To begin to test CHRNA5 variant function, midbrain-like DA neuronal cultures were chosen to mimic the DA neurons of the VTA, thought to be an important member of the reward/addiction circuit3132. The presence of the N398 variant does not interfere with the generation of mature DA neurons, as determined by the expression of neuronal markers (Fig. 1), or of the basic membrane properties, and the capacity for generating action potentials is not different between the two CHRNA5 variants (Fig. 2). Expression of nAChR mRNAs was similarly expressed in both variants. While mRNA levels cannot predict cellular protein levels or assembly into functional receptors, and a complete set of nAChR subunit-selective antibodies is not available, cultures expressed robust levels of mRNAs encoding CHRNA3, 4, 5, and 6, CHRNB2 and 4 as would be expected for central nAChRs (Fig. 1Q). We used 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
