METHODS OF NEUROANATOMY
TYPICAL SEQUENCE OF STEPS IN MANY EXPERIMENTS
Fixation
Alcohol (archaic)
Aldehydes (contemporary)
Slicing
After embedding or freezing
Tools: Cryostat; freezing or rotary microtome; vibratome
Staining
Immersion of sections in various baths
Mounting
Sections onto glass slides, coverslip for clear optics
Microscopy
Light microscopy (brightfield)
Darkfield
Epifluorescence
Electron microscopy - transmitted, scanning
Many methods depart from standard sequence:
E.g., staining en bloc before cutting, or after mounting.
CLASSICAL METHODS FOR NORMAL MORPHOLOGY:
Golgi technique
Inventor: Camillo Golgi (1870's)
Tissue blocks in solution of chromate salts, then heavy metals
Selective, capricious [if all cells were stained it would be useless]
Mechanism
Not understood
Ramon y Cajal: master of the method
Huge contribution
Still correct
Fight with Golgi over the neuron doctrine
Nissl
Inventor: Franz Nissl (1880's)
For seeing all nerve cells (in contrast to Golgi method)
Logic:
Aniline dyes bind to acidic constituents, especially nucleic acids;
Ribosomes on rough ER -> "Nissl bodies" or Nissl substance.
Examples:
Cresyl violet
Thionine
Neutral red
Myelin
Inventor: Weigert, (1880's)
Hematoxylin after pretreating with a mordant that binds well to both fatty
myelin and hematoxylin
Variations: Weil, Loyez
Normal fiber stain
Inventor: Ramon y Cajal (1900)
Method:
Immerse sections in silver nitrate
Reducing agent -> metallic silver (dark)
Likes neurofibrils (neurotubules and filaments)
Impregnates even unmyelinated axons (whereas a myelin stain doesn’t)
Electron Microscopy:
Palay (late 1950's) - fixation protocols critical step
Fine structure (intracellular constituents, synaptic morphology, myelin
structure)
Neuron doctrine: first direct confirmation
ANTEROGRADE VS. RETROGRADE TRACING
The need
Classic methods poor for circuitry questions (what-connects-to-what).
Approaches (2)
"Anterograde tracing"
Where do outputs (efferents) of this structure go?
"Retrograde tracing"
Where do its inputs (afferents) come from?
ANTEROGRADE TRACING:
Marchi technique
"Wallerian” or anterograde degeneration (mid -1800's)
Periph nerve example: transection leads to degeneration of myelin as
well as axon distal to cut
Marchi (~1880's)
Modified myelin stain so selective for degenerating myelin
New application
Make lesion
Do stain
See distribution of degenerating axons
Dawn of experimental neuroanatomy!
Much learned with this method (the only anterograde method for 70
years)
However, it did not permit tracing of unmyelinated axons, or
unmyelinated terminal portion of axonal arbor.
Nauta degeneration (1940's-50's):
Modified normal-fiber (silver impregnation) to selectively stain degenerating
axons.
More sensitive, better picture (terminals, unmyelinated axons)
Reigned 20 years, now obsolete.
Weakness: fiber of passage problem (i.e., inability to tell whether labeling
is attributable to direct effects on cell bodies at the lesion site or instead
to damage to axons that pass through the lesion but originate
elsewhere).
Autoradiography: emerges in 1970's.
Exploits axoplasmic flow (first of many techniques to do so)
Method
Inject tritium-labeled amino acids into a brain site
Cells take them up, incorporate the amino acids into protein
Wait for transport of some of this labeled protein down axons
Fix and cut brain
Coat sections with photographic emulsion, in dark; wait
Develop as for film
Film is "fogged" where tritium is. In brightfield optics see black silver
grains, or in darkfield microscopy, bright stars.
Advantages
More sensitive than Nauta method
No fiber of passage problem (axons passing through injection site
are not labeled since they lack uptake and protein synthetic
machinery)
Disadvantage:
Staining is an indirect "image" (grains are in overlying emulsion,
not the tissue element you are interested in). Fine structure of
terminals lost.
Newer Anterograde Techniques
All solve "image" problem of autoradiography
Horseradish peroxidase (HRP; see below)
Lectins
Plant macromolecules (e.g. wheat germ, kidney bean)
Love glycoproteins
Visualized immunocytochemically (see below)
Biocytin
Fluorescent tracers (e.g., dextran amines)
RETROGRADE TRACING:
Retrograde degeneration (1880's)
Peripheral nerve example: cells with cut axons undergo chromatolysis:
Swelling of soma
Eccentric position of nucleus
Breakup of Nissl substance
Logic of technique:
Make lesion
Wait
Look for nerve cell bodies exhibiting chromatolysis
Problems
Changes are subtle, requires a practiced eye
False negatives when only one of several axonal branches is cut
Retrograde axon transport techniques:
Origins
Kristennson, 1970's:
Several injected exogenous macromolecules underwent retrograde
transport (tetanus toxin, bovine serum albumen, HRP)
This phenomenon not surprising: retro transport as means of
signalling cell about events at terminals [how, e.g., a regenerating
motorneuron, which first shows chromatolysis, returns to normal]
HRP (horseradish peroxidase)
Especially convenient: can exploit its enzymatic activity to visualize
histochemically
Method
Inject HRP
Survival period for transport (200-300mm per day)
Fix, slice brain
Histochemical reaction
Enzyme reduces H202 to water, releasing an oxygen
Expose tissue to peroxide (substrate) and a chromogen (a
chemical which forms an insoluble colored precipitate when
oxidized).
Various chromogens available
Vary in color, sensitivity and stability
Sensitive ones showed that HRP is also transported in
anterograde direction
Fluorescent retrograde tracers
Endless variety
Double labeling (different colors for different brain sites; double-labeled
cells project to both via axonal collaterals)
CARBANOCYANINE DYES
Special case: usable in fixed (as well as living) tissue
Example dyes: DiI, DiO
Logic in fixed tissue:
Fluorescent, lipophilic nature of dye
Apply to neural membrane
Diffusion in plane of membrane (slow! not active transport)
Won't cross aqueous extracellular space
Visualize by epifluorescence microscopy
Unique applications
Embryonic (avoids difficult intra-uterine surgery)
Human (no need to inject dye in living brain)
INTRACELLULAR STAINING OF LIVING CELLS:
In vivo or in vitro (e.g., slices)
Strengths:
"Directed Golgi" - see specific cell types without the capriciousness,
and "dirty background" problems of Golgi
Correlation with physiology
Intracellular dyes (examples):
HRP
Lucifer Yellow (fluorescent)
Biocytin
CHEMICAL NEUROANATOMY:
Anatomist's motivation:
Tissue differentiation (markers analogous to Nissl or myelin stains)
Functional: relationship to specific transmitter systems,
developmentally relevant macromolecules, etc.
Enzyme histochemistry
Exploits intrinsic activity of the protein to visualize it
Examples
Acetylcholinesterase (AChE)
Degradative enzyme for cholinergic transmission
Cytochrome oxidase
Metabolic function
Used to visualize "blobs" in visual cortex
Immunohistochemistry Logic:
Generate antibodies
Isolate macromolecule of interest
Inject it into a species other than the one being studied
Immune system generates antibodies to the foreign
molecule
Expose tissue sections to antibody so that it "sticks" where your
molecule is.
Visualize antibody location
Various methods for tagging with fluorescence or optically
dense markers
Examples shown:
GAD+ cells in cortex
SP+ fibers in dorsal horn of spinal cord
In situ hybridization One problem with immunohistochemistry: does presence of the
molecule in a cell mean synthesis of that molecule by that cell?
Approach: label the mRNA that codes for a specific protein
Method
Radioactively label single-stranded DNA complementary to the
mRNA
Bind to tissue sections
Autoradiography
EXAMPLES OF COMBINATIONS OF ANATOMICAL TECHNIQUES:
Electron microscopy with
- anterograde degeneration
- autoradiography
- immunohistochemistry
- intracellular staining
Immunohistochemistry + axon-transport tracing to learn input, outputs of
chemically identified neurons.
Intracellular recording and injection of retrogradely labeled neurons