BIPN 154 M#2

What is different about the danger in fear and anxiety responses?

Fear: danger is imminent

Anxiety: threat is more diffuse and uncertain

What is the difference between the state of fear and anxiety?

Fear: sets organism up for immediate action against present threats

Anxiety: a lasting state of apprehension of potential future threats

What are the two ways to test anxiety?

Elevated maze test and the light/dark box

What is the timing of fear conditioning to cause a conditioned response?

CS must predict the US

What is extinction in fear conditioning?

When the CS is repeated enough time without predicting the US and thus it stops eliciting the conditioned response

What are the three ways to measure fear in a rodent?

Freezing, increase in blood pressure and hormones

What is the crucial site in the brain for fear conditioning and why?

The lateral amygdala: this is wear auditory and somatosensory information converges

What brain region is responsible for the conditioned response in fear conditioning?

CeA: All inputs converge at the lateral amygdala, but are processed in the CeA that then send the signal to the rest of your brain to cause the conditioned response

What happens in fear conditioning if there is damage to the CeA?

CeA: less expression of CR

What areas of the brain does the CeA project too?

Central gray, Lateral hippocampus, and the Paraventricular nucleus

What happens in fear conditioning when there is damage to the Central Gray? Lateral hippocampus? (regarding BP and Freezing)

Central Gray: no Freeze response

Lateral hippocampus: no BP response

You find a specific output that looks like it may be involved in fear conditioning. Design an experiment that tests this theory?

Mouse Line:

Tool:

Manipulation:

Behavioral task:

Detection:

2: Optogenetics: selectively silence the part of the brain that you believe is important and see if it stops the fear response

Mouse Line: are line specific to brain area you are targeting

Tool: Halorhodopsin

Manipulation: optic cannula and 568nm light

Behavioral task: Fear conditioning (tone followed by shock)

Detection: measure fear response (BP and Freeze)

Why are NMDAr important for fear conditioning?

NMDAr are responsible for the LTP that causes the fear response when the conditioned stimuli is presented

What are the different parts of the CeA?

Central lateral and central medial amygdala

Anxiogenic:

producing anxiety

Anxiolytic:

relieves anxiety

Describe what would happen if you injected a GABAa agonist into the CeL? CeM? during fear response

GABAa agonist: activating inhibition = suppressing neural activity

CeI: still exhibits fear response

CeM: suppressing CeM activity = no freeze response

What causes the CeL/CeM to be Anxiogenic?

CeL: inhibition causes anxiety

CeM: activation causes anxiety

What brain region projects to the Central Amygdala?

BLA

If you activate the pathway of the BLA to CeL what happens? inhibit?

Activate: anxiolytic/ relieves anxiety because activation of the BLA activates the CeL

Inhibit: anxiogenic /produces anxiety

What kind of neurons are in the CeA?

Inhibitory interneurons

Describe the pathway from the BLA-CeL

BLA --> CeL --] CeM(fear off neuron) --] fear go neuron --] fear expressing brain regions

Describe why activation of BLA-CeL is anxiolytic

activated BLA-CeL inhibits the CeM ability to inhibit the neuron that inhibits fear response = less fear response

Describe the pathway of BLA-CeM

BLA --> CeM --] fear go neuron --] fear response

describe why activation of BLA-CeM is anxiogenic

Activated BLA-CeM increase CeM ability to inhibit the neuron that inhibits fear response = bigger fear response

What are the criteria for the ideal model of depression

Face validity: the symptoms

Construct: what caused the disease

Predictive: common treatment

Why is in not necessary for the depressive animal model to exhibit all the abnormalities of depression relevant behaviors?

human patients do not show every possible symptom of depression

What are the different depressive models in animals?

1. chronic mild stress

2. Social defeat stress

3. Chronic restraint stress

4. Learned helplessness: least used

Describe what learned helplessness is and why it is not the ideal model?

This is depressive symptoms induces via uncontrollable and unpredictable foot shocks

repeated presentation of the sam stressor usually leads to adaptation

Describe chronic mild stress depressive model.

Done through unpredictable sequence of various stressors

What anti-depressant can be used to combat the depressive symptoms exhibited in animals after chronic mild stress?

chronic, not acute, treatment with SSRIs

Describe social defeat stress depressive model

You small black mouse in cage with aggressive white mouse for 10 min (they fight), then you separate them but the black mouse will still feel as though the danger is there (bc same cage).

**Must have different white mouse everyday

What can reverse the affects of Social Defeat Stress?

Chronic, not acute, antidepressant administration

How would you test the role of Dopaminergic neurons in the VTA in depression?

Mouse line:

Tools:

manipulation:

1. TH-Cre mouse line

2. Inject virus with halorhosopsin and put optic fiber

3. shine 568nm light

What is the role of Dopaminergic neurons in the VTA in depression?

Dopamine receptor secretions is reduced when animal has undergone Chronic mild stress

If you silence the whole Dopaminergic neuron population in the VTA what are the physiological affect is there?

TST: freezing

SPT: anhedonic behavior

In susceptible animals what is the activity of dopaminergic neurons during the Social defeat stress?

Dopaminergic neurons are activated

Are VTA dopaminergic neurons in the VTA homogeneous or heterogeneous? How do we know?

heterogeneous, meaning based on where they project to they have totally different effects

What are the sites that the VTA projects to?

NAcc and PFC

Describe how each pathway of the VTA is involved in producing depressive behavior

VTA --> NAcc: activation in susceptible animals is involved in depressive behavior

VTA-->PFC: inhibition in susceptible animals is involved in depressive behavior

What pathway from the VTA is involved in social defeat stress?

VTA --> NAcc

What is the mesolimbic pathway? activation/inhibition during sub threshold SDS is associated with depression

VTA --> NAcc

Activation: depression

What is the mesocoritcal pathway? Activation/inhibition during sub threshold SDS is involved in depression

VTA--> PFC

Inhibition: depression

If you have an animal showing depressive behavior after chronic mild stress, how can you alleviate these symptoms via the mesolithic pathway ?

Activating the mesolimbic pathway

How to alleviate depressive behavior induced by social defeat stress ?

Inhibiting the mesolimbic pathway

What kind of receptor are dopaminergic neurons?

Gprotien coupled receptors

What protein do D1 neurons use? D2?

D1: Gs

D2: Gi

What happens to neural activity when activating D1/D2 receptors?

D1: increase in neural activity

D2: decrease in neural activity

Describe the pathway involved in D1 receptors

Gs --> AC --> cAMP production --> PKA --> inc excitability and LTP

Describe the pathway involved in D2 receptors

Gi --> PLC --> IP3 & DAG --> >> supresses neural activity

IP3--> PP1

DAG --> PKC

How can you verify whether activated receptors induce Gs or Gi like activity

Using FRET

Describe FRET: Fluorescence resonance Energy transfer

If a G protein is active their alpha and beta subunit are separated

In FRET: yellow light means close, blue means far, because the beta subunit cannot absorb the energy from the alpha subunit

High FRET: close (not active)

Low FRET: active (far)

How do you measure the blue light in FRET? the yellow light?

You need a fluorescence microscope

Blue: CFP imaging

Yellow: FRET imaging

Design an experiment to see whether the dopamine receptor has activated the Gs pathway or the Gi pathway

Mouseline:

Tracing tool:

Manipulation:

Detection:

1. Mouse line: express Cre recombinase in VTA

2. Tool: Provide construct of Gs tag with CFP (alpha subunit), and provide YFP on beta subunit

3. Manipulation: optic cannulae and 488nm light (to activate)

Detection method: fluorescence miscroscope

Where are D1 receptors mostly found?

Substantia nigra, NAcc, Olfactory bulb

Where are D2 receptors mostly found?

Substantia nigra, NAcc, Ventral tegmental area

D1 MSN and D2 MSN: rewarding or aversive?

D1: rewarding

D2: Aversive

How can you specifically monitor D1 or D2 MSNs?

fiber photometry

Describe Fiber Photometry:

Measures calcium activity in vivo: via GCamp

GCamp: activated by blue light and the sensors are activated with the green filter

How would you specifically monitor D1 (or D2) MSN?

Mouseline:

Tracing tool:

Manipulation:

Behavioral test:

Detection:

Mouse Line: D1-Cre mouse

Tracing tools: Inject GCamp protein into D1 MSN

Manipulation method: optic cannula into NAcc, shine blue light (488nm)

Behavioral: inject cocaine

Detection method: GCamp measurement

When you inject cocaine what happens to the activity in D1 and D2 neurons?

D1: activity increases bc D1 is rewarding

D2: activity decreased bc D2 is aversive

Describe what happens in D1 and D2 receptors during rewarding and aversive experiences?

Rewarding: D1 up and D2 down

Aversive: D1 down and D2 up

Describe the street chamber test?

One chamber with cocaine: if they have D1 activation of MSN during cocaine retrieval, the animal will remain in the spot they received the stimuli (cocaine)

What does a decrease/ increase in AMPA/NMDA ratio mean

Decrease: AMPAr endocytosis (LTD)

Increase: AMPAr exocytosis (LTP)

What is the G2CT-Peptide

It inhibits stressed induced Anhedonic Behaviors

D1 MSN are involved in what kind of induced depressive behavior?

D1 MSN mediated synaptic change is more involved in Chronic Mild Stress and Social Defeat Stress induced depressive behaviors

If you activate them it rescues the social interaction test and Anhedonic behavior (SPT)

Describe LHb activity during reward and aversive experiences

Reward cue: Inhbitied

No reward: excited/activated

How do you measure LHb activity in vivo?

Mouseline:

Tracing tool:

Manipulation:

Behavioral test:

Detection:

Mouseline: LHb-Cre mouse

Tracing tool: AAV-EF1a-DIO-GCamp, Insery GCamp into LHb so it will only be expressed in Cre expressing areas

Manipulation: optic cannulae and 488nm

Behavioral test: bitter taste and shock test (should activate LHb activity

Detection: Fiber photometry

What is the difference in susceptible and resilient animals in LHb activity.

Social interaction in susceptible animals becomes aversive, so LHb activity is high, but in in Resilient animals it is not

Describe LHb activity during the chronic restraint test. How can it be rescued?

LHb activity has increased firing pattern. This can be rescued by one shot of ketamine

Why would you use a retrograde rabies pseudo virus?

Stable expression of transgenes and has minimal impact on the health of neurons

How do you get cell type and projection specific strategy for manipulation of mice?

Retrograde virus expressing FLP recombinase in Cre dependent manor

Describe FLP manipulation

-You have a mouse line that expresses FLP in a Cre dependent manor (DIO- RG- viral construct)

-DIO GFP construct is expressed in FLP dependent manor

Meaning: only neurons expressing Cre that project to neurons expressing FLP will express GFP

If you want to see whether the Ventral Pallidum projects to the LHb where would you injects what viruses?

Inject FLP dependent tracing tool into VP

Inject retrograde Cre-Dependent FLP into the LHb

FLP will retrograde to whatever area is projecting to it, and if the VP is involved Cre will then be there and GFP will be expressed

What kind of neurons are in the VP pathways to LHb and VTA

VP --> LHb = excitatory

VP --> VTA = Inhibitory

If you silence LHb projecting neurons in the VP what happens?

Anti-depressive social interaction phenotype

If you excite the LHb projecting neurons in the VP what happens?

Anti-depressive tail suspension test phenotype

What are the two way to measure neuronal activity ?

fiber photometry, and extracellular recording

What are the two steps in Fiber photometry?

1. Must inject GCamp expressing virus in right cell type

2. Put optic fiber to measure the GCamp signal

What does the Patch Clamp recording record?

AMPAr/NMDAr ratio

high: potentiated

low: depressed

E/I ratio:

increased: neuron is more excitable

decreased: neuron is suppressed

What are the tests for depression?

Sucrose preference test

Tail Suspension

Social Interaction Test

What are the Tests anxiety?

Open field test

Elevated plus maze