Neuroanatomical Techniques I

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Neuroanatomical Techniques I
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Objectives

• Short history of modern neuroanatomy
• Histochemical stains
• Neuronal and axonal tracing
• Immmunohistochemistry and in situ hybridization

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• 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?

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Neuroanatomical techniques

• Gross anatomy and dissection
• Histology
• Histochemical stains
• Immunohistochemical labeling
• Degeneration studies
• Tract tracing HRP, autoradiographic
• In situ hybridization
• Electrophysiological techniques
• Noninvasive imaging CT, MRI, PET
• Brain atlas-digital databases

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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

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Golgi Stain
Jim Conner, UCSD
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Common immunohistochemical stains

• Golgi selective but random neurons and fibers
• Hematoxylin/Eosin cell stain
• Nissl (thionin) cell body stain
• Kluver Barrera mixed cell fiber stain
• Weil myelinated fiber stain
• Acetycholine-esterase

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Anterograde and Retrograde Tracing

• Anterograde tracing identification of
  projections
• Uptake of the tracer by cell body
• Transport along axon
• Axon is labeled
• Retrograde tracingidentification of the origin
  of afferent projections
• Injection of tracer in fiber tract, terminal
  field or peripheral target
• Uptake of the tracer by axons
• Cell body is labeled

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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)
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Chromatolysis
Normal (10x)
Diseased (20x) Anterior horn
motor neurons

• http//cclcm.ccf.org/vm/VM_cases/neuro_cases_PNS_m
  uscle.htm

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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
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Retrograde labeling of spinal motor neurons with
HRP
First introduced by Kristensson Olsson
(1971) LaVail LaVail (1972) 1 40 µm
(TMB) 2 1 µm (TMB) 3 7 µm (TMB) 4 7 µm (DAB)
Van der Want et al.1997
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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 Rabies, GFP recombinant viruses
• Retrograde tracers FB, DiY, Fluorogold,
  Microspheres
• (Transgenic animals)

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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

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Uptake Mechanisms

• Active uptake
• Lectins bind to sugar moieties of membrane
  glycoproteins
• Uptake at nerve terminals FB, microspheres
• Uptake by fibers of passage
• Passive incorporation lipophilic substances
• Intracellular injection

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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

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Detection

• Fluorescence
• Enzyme reaction HRP (WGA-HRP, CTB-HRP)
• Antibodies e.g. CTB
• Streptavidin-HRP conjugate for biotinylated
  tracers e.g. BDA, biocytin

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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

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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

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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

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Transganglionic tracing of sensory axons with CTB
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PHA-L

• Mostly anterograde
• Application 2.5, iontophoresis
• Detection immunohistochemically
• Highly sensitive
• Long transport times (2-7 weeks)
• Not very effective in old animals

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Anterograde tracing with PHA-L
Nigrostriatal projections
Gerfen et al. in Neuroanatomical tract tracing
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FITC/RITC

• Fluoresceine isothiocyanate (FITC) green
  Rhodamine isothiocyanate (RITC) emission gt590 nm
  (red)
• Anterograde and retrograde transport
• Pressure injection of 1-3 aqueous solution

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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

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Lipophilic Carbocyanine Dyes

• DiI label from corpus
• callosum, Hoechst
• counterstain
• DiI (orange)
• callosal DiA
• (green) striatal
• projection neurons

From Vercelli et al. 2000
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Labeling of radial glia
Thanos et al. 2000
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Dextran amines

• 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
• BDA (biotinylated dextran amine)
• FR Fluororuby (tetramethyl rhodamine DA)
• Fluoro-emerald (fluorescein conjugated DA)
• Alexa-dye conjugated DA (488, 594, 632...)

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Biotinylated dextran amine (BDA)

• 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)

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BDA
Reiner et al. 2000
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Anterograde tracing of corticospinal axons
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Biocytin/Neurobiotin

• Application 5 solution, pressure injection or
  iontophoresis
• Fast degradation-short survival time 2-3 days
• Mostly anterograde transport
• Requires glutaraldehyde fixation

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Retrograde tracers

• All anterograde tracers are partially transported
  retrogradely
• Purely retrograde tracers
• Fast Blue UV dye accumulates in soma
• Diamidino Yellow UV dye, accumulates in nucleus
• Microspheres filled latex beads, accumulate in
  soma

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Fast Blue (FB) Diamidino Yellow (DiY)

• Application 1-3 aqueous solution
• Intensity stronger after longer transport times
  but leakage

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Microspheres

• Latex or polystyrene beads filled with
  fluorophores
• Purely retrograde transport
• Cell morphology not well visible, granular
  appearance of labeled cells, permanent, non-toxic

• Uptake mainly by
• terminals, uptake
• by fibers of passage??

Edmund Hollis, UCSD Scale bar 100 µm
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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

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Fluorogold
Fluorescence Immunolabeling
Naumann et al. 2000
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Cell Filling with Lucifer Yellow
Layer V Corticospinal neurons
Ling Wang, UCSD
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Cell filling
Ling Wang, UCSD
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Viruses

• Replication competent neurotropic viruses
• HSV
• Pseudorabies
• Multisynaptic retrograde tracing
• Highly sensitive as viruses replicate after
  transsynaptic transport
• Pathways over several orders of synapses can be
  followed depending on the survival time
• Replication incompetent viruses expressing
  reporter genes
• AAV/Lentivirus expressing GFP

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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
• Complete cell filling necessary
• Compatibility with double labeling/
  electrophysiology
• Stability of labeling
• Spread of tracer at the injection site
• Cost?

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Transgenic Golgi stains

• Crossing of YFP mice with transgenics or KO or
  conditional KO

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GENSAT

• Objective generate BAC-transgenic mice
  expressing GFP or CRE under the control of a gene
  specific promoter

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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.

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Emulsion Autoradiograpy
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Karin Loew, UCSD
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Double labeling

• Immunolabeling followed by in-situ hybridization
  ER? immuno ERß in-situ

Blurton-Jones et al.
Blurton-Jones et al
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Multiplex mRNA detectionDave Kosman (Ethan Bier
and Bill McGinnis labs, UC San Diego)
http//superfly.ucsd.edu/7Edavek/images/quad.html
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Allen Brain Atlashttp//www.brain-map.org
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Controls

• Specificity of probe
• Sequence analysis
• Testing by Northern blot
• Negative controls
• 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

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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

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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

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TSA

• HRP catalyzes deposition of labeled tyramide.
  (biotin, fluorescein, Tyramide reacts with
  tyrosine of endogenous proteins.

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Samples

• Immunohistochemical stains
• Golgi staining
• AchE staining
• WGA-HRP anterograde tracing
• CTB tranganglionic tracing
• Fluororuby retrograde tracing
• BDA anterograde tracing

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Köhler microscope alignment

• To optimize microscopic viewing and even
  illumination of the microscope field.
• 1. Move the condenser lens all the way to the top
  just below the plane of the stage
• 2. Place a microscope slide on the specimen
  stage, turn on the light bring the specimen
  into focus.
• 3. Close the iris diaphragm at bottom of
  microscope and examine the small illuminated
  circle.
• 4. Slowly lower condenser and bring the dark
  blurred edges of the illuminated circle into
  focus
• 5. Use the condenser adjustment screws to center
  the illuminated circle
• 6. Open the iris diaphragm until the illuminated
  circle fills the entire microscopic field

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Stereotactic brain navigation

• Head fixation
• Bregma
• xyz coordinates

Adapted from Wilson JF. Biological Foundations
of Human Behavior. Wadsworth/Thompson Learning,
Belmont, CA, 2003. www.psy.fsu.edu/berkleylab/psb
_2000sp07/2000-CLASS_8-MethEth-07.ppt