Results from the dual-SMAD-derived DA cultures suggest that human DA neurons may co-release glutamate (Fig. 1O,P) and the release of glutamate was likely differentially affected by the N398 or D398 nAChR variants (Fig. 2L,M). Gene expression patterns predict a difference in neurotransmitter response pathways, particularly for glutamate signaling. We considered whether the difference in response between N398 and D398 variant nAChRs could be observed in cultures of purely excitatory neurons, which have been puromycin-selected to increase the uniformity of the cultures. To test this, we differentiated N398 and D398 iPSC into forebrain excitatory neurons using iN protocols27 (Fig. 4A) and studied the impact of nicotine on synaptic transmission. Forebrain excitatory glutamatergic neurons are modulated by nAChRs2; thus we hypothesize that the N398 variant would have a greater functional impact on excitatory neurons. We examined the effects of nicotine on spontaneous excitatory PSCs (sEPSCs) in both D398 and N398 human excitatory neurons. The frequencies of sEPSCs in both N398 and D398 neurons are stable before the application of nicotine (Fig. 4B–D). However, within 30 s of 0.1 μM nicotine exposure, frequencies
