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PSYC 318- Lecture#2
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Psychology Mind Map on PSYC 318- Lecture#2, created by Pascale Bockelmann on 09/01/2017.
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Pascale Bockelmann
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Pascale Bockelmann
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Resource summary
PSYC 318- Lecture#2
NAc
part of limbic system
interface between motivation and movement
Conditioned Place Preference Assay
answers question
do mice find social interactions rewarding?
measures rewarding effects of objects/ experiences
also w/ learning & memory
1. baseline test
animals placed in 2 room chamber
2. conditioning
animals are repeatedly placed in a room with something rewarding
or aversive: conditioned place aversion
3. preference test
mice are placed in the chamber without the stimulus
dependent measure
time spent in the room
do they have positive or negative associations with the environmental cues in that room
OXT receptor activity
somewhere in the animal
necessary for social reward learning
OTR-A
OXT antagonist blocks OXT receptors
mice don't form positive associations of social interactions
Microdialysis
probe that delivers drugs to specific areas of the brain
drug mixed w. florescent indicators
drug ends up in NAc
there are other OXT receptors in the brain
but OXT in NAc are the ones that matter w.r.t. social reward
Immunohistochemistry
used to label OXT containing neurones in the brain
mammal immune system produces antibodies
proteins in blood
bind to antigens
short A.A. sequences
scientists can conjugate antibody to GFP
purpose
marks antigen to be broken down & destroyed
glowing antibody specifically binds to OXT peptide
you can wash antibody over brain slice to identify OXT
How do you remove OXT receptors located on the soma of cells in the NAc required for social reward
make conditional OXT receptor knockout mouse
knock out mouse
completely normal unless certain condition is met
cells where condition is met
gene in question is excised/ deleted from genome of specific cells
most common requirement is presence of enzyme
cre-recombinase
"cre" can be expressed in specific cells through the use of viruses
in this case
use virus to drive "crd" expression in cells whose soma is located in NAc
this triggers deletion of OXT receptor gene from the genome of those cells
Interneurons and projection neurones have different proteins
How do you remove OXT from soma located in the NAc
Glutametergic axon terminals
adding OXT to NAc doesn't change excitability of projection neurons very much but reduces f. of glutamate input
Difference in between 2 Pairie voles
diff. b/w OXT receptor signalling
OXT receptor antagonist
blocks bonding
OXT
peptide made by hypothalamus
released into blood w. pituitary
hormone
axons leaving hypothalamus release OXT
nuropeptide
acts short distances
OXT spray
stronger effect in rodents instead of humans
Is OXT released in the NAc?
sample NAc solution
w. mass spec
determine presence of OXT peptide
problem
we might be detecting OXT only located in axons passing through NAc
presence of OXT peptide in NAc doesn't prove it was specifically released there
solution
1. electron microscopy + immunohistochemistry
test if OXT is localized in LDCV
in axon terminals of NAc
problem
time consuming
gold standard
2.
OXT containing neurones form synaptic connections
a) identify all neurones in the WHOLE brain that form synapses in the NAc
some located in hypothalamus
b) show hypothalamus neurones that project to the NAc contain OXT
i) some hypothalamic neurones containing OXT which DO NOT project NAc
How to label neurones that project to NAc?
Viral Mediated Gene Delivery to label cells (gene therapy)
make a virus " replication - deficient"
remove DNA from a virus
add DNA to a virus
DNA encodes
fluorescent proteins
almost all lab-made viral contains this 'GFP'
used to identify infected cells
optogenetic proteins
1. modified virus injected into animals brain
infects cells it comes into contact with
a) infects cell bodies
AAV
b) axon terminals
rabies
RbV
once in a cell, virus travels retrogradely to the cell body
get's DNA into infected cells nucleus
placed GFP into rabies virus
RbV-GFP labels NAc-projecting neurons
What neurones in the brain enervate the NAc
paraventricular nucleus of hypothalamus projects to NAc
how do PVN neurones manufacture OXT
How to make an antibody for a specific protein
1. synthesize/ purify protein you wan tot label
ex: OXT peptide
2. conjugate protein to a known antigen
attach= conjugate
antigen = protein produces large immune response in the animal
3. inject mammal w. OXT-antigen combo protein
4. wait for animal's immunesystem to create anti-bodies against OXT-antigen combo
5. draw blood from rabbit & purify antibodies that bind specifically to OXT
(not the ones that bind to the unknown antigen)
6. attach newly created oxytocin antibody to dye/ fluorescent marker
this now allows you to label oxytocin
7. wash brain slices with sol. containing antibody and all OXY peptides become visible
Which neurons in the NAc have OXT receptors
could have used immunohistochemistry
made antibodies specifically binding to OXT receptors
wanted a more specific answer
OXT Receptor Reporter Mice
endogenous DNA of animal
foreign DNA added to animal
are OXT receptors located on the soma of the cells in the NAc required for social reward
Lox P
doesn't influence endogenous expression of genes
sets condition to eliminate whatever gene it flanks
excised OXT DNA will be destroyed
Transgenic vs. knockout mice
Transgenic mice have DNA added to them
most GFP-reporter mice are transgeneic mice
knock in/ knock out mice
harder b/c they have DNA added/removed from specific parts of animals normal genome
therefore genome changed in targeted manner
knock out
targeted deletion of a specific gene
knock in
Lox P sites added to specific places
also called " conditional knock-out mice "
DNA b/w loxP sites will be deleted in presence of cre-recombinase
OXT receptor expression in NAc cells is not required for social reward
adeno-associated virus
AAV
only infects cell bodies
used to drive expression crd in NAc neurones in conditional OXT receptor knock-out mice
Brain Slice Electrophysiology
measure voltage change or electric current
adding OXT to NAc doesn't change excitability of projection neurons very much
but it does reduce f. of glutamate input
excitatory synaptic currents mediated by glutamate release onto NAc neurones
do glutamatergic axon terminal in NAc express OXT receptors?
NT receptors are found on axon terminals
also on dendrites, soma, everywhere else on the neurone
receptors alter likelihood a vesicle will release NT
after an action potential occurred
probability a NT will be released (2%-100%)
probability affected by 3 things
1. amount of presynaptic Ca2+
a.k.a. vesicle release triggered by calcium
2. # of vesicle are docked or ready to go
'readily releasable pool of vesicles'
3. # of vesicles in reserve pool
1,2,3 can be changed by g-protein receptor signalling cascades in axon terminal
Do any neurons that project to the NAc from anywhere express OXT receptors
OXT receptor expression in cells that innervate the NAc is required for social reward
RvB infections axon terminals
drives expression of cre (& GFP) in all neurons projecting to NAc
in conditional OXT receptor knock-out mice
which projection to NAc mediates OXT dependent social reward?
OXT receptor expression in dorsal raphe neurons required for social reward
DRN sends serotongeric projections to the NAc
serotonin receptor agonist dosen't change excitability of projection neurons in NAc much
but: does reduce f. of glutamate input
AVV only infects cell bodies
use to drive expression crd & GFP in dorsal rap the neurones
of conditional OXT receptor knock-out mice
summery of how OXT changes social reward in mice
serotonin receptors in NAc required for social reward
test: injected serotonin receptor antagonist
OXT doesn't reduce strength of glutamate input to the NAc in mice where...
OXT receptors were selectively removed from serotonin neurons
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