成年小鼠人工吸气方法

Thomas P. Eiting; Matt  Wachowiak

doi:10.21769/BioProtoc.3024

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Peer-reviewed

Artificial Inhalation Protocol in Adult Mice

成年小鼠人工吸气方法

TE Thomas P. Eiting

MW Matt Wachowiak email

发布: 2018年09月20日第8卷第18期 DOI: 10.21769/BioProtoc.3024 浏览次数: 5474

评审: Oneil G. BhalalaTanveer AhmadEhsan Kheradpezhouh

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参见作者原研究论文

The authors used this protocol in:

Cover of The Journal of Neuroscience, featuring study using the protocol.

Feb 2018

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Abstract

Research in the area of in vivo olfactory physiology benefits from having direct access to the nasal airways through which odorants can be presented. Ordinarily, the passage of odorants through the airways is controlled by respiratory rhythm. This fact makes it difficult to control the timing and strength of an olfactory stimulus, since animals must breathe regularly, and the act of breathing itself also controls odorant presentation. However, using an artificial inhalation preparation allows us to decouple breathing from olfaction. With this technique we present oxygen and anesthetic (if desired) to the lungs directly and independently control odorant access to the nasal passages. This technique allows for direct control of odorant presentation in vivo, enabling more precise control of parameters of stimulation when investigating olfactory processing. This technique may have additional applications, for example in aerosolized drug delivery.

Keywords: Inhalation (吸入)

Background

Olfaction, aerosolized drug delivery, and many facets of airway humidification and homeostasis are governed by active respiration through the nasal passages. Basic research into these areas benefits from being able to actively control respiration in an anesthetized preparation. Our lab focuses in part on how active respiration controls olfactory coding in the olfactory bulb, which is the site of the first synapse in the neurobiology of odor perception. To address questions along this front in anesthetized mice, we employ an artificial respiration strategy that decouples airflow through the nasal passages from gas exchange with the lungs. This type of preparation allows experimentally manipulating nasal airflow while simultaneously delivering oxygenated air (usually incorporating a dilute aerosolized general anesthetic) to the lungs, thus producing a stable in vivo preparation that can last for eight hours or more.

This protocol is modified from an earlier form that has been briefly outlined in previous publications (Wachowiak and Cohen, 2001; Vučinić et al., 2006). Our lab and others have used this or a similar technique to investigate odor processing in the olfactory bulb of anesthetized rodents for several years (e.g., Sobel and Tank, 1993; Wachowiak and Cohen, 2001; Spors et al., 2006; Bathellier et al., 2008; Wachowiak et al., 2013; Rothermel et al., 2014; Economo et al., 2016). Here, we provide a detailed protocol for this method with the goal making it more easily adopted, and we expect that it may even find uses outside of the community working on olfactory physiology in rodents.

Materials and Reagents

Surgical thread, 6-0 Silk Suture Thread (Surgical Specialities, LOOK^TM, catalog number: SP102 )
Micro swabs (Absorbent Points #501) (Henry Schein, catalog number: 9004693 )
Razor blade (Exel Surgical Blades) (Exel International, model: #11, catalog number: 29502 )
Trachea Tubing, 0.034” x 0.050” x 0.008” (A-M systems, catalog number: 801900 )
Sniff Tubing, 0.045” x 0.062” x 0.0085” (A-M systems, catalog number: 802500 )
Sniff Connector, 0.04” ID, 0.085” OD (Dow Corning, Silastic^TM, catalog number: 508-005 )
T-junction tubing connector (about ¼” diameter), connected to flexible tubing (Tygon)
Y-connector (Grainger), 1/16” (for connecting and then splitting the sniff tubing)
Adult M or F mice (we use C57/Bl6, but any strain should work) of healthy weight (~20-40 g)
Super Glue (Henkel, Loctite, catalog number: LOCTITE 404 )
0.5% bupivacaine (Marcaine, Hospira Inc.)
Ketamine/xylazine cocktail
Isoflurane (VetONE Fluriso)
Nair (Church and Dwight Co.)
Denture/Dental Cement (Teets Cold Cure, Co-Oral-Ite Dental Mfg Co.)

Equipment

Isoflurane vaporizer (VetEquip)
3-way solenoid valve (Cole-Parmer, catalog number: EW-01540-11 )
Soldering iron
A vacuum line or other way of generating vacuum
Custom-cut steel headbar
Note: We use one that is roughly omega-shaped, but many shapes would suffice. Should be roughly 0.5 mm thick.
Heating pad
Two flow meters (Aalborg) (one of which is capable of 500 ml/min, the other of which is capable of 1 L/min or more)
Pressure sensor (Honeywell, catalog number: ASCX01DN or similar), mounted to circuit board and connected to Sniff Tubing via Y-connector
Tank of Compressed Oxygen Gas
Very fine surgical scissors (Roboz Surgical Instrument, catalog number: RS-5610 )
Fine Forceps (Fine Science Tools, model: Dumont #5 )
8" x 8" breadboard (ThorLabs)

Procedure

English

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文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Eiting, T. P. and Wachowiak, M. (2018). Artificial Inhalation Protocol in Adult Mice. Bio-protocol 8(18): e3024. DOI: 10.21769/BioProtoc.3024.
Diaz-Quesada, M., Youngstrom, I. A., Tsuno, Y., Hansen, K. R., Economo, M. N. and Wachowiak, M. (2018). Inhalation Frequency Controls Reformatting of Mitral/Tufted Cell Odor Representations in the Olfactory Bulb. J Neurosci 38(9): 2189-2206.