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

• Short history of modern neuroanatomy

• Histochemical stains

• Neuronal and axonal tracing

• Immmunohistochemistry & in situ hybridization

• Genetic labeling of cells and connections

History of modern neuroanatomyRudolf 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

• 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

http://cclcm.ccf.org/vm/VM_cases/neuro_cases_PNS_muscle.htm

Normal (10x) Diseased (20x)

Anterior horn motor neurons

Anterograde tracing with radioactive amino acids

Edwards and Hendrickson in: Neuroanatomical tract tracing

First introduced by Grafstein (1967)

A: terminal fieldB: white matter tract

(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, CTB-HRP)

• 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

• 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

Gerfen et al. in: Neuroanatomical tract tracing

Nigrostriatal projections

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

From: Vercelli et al. 2000

A. DiI label from corpus callosum, Hoechst

counterstainB. DiI (orange)

callosal & DiA (green) striatal projection neurons

Labeling of radial glia

Thanos et al. 2000

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...)

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)

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

Naumann et al. 2000

Fluorescence Immunolabeling

Layer V Corticospinal neurons

Ling Wang, UCSD

Cell Filling with Lucifer Yellow

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?

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.

Karin Loew, UCSD

Dark grains= mRNA; blue= counterstain

In situ Hybridization

In situ Hybridization(bright field detection methods)

Allen Brain Atlashttp://www.brain-map.org

Multiplex mRNA detectionDave Kosman (Ethan Bier and Bill McGinnis labs, UC San Diego)

http://superfly.ucsd.edu/%7Edavek/images/quad.html

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

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