Nicotine Vapor Method to Induce Nicotine Dependence in Rodents

Marsida Kallupi1, Olivier George1

1 Department of Neuroscience, The Scripps Research Institute, La Jolla, California
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 8.41
DOI:  10.1002/cpns.34
Online Posting Date:  July, 2017
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Nicotine, the main addictive component of tobacco, induces potentiation of brain stimulation reward, increases locomotor activity, and induces conditioned place preference. Nicotine cessation produces a withdrawal syndrome that can be relieved by nicotine replacement therapy. In the last decade, the market for electronic cigarettes has flourished, especially among adolescents. The nicotine vaporizer or electronic nicotine delivery system is a battery‐operated device that allows the user to simulate the experience of tobacco smoking without inhaling smoke. The device is designed to be an alternative to conventional cigarettes that emits vaporized nicotine inhaled by the user. This report describes a procedure to vaporize nicotine in the air to produce blood nicotine levels in rodents that are clinically relevant to those that are observed in humans and produce dependence. We also describe how to construct the apparatus to deliver nicotine vapor in a stable, reliable, and consistent manner, as well as how to analyze air for nicotine content. © 2017 by John Wiley & Sons, Inc.

Keywords: nicotine; vapor; dependence; withdrawal

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Table of Contents

  • Introduction
  • Basic Protocol 1: Induction of Nicotine Dependence
  • Support Protocol 1: Analysis of Nicotine Content in the Test Chamber
  • Support Protocol 2: Analysis of Nicotine Content in Blood
  • Support Protocol 3: Somatic Signs of Withdrawal
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Induction of Nicotine Dependence

  • Liquid (−)‐nicotine, >99% by GC (this product is liquid in room temperature; Sigma‐Aldrich)
  • Rats
  • Pyrex gas‐washing bottle (250 ml, Sigma‐Aldrich; see Fig.  ).
  • Drop‐catch bottle (Sigma‐Aldrich).
  • 2000‐ml Erlenmeyer vacuum flask (Fisher Scientific).
  • Sealed Plexiglas chambers (25 in. length × 16 in. height × 21.5 in. width; LABEX).
  • Standard rat cages (18 in. length × 11 in. height × 11 in. width; Allentown)

Support Protocol 1: Analysis of Nicotine Content in the Test Chamber

  • Rats subjected to nicotine vapor exposure, plus control rats exposed only to air (see protocol 1Basic Protocol)
  • 1 μM 2‐phenylimidazole
  • 20% (w/v) NaOH.
  • Dichloromethane (DCM)
  • 6 M HCl
  • Nitrogen source
  • 0.25 pmol ethyl‐nor‐cotinine as internal standard [derived from norcotinine (Sigma‐Aldrich) dissolved in methanol]
  • Ammonium formate
  • Acetonitrile
  • Formic acid
  • Razor blade
  • Heparin‐coated and uncoated 1.5‐ml microcentrifuge tubes
  • Liquid chromatography mass spectroscopy (LC‐MS) system including capillary autosampler, capillary liquid chromatography pump, and microelectrospray interface
  • 1 × 150 mm polyhydroxyethyl‐A column (5‐μm spheres, 100‐Å pore size; available from Poly LC)

Support Protocol 2: Analysis of Nicotine Content in Blood

  • Plastic transparent cylindrical container (30 cm × 29 cm)
  • Video recording device
NOTE: Animals must be habituated to the room and the cages for 1 to 3 days before testing (Watkins, Koob, & Markou, ) in order to avoid misreading exploratory behaviors for the new environment as withdrawal scores. A pause between episodes is required to perform multiple and successive counts of any sign. If ptosis is continuously present, then it is counted only once every minute. The total number of somatic signs is the sum of individual occurrences of each sign.
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Literature Cited

Literature Cited
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