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 official strain name B6;129P2-Olfr16tm2Mom/MomJ; similarly, the SR1-IRES-tauGFP line, stock number 6717 bears the official name B6;129P2-Olfr124tm1Mom/MomJ.</li>
	<li>Regarding the age of the animals: for a better outcome of the protocol, use animals between 2 and 4 weeks of age. In this age group, the dissection is easier (softer bones, firmer olfactory epithelium) and the dendritic knobs are bigger compare to older animals.</li>
</ol>
2. Preparation of Electrodes and Solutions
<ol>
	<li>For the stimulating pipettes: purchase prepulled stimulating pipettes. Otherwise, manually prepare them.
	<ol>
		<li>Using a flame, bend six 1 mm glass pipettes at about 1 cm from the tip. Insert these six pipettes plus a straight 7th in 1.5 cm heat-shrink tubing strengthen by an eyelet.</li>
		<li>Heat shrink the tubing to maintain the barrels attached together. Attach an additional heat-shrink tubing to the other extremity of the barrels. Pull this stimulating pipette with a multi-barrel puller.</li>
		<li>Add some white liquid glue around the eyelet to strengthen the bended tips. Let dry O/N.</li>
	</ol>
	</li>
	<li>Prepare 1 or 2 L of normal Ringer&#8217;s extracellular solution (in mM: NaCl 124, KCl 3, MgSO4 1.3, CaCl2 2, NaHCO3 26, NaH2PO4 1.25, glucose 15; pH 7.6 and 305 mOsm). Keep at 4 &#176;C until use.</li>
	<li>Prepare intracellular stock solution (in mM): KCl 70, KOH 53, methanesulfonic acid 30, EGTA 5, HEPES 10, sucrose 70; pH 7.2 (KOH) and 310 mOsm. Keep at 4 &#176;C until use. Good for several weeks.</li>
	<li>Prepare intracellular recording solution with nystatin extemporaneously (at the last minute) before experiment.
	<ol>
		<li>Weigh 3 mg of nystatin, add 50 &#181;l&#160;of DMSO; vortex 20 sec then sonicate 2-3 min until entirely diluted.</li>
		<li>Add 20 &#181;l of DMSO-nystatin solution in 5 ml&#160;of intracellular stock solution. Vortex 20 sec, then sonicate 3 min. Keep this solution at 4 &#176;C and protect from direct light, nystatin is light-sensitive. This solution can be used for a few hours. Replace every day.</li>
		<li>Once the olfactory epithelium preparation is under the microscope, take some of this solution in a 1 ml&#160;syringe with a flame-elongated yellow tip to fill the electrodes; protect from direct light. Once at RT, the nystatin solution is not stable; replace the solution in the 1 ml syringe every hour or keep it in ice.</li>
	</ol>
	</li>
	<li>Pull recording electrodes with a puller to obtain long neck and small tip (~2 &#181;m) with a resistance of 15-20 M&#8486; with the internal nystatin solution.</li>
	<li>Prepare odorant solution at 0.5 M in DMSO under fume hood; aliquot and keep at -20 &#176;C until use. Dilute odorant in Ringer&#8217;s solution until the desired concentration. Fill the stimulating pipette.</li>
</ol>
3. Preparation of Olfactory Epithelium
Note: OR-IRES-tauGFP mice express the tauGFP under the control of the OR promoter. In these mice, all neurons expressing the OR of interest are labeled with GFP. This protocol is adapted for ORs expressed in all zones. However, dissections and recordings are easier for ORs expressed in the dorsal zone.
<ol>
	<li>Anesthetize the animal by injecting a mix of ketamine and xylazine (150 mg/kg and 10 mg/kg body weight, respectively).&#160;Decapitation can be performed with sharp scissors for young mice or with a properly maintained rodent guillotine for older mice.
	<ol>
		<li>Using ring dissecting scissors make a longitudinal medial incision through the dorsal skin. Remove the skin by pulling it apart. Using the scissors, cut the lower jaws at the jaw joint. Remove the upper front teeth by a coronal cut parallel to the teeth.</li>
		<li>Make a coronal cut of the head behind the eyes and keep only the anterior part of the head. Dip it in ice-cold Ringer solution for the dissection under the scope.</li>
	</ol>
	</li>
	<li>Dissect under the scope: in the ventral side, make a longitudinal cut along the upper jaw/the teeth. Cut the dorsal bones longitudinally, following the dorso-lateral side of the nasal cavity. Remove most bones and palate; transfer the septum and the epithelium attached to it in oxygenated Ringer at RT.</li>
	<li>For the final dissection: Right before starting the recording session, peel away the epithelium from the underlying septum with forceps. Detach the epithelium with forceps and with two 4-5 mm scissor cuts (use microvannas scissors) at the anterior part of the septum where the adhesion is stronger.
	<ol>
		<li>Carefully remove the vomeronasal organ by cutting it out along its dorsal connection to the septal epithelium. Transfer the epithelium to a recording chamber with the mucus layer facing up; keep it flat in the chamber with a harp.</li>
	</ol>
	</li>
	<li>Install chamber under an upright microscope equipped with fluorescence optics and a sensitive camera. Visualize the preparation on the computer screen at high magnification through a 40X water-immersion objective (numerical aperture 0.8) and an extra 2X or 4X magnification achieved by a magnifying lens. Perfuse continuously with oxygenated Ringer at RT (1-2 ml/min).</li>
</ol>
4. Recording Session
<ol>
	<li>Search for fluorescent cell: excite the preparation at 480 nm for EGFP, which emits light in the 530-550 nm range; target one dendritic knob which is reliably visible in fluorescence and under bright field.</li>
	<li>Fill electrode with intracellular solution with nystatin; remove bubbles by gently tapping on the electrode.</li>
	<li>Insert electrode on electrode holder, apply positive pressure in the pipette; Resistance should be 15-20 M&#8486;.</li>
	<li>Bring electrode close to the cell; once resistance reaches ~40 M&#8486;, release positive pressure and apply slight negative pressure to reach a gigaseal.</li>
	<li>Once seal is reached, set the membrane potential at about -75 mV;</li>
	<li>Once the cell is opened, proceed with stimulation protocols, pharmacological treatments. To measure the response to a single odorant stimulation, record 200-500 msec of spontaneous activity, stimulate for 500 msec and measure the response of the cell for up to 10 sec&#160;13. For pharmacological treatments, perfuse the pharmacological agents at the desired concentration20.</li>
</ol>
5. Data Analysis
<ol>
	<li>Analyze currents elicited by odorant stimulation as followed: the maximum amplitude, the rise-time (time necessary to reach 90% of the maximum amplitude, in msec), the total current (area under the curve in pAs), the time at 50% (time between the onset and the offset of the response at 50% of the maximum amplitude, in msec).
	<ol>
		<li>Using the peak transduction currents (maximum amplitude reached) at different concentrations, draw and fit a dose response curve using the Hill equation: I &#160;= Imax/(1 + (K1/2/C)n), where I represents the peak current, Imax the maximum response at saturating concentrations, K1/2 the concentration at which half of the maximum response was reached, C the concentration of odorant and n the Hill coefficient.</li>
	</ol>
	</li>
	<li>Analyze recordings of membrane potential in current clamp for the maximum depolarization, the total potential elicited (area under the curve in mVsec); record spontaneous spiking activity over 30 sec to 1 min recordings; record excitability through spikes elicited by injection of currents (5-10 pA).</li>
</ol>

<ol><li>Lowe, G., Gold, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nonlinear+amplification+by+calcium-dependent+chloride+channels+in+olfactory+receptor+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Odorant-induced+responses+recorded+from+olfactory+receptor+neurons+using+the+suction+pipette+technique%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Electrophysiological+and+biochemical+responses+of+mouse+vomeronasal+receptor+cells+to+urine-derived+compounds%3A+possible+mechanism+of+action%5BTitle%5D)+AND+%22Chem+Senses%22%5BJournal%5D)">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/pubmed?term=((Live+cell+calcium+imaging+of+dissociated+vomeronasal+neurons%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">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/pubmed?term=((Electrophysiological+characterization+of+rat+and+mouse+olfactory+receptor+neurons+from+an+intact+epithelial+preparation%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">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/pubmed?term=((A+novel+multigene+family+may+encode+odorant+receptors%3A+a+molecular+basis+for+odor+recognition%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Odorant+receptor+expression+defines+functional+units+in+the+mouse+olfactory+system%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Mapping+of+class+I+and+class+II+odorant+receptors+to+glomerular+domains+by+two+distinct+types+of+olfactory+sensory+neurons+in+the+mouse%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">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/pubmed?term=((Minigenes+impart+odorant+receptor-specific+axon+guidance+in+the+olfactory+bulb%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">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/pubmed?term=((Axon+guidance+of+mouse+olfactory+sensory+neurons+by+odorant+receptors+and+the+beta2+adrenergic+receptor%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Genes+and+ligands+for+odorant%2C+vomeronasal+and+taste+receptors%5BTitle%5D)+AND+%22Nat+Rev+Neurosci%22%5BJournal%5D)">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/pubmed?term=((SR1%2C+a+mouse+odorant+receptor+with+an+unusually+broad+response+profile%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Odorant+responses+of+olfactory+sensory+neurons+expressing+the+odorant+receptor+MOR23%3A+a+patch+clamp+analysis+in+gene-targeted+mice%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((Odorant+responsiveness+of+embryonic+mouse+olfactory+sensory+neurons+expressing+the+odorant+receptors+S1+or+MOR23%5BTitle%5D)+AND+%22Eur+J+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Uncoupling+stimulus+specificity+and+glomerular+position+in+the+mouse+olfactory+system%5BTitle%5D)+AND+%22Mol+Cell+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Ultrasensitive+detection+of+amines+by+a+trace+amine-associated+receptor%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Expression+patterns+of+odorant+receptors+and+response+properties+of+olfactory+sensory+neurons+in+aged+mice%5BTitle%5D)+AND+%22Chem+Senses%22%5BJournal%5D)">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/pubmed?term=((Postnatal+odorant+exposure+induces+peripheral+olfactory+plasticity+at+the+cellular+level%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Axonal+wiring+in+the+mouse+olfactory+system%5BTitle%5D)+AND+%22Annu+Rev+Cell+Dev+Biol%22%5BJournal%5D)">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/pubmed?term=((Dual+functions+of+mammalian+olfactory+sensory+neurons+as+odor+detectors+and+mechanical+sensors%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Odor+response+properties+of+rat+olfactory+receptor+neurons%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Odorant-binding+proteins+and+xenobiotic+metabolizing+enzymes%3A+implications+in+olfactory+perireceptor+events%5BTitle%5D)+AND+%22Anat+Rec+%28Hoboken%29%22%5BJournal%5D)">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/pubmed?term=((Perireceptor+events+in+olfaction%29%5BTitle%5D)+AND+%22J+Neurobiol%22%5BJournal%5D)">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/pubmed?term=((3-phosphoinositides+modulate+cyclic+nucleotide+signaling+in+olfactory+receptor+neurons%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">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/pubmed?term=((Blocking+adenylyl+cyclase+inhibits+olfactory+generator+currents+induced+by+%27IP%283%29-odors%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">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/pubmed?term=((Modulation+of+spontaneous+and+odorant-evoked+activity+of+rat+olfactory+sensory+neurons+by+two+anorectic+peptides%2C+insulin+and+leptin%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">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/pubmed?term=((Phosphoinositide+3-kinase-dependent+antagonism+in+mammalian+olfactory+receptor+neurons%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Phosphoinositide 3-kinase-dependent antagonism in mammalian olfactory receptor neurons.</a> J Neurosci. 31 (1), 273-280 (2011).</li>
</ol>

The authors declare that they have no competing financial interest.

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><tbody><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>optics</td><td></td><td></td><td></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&#039;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>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 1/10th 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;&amp;nbsp; to prepare recording pipets of about 2&amp;micro;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>stimulation</td><td></td><td></td><td></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&amp;nbsp;</td><td>Toohey Company</td><td>P/N T25-1-900 Single Channel&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>this precision pressure injector&amp;nbsp; controls the pressure ejected in the multibarrel puller; it is controlled manually or by the amplifier by a 5V&amp;nbsp; 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>solutions/perfusion/chemicals</td><td></td><td></td><td></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&amp;nbsp;</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 dihydrate</td><td>Sigma-Aldrich</td><td>C7902</td><td>extracellular solution</td></tr><tr><td>Sodium phosphate monobasic monohydrate (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S9638</td><td>extracellular solution</td></tr><tr><td>Magnesium sulfate heptahydrate (MgSO&lt;sub&gt;4&lt;/sub&gt; 7H&lt;sub&gt;2&lt;/sub&gt;O)</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&amp;prime;,N&amp;prime;-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>Methyl Sulfoxide</td><td>Sigma-Aldrich</td><td>W387509</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 ...

perforated-patch-clamp-recording-mouse-olfactory-sensory-neurons

Research

JoVE Journal

Neuroscience

26.7K Views.  Université de Bourgogne. 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.

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

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs1, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components3-8, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses9-12.
Odorant detection occurs at the surface of the olfactory epithelium on the cilia of OSNs, where a signal transduction cascade leads to opening of ion channels, generating a current that flows into the cilia and depolarizes the membrane13. The EOG is the negative potential recorded extracellularly at the surface of the olfactory epithelium upon odorant stimulation, resulting from a summation of the potential changes caused by individual responsive OSNs in the recording field2. Comparison of the amplitude and kinetics of the EOG thus provide valuable information about how genetic modification and other experimental manipulations influence the molecular signaling underlying the OSN response to odor. 
Here we describe an air-phase EOG recording on a preparation of mouse olfactory turbinates. Briefly, after sacrificing the mouse, the olfactory turbinates are exposed by bisecting the head along the midline and removing the septum. The turbinate preparation is then placed in the recording setup, and a recording electrode is placed at the surface of the olfactory epithelium on one of the medial turbinates. A reference electrode is electrically connected to the tissue through a buffer solution. A continuous stream of humidified air is blown over the surface of the epithelium to keep it moist. The vapor of odorant solutions is puffed into the stream of humidified air to stimulate the epithelium. Responses are recorded and digitized for further analysis.

The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis.

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors&#8212;an essential step toward a model of odor coding in the mammalian olfactory system.

Olfactory receptor activation patterns encode odor identity, but the lack of published data identifying odorant ligands for mammalian olfactory receptors hinders the development of a comprehensive model of odor coding. This protocol describes a method to facilitate high-throughput identification of olfactory receptor ligands and quantification of receptor activation.

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors ...

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

An Effective Manual Deboning Method To Prepare Intact Mouse Nasal Tissue With Preserved Anatomical Organization

The mammalian nose is a multi-functional organ with intricate internal structures. The nasal cavity is lined with various epithelia such as olfactory, respiratory, and squamous epithelia which differ markedly in anatomical locations, morphology, and functions. In adult mice, the nose is covered with various skull bones, limiting experimental access to internal structures, especially those in the posterior such as the main olfactory epithelium (MOE). Here we describe an effective method for obtaining almost the entire and intact nasal tissues with preserved anatomical organization. Using surgical tools under a dissecting microscope, we sequentially remove the skull bones surrounding the nasal tissue. This procedure can be performed on both paraformaldehyde-fixed and freshly dissected, skinned mouse heads. The entire deboning procedure takes about 20-30 min, which is significantly shorter than the experimental time required for conventional chemical-based decalcification. In addition, we present an easy method to remove air bubbles trapped between turbinates, which is critical for obtaining intact thin horizontal or coronal or sagittal sections from the nasal tissue preparation. Nasal tissue prepared using our method can be used for whole mount observation of the entire epithelia, as well as morphological, immunocytochemical, RNA in situ hybridization, and physiological studies, especially in studies where region-specific examination and comparison are of interest.

The mammalian nose is a multi-functional organ with intricate internal structures.  The nasal cavity is lined with various epithelia such as olfactory, ...

Biology

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of available mating partners or threats. In mice, this task is executed primarily by the olfactory system and its underlying subsystems, including the main and accessory olfactory systems. Both have peripheral organs populated by sensory neurons expressing G-protein coupled receptors able to bind chemical cues that reach the nasal cavity. Even though the molecular characteristics of these receptors is well understood, little is known about their cognate specific ligands. The method described here combines in situ hybridization detection of olfactory or vomeronasal receptors with immunodetection of phosphorylated ribosomal protein S6 (pS6) - a marker of neuronal activation. This protocol was devised to identify neurons activated after a single event of exposure to purified or complex chemical stimuli detected by the olfactory organs. Importantly, this technique allows the investigation of neurons triggered in biologically relevant contexts. Ideally, this method should be used to probe the molecular biology of the olfactory system and to study olfactory behaviors.

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

This protocol combines in situ hybridization with immunofluorescence to identify olfactory and vomeronasal receptor genes expressed in olfactory sensory neurons after activation by chemical stimuli in the mouse.

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of ...

Perforated Patch-Clamp Recording of Olfactory Sensory Neurons in Mice

Source: Jarriault, D., et al. <a href="https://app.jove.com/v/52652">Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor.</a> J. Vis. Exp. (2015).
This video demonstrates electrophysiological recording of olfactory sensory neurons (OSNs) in mice using a perforated patch-clamp technique. The olfactory epithelium, containing OSNs with fluorophore-tagged odor receptors, is isolated from a mouse and secured in a recording chamber. A recording pipette is clamped to the dendritic knob of an OSN, and nystatin within the pipette perforates the membrane to form electrical connectivity with the cell. Finally, another pipette is used to deliver an odor stimulant, and the electrophysiological activity of the patched cell is recorded.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

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

Samenvatting

Meer video's verkennen