Neuroanatomical
Techniques
General avenues in neuroanatomy
• Descriptive neuroanatomy:
– What does a structure (cell/cell group/nucleus) look like?
– Where is a structure localized?
– Which neuron connects with what?
• Functional neuroanatomy
– What structure is associated with what function?
– How does manipulation, injury, disease and experience
influence the structure and connectivity of the nervous
system?
Objectives
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Short history of modern neuroanatomy
Histochemical stains
Neuronal and axonal tracing
Immmunohistochemistry & in situ
hybridization
• Genetic labeling of cells and connections
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
Common immunohistochemical stains
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Golgi: sparse, but random
Hematoxylin/Eosin: cell stain
Nissl (thionin): cell body stain
Kluver Barrera: mixed cell fiber stain
Weil: myelinated fiber stain
Acetycholine-esterase; cytochrome oxidase
Golgi Stain
Jim Conner, UCSD
Nissl (thionin) stain
Brainmaps.org
Brain-map.org
Cytochrome oxidase
Adams, Sincich and Horton, J Neuroscience 2007
“Metabolic marker” Mitochondria in dendrites and somata
Anterograde and Retrograde Tracing
• Anterograde tracing: identification of projections
– Uptake of the tracer by cell body
– Transport along axon
– Axon is labeled
• Retrograde tracing: identification of the cells that
give rise to afferent projections
– Injection of tracer in fiber tract, terminal field or
peripheral target
– Uptake of the tracer by axons
– Cell body is labeled
• Anterograde tracing: identification of projections
– Uptake of the tracer by cell bodies (1 or many)
– Transport along axon/s
– Axon/s labeled
• Retrograde tracing: identification of the cells that
give rise to afferent projections
– Injection of tracer in fiber tract, terminal field or
peripheral target
– Uptake of the tracer by axons
– Cell bodies labeled (1 or many)
Retrograde labeling with HRP
First introduced by
Kristensson & Olsson (1971)
LaVail & LaVail (1972)
Spinal cord motor neurons
1: 40 µm (TMB)
2: 1 µm (TMB)
3: 7 µm (TMB)
4: 7 µm (DAB)
Van der Want et al.1997
Brief History of Tracing
• Degeneration techniques:
– Anterograde: Wallerian degeneration
Silver impregnation methods: Nauta 1950,
Nauta and Gygax 1954, Fink and Heimer 1967
– Retrograde chromatolysis
(disintegration of Nissl bodies as a result of injury/disease)
• Autoradiography: anterograde transport of
radioactive amino acids (Grafstein, 1967)
• Retrograde transport of HRP (horseradish
peroxidase) (Kristensson & Olsson, 1971)
Fink-Heimer stain
(Heimer 1999)
Chromatolysis
Normal (10x)
Diseased (20x)
Anterior horn motor neurons
http://cclcm.ccf.org/vm/VM_cases/neuro_cases_PNS_muscle.htm
Anterograde tracing with
radioactive amino acids
First introduced by
Grafstein (1967)
A: terminal field
B: white matter tract
Edwards and Hendrickson
in: Neuroanatomical tract tracing
(Transneuronal) anterograde tracing
with radioactive amino acids
Uptake Mechanisms
• Active uptake:
– Lectins bind to sugar moieties of membrane
glycoproteins
– Uptake at nerve terminals
– Uptake by fibers of passage
• Passive incorporation: lipophilic substances
• Intracellular injection
Types of tracers
• Lipophilic dyes: DiI, DiO, DiA
• Dextran conjugates: BDA, fluororuby…
• Lectins: WGA(wheat germ agglutinin), PHA-L
(Phaseolus vulgaris leuco-agglutinin)
• Bacterial toxins: CTB (cholera toxin beta subunit)
• Biocyctin
• Viruses: pseudorabies, GFP recombinant
viruses…
• Retrograde tracers: FB, DiY, Fluorogold,
Microspheres
• (Transgenic animals)
Application of tracers
• Pressure injection:
glass micropipette
Hamilton syringe
• Iontophorestic injection: charged tracers
– Extracellular and intracellular application
– Electrophysiological measurements can be taken before
tracer application
• Dye Crystals: Carbocyanic dyes, WGA-HRP
Transport
• Diffusion in membrane:
– DiI, DiO, DiA
– Slow, dependent on temperature, fixation
• Active transport through vesicles
– Faster, up to 2 cm/day
– HRP & CTB stay in vesicles-granular appearance
– PHA-L, FB better cell morphology
• Intracellular diffusion
Detection
• Fluorescence
• Enzyme reaction: HRP (WGA-HRP, CTBHRP)
• Antibodies
• Streptavidin-HRP conjugate for biotinylated
tracers e.g. BDA, biocytin
Lectins and Toxins
• High affinity to specific sugars
• Bind to glycoproteins on membrane and are
internalized
– WGA: wheat germ agglutinin
– PHA-L: Phaseolus vulgaris leuco-agglutinin
– Concavalin A, agglutinins from soy bean, lens,
rhicinus…
– CTB: cholera toxin beta subunit
– Tetanus toxin fragment C
• Unmodified, biotinylated or conjugated to HRP or
fluorophors
WGA-HRP
• Retrograde, anterograde and transneuronal
transport
• Very fast transport:
– retrograde: 100 mm/day
– anterograde: 300 mm/day
• Disadvantages:
• wide diffusion
• artefact
• Tissue is fragile due to need of weak fixation
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 populations and species
PHA-L
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Mostly anterograde
Application: 2.5%, iontophoresis
Detection: immunohistochemically
Highly sensitive
Long transport times (2-7 weeks)
Not very effective in old animals
Anterograde tracing with PHA-L
Nigrostriatal projections
Gerfen et al. in:
Neuroanatomical tract tracing
Lipophilic Carbocyanine Dyes
• DiI, DiO, DiA: differ in exc/ems wavelengths
• Anterograde and retrograde transport
• Can be used in vivo (DiI & DiA) and in fixed
tissue (DiI & DiO) for post-mortem labeling
• Best choice for fixed tissue: slow diffusion (2
mm/month)
• Non-toxic
• Slice cultures, cell labeling in vitro, time lapse
videomicroscopy
Lipophilic Carbocyanine Dyes
A. DiI label from corpus
callosum, Hoechst
counterstain
B. DiI (orange)
callosal & DiA
(green) striatal
projection neurons
From: Vercelli et al. 2000
Labeling of radial glia
Thanos et al. 2000
Dextran amines
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Polysaccharides
Soluble in water
Molecular weights from 3,000 -100,000 kD
Anterograde and retrograde transport: uptake by
lesioned fibers and cells
• One of the best tracers
• Conjugated either to biotin or Fluorophores
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BDA (biotinylated dextran amine)
FR: Fluororuby (tetramethyl rhodamine DA)
Fluoro-emerald (fluorescein conjugated DA)
Alexa-dye conjugated DA (488, 594, 632...)
Biotinylated dextran amine
(BDA)
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Anterograde and retrograde transport
Highly sensitive and detailed
Iontophoretic and pressure injection
Visualization using ABC and DAB
Anterograde: MW 10,000 kD
Retrograde: MW 3,000 kD (in sodium citrate -HCl
pH 3)
BDA
Reiner et al. 2000
Biocytin/Neurobiotin
• Application: 5% solution, pressure injection
or iontophoresis
• Fast degradation-short survival time 2-3
days
• Mostly anterograde transport
• Requires glutaraldehyde fixation
Fluorogold
• Application : 1-10%, pressure injection or
iontophoresis
• Retrogradely transported
• Often granular appearance of labeled cell somata
• Antibodies against Fluorogold available
• Exc.: 325 nm, emm.:440 nm
• Labeling for extended time: several months
• Long-term toxicity
Fluorogold
Fluorescence
Immunolabeling
Naumann et al. 2000
Cell Filling with Lucifer Yellow
Layer V Corticospinal neurons
Ling Wang, UCSD
Choosing the Right Tracer
• Points to consider:
– Anterograde or retrograde tracing
– Transport time
– Efficient transport in investigated system:
• Age of animal, species and neuronal population
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Complete cell filling necessary
Compatibility with double labeling/ electrophysiology
Stability of labeling
Spread of tracer at the injection site
Cost?
In situ Hybridization
Method of localizing, either mRNA within the
cytoplasm or DNA within the chromosomes, by
hybridizing the sequence of interest to a complimentary
strand of a nucleotide probe.
In situ Hybridization
Karin Loew, UCSD
Dark grains= mRNA; blue= counterstain
In situ Hybridization
(bright field detection methods)
Allen Brain Atlas
http://www.brain-map.org
Multiplex mRNA detection
Dave Kosman (Ethan Bier and Bill McGinnis labs, UC San Diego)
http://superfly.ucsd.edu/%7Edavek/images/quad.html
• Specificity of probe
Controls
– Sequence analysis
– Testing by Northern blot
• Negative controls:
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RNase treatment pre-hybridization
Addition of an excess of unlabeled probe
Hybridization with sense probe
Tissue known not to express the gene of interest
• Positive Controls:
– Comparison with protein product
– Comparison to probes hybridizing to different part of the same
mRNA
– Tissue known to express the gene of interest
– Poly dT probe or housekeeping gene to check RNA integrity
Immunohistochemistry
• Fixation: formalin, paraformaldehyde,
glutaraldehyde
• ± parafinn embedding
• Tissue cutting: cryostat, sliding microtome,
vibratome
• Tissue penetration: mild detergents
• Blocking of unspecific binding
• Primary antibody binding
• Secondary antibody for detection
Detection Methods
• Horseradisch peroxidase:
– PAP (peroxidase anti peroxidase)
– ABC (avidin-biotin-complex) method:
• secondary antibody is biotinylated,
• detection with streptavidin-HRP complex
• Alkaline phosphatase
– APAAP (alkaline phosphatase anti-alkaline phosphatase
• TSA (tyramide signal amplification) method
=CSA (catalyzed signal amplification)
• Fluorescence
BAC-transgenic mice expressing GFP or CRE
under the control of a gene specific promoter
Expression patterns can be due to promoter
and/or ‘positional effects’
Transgenic “Golgi” stains
• Crossing of YFP
mice with
transgenics or KO
or conditional KO
Combining cell type specificity with tracing and
molecular ‘anatomy’
Example: DRD4…’experiment’
Viruses
• Replication in/competent neurotropic viruses:
– HSV
– Pseudorabies
• Multisynaptic retrograde tracing
• Highly sensitive as viruses can replicate after infection
• Pathways over several orders of synapses can be followed
depending on the survival time
• Cell type specific promoters