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Lab 3.1: Smooth Endoplasmic Reticulum, Spine Apparatus, and Endosomes

NEU466G, Dr. Harris · Class documents

Overview

In this lab you look for smooth endoplasmic reticulum (SER), spine apparatus (SA), and endosomes in 5 more spines of dendrite d03, and you place a stamp on each one you find.

  • Spines: d03sp12, d03sp14, d03sp37_branched, d03sp40 and d03sp42 (see 5 additional spines).
  • What you make: one stamp for each SER, SA or endosome you find, named after its spine.
  • What you hand in: your saved series, uploaded to Canvas.

Each Figure box names a figure in one of the two papers under References. Open both papers before you start. The EM screenshots on this page show examples from other spines of the series, not from your 5 spines.

Smooth endoplasmic reticulum (SER)

  • SER has a flattened, oval appearance surrounded by a membrane, but this shape can be variable. On one section the SER can look like a gray smudge, a small black dot, a snake-like black line, or a large vesicle-like lake.
  • SER is connected across serial sections, though sometimes this continuity can be difficult to see.

Figure: snake-like tubule of SER entering a spine

Spacek & Harris, 1997, Fig. 2c. Open the paper.

Section 15 without traces Section 15 with spine d03sp05 in yellow and the SER stamp d03sern05, a yellow cross
SER in spine d03sp05 on section 15, without traces (left) and with d03sp05 and the SER stamp d03sern05 (right). The view is about 1.3 µm across.

Clip: section 13 in a wider view; the camera closes in on spine d03sp05, and the pointer clicks the up arrow four times, through sections 14 to 17, with the SER stamp d03sern05 (yellow cross) in view.

▶ 10 s · loops

SER in spine d03sp05, sections 13 to 17, stepping up one section at a time.

The example stamps already in the series have longer names than yours: a letter after "ser" or "sa" says where the stamp sits (h for head, n for neck, s for shaft), as in d03sern05 and d03sah19. Your stamps use the short names given below, such as d03ser12.

Spine apparatus (SA)

  • The spine apparatus is a specialized form of the SER.
  • SA is typically seen in large mushroom spines but can also appear in the dendritic shaft.
  • SA appears as flat cisternae (arrows) with electron-dense plates between them (wavy arrows); see the figure below.

Figure: spine apparatus, with cisternae (arrows) and dense plates (wavy arrows)

Spacek & Harris, 1997, Fig. 3b. Open the paper.

Section 70 without traces Section 70 with spine d03sp19 in yellow and the SA stamp d03sah19, a red cross
SA in the head of spine d03sp19 on section 70, without traces (left) and with d03sp19 and the SA stamp d03sah19 (right). The view is about 1.6 µm across.

Endosomes

Endosomes are sorting organelles inside the cell. They take part in the local recycling, sorting, and breakdown of membrane and proteins.

Four major subtypes:

  1. Multivesicular body
  2. Tubule
  3. Smooth vesicle
  4. Coated vesicle or pit

Figure: drawing of endosome types in three spines

Cooney et al., 2002. Open the paper.

Endosome criteria

1. Coated vesicles and pits

  • Vesicles: elliptical, with a gray interior and a clathrin coat (a coat on the cytoplasmic side made of spoke-like protrusions).
  • Pits: elliptical, omega-shaped invaginations of the membrane, with gray interiors and a clathrin coat.

Figure: clathrin-coated pit, with an inset of a clathrin-coated vesicle

Cooney et al., 2002. Open the paper.

Section 108 without traces Section 108 with spine d03sp29 in yellow and the coated pit stamp d03endo29_cp, an orange octagon
A coated pit on spine d03sp29 on section 108, without traces (left) and with d03sp29 and the stamp d03endo29_cp (right). The view is about 2.3 µm across.

Clip: section 108 in a view about 5 µm across; the camera closes in on a coated pit and the pointer points at it, then spine d03sp29 (yellow) and the stamp d03endo29_cp (orange) appear.

▶ 11 s · loops

Closing in on the coated pit of spine d03sp29 on section 108.

2. Endosomal tubule

  • Cylindrical compartment with a smooth outer membrane and a dark, grainy interior.
  • Uniform diameter.
  • May have a clathrin-like protein coat on the end, a sign that a vesicle is budding off.

Figure: endosomal tubule

Cooney et al., 2002. Open the paper.

3. Multivesicular body (MVB)

  • Dark, grainy interior with a variable number of vesicles inside.

Figure: multivesicular body

Cooney et al., 2002. Open the paper.

4. Vesicles (small, large, amorphous)

  • Small and large smooth vesicles: light gray interiors with a smooth outer membrane.
  • Amorphous vesicles: smooth membranes, electron-lucent (pale) interiors, and irregular shapes. They often occur in clumps.

Figure: small and large smooth vesicles, and a clump of amorphous vesicles

Cooney et al., 2002. Open the paper.

5 additional spines

To learn to identify these spine resources, you are assigned 5 more spines. Each is marked by a red p-stamp, and each is already traced, with its synapse (c-trace) and contact flat area (cfa-trace):

p-stamp (Object List group "Assigned") Spine trace Synapse traces
d03p12 d03sp12 d03c12, d03cfa12
d03p14 d03sp14 d03c14, d03cfa14
d03p37_branched d03sp37_branched d03c37a, d03cfa37a, d03c37b, d03cfa37b
d03p40 d03sp40 d03c40, d03cfa40
d03p42 d03sp42 d03c42, d03cfa42

Search these 5 spines, head and neck, for SER, SA, and endosomes. To go to a spine, double-click its p-stamp in the Object List.

The group "Assigned" also holds d03p06, d03p32 and d03p47_branched, the spines you traced in Lab 1.1. They are not part of this lab.

Stamp spine SER and SA

Identify and stamp the SER or SA in each of the 5 spines. Stamp any SER and SA you see in the spine head and the spine neck.

  1. In the Tools window, select the Stamp tool.
  2. Right-click an unused palette square. The Set Attributes dialog opens.
  3. In Name, type the name for what you found, with ## replaced by the spine number:
    • SA: d03sa##. SA in d03sp12 is d03sa12.
    • SER: d03ser##. SER in d03sp12 is d03ser12.
  4. In Stamp radius (microns), type 0.05.
  5. Click OK.
  6. Click on the SER or SA to place the stamp.

Use a separate palette square for each name.

Set Attributes for d03sa12: red, circle, Fill None, Stamp radius 0.05
Set Attributes for d03sa12.

Clip: on section 70 with the Stamp tool, the pointer right-clicks the first palette square, sets the name to d03sa19 and Stamp radius (microns) to 0.05, clicks OK, and clicks inside spine d03sp19 to place the red stamp d03sa19 on the SA.

▶ 16 s · loops

Set the name and the radius, click OK, then click on the SA. Shown in spine d03sp19, not one of your 5 spines.

Stamp spine endosomes

Look for endosomes in each of the 5 spines, and stamp each endosome you find.

Hint

You are likely to find coated pits in your assigned spines.

  1. With the Stamp tool selected, right-click an unused palette square. The Set Attributes dialog opens.
  2. In Name, type d03endo##_cp, with ## replaced by the spine number (cp stands for coated pit). A coated pit in d03sp12 is d03endo12_cp.
  3. In Stamp radius (microns), type 0.05.
  4. Click OK.
  5. Click on the endosome to place the stamp.
Set Attributes for d03endo12_cp: orange, circle, Fill None, Stamp radius 0.05
Set Attributes for d03endo12_cp.

Check your stamps

In the Object List, choose Filter > Group filter... and pick the group "SER, SA, Endo" to see the example stamps. Your new stamps are not in this group; to see them too, type a regex filter such as d03(ser|sa|endo).* in a second Object List (see Lab 1.1).

The Object List filtered by the group SER, SA, Endo, with columns Name, Start, End, Count and Groups, rows d03endo29_cp to d03sern05
The Object List filtered by the group "SER, SA, Endo", with the first and last section of each stamp.

Saving

Save with Ctrl+S (Mac: Cmd+S), back up your .jser file, and upload it to Canvas, as in Lab 1.1.

References

  • Spacek J, Harris KM (1997). Three-dimensional organization of smooth endoplasmic reticulum in hippocampal CA1 dendrites and dendritic spines of the immature and mature rat. Journal of Neuroscience 17(1):190-203. doi:10.1523/JNEUROSCI.17-01-00190.1997
  • Cooney JR, Hurlburt JL, Selig DK, Harris KM, Fiala JC (2002). Endosomal compartments serve multiple hippocampal dendritic spines from a widespread rather than a local store of recycling membrane. Journal of Neuroscience 22(6):2215-2224. doi:10.1523/JNEUROSCI.22-06-02215.2002