activity and the impairments in motoric capacity (e.g. unsteady gait, disturbed balance) were transient and no longer detectable on PND 24-27 nor in the OF and MCSF tests at adult age, suggesting that the animals had recovered over a short period of time. Recovery could have been due to a transient stimulation of glutamate recep-tors, which would not have induced any permanent changes, or to great plas-ticity in the affected areas.

Long-term cognitive impairments Paper II–IV Although the OF test on PND 26-27 showed no differences in motoric func-tion, the results revealed that the BMAA-treated rats in the high dose group (600 mg/kg) did not habituate to the environment (i.e. significantly lowered their activity over time) the way the control rats did (Figure 21). This obser-vation is supported by the report of Dawson et al. [122], who demonstrated higher activity in neonatal rats exposed to BMAA than in controls when retesting in the OF test, and could indicate disturbed cognitive function. Ha-bituation is considered to be a simple form of learning. For rats, it is general-ly demonstrated within 10 min in an OF test and can be used as a non-specific test of ontogeny of learning [218]. The hippocampus is important for cognitive function and appears necessary for reduction in the response to an environment over time, i.e. habituation [263-265]. Together with the dis-tinct uptake of 3H-BMAA in the hippocampus of neonatal mice, this could indicate that neonatal exposure to BMAA may have effects on various cog-nitive functions.

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In order to further evaluate the effects of BMAA on cognitive processes the long-term effects of neonatal BMAA administration was studied in adult rats

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using the MCSF, RAM, WM and OR tests (Paper III and IV). In Paper III, the first MCSF trial at 10 weeks of age did not demonstrate any differences with regard to general activity, exploration, risk assessment, risk taking, or shelter-seeking behavior between the groups neonatally treated with BMAA or vehicle (Figure 22).

Figure 22. Principal component analysis (PCA) based on behavior parameters from the first MCSF trial at 10 weeks of age. The PCA score plot revealed no distinct separation of rats treated with BMAA or vehicle during the neonatal period, indicat-ing that there were no differences in explorative strategies and behavioral profiles between the groups (A). The PCA loading plot illustrating the MCSF variables in-cluded in the analysis (B).

The cognitive part of the test included an aversive stimulus (air puff) at the BRIDGE ENTRANCE (Figure 9) administered at the end of the first MCSF trial and evaluated two weeks later. Eighty-three percent and 63% of the animals in the high- and low-dose BMAA groups, respectively, entered the risk area BRIDGE in the repeated trial two weeks after the air puff expe-rience, in comparison to 29% of the vehicle-treated controls (Figure 23A). This indicates an impaired association between the aversive stimulus and the spatial location in the BMAA-treated rats. In addition, the controls, in con-trast to the BMAA-treated groups, significantly increased their initial activi-ty compared to the first trial (Figure 23B). This could be due to different memory of the first trial and/or an effect of the aversive stimulus.

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Figure 23. The percentage of animals in each group that visited the risk area (BRIDGE) in the first MCSF trial and in the repeated trial two weeks after the expe-rience of a discomforting stimulus (air puff) at the entrance to the risk area. The number on the bars represent the number of animals visiting the bridge (A). Sponta-neous activity in the MCSF test. Bars represent the total distance moved (mean ± SEM) during the first 5-min period in trial one and two (B). **p < 0.01 compared with trial one.

The 5-day acquisition period of the RAM test at 20 weeks of age revealed significant differences that were indicative of impaired spatial learning in BMAA-treated animals (Paper III). The animals in the control group passed the test (collecting all 8 pellets) in 62% of the trials during the acquisition period as compared to 24% of the trials with the BMAA-treated groups (Figure 24). The BMAA-treated animals collected less pellets, had lower percentage of correct choices and displayed an impaired performance in general compared to controls, on all days during the acquisition period in the RAM test (Figure 25). The retention test, performed two weeks after the last acquisition day, revealed no differences in long-term memory between the groups. The RAM test in Paper IV confirmed the long-term effect of BMAA on spatial learning. During the acquisition period, the BMAA-treated rats collected less pellets and showed impaired performance in general compared to vehicle-treated controls. The animals in the control group collected all eight pellets in 54% of the trials during the acquisition period, while the an-imals in the BMAA-treatment groups (50 mg/kg and 200 mg/kg) passed 33% and 6% of the trials, respectively. In contrast to the study in Paper III, the retention trial two weeks after the last acquisition trial also indicated that neonatal exposure to BMAA causes long-term memory impairment in rats. For example, both of the BMAA-treated groups made lower proportion of correct choices relative to acquisition day 5; this contrasted with the con-trols, where the proportion of correct choices in the retention trial increased significantly compared to acquisition day 5 (see Figure 8 in Paper IV).

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In Paper IV, the cognitive function was further studied in the WM test. The acquisition trials in the WM test revealed no significant difference between the controls and both BMAA-treated groups (se Figure 5 in Paper IV). All groups learned the task well, as indicated by reduced escape latencies over the five days of learning. However, the retention test (probe trial) 72 hours after the last learning session, showed impaired long-term memory in BMAA-treated animals, as indicated by the quadrant analysis. In contrast to both BMAA-treated groups, the control group had significantly reduced latency in first visiting the target quadrant in the probe trial compared to the last acquisition trial (se Figure 5 in Paper IV). Furthermore, when analyzing time spent in the different quadrants during the first 30 seconds, both BMAA-treated groups spent significantly more time in the starting quadrant than the control group. Compared to the controls, animals that received BMAA also spent less time in the target quadrant (se Figure 6 in Paper IV). The observed impairments in spatial learning in BMAA-treated animals during the acquisition period of the RAM test but not the WM test could be due to a difference in the type of memory measured (working and reference memory, respectively), important differences in the design of the tests (e.g. different types of reinforcement), but also differences in age as discussed below. The group treated with 50 mg/kg BMAA, demonstrated more ob-vious impairments in performance in the retention trials, in both tests, com-pared to the acquisition periods. Altogether, the results from the WM and the RAM tests demonstrated that there were deficits in spatial learning and in both working and reference memory in adult rats that had been exposed neo-natally to BMAA at lower doses (50 mg/kg and 200 mg/kg).

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Figure 25. The performance of rats treated neonatally with BMAA at 200 or 600 mg/kg or vehicle and tested in the RAM test. The acquisition trials were conducted at 20 weeks of age, and the retention test was conducted two weeks later. *p < 0.05, **p < 0.01 compared to control; £p < 0.05 compared to the first acquisition trial.

The OR test revealed no differences in habituation between the treatment groups and showed an intact recognition memory in adult animals treated neonatally with BMAA. It is not clear to what extent the cognitive processes involved in spatial learning, reference memory, and working memory are the same as those engaged in non-spatial learning and memory. It could be that neonatal exposure to BMAA affects brain areas and/or molecular and cellu-lar mechanisms that are more important for spatial tasks in adult animals. However, the significance of age when being tested in the different beha-vioral tests cannot be ruled out as an important factor in Paper IV. As habit-uation learning and object recognition was tested at a maximum age of 13 weeks and WM and RAM tests were conducted at the maximum ages of 17 and 26 weeks, respectively, the different outcomes could result from BMAA-induced brain changes that progress to more severe impairment with age.

Effects on anxiety-like behavior Paper II–IV In the EPM test on PND 24-25, the rats that received the high dose (600 mg/kg) spent a significantly longer total time and time per visit in the open arms than the other two groups (Paper II), which is generally considered to be a characteristic of low anxiety-like behavior [225, 226]. In contrast, the results from the OF test on PND 26-27 revealed significantly more thigmo-taxis in both groups of BMAA-treated rats, indicating anxiety-like behavior [231]. However, the original interpretation of behavior in the OF test was that an animal displaying high anxiety-like behavior would demonstrate low ambulation and high defecation [230], and these parameters did not differ between the groups. At 13 weeks of age the animals in the high-dose BMAA group still spent significantly longer time per visit on the open arms, which

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suggests that neonatal exposure to BMAA may result in long-term effects on emotional reactivity. In contrast to the observed effects in the EPM test, no differences between the groups were apparent in the OF test, or in the MCSF test where the ratio between the performance in risk and sheltered areas are suggested as an indicator of anxiety-like behavior [233, 235]. Earlier studies using the MCSF test with the EPM as a reference test have demonstrated that the MCSF test is more sensitive than the EPM test in detecting changes in emotional reactivity [234, 235, 266]. Other interpretations of high open arm activity in the EPM test, such as an increased flight response, have been proposed [227]. Furthermore, it cannot be ruled out that the altered cognitive function affected the behavior in the EPM test in rats treated with BMAA at 600 mg/kg. The behavioral studies in Paper IV confirmed that neonatal ex-posure to lower doses of BMAA (200 and 50 mg/kg) have no effect on emo-tional reactivity at adult age.

Early morphological brain changes Paper IV As previously discussed the temporal lobe, including the hippocampus, the entorhinal cortex, and the perirhinal cortex, is important for reference memo-ry and working memory [241]. The performance in spatial tasks, such as the RAM and WM tests, appears to be particularly sensitive to hippocampal dysfunction [248]. Selective hippocampal lesions also produce anxiolytic effects in rats (e.g. increased time spent in open arms of the EPM) [267-269]. Other areas such as the striatum have been suggested to have an impact on performance in spatial tasks through their involvement in acquisition of response sequences (e.g. turning right or left) [241]. In order further investi-gate BMAA-induced neurotoxicity, early morphological changes in the neo-natal brain, including the hippocampus and striatum, were investigated in Paper IV. Rats injected with BMAA at 600 mg/kg on PND 9–10 showed neuronal loss in the retrosplenial and cingulate cortices, areas that also are important for cognitive function [270, 271], and in the hippocampus, as eva-luated on PND 11. In three animals, this effect was mild and confined to occasional neurons. In the fourth rat, there was extensive neuronal loss and cell death in the dentate gyrus and CA1 segment of the hippocampus, with milder lesions in the CA3 and CA4 segments, cingulate cortex, retrosplenial granular cortex, and lateral posterior thalamic nuclei. The neuronal lesions were not associated with any reactive astrogliosis. Other brain regions such as striatum and amygdala did not show any evidence of neuronal cell death. Lower doses of BMAA (50 and 200 mg/kg) did not induce any histopatho-logical abnormalities at the level of the striatum or hippocampus/thalamus. The early neuronal cell death is probably due to an excitotoxic mechanism. BMAA is reported to induce oxidative stress and neuronal degeneration in vitro via an excitotoxic mechanism [94, 96, 104]. The intracellular mechan-

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isms responsible for excitotoxic neuronal death are not fully understood. Excessive activation of glutamate receptors leads to increased intracellular calcium, which triggers a cascade of reactions. This can cause cell death through a number of pathways and can mediate both neuronal apoptosis and necrosis [272]. The BMAA-induced cell death was mainly of apoptotic cha-racter, as shown by typical morphology and positive TUNEL staining (Fig-ure 26B).

Figure 26. Early neuronal cell death in the hippocampus (dentate gyrus) of rats treated with BMAA at 600 mg/kg. A–B TUNEL staining. The vehicle-treated con-trol rat showed normal cellularity of the dentate gyrus and no apoptotic cells in the same region (A). The rat treated with BMAA at 600 mg/kg displayed marked cellu-lar loss in the dentate gyrus, accompanied by numerous apoptotic neurons (B). All rats were treated with vehicle or BMAA (50, 200, 600 mg/kg) or vehicle on PND 9–10 and were evaluated on PND 11. Rats treated with the lower doses of BMAA did not differ from the control.

Figure 27. Hundreds of small proteins could be detected and visualized in the initial screening of sagittal brain sections using MALDI IMS (A and B). Several areas displayed BMAA-induced changes in peak intensities, including the striatum and the hippocampus (B). All rats were treated with vehicle or BMAA (600 mg/kg) on PND 9–10 and evaluated at 23 weeks of age.

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Glucocorticoid levels in serum Paper III The hippocampus is not only important for cognitive function, regulatory systems in the hippocampus also exert inhibitory actions on the hypothalam-ic-pituitary-adrenal (HPA) axis through negative feedback regulation [273]. The HPA axis is important for control of both emotional and cognitive processes. The HPA axis is the primary neuroendocrine axis that mediates the hormonal stress response which in the end stimulates the production and secretion of the glucocorticoids (primarily cortisol in humans and corticoste-rone in rodents). In general stress-induced release of glucocorticoids me-diates several beneficial effects. However, long-term or chronic activation of the HPA axis can have negative effects on many physiological and neurobe-havioral functions, including cognitive and emotional functions [274-276]. The HPA axis is highly susceptible to environmental influences (e.g. mater-nal stress) during fetal and neonatal development which can result in repro-gramming of its function and increased responsiveness to stress. Further-more, glucocorticoids have been shown to induce neuronal degeneration in hippocampus which in turn could lead to further increase in HPA axis activi-ty due to reduced feedback regulation. The glutamatergic system also plays an important role in the regulation of the HPA axis. Systemic administration of glutamate agonists activates the HPA axis in neonatal and adult rodents which increases the secretion of glucocorticoids [277, 278]. In Paper III, glucocorticoid secretion was measured in adult animals neonatally treated with BMAA. The results revealed that there was no difference in basal se-rum corticosterone levels between animals treated with BMAA or vehicle, which suggests that the observed long-term effect of BMAA treatment dur-ing the BGS is not secondary to an altered basal HPA axis function. Howev-er, it cannot be ruled out that there are differences between the groups in stimulated corticosterone release.

Protein expression in the brain Paper V MALDI IMS was used to search for changed protein expression in adult brains of rats treated neonatally with BMAA. In order to identify brain re-gions affected by BMAA an initial screening of sagittal brain sections from animals in the high treatment group (600 mg/kg) and vehicle-treated animals was performed. Hundreds of small proteins could be detected in mass range from 4000 to 20,000 Da (Figure 27B). Several brain regions displayed BMAA-induced changes in peak intensities, including the striatum and the hippocampus. Further studies were focused on coronal sections and revealed the hippocampus as the most affected. About 180 peaks displayed BMAA-induced alterations in hippocampal ion intensities. Visualization of peak ion

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distribution maps revealed that several of these proteins were localized to the CA1 area of the hippocampus, indicating that CA1 was the most affected region in BMAA-treated animals. This was confirmed by the regional analy-sis of BMAA-induced changes that revealed 222 and 102 significantly changed protein peaks in the CA1 and the dentate gyrus, respectively. This is in agreement with the histopathological examination which revealed mor-phological changes and neuronal loss accompanied by astrogliosis in the CA1 segment of hippocampus in adult rats neonatally injected with a high dose of BMAA (see Figure 6 in Paper V). Other regions of the hippocampus, striatum and substantia nigra were histologically unaffected. Previous studies have suggested that CA1 pyramidal neurons of the hippocampus are espe-cially sensitive to BMAA neurotoxicity, showing induction of cell stress markers and cell death following a direct intracranial injection in adult ro-dents [279, 280].

NMDA receptor-dependent long-term potentiation is widely believed to be the neural mechanism behind hippocampus-dependent spatial memory [243]. Pharmacological studies in rodents and studies in genetically modified mice have shown that spatial working memory and spatial reference memory are, at least in part, dependent on different information processing mechan-isms in the hippocampus. For example, specific deletion of the NMDA re-ceptor subunit NR1 in the granule cells of the dentate gyrus resulted in spa-tial working memory impairments in the absence of any effect on spatial reference memory [242]. In contrast, deletion of NR1 in the CA1 subfield of the hippocampus resulted in impairments in both types of memories [281] similar to the BMAA-induced cognitive impairments observed in the present study. Although lower doses of BMAA (50 and 200 mg/kg) caused impair-ments in learning and memory function we have not been able to identify any distinct acute or long-term morphological changes in the brain, including the CA1 region of the hippocampus. However, the MALDI IMS studies (Paper V) revealed protein changes in the striatum and hippocampus in both the studied BMAA-treatment groups, which could indicate more subtle ef-fects of BMAA on neurodevelopmental processes during development. Fur-thermore, our unpublished studies have revealed early changes in neuropep-tide expression in the striatum after exposure to low doses of BMAA (50 or 200 mg/kg).

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Summary of findings

The tissue distribution studies in this thesis demonstrated a less efficient transfer of BMAA to the adult mouse brain with no specific localization in discrete brain regions. In neonatal mice, however, there was an efficient transport across the BBB and a selective uptake of BMAA in discrete brain regions such as the hippocampus and striatum. Moreover, the placental transfer of BMAA on gestation day 14, resulted in a pronounced uptake in the fetal brain. BMAA treatment of neonatal rats during the BGS (PND 9–10) induced dose-dependent acute but transient alterations such as impaired motoric capacity and hyperactivity. The results also revealed that develop-mental exposure to BMAA leads to long-term consequences for adult per-formance in spatial cognitive behavioral tasks. The BMAA-treated animals had impairments in spatial learning and memory whereas no deficits in non-spatial learning and memory were detected, suggesting that neonatal expo-sure to BMAA affects neuronal systems that are more important for spatial tasks in adult animals. Rats injected with BMAA (600 mg/kg) on PND 9–10 showed early neuronal cell death in areas such as the hippocampus and re-trosplenial and cingulate cortices. In adulthood the CA1 region of hippo-campus was damaged showing neuronal loss accompanied by astrogliosis. Lower doses of BMAA (50 and 200 mg/kg) caused impairments in learning and memory function without any distinct acute or long-term morphological changes in the brain. However, the MALDI IMS studies revealed protein changes in the striatum and hippocampus which could indicate more subtle effects of BMAA on neurodevelopmental processes.

The autoradiographic studies showed that ³H-BMAA interacts with mela-nin and neuromelanin to the extent that pigmented cells and tissues, e.g. neuromelanin-containing neurons and the eye, had the highest level in the body after a single administration of ³H-BMAA to mice and frogs (frogs have neuromelanin). The in vivo and in vitro data demonstrated that ³H-BMAA is incorporated into the growing melanin polymer.

Furthermore the tissue distribution studies suggests that ³H-BMAA was associated to and/or incorporated into proteins since the general tissue distri-bution pattern was similar to that of protein-forming amino acids and the main part of the radioactivity in the tissues was retained following acid-extraction.

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Concluding remarks

The results described in this thesis have demonstrated that the mixed gluta-mate receptor agonist and nonprotein amino acid BMAA is a developmental neurotoxin inducing long-term cognitive impairments in adult rodents, and that the targeting of BMAA to discrete brain regions is more selective in rodent fetuses and neonates than in adult animals. These rodent data are sup-ported by a recent study in zebrafish, demonstrating severe neurodevelomen-tal aberrations following exposure to BMAA in the water [282].

Rats injected with BMAA (600 mg/kg) during the development showed acute neuronal cell death in areas such as the hippocampus and retrosplenial and cingulate cortices. In adulthood, the CA1 region of the hippocampus was revealed as the most affected brain region showing neuronal loss accompa-nied by astrogliosis. Lower doses of BMAA (50 and 200 mg/kg) caused impairments in learning and memory function without any distinct acute or long-term morphological changes in the brain. The glutamatergic system is known to be involved in the modulation of many of the events that occur during the BGS and normal CNS development requires an optimum level of glutamate receptor activity. Neonatal exposure to the glutamatergic BMAA could potentially disturb several of these processes, resulting in severe brain dysfunction without any visible morphological changes. The MALDI IMS studies also revealed protein changes in the striatum and hippocampus that could indicate more subtle effects of BMAA on neurodevelopmental processes.

The BMAA-induced changes in the rodent brain presented in this thesis are in agreement with reports showing that neonatal exposure to high doses of glutamate during the BGS induces cognitive impairments in adult ani-mals. However, the in vivo studies on glutamate often uses doses up to 4000 mg/kg injected for several days [50, 283] compared to the highest dose of 600 mg/kg BMAA on two consecutive days used in the present thesis. Neonatal administration of glutamate (2000–4000 mg/kg) has also been reported to induce specific neuronal loss in the CA1 region of the hippocampus in adult rats. However, the lowest dose of glutamate (1000 mg/kg) administered for 5 days induced no cognitive impairments or histological changes in the hippocampus [283, 284]. Although BMAA is much less toxic than glutamate in neuronal cell cultures [96, 285], the present data suggest that BMAA may be a more potent developmental neu-rotoxin than glutamate in vivo. In addition to activation of glutamate recep-

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tors and the subsequent excitotoxicity, BMAA is reported to induce oxida-tive stress and possibly misincorporate into proteins [94, 110]. The autoradi-ographic observations in this thesis support the suggestion that BMAA is associated to and/or incorporated into proteins. Further studies on the bio-chemical effects of BMAA exposure in various brain regions are needed in order clarify the long-term neurotoxicity and behavioral alterations in adult animals.

BMAA is considered to have a relatively low neurotoxic potency in adult rodents. Large amounts (totaling 15500 mg/kg) of BMAA given orally to adult mice over 11 weeks induced no behavioral, neurochemical or neuropa-thological changes [117]. Furthermore, in adult mice fed 28 mg/kg BMAA for 30 days there was no motor, cognitive or neuropathological changes [72]. In the present thesis rat pups subcutaneously injected with BMAA (50 mg/kg) for two days during the development demonstrated impairments in learning and memory function at adult age.

The suggested widespread production of BMAA by cyanobacteria in aq-uatic environments may pose a risk to aquatic wildlife and humans. Human exposure to BMAA may occur through direct contact with water-borne cya-nobacteria, via contaminated drinking water supplies or following food-chain transfer. The BGS in humans starts during the last trimester of preg-nancy and lasts for the first few years of age. Fetuses may be exposed to BMAA indirectly via maternal exposure and neonates from contaminated water or food. The excretion of BMAA in breast milk is not known. The presence of BMAA in mollusks and fish, sampled in the Baltic Sea, destined for human consumption suggests that BMAA may accumulate in aquatic food chains [83]. A recent study in Florida, USA reported levels of BMAA in several aquatic species that are comparable or even higher than those in the flying foxes used for human consumption in Guam [84]. However, there have been a number of conflicting reports on the detection and production of BMAA in cyanobacteria and it should be noted that some research groups have not been able to detect the presence of BMAA in cyanobacteria [78, 79].

The autoradiographic studies in the present thesis demonstrated that BMAA interacts with melanin and neuromelanin to the extent that pigmented cells and tissues had the highest level in the body after a single administration of ³H-BMAA to mice and frogs. The tissue radioactivity could represent the free amino acid or its metabolites. Previous in vitro studies have not demon-strated any significant decrease in BMAA concentration during incubation with homogenates from liver, muscle, kidney and brain suggesting that there is no major metabolism of this compound [118]. Moreover, the in vitro stu-dies confirmed the interaction of 3H-BMAA with melanin. Taken together these findings suggest that the tissue radioactivity observed in the present studies mainly corresponds to unchanged 3H-BMAA or the corresponding

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reversible carbamate form of BMAA [100, 101]. The results in this thesis suggest that the interaction of BMAA with melanin and neuromelanin may result in a reservoir of BMAA in pigmented tissues such as the substantia nigra and the eye in mammals and amphibians. Interestingly, the highest levels of BMAA found in flying foxes on Guam are detected in hair and skin tissue [70, 106] known to be highly melanized in this species [286-288]. The demonstrated interaction of BMAA with melanin pigment suggest that fish and amphibians may contribute to bioaccumulation during algal blooming since these species have considerable amounts of melanin in the body com-pared to mammals [289, 290].

Dietary exposure to BMAA has earlier been proposed to be involved in the etiology of the neurodegenerative disease ALS/PDC [70, 71]. A complex disease like ALS/PDC is likely to have a multifactorial etiology with both genetic and environmental risk factors and no single risk factor has been established. A potential depot of BMAA in melanin- and neuromelanin-containing cells could be a link between the ALS/PDC complex and retinal pigment epitheliopathy seen in more than 50% of the patients. However, further studies are needed to examine possible effects of BMAA interaction with melanin and neuromelanin. Rats and mice lack neuromelanin and can-not be used to study BMAA melanin interaction in the substantia nigra, but the effect of BMAA administration on the retinal pigment epithelium could be investigated in these species.

Finally, this thesis demonstrates that exposure to BMAA during the neo-natal period, at lower doses than previously used for studies in adult animals that found no adverse effects, caused cognitive impairments in adult rats. The melanin interaction could possibly contribute to the neurotoxicity of BMAA, for species with neuromelanin in the brain e.g. humans. Taken to-gether the findings in this thesis indicate that the risk to human health should be considered if the neurotoxin BMAA is significantly biomagnified in food chains.

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Acknowledgements

This work was carried out at the Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden. The work presented herein was financially supported by the Formas Re-search Council, the Helge Ax:son Johnson Foundation, the Facias Founda-tion and NN Jubileumsfond B. Travel grants from the Swedish Pharmaceuti-cal Society and the Helge Ax:son Johnson Foundation are gratefully ac-knowledged. I would like to express my sincere gratitude to all of you who has contri-buted to this thesis. First of all I want to thank my supervisor Eva Brittebo as well as my co-supervisors Nils Gunnar Lindquist and Erika Roman. Thanks for your great support and for always taking time to listen. My co-authors: Cecilia Berg, Anna-Lena Berg, Gunnel Arnerup, Anna-Karin Lindström, Jonas Bergquist, and Malin Andersson. You are all greatly acknowledged. I would also like to thank Bill Webster for valuable comments on this thesis.

Utan tvivel är man inte riktigt klok Tage Danielsson

Visst har det funnits stunder under min doktorandtid då jag tvivlat. Att stänga in sig i veckor på en djuravdelning utan fönster eller stirra 16 timmar på en fallande droppe, kan lätt ha den effekten. Kanske väcktes det även en del tvivel när jag som ny doktorand plötsligt förväntades dissekera en häst-hjärna i ett rum fullt med hästkadaver och badkar fulla av tarmar. Allt detta har dock varit lärorikt, det mesta av tiden på toxen har varit just lärorik och rolig.

Två stora anledningar till att arbetet på toxen fungerar så bra som det gör är avdelningens två hjärtan Raili Engdahl och Lena Norgren. De har sett många doktorander komma och gå men tröttnar aldrig på att hjälpa till och ställer upp i vått och torrt. Ett extra tack till Raili som har hjälpt mig med snittning och djurförsök. Det har blivit många långa och trevliga pass under åren. Tack också till Lennart Dencker för din stora gästfrihet och för ditt

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sätt att leda avdelningen. Sen vill jag uppmärksamma Björn Hellman som först väckte mitt intresse för toxikologi. Hans värdefulla råd att ta några öl och kasta tärning vid valet av ämne för examensarbete resulterade i att jag gjorde mitt arbete på toxen med Björn och hans dåvarande doktorand Maria Andersson som handledare.

Tack Eva för att du antog mig som doktorand. Det har varit många saker som pockat på uppmärksamhet under dina år som prefekt men ändå hittar du alltid tid när jag behöver hjälp. Du har lärt mig mycket under dessa år. Jag vill tacka Nils Gunnar för all hjälp samt för din positiva syn på tillvaron. Dina besök har alltid varit uppfriskande och lärorika. Tack Erika för din kunskap och ditt stora intresse för beteendeforskning. Du är en arbetsmyra som är väldigt lätt att jobba med. Hoppas det kan bli fler samarbeten i fram-tiden. Jag vill även uttrycka min uppskattning till Anne-Lie Svensson som var ett stöd i början av min doktorandtid. Synd att försöken inte gick som de skulle. Tack Malin för ett trevligt samarbete som tillslut ser ut att bära lite frukt. Det är bra för toxen att du kommit in och tillfört nya krafter.

Vill även uppmärksamma Marita Berg för hjälp med mina djurförsök. Tack också till Bengt Meyerson för goda råd och kommentarer på manuskript. Vill likaså tacka Per Andrén och hans forskargrupp med Henrik Wadens-ten och Anna Nilsson i spetsen för ett bra och trevligt samarbete.

Sen har vi alla gamla och nuvarande doktorander utan er skulle det ha varit så mycket tråkigare. Först vill jag nämna de nuvarande kvinnliga doktoran-derna Anna Karlsson, Helén Andersson och Elena Piras som bidrar med mycket till avdelningen. Anna, du har inte bara ett vackert efternamn du är också väldigt lättsam att umgås med. Tack för givande diskussioner och hjälp rörande MALDI IMS. Helén, du har bidragit med många skratt och det är alltid lika roligt att åka på konferens med dig. Jag vet ingen som kan som-na så fort på ett flygplan. Elena, du är alltid lika trevlig att prata med. Hop-pas något av våra samarbeten kan leda till en publikation. Sen har vi de två musketörerna Henrik Alm och Birger Scholz. Henrik, en begåvad musiker som slitigt många timmar i sitt hår och lyser upp han får dricka/prata om riktig öl. Jag tvivlar dock på att du klarar stora pubrundan. Birger, mannen som har haft händerna i flest kakburkar under min tid på toxen. Skämt åsido, du är väldigt omtänksam och din kunskap har drivit fram många projekt under åren. Jag vill verkligen tacka er båda för alla nära, och mysiga stunder som vi upplevt tillsammans framförallt under senaste konferensresan. Sen har vi d'Artagnan, eller rättare sagt Mats Nilsson. Vi har delat rum under de flesta av åren och jag kunde inte önska mig ett bättre sällskap. Jag tror du föredrar att prata men du är ingen dum lyssnare heller. Du ställer dessutom upp i alla lägen. Inte minst när jag missbedömer storleken på fåtöljer. Sist men inte minst har vi Måns Jergil och Kim Kultima. Måns, delar mitt stora

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idrottsintresse och har även haft vänligheten att bjuda på ett par tunnlar på fotbollsplanen. Kim, eller ska jag säga jägarkungen, eller kanske fiskarkungen det har varit kul att samarbeta med dig. Tack till er båda för att ni hjälper till att leda bort tankarna från forskning lite då och då. Sen ska jag inte glömma avdelningens triatlet Michael Stigson. Hoppas på att slå ditt maraton-rekord framöver.

Tack också till mina syskon Erika och Gustav samt till alla mina andra kompisar, ni vet vilka ni är. För att inte behöva skriva flera sidor nöjer jag mig med ett kort tack, ni är alla värda en guldstjärna. Jag lovar att de som vill ha några skrivna rader ska få det vid förfrågan, tvivla inte på det. Slutligen vill jag tacka mina föräldrar Åke och Marianne. Tack för allt stöd och hjälp genom åren. Ni betyder mycket.

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