घ्राण रिसेप्टर न्यूरॉन्स (ORNs) गंध संकेतों एक रिसेप्टर में पहली परिवर्तित वर्तमान है कि बदले में कार्रवाई की क्षमता है कि घ्राण बल्ब में दूसरे क्रम न्यूरॉन्स को अवगत करा रहे हैं से चलाता है. यहाँ हम चूषण विंदुक के लिए एक साथ odorant प्रेरित रिसेप्टर वर्तमान और माउस ORNs से ऐक्शन पोटेंशिअल रिकॉर्ड तकनीक का वर्णन करता है.
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 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.
The authors have nothing to disclose.
यह काम DC009613 एनआईएच, मानव सीमाओं विज्ञान कार्यक्रम और एक मॉर्ले केयर फैलोशिप (जूनियर) द्वारा समर्थित किया गया.
Name of the material | Tipo | 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 |