CSB332H1 Lecture Notes - Lecture 12: Image Stabilization, Confocal Microscopy, Barrel Cortex
14 May 2018
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Lecture 12(b): Altering Synapses II
Visualization of AMPAR Synaptic Plasticity Movement In Vivo:
ð Movement of AMPAR is dependent on NMDAR
o Thus far, it was shown that AMAPR are trafficked to and from the cell surface;
however it was questioned if this is seen in the brain to drive synaptic plasticity
Mouse Barrel Cortex:
• Barrel cortex is an area found within a mouse’s primary somatosensory cortex – somatotopic
map of the animal whiskers
o Capable of identifying which area of the brain is stimulated when stimulating a whisker
§ Each individual whisker is represented as its own anatomical unit or ‘barrel’
ð Ex Vivo experiment – using brain slices; it was shown that AMPAR expression on cell surface
changes in the barrel cortex with frequent stimulation of whiskers
In Vivo Experiment:
• Electroporation:
o Two different electrode (positive & negative) are placed on specific areas over the skull
§ Electroporation induces cell membrane permeability – DNA migrates from
the negative electrode toward the positive electrode and will be taken up
and expressed by cells along the way
• Two Form Microscopy:
o Form of laser scanning microscopy; requires low levels of light to stimulate it and is
capable of penetrating deeper than standard confocal microscopy
§ Looked @ mouse somatosensory barrel – stimulate whisker, see changes in
brain by looking at GluA1 subunit; before, during and after stimulation
• Super Ecliptic pHluorin (SEP):
o PH-sensitive variant of GFP – specific fluorescence at certain pH
§ Intracellular pH, quite acidic (<5.5), prevents SEP fluorescence
§ Extracellular pH is ~neutral permitting SEP fluorescence
• This experiment consisted of taking embryos from a pregnant mouse (
in utero experiment
)
and transfect layers 2 & 3 in the pyramidal cortex (within the barrel cortex) using
electroporation technique with DNA for:
a) AMPAR subunit GluR1 tagged with SEP
b) dsRed2:
§ Morphological DNA that stains the entire neuron; allows the visualization of
the neuron itself
ð Immobilized mouse using a polystyrene ball and on day 70 a craniotomy was performed to
insert a glass coverslip to visualize the fluorescence in the brain
o
In vivo
OIS (optical image stabilization) and two-photon microscopy were used
to observe these changes:
§ OIS: visualizes changes in blood flow
§ Two photon microscopy: visualizes changes in receptor expression
