This work was supported by National Institutes of Health (NIH) grant DA030396. Thanks to members of the Drenan lab for helpful discussion and critique of the manuscript. Special thanks to Mi Ran Kim for technical assistance and Jonathan Thomas Ting for advice regarding adult mouse brain slices.

1. Preparation of Solutions for Brain Slice Preparation and Electrophysiology
<ol>
 <li>Solutions for preparation of brain slices were previously described 3, 4. Prepare N-methyl D-glucamine (NMDG)-based cutting and recovery solution of the following composition (in mM): 93 n-methyl D-glucamine, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 5 Na+ ascorbate, 2 thiourea, 3 Na+ pyruvate, 10 MgSO4&bull;7H2O, 0.5 CaCl2&bull;2H2O. Adjust to 300-310 mOsm with NMDG. Adjust pH to 7.3-7.4 with 10 N HCl.
 <ol class="lalpha">
 <li>Dissolve MgSO4&bull;7H2O and CaCl2&bull;2H2O in Millipore water to make 2 M stock solutions of each.</li>
 <li>Weigh out other components in the order listed and add to a volumetric flask partially filled with Millipore water while stirring to dissolve. Once everything is added, fill volumetric flask with Millipore water.</li>
 <li>Measure the pH and adjust with 10 N HCl.</li>
 <li>Measure the osmolarity and adjust with NMDG if necessary.</li>
 </ol>
 </li>
 <li>Prepare HEPES holding artificial cerebrospinal fluid (aCSF) of the following composition (in mM): 92 NaCl, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 5 Na+ ascorbate, 2 thiourea, 3 Na+ pyruvate, 2 MgSO4&bull;7H2O, 2 CaCl2&bull;2H2O. Adjust to 300-310 mOsm with sucrose. Adjust pH to 7.3-7.4 with 1 N HCl or NaOH.
 <ol class="lalpha">
 <li>Weigh out components in the order listed and add to a volumetric flask partially filled with Millipore water while stirring to dissolve. Once everything is added, fill volumetric flask with Millipore water.</li>
 <li>Measure the pH and adjust with NaOH or HCl if needed.</li>
 <li>Measure the osmolarity and adjust with sucrose if necessary.</li>
 </ol>
 </li>
 <li>Prepare standard aCSF recording solution of the following composition (in mM): 124 NaCl, 2.5 KCl, 1.2 NaH2PO4, 24 NaHCO3, 12.5 glucose, 2 MgSO4&bull;7H2O, 2 CaCl2&bull;2H2O. Adjust to 300-310 mOsm with sucrose. Adjust pH to 7.3-7.4 with 1 N HCl or NaOH.</li>
</ol>
2. Preparation of Acute Brain Slices
<ol>
 <li>Brain slice cutting and recovery are done as previously described 3, 4. Bubble two crystal dishes filled with NMDG recovery solution with 95% oxygen/5% CO2 gas (carbogen), one dish on ice, the other at 33 &deg;C.</li>
 <li>Bubble one crystal dish filled with HEPES holding aCSF with carbogen at room temperature.</li>
 <li>Anesthetize adult mouse (aged 2 to 12 months) with Na+ pentobarbital (200 mg/kg, i.p.). Proceed with procedure when the animal has no response to a toe-pinch.</li>
 <li>If necessary, cut a tail tissue sample for later genotyping of the animal.</li>
 <li>Open the chest cavity, expose the heart, and clamp the descending aorta. Lacerate the right atrium.</li>
 <li>Transcardially perfuse animal with 5-10 ml of 0-4 &deg;C NMDG-recovery solution.</li>
 <li>Decapitate animal, remove the brain and place it in 4 &deg;C NMDG-recovery solution for 1 min.</li>
 <li>On an ice-chilled metal plate, trim the brain in the desired orientation (coronal, parasagittal, horizontal, etc.) to include the area of interest.</li>
 <li>Affix the brain block with superglue to the dry stage surface of a vibrating slicer (DSK-Zero 1; Dosaka), submerge the brain block in 4 &deg;C NMDG-recovery solution, and continuously bubble the solution with carbogen.</li>
 <li>Cut slices of the brain area of interest. Slice thickness is 200-400 &mu;m, depending on the specific brain area of interest, animal age, and experiment to be performed.</li>
 <li>Immediately after cutting, incubate desired slices in carbogenated, 33 &deg;C NMDG-recovery solution for exactly 12 min, then transfer to carbogenated, room temperature HEPES holding solution for an initial period of 60 min. After this 60 min incubation, slices can be used for recording. Slices are maintained in HEPES holding solution throughout the day until they are used for recording.</li>
</ol>
3. Patch Clamp Recording from Neurons in Brain Slices
<ol>
 <li>Pull a standard patch micropipette with a programmable Flaming-Brown puller (Sutter P-97 or equivalent vertical puller). Pipettes that are optimal for giga-ohm seal formation typically have a resistance of 4-6 M&Omega;</li>
 <li>Transfer one slice to the recording chamber (RC-27L; Warner Instruments) on the stage of an upright microscope (Nikon FN-1). Continuously superfuse (1.5 - 2.0 ml/min) the slice with carbogenated, standard recording aCSF heated and maintained at 32 &deg;C. Alternatively, slices can be maintained at room temperature.</li>
 <li>Locate and center the brain area of interest using a 10X air objective (Nikon Plan Fluor 10X; NA 0.3; WD: 16 mm).</li>
 <li>Visualize individual neurons using a 40X near-infrared (IR) water immersion objective (Nikon APO 40XW; NA: 0.80; WD: 3.5 mm) connected to IR-differential interference contrast (DIC) optics and a high-speed near infrared charged coupled device (CCD) video camera (Hamamatsu C-7500). Under IR-DIC observation, healthy neurons exhibit a smooth, light-grey plasma membrane.</li>
 <li>Fill a micropipette with a suitable intracellular recording solution. For most applications, we use the following solution (in mM): 135 potassium gluconate, 5 EGTA, 0.5 CaCl2, 2 MgCl2, 10 HEPES, 2 Mg-ATP, and 0.1 GTP (pH adjusted to 7.25 with Tris base, osmolarity adjusted to 290 mOsm with sucrose). Establish a whole-cell recording from a visualized neuron.</li>
</ol>
4. Local Application of Drugs to Neurons in Slices
<ol>
 <li>Pull a standard patch clamp micropipette as described above. Using a pipette puller such as a Sutter P-97 that produces two identical "sister" tips from the same piece of glass is advantageous when multiple drug solutions are to be used on the same cell. Tip sizes that would have a resistance of ~5 M&Omega; if they were filled with the intracellular solution described above are optimal.</li>
 <li>Backfill the micropipette with a solution of drug diluted in carbogenated, standard recording aCSF. Point the tip downward, and flick the drug-filled micropipette to remove any air bubbles trapped in the column of solution.</li>
 <li>Mount the drug-filled micropipette in a pipette holder connected with tubing to a suitable pressure ejection system, such as a Picospritzer III (General Valve Co.). The pipette holder should be mounted onto a micromanipulator with the same resolution as manipulators commonly used for patch clamp recording (for example, Sutter MP-285). Additionally, the manipulator should allow for diagonal movement into and out of the slice. Manually eject pressure 3 to 4 times into the micropipette in order to build up adequate pressure behind the column of fluid.</li>
 <li>Using the micromanipulator controls, lower the drug pipette into the slice while visualizing the tip under IR-DIC optics. Ideally, one should visually identify and center a neuron to be recorded from, followed by positioning of the drug pipette near the cell prior to lowering the recording micropipette into the slice. Positioning the drug pipette can cause movement of tissue in the vicinity of the recorded cell that could disrupt the recording if the drug pipette is moved into position after establishing a gigaohm seal.</li>
 <li>Using IR video, position the drug micropipette at the top surface of the tissue slice. Execute one pressure ejection and monitor 1) the vicinity of the tip for movement of debris related to the ejection, and 2) the micropipette tip for any signs that the tip is clogged or blocked. If the tip is blocked/clogged, retract the pipette and replace it with a new one.</li>
 <li>Approach the recorded cell diagonally from the top of the slice such that when in final position, the tip of the drug-filled pipette is 1) in the same focal plane (or slightly below) as the recorded cell and the tip of the recording electrode, and 2) 10 to 40 &mu;m from the recorded cell. If the drug-filled pipette tip is above the recorded cell, the force from the pressure ejection could disrupt the gigaohm seal.</li>
 <li>Record the desired cellular response while holding the cell in voltage clamp mode or current clamp mode. We routinely record acetylcholine and/or nicotine-elicited cellular currents while holding neurons in voltage clamp mode. Although pressure ejection can be triggered manually when using a Picospritzer III, for more reproducible results it is advisable to trigger the pressure ejection using the acquisition system (Axon Digidata 1440 and pClamp 10.3) with a digital TTL pulse.</li>
</ol>
5. Controlling the Drug-filled Micropipette with a Piezoelectric Translator
<ol>
 <li>If possible, mount the drug-filled pipette holder to a single-dimension piezoelectric translator (e.g. Piezojena PA-100 or Burleigh PZM-150) that is capable of accepting an analog voltage input (again, from the acquisition system), which is then mounted onto the high-precision micromanipulator (Sutter MP-285). A piezoelectric translator is useful because the movement of the drug-filled pipette into and out of the slice, including the timing and speed of entry/exit, can be more easily controlled and reproduced from experiment to experiment. When studying nAChRs, the desensitizing effects of nicotine leakage from the drug-filled pipette, should they ever occur, are minimized when the pipette is only moved near the cell with a piezoelectric translator for the moment of pressure ejection.</li>
 <li>Using the manually-controlled potentiometer on the piezoelectric voltage control amplifier, dial the voltage to its maximal value.</li>
 <li>Using the micromanipulator, position the drug-filled pipette in the slice where the pipette will eject its contents onto the recorded cell, and retract the pipette by manually reducing the voltage to zero on the piezoelectric voltage control amplifier.</li>
 <li>Using an analog output signal from the recording acquisition system, move the drug-filled pipette into the slice over a period of 250 msec starting 300 msec before the pressure ejection TTL pulse occurs. Retract the pipette over a period of 250 msec, starting 50 msec after the TTL pulse ends.</li>
</ol>

<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>

The method presented in this paper is broadly useful for studying ligand-gated ion channel function in brain slice preparations. However, there are a number of factors that will significantly affect the quality and reproducibility of experimental data that result from utilizing this method. For example, evoked currents are very sensitive to the diameter of the tip of the drug-filled pipette. Small tips will cause difficulty with ejecting the drug solution, and large tips with low resistance will be more likely to disrupt...

<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

In this paper, we describe a useful method to study ligand-gated ion channel function in neurons of acutely isolated brain slices. This method involves the use of a drug-filled micropipette for local application of drugs to neurons recorded using standard patch clamp techniques.

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

local-application-drugs-to-study-nicotinic-acetylcholine-receptor

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Neuroscience

Unit 1

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

SCIENTISTS IN ACTION

18.8K Views.  Purdue University. In this paper, we describe a useful method to study ligand-gated ion channel function in neurons of acutely isolated brain slices. This method involves the use of a drug-filled micropipette for local application of drugs to neurons recorded using standard patch clamp techniques.

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

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2. Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice3,4. Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs3. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice5. These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2&#916;TM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs6.

This article describes how to inject viral vectors into the mouse frontal cortex to test behavioral assays that require GPCR heteromeric formation.

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug application during electrophysiological recording is the puff technique, which can be used to study the effect of pharmacological reagents on neuronal activities in brain slices. The greatest advantage of puff application is that the drug concentration around the recording site increases rapidly, thus preventing desensitization of membrane receptors. Successful use of puff application involves careful attention to the following elements: the concentration of the drug, the parameters of the puff micropipette, the distance between the tip of a puff micropipette and the neuron recorded, and the duration and pressure driving the puff (pounds per square inch, psi). This article describes a step-by-step procedure for recording whole-cell currents induced by puffing gamma-aminobutyric acid (GABA) onto a neuron of a prefrontal cortical slice. Notably, the same procedure can be applied with minor modifications to other brain areas such as the hippocampus and the striatum, and to different preparations, such as cell cultures.

We describe the puff technique, by which pharmacological reagents can be administered during whole-cell patch-clamp recording, and highlight various aspects of the features that are crucial for its success.

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

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

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with subcellular location within cells where this function is carried out. To study the subcellular location of nicotinic ACh receptors (nAChRs) in native brain tissue, an optical method was developed for precise release of nicotine at discrete locations near neuronal membranes during electrophysiological recordings. Patch-clamped neurons in brain slices are filled with dye to visualize their morphology during 2-photon laser scanning microscopy, and nicotine uncaging is executed with a light flash by focusing a 405 nm laser beam near one or more cellular membranes. Cellular current deflections are measured, and a high-resolution three-dimensional (3D) image of the recorded neuron is made to allow reconciliation of nAChR responses with cellular morphology. This method allows for detailed analysis of nAChR functional distribution in complex tissue preparations, promising to enhance the understanding of cholinergic neurotransmission.

This article presents a method for studying nicotinic acetylcholine receptors (nAChRs) in mouse brain slices by nicotine uncaging. When coupled with simultaneous patch clamp recording and 2-photon laser scanning microscopy, nicotine uncaging connects nicotinic receptor function with cellular morphology, providing a deeper understanding of cholinergic neurobiology.

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

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...