Chemotherapy and adjuvant endocrine treatment for breast cancer has been associated with varying degrees of cognitive dysfunction, with 15-50% of women reported to experience subtle cognitive decline. While these treatments may have direct adverse consequences on neurological functioning, cancer diagnosis and treatment is also associated with many health and psychosocial factors that may decrease performance on neuropsychological tests. However, despite a growing body of literature on affected cognitive domains and observable neurological changes after chemotherapy, there has not been a thorough investigation into potentially important psychosocial and physical health mechanisms that may underlie the observed cognitive dysfunction. Therefore, the primary aim of this thesis was to evaluate the relationship between health/ treatment, psychosocial, and endocrine-related factors and cognitive dysfunction after breast cancer treatment. In addition, a smaller secondary aim was to assess the appropriateness of different methods of individual change. Chapter 1 provides a brief overview of the structure and content of the thesis. Chpaters 2 and 3 are review papers that evaluate whether there is evidence that variations in psychosocial adjustment, health and treatment factors result in cognitive changes after chemotherapy. Based on previous research, the mechanisms evaluated are endocrine-related changes (use of adjuvant endocrine treatment and chemotherapy-induced menopause); chemotherapy-induced anaemia; depression; anxiety; fatigue; quality of life; and other treatment factors (e.g. treatment duration, time since treatment, tumour stage, use of concomitant medications and co-morbid medical conditions). The impact of confounding variables such as age of participants, level of baseline functioning and methodological limitations are also considered. These two chapters have been published (refer to Appendix A for a complete list of presentations and publications arising from this thesis). The fourth and fifth chapters are methodological in nature. Chapter 4 describes methods, while Chapter 5 is a brief paper (under review) which examines methodological considerations regarding analysis of individual change in neuropsychological performance over time and across domains for women undergoing treatment for breast cancer. The sixth and seventh chapters involve empirical analyses of the data collected as part of the Cognition in Breast Cancer (CBC) study, a longitudinal study examining the causes of variation in cognitive functioning, health and well-being in women up to 2 years post-chemotherapy. Chapter 6 was an experimental study designed to investigate the acute effects of psychosocial mechanisms on cognitive functioning after chemotherapy in a sample of 157 breast cancer patients. Many of the methodological limitations identified in the review studies were addressed and the neuropsychological performance of two groups was compared, namely recently diagnosed breast cancer patients scheduled for chemotherapy (n = 136) or other forms of treatment (n = 21). Participants were assessed prior to commencing treatment and approximately one month post completion of chemotherapy (or 6 months after the first assessment). Individual cognitive impairment was examined using the Reliable Change Index, while Pearson correlations were utilised in order to investigate the effect of psychosocial and health factors on cognitive change. The results indicated that decline in haemoglobin levels and increased anxiety over the course of chemotherapy significantly predicted impairment in multiple cognitive measures, while change in specific cognitive measures was significantly associated with baseline measures of fatigue, depression and functional well-being. The impact of these findings on rehabilitation strategies for women after chemotherapy was discussed. Chapter 7 investigated whether endocrine-related changes, namely chemotherapy-induced menopause and adjuvant endocrine treatment, resulted in increased cognitive dysfunction. One hundred and thirty-six breast cancer patients were assessed using a comprehensive neuropsychological assessment over three time-points, namely pre-chemotherapy, one month and six months post chemotherapy (or at similar time-points). Linear mixed models evaluated the effects of these two factors, with little evidence found to suggest that endocrine-related factors contribute to cognitive dysfunction in breast cancer patients. Chapter 8 comprises a brief summary and overview of the entire thesis and offers overarching conclusions, strengths and weaknesses, and directions for future research. The findings of the present investigations attempt to elucidate the contributions of potentially important psychosocial and health/ treatment-related mechanisms for cognitive dysfunction after breast cancer treatment. While there was little evidence to suggest endocrine-related changes impacted on cognition, the findings linking chemotherapy-induced anaemia and baseline psychosocial measures may play an important role in identifying and treating at-risk individuals. These findings have potential research implications for the ways data is collected, analysed and presented in empirical research as well as clinical ramifications for how women are affected cognitively as well as psychologically by treatment for breast cancer.

<p>The dietary intake of polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCD) have been estimated for Swedish children. A dietary survey performed in 2003, including 4, 8-9 and 11-12 year olds, and concentrations in individual food items were combined. The food included in the study was mainly of animal origin, consisting of fish and shellfish, dairy products, meat products, eggs, animal and vegetable fats and fats from miscellaneous food products. The medium-bound intake of PBDEs (9 congeners) were estimated to 23.0 ng/day, 30.9 ng/day and 27.7 ng/day for 4, 8-9 and 11-12 years olds respectively. The corresponding estimations for HBCD were 7.94 ng/day 10.7 ng/day and 9.46 ng/day for 4, 8-9 and 11-12 years olds respectively. These results show a higher daily intake for 8-9 year olds compared with the other age groups. However, when estimating the daily intake per kg bw, the intake decreases with age. BDE-47 contributed the most to the total intake of PBDEs, with approximately 40%. The food group contributing the most to the intake of PBDEs and HBCD was fish and shellfish, of which non-Baltic fatty fish was the largest contributor. There were no considerable differences between boys and girls in any of the aspects examined. The result from this study show a lower intake of PBDEs and HBCD in Swedish children compared with children in other studies made in Europe and the United States.</p> / <p>Bromerade flamskyddsmedel används för att skydda brännbara material från att fatta eld, till exempel skyddas textilier och plaster i bland annat elektronik, fordon och möbler. Två typer av bromerade flamskyddsmedel är polybromerade difenyletrar (PBDE) och hexabromocyklododekan (HBCD). Dessa är additiva flamskyddsmedel och blandas i materialet som ska skyddas men binder inte in i produkten och kan därför lätt läcka ut i miljön, vilket också har skett. Halter har påträffats i miljön och i biota långt från plaster där ämnena produceras eller används.</p><p>PBDE och HBCD har visats ha hormonstörande och neurotoxiska effekter i studier på råtta och mus. Thyroxinnivåerna sjunker vid exponering av PBDE och HBCD, vilket skulle kunna leda till sköldkörtelproblem och störd utveckling av bland annat hjärnan om exponering sker perinatalt. De neurotoxiska effekterna inkluderar inlärnings- och minnessvårigheter och ett förändrat beteende med hyper- och hypoaktivitet som följd.</p><p>Human exponering för PBDE och HBCD sker främst via födan och speciellt via animaliska produkter då dessa ämnen är lipofila, bioackumulerande och ofta biomagnifierande vilket gör att de påträffas i högre koncentrationer högre upp i trofinivåerna. Studier från bland annat Sverige och Finland visar att fisk och skaldjur är den största källan till intag av PBDE.</p><p>De flesta intagsberäkningar av PBDE och HBCD baseras på livsmedelskonsumtionen hos vuxna och visar följaktligen endast hur intaget ser ut för den delen av populationen. För barn, som är en av de känsligaste grupperna i populationen, finns inte många studier att tillgå, varken från Sverige eller andra delar av världen. I den här studien har därför intaget av PBDE (summan av 9 kongener) och HBCD beräknats för barn i Sverige.</p><p>I en rikstäckande kostundersökning utförd 2003 deltog barn i åldrarna 4, 8-9 och 11-12 år. De fick i en matdagbok ange sin konsumtion under fyra på varandra följande dagar. Data från denna undersökning kombinerades sedan med haltdata från olika livsmedel för att räkna ut intaget av PBDE och HBCD på individbasis. Undersökningen innefattade främst animaliska livsmedel och innehöll därför fisk och skaldjur, mejeriprodukter, köttprodukter, ägg, animaliskt och vegetabiliskt fett och fett från övriga livsmedel.</p><p>Resultaten visar att födointaget av PBDE var 23,0 ng/dag, 30,9 ng/dag och 27,7 ng/dag för 4, 8-9 respektive 11-12 åringar. Intaget av HBCD beräknades till 7,94 ng/dag, 10,7 ng/dag och 9,46 ng/dag för 4, 8-9 respektive 11-12 åringar. Detta visar att 8-9 åringar har det högsta dagliga intaget av PBDE och HBCD. När intaget beräknas på kroppsvikt däremot, har de yngsta barnen det högsta intaget som sedan sjunker med åldern. Fisk och skaldjur var den största källan till intaget av PBDE och HBCD, trots att konsumtionen av dessa livsmedel var relativt lågt. Det fanns ingen större skillnad mellan pojkar och flickor, varken i intag av PBDE eller av HBCD. Jämfört med de få studier som gjorts i andra länder, är det tydligt att svenska barn har ett lägre intag av PBDE och HBCD.</p><p>Undersökningen tyder också på att intaget av PBDE och HBCD hos svenska barn, utifrån de kunskaper vi har idag, inte utgör någon risk med avseende på de effekter av PBDE och HBCD som påträffats i toxikologiska studier. Däremot är barn i ett känsligt skede i livet och upprepad exponering samt exponering för flera miljögifter samtidigt skulle kunna påverka deras utveckling negativt.</p>

<p>This thesis examines developmental neurotoxic effects of polybrominated diphenyl ethers (PBDEs), PBDE 99, PBDE 153, and the fully brominated PBDE 209, after exposure during the newborn period in rodents.</p><p>Our environment contains vast numbers of contaminants, including the flame retardants, PBDEs. The PBDEs are widely found in the environment and are increasing in human milk. Individuals can be exposed to PBDEs during their whole lifetime, and especially during the lactation period. The neonatal period, coinciding with the lactation period, is characterized in many mammalian species by rapid growth and development of the immature brain. It has been shown that numerous toxicants can induce permanent disorders in brain function when administered to the neonatal mouse during the brain growth spurt (BGS). In mice and rats this period is postnatal, spanning over the first 3-4 weeks of life, while in humans, BGS begins during the third trimester of pregnancy and continues throughout the first two years of life.</p><p>The present studies identified a defined critical period during BGS in mice when the brain is vulnerable to insults of low doses of PBDEs and that it is the presence of PBDEs or their metabolites in the brain during this critical period that is crucial to evoking neurotoxic effects. The effects observed are permanent altered spontaneous behavior, reduced habituation, deficits in learning and memory, and disturbances in the cholinergic system. These effects worsen with age.</p><p>The ability of PBDEs to induce neurotoxic effects does not appear to be gender-, strain- or species-specific, because the neurotoxic effects are induced in rats and male and female mice of different strains.</p><p>The developmental neurotoxic effects of PBDEs are similar to those observed for polychlorinated biphenyls (PCBs) and possible interactive effects of PBDEs and other environmental contaminants are therefore of concern.</p>

This thesis examines developmental neurotoxic effects of polybrominated diphenyl ethers (PBDEs), PBDE 99, PBDE 153, and the fully brominated PBDE 209, after exposure during the newborn period in rodents. Our environment contains vast numbers of contaminants, including the flame retardants, PBDEs. The PBDEs are widely found in the environment and are increasing in human milk. Individuals can be exposed to PBDEs during their whole lifetime, and especially during the lactation period. The neonatal period, coinciding with the lactation period, is characterized in many mammalian species by rapid growth and development of the immature brain. It has been shown that numerous toxicants can induce permanent disorders in brain function when administered to the neonatal mouse during the brain growth spurt (BGS). In mice and rats this period is postnatal, spanning over the first 3-4 weeks of life, while in humans, BGS begins during the third trimester of pregnancy and continues throughout the first two years of life. The present studies identified a defined critical period during BGS in mice when the brain is vulnerable to insults of low doses of PBDEs and that it is the presence of PBDEs or their metabolites in the brain during this critical period that is crucial to evoking neurotoxic effects. The effects observed are permanent altered spontaneous behavior, reduced habituation, deficits in learning and memory, and disturbances in the cholinergic system. These effects worsen with age. The ability of PBDEs to induce neurotoxic effects does not appear to be gender-, strain- or species-specific, because the neurotoxic effects are induced in rats and male and female mice of different strains. The developmental neurotoxic effects of PBDEs are similar to those observed for polychlorinated biphenyls (PCBs) and possible interactive effects of PBDEs and other environmental contaminants are therefore of concern.

Ethanol causes neurotoxicity via several mechanisms at different points in the cycle of dependence, including neuroinflammation and oxidative stress during ethanol exposure as well as excitotoxicity during ethanol withdrawal. The primary therapeutic implication is that ethanol-induced neurotoxicity requires multifunctional pharmacotherapies which reduce all mechanisms. Using an innovative pharmacological high throughput screening method on a large plant extract library we discovered flavonoids with alpha7 nicotinic acetylcholine receptor (nAChR) activity. In addition to their well-known anti-inflammatory and antioxidant properties, this novel activity means they can potentially reduce excitotoxicity and therefore makes them ideal for inhibition of ethanol-induced neurotoxicity. Rhamnetin, the candidate compound, was first found to inhibit lipopolysaccharide induced inflammation in immortalized BV2 microglia, in part, via alpha7 nAChRs. We then established an in vitro model of ethanol induced-neurotoxicity using organotypic hippocampal slice cultures which incorporated both neuroinflammatory and excitotoxic components. Neuroinflammation enhanced excitotoxicity under control conditions but the reverse was observed during ethanol withdrawal. Both mechanisms are important but their interaction is not simple. Finally, rhamnetin was evaluated in this model and found to reduce neuroinflammation and excitotoxicity associated with ethanol withdrawal. In conclusion, the studies herein provide strong evidence for alpha7 nAChRs selective flavonoids as potential pharmacotherapies for the treatment of ethanol-induced neurotoxicity and further implicate neuroinflammation, excitotoxicity, and their interaction as critical mechanisms in this pathology.

Malaria affects millions of people annually especially in third world countries. Increase in resistance and limited research being conducted adds to the global burden of malaria. Mefloquine, known for unwanted adverse reactions and neurotoxicity, is highly lipophilic and is still used as treatment and prophylaxis. Lipid drug delivery systems are commonly used to increase solubility and efficacy and decrease toxicity. The most generally used lipid drug delivery system is liposomes. The lipid bilayer structure varying in size from 25 nm to 100 μm can entrap both hydrophilic and lipophilic compounds. Similar in structure and size to liposomes, Pheroid™ technology consist of natural fatty acids and is also able to entrap lipophilic and hydrophilic compounds. The aim of this study was to formulate liposomes and Pheroid™ vesicles loaded with mefloquine and evaluate the physiochemical characteristic of the formulations followed by efficacy and toxicity studies. Pheroid™ vesicles and liposomes with and without mefloquine were evaluated in size, morphology, pH and entrapment efficacy during three month accelerated stability testing. Optimization of size determination by flow cytometry lead to accurate determination of size for both Pheroid™ vesicles and liposomes. During the three months stability testing, Pheroid™ vesicles showed a small change in size from 3.07 ± 0.01 μm to approximately 3 μm for all three temperatures. Confocal laser scanning microscopic evaluation of the liposomes showed structures uniform in spherical shape and size. No difference in size or structure between the Pheroid™ vesicles with and without mefloquine were obtained. Significant increase (p=0.027) in size from 6.46 ± 0.01 μm to above 10 μm was observed for liposomes at all the temperatures. Clearly formed lipid bilayer structures were observed on micrographs. With the addition of mefloquine to the liposome formulation, a decrease in the amount of bilayer structures and an increase in oil droplets were found. Entrapment efficacy was determined by firstly separating the entrapped drug from the unentrapped drug utilizing a Sephadex®G50 mini column. This was followed by spectrophotometric evaluation by UV-spectrophotometry at 283 nm. Initial entrapment efficacy of both Pheroid™ vesicles and liposomes was above 60%. An increase in entrapment efficacy was observed for Pheroid™ vesicles. The addition of mefloquine to already formulated Pheroid™ vesicles illustrated entrapment efficacy of 60.14 ± 5.59% after 14 days. Formulations loaded with mefloquine resulted in lower pH values as well as a decrease in pH over time. Optimization of efficacy studies utilizing propidium iodide was necessary due to the similarity in size and shape of the drug delivery systems to erythrocytes. A gating strategy was successfully implemented for the determination of the percentage parasitemia. Efficacy testing of mefloquine loaded in Pheroid™ vesicles and liposomes showed a 186% and 207% decrease in parasitemia levels compared to the control of mefloquine. Toxicity studies conducted include haemolysis and ROS (reactive oxygen species) analysis on erythrocytes as well as cell viability on mouse neuroblastoma cells. Pheroid™ vesicles with and without mefloquine resulted in a dose dependent increase in ROS and haemolysis over time. A dose dependent increase in ROS and haemolysis in both liposome formulations were observed, but to a lesser extent. Mefloquine proved to be neurotoxic with similar results obtained when mefloquine was entrapped in liposomes. Pheroid™ vesicles seem to have neuroprotective properties resulting in higher cell viability. Mefloquine could be entrapped successfully in Pheroid™ vesicles and less in liposomes. Pheroid™ vesicles was more stable over a three months accelerated stability testing with more favourable characteristics. The increase in ROS levels of Pheroid™ vesicles could be responsible for the higher efficacy and haemolytic activity. DL-α-Tocopherol in Pheroid™ vesicles possibly acted as a pro-oxidant due to the presence of iron in the erythrocytes. DL-α-Tocopherol showed possible antioxidant properties in the neurotoxicity evaluation resulting in higher cell viability. Even though liposomes illustrated higher efficacy and little haemolysis and ROS production, no difference in neurotoxicity was observed together with unfavourable properties during stability testing makes this drug delivery system less favourable in comparison to Pheroid™ vesicles. Mefloquine was successfully incorporated into Pheroid™ vesicles resulted in high efficacy and showed possible neuroprotection and therefore makes it an ideal system for treatment of malaria. / Thesis (Ph.D. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2011

Endogenous brain compounds and xenobiotics, including the neurotoxins such as the amphetamine analogs 3,4-methylenedioxymethamphetamine (MDMA,Ecstasy), methamphetamine (METH, Speed) and methylenedioxyamphetamine (MDA, active metabolite of MDMA), may be bioactivated by prostaglandin H synthase (PHS) to free radicals that generate reactive oxygen species (ROS). In the absence of adequate antioxidant or repair mechanisms, ROS oxidize macromolecules such as DNA, protein and lipids, which can lead to toxicity. In vitro, we evaluated bioactivation using both purified ovine PHS-1 and cultured cells stably overexpressing either human PHS-1 or hPHS-2 isozymes. We found the neurotransmitter dopamine, its precursors and some metabolites, as well as METH and MDA, can be bioactivated by ovine and/or human PHS in an isozyme-dependent fashion that generates ROS, which oxidize DNA and protein and increase toxicity. This process is blocked by both the PHS inhibitor acetylsalicylic acid (ASA) and the ROS detoxifying enzyme catalase. Our data are the first to reveal isozyme-dependent bioactivation by PHS as a potential mechanism for enhanced susceptibility to both exogenous and endogenous neurotoxins, the latter of which may be particularly important in aging. METH-initiated ROS can also activate redox-sensitive transcription factors such as nuclear factor erythroid 2-related factor 2 (Nrf2), which is involved in the induction of an array of protective mechanisms in both adult and fetal brain. Using Nrf2 knockout mice, we showed Nrf2 has a novel neuroprotective role in METH-initiated oxidative stress, neurotoxicity and functional deficits in both fetal development and adulthood, especially with multiple exposures allowing time for the induction of neuroprotective mechanisms. Our studies are the first to show that Nrf2 afforded protection against both motor coordination deficits and olfactory deficits caused by METH in utero and in adults, suggesting that deficiencies in Nrf2 activation constitute a risk factor for ROS-mediated neurotoxicity in the fetus and adult.

Endogenous brain compounds and xenobiotics, including the neurotoxins such as the amphetamine analogs 3,4-methylenedioxymethamphetamine (MDMA,Ecstasy), methamphetamine (METH, Speed) and methylenedioxyamphetamine (MDA, active metabolite of MDMA), may be bioactivated by prostaglandin H synthase (PHS) to free radicals that generate reactive oxygen species (ROS). In the absence of adequate antioxidant or repair mechanisms, ROS oxidize macromolecules such as DNA, protein and lipids, which can lead to toxicity. In vitro, we evaluated bioactivation using both purified ovine PHS-1 and cultured cells stably overexpressing either human PHS-1 or hPHS-2 isozymes. We found the neurotransmitter dopamine, its precursors and some metabolites, as well as METH and MDA, can be bioactivated by ovine and/or human PHS in an isozyme-dependent fashion that generates ROS, which oxidize DNA and protein and increase toxicity. This process is blocked by both the PHS inhibitor acetylsalicylic acid (ASA) and the ROS detoxifying enzyme catalase. Our data are the first to reveal isozyme-dependent bioactivation by PHS as a potential mechanism for enhanced susceptibility to both exogenous and endogenous neurotoxins, the latter of which may be particularly important in aging. METH-initiated ROS can also activate redox-sensitive transcription factors such as nuclear factor erythroid 2-related factor 2 (Nrf2), which is involved in the induction of an array of protective mechanisms in both adult and fetal brain. Using Nrf2 knockout mice, we showed Nrf2 has a novel neuroprotective role in METH-initiated oxidative stress, neurotoxicity and functional deficits in both fetal development and adulthood, especially with multiple exposures allowing time for the induction of neuroprotective mechanisms. Our studies are the first to show that Nrf2 afforded protection against both motor coordination deficits and olfactory deficits caused by METH in utero and in adults, suggesting that deficiencies in Nrf2 activation constitute a risk factor for ROS-mediated neurotoxicity in the fetus and adult.

This study investigated the neurotoxic effects of rotenone and 6-hyroxydopamine (6 OHDA), two compounds which have been implicated in Parkinson’s disease (PD). PD is a neurodegenerative disorder that results in the impairment of movement. During the disease process, a group of dopamine-containing cells in the brain region called the Substantia Nigra pars compacta (SNc), degenerate. Whilst genetic factors contribute to approximately 5% of PD cases, the causes of the remaining 95% are unknown. What does seem clear is the pivotal role of mitochondrial dysfunction as observed in post-mortem human tissue. Mitochondrial dysfunction leads to energy depletion and the generation of harmful reactive oxygen species (ROS). However, despite the fact that the involvement of mitochondria in the disease process has been well established, the cellular events that lead to, and result from, mitochondrial dysfunction remain poorly understood. Rotenone and 6 OHDA have been implicated in PD for two reasons: (1) both toxins can relatively selectively kill SNc neurons in animal models of PD, and (2) there is evidence for both compounds having a potential causative role in the etiology of the disease in humans. When 6 OHDA is injected into the brain, or rotenone applied systemically, both toxins cause degeneration of SNc neurons. This ability makes them excellent tools for studying mechanisms of PD in animal models. In addition, both toxins inhibit mitochondrial function. Despite extensive use in models of PD, the mechanisms by which each toxin cause cell damage remains elusive. The first part of this study investigated the acute responses of dopaminergic SNc neurons to rotenone exposure (5 nM – 1 µM). The experiments were conducted on brain slices obtained from rats. Electrophysiological recordings (whole-cell patch-clamp technique) were used to detect activation of specific membrane channels as well as cell firing and changes to the membrane potential. In addition, imaging of several fluorescent dyes sensitive to specific cellular events was carried out. In voltage-clamp experiments, acute rotenone (200 nM – 1 µM) application evoked a concentration-dependent outward current which was mediated by tolbutamide-sensitive KATP channels. The current was associated with a drop in cell input resistance (Rm) and, in current-clamp, membrane hyperpolarization and inhibition of spontaneous action potentials. The mechanisms by which rotenone activates KATP channels is controversial, with some studies suggesting activation by ATP depletion and others by elevated reactive oxygen species (ROS). To address this issue, experiments were conducted with high levels of ATP in the pipette solution. Since the rotenone-induced outward current was unaffected by high ATP levels, it was concluded that KATP channel activation was due to oxidative stress. Indeed, the antioxidant Trolox significantly attenuated the current response. Confirmation of elevated ROS production was obtained by recording increased mitochondrial superoxide production, using the fluorescent dye MitoSOX. In addition, rotenone evoked depolarization of mitochondrial membrane potential (ΔΨm). Measurements of intracellular Ca2+ and Na+ were performed using the fluorescent dyes Fura 2 and SBFI, respectively. Rotenone evoked increases to both [Ca2+]i and [Na+]i in a concentration-dependent manner. The rotenone-induced [Ca2+]i rise was unaffected by blocking KATP channels with Cs+. The elevation of [Ca2+]i is particularly important in relation to cell death, since [Ca2+]i overload is known to activate pathways leading to necrosis and apoptosis. There has been growing interest in the synergistic action of rotenone with other toxins/conditions which also enhance [Ca2+]i. This concept was explored in the present study by testing the relationship between the baseline [Ca2+]i level and the rotenone-induced [Ca2+]i increase. Two approaches were taken. Firstly, baseline [Ca2+]i was deliberately raised by activation of voltage-gated calcium channels. When rotenone was applied in the presence of this raised baseline calcium level, the rotenone-induced [Ca2+]i rise was significantly greater. The second approach involved post-hoc analysis of the relationship between the normal cellular variation in baseline [Ca2+]i and the rotenone-induced [Ca2+]i elevation. This analysis also revealed a dependency of the rotenone-induced [Ca2+]i elevation on the baseline calcium level. From this finding, as well as the observation that rotenone evoked ROS production, Transient Receptor Protein subtype M2 (TRPM2) channels were proposed as the likely underlying mechanism. The potentiation of the rotenone-induced [Ca2+]i rise by an elevation in baseline calcium level can be attributed to the calcium-dependence of ROS-sensitive TRPM2 channels, known to respond with increased channel opening to increased [Ca2+]i. Recent findings from our laboratory have confirmed TRPM2 involvement in rotenone toxicity, since blockade of these channels with ACA reduced the rotenone-induced [Ca2+]i rise (K. Chung, unpublished). Imaging using the fluorescent dye propidium iodide (PI) to label cells with compromised membrane integrity was also conducted in acute midbrain slices. SNc neurons were retrograde-labelled with FluoroGold and then exposed to various toxic insults. The detergent Triton-X100 caused an increase in PI labelling, whilst rotenone and high concentrations of glutamate were ineffective over the period of time investigated (up to 40 min). The second part of this study, also conducted on acute rat midbrain slices, investigated the acute responses of SNc neurons to 6 OHDA (0.2 – 2 mM) exposure. Extracellular recordings of action potential firing were conducted on SNc neurons. 6 OHDA evoked rapid inhibition of firing in a similar manner to dopamine (100 µM). In the presence of D2 dopamine receptor blocker sulpiride, the inhibition of firing evoked by 6 OHDA was delayed, and an initial increase of firing was observed. Blockade of the dopamine transporter with nomifensine reduced the 6 OHDA-induced inhibition of firing, and prevented the persistent inhibition of firing after 6 OHDA washout. For comparison, the response to 6 OHDA of non-dopaminergic neurons in the subthalamic nucleus was also studied. In the subthalamic nucleus, 6 OHDA evoked an increase of spontaneous action potential firing. Rapid application of 6 OHDA (using the picospritz application technique) in voltage-clamp recorded SNc neurons evoked an outward current, similar to that observed after dopamine application. In the presence of sulpiride, 6 OHDA induced an inward current, consistent with the initial increase of firing activity observed in extracellular recordings. Microfluorometric experiments with Fura 2, showed that 6 OHDA evokes an increase in [Ca2+]i. Loading cells with the fluorescent dye Lucifer Yellow enabled visualization of 6 OHDA-induced swelling of the cell body and damage to proximal dendrites. Imaging of SNc neurons loaded with dextran-rhodamine revealed 6 OHDA-induced damage of distal dendrites. The last part of the study was performed on organotypic cultures obtained from slices of the ventral midbrain. These cultures were prepared from newborn transgenic mice expressing green fluorescent protein (GFP) under the tyrosine hydroxylase-promoter. This fluorescent marker enabled easy identification of dopamine-containing cells (including SNc neurons). Only preliminary experiments were carried out using this preparation. GFP-positive neurons did not show the classic membrane hyperpolarization in response to dopamine. For comparison, recordings from GFP-positive SNc neurons in acute slices obtained from age-matched animals did show a typical hyperpolarizing response to dopamine. GFP-neurons from organotypic cultures also lacked the Ih current – another characteristic feature of SNc neurons in vivo or in acute brain slices. In addition, atypical responses to CNQX (blocker of NMDA receptors) and baclofen (blocker of GABAB receptors) application were identified in GFP-positive neurons. These results demonstrate that the culturing process used in this study alters the functional ‘phenotype’ of dopaminergic neurons, a change which needs to be considered in future studies using this preparation. Chronic exposure of organotypic cultures to low concentration of rotenone (50 nM) evoked a delayed increase of PI labelling indicative of cell death, however technical limitations prevented detection of PI co-localization with GFP was observed. In conclusion, this study identified several key aspects of 6 OHDA and rotenone toxicity in SNc neurons. The most significant novel findings include evidence for ROS activation of KATP channels, presumed involvement of TRPM2 channels in rotenone-induced [Ca2+]i rise, and dopamine-analogous effects of 6 OHDA. The controversial role of KATP channels in neuroprotection was addressed. Findings from this study suggest therapies targeting this channel alone would be of little benefit. The proposed involvement of TRPM2 channels in rotenone-induced [Ca2+]i overload in SNc neurons is particularly interesting as it provides a mechanism for synergism between rotenone and other factors that disrupt [Ca2+]i homeostasis.

This study investigated the neurotoxic effects of rotenone and 6-hyroxydopamine (6 OHDA), two compounds which have been implicated in Parkinson’s disease (PD). PD is a neurodegenerative disorder that results in the impairment of movement. During the disease process, a group of dopamine-containing cells in the brain region called the Substantia Nigra pars compacta (SNc), degenerate. Whilst genetic factors contribute to approximately 5% of PD cases, the causes of the remaining 95% are unknown. What does seem clear is the pivotal role of mitochondrial dysfunction as observed in post-mortem human tissue. Mitochondrial dysfunction leads to energy depletion and the generation of harmful reactive oxygen species (ROS). However, despite the fact that the involvement of mitochondria in the disease process has been well established, the cellular events that lead to, and result from, mitochondrial dysfunction remain poorly understood. Rotenone and 6 OHDA have been implicated in PD for two reasons: (1) both toxins can relatively selectively kill SNc neurons in animal models of PD, and (2) there is evidence for both compounds having a potential causative role in the etiology of the disease in humans. When 6 OHDA is injected into the brain, or rotenone applied systemically, both toxins cause degeneration of SNc neurons. This ability makes them excellent tools for studying mechanisms of PD in animal models. In addition, both toxins inhibit mitochondrial function. Despite extensive use in models of PD, the mechanisms by which each toxin cause cell damage remains elusive. The first part of this study investigated the acute responses of dopaminergic SNc neurons to rotenone exposure (5 nM – 1 µM). The experiments were conducted on brain slices obtained from rats. Electrophysiological recordings (whole-cell patch-clamp technique) were used to detect activation of specific membrane channels as well as cell firing and changes to the membrane potential. In addition, imaging of several fluorescent dyes sensitive to specific cellular events was carried out. In voltage-clamp experiments, acute rotenone (200 nM – 1 µM) application evoked a concentration-dependent outward current which was mediated by tolbutamide-sensitive KATP channels. The current was associated with a drop in cell input resistance (Rm) and, in current-clamp, membrane hyperpolarization and inhibition of spontaneous action potentials. The mechanisms by which rotenone activates KATP channels is controversial, with some studies suggesting activation by ATP depletion and others by elevated reactive oxygen species (ROS). To address this issue, experiments were conducted with high levels of ATP in the pipette solution. Since the rotenone-induced outward current was unaffected by high ATP levels, it was concluded that KATP channel activation was due to oxidative stress. Indeed, the antioxidant Trolox significantly attenuated the current response. Confirmation of elevated ROS production was obtained by recording increased mitochondrial superoxide production, using the fluorescent dye MitoSOX. In addition, rotenone evoked depolarization of mitochondrial membrane potential (ΔΨm). Measurements of intracellular Ca2+ and Na+ were performed using the fluorescent dyes Fura 2 and SBFI, respectively. Rotenone evoked increases to both [Ca2+]i and [Na+]i in a concentration-dependent manner. The rotenone-induced [Ca2+]i rise was unaffected by blocking KATP channels with Cs+. The elevation of [Ca2+]i is particularly important in relation to cell death, since [Ca2+]i overload is known to activate pathways leading to necrosis and apoptosis. There has been growing interest in the synergistic action of rotenone with other toxins/conditions which also enhance [Ca2+]i. This concept was explored in the present study by testing the relationship between the baseline [Ca2+]i level and the rotenone-induced [Ca2+]i increase. Two approaches were taken. Firstly, baseline [Ca2+]i was deliberately raised by activation of voltage-gated calcium channels. When rotenone was applied in the presence of this raised baseline calcium level, the rotenone-induced [Ca2+]i rise was significantly greater. The second approach involved post-hoc analysis of the relationship between the normal cellular variation in baseline [Ca2+]i and the rotenone-induced [Ca2+]i elevation. This analysis also revealed a dependency of the rotenone-induced [Ca2+]i elevation on the baseline calcium level. From this finding, as well as the observation that rotenone evoked ROS production, Transient Receptor Protein subtype M2 (TRPM2) channels were proposed as the likely underlying mechanism. The potentiation of the rotenone-induced [Ca2+]i rise by an elevation in baseline calcium level can be attributed to the calcium-dependence of ROS-sensitive TRPM2 channels, known to respond with increased channel opening to increased [Ca2+]i. Recent findings from our laboratory have confirmed TRPM2 involvement in rotenone toxicity, since blockade of these channels with ACA reduced the rotenone-induced [Ca2+]i rise (K. Chung, unpublished). Imaging using the fluorescent dye propidium iodide (PI) to label cells with compromised membrane integrity was also conducted in acute midbrain slices. SNc neurons were retrograde-labelled with FluoroGold and then exposed to various toxic insults. The detergent Triton-X100 caused an increase in PI labelling, whilst rotenone and high concentrations of glutamate were ineffective over the period of time investigated (up to 40 min). The second part of this study, also conducted on acute rat midbrain slices, investigated the acute responses of SNc neurons to 6 OHDA (0.2 – 2 mM) exposure. Extracellular recordings of action potential firing were conducted on SNc neurons. 6 OHDA evoked rapid inhibition of firing in a similar manner to dopamine (100 µM). In the presence of D2 dopamine receptor blocker sulpiride, the inhibition of firing evoked by 6 OHDA was delayed, and an initial increase of firing was observed. Blockade of the dopamine transporter with nomifensine reduced the 6 OHDA-induced inhibition of firing, and prevented the persistent inhibition of firing after 6 OHDA washout. For comparison, the response to 6 OHDA of non-dopaminergic neurons in the subthalamic nucleus was also studied. In the subthalamic nucleus, 6 OHDA evoked an increase of spontaneous action potential firing. Rapid application of 6 OHDA (using the picospritz application technique) in voltage-clamp recorded SNc neurons evoked an outward current, similar to that observed after dopamine application. In the presence of sulpiride, 6 OHDA induced an inward current, consistent with the initial increase of firing activity observed in extracellular recordings. Microfluorometric experiments with Fura 2, showed that 6 OHDA evokes an increase in [Ca2+]i. Loading cells with the fluorescent dye Lucifer Yellow enabled visualization of 6 OHDA-induced swelling of the cell body and damage to proximal dendrites. Imaging of SNc neurons loaded with dextran-rhodamine revealed 6 OHDA-induced damage of distal dendrites. The last part of the study was performed on organotypic cultures obtained from slices of the ventral midbrain. These cultures were prepared from newborn transgenic mice expressing green fluorescent protein (GFP) under the tyrosine hydroxylase-promoter. This fluorescent marker enabled easy identification of dopamine-containing cells (including SNc neurons). Only preliminary experiments were carried out using this preparation. GFP-positive neurons did not show the classic membrane hyperpolarization in response to dopamine. For comparison, recordings from GFP-positive SNc neurons in acute slices obtained from age-matched animals did show a typical hyperpolarizing response to dopamine. GFP-neurons from organotypic cultures also lacked the Ih current – another characteristic feature of SNc neurons in vivo or in acute brain slices. In addition, atypical responses to CNQX (blocker of NMDA receptors) and baclofen (blocker of GABAB receptors) application were identified in GFP-positive neurons. These results demonstrate that the culturing process used in this study alters the functional ‘phenotype’ of dopaminergic neurons, a change which needs to be considered in future studies using this preparation. Chronic exposure of organotypic cultures to low concentration of rotenone (50 nM) evoked a delayed increase of PI labelling indicative of cell death, however technical limitations prevented detection of PI co-localization with GFP was observed. In conclusion, this study identified several key aspects of 6 OHDA and rotenone toxicity in SNc neurons. The most significant novel findings include evidence for ROS activation of KATP channels, presumed involvement of TRPM2 channels in rotenone-induced [Ca2+]i rise, and dopamine-analogous effects of 6 OHDA. The controversial role of KATP channels in neuroprotection was addressed. Findings from this study suggest therapies targeting this channel alone would be of little benefit. The proposed involvement of TRPM2 channels in rotenone-induced [Ca2+]i overload in SNc neurons is particularly interesting as it provides a mechanism for synergism between rotenone and other factors that disrupt [Ca2+]i homeostasis.