Dense-cored vesicles, smooth endoplasmic reticulum, and mitochondria are closely associated with non-specialized parts of plasma membrane of nerve terminals: implications for exocytosis and calcium buffering by intraterminal organelles

Abstract

To determine whether there are anatomical correlates for intraterminal Ca2+ stores to regulate exocytosis of dense-cored vesicles (DCVs) and whether these stores can modulate exocytosis of synaptic vesicles, we studied the spatial distributions of DCVs, smooth endoplasmic reticulum (SER), and mitochondria in 19 serially reconstructed nerve terminals in bullfrog sympathetic ganglia. On average, each bouton had three active zones, 214 DCVs, 26 SER fragments (SERFs), and eight mitochondria. DCVs, SERFs and mitochondria were located, on average, 690, 624, and 526 nm, respectively, away from active zones. Virtually no DCVs were within "docking" (i.e., < or = 50 nm) distances of the active zones. Thus, it is unlikely that DCV exocytosis occurs at active zones via mechanisms similar to those for exocytosis of synaptic vesicles. Because there were virtually no SERFs or mitochondria within 50 nm of any active zone, Ca2+ modulation by these organelles is unlikely to affect ACh release evoked by a single action potential. In contrast, 30% of DCVs and 40% of SERFs were located within 50 nm of the nonspecialized regions of the plasma membrane. Because each bouton had at least one SERF within 50 nm of the plasma membrane and most of these SERFs had DCVs, but not mitochondria, near them, it is possible for Ca2+ release from the SER to provide the Ca2+ necessary for DCV exocytosis. The fact that 60% of the mitochondria had some part within 50 nm of the plasma membrane means that it is possible for mitochondrial Ca2+ buffering to affect DCV exocytosis.

Fig. 1

Methods used for data analysis. A,B: An example of the overlay and the photomicrograph used to prepare it. Comparable structures are indicated in the photomicrograph (A) and the overlay (B). SV, synaptic vesicles; DCV, dense-cored vesicle; MT, mitochondrion; AZ, active zone; PM, plasma membrane. C: The method of measuring distances within a bouton in three dimensions. In this example, the shortest distance between a DCV and a mitochondrion is measured by using the Pythagorean theorem, $\mathrm{C}=\sqrt{{\mathrm{A}}^2+{\mathrm{B}}^2}$ , where C is the shortest distance between the DCV and the mitochondrion, A equals the section thickness (70 nm) times number of sections, and B is the distance between the DCV and the projected location of the mitochondrion (*) if it were located in the same section as the DCV. Sections were placed in register by means of fiduciary marks (f). Scale bar = 0.25 μm in A.

Fig. 2

Examples of the synaptic bouton elements analyzed in this study. A: Two separate active zones (AZ1 and AZ2) can be observed in this photomicrograph and were confirmed in serial sections. When totally reconstructed, this bouton contained 207 dense-cored vesicles (DCVs), 26 smooth endoplasmic reticulum fragments (SERFs), and 13 mitochondria (MTs). Although this particular section of the bouton contains mostly synaptic vesicles, two DCVs, three MTs, and a few glycogen bodies are also visible. The rest of the bouton is filled with regular, round synaptic vesicles (SV). The plasma membrane is delimited by small arrows. B: This bouton section contains several MTs and DCVs. In total, the bouton contained 155 DCVs, five SERFs, and six MTs. One active zone (AZ1) and the beginning of another (AZ2) are seen. Note the proximity of some of the DCVs and the MTs to the plasma membrane. Glycogen bodies (GLY) are also present. C: This bouton section contains two SERFs near the plasma membrane. Four MTs and six DCVs can also be observed. The entire bouton contained 82 DCVs, 92 SERFs, and nine MTs. There are also several clumps of GLY. D: This bouton section illustrates one MT and several DCVs, from a total of 672 DCVs, 41 SERFs, and 17 MTs in the bouton. One DCV, indicated by the thick arrow, is located 100 nm away from the active zone. Scale bars = 0.25 μm in A–D.

Fig. 3

Relationships between the numbers of intracellular elements and the volumes of individual boutons. A: DCVs. B: Mitochondria. C: SERFs. The data were fit with linear functions as indicated by the heavy lines. The goodness of fit was calculated using a Pearson coefficient of correlation as indicated in parentheses. The key indicates the location of the bouton on the cell, either at the axon hillock (A) end or the opposite nuclear (N) end. The one solid circle in C is an outlier and was not included in the fit.

Fig. 4

Spatial distributions of dense-cored vesicles (DCVs) within boutons. Distributions of the distances between the DCVs and (A) a nearest active zone (AZ) and (B) other parts of the plasma membrane (PM). C: Normalized density distribution of the DCVs within 100-nm-width concentric cylindrical shells of the plasma membrane. See text for the normalization procedure. D: Proportions of DCVs that were within 100 nm of plasma membrane in each bouton. In this and the following two figures, the insets in A and B are number of elements within 100 nm of a nearest active zone and other parts of the plasma membrane, respectively. The linear function that fits the data in D is indicated by the fitting curve, and the goodness of fit was calculated using a Pearson coefficient of correlation as indicated in parentheses.

Fig. 5

Spatial distributions of smooth endoplasmic reticulum fragments (SERFs) within boutons. Distributions of the distances between the SERFs and (A) a nearest active zone and (B) other parts of the plasma membrane. C: Normalized density distribution of the SERFs within 100-nm-width concentric cylindrical shells of the plasma membrane. D: Proportions of SERFs that were within 100 nm of plasma membrane in each bouton. Figure conventions are described in Figure 4.

Fig. 6

Spatial distributions of mitochondria (MT) within boutons. Distributions of the distances between the MTs and (A) a nearest active zone and (B) other parts of the plasma membrane. C: Normalized density distribution of the MTs within 100-nm-width concentric cylindrical shells of the plasma membrane. D: Proportions of MTs that were within 100 nm of plasma membrane in each bouton. Figure conventions are described in Figure 4.

Fig. 7

The total volume of mitochondria and two different estimates of the total volume of the glycogen bodies are plotted against the total volume of the same bouton. Data located on the same dotted vertical line were from a single bouton. The thicknesses used in calculating total glycogen body volume are indicated in parentheses in the key.