On the path to understanding pacemaking of dopaminergic neurons

Despite many years of investigations by many researchers, the mechanism of pacemaking of midbrain dopaminergic (DA) neurons remains unknown. Several groups found that the Ih current, which is prominent in a majority of these neurons, enhances pacemaking in a subgroup of DA neurons, but this is not its core mechanism. We and others have been able to rule out various types of Cav channels as pacemaking generators. In our hands, NALCN channels do not seem to contribute either, because the (admittedly non-selective) blocker Gd3+ does not reliably affect pacemaking of DA neurons at 100 µM, whereas it stops fast pacemaking of reticulata GABAergic neurons (Lutas et al., 2016). Because we know that slow pacemaking of DA neurons necessitates only a very small inward current (1-6 pA) (Khaliq and Bean, 2008), we tested the hypothesis that their pacemaking is due to many membrane proteins with a very low unitary conductance. To do this, we investigated the effect of 1-(2,4-xylyl) guanidinium (XG) on their firing. This compound had been shown to block a so-called gating pore current in pathological Nav1.4 channels (Sokolov et al., 2010). In the presence of synaptic blockers (including 1 µM sulpiride), XG completely stopped the firing of DA neurons in a concentration-dependent manner (IC50 was 485 µM, Hill coefficient was 1.28), suggesting a one-to-one interaction. The effect of XG was only weakly reversible, but the firing in the partially inhibited neurons (200-600 µM) remained extremely stable after superfusion of the drug, arguing against a toxic effect. The effect of XG was similar in young and adult rat DA neurons, and was also seen in mouse DA neurons (SH and JR). Because the commercially available XG salt is poorly water-soluble, we (RV and JFL) synthesized a much more soluble salt (XG-M) which yielded the same effect (IC50 was 427 µM). XG inhibited the firing of DA neurons without affecting their conductance and without inducing a hyperpolarization, contrary to D2 agonists. In addition, in the presence of XG, DA neurons could be induced to fire with a small amount of positive current (10 to 30 pA), and their action potential properties were unaffected by the drug. Finally, XG did not affect the pacemaking of fast GABAergic pacemakers. Using pacemaker clamp (imposing in voltage clamp the waveform of spontaneous firing), we isolated a small XG-sensitive inward current which operates from ~-60 to ~-20 mV, but is not seen at more hyperpolarized potentials, arguing against a gating pore current. Molecular biology experiments (BL and KJ) also ruled out an editing of mRNAs coding for the main voltage-dependent channels of these neurons in their S4 segments (this could have been the mechanism of a « physiological » gating pore). In ion substitution experiments, we found that the XG-sensitive current is carried by Na+ and Cl- ions, but not Ca2+ or K+ ions. Given these characteristics, we recently checked whether the dopamine transporter would be involved, since it had been shown to depolarize cultured DA neurons (Ingram et al., 2002). However, we recently ruled out this hypothesis. What could be the nature of this elusive XG-sensitive current