Electrophysiology of Airway Nerves

Allen C. Myers1

1 The Johns Hopkins University School of Medicine, Baltimore, Maryland
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 11.10
DOI:  10.1002/0471141755.ph1110s39
Online Posting Date:  December, 2007
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Abstract

Several different electrophysiological approaches have been used to study the pharmacology of the afferent, central, and efferent nervous systems in airways. This unit describes electrophysiological methods used to study nerves in these pathways and includes: (1) extracellular recording of afferent nerve activity in vivo and from the isolated airway in vitro, (2) intracellular and patch clamp recording of identified airway sensory neurons, (3) patch clamp recording of secondary afferent central nervous system neurons, (4) in vitro and in vivo intracellular recording of intact parasympathetic ganglionic neurons, and (5) patch recordings of dissociated parasympathetic ganglionic neurons. Curr. Protoc. Pharmacol. 39:11.10.1‐11.10.27. © 2007 by John Wiley & Sons, Inc.

Keywords: airway; bronchus; trachea; afferent; sensory; parasympathetic

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Recording Extracellular (Single Unit) Action Potentials Evoked from Airway Nerve Terminals in Anesthetized Animals
  • Basic Protocol 2: Recording Extracellular (Single Unit) Action Potentials Evoked from Airway Nerve Terminals in Isolated Airways
  • Basic Protocol 3: Measurement of Pharmacological Responses of Single Cells Using Patch Clamp Electrode Recordings
  • Alternate Protocol 1: Intracellular Recordings of Intact Sensory Ganglionic Neurons
  • Basic Protocol 4: Pharmacological Analysis of Intact Airway Parasympathetic Postganglionic Neurons In Situ
  • Alternate Protocol 2: Patch‐Clamp Recording from Dissociated Single Neurons
  • Basic Protocol 5: Pharmacological Analysis of Synaptic Transmission Between Primary Airway Afferent Nerves and Second‐Order Brainstem Neurons
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Recording Extracellular (Single Unit) Action Potentials Evoked from Airway Nerve Terminals in Anesthetized Animals

  Materials
  • Sprague‐Dawley rats (300‐ to 400‐g)
  • Anesthetic: chloralose (100 mg/ml, Sigma) and urethane (500 mg/ml, Sigma) dissolved in a 2% borax solution
  • Oxygenated physiological saline: e.g., Krebs buffer (units 4.12& 5.26), Lockes solution, or mammalian Ringers solution
  • Mineral oil
  • Capsaicin (400 µg/ml, Sigma) prepared in a vehicle of 10% Tween 80, 10% ethanol, and 80% isotonic saline; dilute with saline to achieve desired concentration
  • 1‐ml disposable syringe
  • 27‐G needles
  • Venous and arterial cannulas made from PE 40–60 tubing, 22‐G luer stub adapters, and 3‐way stopcocks
  • Pressure transducers (Validyne Engineering, MP45–28; P23AA, Statham)
  • Tracheal cannula: 15‐G luer stub adapter 10‐ to 20‐mm in length (commercially prepared cannulas also available from Harvard Apparatus)
  • Mechanical respirator (Ugo Basile, no. 7025)
  • Heating pad
  • Cotton pledgets
  • Solution heating and perfusion system
  • Dissecting platform
  • Fine forceps
  • Micro spring scissors
  • Platinum–iridium hook electrode (Frederick Haer)
  • Amplifier (Grass Technologies, P511K)
  • Audio monitor (Grass Technologies, AM8RS)
  • Oscilloscope (Tektronix, 5103N)
  • Chart recorder (Gould Instrument Systems, TW11)
  • Tape‐format data recorder (Vetter, Rebersburg, PA, 4000 A)
  • Computer
  • Data acquisition system (Biocybernetics, TS‐100)

Basic Protocol 2: Recording Extracellular (Single Unit) Action Potentials Evoked from Airway Nerve Terminals in Isolated Airways

  Materials
  • Guinea pig (male, 100‐ to 300‐g, Hartley Hilltop Laboratory Animals)
  • Pentobarbital (i.p inject 100 mg/kg)
  • Oxygenated physiological saline: e.g., Krebs buffer (see unit 4.12 or ), Lockes solution, or mammalian Ringers solution
  • Paraffin oil
  • Electrolyte solution (3 M NaCl, pH 7.4)
  • Stock solutions of test compounds
  • Electrophysiological setup:
    • Dissection microscope (10× to 80× magnification, VMZ, Olympus)
    • Recording chamber (see Fig. A)
    • Calibrated von Frey fibers (Stoelting, model 18011)
    • Stimulator (Grass Technology, S44)
    • Silver‐silver chloride wire (return electrode)
    • Platinum–iridium hook electrode (Frederick Haer) for recording method 1 (step 5)
    • Glass recording electrodes (thick wall aluminosilicate, 1.0‐mm o.d., 0.5‐mm i.d., 1 to 3 MΩ) for recording method 2 (step 6)
    • Micropipette puller (P87, Sutter Instrument) for recording method 2 (step 6)
    • Electrode holder
    • Headstage (A.M. Systems)
    • AC microelectrode amplifier (A.M. Systems, model 1800)
    • Data acquisition and stimulation hardware (TheNerveOfIt, PHOCIS, Baltimore, MD)
    • Audible baseline monitor (FHC, model 12557)
    • Oscilloscope (Tektronix, TDS 320)
    • Chart recorder (Gould Instrument Systems, TA240)
    • Fast solution perfusion system
    • Fine manual manipulator (World Precision Instruments, M3301R)
    • Vibration isolation table (Newport, Technical Manufacturing)
    • Computer to execute commands and for data storage and analysis
    • Excel
  • Large scissors
  • Two fine‐tipped forceps
  • Sylgard‐lined petri dish (dissecting dish; see recipe)
  • Micro spring scissors
  • Fine, blunt plastic rod (outer diameter, 2 mm)
  • Additional reagents and equipment for dissection of the guinea pig (unit 4.12)

Basic Protocol 3: Measurement of Pharmacological Responses of Single Cells Using Patch Clamp Electrode Recordings

  Materials
  • DiC18(3) or Fast DiI (Molecular Probes)
  • Dimethylsulfoxide (DMSO)
  • Normal saline: 0.9% (w/v) NaCl
  • Animal (guinea pig, rat, or mouse)
  • Ketamine
  • Xylazine
  • 100% CO 2
  • Deep‐welled dissecting dish filled with gassed Krebs buffer at room temperature
  • Enzyme solution: 10 mg collagenase type 1A (Sigma) and 10 mg dispase II (Boehringer Mannheim) in 5 ml calcium‐ and magnesium‐free Hanks' balanced salt solution (CMF‐HBSS)
  • L15 medium (Invitrogen) containing 10% fetal bovine serum (JRH Biosciences)
  • Electrolyte solution (in mM: 140 KCl, 1 CaCl 2, 2 MgCl 2, 10 HEPES, 11 EGTA and 10 dextrose; titrated to pH 7.3 with KOH; 304 mOsmol/liter)
  • Gramicidin (optional)
  • Dimethylsulfoxide (DMSO), optional
  • Oxygenated physiological saline: e.g. Krebs buffer (units 4.12& 5.26), Lockes solution, or mammalian Ringers solution
  • Stock solutions of test compounds
  • Electrophysiological setup
    • Patch pipets (also see units 11.2& 11.8)
    • Axopatch 200B or Multiclamp 700A patch‐clamp amplifier (Axon Instruments)
    • Data acquisition and stimulation interface (Axon Instruments, Digidata 1200B)
    • Oscilloscope (Tektronix, or computer simulated, e.g., Axograph software)
    • Fast solution perfusion system
    • Micromanipulator (Sutter Instruments, MP‐285)
    • Vibration isolation table (Newport, Technical Manufacturing)
    • Computer to execute commands and for data storage and analysis
    • Excel (Microsoft)
    • Prism (GraphPad Software for statistics)
    • Software for data analysis and graphing includes: pClamp (for PC, Axon Instruments) or Axograph (for Macintosh, Axograph X)
    • Origin (MicroCal; for pClamp analysis)
    • Recording chamber to hold 15‐mm coverslips (Warner Instrument)
    • Fluorescence microscope with 40× to 60× water immersion objective
    • Fluorescence microscope equipped with 560‐nm excitation filter and 480‐nm emission filter
    • Micropipet puller (P‐87; Sutter Instruments)
    • Microforge for fire polishing patch pipet
    • Fire‐polished Pasteur pipets (0.8‐, 0.4‐, and 0.2‐mm bore size)
    • Patch electrodes
    • Recording microelectrode
    • Electrode holder
  • Syringes and 27‐G and 18‐G needles
  • Recovery cage
  • Large scissors
  • Forceps
  • Sylgard‐lined petri (dissecting) dish (see recipe)
  • Lysine‐coated 15‐mm glass coverslips ( appendix 3D)
  • 1‐ml syringe
  • PE tubing
  • Additional reagents and solutions for euthanizing an animal by asphyxiation (Donovan and Brown, )

Alternate Protocol 1: Intracellular Recordings of Intact Sensory Ganglionic Neurons

  • Isolated tissue (ganglia with vagus nerve attached; protocol 3)
  • Fixative (e.g., 4% formaldehyde)
  • Electrophysiological setup
    • Intracellular pipets (see unit 11.1)
    • Current clamp amplifier (Axon Instruments, Axoclamp 2‐A)
    • Data acquisition and stimulation hardware (Axon Instruments, Digidata 1200B)
    • Oscilloscope (Tektronix, or computer simulated by Axograph software)
    • Fast solution perfusion system
    • Micromanipulator (MP‐285; Sutter Instruments)
    • Vibration isolation table (Newport, Technical Manufacturing)
    • Computer to execute commands and for data storage and analysis
    • Excel (Microsoft)
    • Origin (MicroCal; for pClamp analysis)
    • Prism (GraphPad Software for statistics)
    • Sylgard‐lined recording chamber (Warner Instrument; see recipe)
    • Fluorescence microscope with long working distance objective (100× to 200× magnification)
    • Fluorescence microscope equipped with 560‐nm excitation filter and 480‐nm emission filter
    • Stimulator (Grass Technology, S44)
    • Recording microelectrode
  • Additional reagents and equipment for dye diffusion ( protocol 3, steps 1 to 11)

Basic Protocol 4: Pharmacological Analysis of Intact Airway Parasympathetic Postganglionic Neurons In Situ

  Materials
  • Oxygenated physiological saline: e.g., Krebs buffer (units 4.12& 5.16), Lockes solution, or mammalian Ringers
  • Pentobarbital
  • 150‐ to 200‐g guinea pig
  • Stock solutions of test compounds
  • Electrolyte solution (3 M KCl, pH 7.4)
  • Electrophysiological setup
    • Axoclamp 2A current‐ and voltage‐clamp amplifier (Axon Instruments)
    • Data acquisition and stimulation hardware (Axon Instruments, Digidata 1200B)
    • Oscilloscope (Tektronix, or computer simulated by Axograph software)
    • Fast solution perfusion system, heater
    • Micromanipulator (Sutter Instruments, MP‐285)
    • Vibration isolation table (Newport, Technical Manufacturing)
    • Computer to execute commands and for data storage and analysis
    • Sylgard‐lined recording chamber
    • Intracellular pipets (also see units 11.2& 11.8)
    • Upright, fixed stage microscope with long working distance objective (100× to 200× magnification)
    • Stereomicroscope (20× to 80× magnification), preferably with transmitted light
    • Stimulator (Grass Technology, S44)
    • Suction electrode
    • Recording electrode (filled with electrolyte solution)
    • Sylgard‐line petri dish (dissecting dish; see recipe)
    • Fine‐tipped no. 5 forceps
    • Micro spring scissors

Alternate Protocol 2: Patch‐Clamp Recording from Dissociated Single Neurons

  • Rats (2‐week‐old Wistar) or adult mice
  • Enzyme solution (see protocol 3)
  • Collagenase
  • Trypsin or dispase
  • L15 medium (Invitrogen) containing 10% fetal bovine serum (FBS; JHR Biosciences)
  • Fire‐polished Pasteur pipet
  • Culture dish
  • Sylgard‐coated petri dish (see recipe)
  • Additional reagents and equipment for patch‐clamp recording ( protocol 2)

Basic Protocol 5: Pharmacological Analysis of Synaptic Transmission Between Primary Airway Afferent Nerves and Second‐Order Brainstem Neurons

  Materials
  • Animal of choice
  • Ketamine
  • Xylazine
  • Oxygenated artificial cerebrospinal fluid (aCSF; high and normal sucrose, see recipes), ice‐cold
  • 95% O 2/5% CO 2
  • Stock solutions of test compounds
  • Whole‐cell CsF (cesium fluoride) recording solution (see recipe)
  • Vibratome 1000 (Technical Products International)
  • Electrophysiological setup:
    • Axopatch 1D patch‐clamp amplifier (Axon Instruments)
    • Data acquisition and stimulation interface (Axon Instruments, Digidata 1322A)
    • Oscilloscope (Tektronix)
    • Fast solution perfusion system
    • Micromanipulator (Sutter Instruments, MP‐285)
    • Vibration isolation table (Newport, Technical Manufacturing)
    • Computer to execute commands and for data storage and analysis
    • Excel (Microsoft)
    • Software used for data analysis and graphing includes: pClamp (for PC, both from Axon Instruments) or Axograph (Axograph X)
    • Mini Analysis program (Syaptosoft)
    • Recording chamber for brain slice (Warner Instrument)
    • Fluorescence microscope with 40× to 60× water immersion objective
    • Fixed‐stage fluorescence microscope (e.g., Olympus BX50WI microscope, Olympus Optical)
    • Nomarski optics
    • Borosilicate glass pipets
    • Bipolar tungsten electrodes
    • Silk mesh
  • Patch pipets (also see units 11.2& 11.8)
  • Additional reagents and equipment for DiI or Fast DiI instillation ( protocol 3)
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Figures

Videos

Literature Cited

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