Lecture Gulmez Flashcards
(25 cards)
Fear learning and extinction
Learning objectives
- Describe how fear memory can be bahaviorally analyzed in animals
- Describe neural representations of memories in the brain
- Explain how neural activity can be assessed in mice across different experimental conditions (freely moving, living animal vs post-mortem analysis), and how these methods can be used to answer specific research questions
- Contextual fear conditioning (maybe more interest in hippocampus): test triggers fear response
- Tone test (novel context, odor, floor) (more interest in auditory cortex to amygdala projections) –> pre-tone vs post-tone freezing shows fear response ( sometimes the frequency of the tone can have a different effect)
Fear
- emotional response that follows an aversive experience such as threat or danger
–> novel stimuli
- novelty
- ambiguity
- uncertainty
one of oldest and strongest responses as it is beneficial for survival (prepares for future threats as you remember better)
Fear learning - classical (Pavlovian) conditioning
Conditioned stimulus (CS): tone or light
Unconditioned stimulus (US): footshock
results in generalized fear to CS (increased freezing behavior by the end of the training)
During fear expression test there is no more foodshock, but fear response is still showed upon conditioned stimulus
Fear potentiated startle
Day 1: classical pavlovian fear conditioning
Day 2: animals hear auditory cue (they show fear response)
then they see light again and effect of tone is amplified (fear response is amplified - association)
Fear learning- Freezing chambers
Training:
White light
Background noise (optional)
Odor
Flat shock grid
Fear learning - operant conditioning
instrumental (operant) conditioning –> behavioral response paired with CS (reward or punishment)
lever pressing –> reward (increase in behavior - reinforcement)
lever pressing –> punishment (decrease in behavior - avoidance)
Fear extinction
- fear conditioning (association) e.g. tone, box and shock
- fear MEMORY recall
- extinction training (associated cue, but without footshock (you see an extinction of the fear response) –> exposure therapy
- extinction retention
Theories on fear extinction
A NEW INHIBITORY LEARNING
- currently general consensus
- competes with and suppresses the fear memory rather than erasing it
Evidence:
Behavioral evidence:
- Fear can reemerge
Neuronal evidence:
- Fear and extinction learnings activate different brain networks (not same but opposite directions)
- Extinction triggers neural signaling pathways similar to memory acquisition
ERASURE OR UNLEARNING OF CONDITIONED FEAR
Original fear memory is de-potentiated and eventually removed
- Neural evidence:
Extinction learning triggers reversal of fear induced synaptic potentiation in the amygdala
Return of fear
a relapse of fear can happen–> Extinction recall
- reminder of shock, the fear response even amplifies compared to before
- Extinction is context dependent (problem in exposure therapy, where extinction might be associated only with that concrete setting)
- Passing of time (spontaneous recovery of the fear)
(??) a new CS-US pairing –> re-learn - different tone or light but strong response
Neuronal circuits of fear and extinction memory
- Medial prefrontal cortex (mPFC) - executive functions
- Prelimbic area: fear
- Infralimbic area: extinction
- Amygdala - fear center
- Hippocampus - spatial navigation and episodic memory
Homologous regions exist in rodent brains
Memory engrams (physical memory representations in the brain)
Neurons activated during learning and recall
Building blocks of memories in the brain
In each brain region there is only a subset of neurons activated
——————————————————————Activated during LEARNING
During memory CONSOLIDATION Synaptic plasticity is potentiated, connectivity with each other, increased spine density, changes on transcriptional and epigenetic level
In RECALL the same subset of neurons is reactivated
triggering these neurons leads to fear response, but inactivating these neurons leads to NO fear response even if animals are in fear context
Long-lasting physical changes
Semon (1859-1918)
- Persistence
- Can be awaken by cues
- Content
- Dormancy
At diffent scales
Tools to label activated neurons in vivo
4 levels:
Temporal control - How to label activated neurons within a certain time window
Transgene delivery - How to deliver these synthetic constructs
Spatial control - How to selectively label activated neurons, but not others
Tag - What kind of a tag should be expressed in the activated neurons
TRAP system example
Transgenic mouse linepromisses both spatial and temporal control of labeling these neurons.
There is a gene (Arc gene) that is only present in neurons that have recently been activated
Researchers have put a CreER T2 gene onder to control of this Arc gene, so whenever there in an activation CreEr is expressed
Only in the presence of neuronal activity and 4-TM CreRT T2 can go to nucleus it makes a recombination that leads to creation of tdtomato
available in transgenic mouse line, or virus-mediated
Summary
- Fear memories are well retained
- Fear learning is rapidly acquired by conditioning
- Fear can be extinguished through extinction trials, but may return over time
- mPFC-Amygdala-Hippocampus circuits are key for fear/extinction modulation
- Engram neurons may hold representations of fear/extinction memories in brain
- Technology available to target and lable engram neurons in vivo
Neuronal activity assassment
What is the aim of the experiment?
- single time point or longitudinal analysis?
- Post-mortem tissue or living animal?
- Freely moving animal or head-fixed?
- How much resolution is required?
- Single cell, or population level analysis?
Tools to capture and image or record from activated neuronal ensembles
–> no manipulation
Post-mortem analysis of neuronal activity
Combination of reporter protein expression with immunodetection of endogenous proteins
- spectra of Green Fluorescent protein (GFP)
in order to label the activated cells and then
Transcardiac perfusions & brain dissection to sget snapshot of activated cells in that moment - combination of reporter protein expression with immunodetection of endogenous proteins
Immunofluorescence: Antibody tagged to a fluorophore
Immunostaining (look video explaining it) DAPI, cFos, tdTomato
When would you prefer one or the other
cFos very fast and 90 min already starts decaying (only short-term) - 1-time analysis
tdTomato (indefinite labelling –> you do not loose information)
Fiber photometry
Activity of a specific neuronal population in living animals, in real time
- In vivo Ca 2+ imaging techniques-based on fluorescent sensors
Genetically encoded Ca 2+ sensor (GCaMP)
fluorescent sensor gets activated through calcium
Animals are implanted with optic fiber, from cells with high calcium you get emission from GFP back
Temporal specificity –> temporally-regulated promoter (e.g. activity-dependent promoters)
Spatial specificity –> AAV delivery site, cell-type specific promoters
Multi-fiber photometry –> up to 8 sites
Correct fluorescence signal levels (time-locked) calcium is happening in the moment when stimulus happens
Fiber photometry Advantage/Disatvantage
Advantage:
- suitable for longitudinal study
- specific targeting of a certain brain region/cell type
- In vivo recording from deep structures
Disatvantages:
- Limited spatial resolution
- (Minimally) invasive procedure
- Restricted to fluorescent indicators (i.e. no electrophysiology)
Miniature microscopes
Miniature, lightweight, and portable fluorescence microscopes
Imaging neuronal activity in specific brain regions in freely moving animals
you see all cells that are emitting calcium indicator –> changes in calcium signal detectable
Rely on fluorescence imeging (similar to fiber photometry), e.g. calcium sensor
Long-term examine te same population of cells
Miniature microscopes
Advantage:
- suitable for longitudinal study
- freely moving animal
- spatial resolution (cells)
Disatvantages:
- Limited imaging depth (near the brain surface)
- Lower spatial resolution in densely packed regions
- Restricted to fluorescent indicators (no electrophysiology)
- elaborate surgical operation
Two-photon microscopy
The most resolution
Uses a focused laser beam –>excites fluorescent molecules in a tissue
Uses two photons to penetrate deeper structures
High spatial and temporal resolution
Cranial window surgery:
stereotactic device –> skin on top is removed and microscopy imaging window is inserted. A ring is also inserted and they are connected to the microscopy apparatus
Two-photon microscopy advantage and disadvantage
Advantages:
- deep tissue imaging
- high spatial resolution
- reduced photodamage
- longitudinal study
Disadvantages:
- complex setup and cost
- slower imaging speed
- invasive preparation (cranial window)
- mostly anesthetized mice
Low temporal, high spatial resolution
