Some presynaptic terminals in the central nervous system appear non-functional or silent. Here we present a protocol for identifying silent presynaptic terminals. This protocol can be applied to identifying treatments and manipulations that are suspected of silencing or awakening terminals.
The protocol combines vital dye labeling of synaptic vesicles during neuronal depolarization with subsequent labeling of fixed cells with an antibody that labels all presynaptic terminals, both silent and active.Synapsis. Co labeled with dye and antibody are active. Synapsis labeled only by antibody are silent.
Hi, I'm Amanda Taylor from the laboratory of Dr.Steve Minick in the Department of Psychiatry at Washington University School of Medicine. I'm Shahin also from the Manic Lab. Today we would like to show your procedure for visualizing silent presynaptic terminals using cultured hippocampal neurons.
We use this procedure in our laboratory to study how electrical activity and the other single noling pathways to modulate neurotransmitter release. So let's get started. In a sterile laminar flow hood, use HIPA can't buy from postnatal day zero to three rats or mice to prepare dissociated cell cultures.
According to our published protocol, our cultures are mixed glial Cultures derived from the same dissection cells should be plated in sterile 35 millimeter dishes. Each containing a number zero cover slip that was coated with five milligrams per milliliter of collagen plate. The neurons at an approximate density of 500 cells per centimeter squared.
12 rat pups will typically yield 30 35 millimeter dishes. Allow the cultures to grow in a culture incubator for 10 to 14 days. For synaptic development and maturation, add the anti-mitotic a C to arrest glial growth at day in vitro four.
Also at day in vitro five, perform a half medium exchange with neuro basal medium plus B 27 supplement to enhance neuronal survival during this maturation period. Introduce treatments that alter the number of presynaptically silent synapses. We find that one convenient way to silence a large percentage of glutamate synapses is a four hour treatment with 30 millimolar potassium.
When the cultures are mature, neurons should have a relatively low density with well separated and extensive neurotic processes. Remove the cultures from the incubator and bring them to the bench. Replace the culture medium with a buffered saline solution containing 45 millimolar potassium chloride and 10 micromolar FM 1 43 FX die.
This solution should also contain NBQX and A PV to prevent recurrent signaling. It is important that the FM 1 43 FX stock solution and experiments be kept in the dark to prevent photobleaching. So cover the culture dishes with foil for all subsequent incubation steps.
Incubate for exactly two minutes at room temperature. Remove the dye solution and wash for only three to five seconds in heaps buffered saline with 500 micromolar adversive.Seven. To remove non-specific dye timing in this step is critical.
As prolonged exposure to adv adverse F seven will remove all the FM 43 FX labeling. Then wash the cells and heaps buffered saline without adv adverse. Seven for five two minute intervals in a chemical fume hood.
Fix the cultures in 4%paraldehyde and 0.2%glutaraldehyde in PBS for 10 minutes. Wash the cells briefly with PBS. The cells can now be moved out of the fume hood.
Add blocking solution containing 4%normal goat serum and 0.04%tritton X to reduce background and perme the cells for immuno staining. Incubate for 15 minutes. Stain the cells with vlu T one antibody diluted to one to 2000 in blocking solution.
Incubate the cultures in antibody with gentle rocking for three hours. Wash the cells four times with PBS. Incubate the cells with Alexa.
6 47 conjugated anti Guinea pig antibody diluted to one to 500 in blocking solution. Maintain the cultures in the secondary antibody with gentle rocking for 30 minutes. Wash the cells four more times with PBS.
Place a small drop of floor amount on a clean glass slide using forceps. Pick up a cover slip and gently lower it onto the drop of floor amount cells facing the slide. Allow the slide to dry overnight.
Prepare a 60 x oil objective on a confocal laser scanning microscope. Place a slide on the stage so that the cover slip is facing the objective. Find a field of neurites that is not overly dense and that is away from the soma which can retain residual dye.
Zoom to a field size of 51 by 51 microns. Using image acquisition software. Acquire a ZS stack covering a depth of 7.8 microns over 27 steps of 0.3 microns for each step.
Scan first for the VLU T one antibody to identify all glutamatergic synapses in the field. Then re-scan for FM 1 43 FX fluorescence to analyze images after acquisition. Use analysis software to convert the Z tack into a composite single plane image based on the maximum intensity reading from any plane at each pixel set threshold.
To reduce background, identify VG glu T one terminals without reference to the FM 1 43 effects stain. And mark these puncta as regions of interest. We typically select 10 PUNTA per field.
Repeat thresholding and puncta selection on the FM 1 43 effects image and superpose the images to identify silent synapses. An absolute pixel criterion, or a percentage pixel criterion can be used to distinguish active FM 1 43 positive from inactive FM 1 43 negative synapses. In our cultures, we find that 70 to 80%of glutamate synapses defined by V glut one staining exhibit detectable FM 1 43 staining in overlaid images of green FM 1 43 fluorescence and red V glut one staining.
This is evident as an abundance of yellow puncta synapses with co localized markers. These are indicated by the arrows. Approximately 20 to 30%of red V glut one positive puncta will fail to have green FM 1 43 fluorescence above baseline.
These are the inactive presynaptic terminals and an example is indicated by the arrowhead. A third population of synapses will also be observed. These are positive for green FM 1 43 fluorescence, but devoid of red B glut one staining.
These are active GABAergic synapses and an example is indicated by the asterisk. These are excluded from most of our analyses, but can be explicitly studied using a vesicular GABA transporter antibody. In place of the VLU T one antibody, We have just shown you how to visualize presynaptically silent synapses using FM 1 43 and VLU one immuno staining.
When performing this procedure, it is important to remember that FM 1 43 FX is sensitive to both the time in the adver sub seven wash and the concentration of the treating X 100 included in the blocking solution. So that's it. Thanks for watching and good luck with your experiments.
