CSB332H1 Lecture Notes - Lecture 16: Cholera Toxin, Middle Cerebral Artery, Sprouting

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14 May 2018
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CTB Retrograde Tracer
Lecture 16(b): Neural Stem Cell Transformation
Cortical (cortex) Neurons:
Layer 2 & 3:
o Neurons have glutamatergic synaptic
connections with other groups of neurons in
the same layer – will send callosal fibers; cross
the midbrain and go to the opposite side of the cell body
Layer 5:
o Neurons are found deeper within the brain and make synaptic connections to other
groups of nuclei within different brain structures
Experimental Model:
Stroke was produced in the whisker (barrel) field of the somatosensory cortex in both young
and aged rats which induces a region of axonal sprouting in peri-infarct somatosensory cortex
o Neurons found within the infarct region will undergo apoptosis however, neurons
found in the pre-infarct (penumbra): some can sprout, and some cannot
§ Neurons that resprout are likely to regain functional connectivity (survive)
Investigators must show that sprouting occurs in living tissue did this by using
fluorescently labeled Cholera Toxin B (CTB) which is a retrograde tracer
o Injected CTB will be taken up by the axons which is then carried back
to the cell body; CTB will be mark neurons that is sending projections
to a particular area of the brain
Experiment:
Stroke was produced in the somatosensory cortex by permanently occluding two anterior
branches: the distal middle cerebral artery over the parietal cortex
o Grey box area: infarct zone – neurons in this region undergo apoptosis
o Green: penumbranearby healthy somatosensory neurons
§ Can the neurons in the penumbra functionally
compensate for the neurons in the infarct zone?
Would they make new synaptic connections and
become involved in with new pathways to
compensate their loss?
CTB Retrograde Injections:
o (1) at the time of stroke; injected in the penumbra region
with a specific color (i.e. red)
o (2) 7 or 21 days after; injected in the same region (penumbra)
with a different color (i.e. green)
ð If the second color is taken up to the cell body; but was not taken up during the first injection
o Implies that the neuron was capable of sprouting, and established a new projection
pattern after the stroke
ð Individual cells will be isolated via laser capture technology; and analyzed to compare
different transcripts with cells that were unable to sprout
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Document Summary

Neurons that resprout are likely to regain functional connectivity (survive) If the second color is taken up to the cell body; but was not taken up during the first injection: implies that the neuron was capable of sprouting, and established a new projection pattern after the stroke. Individual cells will be isolated via laser capture technology; and analyzed to compare different transcripts with cells that were unable to sprout. Neurons that were double labeled (red & green) were those that did not change their projection to the injection after stroke non sprouting. Neurons that were labeled with green (but not red) were those that established a new projection after stroke sprouting. Compared the gene expression/rna profiles of the non-sprouting neurons versus sprouting neurons. Aged animals upregulated, in neurons: (1) atrx stabilize neurons (2) igf-1 survival factor for neurons (3) neurite growth inhibitors:

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