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

Summary

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.

Abstract

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 & 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 body^2-4.

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 ORNs^7,8. 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.

Protocol

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…

Discussion

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…

Materials

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