Adeno‐Associated Viral Vectors for Anterograde Axonal Tracing with Fluorescent Proteins in Nontransgenic and Cre Driver Mice

Julie A. Harris1, Seung Wook Oh1, Hongkui Zeng1

1 Allen Institute for Brain Science, Seattle, Washington
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 1.20
DOI:  10.1002/0471142301.ns0120s59
Online Posting Date:  April, 2012
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Harnessing the natural ability of viruses to infect post‐mitotic cells such as neurons has provided an explosion of new methods to manipulate and reconstruct neural circuits in vivo. Here we describe the use of recombinant adeno‐associated viral vectors (rAAV) for axonal tract tracing in nontransgenic and transgenic Cre driver mice. Two protocols are presented for stereotactic‐guided placement of rAAV vectors into the live mouse brain using iontophoretic or nanoliter pressure injections. The methods discussed here will result in expression of fluorescent proteins in cell bodies, dendrites, and axons in targeted neurons, and can be easily adapted to address various experimental questions. Curr. Protoc. Neurosci. 59:1.20.1‐1.20.18. © 2012 by John Wiley & Sons, Inc.

Keywords: AAV; rAAV; viral tracer; neural circuits; anterograde tracer; Cre‐dependent virus

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Stereotaxic Injections of rAAV into Adult Mouse Brain Using Iontophoresis
  • Alternate Protocol 1: Pressure Injections of rAAV Using a Nanoliter Microdispenser (Nanoject II)
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Stereotaxic Injections of rAAV into Adult Mouse Brain Using Iontophoresis

  • 70% (v/v) ethanol in spray bottle
  • High titer purified rAAV particles (titer should ideally be >1012 GC/ml; small volume aliquots should be stored at −80°C to prevent repeated freeze/thaw cycles, which significantly affect concentration and infection efficiency)
  • Isoflurane (see note on General Anesthetics)
  • Mice (see Strategic Planning)
  • Eye ointment (e.g., Puralube)
  • Betadine
  • Coordinates for target brain regions (see Strategic Planning)
  • Quatricide (Pharmacal Research Laboratories)
  • Saline
  • 4% paraformaldehyde (PFA)
  • Glass pipets (fire‐polished, 100‐mm length, 1.2‐mm outer diameter, 0.68‐mm inner diameter; e.g., World Precision Instruments, cat. no. 1B120F‐4)
  • “Marker” glass pipet, pulled, broken, and marked with Sharpie for measuring coordinates
  • Micropipet puller
  • Vortex mixer
  • Centrifuge
  • Dissecting microscope (for measuring pipet length)
  • Microfil (World Precision Instruments, cat. no. MF28G67‐5)
  • 1‐ml syringes
  • Stereotaxic frame with alignment system (Kopf, Model no. 1900)
  • Electrode holder
  • Absorption spears, optional
  • Digital stereotaxic coordinate readout system (Anilam Wizard, Model 550 Grinding)
  • Fiber optic light source
  • Iontophoresis Midgard current source
  • Surgical instruments (sterile, or sterilized in an autoclave before use) including:
    • Scalpel handle no. 3
    • Scalpel blades no. 10
    • Suture needle holder
    • Suture silk with needle attached: size 4‐0, 30” silk black braided thread; RB‐1, 17 mm, half circle taper needle
    • Fine forceps (Roboz Surgical Instrument, cat. no. RS‐4955)
    • Small scissors (Roboz Surgical Instrument, cat. no. RS‐5910)
    • Microprobe, 45°
    • Micro drill
    • Extra drill bits (0.9 mm)
  • Surgery record sheet for each mouse
  • Isoflurane anesthesia delivery system (see note on General Anesthetics)
  • Anesthesia chamber
  • Oxygen tank
  • Isoflurane nose cone (see note on General Anesthetics)
  • Mouse ear bars
  • Sterile cotton‐tipped applicators
  • Stereomicroscope
  • Black felt tip marker (Sharpie)
  • Silver wire
  • Bulldog clamp
  • Heat source for use during recovery from surgery
  • Biohazard bin
  • Timer
NOTE: Personal protective equipment should be used for this protocol.

Alternate Protocol 1: Pressure Injections of rAAV Using a Nanoliter Microdispenser (Nanoject II)

  • Mineral oil
  • Nanoject II System (Drummond Scientific, cat. no. 3‐000‐204;)
  • Syringe holder
  • Syringe pump adaptor (e.g., Kopf Model 1972, needed to attach injector head to syringe holder)
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Literature Cited

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