Discussion paper 9 Flashcards by Lauren Mills

The focus of this discussion paper is examining…

The mechanisms which lead to synaptic refinement

How well did you know this?

Not at all

Perfectly

Cx3cr1 is only expressed in…

The CNS, specifically in microglia

How well did you know this?

Not at all

Perfectly

The ligand for cx3cr1 exists in 2 forms…

Membrane-bound and released form

How well did you know this?

Not at all

Perfectly

cx3cr1 is required for…

migration of microglia

How well did you know this?

Not at all

Perfectly

The ligand for cx3cr1 is called…

Fractalkine

How well did you know this?

Not at all

Perfectly

What is the model organism used in this study?

Mouse

How well did you know this?

Not at all

Perfectly

Although cx3cr1 is only expressed in microglia in the CNS, it is also expressed in…

macrophages in the rest of the body

How well did you know this?

Not at all

Perfectly

The region where Ca1 dendrites reside is called..

The stratum radiatum

How well did you know this?

Not at all

Perfectly

What is the underlying question of this figure?

Do microglia engulf synapses?

How well did you know this?

Not at all

Perfectly

When gathering data for this figure, what was the greatest challenge and how did the authors overcome this experimentally?

With synapses being very small, it would be very
challenging to visualize them if engulfed by microglia = challenge

overcome by using STED, super resolution microscopy

How well did you know this?

Not at all

Perfectly

What is being examined in this figure?

Microglia in green, trying to examine if PSD95 (postsynaptic marker, red) is being engulfed by microglia

How well did you know this?

Not at all

Perfectly

Describe why STED gives a higher resolution image

It depletes the fluoresence of the perimeter of the sample to leave the middle bit to fluoresce, leaving a center focal spot active to emit fluo

How well did you know this?

Not at all

Perfectly

Snap25 in this paper is used as…

A presynaptic marker

How well did you know this?

Not at all

Perfectly

What does Snap25 do?

It is a vesicle-docking protein

How well did you know this?

Not at all

Perfectly

What is being marked by the large and small dots here?

large - microglia
small - psd95

How well did you know this?

Not at all

Perfectly

What are PM and CCP in this image

Plasma membrane and clathrin-coated pit

How well did you know this?

Not at all

Perfectly

Describe the role of clathrin

A vesicle coat protein, helps form vesicles and transport molecules within cells

How well did you know this?

Not at all

Perfectly

Does clathrin stay on the vesicle the whole time during trafficking?

Usually falls off

How well did you know this?

Not at all

Perfectly

What is the conclusion of this figure?

Microglia contain pre and post synaptic material

Based on these figures, is cx3cr1 required for engulfment of synapses by microglia?

Do microglia engulf synapses when cx3cr1 is knocked out?

Yes, contain psd95 in the knockout (evidence it has been engulfed)

What is being shown in this image? What is it suggestive of?

Showing that PSD-95 increased in homozygous knockout - potentially evidence that synapses are not being cleared

What is the take-home message of figure 1? (2)

microglia engulf pre and post synaptic material

cx3cr1 is not required for engulfing

What is the underlying question of this figure?

Are the number of dendritic spines altered in cx3cr1 knockout?

What kind of cells are being examined here?

Ca1 neurons

What kinds of dendrites are being examined here?

Ca1 pyramidal neurons

What is the assay being used in this figure?

Quantification of dendritic spines

Explain what is happening here

In knockout, number of spines is pretty much the same except around P15, where there is a transient increase in spines

What does the transient increase in spines at P15 tell us about the function of cx3cr1 in synapse maturation?

Synapse maturation may be mediated in a cx3cr1 dependent OR independent manner

What is the underlying question here?

Are there physiological defects associated with the cx3cr1 knockout?

What is the approach used here? Why is TTX used?

Doing whole-cell ephys from Ca1 neurons TTX blocks sEPSP (spontaneous action potential), used to examine if knockout is capable of producing these (if there is a significant decrease, this means it is capable of firing sEPSP)

What is the takeaway of this image?

the knockout (right column) is incapable of firing sEPSP, means that there is a physiological defect in these neurons

When TTX is applied, what kind of activity remains?

only mEPSP (mini excitatory post synaptic potentials)

Based on the findings here, what do the authors suggest is happening in the cx3cr1 knockout?

Since mEPSPs occur earlier in development than sEPSP, the authors suggest that these synapses are immature

What is the underlying question of this figure?

What is the underlying mechanism being disrupted which results in defective synapses in the cx3cr1 knockout

Based on this data, what mechanism do the authors propose is responsible for synaptic defects in cx3cr1 knockout?

Suggest there is a defect in migration of microglia to the ca1 neurons

Does this paper provide direct evidence that microglia are responsible for synaptic pruning?

No, something else could be doing the pruning and microglia could just be engulfing

What is the proposed mechanism for how microglia may be targeting synapses?

Using the complement immune system pathway

The complement pathway could possibly be involved in identifying...

Low-activity connections

How could you genetically ablate microglia?

Use the cx3cr1 gene and the cre-lox system to express diptheriatoxin (DTA) to genetically ablate only microglia

Normally, the complement immune system is involved in clearing...

Dead cells

What is being demonstrated by this graph? (2)

That following ablation of microglia with dta, there are fewer spines being removed and decreased formation of spines REQUIRED FOR ELIMINATION AND FORMATION OF SPINES

How is it hypothesized that microglia are required for formation of new dendritic spines?

Secrete BDNF which helps form new spines

Is formation of new spines ever required in postnatal development?

Yes -- learning and memory

(1) can induce new spines in adult life

Exposure to novel experiences/rapid learning