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

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

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

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

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

Novel Platform of Cardiomyocyte Culture and Coculture via Fibroblast-Derived Matrix-Coupled Aligned Electrospun Nanofiber

For cardiac tissue engineering, much attention has been given to the artificial cardiac microenvironment in which anisotropic design of scaffold and extracellular matrix (ECM) are the major cues. Here we propose poly­(l-lactide-<i>co</i>-caprolactone) and fibroblast-derived ECM (PLCL/FDM), a hybrid scaffold that combines aligned electrospun PLCL fibers and FDM. Fibroblasts were grown on the PLCL fibers for 5–7 days and subsequently decellularized to produce PLCL/FDM. Various analyses confirmed aligned, FDM-deposited PLCL fibers. Compared to fibronectin (FN)-coated electrospun PLCL fibers (control), H9c2 cardiomyoblast differentiation was significantly effective, and neonatal rat cardiomyocyte (CM) phenotype and maturation was improved on PLCL/FDM. Moreover, a coculture platform was created using multilayer PLCL/FDM in which two different cells make indirect or direct cell–cell contacts. Such coculture platforms demonstrate their feasibility in terms of higher cell viability, efficiency of target cell harvest (>95% in noncontact; 85% in contact mode), and molecular diffusion through the PLCL/FDM layer. Coculture of primary CMs and fibroblasts exhibited much better CM phenotype and improvement of CM maturity upon either direct or indirect interactions, compared to the conventional coculture systems (transwell insert and tissue culture plate (TCP)). Taken together, our platform should be very useful and have significant contributions in investigating some scientific or practical issues of crosstalks between multiple cell types

Inositol 1,4,5-trisphosphate 3-kinase A overexpressed in mouse forebrain modulates synaptic transmission and mGluR-LTD of CA1 pyramidal neurons

<div><p>Inositol 1,4,5-trisphosphate 3-kinase A (IP₃K-A) regulates the level of the inositol polyphosphates, inositol trisphosphate (IP₃) and inositol tetrakisphosphate to modulate cellular signaling and intracellular calcium homeostasis in the central nervous system. IP₃K-A binds to F-actin in an activity-dependent manner and accumulates in dendritic spines, where it is involved in the regulation of synaptic plasticity. IP₃K-A knockout mice exhibit deficits in some forms of hippocampus-dependent learning and synaptic plasticity, such as long-term potentiation in the dentate gyrus synapses of the hippocampus. In the present study, to further elucidate the role of IP₃K-A in the brain, we developed a transgenic (Tg) mouse line in which IP₃K-A is conditionally overexpressed approximately 3-fold in the excitatory neurons of forebrain regions, including the hippocampus. The Tg mice showed an increase in both presynaptic release probability of evoked responses, along with bigger synaptic vesicle pools, and miniature excitatory postsynaptic current amplitude, although the spine density or the expression levels of the postsynaptic density-related proteins NR2B, synaptotagmin 1, and PSD-95 were not affected. Hippocampal-dependent learning and memory tasks, including novel object recognition and radial arm maze tasks, were partially impaired in Tg mice. Furthermore, (R,S)-3,5-dihydroxyphenylglycine-induced metabotropic glutamate receptor long-term depression was inhibited in Tg mice and this inhibition was dependent on protein kinase C but not on the IP₃ receptor. Long-term potentiation and depression dependent on N-methyl-d-aspartate receptor were marginally affected in Tg mice. In summary, this study shows that overexpressed IP₃K-A plays a role in some forms of hippocampus-dependent learning and memory tasks as well as in synaptic transmission and plasticity by regulating both presynaptic and postsynaptic functions.</p></div

Basal physiological properties of hippocampal CA1 synapses in IP₃K-A Tg young mice.

<p>(A), Input-output relationship of basal evoked synaptic transmission. Left, the field EPSP slopes were measured in response to six-step incremental stimulus current intensity per hippocampal slice. Scale bars: 0.5 mV, 5msec. Right, linear regression (LR) was performed on the slope values from each slice in the left I-O plot. Mean ± SEM. (WT, 1.92 ± 0.05, n = 86 slices, 19 mice; Tg, 2.10 ± 0.06, n = 97, 18; *<i>p</i> < 0.05). (B), Paired-pulse ratio of fEPSP responses. The ratio of the 2^nd fEPSP slope (R2) to the 1^st fEPSP slope (R1) was measured at the CA3-CA1 synapse of the hippocampal slice. WT, n = 85 slices, 19 mice; Tg, n = 94, 18; *<i>p</i> < 0.05 at the five intervals measured. Scale bars: 0.5 mV, 30 msec. (C-G), mEPSC properties. (C), Scale bars for all sample traces: 50 μA, 50 msec. (D), Measurements of amplitude. Mean ± SEM. (WT, 13.5 ± 0.2 pA; Tg, 15.1 ± 0.4 pA; **<i>p</i> < 0.001). (E), Frequency (WT, 1.87 ± 0.21 Hz; Tg, 1.91 ± 0.36 Hz; <i>p</i> = 0.92). (F), Rise time (WT, 1.66 ± 0.02 msec; Tg, 1.65 ± 0.03 msec; <i>p</i> = 0.74). (G), Decay time constant (WT, 2.36 ± 0.03 msec; Tg, 2.39 ± 0.04 msec; <i>p</i> = 0.57). The mEPSCs were measured from WT (n = 24 cells, 4 mice) and Tg mice (n = 22, 3).</p

Subcellular localization of IP₃K-A and increased number of synaptic vesicles in IP₃K-A Tg mice.

<p>(A), Distribution pattern of IP₃K-A in the adult mouse brain subcellular fractions. PSD-95 and synaptophysin (SynPhy) were used as controls. H, homogenates; P1, nuclei and large debris; P2, crude synaptosomes; S2, supernatant after P2 precipitation; S3, cytosol; P3, light membranes; LP1, synaptosomal membranes; LS2, synaptosomal cytosol; LP2, synaptic vesicle-enriched fraction. (B), Representative EM images of hippocampal CA1 synapses in WT and Tg mice. Scale bar: 100 mm. (C), Quantification of the number of synaptic vesicles per area (WT, n = 60 synapses, 3 mice; Tg, n = 60, 3). Presynaptic terminals with well-defined postsynaptic density (arrows) were chosen for analysis. Dashed lines indicated the presynaptic area for the analysis of synaptic vesicle (arrowheads) density per area.</p