Name: perforated patch-clamp recording of mouse olfactory sensory neurons in intact neuroepithelium: functional analysis of neurons expressing an identified odorant receptor
Uploaded: 2015-07-13T13:00:00
Description: Watch this Scientific Journal Video about Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant

Authors would like to thank Peter Mombaerts for the generous gift of OR-GFP mice; Anne Lefranc and the CSGA animal facility for excellent animal care. Funding was provided by CNRS through an ATIP and ATIP Plus grants, by Conseil R&#233;gional de Bourgogne (FABER and PARI grants), by Universit&#233; de Bourgogne (BQR program).

This protocol follows the animal care guidelines of the Universit&#233; de Bourgogne and was approved by the Universit&#233; de Bourgogne ethics committee.
1. Animals
<ol>
	<li>Use genetically engineered OR-IRES-tauGFP mice available at the Jackson Laboratory. These mice were developed in Dr. Peter Mombaerts&#8217; laboratory in order to analyze axon targeting and development of the olfactory system19. For example, the MOR23-IRES-tauGFP line, stock number 006643, bears the officia...

<ol>
	<li>Lowe, G., Gold, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+amplification+by+calcium-dependent+chloride+channels+in+olfactory+receptor+cells.">Nonlinear amplification by calcium-dependent chloride channels in olfactory receptor cells.</a> Nature. 366 (6452), 283-286 (1993).</li><li>Ponissery Saidu, S., Dibattista, M., Matthews, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-induced+responses+recorded+from+olfactory+receptor+neurons+using+the+suction+pipette+technique.">Odorant-induced responses recorded from olfactory receptor neurons using the suction pipette technique.</a> J Vis Exp. (62), e3862 (2012).</li><li>Moss, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+and+biochemical+responses+of+mouse+vomeronasal+receptor+cells+to+urine-derived+compounds:+possible+mechanism+of+action.">Electrophysiological and biochemical responses of mouse vomeronasal receptor cells to urine-derived compounds: possible mechanism of action.</a> Chem Senses. 23 (4), 483-489 (1998).</li><li>Kaur, A., Dey, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+calcium+imaging+of+dissociated+vomeronasal+neurons.">Live cell calcium imaging of dissociated vomeronasal neurons.</a> Methods Mol Biol. 1068, 189-200 (2013).</li><li>Ma, M., Chen, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+characterization+of+rat+and+mouse+olfactory+receptor+neurons+from+an+intact+epithelial+preparation.">Electrophysiological characterization of rat and mouse olfactory receptor neurons from an intact epithelial preparation.</a> J Neurosci Methods. 92 (1-2), 31-40 (1999).</li><li>Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+multigene+family+may+encode+odorant+receptors:+a+molecular+basis+for+odor+recognition.">A novel multigene family may encode odorant receptors: a molecular basis for odor recognition.</a> Cell. 65 (1), 175-187 (1991).</li><li>Bozza, T., Feinstein, P., Zheng, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant+receptor+expression+defines+functional+units+in+the+mouse+olfactory+system.">Odorant receptor expression defines functional units in the mouse olfactory system.</a> J Neurosci. 22 (8), 3033-3043 (2002).</li><li>Bozza, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+class+I+and+class+II+odorant+receptors+to+glomerular+domains+by+two+distinct+types+of+olfactory+sensory+neurons+in+the+mouse.">Mapping of class I and class II odorant receptors to glomerular domains by two distinct types of olfactory sensory neurons in the mouse.</a> Neuron. 61 (2), 220-233 (2009).</li><li>Vassalli, A., Rothman, A., Feinstein, P., Zapotocky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minigenes+impart+odorant+receptor-specific+axon+guidance+in+the+olfactory+bulb.">Minigenes impart odorant receptor-specific axon guidance in the olfactory bulb.</a> Neuron. 35 (4), 681-696 (2002).</li><li>Feinstein, P., Bozza, T., Rodriguez, I., Vassalli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+guidance+of+mouse+olfactory+sensory+neurons+by+odorant+receptors+and+the+beta2+adrenergic+receptor.">Axon guidance of mouse olfactory sensory neurons by odorant receptors and the beta2 adrenergic receptor.</a> Cell. 117 (6), 833-846 (2004).</li><li>Mombaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+and+ligands+for+odorant,+vomeronasal+and+taste+receptors.">Genes and ligands for odorant, vomeronasal and taste receptors.</a> Nat Rev Neurosci. 5 (4), 263-278 (2004).</li><li>Grosmaitre, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SR1,+a+mouse+odorant+receptor+with+an+unusually+broad+response+profile.">SR1, a mouse odorant receptor with an unusually broad response profile.</a> J Neurosci. 29 (46), 14545-14552 (2009).</li><li>Grosmaitre, X., Vassalli, A., Mombaerts, P., Shepherd, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant+responses+of+olfactory+sensory+neurons+expressing+the+odorant+receptor+MOR23:+a+patch+clamp+analysis+in+gene-targeted+mice.">Odorant responses of olfactory sensory neurons expressing the odorant receptor MOR23: a patch clamp analysis in gene-targeted mice.</a> Proc Natl Acad Sci U S A. 103 (6), 1970-1975 (2006).</li><li>Lam, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant+responsiveness+of+embryonic+mouse+olfactory+sensory+neurons+expressing+the+odorant+receptors+S1+or+MOR23.">Odorant responsiveness of embryonic mouse olfactory sensory neurons expressing the odorant receptors S1 or MOR23.</a> Eur J Neurosci. 38 (2), 2210-2217 (2013).</li><li>Zhang, J., Huang, G., Dewan, A., Feinstein, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncoupling+stimulus+specificity+and+glomerular+position+in+the+mouse+olfactory+system.">Uncoupling stimulus specificity and glomerular position in the mouse olfactory system.</a> Mol Cell Neurosci. 51 (3-4), 79-88 (2012).</li><li>Zhang, J., Pacifico, R., Cawley, D., Feinstein, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasensitive+detection+of+amines+by+a+trace+amine-associated+receptor.">Ultrasensitive detection of amines by a trace amine-associated receptor.</a> J Neurosci. 33 (7), 3228-3239 (2013).</li><li>Lee, A. C., Tian, H., Grosmaitre, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+patterns+of+odorant+receptors+and+response+properties+of+olfactory+sensory+neurons+in+aged+mice.">Expression patterns of odorant receptors and response properties of olfactory sensory neurons in aged mice.</a> Chem Senses. 34 (8), 695-703 (2009).</li><li>Cadiou, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+odorant+exposure+induces+peripheral+olfactory+plasticity+at+the+cellular+level.">Postnatal odorant exposure induces peripheral olfactory plasticity at the cellular level.</a> J Neurosci. 34 (14), 4857-4870 (2014).</li><li>Mombaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+wiring+in+the+mouse+olfactory+system.">Axonal wiring in the mouse olfactory system.</a> Annu Rev Cell Dev Biol. 22, 713-737 (2006).</li><li>Grosmaitre, X., Santarelli, L. C., Tan, J., Luo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+functions+of+mammalian+olfactory+sensory+neurons+as+odor+detectors+and+mechanical+sensors.">Dual functions of mammalian olfactory sensory neurons as odor detectors and mechanical sensors.</a> Nat Neurosci. 10 (3), 348-354 (2007).</li><li>Duchamp-Viret, P., Chaput, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odor+response+properties+of+rat+olfactory+receptor+neurons.">Odor response properties of rat olfactory receptor neurons.</a> Science. 284 (5423), 2171-2174 (1999).</li><li>Heydel, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-binding+proteins+and+xenobiotic+metabolizing+enzymes:+implications+in+olfactory+perireceptor+events.">Odorant-binding proteins and xenobiotic metabolizing enzymes: implications in olfactory perireceptor events.</a> Anat Rec (Hoboken). 296 (9), 1333-1345 (2013).</li><li>Pelosi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perireceptor+events+in+olfaction).">Perireceptor events in olfaction).</a> J Neurobiol. 30 (1), 3-19 (1996).</li><li>Spehr, M., Wetzel, C. H., Hatt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-phosphoinositides+modulate+cyclic+nucleotide+signaling+in+olfactory+receptor+neurons.">3-phosphoinositides modulate cyclic nucleotide signaling in olfactory receptor neurons.</a> Neuron. 33, 731-739 (2002).</li><li>Chen, S., Lane, A. P., Bock, R., Leinders-Zufall, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+adenylyl+cyclase+inhibits+olfactory+generator+currents+induced+by+'IP(3)-odors.">Blocking adenylyl cyclase inhibits olfactory generator currents induced by 'IP(3)-odors.</a> J Neurophysiol. 84 (1), 575-580 (2000).</li><li>Savigner, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+spontaneous+and+odorant-evoked+activity+of+rat+olfactory+sensory+neurons+by+two+anorectic+peptides,+insulin+and+leptin.">Modulation of spontaneous and odorant-evoked activity of rat olfactory sensory neurons by two anorectic peptides, insulin and leptin.</a> J Neurophysiol. 101 (6), 2898-2906 (2009).</li><li>Ukhanov, K., Brunert, D., Corey, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoinositide+3-kinase-dependent+antagonism+in+mammalian+olfactory+receptor+neurons.">Phosphoinositide 3-kinase-dependent antagonism in mammalian olfactory receptor neurons.</a> J Neurosci. 31 (1), 273-280 (2011).</li></ol>

The ability of this protocol to correctly monitor the properties of healthy OSNs depends heavily on the quality of the preparation. Therefore, the dissection steps are critical. First it is critical to pay attention to the quality (pH, osmolarity), oxygenation and temperature (ice-cold but not frozen) of the dissection medium. Second, the manipulation of the epithelium with dissecting tools must be as limited as possible to avoid damages. Finally, it is critical to obtain a preparation as flat as possible in order to acc...

Olfactory sensory neurons (OSN) represent the first step of olfactory perception. Located in the olfactory epithelium lining the nasal cavity in rodents, they transform the chemical information of odorants into action potentials sent through their axon to the brain. To better understand the olfactory coding mechanisms, it is necessary to characterize the transduction and membrane properties of OSNs. Until recently, most of the techniques used to characterize the properties of mammalian OSNs were carried out on dissociated OSNs1-4. The dissociation process uses various mechanical and chemical (i.e., enzymes) processes to free the OSNs from their environment. These processes induce a low number of available cells for recordings. This low number can be even more critical in the case of GFP labelled cells. Dissociation also removes the local cell-to-cell interactions between OSNs and other cells of the olfactory epithelium that may enhance survival and modulation of OSNs&#39; properties. In order to bypass the dissociation procedure, an intact preparation was developed5.
Each OSN expresses one olfactory receptor (OR) selected from a large multigene family6. There are ~1,000 ORs expressed in the main olfactory epithelium in the mouse. Due to the large number of OR in wild type animals, the chances to record OSNs expressing the same OR are very low. To overcome these limitations, gene targeted mice are available&#160;in which all OSNs expressing an identified OR are labeled with a fluorescent protein7-9. These labeled OSNs were used to do functional analysis in dissociated preparations7,10,11 with the drawbacks mentioned earlier. An intact epithelium preparation5 from genetically labeled mice therefore circumvents these issues. It allows the monitoring of the activity of OSNs expressing precisely defined ORs in an environment as close to in vivo as possible. Besides, patch-clamp recordings of OSNs also allow precise analysis of membrane properties, transduction pathway pharmacology, ligand/OR interactions. All these topics can hardly be analyzed using extracellular recordings. We used this technique to monitor the responses of OSNs expressing the odorant receptors SR1 and MOR2312,13. The feasibility of the technique was confirmed by other groups on MOR23 expressing OSNs14 as well as on other ORs expressing neurons15,16. The monitoring of a defined population of OSNs can lead to the analysis of their properties in many different contexts such as development14, aging17, odorant induced plasticity18, and the role of variations in the odorant receptor&#8217;s sequence in odor coding15. This protocol thus provides a powerful tool to monitor the functional properties of defined OSNs at the membrane level.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td colspan="4">heavy equipment</td>
		</tr>
		<tr>
			<td>vibration table with Faraday cage</td>
			<td>TMC</td>
			<td>63-500 SERIES</td>
			<td>required : isolates the recording system from vibrations induced by the environment (movements of experimenter, vibrations of equipment such as fans for cooling computers, etc); can also be purchased with a Faraday cage, or equipped by a custom made Faraday cage; this cage is recommended to avoid electric noise from the environment</td>
		</tr>
		<tr>
			<td colspan="4">optics</td>
		</tr>
		<tr>
			<td>microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td>upright microcope equipped with epifluorescence; fixed or moving stage depending on the user&#39;s preference</td>
		</tr>
		<tr>
			<td>objectives</td>
			<td>Olympus</td>
			<td>LUMPLFL40XW</td>
			<td>at least 2 objectives required: a 4x or 10x for coarse approach to the cell; and a 40x immersion long distance example Olympus LUMPLFL40XW/IR/0,8/WD:3.3 MM</td>
		</tr>
		<tr>
			<td>magnifier</td>
			<td>Olympus</td>
			<td>U-TVCAC</td>
			<td>ABSOLUTELY REQUIRED: placed in the light path between the objective and the camera; allows to magnify the image on the screen in order to reach precisely the knob with the recording electrode</td>
		</tr>
		<tr>
			<td>camera</td>
			<td>Olympus</td>
			<td>DP72</td>
			<td>a good camera is required to see the neurons in fluorescence as well as in bright field; the controlling software is simple and allows to take pictures and do live camera image to monitor the approach of the electrode to the cell. An ultrasensitive camera is not necessary</td>
		</tr>
		<tr>
			<td>filters</td>
			<td>Olympus/Chroma</td>
			<td></td>
			<td>depending on the fluorescent protein used in the mice; example for GFP: excitation : BP460-490: emission: HQ530/50m</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">recording electrodes /system</td>
		</tr>
		<tr>
			<td>amplifier</td>
			<td>HEKA</td>
			<td>EPC10 USB</td>
			<td>monitors the currents flowing through the recording electrode and also controls the puffing by sending a TTL signal to the spritzer; the EPC10 setup is controled by computer</td>
		</tr>
		<tr>
			<td>software</td>
			<td>HEKA</td>
			<td>Patchmaster</td>
			<td>controls the amplifier during the experiment</td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Sutter</td>
			<td>MP225</td>
			<td>precision micromanipulator, allows precise movements down to 1/1Oth of a micrometer; this model is very stable; avoid hydraulic manipulators that may drift</td>
		</tr>
		<tr>
			<td>electrode puller</td>
			<td>sutter</td>
			<td>P97</td>
			<td>with a FT345-B wide trough filament;&#160; to prepare recording pipets of about 2&#181;m diameter with a long tip to reach the cells; the resistance should be 15 to 20Mohm with perforated patch clamp solution</td>
		</tr>
		<tr>
			<td>glass</td>
			<td>sutter</td>
			<td>BF120-69-10</td>
			<td>in our recording conditions, this glass is ideal for recording pipets</td>
		</tr>
		<tr>
			<td>recording chamber</td>
			<td>warner instruments</td>
			<td>RC-26G</td>
			<td>a chamber is needed to set the preparation under the microscope. To maintain the preparation in the center of the chamber, a net/anchor should be used.</td>
		</tr>
		<tr>
			<td colspan="4">stimulation</td>
		</tr>
		<tr>
			<td>glass</td>
			<td>WPI</td>
			<td>TW100F-4</td>
			<td>attached in groups of 7, these pipettes are used to prepare prepulled stimulating pipettes</td>
		</tr>
		<tr>
			<td>multibarrel puller</td>
			<td>MDI</td>
			<td>PMP-107-Z</td>
			<td>by association of pull and twist, this puller allows us to prepare puffing electrodes with 7 barrels</td>
		</tr>
		<tr>
			<td>precision pressure injector&#160;</td>
			<td>Toohey Company</td>
			<td>P/N T25-1-900 Single Channel&#160;&#160;&#160;</td>
			<td>this precision pressure injector&#160; controls the pressure ejected in the multibarrel puller; it is controlled manually or by the amplifier by a 5V&#160; TTLs</td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Narishige</td>
			<td>YOU-1</td>
			<td>a coarse manipulator is enough to bring the puffing electrode close to the recording site</td>
		</tr>
		<tr>
			<td>tubings</td>
			<td>N/A</td>
			<td></td>
			<td>tygon tubing to bring the pressure from the puffer to the puffing pipette</td>
		</tr>
		<tr>
			<td colspan="4">solutions/perfusion/chemicals</td>
		</tr>
		<tr>
			<td>vacuum pump</td>
			<td>gardner denver</td>
			<td>300 series</td>
			<td>a vibrating membrane pump is more quiet and efficient than other types of pumps</td>
		</tr>
		<tr>
			<td>perfusion system</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>gravity perfusion system with polyethlylen tubing to bring in and out the external solution from the recording chamber</td>
		</tr>
		<tr>
			<td>nystatin</td>
			<td>Sigma-Aldrich</td>
			<td>N3503</td>
			<td>mandatory to perpare internal solution for perforated patch clamp</td>
		</tr>
		<tr>
			<td>DIMETHYL SULFOXIDE</td>
			<td>Sigma-Aldrich</td>
			<td>D5879</td>
			<td>used to disolve nystatin for internal solution for perforated patch&#160;</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9625</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P4504</td>
			<td>intracellular/extracellular solution</td>
		</tr>
		<tr>
			<td>Calcium chloride di hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C7902</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic monohydrate (NaH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate (MgSO4 7H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>63140</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6297</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>EGTA (Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid)</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td>internal solution</td>
		</tr>
		<tr>
			<td>Potassium hydroxyde</td>
			<td>Sigma-Aldrich</td>
			<td>P1767</td>
			<td>internal solution</td>
		</tr>
		<tr>
			<td>MethylSulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>47,135-6</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>Hepes-Na</td>
			<td>Sigma-Aldrich</td>
			<td>H7006</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>intracellular solution</td>
		</tr>
	</tbody>
</table>

perforated patch-clamp recording of mouse olfactory sensory neurons in intact neuroepithelium: functional analysis of neurons expressing an identified odorant receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

The outcome of this protocol depends on the quality of the dissection. This dissection steps must be short (less than 10 to 15 min) and precise (i.e., to avoid damages of the epithelium). The Figure 1 illustrates how an ideal preparation looks like at different magnification levels. At a low magnification under bright field the different cell types (such as knobs of OSNs, supporting cells) are distinguishable (Figure 1A). At the highest magnification level, typically 80X to 160X...

Watch this Scientific Journal Video about Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

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Neuroscience

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