Differential Effects of Parkinsonian Toxins and L-DOPA on Locus Coeruleus and Substantia Nigra Neurons: Implications for Parkinson’s Disease

Abstract

Parkinson’s disease (PD) is traditionally known for its characteristic motor symptoms, caused by degeneration of dopamine (DA)-producing neurons of the Substantia Nigra pars compacta (SNc). However, PD patients also suffer from many non-motor symptoms (NMS; eg. sleep disturbance, autonomic dysfunction, depression and anxiety), which cause significant disability and impair quality of life. These NMS often occur many years before the onset of motor symptoms, and are associated with degeneration of lower brain stem nuclei, including noradrenaline (NA)-producing neurons in the Locus Coeruleus (LC). By tracking the appearance of Lewy bodies in PD, it has been hypothesized (‘Braak hypothesis’) that degeneration ascends through the brain in the caudal-to-rostral direction. Located in the pons, LC neurons are the first catecholaminergic neurons to show signs of Lewy body accumulation. This is consistent with the early development of NMS attributed to LC degeneration, before the onset of motor symptoms and nigral pathology. Post mortem evidence has revealed that neuronal loss in the LC can actually exceed that seen in the SNc. Together, these observations suggest that LC neurons are more vulnerable to degenerative processes than SNc neurons. In addition, animal models have demonstrated that prior lesioning of the LC renders SNc neurons more vulnerable to damage. This indicates that LC degeneration may have an important role not only in the development of NMS, but also in the development of nigral pathology. The majority of PD cases are idiopathic, but there is strong evidence that neurodegeneration involves mitochondrial inhibition and oxidative stress. Exposure to environmental toxins (such as the pesticide, rotenone) has been associated with the development of PD, and rare cases have shown a causal link (MPTP/MPP+) to the disease. There is also evidence that endogenous toxins (DA-derived 6-OHDA) may be involved in disease progression. These parkinsonian toxins (rotenone, MPTP/MPP+, 6-OHDA) reproduce many behavioural, biochemical and pathological features of PD, and cause relatively selective degeneration of nigral dopaminergic neurons. However, the cellular mechanisms of action of these toxins have not been fully characterized, and little is known about how these toxins affect noradrenergic LC neurons. Primarily using a combination of electrophysiology and microfluorometry, this study characterized the effects of rotenone on the electrophysiological properties and intracellular Ca2+ levels of noradrenergic LC neurons in brain slices, and directly compared these responses to those of nigral dopaminergic and non-dopaminergic (non-DA) neurons. The effects of MPP+ and 6-OHDA were also compared between these groups of neurons. Rotenone caused dose-dependent inhibition of firing in LC neurons and evoked a net outward current mediated by ATP-sensitive K+ (KATP) channels. When KATP channels were blocked, rotenone increased the firing of these neurons and evoked an inward current. Inward and outward currents were associated with an increase of intracellular [Ca2+], mediated by oxidative stress-gated TRPM2-like channels. These changes were also associated with depolarization of mitochondrial membrane potential (Ψm). The responses of LC neurons were smaller than those of nigral dopaminergic neurons, suggesting that LC neurons are less susceptible to metabolic and oxidative stress evoked by rotenone. Rotenone also evoked a larger increase of extracellular [H2O2] in the SNc compared to LC or SNr regions, as measured using fast-scan cyclic voltammetry. Both MPP+ and 6-OHDA inhibited the firing of both LC and SNc neurons, but increased the firing of nigral non-DA neurons, likely via TRPM2-like channels. MPP+ inhibited the activity of LC and SNc neurons by indirectly activating α2 and D2 auto-receptors respectively. This effect was attributed to release of NA/DA, since MPP+ did not directly interact with D2 receptors, as shown in membrane binding assays. Additionally, MPP+ caused long-lasting inhibition of nigral dopaminergic neurons by activating KATP and Ca2+-activated K+ (KCa) channels. Prolonged inhibition via KATP/KCa channels was not observed in LC neurons. 6-OHDA directly activated D2 (and likely α2) receptors, but increased the firing of SNc and LC neurons when these receptors were blocked. Excitation was transient in the SNc, before the onset of long-lasting inhibition of firing, which was dependent on uptake of the toxin. Together, these results showed that rotenone, MPP+ and 6-OHDA caused larger, and more prolonged inhibitory effects in nigral dopaminergic neurons than either LC or nigral non-DA neurons. Since these toxicants eventually lead to the demise of SNc neurons, these results suggest that chronic inhibition of firing represents an early stage of degeneration. This study also investigated the effects of L-DOPA (levodopa), which remains the ‘gold standard’ for the treatment of PD despite in vitro evidence that the drug might be toxic to remaining dopaminergic neurons. The classical D2-mediated, inhibitory effect of L-DOPA, via conversion to DA in nigral dopaminergic neurons, has been well-documented. However, the effects of L-DOPA on LC and nigral non-DA neurons have not previously been shown. An oxidation product of L-DOPA, called TOPA quinone, activated AMPA/kainate receptors in all three groups of neurons, and the drug produced long-lasting excitation in both nigral dopaminergic neurons (when D2 receptors were blocked) and nigral non-DA neurons (on its own). The ‘late phase’ of excitation in nigral dopaminergic neurons was mediated by newly synthesized DA, which potentially elevated firing frequency by blocking KCa-mediated afterhyperpolarization. When slices were treated with a low dose of rotenone, L-DOPA activated KATP channels in both SNc and LC neurons. A higher dose of L-DOPA was required to activate KATP channels in nigral non-DA neurons. This indicates that L-DOPA is able to induce metabolic and/or oxidative stress more readily in catecholaminergic neurons. Overall, metabolic and oxidative stress evoked by parkinsonian toxins and L-DOPA have profound effects on the activity of neurons in the LC and Substantia Nigra. Both chronic inhibition of firing and prolonged rise of Ca2+ evoked in nigral dopaminergic neurons are likely to be detrimental for their survival. Contrary to my initial hypothesis, these results indicate that LC neurons are less susceptible to the effects of parkinsonian toxins compared to nigral dopaminergic neurons, suggesting that other factors may contribute to their earlier degeneration in PD.