Nicotinic Acetylcholine Receptor Assays

Brendan J. Canning1

1 The Johns Hopkins Medical Institutions, Baltimore, Maryland
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 4.12
DOI:  10.1002/0471141755.ph0412s04
Online Posting Date:  May, 2001
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Abstract

Nicotinic acetylcholine receptors (nAChRs) in peripheral tissues are localized almost exclusively to autonomic nerves and the motor end plates of striated musculature. Pharmacologic analyses of nicotinic receptor antagonist potencies can be conducted by assessing the ability of these compounds to inhibit responses elicited by preganglionic autonomic nerve stimulation or stimulation of the motor nerves innervating striated muscle in isolated tissue preparations. In addition, in some isolated tissues innervated by autonomic nerves, nicotinic receptor mediated responses can be elicited by exogenously administered agonists, and the effects of antagonists on these responses can be assessed using pharmacologic analyses. This unit describes the guinea pig trachea/esophagus preparation, in which nicotinic receptor pharmacology can be studied at synapses of the parasympathetic and sympathetic nervous system and the striated musculature of the esophagus. In addition, a preparation whereby the nicotinic receptors of the striated musculature of the diaphragm can be studied is described as are techniques for studying exogenous nicotinic agonistmediated effects in two smooth muscle preparations.Nicotinic acetylcholine receptors (nAChRs) in peripheral tissues are localized almost exclusively to autonomic nerves.

     
 
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Table of Contents

  • Basic Protocol 1: The Innervated Guinea Pig Tracheal/Esophageal Isolated Tissue Preparation to Study Receptors on Autonomic Nerves
  • Alternate Protocol 1: Nicotinic Agonist–Mediated Effects: Isolated Guinea Pig Trachealis and Ileum
  • Basic Protocol 2: Nicotinic Receptors in Striated Muscle: The Isolated Diaphragm/phrenic Nerve Preparation
  • Alternate Protocol 2: Nicotinic Receptors in Striated Muscle: The Isolated, Innervated Guinea Pig Esophagus
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: The Innervated Guinea Pig Tracheal/Esophageal Isolated Tissue Preparation to Study Receptors on Autonomic Nerves

  Materials
  • Male or female guinea pigs (200 to 400 g), any strain
  • Krebs buffer (see recipe), 37°C, gassed with Carbogen gas (95% O 2/5% CO 2; also see unit 4.3)
  • Compounds appropriate for pretreatment (see recipe), dissolved in Krebs buffer: indomethacin (required for all studies), atropine, histamine or prostaglandin F , phentolamine, propranolol, papaverine, tetrodotoxin, or barium chloride
  • Test compounds of interest (antagonists, ganglionic blockers) dissolved in Krebs buffer with vehicles (water or ethanol) as controls
  • Deep‐welled dissecting dish with Sylgard‐coated bottom (for crude dissections; see recipe)
  • Surgical instruments:
  •  Large scissors and forceps for cutting bone
  •  Fine scissors and forceps for blunt and fine dissection
  •  4–0, 5–0, or 6–0 surgical silk
  •  18‐ and 30‐G dissecting pins (1/2‐in. or 1.75‐cm hypodermic needles are ideal)
  • Equipment for nerve stimulation (Fig. ) including:
  •  Dissecting microscope with retracting arm
  •  Water‐jacketed dissecting dish with Sylgard‐coated bottom (see recipe)
  •  Small water‐jacketed heating coil
  •  Thermostatted water circulator with tubing (Cole Parmer)
  •  Peristaltic pump with tubing (Cole Parmer)
  •  Bubbler and source of Carbogen gas (95% O 2/5% CO 2)
  •  Fiber‐optic light source
  •  Polygraph with isometric force transducers (also see units 4.3 & 4.4; Grass Instruments)
  •  Stimulator (Grass Instruments Model S44) with suction electrodes (World Precision Instruments Model MPH‐RW 1.2; also see Fig. )
  • Equipment for electrical field stimulation (EFS) including (see units 4.6 & 4.8):
  •  Water‐jacketed organ baths
  •  Tissue holders with platinum ring electrodes
  •  Med‐Lab Stimusplitter (Med‐Lab Instruments)
  • Additional reagents and equipment for maintaining and measuring response in isolated tissue preparations (units 4.3, 4.4 & 4.6)

Alternate Protocol 1: Nicotinic Agonist–Mediated Effects: Isolated Guinea Pig Trachealis and Ileum

  • Isolated tracheal strips (unit 4.6) or ileum (unit 4.8), prepared and placed in an organ bath
  • Nicotine or other nicotinic agonists: e.g., 1,1‐dimethyl‐4‐phenylpiperazine (DMPP)
  • Additional reagents and equipment for maintaining and measuring response in isolated tissue preparations (unit 4.3, & ) and generating dose‐response curves (unit 4.1)

Basic Protocol 2: Nicotinic Receptors in Striated Muscle: The Isolated Diaphragm/phrenic Nerve Preparation

  Materials
  • Male or female guinea pigs (any strain, 400 to 600 g) or rats (any strain, 200 to 400 g)

Alternate Protocol 2: Nicotinic Receptors in Striated Muscle: The Isolated, Innervated Guinea Pig Esophagus

  • 0–0 surgical silk sutures
  • 15‐G cannula
  • Polygraph with pressure transducers
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Figures

  •   FigureFigure 4.12.1 Nerves and ganglia associated with the isolated, innervated guinea pig trachea/esophagus preparation. The trachea and esophagus are innervated by both sympathetic and parasympathetic nerves. Preganglionic parasympathetic fibers innervating the preparation are carried by the vagus nerves. The vagi also carry vagal afferent nerves that have cell bodies in the jugular and nodose ganglia, many of which reach the preparation via the superior laryngeal nerve. The vagus nerves also carry motor fibers innervating the striated muscle of the esophagus. The cervical sympathetic nerves carry preganglionic fibers that innervate neurons in the superior cervical sympathetic ganglia (SCG). Postganglionic sympathetic axons project from the SCG to the airways and mediate relaxations of the trachealis. The hypoglossal, accessory, pharyngeal, and glossopharyngeal nerves do not innervate the airways and provide little, if any, innervation to the esophagus. The diagram is drawn from a ventral view of the left side of a guinea pig.
  •   FigureFigure 4.12.2 Schematic diagram of the apparatus utilized to study the isolated, innervated guinea pig trachea/esophagus preparation. The buffer is warmed to 37°C by passing it through a water‐jacketed heating coil prior to its delivery into the dish.
  •   FigureFigure 4.12.3 An illustration of the isolated, innervated guinea pig trachea/esophagus preparation. Two ring segments prepared for isometric tension measurement are illustrated at two sites along the length of the trachea, to emphasize that isometric tension can be measured in any portion of the trachea. The advantage of utilizing the rostral portion of the trachea (rings 1 to 10 caudal to the larynx), however, is that the preganglionic vagal fibers innervating this portion of the trachea reach the airways by distinct extrinsic nerves (the recurrent laryngeal nerves) from those carrying the vagal afferent fibers. In addition, the sympathetic innervation from the superior cervical ganglia is concentrated in the rostral portion of the trachealis. See for details of dissection.
  •   FigureFigure 4.12.4 Schematic diagrams of a suction electrode. (A) A typical suction electrode consists of a chamber with holes through its core and side. The core contains conducting wire (e.g., silver chloride) connected to a stimulator. At the terminal end, capillary tubing is inserted and wrapped with another length of conducting wire, also connected to the stimulator. The chamber and capillary tubing are filled with buffer. (B) Illustration of the electrode tip, including the wires on the inside and outside of the capillary tubing, both connected to stimulator leads. Note arrangement of anode and cathode. Note the constriction in the capillary tube, which is formed by pulling following flame heating. (C) Once filled with buffer, the electrode is placed near the free end of a nerve, which is pulled into the capillary tubing with suction (applied by a syringe). The constriction in the capillary tube causes the cut end of the nerve to become wedged in the lumen upon suction, creating a high resistance to current but permitting a high stimulus intensity to be delivered to the nerve trunk. (D) Schematic diagram of the effects of nerve stimulation on an end organ. Preganglionic or motor fibers in a nerve trunk, when stimulated with sufficient intensity, will generate action potentials that conduct without decrement to the preganglionic nerve terminals in the autonomic ganglia (or to the neuromuscular junction). Upon depolarization of the preganglionic nerve terminal, acetylcholine (A) is released, which binds to nicotinic receptors (NR) on the ganglion neurons. If enough nicotinic receptors are activated and the neuron reaches the depolarization threshold for action potential formation, the action potential in the postganglionic axons conducts without decrement to the postganglionic nerve terminals, which release neurotransmitter (N) onto receptors in the end organ, which can be measured as a change in end organ activity (e.g., muscle contraction or relaxation, or secretion).
  •   FigureFigure 4.12.5 Schematic diagrams illustrating the differences between (A) frequency‐response and (B) voltage‐response curves. Panel A illustrates stimulation of an extrinsic nerve at optimal stimulus intensities (S.I.), which faithfully elicits action potentials in all axons carried by the extrinsic nerve for the duration of the stimulus (denoted by the thick, horizontal bars). Increasing the stimulation frequency (stimulus intensity and duration constant) will increase the concentration of acetylcholine (A) at the synapse, which will in turn increase activity in the post‐ganglionic nerve, thus increasing neurotransmitter (N) release. This increase can be measured as an increase in end‐organ response. Panel B shows that axons vary in their sensitivity to stimulation due to a number of biophysical factors (e.g., membrane resistance, resting membrane potential, and threshold for action potential formation). At suboptimal stimulus intensities, axons do not always generate action potentials with each stimulator pulse. Increasing the stimulus intensity (voltage or pulse duration) while keeping stimulation frequency and duration constant will elicit action potentials in the nerve axons with greater fidelity, increasing the concentration of acetylcholine (A) at the ganglionic synapse, resulting in an increase in postganglionic nerve activity and an increase in end‐organ response (Canning and Undem, ; Undem et al., ; also see Fig. ).
  •   FigureFigure 4.12.6 Concentration‐dependent inhibition of vagally‐mediated cholinergic contractions of the guinea pig trachea mediated by (A) hexamethonium and (B) trimethaphan. Data are presented as a percentage of the maximum contraction elicited by 1 mM carbachol. Each dose was added 20 min prior to subsequent stimulation. Each point is the mean ± SEM of five observations.
  •   FigureFigure 4.12.7 Changes in guinea pig esophageal pressure elicited by varying the (A) voltage or (B) frequency of vagus nerve stimulation. Train duration was constant at 10 sec. In panel A, frequency was constant at 24 Hz. In panel B, voltage was constant at 50 V. Each point is the mean ± SEM of six or seven observations (see text and Canning and Undem, for details). These responses are sensitive to nAChR antagonists (see Table ).

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Literature Cited

Literature Cited
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   Canning, B.J. and Undem, B.J. 1993b. Evidence that distinct neuronal pathways mediate parasympathetic contractions and relaxations of guinea pig trachealis. J. Physiol. (Lond.) 481:25‐40.
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Key References
   Canning and Undem, 1993a. See above.
  Describes the initial method and use of the isolated, innervated guinea pig tracheal preparation.
   Canning and Undem, 1993b. See above.
  Provides data relating to the potency of selected ganglionic blockers in the guinea pig trachea.
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