3.2.1 KAR-EPSCs of Mf-CA3 synapses are reduced in APP/PS1 mice
While AMPARs and NMDARs mediate a large part of EPSCs in CA3 PCs, KARs also play distinct roles in regulating the activity of CA3 circuits. KARs are found to be abundantly expressed in the stratum lucidum of the CA3 region, both pre- and postsynaptically at Mf synapses (Darstein et al., 2003; Ruiz, Sachidhanandam, Utvik, Coussen, & Mulle, 2005). Knockout studies have demonstrated that KARs may also play important role in hippocampus-dependent memory (Ko, Zhao, Toyoda, Qiu, &
Zhuo, 2005; Rebola et al., 2017). Moreover, there have been many pieces of evidence showing that KARs can modulate slow AHP and neuronal excitability in pyramidal neurons (Fisahn, Heinemann, & McBain, 2005; Melyan, Lancaster, & Wheal, 2004;
Melyan, Wheal, & Lancaster, 2002; Ruiz et al., 2005). Therefore, we hypothesized that there may be dysfunction of KARs at Mf-CA3 synapses in APP/PS1 mice.
We first recorded evoked Mf-EPSCs from CA3 PCs in the presence of 20µM D-APV, 10µM Bicuculline, 3µM CGP 55845 at 0.1Hz, 1Hz and 3Hz respectively. These Mf-EPSCs corresponded to conductance of AMPA and Kainate receptors (noted as AMPAR+KAR EPSCs). We then added additional 25µM LY303070 to isolate the KAR component of Mf-EPSCs during 0.1Hz, 1Hz, and 3Hz of Mf stimulation. Minimal stimulation was achieved, and the failure rate and AMPAR-EPSC amplitude at 0.1Hz was compared between genotypes to ensure that comparable strength of stimulation was used (For failure rates of AMPAR-EPSCs at 0.1 Hz, WT 13.05 ± 3.54%, n=13;
APP/PS1 19.06 ± 6.41%, n=12; Unpaired t-test p=0.4110; For amplitudes of AMPAR-EPSCs at 0.1 Hz, WT 64.46 ± 8.86pA, n=13; APP/PS1 55.91 ± 9.41pA, n=12; Unpaired t-test p=0.5143; Figure 3.1). The AMPAR-EPSCs were comparable between the two groups (At 1Hz, WT 219.0 ± 20.25pA, n=13; APP/PS1 mice 197.8 ± 33.93pA, n=12;
t-test p=0.5901; At 3Hz, WT 298.0 ± 21.23pA, n=13; APP/PS1 mice 266.9 ± 41.72pA, n=12; t-test p=0.5032; Figure 3.1).
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We observed that the KAR-EPSCs were significantly reduced, and the KAR/AMPAR ratio was significantly decreased in APP/PS1 mice (At 1Hz, WT 0.058
± 0.005, n=13; APP/PS1 0.032 ± 0.004, n=12; t-test p=0.0003; At 3Hz, WT 0.054 ± 0.004, n=13; APP/PS1 0.030 ± 0.004, n=12; t-test p=0.0005; Figure 3.1). The KAR-EPSCs at 0.1Hz were not shown due to their very small amplitudes and corresponding large errors.
Figure 3.1. KAR-mediated EPSCs at Mf-CA3 synapses are significantly reduced in 6-month APP/PS1 mice.
A. Representative traces illustrating evoked Mf-EPSCs of AMPAR and KAR during 1Hz stimulation, and was an average of 30 sweeps. 20µM D-APV, 10µM Bicuculline, 3µM CGP 55845 was present through the experiment to isolate AMPAR+KAR currents, and for KAR response recording, additional 25µM LY303070 was present. In the graph, black color was used for WT and red for APP/PS1 mice. B. Dot plot displaying the ratio of KAR-EPSCs amplitudes over AMPAR-EPSCs amplitudes at 1Hz, and there was a significant reduction in mean KAR/AMPAR ratio in the APP/PS1 mice comparing to WT littermates. Black dots represent WT (0.058 ± 0.005, n=13, 9 mice) and red dots represent APP/PS1 (0.032 ± 0.004, n=12, 6 mice), t-test p=0.0003. *** represents p<0.001. C. Box plot displaying the amplitude of AMPAR-EPSCs at 1Hz observed in Mf-CA3 synapses in WT (219.0 ± 20.25pA, n=13, 9 mice) and APP/PS1 mice (197.8
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± 33.93pA, n=12, 6 mice). t-test p=0.5901. D. Representative traces illustrating evoked Mf-EPSCs of AMPAR and KAR during 3Hz stimulation, and was an average of 30 sweeps. In the graph, black color was used for WT and red for APP/PS1 mice. E. Dot plot displaying the KAR/AMPAR ratio at 3 Hz, and there was a significant reduction in mean KAR/AMPAR ratio in the APP/PS1 mice comparing to WT littermates. Black dots represent WT (0.054 ± 0.004, n=13, 9 mice) and red dots represent APP/PS1 (0.030
± 0.004, n=12, 6 mice), t-test p=0.0005. *** represents p<0.001. F. Box plot displaying the amplitude of AMPAR-EPSCs at 3 Hz observed in Mf-CA3 synapses in WT (298.0
± 21.23pA, n=13, 9 mice) and APP/PS1 mice (266.9 ± 41.72pA, n=12, 6 mice).
Unpaired t-test p=0.5032. G. Box plot illustrating comparable failure rates of AMPAR-EPSCs at 0.1 Hz between WT (13.05 ± 3.54%, n=13) and APP/PS1 mice (19.06 ± 6.41%, n=12). Unpaired t-test p=0.4110. H. Box plot illustrating comparable amplitudes of AMPAR-EPSCs at 0.1 Hz between WT (64.46 ± 8.86pA, n=13) and APP/PS1 mice (55.91 ± 9.41pA, n=12). Unpaired t-test p=0.5143.
These results suggest that there are less functional KARs at the postsynaptic sites of Mf-CA3 synapses. However, whether the number/functional state of extrasynaptic KARs is affected or not is unclear.
3.2.2 KAR-EPSCs of Mf-CA3 synapses is also reduced in PS KO mice
Previous work of our team has demonstrated thatN-Cadherin (NCad) can recruit and stabilize KARs by interacting with the GluK2a C-terminal domain, and overexpression of a dominant-negative form of NCad or knockdown of NCad in CA3 PCs could lead to a strong reduction in the amplitude of KAR-EPSCs (Fievre et al., 2016). We suspect that similar mechanism is responsible for the reduction of KAR-EPSCs in APP/PS1 mice. APP/PS1 transgenic mice express a mutated form of Presenilin (i.e. PSEN1 delta E9), which is the catalytic component of the γ-secretase complex. Mutated PS1 could induce loss of function of γ-secretase, potentially leading to accumulation of NCad lacking the extracellular fragment at the cell membrane.
Therefore, we hypothesized that this mutated PS1 gene in APP/PS1 mice could cause accumulation of truncated NCad at the membrane and thus impair the presence or function of KARs at Mf-CA3 synapses. To investigate this question, we took advantage of existing mouse lines at the local institute and generated conditional PS KO mice by injecting 500nl of AAV2.9 Syn Cre-GFP in hippocampal CA3 region of one hemisphere of PS1-floxed/PS2 KO mice. We patched the GFP-positive CA3 PCs
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as the PS KO cells, and non-fluorescent neurons from the other hemisphere were used as controls.
KAR and AMPAR-mediated EPSC recordings were performed as previously described in Chapter 3.3.5. Again, we observed an significant reduction of the KAR/AMPAR ratio in APP/PS1 mice (At 1Hz, control 0.030 ± 0.005, n=9; PSKO 0.014
± 0.005, n=10; t-test p=0.0447; At 3Hz, control 0.027 ± 0.005, n=9; PSKO 0.009 ± 0.004, n=10; t-test p=0.0056; Figure 3.2), while the AMPAR-EPSCs were comparable (At 1Hz, control 227.6 ± 24.85pA, n=9; PSKO 178.8 ± 16.27pA, n=10; t-test p=0.1121;
At 3Hz, control 317.5 ± 26.06pA, n=9; PSKO 243.8 ± 24.27pA, n=10; t-test p=0.0539;
Figure 3.2). The failure rates and AMPAR-EPSC amplitudes at 0.1Hz were also comparable between genotypes (For failure rates of AMPAR-EPSCs at 0.1 Hz, control 13.05 ± 3.54%, n=9; PSKO 19.06 ± 6.41%, n=10; t-test p=0. 6673; For amplitudes of AMPAR-EPSCs at 0.1 Hz, control 65.67 ± 15.18pA, n=9; PSKO 74.07 ± 12.07pA, n=10; t-test p=0.5143; Figure 3.2).
Figure 3.2. KAR-mediated EPSCs at Mf-CA3 synapses are significantly reduced in PSKO mice.
A. Representative traces illustrating evoked Mf-EPSCs mediated by AMPARs and KARs at 1Hz stimulation, average of 30 sweeps. 20µM D-APV, 10µM Bicuculline,
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3µM CGP 55845 was present throughout the experiment to isolate AMPAR+KAR currents. For KAR-EPSCs recording, 25µM LY303070 was added to the aCSF. In the graph, black color was used for control and blue for PSKO mice. B. Dot plot displaying the ratio of KAR-EPSCs amplitudes over AMPAR-EPSCs amplitudes at 1Hz. There was a significant reduction in mean KAR/AMPAR ratio in PSKO mice compared to controls. Black dots represent control (0.030 ± 0.005, n=9, 9 mice) and blue dots represent PSKO (0.014 ± 0.005, n=10, 9 mice), t-test p=0.0447. * represents p<0.05.
C. Box plot displaying the amplitude of AMPAR-EPSCs at 1Hz observed in Mf-CA3 synapses in control (227.6 ± 24.85pA, n=9, 9 mice) and PSKO mice (178.8 ± 16.27pA, n=10, 9 mice). t-test p=0.1121. D. Representative traces illustrating evoked Mf-EPSCs mediated by AMPARs and KARs during 3Hz stimulation, average of 30 sweeps. In the graph, black color was used for control and blue for PSKO mice. E. Dot plot displaying the KAR/AMPAR ratio at 3 Hz, indicating a significant reduction in mean KAR/AMPAR ratio in PSKO mice as compared to controls. Black dots represent control (0.027 ± 0.005, n=9, 9 mice) and blue dots represent PSKO (0.009 ± 0.004, n=10, 9 mice), t-test p=0.0056. ** represents p<0.01. F. Box plot displaying the amplitude of AMPAR-EPSCs at 3 Hz observed in Mf-CA3 synapses in control (317.5
± 26.06pA, n=9, 9 mice) and PSKO mice (243.8 ± 24.27pA, n=10, 9 mice). Unpaired t-test p=0.0539. G. Box plot illustrating comparable failure rates of Mf-EPSCs at 0.1 Hz between control (13.05 ± 3.54%, n=9) and PSKO mice (19.06 ± 6.41%, n=10).
Unpaired t-test p=0. 6673. H. Box plot illustrating comparable amplitudes of AMPAR-EPSCs at 0.1 Hz between control (65.67 ± 15.18pA, n=9) and PSKO mice (74.07 ± 12.07pA, n=10). Unpaired t-test p=0.5143. I. Epifluorescence microscopy (490nm channel) showing the sparse infection of AAV-Cre-GFP in the hippocampal CA3 region.
So far, we observed a significant reduction in KAR-EPSCs in CA3 PCs in both APP/PS1 mice and PS KO mice. This suggest that the mutated PS might be the common cause of this phenotype. To hypothesize one step further, the lack of normal NCad resulted from dysfunctional γ-secretase might underlie the dysfunction or inadequate presence of KARs at Mf-CA3 synapses.
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