Name: odorant-induced responses recorded from olfactory receptor neurons using the suction pipette technique
Uploaded: 2012-04-05T12:00:00
Description: Watch this Scientific Journal Video about Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique at JoVE.com

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...

<ol>
	<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 ...

Research

JoVE Journal

Neuroscience

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 ...

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific ...

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

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 ...