Axonal Tracing Flashcards
Neuronal tracing methods give ?
detailed descriptions of neuron projections in the brain
and allow thes projections to mapped giving info on function and identification
Anterograde tracing
establishes where neurons send their axons
retrograde tracing
establishes from which neurons axons originate
Cytoskeleton polymers
microtubules - alpha/beta tubulin , beta III tubulin in neurons. 25nm
microfliaments - actin monomers 3-5nm
intermeidate filaments - form neurofilaments 10nm
Cytoskeleton polmer functions
Microtubules - 25nm diameter hollow tubes interspersed between neurofilaments
occur in individual units or in groups or 2-3.
•Formed by the polymerization of a dimer of two globular proteins; alpha and beta tubulin
• Microtubules form the mitotic spindle during cell division.
• Axon transport.
Intermediate Filaments -10 nm diameter
• Neurofilaments occur individually, scattered or loosely aggregated in small bundles
• bundles of neurofilaments = neurofibrils
• Extend as parallel fibers through cell body into dendrites (forming main component
of cytoskeleton – cytokeratin family, hence very stable).
• Clinical correlate: Alzheimers disease is linked with changes in intermediate
filaments
Microfilaments - 3 -5 nm diameter. Actin monomers.
• Form a dense supportive network underneath plasma membrane.
• Formation of new cell processes and retraction of older ones.
• Cell movement & signal transduction systems use actin
cytoskeleton as scaffold
Fast anterograde transport
away from cell body
transport vesicles containg NT
mediated by kinesin motor protein and microtubules
200-400mm/day
Slow anterograde transport
bulk movement of cytoplasm (1-5 mm/day).
• Transport of cytoskeletal elements, neurofilaments,
mitochondria and other organelles.
Fast retrograde transport
Returns used materials from the axon terminal to cell body
for recycling (100-200 mm/day).
• Transports nerve growth factor and neurotropic viruses and
toxins (herpes simplex, polioviruses, and tetanus toxin).
• Mediated by microtubules and dynein motor protein.
Microtubule orientation
same orientation in axons
mixed in dendrites
Silver impregnation method
frank & heimer method
damaged neurons
Argyrophilic-Silver affinity= Damaged neuron
Marchi method
damaged myelin
first he tissue specimen is first fixed in a solution of potassium bichromate (Müller’s fluid), which prevents normal myelin
sheets from being stained with osmic acid; osmic acid is then applied as a definitive black stain for abnormal
nerve fibers
Wallerian degeneration (Anterograde)
Process that results when a nerve fibre is cut orcrushed, in which the part of the axon separated from the neuron’s cell body degenerates.
Occurs toward the distal end of the axon.
Takes place in both the PNS and the CNS.
Characterized by successive fragmentation and disappearance of axons and myelin
sheaths and by secondary proliferation of Schwann cells.
Retrograde degeneration what happens to the neuron ?
Chromatolysis: The dissolution of the Nissl bodies in the cell body of a neuron (E).
Also occurs in wallerian degeneration if injury is close to cell body
Swelling of the cell body
Flattening and displacement of the nucleus to the periphery
Transneuronal degeneration what happens to neuron ?
The death of neurons resulting from the disruption of
input from or output to other nearby neurons
Antrograde tracing methods
frank & hemier
marchi
autoradiogrpahy with radiolabelled aa
anterograde tracers
Reterograde tarcing methods
nissl reaction
retrogarde tracer
toxins and virus
fluroescent
Di I, Dioctadecyl-Tetramethylindocarbocyanine
red orange fluroscent dye that is weakly fluroscent until incorporated into membrane
used to long term staining of neurons
Lipophilic membrane targeted fluorescent dyes.
Stain that diffuses laterally to stain the entire cell.
Several derivatives with different excitation and
emission wavelengths
Wheat germ agluttin -HRP
bind to oligosaccharides & endocytosis
cholera toxin B-HRP
bind to specific lectin or toxin receptors
Colloidal Gold-labelled tracers
WGA-GOLD and CTB-GOLD
retrograde
pressure injection only not suitable fro cut nerves
detection by sliver intensification, a photographic method whereby growing shells of metallic silver are
deposited over colloidal gold particles
HRP detection
soluble chromogen oxidised to detectable product by action of HRP on its substrate hydrogen peroxidase
Unconjugated cholera toxin B
detected by immunhistochem (Cholera B antibodies)
admin by iontoporesis, pressure injection
long lasting
Major advantage is labelling of distal
dendrites and axon in addition to soma and proximal dendrite
Phytohaemagglutinin –leucoagglutinin (PHA-L)
extract of red kidney bean
antergrade tracing - fast axonal transport
administered by iontophoresis
detected immunocytochemically
reveals fine details of axonal projections
Biocytin
anterograde tracing combo of lysine and biotin detection using EM & LM using advin biotin methods degraded fast admin pressure injection
Transneuronal tracers
herpes simplex
• Based on ability of virus to invade and replicate in neurons and
subsequently infect synaptically connected neurons.
• Pass transneuronally and replicate in synaptically linked populations.
• Signal increase with time.
• Detected by immunocytochemistry or by tritiatedthymidine autoradiography
