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Mitochondrial support of persistent presynaptic vesicle mobilization with age-dependent synaptic growth after LTP

  1. Heather L Smith
  2. Jennifer N Bourne
  3. Guan Cao
  4. Michael A Chirillo
  5. Linnaea E Ostroff
  6. Deborah J Watson
  7. Kristen M Harris  Is a corresponding author
  1. Center for Learning and Memory, Institute for Neuroscience, University of Texas at Austin, United States
  2. University of Colorado Denver - Anschutz Medical Campus, United States
  3. New York University, New York
Research Article
Cite this article as: eLife 2016;5:e15275 doi: 10.7554/eLife.15275
10 figures and 1 table

Figures

Stable responses during control stimulation and expression of LTP following TBS in S. radiatum of area CA1 in acute hippocampal slices.

(A) P15 slice in interface chamber. Recording electrode (R) between two stimulating electrodes (S1 and S2) separated by more than 500 µm. Rectangles indicate the regions from which tissue was sampled for 3DEM. (B) Average responses after LTP was induced by TBS and average responses to control stimulation (Con) in P15 slices (n = 2 animals, modified from Figure 1 of Watson et al., 2016). (Structural analyses were also obtained at 5 min and 30 min following TBS using experiments from Watson et al., 2016). (C) Average responses after LTP was induced by TBS and average responses to control stimulation (Con) from identically prepared slices at P55-70 (n = 2 animals; modified from Figure 1 of Bourne and Harris, 2011). Red triangles indicate when TBS was applied. Arrows indicate times when the sample traces were obtained for the Con and TBS conditions.

https://doi.org/10.7554/eLife.15275.002
Maintenance of SSBs and increase in MSBs following induction of LTP by TBS at P15.

(A) Spine density (#/µm length of reconstructed dendrite) from perfusion-fixed (PF) decreased under control stimulation (Con) and increased at 120 min after TBS induction of LTP (F (2, 60)=26.45, p<0.0001, adapted from Watson et al., 2016). Example electron micrographs and 3D reconstructions of: (B) SSB with one PSD (red arrow), (C) MSB with two synapses (red arrows), and D) NSB (black arrow) with 16 vesicles but no postsynaptic partner. Reconstructions have PSDs (red), synaptic vesicles (green), and axonal plasma membranes (translucent gray). In D (for B–D), the scale bar is 0.25 µm and the scale cube is 0.25 µm on each side. (E) Overall, the frequency (boutons/µm3 of unbiased bricks) of presynaptic boutons was significantly greater at 120 min following TBS induction of LTP, relative to PF and Con conditions (F = 11.312, df = 2, p<0.01). There were fewer SSBs under control conditions compared to PF, whereas SSB frequencies were maintained at PF levels following TBS and were significantly greater than controls (F1,6 = 14.6, p=0.0087). MSB frequencies were greater following TBS than controls or PF (F1,6 = 10.1, p=0.012). The NSB frequencies did not differ significantly across conditions (F1,6 = 0.32, p=0.59). Two unbiased bricks (see Materials and methods) were analyzed per series for a total of 546 boutons across conditions (Table 1). Significant post hoc differences are indicated as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

https://doi.org/10.7554/eLife.15275.003
Presynaptic boutons with mitochondria had more vesicles than those without mitochondria in PF hippocampal area CA1 at P15.

(A) Three neighboring presynaptic boutons located along the same axon (translucent gray). Bouton 1 (b1) was an MSB with two postsynaptic partners (PSDs, red) that contained a mitochondrion (navy), 303 nondocked vesicles (green), and 12 docked vesicles (blue, inset). Nearest distances from each PSD to the mitochondrion were less than 1 µm (Dm1a, 1b). For b2, the distance from the edge of the PSD to the nearest mitochondrion was 1.4 µm (Dm2, blue line), and it contained 124 nondocked vesicles and nine docked vesicles. The b3 had 82 nondocked vesicles and two vesicles docked at the PSD which was 4.4 µm from the nearest mitochondrion (b4, nonsyn mito, Dm3, orange line) and 5.3 µm from the end of the axon reconstruction (D3end). Scale cube is 0.5 µm on each side. (B) Representative EMs from four regions along an axon illustrating boutons with PSDs (red arrows), nondocked vesicles (green arrows), and docked vesicles (blue arrows) and a nonsynaptic inter-bouton region containing a mitochondrion (nonsyn mito). Scale bar is 0.25 µm in b4 for all four micrographs. (C) Of all the axonal boutons, 76% were SSBs and 24% were MSBs. Absolute numbers of boutons are in corresponding bars that also indicate the presence (black) or absence of a mitochondrion (gray). (D) Overall, the PSD areas did not differ between synapses on MSBs and SSBs (n = 216, F1,215 = 0.026, p=0.87). However, PSD areas were larger on SSBs when mitochondria were present (n = 140, F1,138 = 15.4, p<0.001), but on MSBs this difference did not reach significance (n = 75, F1,74 = 2.791, p=0.10). (E) The number of docked vesicles per PSD did not differ between MSBs and SSBs with or without mitochondria (n = 177, F1,175 = 0.26, p=0.61); however, the number of docked vesicles per PSD was greater for both types of boutons with mitochondria than those without mitochondria (n = 177, F1,175 = 65.1, p<0.001). (F) Boutons with mitochondria had more nondocked vesicles whether they were SSBs or MSBs (n = 180, F1,178 = 16.2). Since the pool of nondocked vesicles could not be attributed to a specific PSD, the MSBs had more nondocked vesicles than SSBs, which reached significance for those boutons without mitochondria (gray line above the bars). (G) Axon segments containing mitochondria somewhere along their lengths were analyzed and the amount of axon volume with mitochondria was significantly less than the amount of axon volume without mitochondria (n = 81, ANOVA, F1,161 = 104.1, p<0.0001). (H) Of all axonal mitochondria in the perfusion fixed condition from the brick analyses (see Figure 2E), 51% of mitochondria resided in nonsynaptic regions (Nonsyn), while 49% resided in synaptic boutons (Syn). (I) Axonal and mitochondrial diameters were correlated and this relationship did not differ significantly between mitochondria located in synaptic or nonsynaptic regions of the axons from (H) (n = 75, ANCOVA, F1,72 = 1.91, p=0.17). Significant post hoc differences are indicated as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

https://doi.org/10.7554/eLife.15275.004
Synapses had fewer vesicles and PSD areas were smaller as distance from an axonal mitochondrion increased.

(A) Nondocked vesicles versus mitochondrial distance (Dm) had a statistically significant breakpoint at 2.97 ± 0.67 µm (t(165) = 4.4, p<0.001). (B) The breakpoint for docked vesicles per PSD area versus mitochondrial distance was 3.06 ± 0.62 µm (t(161) = 4.92, p<0.001). (C) The breakpoint for PSD area per bouton (ΣPSD across MSBs) vs. Dm was 3.02 ± 1.24 µm (t(165) = 2.44,p=0.015). The mitochondria had lengths and hence were considered to be inside the bouton when any portion of the mitochondrion was within the vesicular domain and distances from the center of the PSD were shorter than the solid gray line positioned at 0.91 µm (in AC), which was the longest measured distance from the edge of a PSD to a mitochondrion located within the vesicle cloud of a presynaptic bouton. Breakpoints in A–C are shown as mean (dashed line) ± s.e.m. (gray shading) from the regression analysis. (D) The mean number of nondocked vesicles was greater in boutons with a mitochondrion (mito) than at Dm ≤ 3 µm, which was greater than Dm > 3 µm (n = 177, F2,175 = 46.3, p<0.001). (E) The mean numbers of docked vesicles per PSD area was greater in boutons with a mitochondrion than at Dm ≤ 3 µm, which was greater than Dm > 3 µm (n = 183, F2,181 = 55.0, p<0.001). (F) The mean ∑PSD area was also greater when presynaptic boutons had a mitochondrion than when Dm ≤ 3 µm, which was greater than Dm > 3 µm (n = 175, F2,175 = 19.6, p<0.001). All these data are from perfusion-fixed hippocampus at P15. y-Axis labels in AC are also for D–F, and similarly the x-axis labels at the bottom apply to all graphs above them. Significant post hoc differences are indicated as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

https://doi.org/10.7554/eLife.15275.005
The relative distribution of mitochondrial distances from synapses was unchanged for control and LTP conditions at P15.

Representative axons are shown from the control (Con) and TBS sites for each time point (5’, 30’, 120’ = minutes) after the induction of LTP at P15. Each axon is oriented to illustrate sequentially a presynaptic bouton with a mitochondrion (mito), boutons with PSDs located within 3 µm of a mitochondrion (Dm £ 3) or beyond (Dm > 3). For each time and condition, a 3D reconstruction of the axons (gray) illustrates PSDs (red), nondocked vesicles (green), and mitochondria (navy). Sample EMs are positioned beneath each bouton to illustrate their PSDs (red arrows in postsynaptic side), presynaptic boutons (green B), and in the leftmost column the mitochondria (blue arrows). Pie charts indicate the proportions of boutons that reside in each category (Mito, Dm £ 3, Dm > 3) for the corresponding condition and time point. Scale cube (dark gray) in top row is 0.5 µm on each side for all 3D reconstructions, and scale bar is 0.5 µm for all EMs.

https://doi.org/10.7554/eLife.15275.006
Mobilization of presynaptic vesicles was restricted to presynaptic boutons with or near mitochondria.

Representative 3D reconstructions of boutons with mitochondria (navy, top row) and without mitochondria (second row) with PSDs (red), docked vesicles (blue), and nondocked vesicles (green) at (A) 5 min, (B) 30 min, and (C) 120 min post-TBS. Light blue boxes enclose the control conditions, and red boxes enclose the TBS conditions at each time point. Total vesicle number is at top left corner, and PSDs with docked vesicles are reconstructed in bottom right corner of each box. At 5 min, Con conditions had (A1) more docked vesicles (F2,94 = 20.4, p<0.001) and more (A2) nondocked vesicles (F2,126 = 14.7, p<0.01) in presynaptic boutons with mitochondria (Mito) or at Dm ≤ 3 µm (blue bars and stars), but there were no LTP effects. At 30 min, (B1) docked vesicles decreased post-TBS, relative to Con and PF conditions only when presynaptic boutons contained a mitochondrion (F1,138 = 13.7, p<0.001, red stars). In addition, Con boutons with Mito or Dm ≤ 3 had more docked vesicles (F2,91 = 10.9, p<0.001) and nondocked vesicles (F2,132 = 10.1, p<0.001, blue stars and bars). At 120 min, (C1) docked vesicles were reduced in boutons with mitochondria (F1,106 = 13.9, p<0.001) and when Dm £ 3 (F1,102 = 9.06, p<0.01) relative to control and PF conditions. At 120 min when Dm ≤ 3, boutons have more docked vesicles in Cons (F2,63 = 4.27, p=0.018). Nondocked vesicles (C2) were reduced in boutons with mitochondria relative to Con and PF levels (F1,122 = 20.2, p<0. 01). Furthermore, Con boutons contain more nondocked vesicles when mitochondria are present (F2,81 = 6.93, p<0.01). Significant post hoc differences are indicated as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

https://doi.org/10.7554/eLife.15275.007
Effect of synapse size and mitochondrial presence on vesicle mobilization following TBS at P15.

Synapses with mitochondria located among the presynaptic vesicles (Mito) were compared to those without mitochondria (>1 µm to Mito). At 30 min following TBS, boutons with mitochondria had (A) fewer docked vesicles (F1,81 = 61.4, p<0.001) and (B) fewer nondocked vesicles (F1,108 = 5.03, p=0.021) relative to control conditions. In contrast, boutons with no mitochondria showed no significant differences in (C) docked (F1,169 = 2.64, p=0.11) or (D) nondocked vesicles (F1,227 = 0.17, p=0.69) between TBS and control conditions at 30 min. Furthermore, under control conditions, (A) fewer boutons with mitochondria had zero docked vesicles than (B) boutons with no mitochondria (F; F1,107 = 3.87, p<0.05), but this mitochondria-related difference (F1,145 = 0.57, p=0.45) did not hold in the TBS condition. At 120 min following TBS, boutons with mitochondria had (E) fewer docked (F1,44 = 12.4, p=0.001008) and (F) fewer nondocked vesicles (F1,63 = 7.84, p=0.007) relative to control conditions across the distribution of synapse sizes. In contrast, boutons with no mitochondria showed no significant differences between the TBS and control conditions in the number of (G) docked vesicles (F1,116 = 0.41, p=0.53) or (H) nondocked vesicles (F1,169 = 1.71, p=0.19). Furthermore, fewer boutons (E) with mitochondria lacked docked vesicles than those (F) without mitochondria (F1,92 = 4.2, p<0.05) at 120 min following TBS, whereas this difference did not reach significance under the control conditions (F1,72 = 3.01, p=0.08). In addition, (G) more of the boutons without mitochondria lacked docked vesicles at 120 min after TBS than under control conditions (F1,118 = 4.9, p<0.05). The x-axis represents individual PSD areas for docked vesicles (A, C, E, G) and summed PSD area for nondocked vesicles to account for MSBs (B, D, F, H). The results of the ANCOVAs are indicated in the lower left corner of each graph, whereas the results from ANOVAs concerning the differences among conditions for boutons lacking docked vesicles are summarized in this figure legend.

https://doi.org/10.7554/eLife.15275.008
Vesicle mobilization was also greatest in presynaptic boutons that contained a mitochondrion at 120 min following TBS in adult hippocampus.

(AB) Representative EMs of presynaptic boutons (green B) from 120 min control (A1 mito, A2 no mito) and TBS (B1 mito, B2 no mito) conditions (red arrows, PSDs in A1, B2, scale bar is 0.5 µm). (C) Overall, the total number of boutons per µm3 was decreased under TBS conditions relative to control (n = 6 vol bricks per condition, see Table 1, F1,8 = 30.8, p<0.001). There were fewer single synaptic boutons (SSB, n = 6, F1,8 = 22.6, p<0.01) and nonsynaptic boutons (NSB, n = 6, F1,8 = 13.9, p<0.01) 2 hr after the onset of TBS, but the multisynaptic boutons were unchanged (MSB, n = 6, F1,8 = 0.22, p=0.65). Proportions of boutons that contained mitochondria (Mito) or were Dm ≤ 3 or Dm > 3 from a mitochondrion under control (D) and TBS (E) conditions were unchanged in the LTP relative to the control condition (n = 518, χ2=2.98, p=0.23). Representative reconstructions of synapses from adult hippocampal area CA1 at the 120 min (PSDs, red, nondocked vesicles, green, and docked vesicles, blue, lower right corner) for boutons (F1) with mitochondria (navy, Mito) and (G1) without mitochondria (No Mito). (F2) Docked vesicles decreased at 120 min after TBS relative to Con in boutons with mitochondria (F1,244 = 27.2, p<0.001), and (G2) to a lesser degree in boutons without mitochondria (F1,207 = 4.72, p<0.05) across synapses of all sizes. (F3) Nondocked vesicles also had a pronounced decrease in the TBS condition relative to Con in boutons with mitochondria (F1,297 = 112.1, p<0.001) and (G3) to a lesser degree in boutons without mitochondria (F1,250 = 35.8, p<0.001).

https://doi.org/10.7554/eLife.15275.009
Changes in mitochondrial dimensions in P15 and adult hippocampal axons.

(AB) Representative 3D reconstructions of axons from P15 and adult (Adult) control and TBS conditions at 120 min. (Total axon segment lengths – Lt, black lines – mitochondrial dimensions, length – L, diameter – D, and scale cube is 0.5 µm per side.) (C) Mitochondrial volumes were smaller at P15 and larger in adults in the 120 min TBS vs. the control condition (F1,919 = 74.4, p<0.001, n = all mitochondria for all measured axons). (D) Neither P15 nor adult mitochondria showed differences in diameter between conditions (n = 320, F1,316 = 0.37, p=0.55), although adult mitochondria were wider than P15 mitochondria (n = 320, F1,316 = 37.5, p<0.001). (E) P15 mitochondria were shorter and adult mitochondria were longer at 120 min after TBS vs. the control condition (n = 320, F1,316 = 30.0, p<0.001). (F) Mitochondria per axon segment length (#mito/µm) was greater in adults than P15 in the control condition (F1,349 = 73.2, p<0.001, blue bar and stars), and the adult mitochondria frequency was significantly reduced in the 120 min post TBS versus control condition (F1,349 = 45.6, p<0.001). (Numbers at the base of each bar indicate the total number of mitochondria in each group. All the mitochondrial volumes were measured. See Table 1 and Materials and methods for sampling details.).

https://doi.org/10.7554/eLife.15275.010
Synaptic and nonsynaptic mitochondria exhibited non-orthodox configurations after TBS-induction of LTP at both ages.

(A1) The orthodox mitochondrial configuration had cristae with uniformly thin widths (<20 nm) and homogeneously gray matrices. (A2) The condensed mitochondrial configuration had wider and more variable cristae (>30 nm, red arrows) and less uniform matrices. (A3) Mitochondria with an intermediate configuration had cristae ranging in width from 20 to 30 nm and nonuniform matrices. (A4) Long mitochondria often had a mixed appearance showing distinct regions of orthodox, condensed, or intermediate configurations along their lengths. (A5) Swollen mitochondria were more than two times wider than other mitochondria. (Cristae are labeled with red arrows and the matrix is labeled with blue arrows in the micrographs of A1-5. Scale bar in A5 is 0.25 µm for A1-5.) (B) Under control conditions, 44% of P15 mitochondria and 82% of adult mitochondria were synaptic and located among the presynaptic vesicles (X2 = 37.5, df = 1, p<0.0001). These proportions did not differ significantly between TBS and control conditions at either age. (C) Comparison of mitochondrial configurations showed no significant differences between synaptic and nonsynaptic mitochondria or between P15 and adult ages under control conditions, or in the (D) TBS condition. Significant differences between Con and TBS are discussed in the text. (Pie chart colors in C and D match colors of the mitochondrial configuration boxes in A, with solid shades for synaptic and hatched shades for nonsynaptic mitochondria.) (E) At both ages, the cristae were wider in the TBS condition for both synaptic and nonsynaptic mitochondria.

https://doi.org/10.7554/eLife.15275.011

Tables

Table 1

Sample sizes for axons, boutons, vesicles, and mitochondria under each condition. Table 1 provides total n’s for each of the figures. Dashes mean that category was not used in a particular figure. Figure 2: Number of boutons entering the inclusion volume of the unbiased bricks that were identified as single, multi-, or nonsynaptic boutons. Figures 34: Number of axon segments traced from the presynaptic boutons of dendritic spines on dendrites that were analyzed in Watson et al. (2016). Vesicles were counted and mitochondrial distances measured for all of the boutons along these axons as long as the boutons were complete and nearer to a mitochondrion than to the edge of the image stack. Axons in parentheses contained mitochondria and were used for Figure 3G. Figures 57: Boutons were the presynaptic partners of the dendrites analyzed in Watson et al. (2016). All boutons were used except for those that were incomplete or fewer than three microns from the edge of the image stack. Figure 8: All boutons from Bourne et al. (2013) were used except for those that were less than three microns from the edge of an image stack. Two additional bricks were analyzed from each dataset of the adults, for a total of six bricks in each condition for the adults. Figure 9: Axon lengths are µm (mean ± sd). Figure 9C: Every complete mitochondrion encountered along every axon from the 2-hr P15 and adult series was traced to compute volumes. Figure 9D and E: Every fourth mitochondrion was measured to obtain the lengths and diameters for a subset of these mitochondria. A total of 10 presynaptic mitochondria were traced from each series, for a total of 80 mitochondria per condition. Figure 9F: Mitochondria frequencies were determined for all axon segments greater than 10 microns long at P15. To obtain comparable measurements in the adults, mitochondria frequencies were measured from 10 axon segments that were at least 10 microns from each series for a total of 80 axons. Figure 10: Morphological conformations were identified for all presynaptic mitochondria that fell within the original unbiased bricks at both ages for a total of 4 per condition at P15 and in adults (used the four original bricks from Bell et al., 2014). Mitochondria were identified within the sample volumes beginning on the first section of each of the sample volumes used and numbered in the order in which they were found. The widths of each of their cristae were measured for every third mitochondrion at P15 and every fourth mitochondrion in adults to obtain comparable sample sizes.

https://doi.org/10.7554/eLife.15275.012

Age

P15

Adult

Time

Perfused

5 min

30 min

120 min

120 min

Condition

R88

R89

CON

LTP

CON

LTP

CON

LTP

CON

LTP

# dendrites

9

8

19

20

23

28

24

22

8

8

# bricks

2

2

4

4

6, 4

6, 4

Total Brick Volume (µm2)

54

65

-

147

125

165, 99

222, 145

Axons (total n)

Figure 3, 4

85 (44)

77 (37)

-

Figure 5, 6

132

108

143

184

88

144

Figure 7

143

184

88

144

-

Figure 8

-

225

289

Figure 9

71

121

80

80

Figure 9 axon length

12.5 ± 2.9

13.0 ± 2.9

11.0 ± 1.4

12.6 ± 1.7

Boutons (total n)

Figure 2

89

84

165

208

Figure 3, 4

77

113

Figure 5, 6

132

108

143

184

88

144

Figure 7

143

184

88

144

-

Figure 8

-

-

-

-

225

289

Mitochondria (total n)

Figure 3H,I

36

39

-

Figure 9C

-

-

128

196

265

334

Figure 9D,E

-

-

-

80

80

80

80

Figure 9F

71

121

80

80

Figure 10A–C

88

105

161

159

Figure 10D

33

36

40

40

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