Reduction of Ca_v1.3 channels in dorsal hippocampus impairs the development of dentate gyrus newborn neurons and hippocampal-dependent memory tasks

<div><p>Ca_v1.3 has been suggested to mediate hippocampal neurogenesis of adult mice and contribute to hippocampal-dependent learning and memory processes. However, the mechanism of Ca_v1.3 contribution in these processes is unclear. Here, roles of Ca_v1.3 of mouse dorsal hippocampus during newborn cell development were examined. We find that knock-out (KO) of Ca_v1.3 resulted in the reduction of survival of newborn neurons at 28 days old after mitosis. The retroviral eGFP expression showed that both dendritic complexity and the number and length of mossy fiber bouton (MFB) filopodia of newborn neurons at ≥ 14 days old were significantly reduced in KO mice. Both contextual fear conditioning (CFC) and object-location recognition tasks were impaired in recent (1 day) memory test while passive avoidance task was impaired only in remote (≥ 20 days) memory in KO mice. Results using adeno-associated virus (AAV)-mediated Ca_v1.3 knock-down (KD) or retrovirus-mediated KD in dorsal hippocampal DG area showed that the recent memory of CFC was impaired in both KD mice but the remote memory was impaired only in AAV KD mice, suggesting that Ca_v1.3 of mature neurons play important roles in both recent and remote CFC memory while Ca_v1.3 in newborn neurons is selectively involved in the recent CFC memory process. Meanwhile, AAV KD of Ca_v1.3 in ventral hippocampal area has no effect on the recent CFC memory. In conclusion, the results suggest that Ca_v1.3 in newborn neurons of dorsal hippocampus is involved in the survival of newborn neurons while mediating developments of dendritic and axonal processes of newborn cells and plays a role in the memory process differentially depending on the stage of maturation and the type of learning task.</p></div

Effects of Ca_v1.3 KO on developments of dendrites, spines and MFB filopodia of DG newborn neurons.

<p>(A) Confocal images of GFP (+) neurons at 14 and 28 days after GFP-retroviral infection. <i>Scale bar</i>, 50 μm. (B-E) Quantification of dendritic development. *, **, *** indicate <i>p</i> < 0.05, <i>p</i> < 0.01, <i>p</i> < 0.001, respectively. (B) Total number of dendritic branching points at 14 and 28 days after viral infection. (Day 14, WT, 7.64 ± 0.41, n = 62, KO, 6.55 ± 0.25, n = 102, <i>p</i> = 0.017; Day 28, WT, 5.71 ± 0.20, n = 107, KO, 4.20 ± 0.18, n = 120, <i>p</i> < 0.00001, n = 3 animals per group). Two-way ANOVA, F_G = 26.96, <i>p</i> = 0.000; F_T = 73.08, <i>p</i> = 0.000; F_G+T = 0.68, <i>p</i> = 0.001. (C) Total dendritic length measurement at 14 and 28 days after viral injection. (Day 14, WT, 328.35 ± 14.57 μm, n = 69, KO, 362.34 ± 45.06 μm, n = 100, <i>p</i> = 0.12; Day 28, WT, 552.90 ± 19.15 μm, n = 107, KO, 466.34 ± 19.97 μm, n = 119, n = 4 animals per group, <i>p</i> = 0.002). Two-way ANOVA, F_G = 1.96, <i>p</i> = 0.162; F_T = 76.56, <i>p</i> = 0.000; F_G+T = 10.31, <i>p</i> = 0.001. (D-E) Number of dendritic crossings in Sholl analysis at 14 (D) and 28 days (E) after viral infection. (Day 28: 10 μm, WT, 1.29 ± 0.07, KO, 1.13 ± 0.04, <i>p</i> = 0.022; 20 μm, WT, 1.70 ± 0.10, KO, 1.39 ± 0.07, <i>p</i> = 0.011; 30 μm, WT, 2.30 ± 0.13, KO, 1.81 ± 0.09, <i>p</i> = 0.001; 40 μm, WT, 3.13 ± 0.16, KO, 2.44 ± 0.11, <i>p</i> = 0.001; 50 μm, WT, 3.69 ± 0.18, KO, 3.03 ± 0.13, <i>p</i> = 0.004; 60 μm, WT, 3.75 ± 0.18, KO, 3.15 ± 0.14, <i>p</i> = 0.010; 70 μm, WT, 3.73 ± 0.19, KO, 3.21 ± 0.15, <i>p</i> = 0.025; 80 μm, WT, 3.65 ± 0.16, KO, 2.98 ± 0.14, <i>p</i> = 0.005; 90 μm, WT, 3.49 ± 0.15, KO, 2.96 ± 0.15, <i>p</i> = 0.013; WT, n = 107 cells, KO, n = 122 cells, n = 4 animals per group). Two-way ANOVA, F_G = 10.54, <i>p</i> = 0.001; F_T = 27.18, <i>p</i> = 0.000; F_D = 92.87, <i>p</i> = 0.000; F_G+T = 34.97, <i>p</i> = 0.000; F_G+D = 1.23, <i>p</i> = 0.27; F_T+D = 23.76, <i>p</i> = 0.000; F_G+T+D = 0.92, <i>p</i> = 0.504. (F) <i>Left</i>, representative image (60x) of newborn neurons at 28 days after GFP-retroviral infection. Red, DAPI. White rectangle shows a distal dendritic region of a newborn neuron of Ca_v1.3 WT mice for spine analysis. <i>Right</i>, exemplary high magnification (60x/6x-zoom) images (<i>top</i>) and 3D reconstruction images (<i>bottom</i>) of a distal dendritic region of a newborn neuron of WT and Ca_v1.3 KO mice. White arrows indicate stubby spines, yellow arrows indicate mushroom spines and red arrows indicate thin spines. <i>Scale bar</i>, 50 μm (60x), 5 μm (60x/6x-zoom) and 2 μm (3D image). (G) Spine density plot for each type of spines. (Thin spines, WT, 0.82 ± 0.07 spines/μm, KO, 0.83 ± 0.06 spines/μm, <i>p</i> = 0.434; stubby spines, WT, 1.10 ± 0.07 spines/μm, KO, 0.95 ± 0.05 spines/μm, <i>p</i> = 0.064; mushroom spines, WT, 0.14 ± 0.017 spines/μm, KO, 0.20 ± 0.06 spines/μm, <i>p</i> = 0.409, WT, n = 28 cells, KO, n = 29 cells, n = 2 animals per group). (H) <i>Top</i>, confocal images of CA3 region axonal fibers of newborn neurons at 28 days after GFP expressing retrovirus injection. Red, DAPI. <i>Bottom</i>, high magnification images of axonal boutons near CA3 pyramidal cell layer. White and yellow arrows indicate boutons and filopodia, respectively. <i>Insets</i>, 3D image of bouton and filopodia. <i>Scale bars</i>, 50 μm (40x), 10 μm (40x/6x-zoom), 5 μm (<i>insets</i>). (I) Size of mossy fiber boutons (WT, 11.52 ± 0.47, n = 84 boutons; KO, 10.07 ± 0.45, n = 70 boutons, n = 3 animals per group, <i>p</i> = 0.029). (J) Total number of filopodia of axonal boutons (WT, 3.98 ± 0.25, n = 53 boutons, KO, 3.2 ± 0.19, n = 65 boutons, n = 3 animals per group, <i>p</i> = 0.010) and (K) the length of filopodia of axonal boutons (WT, 25.99 ± 2.02 μm, n = 53 boutons, KO, 19.16 ± 1.29 μm, n = 65 boutons, n = 3 animals per group, <i>p</i> = 0.004).</p

Impairments of hippocampus-dependent memory tasks in Ca_v1.3 KO mice.

<p>(A) Scheme of CFC learning and memory tests. Both recent and remote CFC memories were assessed in the same chamber at Days 0, 1, 2 and 23. (B) Freezing responses of CFC memory tasks. (Day 0, WT, 0 ± 0, n = 5, KO, 0 ± 0, n = 5; Day 1, WT, 39.80 ± 3.33%, n = 15, KO, 8.39 ± 1.40%, n = 11, <i>p</i> < 0.00001; Day 2, WT, 59.59 ± 5.60%, n = 13, KO, 47.18 ± 2.74%, n = 9, <i>p</i> = 0.098; Day 23, WT, 56.75 ± 4.58%, n = 10, KO, 38.74 ± 14.30%, n = 6, p = 0.305). *, **, *** indicate <i>p</i> < 0.05, <i>p</i> < 0.01, <i>p</i> < 0.001, respectively, unless otherwise mentioned. Two-way ANOVA, F_G = 18.24, <i>p</i> = 0.000; F_T = 17.88, <i>p</i> = 0.000; F_G+T = 2.31, <i>p</i> = 0.106. (C) Scheme of PA tasks. (D) Latency of entrance to dark room of PA tasks. (Day 0, WT, 13.13 ± 2.88 s, n = 24, KO, 13.12 ± 1.75 s, n = 24, <i>p</i> = 0.990; Day 1, WT, 268.89 ± 16.18 s, n = 24, KO, 239.34 ± 20.72 s, n = 24, <i>p</i> = 0.267; Day 21, WT, 241.38 ± 28.90 s, n = 13, KO, 294.95 ± 5.05 s, n = 13, <i>p</i> = 0.080; Day 42, WT, 232.67 ± 34.10 s, n = 9, KO, 107.22 ± 27.86 s, n = 12, <i>p</i> = 0.010; Day 63, WT, 224.59 ± 34.76 s, n = 7, KO, 46.29 ± 10.71 s, n = 10, <i>p</i> = 0.000). Two-way ANOVA, F_G = 2.45, <i>p</i> = 0.12; F_T = 17.47, <i>p</i> = 0.00; F_G+T = 2.33, <i>p</i> = 0.08. (E) Schemes of OR and OLR tasks. (F) Preference index measurement of OR/OLR tasks. (OR task: WT, 76.41 ± 1.66%, n = 11, KO, 72.54 ± 4.0%, n = 9, <i>p</i> = 0.339; OLR task: WT, 55.19 ± 4.04%, n = 11, KO, 42.89 ± 4.13%, n = 9, <i>p</i> = 0.048).</p

Expression of Ca_v1.3 in adult hippocampal area.

<p>(A) Ca_v1.3 expression in dorsal hippocampal area. Ca_v1.3 is shown in red and DAPI, a nuclear maker, is shown in blue. <i>Scale bars</i>, 200 μm (10x) and 50 μm (40x). (B) Images of developmental profiling of Ca_v1.3 expression in adult hippocampal newborn neurons. Confocal images of adult hippocampal newborn neurons, infected with GFP-retrovirus and stained with Ca_v1.3 antibody (red), were taken at 3, 7, 14 and 28 days after infection. White arrows indicate newborn cells infected with retrovirus. <i>Scale bars</i>, 50μm (40x) and 10 μm (40x/6x-zoom). (C) Ca_v1.3 antibody fluorescent intensity of newborn neurons (GFP (+), filled bar) and control mature neurons (GFP (-), open bar) of dorsal hippocampus shown at (B). A.U. indicates arbitrary unit. (Day 3, GFP(+), 658.10 ± 41.58, n = 9, GFP(-), 1302.51 ± 40.98, n = 50; Day 7, GFP(+), 558.19 ± 61.26, n = 9, GFP(-), 1149.03 ± 126.35, n = 50; Day 14, GFP(+), 950.79 ± 83.09, n = 7, GFP(-), 1264.75 ± 97.98, n = 50; Day 28, GFP(+), 1217.75 ± 55.34, n = 13, GFP(-), 1470.64 ± 115.84, n = 50; <i>p</i>(Day 3) < 0.000, <i>p</i>(Day 7) = 0.000, <i>p</i>(Day 14) = 0.035, <i>p</i>(Day 28) = 0.041). Two-way ANOVA, F_G = 66.17, <i>p</i> = 0.000; F_T = 15.22, <i>p</i> = 0.000; F_G+T = 3.20, <i>p</i> = 0.031. (D) Normalized Ca_v1.3 antibody fluorescent intensity of newborn neurons to that of mature neurons. (Day 3, 49.52 ± 3.61%, n = 9; Day 7, 48.26 ± 3.08%, n = 9; Day 14, 73.42 ± 5.94%, n = 7; Day 28, 83.76 ± 3.58%, n = 13; <i>p</i>(Day 3–7) = 0.795, <i>p</i>(Day 7–14) = 0.001, <i>p</i>(Day 14–28) = 0.138). One-way ANOVA, F = 20.913, <i>p</i> = 0.000. (E) Comparison of Ca_v1.3 expression among DG, CA1 and CA3 regions of dorsal hippocampus shown at (A) (each, n = 10). (DG, 1851.50 ± 54.44, n = 10; CA1, 2072.08 ± 38.63, n = 10; CA3, 2298.10 ± 115.40, n = 10; <i>p</i>(DG-CA1) = 0.004, <i>p</i>(CA1-CA3) = 0.080, <i>p</i>(DG-CA3) = 0.003). One-way ANOVA, F = 8.42, <i>p</i> = 0.001. *, **, *** indicate <i>p</i> < 0.05, <i>p</i> < 0.01, <i>p</i> < 0.001, respectively.</p

Proliferation and survival of DG newborn cells of dorsal hippocampus in Ca_v1.3 KO mouse.

<p>(A) Confocal images of BrdU (+) cells (red) and NeuN (+) cells (green) in Ca_v1.3 KO and WT mouse. Images are acquired at 1, 14 and 28 days after BrdU injection. <i>Scale bar</i>, 50 μm. (B) Number of BrdU (+) cells. (Day 1, WT, 394.667 ± 30.78 cells, n = 8; KO, 387 ± 49.05, n = 6, <i>p</i> = 0.660; Day 14, WT, 376.6 ± 45.85 cells, n = 6; KO, 35.8 ± 13.22 cells, n = 6, <i>p</i> = 0.472; Day 28, WT, 219 ± 13.61 cells, n = 7; KO, 159.83 ± 23.70 cells, n = 6, <i>p</i> = 0.046). * indicates <i>p</i> < 0.05. Two-way ANOVA, F_G = 3.80, <i>p</i> = 0.061; F_T = 59.12, <i>p</i> = 0.000; F_G+T = 0.84, <i>p</i> = 0.444. (C) Number of BrdU (+) cells of KO mice normalized to that of WT mice at given day. (Day 1, WT, 100 ± 7.80%, n = 8, KO, 98.06 ± 12.43%, n = 10; Day 14, WT, 100 ± 12.17%, n = 6, KO, 93.95 ± 3.50%, n = 6; Day 28, WT, 100 ± 6.73%, n = 7, KO, 72.98 ± 11.85%, n = 6, <i>p</i> = 0.046). Two-way ANOVA, F_G = 4.61, <i>p</i> = 0.040; F_T = 1.82, <i>p</i> = 0.179; F_G+T = 1.90, <i>p</i> = 0.168. (D) Number of BrdU (+) cells per DG area at Day 28. (WT, 21.24 ± 1.22 cells/mm², n = 7; KO, 15.47 ± 2.12, n = 6, <i>p</i> = 0.032). (E) <i>Left</i>, example images for area measurements of DG (white dot line) and GCL (yellow line). <i>Right</i>, NeuN (+) cells (green) of DG in Ca_v1.3 KO and WT mice. <i>Scale bars</i>, 100 um (10x), 50 μm (40x), 10 μm (<i>insets</i>, 40x/5x-zoom). (F) DG area (WT, 9.92 ± 0.19 mm², n = 6, KO, 9.58 ± 0.18 mm², n = 6, <i>p</i> = 0.833), (G) GCL area (WT, 1.85 ± 0.063 mm², n = 6, KO, 1.91 ± 0.05 mm², n = 7, <i>p</i> = 0.445) and (H) Density of NeuN (+) cells in GCL (WT, 6071 ± 691.88 cells/mm², n = 11, KO, 6304.71 ± 339.34 cells/mm², n = 12, <i>p</i> = 0.897).</p

Effects of AAV- and retrovial-Ca_v1.3 KD on both recent and remote memories of CFC.

<p>(A) Experimental scheme of recent and remote memory tests of CFC using AAV mediated Ca_v1.3 KD in dorsal hippocampus. (B) <i>Top</i>, Representative images of GFP expression of AAV-Ca_v1.3 KD cells in dorsal hippocampus. <i>Scale bars</i>, 500 μm and 200 μm (<i>insets</i>). <i>Bottom</i>, representative images of expression of GFP (+) AAV-Ca_v1.3 KD control into the dorsal hippocampus of F1 mouse at 2 week of infection. (C) Freezing responses in AAV-Ca_v1.3 KD and control mice. (Day 0, Control, 0 ± 0, n = 5, KD, 0 ± 0, n = 5; Day 1, Control, 27.25 ± 3.57%, n = 19, KD, 11.31 ± 3.55%, n = 17, <i>p</i> = 0.003; Day 2, Control, 53.74 ± 5.58%, n = 8, KD, 57.13 ± 6.65%, n = 7, <i>p</i> = 0.697; Day 23, Control, 55.96 ± 5.20%, n = 8, KD, 18.00 ± 8.11%, n = 7, <i>p</i> = 0.001). ** indicates <i>p</i> < 0.01. (D) Experimental scheme of recent and remote memory tests of CFC using retrovirus mediated Ca_v1.3 KD in dorsal hippocampus. Two-way ANOVA, F_G = 15.09, <i>p</i> = 0.000; F_T = 27.49, <i>p</i> = 0.000; F_G+T = 6.12, <i>p</i> = 0.004. (E) <i>Top</i>, Representative images of GFP expression of retroviral-Ca_v1.3 KD cells in DG of dorsal hippocampus at 28 days after infection. <i>Scale bars</i>, 200μm and 50μm (<i>insets</i>). <i>Bottom</i>, representative images of expression of GFP (+) retrovirus-Ca_v1.3 KD control into the dorsal hippocampus of F1 mouse at 4 week of infection. (F) Freezing responses in retroviral-Ca_v1.3 KD and control mice. (Day 0, Control, 0 ± 0, n = 5, KD, 0 ± 0, n = 5; Day 1, Control, 24.46 ± 2.63%, n = 14, KD, 12.80 ± 1.88%, n = 15, <i>p</i> = 0.003; Day 2, Control, 58.40 ± 4.54%, n = 14, KD, 52.33 ± 3.96%, n = 15, <i>p</i> = 0.321; Day 23, Control, 60.66 ± 7.09%, n = 9, KD, 52.78 ± 6.00%, n = 9, <i>p</i> = 0.409). Two-way ANOVA, F_G = 4.33, <i>p</i> = 0.041; F_T = 32.07, <i>p</i> = 0.000; F_G+T = 0.14, <i>p</i> = 0.866.</p