Name: spectral confocal imaging of fluorescently tagged nicotinic receptors in knock-in mice with chronic nicotine administration
Uploaded: 2012-02-10T12:00:00
Description: Watch this Scientific Journal Video about Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration at JoVE.com

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.

1. مضخة غرس <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قبل زرع مضخة، وملء وإعداد Alzet مصغرة التناضحي مضخات (Alzet، موديل 2002، كوبرتينو، الولايات المتحدة الأمريكية) والحرص على عدم إدخال فقاعات الهواء. هذا نموذج مصغر من التناضحي المضخة حل بمعدل 0،5 ميكروليتر / ساعة لمد?...

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W., Wonnacott, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+doesn't+nicotinic+ACh+receptor+immunoreactivity+knock+out.">Why doesn't nicotinic ACh receptor immunoreactivity knock out.</a> Trends Neurosci. 28, 343-345 (2005).</li><li>Moser, N., Mechawar, N., Jones, I., Gochberg-Sarver, A., Orr-Urtreger, A., Plomann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+suitability+of+nicotinic+acetylcholine+receptor+antibodies+for+standard+immunodetection+procedures.">Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures.</a> J. Neurochem. , (2007).</li><li>Whiteaker, P., Cooper, J. F., Salminen, O., Marks, M. J., McClure-Begley, T. D., Brown, R. W., Collins, A. C., Lindstrom, J. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotine+reinforcement+and+cognition+restored+by+targeted+expression+of+nicotinic+receptors.">Nicotine reinforcement and cognition restored by targeted expression of nicotinic receptors.</a> Nature. 436, 103-107 (2005).</li><li>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. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+on+nicotine+dose+selection+for+in+vivo+research.">Guidelines on nicotine dose selection for in vivo research.</a> Psychopharmacology. 190, 269-319 (2007).</li><li>Lang, T., Rizzoli, S. 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<p class="jove_content"&gt; استخدام مستقبلات فلوري في نموذج الفأر المغلوب في لتحديد كمية وتوطين قناة أيون معين يوفر عددا من المزايا. وعلى النقيض من البروتينات مثل الأكتين، وهو ما يعبر عنه بتواجد مطلق في جميع الخلايا، والقنوات الأيونية الموجودة في أعداد أقل بكثير، والتعبير عنها يختلف بين الأنواع الفرعية الخلايا ا...

<table border="1" style=";text-align:right;direction:rtl"><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> مصغرة التناضحي مضخات </td><td> Alzet </td><td> موديل 2002 </td><td></td></tr><tr><td> ملحي </td><td> Teknova </td><td> S5819 </td><td></td></tr><tr><td> (-) طرطرات الهيدروجين النيكوتين ملح </td><td> سيغما </td><td> N5260 </td><td></td></tr><tr><td> قطرات عين </td><td> شركة نوفارتيس </td><td> المسيل للدموع جل </td><td></td></tr><tr><td> Vetbond الغراء </td><td> 3M </td><td> 1469SB </td><td></td></tr><tr><td> هيبارين الصوديوم ملح </td><td> سيغما </td><td> H4784 </td><td></td></tr><tr><td> 10X PBS </td><td> إينفيتروجن </td><td> 70011 </td><td></td></tr><tr><td> الكيتامين </td><td> Wyetح صحة الحيوان </td><td> 0856-4403-01 </td><td></td></tr><tr><td> medatomidine هيدروكلوريد </td><td> فايزر </td><td> 1950673 </td><td></td></tr><tr><td> 23G فراشة إبرة </td><td> بيكتون ديكنسون </td><td> 367253 </td><td></td></tr><tr><td> بارافورمالدهيد </td><td> علوم المجهر الإلكتروني </td><td> 15710 </td><td></td></tr><tr><td> تضمين قالب من البلاستيك </td><td> VWR </td><td> 18986-1 </td><td></td></tr><tr><td> أكتوبر مركب تركيب </td><td> الأنسجة تيك </td><td> 4583 </td><td></td></tr><tr><td> Mowiol 4-88 </td><td> EMD-Calbiochem </td><td> 475904 </td><td> درجة الحموضة 8.5 </td></tr></table>

spectral confocal imaging of fluorescently tagged nicotinic receptors in knock-in mice with chronic nicotine administration

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 F&ouml;rster 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 &alpha;4 nAChR subunits (&alpha;4YFP), 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 &alpha;4YFP 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 &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

Watch this Scientific Journal Video about Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration at JoVE.com

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

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.

متحد البؤر التصوير الطيفي من المستقبلات النيكوتينية الموسومة Fluorescently في نوك في الفئران مع الإدارة النيكوتين المزمن

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

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Neuroscience

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Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

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Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Quantifying Infra-slow Dynamics of Spectral Power and Heart Rate in Sleeping Mice

قياس ديناميات تحت البطء من الطاقة الطيفية ومعدل ضربات القلب في الفئران النائمة

Three vigilance states dominate mammalian life: wakefulness, non-rapid eye movement (non-REM) sleep, and REM sleep. As more neural correlates of behavior ...

Automated Gait Analysis in Mice with Chronic Constriction Injury

تحليل مشيه الآلي في الفئران مع الإصابات المزمنة انقباض

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

في فيفو حاجز الدم في الدماغ نفاذية مقايسة في الفئران باستخدام المسمى فلوريسسينتلي تتبع

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

مجهر ريفليكتوميتريك الطيفية على محاور عصبية ميليناتيد في الموقع

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

بيولوجيا الخلية كمية من نيوروديجينيريشن في المورفولوجية من خلال تحليل غير منحازة للبروتينات فلوريسسينتلي المعلمة باستخدام إيماجيج

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine

التدقيق وظيفة مستقبلات النيكوتين أستيل شرائح المخ الماوس عن طريق التحلل الضوئي فلاش الليزر النيكوتين فوتواكتيفاتابل

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with ...