BIOC14H3 Lecture : BIOC14 : Lec 16 : Special Guest Lecture - Dr. Kim

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BIOC14: Lec 16
Special Guest Lecture – Dr. Kim
Legend:
Mocs = molecules
Ppl = people
Btn = between
Nt = neurotransmitter
Cxn = connection
Fxn = function
Genetic approach to study neuronal function and animal behavior
One of the most fundamental questions in contemporary neuroscience
is which types of neurons in the brain are related to which behavior;
one of the most effective way of studying this question is by disrupting
selective group of neurons and see what happens to behavior &
physiology
Not something unique to neuroscience; used daily, which cable
controls the DVD player, unplug cable one by one and see what
happens etc;
Genetic tools that were developed by Dr.Kim that inhibit neuron
activity: neuronal silencing
Neural preservation approach can be executed in diff ways;
oInjecting neurotoxins or drugs into much smaller focal sites to
study the effects of manipulating those set of neurons on
behavior; we used to think that the set of neurons are
homogeneous BUT that’s not the case; genetic architecture of
neurons is more complex than anatomical architecture
oEx) hippocampus CA3 subfield is composed of several
genetically distinct subdomains each of which express diff set of
genes
oNeurons in yellow express diff set of genes compared to neurons
in green territory
oWhen you inject drug/neurotoxin into ventral side of this
structure, you actually target many different types of neurons
oTo tackle a particular type of neuron and its function, ppl started
thinking about a way to deliver mocs only this one type of
neuron w/out affecting others; achieved this by combining cell-
type specific promoters with the gene that you want to deliver
develop transgenic animals; this approach has its own
limitations even though genetic approaches itself is highly
reliable and much less invasive than anatomical approach
NONETHELESS HAS OWN LIMITATION:
Limitation: the driver gene that you use may be specific to
a certain structure like the hippocampus, but when you
look at the entire brain it becomes diff story the driver
gene isn’t only expressed in the hippocampus but also in
other regions
If you use gene A that is only active in a certain subregion
of the hippo but when you look at the entire brain, gene A
is expressed in other regions too
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oYou can add specificity to the genetic approach by
adding another layer of gene, gene B.
oNow, you have territory that expresses ONLY GENE
A, territory that expresses ONLY GENE B and you also have
region that expresses BOTH GENE A AND GENE B
combinatorial gene expression provides unique genetic access
oNext challenge is to produce transgene of interest
only in this specific region using combinatorial approach
To really take advantage of this combinatorial expression pattern in
real mouse genetic experiments, use DNA recombinase technology
DNA recombinase technology: enzymes called Cre recognize DNA site
called LoxP and excise out intervening sequence
oAnother kind of recombinase called Flp which recognizes DNA
sequences called FRT and excise out intervening sequence
oCan really use this DNA recombinase technology to control gene
expression and to achieve this you need 3 major components:
Very strong promoter, broadly active
Gene that you want to activate
And between the above two elements, you have
transcriptional stop sequence flanked by 2 LoxP sites; and
because this sequence actually blocks transcription by
promoter, for now, expression of transgene is OFF; when
you provide Cre recombinase, you excise out
transcriptional stop sequence & expression of transgene
is ON
Can engineer this array of sequences into mouse
chromosome so that every single cell has this array of
sequence and have the potential to be expressed, it’s just
a matter of providing Cre recombinase to a specific group
of cells that you want to manipulate
Ex) when you combine transgene allele with Cre allele to
be expressed only in neurons expressing gene A (shown
in yellow), you excise out stop factor and therefore you
introduce transgene only in yellow territory; can increase
cell type specific cell activity by adding another stop
sequence flanked by two Ftr groups; because there are 2
stop sequences, removing first stop sequence by Cre isn’t
enough to turn on gene expression; have to get rid of
both stop sequences by Cre and Flp to really turn-on
downstream gene
If you combine Cre and Flp with this, you’ll introduce gene
of interest only in the intersectional population of neurons
where the 2 gene expression domain overlay
You can introduce pretty much anything you want, as far
as the protein; you can label these neurons as
overlapping domain by expressing fluorescent protein or
you can deliver a protein that really modulates neuron
activity (turn on/off ap); or simply you can kill them by
delivering neurotoxin
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Dr. Kim’s lab is taking advantage of 3 diff mocs (neuron modulators)
for modulating neuron activity; to really control action potential and
neurotransmission in a very selective group of neurons
o3 mocs (have own strengths and weaknesses):
tetradotoxin
mutated version of human muscarinic receptor HM4E
tetanus toxin
tetrodotoxin:
trigger ap or suppress ap in response to light
must implant the optic fibre into brain (invasive) and if you want to
manipulate multiple groups of neurons that are dispersed throughout the
brain; use multiple fibres
tetanus toxin
really slow approach; blocks synaptic transmission
not reversible
highly efficient for studying animal behavior
focus for today’s lecture
HM4E:
approach in the middle of tetrodotoxin and tetrus toxin
can modulate neuron activity by injecting synthetic ligand called CNO
oinject animal w/
artificial ligand, ligand moves across BBB and bind to neuron
that expresses HM4E and control neuron activity
Tetanus Toxin (FOCUS):
picture: pre-synaptic terminal and post-synaptic membrane
during normal synaptic transmission process, nt release is achieved by
membrane fusion event btn synaptic vesicles and pre-synaptic
membrane
for fusion event to occur efficiently, should have protein complex
protein complex is formed btn vesicle protein, provided by vesicle, and
another membrane protein provided by pre-synaptic membrane; put 2
membrane structures close together
when you give tetanus toxin to neuron, tetanus toxin cleaves the
protein BANF 2 and therefore this membrane protein complex
formation is broken down and as a result membrane fusion process is
suppressed and nt release is blocked
Dr. Kim developed genetic switch that expresses tetanus toxin at
overlapping regions of 2 gene expression domains = highly selective;
also depends on what type of Cre and Flp recombinase you use; and
blocks vesicular neurotransmission process
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Document Summary

If you use gene a that is only active in a certain subregion of the hippo but when you look at the entire brain, gene a is expressed in other regions too. Now, you have territory that expresses only gene. You can add specificity to the genetic approach by o adding another layer of gene, gene b. o. A, territory that expresses only gene b and you also have region that expresses both gene a and gene b combinatorial gene expression provides unique genetic access o. If you combine cre and flp with this, you"ll introduce gene of interest only in the intersectional population of neurons where the 2 gene expression domain overlay. Wanted circuit to be specific neuron type and at the same time wanted animal to be viable so he can analyze in adult animals. Wanted the silencing to affect a phenotype that you can measure quantitative way (electrophysiology to quantify efficiency of silencing)

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