005 Neurocytological and Imaging Techniques

Question 1

Cationic Stains

Answer 1

(AKA Nissl Stains)
 Stains that act as a weak base
 Stain acidic cellular components
• Including nucleic acids
 Good at identifying neuron cell bodies
• Show nuclei, nucleolus and Nissl Substance
 Do NOT stain processes well (i.e. axons and dendrites)
 White matter stains dark; gray matter stains light

Question 2

Myelin Stains

Answer 2

 Bind to the phospholipids of myelin
 Hydrophobic stains (lipid based)
 Good at staining myelinated processes (i.e. nerve fibers)
• Commonly used to show tracts in the CNS
 Do NOT typically stain neuron cell bodies
 *usually look like a photographic negative of a Nissl stained section
 White matter stains light; gray matter stains dark

Question 3

Golgi Stains

Answer 3

 Selectively stain only a few cells in a section (about 10%)
 Mechanism is still not entirely understood
 The cells that do stain are completely filled
• Give the full architecture of only a few cells in a section

Question 4

o Histochemical/immunocytological stains

Answer 4

 Histochemical
• Uses a chemical
 Immunocytological
• Uses an antibody
 Selectively shows a specific structure/substance within a cell
 A dye is attached to the chemical/antibody so it can be viewed
 Reveals patterns NOT seen with other stains

Question 5

Reduced silver stains

Answer 5

 Metallic Stain
 Have affinities for certain components in cells
 Most commonly used to stain glial cells in the nervous system
 Appear black in the light microscope because the metal salts deposited in tissues block light

Question 6

• describe the common experimental methods used for determining connections in the nervous system,

Answer 6

degenerative methods, axonal transport methods, and lipophilic tracers

Question 7

Degenerative methods for determing connections

Answer 7

 Researchers make a lesion in a known location then look to find where signs of degeneration are located
• This allows inference as to where the rest of the nerve cell is
• The cell body that shows degeneration after an axon is cut can be inferred to be where the axons come from
 This works because when a nerve cell, especially an axon, is injured the entire nerve cell shows signs of degeneration
 Retrograde Degeneration
• The cell body located proximal to an injured axon undergoes changes, called axon reaction
• The degeneration is going backward compared to the direction of nerve impulses
• The changes can be seen with Nissl stains
 Anterograde degeneration
• Wallerian degeneration
o When an axon is injured, the axon distal to the injury undergoes degenerative changes
• The degeneration is going in the same direction of the direction of flow of nerve impulses
• The axons that show degeneration after an axon is cut can be inferred to be where the axon is going
o The most distal place where degeneration occurs is where the axon synapses

Question 8

Axonal transport methods for determining connections

Answer 8

 Tracer is injected into a known location
 Cells take up the tracer and move the tracer within the cell via axonal transport
 Causes NO damage to the cell
 Wherever the tracer is found can be inferred to be where the cell is

Question 9

anterograde axonal transport methods

Answer 9

• Uses tracer that is absorbed by the cell body and transported to the axon terminal
• Used to infer where axons go (synapses)

Question 10

retrograde axonal transport methods

Answer 10

• Uses tracer that is absorbed by axon terminal and transported back to the cell body
• Used to infer where axons come from (cell body)

Question 11

Techniques to make tracers viewable under a microscope

Answer 11

• Autoradiography
o ANTEROGRADE method ONLY
o Radioactive tracer
o Seen by the cell as a nutrient
o Injected into a known region
o Cell bodies absorb the tracer and transport it to their axon terminals
o Sectioins of tissue are then coated with a sensitive emulsion
 Developed like a photograph to make a viewable image
• Fluorescent dyes
o RETROGRADE method ONLY
o Substances that fluoresces under a known wavelength of light
o Dye injected into a known region
o Axon terminal absorb the dye and transport it to the cell body
o Sections are examined in the specific wavelength of light
• Horseradish peroxidase (HRP)
o BOTH anterograde and retrograde methods
o Natural enzyme found in horseradish plants
o Absorbed by nerve cells, both at the cell body and axon terminals
o Injected into known locations
o HRP is axonally transported
o HRP is located by an immunologic assay
o HRP can be transported across a synapsed to the next neuron
 Allows for tracing of neurons in circuits

Question 12

Lipophilic tracer method for establishing connections

Answer 12

 E.g. DiI
 BOTH anterograde and retrograde methods
 Dyes that diffuse through lipid moieties (e.g. myelin) of cells
 Hydrophobic so it does NOT leak into surrounding tissues
 Dye is introduced and give time to diffuse
 Usually visualized with fluorescent methods
 The tissue does NOT have to be alive
• Will work when alive because they are nontoxic
• Tissue is commonly harvested from the animal first
• Lipophilic tracers will work even after the tissue has been fixed
o Fixation=process to prevent putrefaction of the harvested tissue by denaturing/crosslinking proteins
o Fixation only changes proteins, not lipids

Question 13

o Skull plain film x-rays

Answer 13

 Standard x-rays
 Give information about the skull (i.e. skull fractures)
 Can sometimes give indirect information about the CNS
 SOME ionizing radiation is delivered to the patient

Question 14

o Cerebral angiography

Answer 14

 Radiopaque dye is injected into blood stream
 Standard exrays are taken at appropriate times to view blood vasculature
 Gives information on the status of the blood vascular systems (aneurysms)
 SOME ionizing radiation is delivered to the patient

Question 15

o Pneumoencephalography

Answer 15

 Air injected into spaces that are normally occupied by the CSF
 An x-ray is then taken
 Shows the ventricles of the brain and can check for blockage of CSF flow
 NOT used that often anymore
 Can causes very bad headaches
 SOME ionizing radiation is delivered to the patient

Question 16

o Computerized (axial) tomography (CT (CAT))

Answer 16

 Multiple x-ray images are put together by computers to create images of sections down to 1 mm thick
 Radioopaque media injected to enhance contrast (e.g. to view blood vessels)
 Excellent views of hard structures, like bone, obtained
 Quite a bit of detail of soft structures obtained
 Relatively inexpensive compared to other processes showing the structure of soft tissues
 Quite a bit of ionizing radiation is delivered to the patient

Question 17

o Magnetic Resonance Imaging

Answer 17

 Uses strong magnetic field to cause atoms (usually Hydrogen atoms in water) to alter their spin
 Change in spin creates a signal that is detectable
 A computer then creates images of sections down to about 3 mm thick or less
 Unlike x-rays, which are based on true densities of tissues, MRIs are based on relative amounts of water
 NO ionizing radiation delivered to patient
 Excellent detail of soft tissue
 EXPENSIVE

Question 18

Functional MRI

Answer 18

 Based upon the fact that oxygenated and deoxygenated hemoglobin have different magnetic properties that can be detected by an MRI
 The images formed are based upon relative amounts of oxygenated and deoxygenated blood
 Tells the relative metabolic activity of various tissues
• More active means more blood means more oxygenated blood

Question 19

o Positron emission tomography (PET)

Answer 19

 Radioactively labeled tracers injected into patient
• Usually 2-deoxyglucose
 Tracer taken up by cells but not metabolized
 Positrons emitted by radioactive tracer are detected and used to create an image
 Requires an on-site cyclotron
• VERY EXPENSIVE TECHNIQUE
 Image based on relative amount of tracer taken up by cells
• Can infer metabolic activity of cells
• More active means more glucose taken up by cells, but the cells cannot use the tracer
 SOME ionizing radiation delivered to patient

Question 20

o Single photon/positron emission computerized tomography (SPECT)

Answer 20

 Uses a radioactively labeled tracer (similar to PET scans)
 Can infer metabolic activity of the tissues (similar to PET scans)
 Technique NOT too expensive
 Images do NOT have great resolution
 SOME ionizing radiation delivered to the patient

Question 21

• describe some limitations of the current state-of-the-art of imaging techniques

Answer 21

o They are slow
 Measured in the realm of minutes
 Neural events are in the realm of milliseconds
 Can measure/infer metabolism but NOT cognitive function

Question 22

• describe the axon reaction

Answer 22

o name for changes in the neuron that occur proximal to the injury (on the soma side)
o In larger neurons, if only the axon is damaged, the cell can sometimes survive through a series of steps
o Several steps
 Axon degenerates to the next proximal Node of Ranvier
 Chromatolysis occurs about 24-48 hours after injury
• Nissl substance changes into finely dispersed granulations
• Rough ER breaks up with an increase in the number of free ribosomes
• Represents the protein producing machinery gearing up to heal the cell
 The nucleus becomes eccentric in the soma
 The soma swells to reach a maximum about 10-20 days after injury
 The nucleolus enlarges which implies increased rRNA production

Question 23

• describe wallerian degeneration

Answer 23

o the name for changes in the nerve fiber distal to the injury
o several steps
 the axon swells and becomes irregular in the first 24 hours
 the axon then breaks into fragments after about 3-5 days
 during this same time (1-3 days after injury) the myelin changes into short elliptical segments
 Next Schwann cell proliferation begins to occur
• The new Schwann cells fill the space vacated by degenerating axon of the endoneurium
 While all of this occurs, the debris of the axon and myelin sheath is phagocytosed by histiocytes (hisitocytes are connective tissue macrophages)
 Bands of von Bugner form
• Tubular formations of endoneurium filled with Schwann cells in the distal stump of a degenerated nerve

Question 24

Axon regeneration in the PNS

Answer 24

 Axon regeneration can occur if
• Either the connective tissue is NOT disrupted with injury
• The ends of the severed nerve are re-apposed (e.g. surgically)
 Stages
• Axons start to regenerate from the last intact internode of Ranvier and invade the region of the bands of von Bunger by about day 4
• The tip end of the regenerating axons form growth cones that guide the axon as it continues to elongate
• Axons continue to grow and hopefulle follow endoneurial tubes at a rate of 1.5 mm per day
o If growing axons miss the bands of con Bunger they may continue to grow and rollup to form whorls that become neuroma (which are pain producing)
• The axons become remyelinated (if myelinated in the first place)
 *unmyelinated neurons have a very low rate of successful regeneration
 At best, regenerated nerve fibers function no better than 80% as efficient as originals

Question 25

Axon regeneration in the CNS

Answer 25

 Starts similarly to the PNS, but there is effectively NO regeneration of CNS axons
 Stages
• Astrocytes invade the region to form an astrocytic scar
• Wallerian degeneration occurs in the regions of fibers distal to injust but with oligodendrocytes instead of Schwann cells
• Microglia phagocytose the debris
o Instead of histiocytes because CNS does not have connective tissue
• Axons begin to regenerate by forming growth cones, but stop growing after about 2 weeks
o Appears to be a signal from oligodendrocytes that stops the growth

Question 26

• describe neural plasticity

Answer 26

o The ability of the nervous system to change to allow at least some functional recovery to occur, even without anatomical regeneration
o Involves the taking over of the functions of the damaged region by other regions that have NOT been damaged
 Done through a reorganization of connections on the synaptic level
 Accompanied by some synaptic structural changes
o Example
 Rehabilitation of patients after a stroke