Fig 1.
Comparison of mitochondrial number at MF and SC presynaptic bouton profiles.
(A) A transmission electron micrograph of hippocampal stratum lucidum in area CA3 showing large cross-sectional mossy fiber bouton profiles (MFB: highlighted in yellow) establishing synaptic contacts with large complex spines of CA3 pyramidal neuron dendrites at multiple sites. Asterisk: spine, D: dendrite, Scale bar: 1 μm. (B) A transmission electron micrograph of hippocampal stratum radiatum in area CA1 showing cross-sectional dendrite profiles of CA1 pyramidal neurons and the neuropil in-between, which contains abundant cross-sectional presynaptic bouton and dendritic spine profiles. The cross-sectional areas of the bouton profiles are highlighted in blue. Arrows indicate cross-sectional bouton profiles with or without mitochondria. Asterisk: spine, D: dendrite, Scale bar: 1 μm. (C) Quantification of the number of mitochondria per cross-sectional bouton profile. Note that the cross-sectional MF bouton profiles contain many mitochondria, in contrast to the scarcity of mitochondria in the cross-sectional SC bouton profiles (MF: 10.22 ± 0.39, n = 45; SC: 0.84 ± 0.08, n = 45; MF vs. SC, p < 0.0001, unpaired t-test). (D) Quantification of the size of cross-sectional bouton profile area. The cross-sectional MF bouton profile areas are larger relative to the cross-sectional areas of SC bouton profiles (MF: 5.91 ± 0.49 μm2, n = 48; SC: 0.77 ± 0.07 μm2, n = 48; MF vs. SC, p < 0.0001, unpaired t-test). (E) Quantification of the number of mitochondria per μm2 of cross-sectional bouton area. The density of mitochondria is higher at MF boutons than SC boutons (MF: 2.52 ± 0.23 mito/μm2, n = 42; SC: 0.95 ± 0.12 mito/μm2, n = 44; MF vs. SC, p < 0.0001, unpaired t-test). (F) The fraction of cross-sectional CA1 bouton profiles containing or lacking mitochondria. The data are presented as mean ± SEM (**** p < 0.0001, Student’s t-test).
Fig 2.
Differential effects of mitochondiral NCX inhibitors on PTP at MF and SC synapses.
PTP was induced by high frequency stimulation (HFS: 16 pulses at 100 Hz, 4 times delivered at 0.33 Hz) in acute hippocampal slices of wild-type mice at 2 months of age. (A & B) Mean value of time-dependent changes in the fEPSP slope was recorded in the absence (A) or presence (B) of CGP37157 (20 μM), a mitochondrial NCX inhibitor, in the hippocampal MF pathway. The insets represent traces recorded before and immediately after HFS. DCG-4 (2 μM) was applied at the end of all experiments to confirm the recording of MF synapses. (C) The summary bar graph for the mean magnitude of PTP shows that PTP is reduced by CGP37157 treatment at MF synapses (Control: 219.1 ± 12.0%; +CGP: 157.1 ± 5.2%; p = 0.00013, paired t-test). (D) The effect of CGP37157 on basal transmission. Treatment of CGP37157 for 15 min has no effect on the fEPSP slope at MF synapses. (E & F) Mean value of time-dependent changes in the fEPSP slope was recorded in the absence (E) or presence (F) of TPP+ (2 μM), another specific inhibitor of mitochondrial NCX, in the hippocampal MF pathway. (G) The summary bar graph for the mean magnitude of PTP further shows that PTP induction is reduced at MF synapses (Control: 194.0 ± 5.2%; +TPP: 147.2 ± 2.7%; p < 0.0001, paired t-test). (H) The effect of TPP+ on basal transmission. Treatment of TPP+ for 15 min has no effect on the fEPSP slope at MF synapses. (I & J) Mean value of time-dependent changes in the fEPSP slope before and after the PTP induction in the hippocampal SC pathway. The insets represent traces recorded before and immediately after HFS. The effect of treatment with CGP37157 on PTP of fEPSPs at the hippocampal SC pathway shows that inhibition of mitochondrial Ca2+ release had no effect on PTP induction. (K) The summary bar graph for the mean magnitude of PTP shows that PTP induction is not different at SC synapses in the absence or the presence of CGP37157 treatment (Control: 234.7 ± 9.8%; +CGP: 222.7 ± 6.9%; p = 0.17, paired t-test). (L) The effect of CGP37157 on basal transmission. Treatment of CGP37157 for 15 min has no effect on the fEPSP slope at SC synapses. All data represent mean ± SEM (*** p < 0.001, **** p < 0.0001, NS: not significant; Student’s t-test). The value in parentheses indicates the number of hippocampal slices (left) and the number of mice (right) used in each experiment. Scale bar: 10 ms, 0.5 mV.
Table 1.
Contributions of mitochondrial and ER Ca 2+ to PTP induction in the MF and SC synapses.
Fig 3.
Inhibition of mitochondrial Ca2+ release reduces PTP at SC synapses by disynaptic MF stimulation.
(A) A Schematic representation of a hippocampus showing the electrode location for stimulating (Sti.) MF synaptic input and recording fEPSPs (Rec.) in the stratum radiatum. (B & C) The magnitude of PTP induced by HFS is substantially less at SC synapses after 15 min treatment of CGP37157 (20 μM). The insets represent traces recorded before and immediately after HFS. Scale bar: 15 ms, 0.5 mV. DCG-4 (2 μM) was applied at the end of each experiment to confirm that MFs were stimulated. (D) Bar graphs show the mean magnitude of disynaptic PTP inductions before and after CGP37157 application (Control: 230.4 ± 8.7%; +CGP: 171.7 ± 5.8%; p < 0.0001, paired t-test). (E) The effects of CGP37157 treatment on basal transmission. The magnitude of basal transmission is similar in the absence or presence of CGP37157. All data are mean ± SEM (**** p < 0.0001; Student’s t-test). The value in parentheses indicates the number of hippocampal slices (left) and the number of mice (right) used in each experiment.
Fig 4.
Depletion of ER Ca2+ differentially affects PTP at the hippocampal MF and SC synapses.
(A & B) After applying thapsigargin (TG: 2 μM) for 40 min, which depletes ER Ca2+ by irreversibly blocks SERCA activity, the magnitude of PTP by HFS is substantially decreased at MF synapses. The insets represent traces recorded before and immediately after HFS. Scale bar: 10 ms, 0.5 mV. DCG-4 (2 μM) was applied at the end of all experiments to confirm the recording of MF synapses. (C) The summary bar graph for the mean magnitude of PTP induction in the absence or presence of TG at MF synapses (Control: 216.7 ± 13.4%; +TG: 149.8 ± 8.4%; p = 0.00045, paired t-test). (D) Effects of TG treatment on basal transmission. Treatment of TG for 40 min has no effect on the fEPSP slope at MF synapses. (E & F) Similarly, mean value of time-dependent changes in the fEPSP slope was recorded in the absence (E) or presence (F) of TG at hippocampal SC synapses. Note that TG treatment has no effect on PTP induction. (G) The summary bar graph for the mean magnitude of PTP induction in the presence of TG at SC synapses (Control: 232.5 ± 9.9%; +TG: 218.7 ± 8.8%; p = 0.34, paired t-test). (H) Effects of TG treatment on basal transmission. Treatment of TG for 40 min has no effect on the fEPSP slope at SC synapses. All data are mean ± SEM (*** p < 0.001, NS: not significant; Student’s t-test). The value in parentheses indicates the number of hippocampal slices (left) and the number of mice (right) used in each experiment.
Fig 5.
Inhibition of mitochondrial Ca2+ release selectively reduces synaptic facilitation at MF synapses.
(A) Representative EPSCs recorded before and after treatment of CGP37157 (20 μM) on frequency facilitation elicited by 20 Hz stimulus train at MF synapses. Scale bar: 100 ms, 100 pA. (B) Inhibition of mitochondrial Ca2+ release by CGP37157 significantly reduces synaptic facilitation at MF synapses (F1, 16 = 10.23, p = 0.0056; two-way ANOVA). (C) Summary graph of the mean magnitude of the 10th stimulus train of frequency facilitation at MF synapses. The inhibition of mitochondrial Ca2+ release by CGP37157 significantly reduces the mean magnitude (157.9 ± 6.0%), relative to the control (292.4 ± 30.1%, p = 0.0008; paired t-test). (D) Representative EPSCs recorded before and after treatment of CGP37157 (20 μM) on frequency facilitation elicited by 20 Hz stimulus train at SC synapses. Scale bar: 100 ms, 100 pA. (E) Inhibition of mitochondrial Ca2+ release by CGP37157 has no effect on synaptic facilitation at SC synapses. (F) Summary graph of the mean magnitude of the 10th stimulus train of frequency facilitation at SC synapses. Inhibition of mitochondrial Ca2+ release by CGP37157 does not significantly reduce the mean magnitude (159.9 ± 10.4%), relative to the control (161.7 ± 6.8%, p = 0.776; paired t-test). All data represent mean ± SEM (** p < 0.01, *** p < 0.001, NS: not significant; two-way ANOVA for panels B and E, Student's t-test for panels C and F). The value in parentheses indicates the number of hippocampal neurons (left) and the number of mice (right) used in each experiment.