WGA‐Alexa Conjugates for Axonal Tracing

Sabrina L. Levy1, Joshua J. White2, Elizabeth P. Lackey2, Lindsey Schwartz1, Roy V. Sillitoe3

1 Jan and Dan Duncan Neurological Research Institute of Texas Children's Hospital, Houston, Texas, 2 Department of Neuroscience, Baylor College of Medicine, Houston, Texas, 3 Program in Developmental Biology, Baylor College of Medicine, Houston, Texas
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 1.28
DOI:  10.1002/cpns.28
Online Posting Date:  April, 2017
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Anatomical labeling approaches are essential for understanding brain organization. Among these approaches are various methods of performing tract tracing. However, a major hurdle to overcome when marking neurons in vivo is visibility. Poor visibility makes it challenging to image a desired neuronal pathway so that it can be easily differentiated from a closely neighboring pathway. As a result, it becomes impossible to analyze individual projections or their connections. The tracer that is chosen for a given purpose has a major influence on the quality of the tracing. Here, we describe the wheat germ agglutinin (WGA) tracer conjugated to Alexa fluorophores for reliable high‐resolution tracing of central nervous system projections. Using the mouse cerebellum as a model system, we implement WGA‐Alexa tracing for marking and mapping neural circuits that control motor function. We also show its utility for marking localized regions of the cerebellum after performing single‐unit extracellular recordings in vivo. © 2017 by John Wiley & Sons, Inc.

Keywords: cerebellum; circuit tracing; connectivity; topography; WGA‐Alexa

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Table of Contents

  • Introduction
  • Basic Protocol 1: Tracing Short‐Range Topographic Circuits Within the Cerebellum
  • Basic Protocol 2: Tracing Long‐Range Topographic Projections From the Spinal Cord to the Cerebellum
  • Basic Protocol 3: Local and Rapid Marking of Neural Tissue After In Vivo Extracellular Single‐Unit Recordings
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Tracing Short‐Range Topographic Circuits Within the Cerebellum

  • 70% (v/v) ethanol
  • Isoflurane
  • Mice
  • Eye ointment (e.g., Celluvisc, NDC code 0023‐4554‐30)
  • Depilatory cream (e.g., Nair) or electric hair trimmers
  • Povidone‐iodine (Betadine Solution Swab Aids; Purdue Pharma, cat. no. 67618‐152‐01)
  • Mineral oil (e.g., MP Biomedicals, cat. no. 194836)
  • Phosphate‐buffered saline, pH 7.4 (PBS; e.g., Sigma, cat. no. P4417)
  • Neural tracer:
    • 2% (v/v) WGA‐Alexa 488 (e.g., Thermo Fisher Scientific, cat. no. W11261) in PBS
    • 2% (v/v) WGA‐Alexa 555 (e.g., Thermo Fisher Scientific, cat. no. W32464) in PBS
  • 3M Vetbond tissue adhesive (e.g., Fisher Scientific, cat. no. NC0304169)
  • Analgesic (0.6 mg/kg buprenorphine; e.g., Reckitt Benckiser Pharmaceuticals, cat. no. 12496‐0757‐5)
  • Anesthetic for perfusion (1.25% [w/v] Avertin solution injected at 25 µl/g body weight; e.g., Sigma, cat. no. 75‐80‐9)
  • 4% (w/v) paraformaldehyde (PFA; e.g., Sigma, cat. no. 441244)
  • 15% and 30% (w/v) sucrose (e.g., Fisher Scientific, cat. no. S5‐3)
  • Optimal cutting temperature (OCT) compound (e.g., VWR, cat. no. 25608‐930)
  • Vectashield Antifade Mounting Medium with DAPI (e.g., Vector Labs, cat. no. H‐1200)
  • Glass capillaries (1.5 mm o.d. × 0.86 mm i.d. × 75 mm and 1.5 mm o.d. × 0.86 mm i.d. × 100 mm length; e.g., Harvard Apparatus, cat. nos. 30‐00056 and 30‐0057, respectively)
  • Micropipette puller (e.g., Narishige PC‐10 or Sutter Instruments Model P‐1000 Flaming/Brown Micropipette Puller)
  • Petri dish with sticky base
  • Heating pad, DC current rechargeable (e.g., Kent Scientific, cat. no. DCT‐15)
  • Surgical microscope (e.g., Zeiss Stemi 2000 with Schott Ace I light source)
  • Rodent stereotactic frame (e.g., David Kopf Instruments) with non‐rupture mouse ear bars (e.g., Kopf, cat. no. 922)
  • Heat lamp (if the recovery chamber is not heated)
  • Surgical tools:
    • Scalpel with No. 10 disposable blades (e.g., Harvard Apparatus, cat. nos. PY2 52‐3514 and PY2 72‐6145, respectively)
    • Small scissors (e.g., Fine Science Tools, cat. no. 14082‐09)
    • #55 forceps (e.g., Fine Science Tools, cat. no. 11255‐20)
    • Dumont AA forceps (e.g., Fine Science Tools, cat. no. 11210‐10)
    • Bulldog clamps (e.g., Fine Science Tools, cat. no. 18039‐45)
    • Bone drill (e.g., Ideal Micro‐Drill, cat. no. 726065)
    • 2‐mm drill bits (e.g., Ideal Micro‐Drill, cat. no. 726066)
  • Inhalation anesthetic delivery system including:
    • Isoflurane vaporizer (e.g., Matrx VIP 3000 Calibrated Vaporizer)
    • Anesthesia chamber, 1.5‐liter (e.g., Midmark)
    • Oxygen tank
    • Scavenge system
    • Nose cone
  • Nanoject II (e.g., Drummond Scientific, cat. no. 3‐000‐204) with 3.5‐in. capillary tubes (e.g., Drummond Scientific, cat. no. 3‐000‐203‐G/X; Fig. )
  • Sharpened soft lead pencil (for marking the skull)
  • Cotton‐tipped applicators
  • Cauterizer
  • Parafilm
  • Timer
  • 7‐mm stainless steel wound clips (e.g., Fine Science Tools, cat. no. 12032‐07)
  • Recovery chamber with heating surface
  • 1‐ml syringe with 27‐G needle
  • KimWipes
  • Cryostat (e.g., Microm HM550)
  • 24‐well tissue culture plate
  • Glass microscope slides (e.g., Fisher Scientific, cat. no. 12‐550‐16 A) and coverslips
  • Epifluorescent or confocal microscope
  • Additional reagents and equipment for transcardial perfusion (unit 1.1; Gerfen, ) and free‐floating immunohistochemistry (White et al., )
NOTE: Personal protective equipment should be used for this protocol. Since this is a sterile surgery, carry out the tracing using sterile technique which involves using autoclaved tools and wearing sterile personal protective equipment including a gown, gloves, and mask.

Basic Protocol 2: Tracing Long‐Range Topographic Projections From the Spinal Cord to the Cerebellum

  • WGA‐Alexa 488 or WGA‐Alexa 555 (e.g., Thermo Fisher Scientific, cat. nos. W11261 and W32464, respectively)
  • 0.9% (w/v) saline
  • Phosphate‐buffered saline, pH 7.4 (PBS; e.g., Sigma, cat. no. P4417)
  • 4% (w/v) paraformaldehyde (PFA; e.g., Sigma, cat. no. 441244)
  • 15% and 30% (w/v) sucrose (e.g., Fisher Scientific, cat. no. S5‐3)
  • Vectashield (e.g., Vector Labs, cat. no. H‐1200) or FloroGel
  • Borosilicate glass electrodes (1.5 mm o.d. × 0.86 mm i.d. × 100 mm; e.g., Harvard Apparatus, cat. no. GC150F‐10)
  • Micropipette puller
  • Cryostat
  • Glass microscope slides and coverslips
  • Epifluorescent or confocal microscope
  • Additional reagents and equipment for performing rodent craniotomies ( protocol 1), electrophysiology (unit 6.1; Finkel & Bookman, ), and transcardial perfusion (unit 1.1; Gerfen, )
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