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, 12 (1), 48

Presenilins Regulate Synaptic Plasticity and Mitochondrial Calcium Homeostasis in the Hippocampal Mossy Fiber Pathway

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Presenilins Regulate Synaptic Plasticity and Mitochondrial Calcium Homeostasis in the Hippocampal Mossy Fiber Pathway

Sang Hun Lee et al. Mol Neurodegener.

Abstract

Background: Presenilins play a major role in the pathogenesis of Alzheimer's disease, in which the hippocampus is particularly vulnerable. Previous studies of Presenilin function in the synapse, however, focused exclusively on the hippocampal Schaffer collateral (SC) pathway. Whether Presenilins play similar or distinct roles in other hippocampal synapses is unknown.

Methods: To investigate the role of Presenilins at mossy fiber (MF) synapses we performed field and whole-cell electrophysiological recordings and Ca2+ imaging using acute hippocampal slices of postnatal forebrain-restricted Presenilin conditional double knockout (PS cDKO) and control mice at 2 months of age. We also performed quantitative electron microscopy (EM) analysis to determine whether mitochondrial content is affected at presynaptic MF boutons of PS cDKO mice. We further conducted behavioral analysis to assess spatial learning and memory of PS cDKO and control mice at 2 months in the Morris water maze.

Results: We found that long-term potentiation and short-term plasticity, such as paired-pulse and frequency facilitation, are impaired at MF synapses of PS cDKO mice. Moreover, post-tetanic potentiation (PTP), another form of short-term plasticity, is also impaired at MF synapses of PS cDKO mice. Furthermore, blockade of mitochondrial Ca2+ efflux mimics and occludes the PTP deficits at MF synapses of PS cDKO mice, suggesting that mitochondrial Ca2+ homeostasis is impaired in the absence of PS. Quantitative EM analysis showed normal number and area of mitochondria at presynaptic MF boutons of PS cDKO mice, indicating unchanged mitochondrial content. Ca2+ imaging of dentate gyrus granule neurons further revealed that cytosolic Ca2+ increases induced by tetanic stimulation are reduced in PS cDKO granule neurons in acute hippocampal slices, and that inhibition of mitochondrial Ca2+ release during high frequency stimulation mimics and occludes the Ca2+ defects observed in PS cDKO neurons. Consistent with synaptic plasticity impairment observed at MF and SC synapses in acute PS cDKO hippocampal slices, PS cDKO mice exhibit profound spatial learning and memory deficits in the Morris water maze.

Conclusions: Our findings demonstrate the importance of PS in the regulation of synaptic plasticity and mitochondrial Ca2+ homeostasis in the hippocampal MF pathway.

Keywords: Calcium; Mitochondria; Mossy fiber; Presenilin; Synaptic plasticity.

Figures

Fig. 1
Fig. 1
Impaired long-term potentiation at hippocampal MF synapses in PS cDKO mice. a Normal synaptic transmission in PS cDKO mice at 2 months of age. The synaptic input–output relationship was obtained by plotting the fiber volley (FV) amplitude against the initial slope of the evoked fEPSP. Each point represents data averaged across all slices for a narrow bin of FV amplitude. Right panel shows representative traces of fEPSPs evoked by various stimulation intensities. b Impaired LTP induced by 5 TBS in PS cDKO mice. Superimposed traces are averages of four consecutive responses 1 min before (thin line) and 50 min after (thick line) TBS induction. DCG IV (2 μM) was applied at the end of all experiments to confirm the recording of MF synapses. All data represent means ± SEM. The number of hippocampal slices (left) and mice (right) used in each experiment is indicated in parentheses
Fig. 2
Fig. 2
Impaired short-term plasticity at hippocampal MF synapses in PS cDKO mice. a Representative traces of fEPSPs evoked by two consecutive stimuli with a 60 ms inter-stimulus interval. b Average PPF plotted as a function of the inter-stimulus intervals (20-2000 ms) shows reduced PPF in PS cDKO mice (F1, 17 = 14.71; p = 0.0013; two-way ANOVA). c Synaptic facilitation elicited by stimulus trains is impaired in a frequency-dependent manner in PS cDKO mice (1 Hz: F1, 13 = 11.64, p = 0.005; 5 Hz: F1, 13 = 13.33, p = 0.003; 10 Hz: F1, 15 = 9.67, p = 0.008; 20 Hz: F1, 15 = 9.71, p = 0.008; two-way ANOVA). fEPSP slopes shown are normalized to the slope of the first fEPSP of the stimulus train. All data represent means ± SEM (** p < 0.01; two-way ANOVA). The number of hippocampal slices (left) and mice (right) used in each experiment is indicated in parentheses
Fig. 3
Fig. 3
Impaired PTP at hippocampal MF synapses in PS cDKO mice. a & b Representative data showing the time course of PTP and the effect of CGP37157 (20 μM) on PTP of EPSCs obtained by whole-cell patch recording at the hippocampal MF synapses in control and PS cDKO mice. DCG IV (2 μM) was applied at the end of each experiment to confirm that MF synapses were recorded. The insets represent EPSC traces recorded the baseline (1) and immediately after HFS induction (2 & 3). Scale bar: 20 ms, 200 pA. c Summary bar graphs of the mean magnitude of PTP in the course of whole-cell patch recording in control and PS cDKO mice. PTP is impaired at MF synapses in PS cDKO mice. d Depicted EPSC trace for the estimation of latency, 10–90% rise time and decay time constant (τ). e Histogram of latency between stimulus onset and EPSC response was analyzed during 1 min baseline recordings of EPSC (n = 72 in 6 PTP recordings) in both control and PS cDKO mice. Latencies ranged from 1.6 to 2.5 ms with means of 1.93 ± 0.03 and 2.01 ± 0.03 ms in the control and PS cDKO mice, respectively. f The 10–90% rise time for control EPSCs (2.27 ± 0.06 ms range from 1.19 to 3.13 ms, n = 72) and PS cDKO EPSCs (2.29 ± 0.05 msec range from 1.38 to 3.14 ms, n = 72) is shown. g Relationship of 10–90% decay time constant (τ) and Δ amplitude of EPSCs. The distribution was well fitted with linear function (Black dots for control: y = 0.019 x + 6.288, n = 72; red dots for PS cDKO: y = 0.016 x + 7.005, n = 72). All data represent means ± SEM (** p < 0.01, NS: not significant; Student’s t-test). The values in parentheses indicate the number of hippocampal neurons (left) and the number of mice (right) used in each experiment
Fig. 4
Fig. 4
Normal mitochondrial content at presynaptic MF boutons of PS cDKO mice. a Transmission electron micrographs of hippocampal stratum lucidum in area CA3 show large presynaptic mossy fiber boutons (MFB: highlighted in yellow) establishing synaptic contacts with large complex spines (asterisks) of CA3 pyramidal neuron dendrites (D). The arrowheads indicate mitochondria in the MFB. Scale bars: 1 μm. The randomized samples were obtained from at least 10 micrographs from each of the 4 mice per genotype. b-d Quantification of the number of mitochondria per bouton (control: 10.02 ± 0.33, PS cDKO: 9.51 ± 0.33, n = 59 boutons), total mitochondrial area per bouton (control: 0.42 ± 0.04 μm2, n = 44; PS cDKO: 0.49 ± 0.04 μm2, n = 42), and bouton area (control: 6.04 ± 0.45 μm2, n = 59; PS cDKO: 6.41 ± 0.58 μm2, n = 42). The data are presented as means ± SEM (NS: not significant, Student’s t-test)
Fig. 5
Fig. 5
Reduced cytosolic Ca2+ increases induced by repetitive stimulation in hippocampal DG granule neurons of PS cDKO mice. a (Left) DIC image of DG granule neurons in an acute hippocampal slice in the course of whole-cell recording. (Right) Fluorescence image obtained from a soma of DG granule neurons loaded with 100 μM Fura 2 via a whole-cell patch pipette. Scale bars: 20 μm. b-d Electrophysiological characteristics of mature DG granule neurons: mean values for resting membrane potential (Control: −78.25 ± 0.94 mV; PS cDKO: −77.83 ± 0.41 mV), threshold currents for triggering of action potential (Control: 125.7 ± 12.3 pA; PS cDKO: 111.4 ± 11.9 pA) and input resistance (Control: 200.4 ± 16.5 MΩ; PS cDKO: 211.8 ± 13.4 MΩ). e Resting Ca2+ level (Control: 62.1 ± 4.1 nM; PS cDKO: 82.9 ± 7.4 nM) is not significantly different between control and PS cDKO mice. f The amplitude of ∆[Ca2+]i elicited by 10 repetitive stimulation (depolarizing pulses of 2 ms duration; from −80 to 0 mV) at 5, 10, 20 Hz is reduced in PS cDKO granule neurons. g Representative Ca2+ transients evoked by 10 repetitive stimulation at various frequencies (1, 5, 10, and 20 Hz). Scale bars: 2 s, 100 nM. All data represent means ± SEM (* p < 0.05, ** p < 0.01, NS: not significant; Student’s t-test). The values in parentheses indicate the number of neurons (left) and the number of mice (right) used in each experiment
Fig. 6
Fig. 6
Inhibition of mitochondrial Ca2+ release mimics and occludes deficits of mitochondrial Ca2+ homeostasis in hippocampal DG granule neurons of PS cDKO mice. a Representative Ca2+ transients evoked by 10 repetitive stimulation (depolarizing pulses of 2 ms duration; from −80 to 0 mV) at 20 Hz recorded in the absence or presence of CGP37157. Scale bar: 2 s, 100 nM. b Summary bar graph of the amplitude of ∆[Ca2+]i shows significant reduction in PS cDKO granule neurons (250.7 ± 19.7 nM, unpaired t-test, p < 0.001) and control granule neurons treated with CGP37157 (289.2 ± 12.1 nM, paired t-test, p < 0.001), relative to untreated control neurons (360.1 ± 11.4 nM). Bar graphs represent means ± SEM (*** p < 0.001; Student’s t-test, NS: not significant). c The amplitude of ∆[Ca2+]i elicited by PTP inducing stimulation (16 pulses at 100 Hz, 4 times delivered at 0.33 Hz; from −80 to 0 mV) is significantly reduced in PS cDKO granule neurons and control granule neurons treated with CGP37157, relative to untreated control neurons (control vs PS cDKO: F1, 15 = 12.56, p = 0.0029, control vs control + CGP: F1, 14 = 28.46, p = 0.0001; two-way ANOVA). CGP37157 treatment does not significantly reduce ∆[Ca2+]i in PS cDKO neurons (F1, 16 = 0.76, p = 0.40; two-way ANOVA). The insets are averaged Ca2+ transients. The values in parentheses indicate the number of neurons (left) and the number of mice (right) used in each experiment
Fig. 7
Fig. 7
Impaired spatial learning and memory in PS cDKO mice at 2 months of age. a Escape latency of PS cDKO mice (n = 6) and controls (n = 6) gradually decreases during 13 days of training in the hidden platform water maze task, and the latency is significantly higher in PS cDKO mice (F1, 10 = 22.69, p < 0.001; two-way ANOVA). Path length is also gradually decreased for both PS cDKO and control mice during training, and the path length of PS cDKO mice is significantly longer, relative to control mice (F1, 10 = 9.62, p < 0.05; two-way ANOVA). b Two post-training probe trials were performed on days 7 and 13. Both PS cDKO and control mice show similar target quadrant occupancy on day 7, but PS cDKO mice show significantly reduced target quadrant occupancy on day 13 (p < 0.01; Student’s t-test). Post-hoc power analysis showed that 6 mice results in 99%, 99% and 98% power for latency, path length, and quadrant occupancies, respectively, at day 13. AR: adjacent right quadrant, T: target quadrant, AL: adjacent left quadrant, OP: opposite quadrant. All data are means ± SEM. The asterisks denote statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001)

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