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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  •   FigureFigure 1.20.1 Setup of the surgical area. Before beginning surgeries, ensure that everything you will need is at hand. In the image is shown the (A) stereotaxic frame and components, (B) all surgical instruments including micro drill, (C) microscope, (D) light source, (E) digital readout coordinate system, (F) current source, and (G) pipet holder.
  •   FigureFigure 1.20.2 Setup for iontophoresis. (A) The current source should be turned on and appropriate settings confirmed before use. (B) A silver wire is placed into the pipet below the surface of the viral suspension, and the positive (red) lead is clipped to the other end of the wire (arrow). To complete the circuit, the negative (black) lead is clipped to a small bulldog clamp (asterisk) that should be attached directly to the skin of the mouse at the incision site during surgery.
  •   FigureFigure 1.20.3 Typical infection after an iontophoretic injection of rAAV‐EGFP. Example images are shown from a mouse that received an injection of rAAV2/1‐hSynapsin.EGFP.WPRE.bGH into the somatosensory cortex using iontophoresis. After a 2‐week survival, native (unenhanced) fluorescence throughout the entire brain was imaged using a 2‐photon microscope integrated with a Vibratome sectioning serially at 100‐µm thickness (TissueCyte 1000, TissueVision; Ragan et al., ). Images were obtained at 20× and tiles stitched together to view the entire section. Red‐colored background is autofluorescence that was enhanced in order to see basic tissue structures. There is substantial cross‐over from the green to red channel because of the brightness of the rAAV‐EGFP, so the infection area appears mostly yellow in this example.
  •   FigureFigure 1.20.4 Typical injection sites and rAAV‐EGFP labeling of cell bodies and axons using 2‐photon imaging. Two examples at the center of the infected area are shown for injections into the somatosensory cortex (A) and medial septum (B). Images of native (unenhanced) fluorescence were obtained after 2‐week survival times using a 2‐photon microscope as described for Figure (TissueCyte 1000, TissueVision; Ragan et al., ). (C, F) Magnified views of the infection sites show cell bodies filled with EGFP after infection. Axons from somatosensory cortex neurons are also brightly labeled by EGFP in the ipsilateral striatum (D, small box in A), and in more caudal thalamic targets (E). Projections from EGFP‐expressing medial septal neurons can be easily seen in the diagonal band nucleus (G, small box in B), and in more distal hippocampal subregions (H). Note the good resolution and detection of even single axon fibers (arrows in E, G), in addition to large axon bundles in tracts (arrowheads in A and D), and target areas with substantial terminal fields (e.g., D and H).
  •   FigureFigure 1.20.5 Enhancement of native fluorescence signal by immunohistochemistry. Alternate sections from one mouse that received an iontophoretic injection of rAAV‐EGFP into the motor cortex are shown. All images were obtained at 10× magnification using an epifluorescent microscope, with the resulting tiles stitched together to view entire sections (VS120, Olympus). (A, C) Sections show results of imaging the native GFP fluorescence. Note the bright saturated area over the injection site and the labeled fibers exiting the corpus callosum into the contralateral cortex in the overview image. (B, D) To enhance this signal, immunohistochemical staining was performed using an antibody directed against EGFP on alternate sections. The overview image (B) shows a similar bright saturated injection site and labeled fibers in the corpus callosum and contralateral cortex. (D) However, when viewing images at full magnification, anti‐EGFP is able to greatly enhance signals (compare C and D, small boxes in A and B).
  •   FigureFigure 1.20.6 Ideal results when injecting Cre‐dependent rAAV vectors into specific targets of Cre driver mice. Images were obtained at 4× magnification on an epifluorescent microscope, and tiles were stitched together to view the entire section (VS120, Olympus). A mixture of rAAV2/1‐hSynapsin.EGFP.WPRE.bGH and rAAV2/1‐CAG.FLEX.tdTomato.WPRE.bGH was injected into either the somatosensory cortex (A) or the ventromedial hypothalamus (VMH) (C) of Nr5a1‐Cre mice, which specifically express Cre in layer 4 of sensory cortex and VMH. The location of the targeted site was confirmed by overlaying the closest match from the Allen Reference Atlas. (B) At full magnification over the injection site in somatosensory cortex, many cell bodies are expressing EGFP, while a subset specifically in layer 4 express tdTomato. (D) In the VMH injection, there are cells expressing EGFP in both VMH and just dorsal to that in DMH. However, tdTomato expression is specific to Cre‐expressing cells in the VMH, allowing for specific axon mapping from these cells.


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