Interactions between calcium channels and SK channels in midbrain dopamine neurons and their impact on pacemaker regularity: Contrasting roles of N- and L-type channels

Abstract

Although small-conductance Ca²⁺-activated K⁺ (SK) channels and various types of voltage-gated Ca²⁺ (Ca_v) channels have been described in midbrain dopaminergic neurons, the nature of their interactions is unclear. More particularly, the role of various Ca_v channel types in either promoting irregularity of firing (by generating an inward current during SK channel blockade) or promoting regularity of firing (by providing the source of Ca²⁺ for the activation of SK channels) has not been systematically explored. We addressed this question using intracellular and extracellular recordings from substantia nigra, pars compacta (SNc), dopaminergic neurons in rat midbrain slices. Neurons were pharmacologically isolated from their differences. When examining the ability of various Ca_v channel blockers to inhibit the SK-mediated afterhyperpolarization (AHP), we found that only the N-type Ca_v channel blocker ω-conotoxin-GVIA was able to reduce the apamin-sensitive AHP, but only partially (~40%). Specific blockers of L, P/Q, T or R channels had no effect on this AHP. Combining ω-conotoxin-GVIA and other specific blockers did not yield greater block and even the broad Ca_v blocker Cd²⁺ induced a submaximal (~75%) effect. Extracellular recordings examining firing regularity yielded congruent results: none of the specific blockers was able to increase firing irregularity to the extent that the specific SK blocker apamin did. The irregularity of firing observed with apamin could only be reversed by blocking L-type Ca²⁺ channels. Thus various sources of Ca²⁺ appear to be required for SK channel activation in SNc neurons (some of them still unidentified), ensuring robustness of pacemaking regularity.

Introduction

Dopaminergic neurons of the substantia nigra, pars compacta (SN_C), sustain important physiological functions such as the control of motricity, motivation and cognition. In vivo, these neurons exhibit a variety of firing patterns, switching from a regular “pacemaker” pattern to irregular firing and/or bursting. This pattern is absent in ex vivo recordings. Indeed, only a highly regular pacemaker-like pattern occurs spontaneously in mature dopaminergic neurons recorded ex vivo, suggesting that afferent inputs play an important role in the control of bursting (Grace and Onn, 1989). The regularity of the slow pacemaking is disrupted to a variable extent by bath application of apamin, a selective blocker of small-conductance Ca²⁺-activated K⁺ (KCa_{2. X} or SK) channels (Shepard and Stump, 1999, Wolfart and Roeper, 2002, Johnson and Wu, 2004). SK channels mediate a medium duration AHP which is very prominent in dopaminergic neurons recorded ex vivo. Because SK channels are solely gated by the intracellular Ca²⁺ concentration in their vicinity, the source of Ca²⁺ which activates these channels is of utmost importance to the pacemaking. Generally speaking, this source appears to be quite variable depending on neuronal types. Indeed, SK channels have been reported to be selectively coupled to P/Q-type Ca_v channels in rat cerebellar Purkinje neurons (Womack et al., 2004) while they are coupled to N-type Ca_v channels in rat vestibular nucleus neurons (Smith et al., 2002), rat striatal cholinergic interneurons (Goldberg and Wilson, 2005) and rat superior cervical ganglion (Davies et al., 1996). In this last preparation, a contribution of Ca²⁺ derived from intracellular stores was also suggested. A recent study by our group also showed that N-type Ca²⁺ channels play a major role in the activation of SK channels in rat serotonergic neurons (Alix et al., 2014). In the case of SNc dopaminergic neurons, a previous study has suggested that T-type channels are the main source of Ca²⁺ for SK channel activation in juvenile mice but N-type Ca²⁺ channels also appeared to be involved (Wolfart and Roeper, 2002). Another study in dissociated rat dopaminergic neurons suggests the involvement of N- and P-type Ca_v channels in the activation of calcium-activated K⁺ currents (Cardozo and Bean, 1995).

The aim of this study was to further explore the nature of the interaction between SK channels and various types of Ca_v channels in SNc dopaminergic neurons. Theoretically this interaction could have two opposite consequences on the spontaneous firing pattern: on the one hand, an increase in intracellular Ca²⁺ through Ca_v activation could induce the activation of SK channels and lead to a slowing of firing activity but, on the other hand, an increase of Ca²⁺ entry could also result in membrane depolarization and increase in firing rate when SK channels are not activated. Using intracellular recording techniques, we quantified the effect of specific antagonists of the different types of Ca_v channels on the amplitude of the apamin-sensitive AHP. Using extracellular recordings, the influence of Ca_v channel blockers on the regularity of firing was investigated and compared with the effect of apamin. Finally we investigated the ability of various Ca_v channel blockers to reverse apamin-induced irregularity.