Human induced pluripotent stem cells (hiPSCs) offer the potential to study otherwise inaccessible cell types. Critical to this is the directed differentiation of hiPSCs into functional cell lineages. This is of particular relevance to research into neurological disease, such as Parkinson’s disease (PD), in which midbrain dopaminergic neurons degenerate during disease progression but are unobtainable until post-mortem. Here we report a detailed study into the physiological maturation over time of human dopaminergic neurons in vitro. We first generated and differentiated hiPSC lines into midbrain dopaminergic neurons and performed a comprehensive characterisation to confirm dopaminergic functionality by demonstrating dopamine synthesis, release, and re-uptake. The neuronal cultures include cells positive for both tyrosine hydroxylase (TH) and G protein-activated inward rectifier potassium channel 2 (Kir3.2, henceforth referred to as GIRK2), representative of the A9 population of substantia nigra pars compacta (SNc) neurons vulnerable in PD. We observed for the first time the maturation of the slow autonomous pace-making (<10 Hz) and spontaneous synaptic activity typical of mature SNc dopaminergic neurons using a combination of calcium imaging and electrophysiology. hiPSC-derived neurons exhibited inositol tri-phosphate (IP3) receptor-dependent release of intracellular calcium from the endoplasmic reticulum in neuronal processes as calcium waves propagating from apical and distal dendrites, and in the soma. Finally, neurons were susceptible to the dopamine neuron-specific toxin 1-methyl-4-phenylpyridinium (MPP+) which reduced mitochondrial membrane potential and altered mitochondrial morphology. Mature hiPSC-derived dopaminergic neurons provide a neurophysiologically-defined model of previously inaccessible vulnerable SNc dopaminergic neurons to bridge the gap between clinical PD and animal models.

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The intracellular second messenger cyclic ADP-ribose (cADPR) induces Ca 2+ release through the activation of ryanodine receptors (RyRs). Moreover, it has been suggested that cADPR may serve an additional role to modulate sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) pump activity, but studies have been complicated by concurrent actions on RyR. Here, we explore the actions of cADPR in Xenopus oocytes, which lack RyRs. We examined the effects of cADPR on the sequestration of cytosolic Ca 2+ following Ca 2+ transients evoked by photoreleased inositol 1,4,5-trisphosphate (InsP 3 ), and by Ca 2+ influx through expressed nicotinic acetylcholine receptors (nAChR) in the oocytes membrane. In both cases the decay of the Ca 2+ transients was accelerated by intracellular injection of a non-metabolizable analogue of cADPR, 3-Deaza-cADPR, and photorelease of cADPR from a caged precursor demonstrated that this action is rapid (a few s). The acceleration was abolished by pre-treatment with thapsigargin to block SERCA activity, and was inhibited by two specific antagonists of cADPR, 8-NH 2 -cADPR and 8-br-cADPR. We conclude that cADPR serves to modulate Ca 2+ sequestration by enhancing SERCA pump activity, in addition to its well established action on RyRs to liberate Ca 2+ .

In addition to its well established function in activating Ca 2؉release from the endoplasmic reticulum (ER) through ryanodine receptors (RyR), the second messenger cyclic ADP-ribose (cADPR) also accelerates the activity of SERCA pumps, which sequester Ca 2؉ into the ER. Here, we demonstrate a potential physiological role for cADPR in modulating cellular Ca 2؉ signals via changes in ER Ca 2؉ store content, by imaging Ca 2؉ liberation through inositol trisphosphate receptors (IP 3 R) in Xenopus oocytes, which lack RyR. Oocytes were injected with the non-metabolizable analog 3-deaza-cADPR, and cytosolic [Ca 2؉ ] was transiently elevated by applying voltage-clamp pulses to induce Ca 2؉ influx through expressed plasmalemmal nicotinic channels. We observed a subsequent potentiation of global Ca 2؉ signals evoked by strong photorelease of IP 3 , and increased numbers of local Ca 2؉ puffs evoked by weaker photorelease. These effects were not evident with cADPR alone or following cytosolic Ca 2؉ elevation alone, indicating that they did not arise through direct actions of cADPR or Ca 2؉ on the IP 3 R, but likely resulted from enhanced ER store filling. Moreover, the appearance of a new population of puffs with longer latencies, prolonged durations, and attenuated amplitudes suggests that luminal ER Ca 2؉ may modulate IP 3 R function, in addition to simply determining the size of the available store and the electrochemical driving force for release.

Summary Liberation of Ca2+ from the endoplasmic reticulum (ER) through inositol trisphosphate receptors (IP3R) is modulated by the ER Ca2+ content, and overexpression of SERCA2b to accelerate Ca2+ sequestration into the ER has been shown to potentiate the frequency and amplitude of IP3-evoked Ca2+ waves in Xenopus oocytes. Here, we examined the effects of SERCA overexpression on the elementary IP3-evoked puffs to elucidate whether ER [Ca2+] may modulate IP3R function via luminal regulatory sites in addition to simply determining the size of the available store and electrochemical driving force for Ca2+ release. SERCA2b and Ca2+ permeable nicotinic plasmalemmal channels were expressed in oocytes, and hyperpolarizing pulses were delivered to induce Ca2+ influx and thereby load ER stores. Puffs evoked by photoreleased IP3 were significantly potentiated in terms of numbers of responding sites, frequency and amplitude following transient Ca2+ influx in SERCA-overexpressing cells, whereas little change was evident with SERCA overexpression alone or following Ca2+ influx in control cells not overexpressing SERCA. Intriguingly, we observed the appearance of a new population of puffs that arose after long latencies and had prolonged durations supporting the notion of luminal regulation of IP3R gating kinetics.