Name: local application of drugs to study nicotinic acetylcholine receptor function in mouse brain slices
Uploaded: 2012-10-29T14:00:00
Description: Watch this Scientific Journal Video about Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices at JoVE.com

Эта работа была поддержана Национальным институтом здоровья (NIH) гранты DA030396. Благодаря членам лаборатории Drenan за полезные обсуждения и критику рукописи. Особая благодарность Ми Ран Ким технической помощи и Джонатан Томас Ting для консультаций по вопросам взрослого кусочки мозга мыши.

1. Подготовка решения для мозга Подготовка Slice и электрофизиологии <ol><li> Решения для подготовки срезах мозга были описаны 3, 4. Подготовка N-метил-D-глюкамин (NMDG)-основанной резки и восстановления данных следующего состава (в мМ): 93 N-метил-D-глюкамин, 2,5 KCl, 1,2 NaH 2 4 PO, 30 NaHCO 3, 20 HE...

<ol>
	<li>Tapper, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotine+activation+of+?4*+receptors:+sufficient+for+reward,+tolerance,+and+sensitization.">Nicotine activation of ?4* receptors: sufficient for reward, tolerance, and sensitization.</a> Science. 306, 1029-1032 (2004).</li><li>Drenan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+activation+of+midbrain+dopamine+neurons+via+sensitized,+high-affinity+?6*+nicotinic+acetylcholine+receptors.">In vivo activation of midbrain dopamine neurons via sensitized, high-affinity ?6* nicotinic acetylcholine receptors.</a> Neuron. 60, 123-136 (2008).</li><li>Zhao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+channelrhodopsin-2+transgenic+mice+for+optogenetic+dissection+of+neural+circuitry+function.">Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function.</a> Nat. Methods. 8, 745-7452 (2011).</li><li>Peca, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shank3+mutant+mice+display+autistic-like+behaviours+and+striatal+dysfunction.">Shank3 mutant mice display autistic-like behaviours and striatal dysfunction.</a> Nature. 472, 437-442 (2011).</li><li>Zhou, F. M., Wilson, C. J., Dani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+interneuron+characteristics+and+nicotinic+properties+in+the+striatum.">Cholinergic interneuron characteristics and nicotinic properties in the striatum.</a> J. Neurobiol. 53, 590-605 (2002).</li><li>Pidoplichko, V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotine+activates+and+desensitizes+midbrain+dopamine+neurons.">Nicotine activates and desensitizes midbrain dopamine neurons.</a> Nature. 390, 401-404 (1997).</li><li>Nashmi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+nicotine+cell+specifically+upregulates+functional+?4*+nicotinic+receptors:+basis+for+both+tolerance+in+midbrain+and+enhanced+long-term+potentiation+in+perforant+path.">Chronic nicotine cell specifically upregulates functional ?4* nicotinic receptors: basis for both tolerance in midbrain and enhanced long-term potentiation in perforant path.</a> J. Neurosci. 27, 8202-8218 (2007).</li><li>Xiao, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+nicotine+selectively+enhances+?4?2*+nicotinic+acetylcholine+receptors+in+the+nigrostriatal+dopamine+pathway.">Chronic nicotine selectively enhances ?4?2* nicotinic acetylcholine receptors in the nigrostriatal dopamine pathway.</a> J. Neurosci. 29, 12428-12439 (2009).</li><li>Cohen, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinic+cholinergic+mechanisms+causing+elevated+dopamine+release+and+abnormal+locomotor+behavior.">Nicotinic cholinergic mechanisms causing elevated dopamine release and abnormal locomotor behavior.</a> Neuroscience. 200, 31-41 (2012).</li><li>Drenan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+modulation+of+locomotion+and+striatal+dopamine+release+is+mediated+by+&#945;6&#946;4*+nicotinic+acetylcholine+receptors.">Cholinergic modulation of locomotion and striatal dopamine release is mediated by &#945;6&#946;4* nicotinic acetylcholine receptors.</a> J. Neurosci. 30, 9877-9889 (2010).</li><li>Grady, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+differences+determine+the+relative+selectivity+of+nicotinic+compounds+for+native+&#945;4&#946;2*-,+&#945;6&#946;2*-,+&#945;3&#946;4*-+and+&#945;7-nicotine+acetylcholine+receptors.">Structural differences determine the relative selectivity of nicotinic compounds for native &#945;4&#946;2*-, &#945;6&#946;2*-, &#945;3&#946;4*- and &#945;7-nicotine acetylcholine receptors.</a> Neuropharmacology. 58, 1054-1066 (2010).</li><li>Drenan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+trafficking,+pentameric+assembly,+and+subunit+stoichiometry+of+neuronal+nicotinic+acetylcholine+receptors+containing+fluorescently+labeled+&#945;6+and+&#946;3+subunits.">Subcellular trafficking, pentameric assembly, and subunit stoichiometry of neuronal nicotinic acetylcholine receptors containing fluorescently labeled &#945;6 and &#946;3 subunits.</a> Mol. Pharmacol. 73, 27-41 (2008).</li><li>Keath, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+modulation+by+nicotine+of+substantia+nigra+versus+ventral+tegmental+area+dopamine+neurons.">Differential modulation by nicotine of substantia nigra versus ventral tegmental area dopamine neurons.</a> J. Neurophysiol. 98, 3388-3396 (2007).</li><li>Mansvelder, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bupropion+inhibits+the+cellular+effects+of+nicotine+in+the+ventral+tegmental+area.">Bupropion inhibits the cellular effects of nicotine in the ventral tegmental area.</a> Biochem. Pharmacol. 74, 1283-1291 (2007).</li><li>Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+largest+group+of+superficial+neocortical+GABAergic+interneurons+expresses+ionotropic+serotonin+receptors.">The largest group of superficial neocortical GABAergic interneurons expresses ionotropic serotonin receptors.</a> J. Neurosci. 30, 16796-16808 (2010).</li><li>Margolis, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+in+the+identification+of+midbrain+dopamine+neurons.">Reliability in the identification of midbrain dopamine neurons.</a> PLoS One. 5, e15222 (2010).</li><li>Margolis, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ventral+tegmental+area+revisited:+is+there+an+electrophysiological+marker+for+dopaminergic+neurons.">The ventral tegmental area revisited: is there an electrophysiological marker for dopaminergic neurons.</a> J. Physiol. 577 (Pt 3), 907-924 (2006).</li><li>Couey, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distributed+network+actions+by+nicotine+increase+the+threshold+for+spike-timing-dependent+plasticity+in+prefrontal+cortex.">Distributed network actions by nicotine increase the threshold for spike-timing-dependent plasticity in prefrontal cortex.</a> Neuron. 54, 73-87 (2007).</li><li>Yang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinctive+nicotinic+acetylcholine+receptor+functional+phenotypes+of+rat+ventral+tegmental+area+dopaminergic+neurons.">Distinctive nicotinic acetylcholine receptor functional phenotypes of rat ventral tegmental area dopaminergic neurons.</a> J. Physiol. 587, 345-361 (2009).</li><li>Endo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinic+acetylcholine+receptor+subtypes+involved+in+facilitation+of+GABAergic+inhibition+in+mouse+superficial+superior+colliculus.">Nicotinic acetylcholine receptor subtypes involved in facilitation of GABAergic inhibition in mouse superficial superior colliculus.</a> J. Neurophysiol. 94, 3893-3902 (2005).</li><li>Bouzat, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+structural+bases+of+activation+of+Cys-loop+receptors.">New insights into the structural bases of activation of Cys-loop receptors.</a> J. Physiol. Paris. , (2011).</li></ol>

Метод, представленный в этой статье в целом полезны для изучения лигандами ионных каналов функцию в подготовке среза мозга. Тем не менее, существует ряд факторов, которые существенно влияют на качество и воспроизводимость экспериментальных данных, которые являются результатом исполь...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>N-Methyl D-glucamine</td>
 <td>Sigma</td>
 <td>M2004</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>Sigma</td>
 <td>P3911</td>
 </tr>
 <tr>
 <td>NaH2PO4</td>
 <td>Sigma</td>
 <td>S9638</td>
 </tr>
 <tr>
 <td>NaHCO3</td>
 <td>Sigma</td>
 <td>S6014</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma</td>
 <td>H3375</td>
 </tr>
 <tr>
 <td>glucose</td>
 <td>Sigma</td>
 <td>G5767</td>
 </tr>
 <tr>
 <td>Na+ ascorbate</td>
 <td>Sigma</td>
 <td>A4034</td>
 </tr>
 <tr>
 <td>thiourea</td>
 <td>Sigma</td>
 <td>T8656</td>
 </tr>
 <tr>
 <td>Na+ pyruvate</td>
 <td>Sigma</td>
 <td>P2256</td>
 </tr>
 <tr>
 <td>MgSO4&bull;7H2O</td>
 <td>Sigma</td>
 <td>230391</td>
 </tr>
 <tr>
 <td>CaCl2&bull;2H20</td>
 <td>Sigma</td>
 <td>223506</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Sigma</td>
 <td>S9625</td>
 </tr>
 <tr>
 <td>Na+ pentobarbital</td>
 <td>Vortech Pharmaceuticals</td>
 <td>76351315</td>
 </tr>
 <tr>
 <td>potassium gluconate</td>
 <td>Sigma</td>
 <td>G4500</td>
 </tr>
 <tr>
 <td>EGTA</td>
 <td>Sigma</td>
 <td>E3889</td>
 </tr>
 <tr>
 <td>Mg-ATP</td>
 <td>Sigma</td>
 <td>A9187 </td>
 </tr>
 <tr>
 <td>GTP</td>
 <td>Sigma</td>
 <td>G8877 </td>
 </tr>
 <tr>
 <td>DSK-Zero 1 Vibrating slicer</td>
 <td>Ted Pella, Inc.</td>
 <td></td>
 </tr>
 <tr>
 <td>P-97 Flaming/Brown micropipette puller</td>
 <td>Sutter</td>
 <td></td>
 </tr>
 <tr>
 <td>RC-27 Recording chamber</td>
 <td>Warner</td>
 <td></td>
 </tr>
 <tr>
 <td>TC-344B Perfusion heater controller</td>
 <td>Warner</td>
 <td>640101</td>
 </tr>
 <tr>
 <td>SH-27B Solution heater</td>
 <td>Warner</td>
 <td>640102</td>
 </tr>
 <tr>
 <td>Nikon FN-1</td>
 <td>Nikon</td>
 <td></td>
 </tr>
 <tr>
 <td>C-7500 CCD Video camera</td>
 <td>Hamamatsu</td>
 <td></td>
 </tr>
 <tr>
 <td>Picospritzer III</td>
 <td>General Valve Co.</td>
 <td></td>
 </tr>
 <tr>
 <td>MP-285 Micromanipulator </td>
 <td>Sutter </td>
 <td></td>
 </tr>
 <tr>
 <td>PA-100 Piezoelectric translator </td>
 <td>piezosystem jena, Inc.</td>
 <td></td>
 </tr>
 <tr>
 <td>12V40 piezo amplifier</td>
 <td>piezosystem jena, Inc.</td>
 <td></td>
 </tr>
 <tr>
 <td>Axopatch 200B</td>
 <td>Molecular Devices Corp.</td>
 <td></td>
 </tr>
 <tr>
 <td>Digidata 1440A</td>
 <td>Molecular Devices Corp.</td>
 <td></td>
 </tr>
 </tbody>
 </table>

local application of drugs to study nicotinic acetylcholine receptor function in mouse brain slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.
Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as &alpha;4 L9&prime;A mice 1 and &alpha;6 L9&prime;S mice 2, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.
In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.

In our experiments, we routinely record from dopamine (DA)-producing neurons of the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). In voltage-clamp mode, pressure application of acetylcholine or nicotine to these cells will typically result in a rapid, inward cation current that reaches peak within 100-200 msec (Figure 1A-B). Decay of the current is largely dictated by diffusion of the drug from the site of action, and whether enzymes in the slice are present to metabolize the dru...

Watch this Scientific Journal Video about Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices at JoVE.com

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

В этой статье мы описываем полезный метод для изучения лигандами ионных каналов функции в нейронах остро изолированных кусочков мозга. Этот метод включает в себя использование наркотиков заполненные микропипетки для местного применения препаратов на нейроны, записанные с помощью стандартных методов зажим патч.

Местное применение наркотиков по изучению никотиновых рецепторов ацетилхолина функции в срезах мозга мыши

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

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Drugs Acting on Autonomic Nervous System: Cholinergic Agonists and Antagonists Agents

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