Increased nicotine response in iPSC-derived human neurons carrying the CHRNA5 N398 allele.

Functionally mature midbrain-like DA neurons present in both D398 and N398 DA cultures.Representative iPSC cultures from (A) D398 and (B) N398 groups stained positively for pluripotency markers TRA-1-60 (green) and Oct4 (red). The majority of cells in iPSC-derived DA cultures expressed both MAP2+ (green) cells and TH (red) in both D398 (C) and N398 (D) groups. (E,F) Punctate staining indicates the presence of DAT (green), likely localized to synapses and surrounding TH+ cells. (G,H) Few TH+ were labeled with a pulse of EdU (green), indicating that most cells were postmitotic. (I,J) HPLC traces showing release of DA into culture medium with 1 mM nicotine. (K–P) Identification of alternative neuronal subtypes in mDA cultures. Cultures were stained with TH (red) and either (K,L) 5HT, (M,N) GAD6, or (O,P) VGluT1 (green). (Q) qPCR analysis of nAChR subunit-encoding mRNAs a3,a4,a5,a6, b2, b4, of D398 (red) or N398 (blue), for either iPSC (lighter) or DA (darker) cultures. mRNA levels are normalized to GAPDH and human fetal VTA RNA. Cultures were also assayed by qPCR for mRNAs encoding (R) TH and the midbrain marker PITX3. For all qPCR assays, DA neurons were different from iPSC cultures (ANOVA, p<0.05) but there was no difference between N398 and D398.

Spontaneous postsynaptic activity is increased in N398 DA cultures.(A,B) Spontaneous action potentials of D398 and N398 DA neurons. (C) Membrane capacitance, (D) Resting membrane potentials (RMP), (E) Input resistance of cell membrane (Rm), and (F) Spontaneous firing frequency of D398 and N398 DA neurons. (G,H) Repetitive action potentials from depolarizing current injections in D398 and N398 DA neurons. (I) Interstimulus intervals of induced action potentials of D398 and N398 DA neurons. (J,K) ICC of TH (Red) and Synapsin (Green) for D398 and N398 DA neurons. (L) Spontaneous postsynaptic currents of D398 and N398 DA neurons. (M) Frequency and amplitudes of postsynaptic current responses from D398 and N398 DA neurons.

Gene expression differences in iPSC derived DA neurons exhibit patterns of differential response.(A) Hierarchical clustering indicates that D398 and N398 DA cultures were more similar to mDA cultures than to iPSC or iPSC-derived NSC (NSC0), or NSC differentiating into glutamatergic neurons (5 days of differentiation, NSC5). The dendrogram shows relative distances (based on the Jenson-Shannon divergence metric) for the top 500 genes varying by group. (B) Expression patterns for genes characteristic of midbrain DA neurons. The mean FPKM is plotted ± the 95% confidence interval, n = 3/group. (C) RNAseq volcano plot. The horizontal axis shows the log2 fold-change, comparing N398 to D398. The vertical axis shows the false discovery rate (FDR) as the −log10 of the value. Points are plotted as individual genes not significantly different (grey dots) or significantly different (black dots) at 1% FDR (horizontal dashed line) and at least 2-fold different (vertical dashed lines). Colored dots indicate the nAChR receptor neuronal markers (green), GO dopamine differentiation genes (blue) and differentially-regulated genes belonging to the significantly-enriched pathways relatively increased in N398 (red). (D) Pathway (KEGG) analysis identifies three functional groups as enriched in the list of genes increased in N398. Bar colors indicate enrichment in N398 (red) or D398 (blue). The bar length indicates the FDR, and the color intensity (alpha) indicates the number of genes enriched. See Supplemental Table 4 for additional functional analyses.

Excitatory N398 neurons exhibit increased response to nicotine followed by desensitization.(A) Glutamatergic iN cultures are positive for MAP2 (green), vesicular glutamate transporter 1 (VGluT1, left, red), and synapsin (Syn, right, red) as detected by ICC. (B) Example traces from a patched neuron to show changes in response upon introduction of 0.1 μM nicotine (indicated by bar). Patched neurons exhibited increased frequency (C) and amplitude (D) during initial exposure to 0.1 μM nicotine. By 60 s after nicotine addition, frequency trended lower in N398 cells, although not significantly. Plotting the initial response to 0.1 μM nicotine for individual cells, it is clear that the changes in frequency (E) or amplitude (F) in N398 was not due to a single cell or subject. D398 samples are plotted as circles and N398 as squares. Color denotes cells from individual subjects (see key and Supplemental Table 1). However, subsequent additions of increasing doses of nicotine had reduced initial frequency (G) and amplitude (H) in N398 compared with D398 cells. N398 was different in frequency response from D398 as assessed by a Tukey post-hoc test of a general linear mixed-effects model with repeated measures (p = 0.00046, n = 115 cells per genotype; 5 cells per culture; 5–7 cultures per individual cell line).