fetal human VTA RNA as a reference, so we can conclude that the relative quantities of these mRNAs in cultures were within 2-3-fold of that found in developing midbrain DA neurons. However, it appears that N398 neurons are receiving more synaptic inputs, as revealed by an increased PSC amplitude and frequency in N398 compared with D398 (Fig. 2M), consistent with a possible increase in excitability of N398 neurons, or reduced input from inhibitory neurons, as suggested by the relative higher frequency of spontaneous action potentials. This correlates with results from others suggesting that while the α5 subunit does not contribute to nicotine binding with nAChRs, the loss or alteration of α5 contributes to a shift in nicotine response, specifically in VTA DA neurons11. We ascribed the increased network activity to interactions among the mixtures of neuronal subtypes in the culture. The dual-SMAD inhibition method was used to generate cultures of relatively high purity DA neurons (≥75% of MAP2+ cells co-expressed TH) with a smaller number of other neuron subtypes (Fig. 1K–P).
