متحد البؤر التصوير الطيفي من المستقبلات النيكوتينية الموسومة Fluorescently في نوك في الفئران مع الإدارة النيكوتين المزمن
Summary
قمنا بتطوير تقنية جديدة لتحديد كمية النيكوتين التغييرات مستقبلات أستيل داخل المناطق التحت خلوية من أنواع فرعية محددة من الخلايا العصبية الجهاز العصبي المركزي إلى فهم أفضل للآليات لإدمان النيكوتين عن طريق استخدام مزيج من الأساليب بما في ذلك وضع علامات بروتين فلوري من مستقبلات باستخدام الضربة القاضية في نهج وطيفية مبائر التصوير.
Abstract
Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.
Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons.
Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using Förster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged α4 nAChR subunits (α4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.
In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from α4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain α4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of α4YFP receptors in the medial perforant path of the hippocampus.
Protocol
Discussion
Disclosures
The authors have nothing to disclose.
Acknowledgements
وأيد أنتوني Renda من قبل جامعة فيكتوريا جائزة زمالة دراسات عليا. وأيد هذا البحث من قبل العلوم الطبيعية والهندسة مجلس البحوث كندا اكتشاف غرانت، NARSAD جائزة الباحث الشاب (لRN)، ومؤسسة فيكتوريا – Myre وينيفرد سيم الصندوق، وهو مؤسسة الكندية للحصول على منحة للابتكار، والمعرفة كولومبيا البريطانية صندوق التنمية والعلوم الطبيعية والهندسة مجلس البحوث من أدوات البحث وكندا، والمنح والتهوية. نشكر جيليان مكاي، بارنز كريستينا، سوليفان ارييل ماكدونالد جينيفر ومورجادو دانيال لتربية الماوس ممتازة.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| mini-osmotic pumps | Alzet | model 2002 | |
| saline | Teknova | S5819 | |
| (-)-nicotine hydrogen tartrate salt | Sigma | N5260 | |
| eye drops | Novartis | Tear-Gel | |
| Vetbond glue | 3M | 1469SB | |
| heparin sodium salt | Sigma | H4784 | |
| 10x PBS | Invitrogen | 70011 | |
| ketamine | Wyeth Animal Health | 0856-4403-01 | |
| medatomidine hydrochloride | Pfizer | 1950673 | |
| 23G butterfly needle | Becton Dickinson | 367253 | |
| paraformaldehyde | Electron Microscopy Sciences | 15710 | |
| plastic embedding mold | VWR | 18986-1 | |
| O.C.T. Mounting Compound | Tissue-Tek | 4583 | |
| Mowiol 4-88 | EMD-Calbiochem | 475904 | pH 8.5 |
References
- Perry, D. C., Davila-Garcia, M. I., Stockmeier, C. A., Kellar, K. J. Increased nicotinic receptors in brains from smokers: membrane binding and autoradiography studies. J. Pharmacol. Exp. Ther. 289, 1545-1552 (1999).
- Schwartz, R. D., Kellar, K. J. Nicotinic cholinergic receptor binding sites in the brain: regulation in vivo. Science. 220, 214-216 (1983).
- Marks, M. J., Burch, J. B., Collins, A. C. Effects of chronic nicotine infusion on tolerance development and nicotinic receptors. J. Pharmacol. Exp. Ther. 226, 817-8125 (1983).
- Carlezon, W. A. J., Nestler, E. J. Elevated levels of GluR1 in the midbrain: a trigger for sensitization to drugs of abuse. Trends Neurosci. 25, 610-615 (2002).
- Fitzgerald, L. W., Ortiz, J., Hamedani, A. G., Nestler, E. J. Drugs of abuse and stress increase the expression of GluR1 and NMDAR1 glutamate receptor subunits in the rat ventral tegmental area: common adaptations among cross-sensitizing agents. J. Neurosci. 16, 274-2782 (1996).
- Saal, D., Dong, Y., Bonci, A., Malenka, R. C. Drugs of abuse and stress trigger a common synaptic adaptation in dopamine neurons. Neuron. 37, 577-5782 (2003).
- Tsien, R. Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509-544 (1998).
- Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., Prasher, D. C. Green fluorescent protein as a marker for gene expression. Science. 263, 802-805 (1994).
- Feng, G., Mellor, R. H., Bernstein, M., Keller-Peck, C., Nguyen, Q. T., Wallace, M. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron. 28, 41-51 (2000).
- Livet, J., Weissman, T. A., Kang, H., Draft, R. W., Lu, J., Bennis, R. A. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature. 450, 56-62 (2007).
- Nashmi, R., Dickinson, M. E., McKinney, S., Jareb, M., Labarca, C., Fraser, S. E. Assembly of α4β2 nicotinic acetylcholine receptors assessed with functional fluorescently labeled subunits: effects of localization, trafficking, and nicotine-induced upregulation in clonal mammalian cells and in cultured midbrain neurons. J. Neurosci. 23, 11554-11567 (2003).
- Nashmi, R., Xiao, C., Deshpande, P., McKinney, S., Grady, S. R., Whiteaker, P. Chronic nicotine cell specifically upregulates functional α4* nicotinic receptors: basis for both tolerance in midbrain and enhanced long-term potentiation in perforant path. J. Neurosci. 27, 8202-8218 (2007).
- Dickinson, M. E., Bearman, G., Tilie, S., Lansford, R., Fraser, S. E. Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy. BioTechniques. 31, 1272-1278 (2001).
- Nashmi, R., Fraser, S. E., Lester, H., Dickinson, M. E., Periasamy, A., Day, R. N. . Molecular imaging: fret microscopy and spectroscopy. , 180-192 (2005).
- Zimmermann, T., Rietdorf, J., Girod, A., Georget, V., Pepperkok, R. Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair. FEBS Lett. 531, 245-249 (2002).
- Larson, J. M. The Nikon C1si combines high spectral resolution, high sensitivity, and high acquisition speed. Cytometry A. 69, 825-8234 (2006).
- Melvin, N. R., Sutherland, R. J. Quantitative caveats of standard immunohistochemical procedures: implications for optical disector-based designs. J. Histochem. Cytochem. 58, 577-5784 (2010).
- Jones, I. W., Wonnacott, S. Why doesn’t nicotinic ACh receptor immunoreactivity knock out. Trends Neurosci. 28, 343-345 (2005).
- Moser, N., Mechawar, N., Jones, I., Gochberg-Sarver, A., Orr-Urtreger, A., Plomann, M. Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures. J. Neurochem. , (2007).
- Whiteaker, P., Cooper, J. F., Salminen, O., Marks, M. J., McClure-Begley, T. D., Brown, R. W., Collins, A. C., Lindstrom, J. M. Immunolabeling demonstrates the interdependence of mouse brain a4 and b2 nicotinic acetylcholine receptor subunit expression. The Journal of Comparative Neurology. 499, 1016-1038 (2006).
- Marks, M. J., McClure-Begley, T. D., Whiteaker, P., Salminen, O., Brown, R. W. B., Cooper, J., Collins, A. C., Lindstrom, J. M. Increased nicotinic acetylcholine receptor protein underlies chronic nicotine-induced up-regulation of nicotinic agonist binding sites in mouse brain. The Journal of Pharmacology and Experimental Therapeutics. , 337-3187 (2011).
- Marks, M. J., Rowell, P. P., Cao, J. Z., Grady, S. R., McCallum, S. E., Collins, A. C. Subsets of acetylcholine-stimulated 86[Rb]+ efflux and 125[I]-epibatidine binding sites in C57BL/6 mouse brain are differentially affected by chronic nicotine treatment. Neuropharmacology. 46, 1141-1157 (2004).
- King, S. L., Caldarone, B. J., Picciotto, M. R. Beta2-subunit-containing nicotinic acetylcholine receptors are critical for dopamine-dependent locomotor activation following repeated nicotine administration. Neuropharmacology. 47, 132-139 (2004).
- Robinson, S. F., Marks, M. J., Collins, A. C. Inbred mouse strains vary in oral self-selection of nicotine. Psychopharmacology (Berl). 124, 332-339 (1996).
- Sparks, J. A., Pauly, J. R. Effects of continuous oral nicotine administration on brain nicotinic receptors and responsiveness to nicotine in C57Bl/6 mice. Psychopharmacology (Berl). , 141-145 (1999).
- Rahman, S., Zhang, J., Engleman, E. A., Corrigall, W. A. Neuroadaptive changes in the mesoaccumbens dopamine system after chronic nicotine self-administration: a microdialysis study. Neuroscience. 129, 415-4124 (2004).
- Picciotto, M. R., Zoli, M., Rimondini, R., Lena, C., Marubio, L. M., Pich, E. M. Acetylcholine receptors containing the β2 subunit are involved in the reinforcing properties of nicotine. Nature. 391, 173-177 (1998).
- Fowler, C. D., Lu, Q., Johnson, P. M., Marks, M. J., Kenny, P. J. Habenular α5 nicotinic receptor subunit signalling controls nicotine intake. Nature. 471, 597-601 (2011).
- Maskos, U., Molles, B. E., Pons, S., Besson, M., Guiard, B. P., Guilloux, J. P. Nicotine reinforcement and cognition restored by targeted expression of nicotinic receptors. Nature. 436, 103-107 (2005).
- Matta, S. G., Balfour, D. J., Benowitz, N. L., Boyd, R. T., Buccafusco, J. J., Caggiula, A. R., Craig, C. R., Collins, A. C., Damaj, M. I., Donny, E. C., Gardiner, P. S., Grady, S. R., Heberlein, U., Leonard, S. S. Guidelines on nicotine dose selection for in vivo research. Psychopharmacology. 190, 269-319 (2007).
- Lang, T., Rizzoli, S. O. Membrane protein clusters at nanoscale resolution: more than pretty pictures. Physiology (Bethesda). 25, 116-1124 (2010).
