Discussion paper 9 Flashcards
The focus of this discussion paper is examining…
The mechanisms which lead to synaptic refinement
Cx3cr1 is only expressed in…
The CNS, specifically in microglia
The ligand for cx3cr1 exists in 2 forms…
Membrane-bound and released form
cx3cr1 is required for…
migration of microglia
The ligand for cx3cr1 is called…
Fractalkine
What is the model organism used in this study?
Mouse
Although cx3cr1 is only expressed in microglia in the CNS, it is also expressed in…
macrophages in the rest of the body
The region where Ca1 dendrites reside is called..
The stratum radiatum
What is the underlying question of this figure?
Do microglia engulf synapses?
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
What is being examined in this figure?
Microglia in green, trying to examine if PSD95 (postsynaptic marker, red) is being engulfed by microglia
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
Snap25 in this paper is used as…
A presynaptic marker
What does Snap25 do?
It is a vesicle-docking protein
What is being marked by the large and small dots here?
large - microglia
small - psd95
What are PM and CCP in this image
Plasma membrane and clathrin-coated pit
Describe the role of clathrin
A vesicle coat protein, helps form vesicles and transport molecules within cells
Does clathrin stay on the vesicle the whole time during trafficking?
Usually falls off
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?
No
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
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
