Upregulation of ␤2 subunit-containing (␤2*) nicotinic acetylcholine receptors (nAChRs) is implicated in several aspects of nicotine addiction, and menthol cigarette smokers tend to upregulate ␤2* nAChRs more than nonmenthol cigarette smokers. We investigated the effect of long-term menthol alone on midbrain neurons containing nAChRs. In midbrain dopaminergic (DA) neurons from mice containing fluorescent nAChR subunits, menthol alone increased the number of ␣4 and ␣6 nAChR subunits, but this upregulation did not occur in midbrain GABAergic neurons. Thus, chronic menthol produces a cell-type-selective upregulation of ␣4* nAChRs, complementing that of chronic nicotine alone, which upregulates ␣4 subunit-containing (␣4*) nAChRs in GABAergic but not DA neurons. In mouse brain slices and cultured midbrain neurons, menthol reduced DA neuron firing frequency and altered DA neuron excitability following nAChR activation. Furthermore, menthol exposure before nicotine abolished nicotine reward-related behavior in mice. In neuroblastoma cells transfected with fluorescent nAChR subunits, exposure to 500 nM menthol alone also increased nAChR number and favored the formation of (␣4) 3 (␤2) 2 nAChRs; this contrasts with the action of nicotine itself, which favors (␣4) 2 (␤2) 3 nAChRs. Menthol alone also increases the number of ␣6␤2 receptors that exclude the ␤3 subunit. Thus, menthol stabilizes lower-sensitivity ␣4* and ␣6 subunitcontaining nAChRs, possibly by acting as a chemical chaperone. The abolition of nicotine reward-related behavior may be mediated through menthol's ability to stabilize lower-sensitivity nAChRs and alter DA neuron excitability. We conclude that menthol is more than a tobacco flavorant: administered alone chronically, it alters midbrain DA neurons of the nicotine reward-related pathway.

Understanding why the quit rate among smokers of menthol cigarettes is lower than non-menthol smokers requires identifying the neurons that are altered by nicotine, menthol, and acetylcholine. Dopaminergic (DA) neurons in the ventral tegmental area (VTA) mediate the positive reinforcing effects of nicotine. Using mouse models, we show that menthol enhances nicotine-induced changes in nicotinic acetylcholine receptors (nAChRs) expressed on midbrain DA neurons. Menthol plus nicotine upregulates nAChR number and function on midbrain DA neurons more than nicotine alone. Menthol also enhances nicotine-induced changes in DA neuron excitability. In a conditioned place preference (CPP) assay, we observed that menthol plus nicotine produces greater reward-related behavior than nicotine alone. Our results connect changes in midbrain DA neurons to menthol-induced enhancements of nicotine reward-related behavior and may help explain how smokers of menthol cigarettes exhibit reduced cessation rates.

Retrospective epidemiological studies show an inverse correlation between susceptibility to Parkinson's disease and a person's history of tobacco use. Animal model studies suggest nicotine as a neuroprotective agent and nicotinic acetylcholine (ACh) receptors (nAChRs) as targets for neuroprotection, but the underlying neuroprotective mechanism(s) are unknown. We cultured mouse ventral midbrain neurons for 3 weeks. Ten to 20% of neurons were dopaminergic (DA), revealed by tyrosine hydroxylase (TH) immunoreactivity. We evoked mild endoplasmic reticulum (ER) stress with tunicamycin (Tu), producing modest increases in the level of nuclear ATF6, phosphorylated eukaryotic initiation factor 2␣, nuclear XBP1, and the downstream proapoptotic effector nuclear C/EBP homologous protein.We incubated cultures for 2 weeks with 200 nM nicotine, the approximate steady-state concentration between cigarette smoking or vaping, or during nicotine patch use. Nicotine incubation suppressed Tu-induced ER stress and the unfolded protein response (UPR). Study of mice with fluorescent nAChR subunits showed that the cultured THϩ neurons displayed ␣4, ␣6, and ␤3 nAChR subunit expression and ACh-evoked currents. Gene expression profile in cultures from TH-eGFP mice showed that the THϩ neurons also express several other genes associated with DA release. Nicotine also upregulated ACh-induced currents in DA neurons by ϳ2.5-fold. Thus, nicotine, at a concentration too low to activate an appreciable fraction of plasma membrane nAChRs, induces two sequelae of pharmacological chaperoning in the ER: UPR suppression and nAChR upregulation. Therefore, one mechanism of neuroprotection by nicotine is pharmacological chaperoning, leading to UPR suppression. Measuring this pathway may help in assessing neuroprotection.

Multi-day tracking of cells in culture systems can provide valuable information in bioscience experiments. We report the development of a cell culture imaging system, named EmSight, which incorporates multiple compact Fourier ptychographic microscopes with a standard multiwell imaging plate. The system is housed in an incubator and presently incorporates six microscopes. By using the same low magnification objective lenses as the objective and the tube lens, the EmSight is configured as a 1:1 imaging system that, providing large field-of-view (FOV) imaging onto a low-cost CMOS imaging sensor. The EmSight improves the image resolution by capturing a series of images of the sample at varying illumination angles; the instrument reconstructs a higher-resolution image by using the iterative Fourier ptychographic algorithm. In addition to providing high-resolution brightfield and phase imaging, the EmSight is also capable of fluorescence imaging at the native resolution of the objectives. We characterized the system using a phase Siemens star target, and show four-fold improved coherent resolution (synthetic NA of 0.42) and a depth of field of 0.2 mm. To conduct live, long-term dopaminergic neuron imaging, we cultured ventral midbrain from mice driving eGFP from the tyrosine hydroxylase promoter. The EmSight system tracks movements of dopaminergic neurons over a 21 day period. "Pharmacological chaperoning of nicotinic acetylcholine receptors reduces the endoplasmic reticulum stress response," Mol. Pharmacol. 81(6), 759-769 (2012). 7. http://www.zeiss.com/microscopy/en_us/products/microscope-components/incubation.html. 8. http://www.nikoninstruments.com/Products/Live-Cell-Screening-Systems/BioStation-CT. 9. http://www.essenbioscience.com/essen-products/incucyte/. 10. A. W. Lohmann, R. G. Dorsch, D. Mendlovic, Z. Zalevsky, and C. Ferreira, "Space-bandwidth product of optical signals and systems," J. Opt. Soc. Am. A 13(3), 470-473 (1996). 11. G. Zheng, X. Ou, and C. Yang, "0.5 gigapixel microscopy using a flatbed scanner," Biomed. Opt. Express 5(1), 1-8 (2014). super-resolution in portable lensless on-chip microscopy using a fiber-optic array," Lab Chip 11(7), 1276-1279 (2011). 17. A. Greenbaum, U. Sikora, and A. Ozcan, "Field-portable wide-field microscopy of dense samples using multiheight pixel super-resolution based lensfree imaging," Lab Chip 12(7), 1242-1245 (2012). 18. A. F. Coskun, I. Sencan, T. W. Su, and A. Ozcan, "Wide-field lensless fluorescent microscopy using a tapered fiber-optic faceplate on a chip," Analyst (Lond.) 136(17), 3512-3518 (2011). 19. A. F. Coskun, I. Sencan, T. W. Su, and A. Ozcan, "Lensless wide-field fluorescent imaging on a chip using compressive decoding of sparse objects," Opt. Express 18(10), 10510-10523 (2010). 20. G. Zheng, R. Horstmeyer, and C. Yang, "Wide-field, high-resolution Fourier ptychographic microscopy," Nat.Photonics 7(9), 739-745 (2013). 21. X. Ou, R. Horstmeyer, C. Yang, and G. Zheng, "Quantitative phase imaging via Fourier ptychographic microscopy," Opt. Lett....

Dopaminergic neurons in the substantia nigra pars compacta (SNc) degenerate in Parkinson’s disease. These neurons robustly express several nicotinic acetylcholine receptor (nAChR) subtypes. Smoking appears to be neuroprotective for Parkinson’s disease but the mechanism is unknown. To determine whether chronic nicotine-induced changes in gene expression contribute to the neuroprotective effects of smoking, we develop methods to measure the effect of prolonged nicotine exposure on the SNc neuronal transcriptome in an unbiased manner. Twenty neurons were collected using laser-capture microscopy and transcriptional changes were assessed using RNA deep sequencing. These results are the first whole-transcriptome analyses of chronic nicotine treatment in SNc neurons. Overall, 129 genes were significantly regulated: 67 upregulated, 62 downregulated. Nicotine-induced relief of endoplasmic reticulum (ER) stress has been postulated as a potential mechanism for the neuroprotective effects of smoking. Chronic nicotine did not significantly affect the expression of ER stress-related genes, nor of dopamine-related or nAChR genes, but it did modulate expression of 129 genes that could be relevant to the neuroprotective effects of smoking, including genes involved in (1) the ubiquitin–proteasome pathway, (2) cell cycle regulation, (3) chromatin modification, and (4) DNA binding and RNA regulation. We also report preliminary transcriptome data for single-cell dopaminergic and GABAergic neurons isolated from midbrain cultures. These novel techniques will facilitate advances in understanding the mechanisms taking place at the cellular level and may have applications elsewhere in the fields of neuroscience and molecular biology. The results give an emerging picture of the role of nicotine on the SNc and on dopaminergic neurons.