Electrophysiological characterization of the novel effects of the Uptake-2 blocker Decynium-22 on dopaminergic neurons in the Substantia Nigra pars compacta

Abstract

Parkinson’s disease (PD) is an incurable, progressive neurological disorder characterised by motor symptoms which are frequently managed with pharmacotherapy. L-DOPA (Levodopa) is the ‘gold standard’ drug which alleviates many symptoms, by replenishing the neurotransmitter dopamine (DA) that is greatly diminished in PD due to degeneration of DAergic neurons in the substantia nigra pars compacta (SNc). However, after prolonged treatment, a ‘wearing-off’ phenomenon begins to manifest, where L-DOPA becomes less effective. This prompts doctors to prescribe higher doses of the drug which often leads to additional side-effects. There are two ways to enhance the therapeutic effects of L-DOPA: (a) by enhancing the delivery of the drug to the brain; and (b) by reducing the clearance of DA released from DA-producing neurons. Apart from the conventional dopamine transporter (DAT), a novel class of transporters known as Uptake-2 (which include OCT3 and PMAT) have recently been proposed to remove L-DOPA-derived DA. Therefore, inhibiting the function of these Uptake-2 transporters potentially constitutes a new strategy for treating PD. The aim of the present study was to determine the role of Uptake-2 in controlling extracellular DA levels in the SNc after L-DOPA administration. The potent Uptake-2 blocker decynium-22 (D-22) was used. Initial electrochemical experiments (fast-scan cyclic voltammetry and fast-scan controlled–adsorption voltammetry) showed that this drug interferes with DA detection, precluding the use of electrochemistry for testing the effects of D-22 on extracellular DA levels. D2 receptor-mediated inhibitory post-synaptic currents (D2-IPSCs) measured electrophysiologically (with the whole-cell patch-clamp technique) were instead used as a ‘tool’ to indirectly assess extracellular DA released in the SNc by local electrical stimulation in mouse midbrain slices following L-DOPA administration. Surprisingly, D-22 (at 5 and 25 μM) abolished D2-IPSC (most likely by blocking GIRK channels), and had a number of other ‘off- target’ effects on nigral DAergic neurons, including inhibition of their spontaneous firing and Ih current. These effects were unrelated to Uptake-2 inhibition. My results show that recording of synaptic currents evoked by stimulated DA release cannot be used to measure changes in extracellular levels of this neurotransmitter. Identification of novel ‘off-target’ effects of D-22 is important, as this drug has been frequently used in previous studies to determine the role of Uptake-2 in the removal of DA and other analytes from the extracellular space. Therefore, this drug should be used with caution when studying its effects on Uptake-2.