Neuroanatomical Techniques

Neuroanatomical Techniques

Presentation by
Armin Blesch, Ph.D.
Harvey Karten, M.D.

Objectives

Neuroanatomical techniques
History of modern neuroanatomy
Rudolf  Albert von Kölliker  (1817-1905)
nucleus  of Kölliker (Rexed  lamina X), continuity of axon and neuron
Heinrich  Wilhelm Gottfried Waldeyer (1837-1921)
Introduced  the term “neuron”  and “chromosome”

Camilio  Golgi   (1843-1926)
Golgi  method; Golgi cells;  Golgi apparatus; Golgi  tendon organ; Golgi-Mazzoni  corpuscle
Santiago  Ramon y Cajal (1852-1934)
Cajal's gold-sublimate method for astrocytes
horizontal  cell of Cajal (Retzius-Cajal cell in cortex
interstitial  nucleus of Cajal

Golgi Stain

Common immunohistochemical stains
Golgi: selective random neuron and fibers
Hematoxylin/Eosin: cell stain
Nissl (thionin): cell body stain
Kluver Barrera: mixed cell fiber stain
Weil: myelinated fiber stain
Acetycholine-esterase
Anterograde and Retrograde Tracing
Brief History of Tracing
(Grafstein, 1967)
(Kristensson & Olsson, 1971)
Fink-Heimer stain
(Heimer 1999)

Chromatolysis

http://cclcm.ccf.org/vm/VM_cases/neuro_cases_PNS_muscle.htm
Anterograde tracing with radioactive amino acids
Edwards and Hendrickson
in: Neuroanatomical tract tracing
Retrograde labeling of spinal motor neurons with HRP
Van der Want  et al.1997
Types of tracers
Application of tracers
Uptake Mechanisms
Active uptake:
Passive incorporation: lipophilic substances
Intracellular injection
Transport
Detection
Fluorescence
Enzyme reaction: HRP (WGA-HRP, CTB-HRP)
Antibodies e.g. CTB
Streptavidin-HRP conjugate for biotinylated tracers e.g. BDA, biocytin
Lectins and Toxins
WGA-HRP
Cholera Toxin beta subunit (CTB)
Retrograde, anterograde and transganglionic
Detection: antibody, HRP conjugate, conjugated to fluorophor
Application: 1 % aqueous solution, iontophoresis or pressure injection
Different efficiency in labeling among different neuronal populatioins and species
Transganglionic tracing of sensory axons with CTB
PHA-L
Anterograde tracing with PHA-L
Gerfen et al. in:
Neuroanatomical tract tracing
FITC/RITC
Fluoresceine isothiocyanate (FITC): green Rhodamine isothiocyanate (RITC): emission >590 nm (red)
Anterograde and retrograde transport
Pressure injection of 1-3% aqueous solution
Lipophilic Carbocyanine Dyes
Lipophilic Carbocyanine Dyes
Labeling of radial glia
Thanos et al. 2000
Dextran amines
Biotinylated dextran amine (BDA)
BDA
Reiner et al. 2000
Anterograde tracing of corticospinal axons
Biocytin/Neurobiotin
Application: 5% solution, pressure injection or iontophoresis
Fast degradation-short survival time 2-3 days
Mostly anterograde transport
Requires glutaraldehyde fixation
Retrograde tracers
All anterograde tracers are partially transported retrogradely
Purely retrograde tracers:
Fast Blue (FB)
Diamidino Yellow (DiY)
Microspheres
Edmund Hollis, UCSD
Scale bar 100 µm
Fluorogold
Fluorogold
Naumann et al. 2000
Ling Wang, UCSD
Cell filling
Viruses
Choosing the Right Tracer
Transgenic “Golgi” stains

GENSAT
 Objective: generate BAC-transgenic mice expressing GFP or CRE under the control of a gene specific promoter
 In situ Hybridization
 Emulsion Autoradiograpy
 Double labeling

Blurton-Jones et al
Blurton-Jones et al

Multiplex mRNA detection
Dave Kosman (Ethan Bier and Bill McGinnis labs, UC San Diego)
http://superfly.ucsd.edu/%7Edavek/images/quad.html
Immunohistochemistry
 Detection Methods
 TSA

Neuroanatomical Techniques.PPT