This work was supported by NIH DC009613, the Human Frontiers Science Program and a Morley Care Fellowship (to JR).

1. The Recording Setup
The recording chamber is mounted on a Nikon TE2000U Eclipse inverted microscope with phase contrast optics which is fitted on an air table and electrically shielded using a Faraday cage. The Plexiglass recording chamber consists of two sections partially separated by a barrier and glued onto a silanized glass slide. One section of the chamber is used to settle the cells while the other is used for stimulus-exposure during the recording to minimize premature exposure of set...

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	<li>Reisert, J., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on:+Information+and+coding+in+mammalian+sensory+physiology:+Response+kinetics+of+olfactory+receptor+neurons+and+the+implications+in+olfactory+coding.">Perspectives on: Information and coding in mammalian sensory physiology: Response kinetics of olfactory receptor neurons and the implications in olfactory coding.</a> J. Gen. Physiol. 138, 303-310 (2011).</li><li>Kaupp, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+signalling+in+vertebrates+and+insects:+differences+and+commonalities.">Olfactory signalling in vertebrates and insects: differences and commonalities.</a> Nat. Rev. Neurosci. 11, 188-200 (2010).</li><li>Tirindelli, R., Dibattista, M., Pifferi, S., Menini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+pheromones+to+behavior.">From pheromones to behavior.</a> Physiol. Rev. 89, 921-956 (2009).</li><li>Kleene, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electrochemical+basis+of+odor+transduction+in+vertebrate+olfactory+cilia.">The electrochemical basis of odor transduction in vertebrate olfactory cilia.</a> Chem. Senses. 33, 839-859 (2008).</li><li>Baylor, D. A., Lamb, T. D., Yau, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+retinal+rods+to+single+photons.">Responses of retinal rods to single photons.</a> J. Physiol. 288, 613-634 (1979).</li><li>Wang, J., Kefalov, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+Suction+Recordings+from+Mouse+Cone+Photoreceptors.">Single-cell Suction Recordings from Mouse Cone Photoreceptors.</a> J. Vis. Exp. (35), e1681 (2010).</li><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=The+spatial+distributions+of+odorant+sensitivity+and+odorant-induced+currents+in+salamander+olfactory+receptor+cells.">The spatial distributions of odorant sensitivity and odorant-induced currents in salamander olfactory receptor cells.</a> J. Physiol. 442, 147-168 (1991).</li><li>Reisert, J., 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=Na+-dependent+Ca2++extrusion+governs+response+recovery+in+frog+olfactory+receptor+cells.">Na+-dependent Ca2+ extrusion governs response recovery in frog olfactory receptor cells.</a> J. Gen. Physiol. 112, 529-535 (1998).</li><li>Reisert, J., 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=Adaptation+of+the+odour-induced+response+in+frog+olfactory+receptor+cells.">Adaptation of the odour-induced response in frog olfactory receptor cells.</a> J. Physiol. 519, 801-813 (1999).</li><li>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=A+compact+modular+flow+heater+for+the+superfusion+of+mammalian+cells.">A compact modular flow heater for the superfusion of mammalian cells.</a> J. Physiol. 518P, 13 (1999).</li><li>Reisert, J., 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=Simultaneous+recording+of+receptor+current+and+intraciliary+Ca2++concentration+in+salamander+olfactory+receptor+cells.">Simultaneous recording of receptor current and intraciliary Ca2+ concentration in salamander olfactory receptor cells.</a> J. Physiol. 535, 637-645 (2001).</li></ol>

The suction pipette technique is a electrophysiological method which is used to record the odor-induced slow receptor current and the fast biphasic action potentials from an ORN simultaneously. Since the plasma membrane of the cell is not breached, this method leaves the intracellular milieu undisturbed ensuring that the odorant responses are not altered due to changes of the cytoplasmic ion concentrations or dilution of intracellular factors. Cells can be recorded from for long durations (up to 4 h in frog and 1 h in m...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the material</td>
 <td>Type</td>
 <td>Company</td>
 <td>Catalogue / Model number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Air table</td>
 <td>equipment</td>
 <td>Newport</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Air Pump</td>
 <td>equipment</td>
 <td>Newport</td>
 <td>ACGP</td>
 <td></td>
 </tr>
 <tr>
 <td>Pipette Puller </td>
 <td>equipment</td>
 <td>Sutter</td>
 <td>P-97</td>
 <td></td>
 </tr>
 <tr>
 <td>Borosilicate glass</td>
 <td>equipment</td>
 <td>WPI</td>
 <td>1B150-4</td>
 <td></td>
 </tr>
 <tr>
 <td>Nikon Eclipse Inverted microscope</td>
 <td>equipment</td>
 <td>Nikon</td>
 <td>TE2000U</td>
 <td>Equipped with Hg lamp, GFP filter and objectives 20X and 5X at least</td>
 </tr>
 <tr>
 <td>Amplifier PC-501A</td>
 <td>equipment</td>
 <td>Warner</td>
 <td> 64-0008</td>
 <td>Headstage 1 G&Omega;</td>
 </tr>
 <tr>
 <td>Diamond knife</td>
 <td>Equipment</td>
 <td>Custom-made</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Digitizer Mikro1401 A/D</td>
 <td>equipment</td>
 <td>Cambridge Electronic Design</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Filter unit 3382</td>
 <td>equipment</td>
 <td>Krohn Hite corporation</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Signal</td>
 <td>software</td>
 <td>Cambridge Electronic Design</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Molded Ag/AgCl Pellet</td>
 <td>equipment</td>
 <td>WPI</td>
 <td>64-1297</td>
 <td></td>
 </tr>
 <tr>
 <td>Pipette holder </td>
 <td>equipment</td>
 <td>Warner</td>
 <td>64-0997</td>
 <td>Custom modified to fit 
 headstage</td>
 </tr>
 <tr>
 <td>Recording chamber</td>
 <td>Equipment</td>
 <td>Custom-made</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td> Micromanipulator 
 MP85-1028</td>
 <td>equipment</td>
 <td>Sutter Instrument</td>
 <td> Micromanipulator 
 MP85-1028</td>
 <td></td>
 </tr>
 <tr>
 <td>Mineral oil</td>
 <td>Solution</td>
 <td>Sigma</td>
 <td>330779-1L</td>
 <td></td>
 </tr>
 <tr>
 <td>Oscilloscope TDS 1001</td>
 <td>equipment </td>
 <td>Tektronix</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Three-barreled square glass tube</td>
 <td>Equipment</td>
 <td>Warner</td>
 <td>64-0119</td>
 <td>0.6 mm ID , 5 cm long</td>
 </tr>
 <tr>
 <td>Valve </td>
 <td>equipment</td>
 <td>The Lee Company</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Valvelink 8.2</td>
 <td>equipment</td>
 <td>Automate Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>SF-77B Perfusion fast step</td>
 <td>equipment</td>
 <td>Warner</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique at JoVE.com

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

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 ...

odorant-induced-responses-recorded-from-olfactory-receptor-neurons

Research

JoVE Journal

Neuroscience

10.7K vistas.  Monell Chemical Senses Center. Neuronas olfativas del receptor (ORNs) convertir señales de olor primero en un receptor de corriente que a su vez desencadena los potenciales de acción que se transmiten a las neuronas de segundo orden en el bulbo olfatorio. Aquí se describe la técnica de pipeta de succión de grabar simultáneamente el receptor de odorizantes de la corriente inducida y los potenciales de acción de ORNs ratón.