CSB332H1 Lecture Notes - Lecture 14: Dopaminergic Cell Groups, Lentivirus, Channelrhodopsin
14 May 2018
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VTA synapse projected to post-synaptic cell (NAcc)
Green = TH | Red = ChR2
Lecture 14(a): Molecular Methods Studying Neurons
McGill Electrode Experiments:
• During the 1950’s, a common technique to study the function of neurons was to:
o Implant an electrode and stimulate a specific brain area (hypothalamus) to stimulate
those neurons and to record what kind of behavior the animal exhibits:
§ Used to study – motivation, sleep and arousal
• Unintentionally, scientists ‘missed their target’ and placed the electrode in the wrong place
o Electrode ON – animal would remain in one area
o Electrode OFF – animal would move and investigate other areas
§ Turning the electron back ON – animal would return to the same area it
received the stimulus
ð Electrophysiology allows us to study different techniques, but still has
profound limitations
o Precision of electrode stimulation is not high enough to rule out
other biological effects
§ We cannot stimulate a specific neuron; adjacent neurons will also be activated by
the sphere of electrical activity – incapable to definitely prove that a specific
response observed is due to specific cells being stimulated
Delivery via Viral Vectors:
• Different viral vectors can be used to infect neurons – how long they remain within the system
is variable on which one we use: some are can remain for years and continue to replicate and
re-infect if found in appropriate conditions
o Shingles – chicken pox virus is resident in people who have had chicken pox, therefore,
in immune suppression condition, the virus can replicate and infect the body again
ð Adenoviruses, AAV, and lentivirus remain in the body for years | HSV does not
Ventral Tegmental Area (VTA) System:
• VTA is a dopaminergic system, and
solely
expresses Tyrosine Hydroxylase (TH):
o TH is found in the cell body that function to convert tyrosine into dopamine
§ Dopamine is packaged into vesicles & released to postsynaptic cell (i.e. NAcc)
ð Channel Rhodopsin tagged fused with the report gene
mCherry was delivered to TH-containing neurons
(i.e. VTA) using a virus vector
ð TH was targeted via an antibody using GFP tag
o Merging the images displays that VTA has
both TH and ChR2
VTA is shown to
have ChR2 (red)
and TH (green)
- solely in VTA;
not in another
neuron
Document Summary
Turning the electron back on animal would return to the same area it received the stimulus. Electrophysiology allows us to study different techniques, but still has profound limitations: precision of electrode stimulation is not high enough to rule out other biological effects. We cannot stimulate a specific neuron; adjacent neurons will also be activated by the sphere of electrical activity incapable to definitely prove that a specific response observed is due to specific cells being stimulated. Adenoviruses, aav, and lentivirus remain in the body for years | hsv does not. Ventral tegmental area (vta) system: vta is a dopaminergic system, and solely expresses tyrosine hydroxylase (th), th is found in the cell body that function to convert tyrosine into dopamine. Dopamine is packaged into vesicles & released to postsynaptic cell (i. e. nacc) Channel rhodopsin tagged fused with the report gene mcherry was delivered to th-containing neurons (i. e. vta) using a virus vector.
