Electrophysiological Measurement of Cannabinoid‐Mediated Synaptic Modulation in Acute Mouse Brain Slices

Rita Báldi1, Dipanwita Ghosh2, Brad A. Grueter2, Sachin Patel3

1 Department of Psychiatry, Vanderbilt University Medical Center, Nashville, Tennessee, 2 Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, Tennessee, 3 Department of Molecular Physiology & Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 6.29
DOI:  10.1002/cpns.8
Online Posting Date:  April, 2016
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Abstract

Endocannabinoids (eCBs) are a class of bioactive lipids that mediate retrograde synaptic modulation at central and peripheral synapses. The highly lipophilic nature of eCBs and the pharmacological tools available to interrogate this system require unique methodological consideration, especially when applied to ex vivo systems such as electrophysiological analysis in acute brain slices. This unit provides protocols for measuring cannabinoid and eCB‐mediated synaptic signaling in mouse brain slices, including analysis of short‐term, long‐term, and tonic eCB signaling modes, and the unique considerations for working with eCBs and TRPV1/cannabinoid ligands in acute brain slices. © 2016 by John Wiley & Sons, Inc.

Keywords: endocannabinoid; electrophysiology; mouse; cannabinoid; CB1 receptor

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Cannabinoid Receptor Modulation of Synaptic Transmission
  • Basic Protocol 2: Evaluation of Tonic Endocannabinoid Signaling
  • Basic Protocol 3: Evaluation of Endocannabinoid Short‐Term Synaptic Depression (DSE/DSI)
  • Basic Protocol 4: Evaluation of Endocannabinoid Long‐Term Synaptic Depression
  • Support Protocol 1: Preparation of Brain Slices Using Sucrose Neuroprotection
  • Support Protocol 2: Preparation of Brain Slices Using NMDG Neuroprotection
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Cannabinoid Receptor Modulation of Synaptic Transmission

  Materials
  • ACSF (see recipe)
  • CB1 agonists (Table 6.29.1; e.g., Cayman Chemical, Tocris)
  • Freshly prepared acute brain slices containing region of interest (see Support Protocols protocol 51 and protocol 62)
  • Electrophysiological recording equipment for whole‐cell or field‐potential recording
Table 6.9.1   MaterialsDrug Affinities and Final Concentrations in Acute Brain Slices for Detecting CB1‐Dependent Synaptic Depression a

Affinity (K i in nM)
Drug CB1 CB2 Final concentration (μM)
CP55940 0.5‐5 0.7‐2.8 0.1‐10
Win‐55212‐2 b 1.9‐123 0.3‐16 0.1‐10
THC c 5‐80 3‐75 1‐10

 aFrom Pertwee et al. ( ).
 bSeveral reports have identified non‐CB1‐dependent synaptic depression with this compound (Nemeth et al., ). It is critical to verify CB1 dependence by preincubating with a CB1 antagonist or using CB1 knockout mice.
 cTetrahydrocannabinol (THC) is a partial agonist at CB1 receptors, and in some cases can antagonize the effects of full agonists (Roloff and Thayer, ), including the eCB 2‐AG. THC is not generally recommended for evaluating CB1 signaling, unless specific experimental questions require its use.

Basic Protocol 2: Evaluation of Tonic Endocannabinoid Signaling

  Materials
  • ACSF (see recipe)
  • HEPES holding ACSF (see recipe)
  • Drugs of interest:
  • CB1 antagonist: AM251 or SR141716 (5‐10 μM; Cayman Chemical or Tocris Bioscience)
  • Monoacylglycerol lipase (MAGL) inhibitor: JZL‐184 (500‐1000 nM; Cayman Chemical)
  • Fatty acid amide hydrolase (FAAH) inhibitor: PF‐3845 (1 μM; Cayman Chemical) or URB597 (1 μM; Cayman Chemical)
  • Gq‐coupled GPCR agonists: e.g., M1/3 muscarinic agonist Oxotremorine‐M (1 μM; Tocris Bioscience or Sigma‐Aldrich)
  • Freshly prepared acute brain slices containing region of interest (see Support Protocols protocol 51 and protocol 62)
  • Electrophysiological recording equipment for whole‐cell or field‐potential recording

Basic Protocol 3: Evaluation of Endocannabinoid Short‐Term Synaptic Depression (DSE/DSI)

  Materials
  • ACSF (see recipe)
  • CB1 antagonist: AM251 or SR141716 (5‐10 μM; e.g., Cayman Chemical, Tocris)
  • Freshly prepared acute brain slices containing region of interest (see Support Protocols protocol 51 and protocol 62)
  • Electrophysiological recording equipment for whole‐cell recording

Basic Protocol 4: Evaluation of Endocannabinoid Long‐Term Synaptic Depression

  Materials
  • ACSF (see recipe)
  • CB1 antagonist: AM251 or SR141716 (5‐10 μM; e.g., Cayman Chemical, Tocris)
  • Freshly prepared acute brain slices containing region of interest (see Support Protocols protocol 51 and protocol 62)
  • Electrophysiological recording equipment for whole‐cell or field‐potential recording

Support Protocol 1: Preparation of Brain Slices Using Sucrose Neuroprotection

  Materials
  • Sucrose ACSF (see recipe)
  • ACSF (see recipe)
  • Mouse
  • 5% isoflurane
  • Carbogen supply, tubing, bubble stones
  • Fully automated vibratome (e.g., Leica VT 1200)
  • Razor blade
  • 50‐ml beaker
  • Slice holding chamber
  • Dissection tools: large and small scissors, forceps, rongeur brain slicer, razor blades, spoon
  • Super Glue
  • Fine brush and/or plastic transfer pipet with cut end to give a wide opening
  • Additional reagents and equipment for anesthesia and euthanasia ( appendix 4B & )

Support Protocol 2: Preparation of Brain Slices Using NMDG Neuroprotection

  Additional materials (also see protocol 5)
  • NMDG ACSF (see recipe)
  • HEPES holding ACSF (see recipe)
  • Water baths at 32°‐34°C and 24°C
  • Perfusion pump (optional; alternatively: syringe filled with cold oxygenated NMDG ACSF right before the perfusion)
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Figures

Videos

Literature Cited

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