Lab 1.2: The Postsynaptic Density¶

NEU466G, Dr. Kirk · Class documents

Overview¶

Synapses can be identified by the presence of a darkly stained postsynaptic density (PSD) in the dendritic spine with small synaptic vesicles in the presynaptic axon directly opposing it. In the electron microscope image below, the red arrows are pointing at the PSDs in dendritic spine heads; vesicles are visible in the opposing presynaptic axon.

This lab will build on the work you did tracing spines from Lab 1.1 by adding the synapses to your fully reconstructed spines. A trace of the PSD area supplies the area of the synapse. Here are the names of the traces you will be adding and their colors:

Before we detail how to do this using the tools in PyReconstruct, we will review the three types of PSD orientation in 3D and how this impacts their 2D projection for a single EM section. This will highlight how the same object can look different in 2D depending on the cutting plane. The PSDs you will be tracing in Lab 1.2 are all cross section PSDs. These are the most simple and straightforward to trace and grasp how the synapse size is calculated.

Three types of PSD orientation¶

In the following cartoons, the dashed lines indicate a single section (sections are typically around 50 nm; these drawings are not to scale). The cartoons indicate how, depending on the orientation of the spine with respect to the shaft, a synapse can be sectioned in different orientations. (Cartoons by P.H. Parker.)

1. Cross section¶

Cross section PSDs occur when the section is cut perpendicular to the synapse. Sometimes you can clearly see the synaptic cleft between the spine and axon membranes. Right, EM image of spine (S) with perfectly cross-sectioned synapse (SynapseWeb Atlas, 1.6.04).

2. Oblique¶

Oblique PSDs occur when the section is cut at an angle to the synapse such that the PSD "slopes" across more than one section. The PSD will appear as a larger smear and you will not see the extracellular space in the synaptic cleft.

3. En face¶

En face PSDs occur when the section is cut parallel to the synapse, so that the entire "face" of the synapse is visible in one section. Note that the entire PSD is contained in the section. En face is a French phrase meaning "facing forward" or "in front".

Two types of tracing tools¶

The two basic types of tracing tools in PyReconstruct are closed and open. Both open and closed traces will be needed to trace synapses.

Tracing tool	Purpose	Flat area calculation	Tools icon
Closed Trace	Closed traces (like circles)	Area of the closed trace
Open Trace	Open traces (like lines)	Length times section thickness

Two types of PSD traces: contact vs contact flat area¶

In this context the word "Contact" refers to synaptic contact.

Contact traces (c-traces; red): creates a 3D reconstruction of the PSD.

Contact flat area traces (cfa-traces): quantifies the synaptic area.

Trace name	Purpose	Color	Tools icon
d##c##	3D visualization in PyReconstruct	red
d##cfa##	Synapse area calculation	green

To create these, right click a trace palette square and edit the name and color:

Tracing PSDs: cross-sectioned¶

(Example from series CLZBJ d04p03.)

Serial section EM images of spine trace (yellow), c-trace to create 3D reconstruction (red), and cfa-trace for synapse flat area quantification (green).

Section 1: No PSD.

Section 2: The PSD is traced with an open cfa-trace line (green) along the length of the PSD. The cfa-trace is encapsulated by a closed c-trace (red) extending outside of the spine membrane and protruding slightly into the axon.

Sections 3, 4: Traced as above on each section.

Section 5: The PSD is no longer present/visible, so no traces are added.

Calculating flat area of synapses¶

1. Filter your object list for d03 synapses (Lists > Object list (Ctrl+Shift+O; Mac: Cmd+Shift+O) > Filter > Regex filter... > replace the .* with d03cfa.* > OK).

2. You should see the flat areas listed for your synapses. The unit of measurement is square microns (µm²). Be sure you're looking at the flat area, not volume. (If you don't see Flat Area listed, go to the menu bar in the Object List window > Columns > Set columns... > make sure Flat area is checked > Click OK.) Here is a screen cap from the Object List:

   

3. You can export your Object List by selecting List > Export > choose a name for the file > save. It will save it as a Comma separated file (.csv) that you can open with Excel. You can also select the listed flat area values in the Object List and Ctrl+C (Mac: Cmd+C) to copy the values and paste into a spreadsheet.

4. For a cross sectioned synapse, flat area is calculated by multiplying the length of the cfa-trace by the section thickness. Flat area = Σ_{all sections} [Σ_{open traces} length × thickness + Σ_{closed traces} area]

References¶

• Legacy Reconstruct User Manual, Chapter 9, p. 106.
• SynapseWeb Atlas, 1.6.04.