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The midbrain dopamine system is involved in many aspects of purposeful behaviour and, when compromised, can have devastating effects on movement and cognition as seen in diseases like Parkinson's. In the healthy brain, dopamine neurons are thought to play particularly important roles in learning by signalling errors in reward prediction. The objective of this thesis was to investigate the diversity in the functional properties of the midbrain dopamine system, and how this is altered through genetic variation of relevance to Parkinson's and development of cell phenotype. This objective was addressed with a combination of behavioural experiments, in vivo single-cell recording and labelling (both in anaesthetised as well as awake rodents), immunofluorescence labelling, retrograde tracing and stereology. In a first set of experiments, it was demonstrated that chronic as well as acute genetic challenges can alter the firing patterns of midbrain dopamine neurons. Using a novel bacterial artificial chromosome-transgenic rat model, it was shown that the R1441C mutation in human leucine-rich repeat kinase 2, which is linked to Parkinson's, leads to motor deficits and an age-dependent reduction in the in vivo firing variability and burst firing of substantia nigra pars compacta (SNc) dopamine neurons. These findings help reveal processes of early, pre-degenerative dysfunction in dopamine neurons in Parkinson's. Similar effects on firing variability and burst firing of SNc dopamine neurons were found in a mouse model with conditional knock- out of the transcription factors Forkhead box A1 and A2 (FoxA1/2) in midbrain dopamine neurons. These findings indicate that FoxA1/2 are not only crucial for the early development of dopamine neurons, but also their function in the mature brain. In a second set of experiments in wildtype mice, it was demonstrated that midbrain dopamine neurons (located in SNc and ventral tegmental area) show diverse expression of the molecular markers Calbindin, Calretinin, Aldh1a1, Sox6, Girk2, SatB1 and Otx2. It was found that selective expression of these markers is of use for discriminating between midbrain dopamine neurons that project to dorsal striatum or nucleus accumbens. To elucidate whether the diverse molecular marker expression would map onto dopamine neurons whose firing correlates with distinct behavioural events, midbrain dopamine neurons were recorded and labelled in head-fixed awake mice either exposed to neutral sensory stimuli or performing a classical conditioning paradigm. The population activity of midbrain dopamine neurons was not modulated by neutral sensory stimuli. Interestingly, fewer than 50% of identified dopamine neurons showed phasic firing increases following reward- predicting cue and/or reward delivery, despite the common assumption that most (if not all) midbrain dopamine neurons signal reward prediction errors. Instead, firing was modulated by other explanatory factors, such as licking, or showed no modulation during the task. Response types of midbrain dopamine neurons were not correlated with their anatomical location nor the selective or combinatorial expression of the markers Aldh1a1, Calbindin and Sox6. In conclusion, the first set of experiments identified how different genetic burdens can alter the in vivo firing of midbrain dopamine neurons, and provide new insights into how circuits can change in pathological or compensatory ways at early disease stages in Parkinson's. The second set of experiments revealed striking heterogeneity of midbrain dopamine neurons in the intact system, and established further a functional diversity in the response types of identified midbrain dopamine neurons that is only partially consistent with canonical reward prediction error signalling.

Parkinson’s disease is an age-related neurodegenerative disorder. The major symptoms of Parkinson’s disease are closely linked to the pathology of the disease. The main pathology of Parkinson’s disease consists of the degeneration of neurons of the substantia nigra pars compacta (SNpc), which leads to reduced amounts of dopamine in the brain. One of the treatment strategies in Parkinson’s disease is to conserve dopamine by inhibiting the enzymes responsible for its catabolism. The monoamine oxidase (MAO) B isoform catalyses the oxidation of dopamine in the central nervous system and is therefore an important target for Parkinson’s disease treatment. Inhibition of MAO-B provides symptomatic relief for Parkinson’s disease patients by increasing endogenous dopamine levels as well as enhancing the levels of dopamine after administration of levodopa (L-dopa), the metabolic precursor of dopamine. Recent studies have shown that phthalide can be used as a scaffold for the design of reversible MAO inhibitors. Although phthalide is a weak MAO-B inhibitor, substitution on the C5 position of phthalide yields highly potent reversible MAO-B inhibitors. In the present study, sesamol and benzodioxane were used as scaffolds for the design of MAO inhibitors. The structures of sesamol and benzodioxane closely resemble that of phthalide, which suggests that these moieties may be useful for the design of MAO inhibitors. This study may be viewed as an exploratory study to discover new scaffolds for MAO inhibition. Since substitution at C5 of phthalide with a benzyloxy side chain yielded particularly potent MAO inhibitors, the sesamol and benzodioxane derivatives possessed the benzyloxy substituent in the analogous positions to C5 of phthalide. These were the C5 and C6 positions of sesamol and benzodioxane, respectively. The sesamol and benzodioxane derivatives were synthesised by reacting sesamol and 6- hydroxy-1,4-benzodioxane, respectively, with an appropriate alkyl bromide in the presence of potassium carbonate (K2CO3) in N,N-dimethylformamide (DMF). 6-Hydroxy-1,4- benzodioxane, in turn, was synthesised from 1,4-benzodioxan-6-carboxaldehyde. The structures of the compounds were verified with nuclear magnetic resonance (NMR) and mass spectrometry (MS) analyses, while the purities were estimated by high-pressure liquid chromatography (HPLC). Sixteen sesamol and benzodioxane derivatives were synthesised. To determine the inhibition potencies of the synthesised compounds the recombinant human MAO-A and MAO-B enzymes were used. The inhibition potencies were expressed as the corresponding IC50 values. The results showed that the sesamol and benzodioxane derivatives are highly potent and selective inhibitors of MAO-B and to a lesser extent MAOA. The most potent MAO-B inhibitor was 6-(3-bromobenzyloxy)-1,4-benzodioxane with an IC50 value of 0.045 μM. All compounds examined displayed selectivity for the MAO-B isoform over MAO-A. Generally the benzodioxane derivatives were found to be more potent inhibitors of human MAO-A and MAO-B than the sesamol derivatives. The reversibility and mode of MAO-B inhibition of a representative derivative, 6-(3- bromobenzyloxy)-1,4-benzodioxane, was examined by measuring the degree to which the enzyme activity recovers after dialysis of enzyme-inhibitor complexes, while Lineweaver- Burk plots were constructed to determine whether the mode of inhibition is competitive. Since MAO-B activity is completely recovered after dialysis of enzyme-inhibitor mixtures, it was concluded that 6-(3-bromobenzyloxy)-1,4-benzodioxane binds reversibly to the MAO-B enzyme. The Lineweaver-Burk plots constructed were linear and intersected on the y-axis. Therefore it may be concluded that 6-(3-bromobenzyloxy)-1,4-benzodioxane is a competitive MAO-B inhibitor. To conclude, the C6-substituted benzodioxane derivatives are potent, selective, reversible and competitive inhibitors of human MAO-B. These compounds are therefore promising leads for the future development of therapy for Parkinson’s disease. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2015

Parkinson’s disease is an age-related neurodegenerative disorder. The major symptoms of Parkinson’s disease are closely linked to the pathology of the disease. The main pathology of Parkinson’s disease consists of the degeneration of neurons of the substantia nigra pars compacta (SNpc), which leads to reduced amounts of dopamine in the brain. One of the treatment strategies in Parkinson’s disease is to conserve dopamine by inhibiting the enzymes responsible for its catabolism. The monoamine oxidase (MAO) B isoform catalyses the oxidation of dopamine in the central nervous system and is therefore an important target for Parkinson’s disease treatment. Inhibition of MAO-B provides symptomatic relief for Parkinson’s disease patients by increasing endogenous dopamine levels as well as enhancing the levels of dopamine after administration of levodopa (L-dopa), the metabolic precursor of dopamine. Recent studies have shown that phthalide can be used as a scaffold for the design of reversible MAO inhibitors. Although phthalide is a weak MAO-B inhibitor, substitution on the C5 position of phthalide yields highly potent reversible MAO-B inhibitors. In the present study, sesamol and benzodioxane were used as scaffolds for the design of MAO inhibitors. The structures of sesamol and benzodioxane closely resemble that of phthalide, which suggests that these moieties may be useful for the design of MAO inhibitors. This study may be viewed as an exploratory study to discover new scaffolds for MAO inhibition. Since substitution at C5 of phthalide with a benzyloxy side chain yielded particularly potent MAO inhibitors, the sesamol and benzodioxane derivatives possessed the benzyloxy substituent in the analogous positions to C5 of phthalide. These were the C5 and C6 positions of sesamol and benzodioxane, respectively. The sesamol and benzodioxane derivatives were synthesised by reacting sesamol and 6- hydroxy-1,4-benzodioxane, respectively, with an appropriate alkyl bromide in the presence of potassium carbonate (K2CO3) in N,N-dimethylformamide (DMF). 6-Hydroxy-1,4- benzodioxane, in turn, was synthesised from 1,4-benzodioxan-6-carboxaldehyde. The structures of the compounds were verified with nuclear magnetic resonance (NMR) and mass spectrometry (MS) analyses, while the purities were estimated by high-pressure liquid chromatography (HPLC). Sixteen sesamol and benzodioxane derivatives were synthesised. To determine the inhibition potencies of the synthesised compounds the recombinant human MAO-A and MAO-B enzymes were used. The inhibition potencies were expressed as the corresponding IC50 values. The results showed that the sesamol and benzodioxane derivatives are highly potent and selective inhibitors of MAO-B and to a lesser extent MAOA. The most potent MAO-B inhibitor was 6-(3-bromobenzyloxy)-1,4-benzodioxane with an IC50 value of 0.045 μM. All compounds examined displayed selectivity for the MAO-B isoform over MAO-A. Generally the benzodioxane derivatives were found to be more potent inhibitors of human MAO-A and MAO-B than the sesamol derivatives. The reversibility and mode of MAO-B inhibition of a representative derivative, 6-(3- bromobenzyloxy)-1,4-benzodioxane, was examined by measuring the degree to which the enzyme activity recovers after dialysis of enzyme-inhibitor complexes, while Lineweaver- Burk plots were constructed to determine whether the mode of inhibition is competitive. Since MAO-B activity is completely recovered after dialysis of enzyme-inhibitor mixtures, it was concluded that 6-(3-bromobenzyloxy)-1,4-benzodioxane binds reversibly to the MAO-B enzyme. The Lineweaver-Burk plots constructed were linear and intersected on the y-axis. Therefore it may be concluded that 6-(3-bromobenzyloxy)-1,4-benzodioxane is a competitive MAO-B inhibitor. To conclude, the C6-substituted benzodioxane derivatives are potent, selective, reversible and competitive inhibitors of human MAO-B. These compounds are therefore promising leads for the future development of therapy for Parkinson’s disease. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2015

Dopamine (DA) neurons of the substantia nigra pars compacta (SNc) have a key role in regulation of voluntary movement control. Their death is a hallmark of Parkinson’s disease, characterised by inhibited motor control, including muscle rigidity and tremor. Excitatory input to SNc-DA neurons is primarily from the subthalamic nucleus, and in PD these afferents display a higher frequency firing, as well as increased burst firing, which could cause increased excitatory activity in SNc-DA neurons. NMDA receptors (NMDARs) bind the excitatory neurotransmitter glutamate, and are essential for learning and memory. In SNc-DA neurons, NMDARs have a putative triheteromeric subunit arrangement of GluN1 plus GluN2B and/or GluN2D. Wild type (WT) mice, and those lacking the gene for GluN2D (Grin2D-null), were used to explore its role in various aspects of DA neuronal function and dysfunction using patch-clamp electrophysiology, viability assaying, and immunofluorescence. Pharmacological intervention using subunit-specific inhibitors ifenprodil and DQP-1105 on elicited NMDAR-EPSCs suggested a developmental shift from primarily GluN2B to GluN2B/D. Activity dependent regulation was assessed by high frequency burst stimulation of glutamatergic afferents: in comparison to controls, significant downregulation of NMDARs was observed in SNc-DA neurons, though no differences were observed based on genotype. This regulatory function may be a neuroprotective or homeostatic response. Ambient extracellular glutamate elicits tonic NMDAR activity in SNc-DA neurons, which may be important for maintaining basal levels of excitability: the role of GluN2D was assessed by recording the deflection in baseline current caused by application of competitive NMDAR antagonist D-AP5. There was a significantly larger NMDAR-mediated current in WT vs Grin2D-null mice, indicating that GluN2D has a role in binding ambient glutamate. Dysfunction of glutamate uptake could be a secondary pathophysiological occurrence in the SNc, leading to increased ambient glutamate: the effect of this was explored by application of the competitive glutamate transporter blocker TBOA. Here, the NMDAR-mediated portion of this current was significantly higher in WT mice in comparison to Grin2D-null. Interestingly, dose-response data obtained from bath application of NMDA showed significantly larger currents in Grin2D-null animals vs WT, but only at the top of the response curve (~1-10 mM), which may indicate a capability for larger conductance in Grin2D-null animals at high NMDAR saturation due to replacement of GluN2D with GluN2B. GluN2D may therefore be neuroprotective, by attenuating peak current flow in response to very high agonist concentrations. Lastly, GluN2D has been found to decrease NMDAR open probability under hypoxic conditions, potentially conferring resistance to hypoxia / ischemia related excitotoxicity. Therefore, low (15% O2 / 80% N2 / 5% CO2) vs high (95% O2 / 5% CO2) oxygen conditions were used along with immunofluorescent propidium iodide cell death assaying and immunofluorescent labeling for DA neurons in order to compare levels of DA neuronal death in the SNc based on oxygen status and genotype. Whilst there was a significant submaximal effect based on O2 status, genotype did not confer a practical resistance under these conditions. In summary, NMDARs have diverse roles in SNc-DA neurons which may both serve to maintain normal function and protect the cell against potentially pathological conditions.

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