Spine Formation Pattern of Adult-Born Neurons Is Differentially Modulated by the Induction Timing and Location of Hippocampal Plasticity

<div><p>In the adult hippocampus dentate gyrus (DG), newly born neurons are functionally integrated into existing circuits and play important roles in hippocampus-dependent memory. However, it remains unclear how neural plasticity regulates the integration pattern of new neurons into preexisting circuits. Because dendritic spines are major postsynaptic sites for excitatory inputs, spines of new neurons were visualized by retrovirus-mediated labeling to evaluate integration. Long-term potentiation (LTP) was induced at 12, 16, or 21 days postinfection (dpi), at which time new neurons have no, few, or many spines, respectively. The spine expression patterns were investigated at one or two weeks after LTP induction. Induction at 12 dpi increased later spinogenesis, although the new neurons at 12 dpi didn’t respond to the stimulus for LTP induction. Induction at 21 dpi transiently mediated spine enlargement. Surprisingly, LTP induction at 16 dpi reduced the spine density of new neurons. All LTP-mediated changes specifically appeared within the LTP–induced layer. Therefore, neural plasticity differentially regulates the integration of new neurons into the activated circuit, dependent on their developmental stage. Consequently, new neurons at different developmental stages may play distinct roles in processing the acquired information by modulating the connectivity of activated circuits via their integration.</p> </div

MML-LTP induction at 21 dpi mediates only spine enlargement of new neurons specifically in MML.

<p>(<b>A</b>) Experimental schedules for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045270#pone-0045270-g004" target="_blank">Figure 4</a>. Insets are samples of evoked field potential traces that are recorded at pre-HFS, 1 day, and 7 days post-HFS. (<b>B</b>) PS amplitudes of the DG obtained from rats used for experiments in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045270#pone-0045270-g004" target="_blank">Figure 4</a>. Pre- and post-HFS delivery are indicated by “pre” and “post”, respectively. (<b>C</b>) Fluorescence micrographs of F-actin signal (phalloidin-TRITC, red) in control (<b>C1</b>) and LTP-induced DG (<b>C2</b>) of 28-dpi rats. Nuclear signal is shown in blue (DRAQ5). (<b>D</b>) Synaptophysin (red) and nucleus (DRAQ5, blue) signals of control (<b>D1</b>) and LTP-induced hemispheres (<b>D2</b>). Scale bars for (<b>C</b>) and (<b>D</b>), 50 µm. (<b>E, F</b>) Representative z-stack images of dendritic segments of new neurons at 28 dpi in control (<b>E1, F1</b>) and LTP hemispheres (<b>E2, F2</b>). (<b>E1, 2</b>) and (<b>F1, 2</b>) represent micrographs of OML and MML, respectively. A dendritic segment within the LTP-induced layer is shown in (<b>F2</b>) only, indicated by red characters. Scale bar, 2 µm. (<b>G</b>) Graphs of average F-actin intensity in each DG layer in arbitrary units (AU). *, <i>P</i><0.05 from Student’s t-test. (<b>H</b>) Graph shows MML-to-OML ratio (MML/OML) of average synaptophysin intensity. (<b>I</b>) Spine number per 10-µm dendritic fragment in each layer is graphed. (<b>J</b>) Graph shows MML-to-OML ratio (MML/OML) of spine density in control and LTP hemispheres. (<b>K</b>) LTP induction enlarges spines expressed within the LTP-induced layer. Average cross-sectional area of spines is indicated. ⁺, <i>P</i><0.063 from Student’s t-test; **, P<0.011 from Mann-Whitney U-test. (<b>G–K</b>) Dendritic fragments for spine analyses: control hemisphere, n = 16, LTP hemisphere, n = 16 from 3 animals. Data from the LTP-induced layer are indicated by red color in each graph. n.s. indicates no significant difference or variance. <i>P</i> values from post-hoc Fisher’s and Scheffe’s test are shown in (<b>G</b>) and (<b>K</b>), respectively.</p

Functional integration of new neurons is enhanced by MML-LTP induction at 12 days of age.

<p>(<b>A</b>) Experimental schedules (<b>B, C</b>) PS amplitudes of the DG obtained from rats under the “HFS 12d” and “HFS 28d” conditions are shown in panels <b>B</b> and <b>C</b>, respectively. Pre- and post-HFS delivery are indicated by “pre” and “post”, respectively. (<b>D</b>) PS amplitudes of the DG obtained from rats under the “HFS/HFS” condition. Both HFS(500) at day 12 and day 28 increased PS amplitude. (<b>E</b>) Representative z-stack images of BrdU⁺ cells of the control hemisphere and HFS(500)-induced hemisphere in the “HFS/HFS” condition. Signals of BrdU, Zif268, and NeuN are shown as green, red, and blue, respectively. The upper side and lower side of each image are the molecular layer and the hilus region, respectively. Arrowheads in each panel indicate the nucleus of the same BrdU⁺ cell. Scale bar, 10 µm. (<b>F</b>) HFS(500)-delivered hemisphere to control the hemisphere ratio (HFS/Control) of Zif268 expression in BrdU⁺ cells. The data were obtained from three animals in each condition. <i>P</i> values from post-hoc Fisher’s test are shown in the graph.</p

Spinogenesis of new neurons during the first 4 weeks after birth in adult DG.

<p>(<b>A</b>) Anatomical organization of the entorhinal-hippocampal DG pathway. Abbreviations: Rec, recording electrode; Stim, stimulating electrode; MPP and LPP, medial and lateral perforant pathway, respectively; MML and OML, middle and outer molecular layer, respectively; GCL, granule cell layer. (<b>B</b>) Side views photos showing needle tip, shaped by electrical grinder, of Hamilton syringes used for RV injection, at 90 degree orientations with respect to each other. (<b>C</b>) Representative z-stack images of morphologies and dendritic segments from newly born neurons at 12, 16, 18, 21, and 28 dpi. New neurons were identified by RV-mediated labeling with GFP-actin (green). Blue indicates nuclear distribution of individual cells (DRAQ5 staining). ML, molecular layer; GCL, granule cell layer. Selected regions in low magnification images (within squares) at 12, 16, and 18 dpi are shown below in high magnification. Scale bars: <b>a-c</b>, 50 µm; <b>d</b>, 5 µm; <b>e-j</b>, 2 µm. (<b>D</b>) Spines of new neurons are contacted by presynaptic terminals. Z-stack images are observations of dendritic fragments from discrete cells at 21 dpi. GFP-actin signal allows visualization of spines (green). The presynaptic marker synaptophysin is shown in red. Nearly all spines labeled with GFP-actin contact presynaptic terminals. Scale bar, 2 µm. (<b>E</b>) Immunoelectron microscopy showed synapse formation of GFP-actin-positive spines on 28 dpi neurons as defined by containing postsynaptic density and contacting with synaptic vesicles containing structure. <b>a</b> and <b>b</b>, typical images of dendritic spines of new neurons. <b>c</b>, Images of a filopodial protrusion were taken by tilting function (left, middle, and right photo: −50°, 0°, and +40°, respectively). Scale bar, 0.5 µm. (<b>F</b>) Density of dendritic spines on new neurons labeled with GFP-actin-RV. Line graph shows the density of protrusions on dendritic fragments of new neurons at 12, 16, 18, and 28 dpi. The protrusions density is expressed as number of protrusions per 10-µm dendritic length. (<b>G-J</b>) HFS(500)-mediated Zif268 expression in new neurons at 12 and 28 dpi. (<b>G</b>) Experimental schedule. HFS(500) was unilaterally delivered to MPP, and brains were dissected at 1 h after the initiation of HFS. (<b>H</b>) Immunohistochemistry with anti-Zif268 antibody. Left, HP of control hemisphere; right, ipsilateral hemisphere treated with HFS. Scale bar, 0.5 mm. (<b>I</b>) Triple staining for Zif268 (red), GFP (green), and DAPI (nucleus, blue). Arrowheads in GFP/Zif268 and Nucleus/Zif268 photos in each panel indicate the soma of the same GFP-labeled new neurons. Scale bar, 10 µm. (<b>J</b>) Percentage of Zif268-positive new neurons in control and HFS-delivered DG. At 12 dpi, Zif268⁺ and GFP⁺ double-positive cells were not detected (n.d.). At 12 dpi, n = 3 animals; control hemisphere, n = 15, 12, and 17 GFP+ cells in each animal; HFS-delivered hemisphere, n = 13, 12, and 7 GFP+ cells in each animal. At 28 dpi, n = 3 animals; control hemisphere, n = 19, 4, and 18 GFP+ cells in each animal; HFS-delivered hemisphere, n = 22, 8, and 15 GFP+ cells in each animal. <i>P</i> values from Student’s <i>t</i>-test are shown in the graph.</p

NMDAR activity during HFS is required for LTP-mediated changes in the spinogenesis of new neurons.

<p>(<b>A</b>) Experimental schedule for CPP pretreatment. Pretreatment with CPP i.p. blocks LTP of PS amplitude in the DG, as previously described (Kitamura <i>et al</i>. 2009). Insets are samples of evoked field potential traces recorded at −1, 1, and 7 days post-HFS. (<b>B</b>) PS amplitude of rats used in this study at 1 day before, 1 day after, and 7 days after the HFS(500) delivery (−1 d, 1 d, and 7 d, respectively). CPP (10 mg/kg) was injected i.p. 2 h before the initiation of HFS delivery. (<b>C</b>) At 12 dpi, CPP was injected i.p. 2 h before HFS(500). Representative z-stack images of dendritic segments of new neurons at 28 dpi in control (<b>C1, C3</b>) and HFS-treated hemispheres (<b>C2, C4</b>) with CPP i.p. administration. (<b>C1, 2</b>) and (<b>C3, 4</b>) represent micrographs of OML and MML, respectively. Scale bars for (<b>C</b>), (<b>F</b>), and (<b>I</b>), 2 µm. (<b>F</b>) At 16 dpi, CPP was injected i.p. 2 h before HFS(500). Representative z-stack images of dendritic segments of new neurons at 28 dpi in control (<b>F1, F3</b>) and of HFS-treated hemispheres (<b>F2, F4</b>) with CPP i.p. administration. (<b>F1, 2</b>) and (<b>F3, 4</b>) represent micrographs of OML and MML, respectively. (<b>I</b>) At 21 dpi, CPP was injected i.p. 2 h before HFS(500). Representative z-stack images of dendritic segments of new neurons at 28 dpi observed in control (<b>I1, 3</b>) and HFS-treated hemispheres (<b>I2, 4</b>) with CPP i.p. administration. (<b>I1, 2</b>) and (<b>I3, 4</b>) represent micrographs of OML and MML, respectively. Dendritic segments within the HFS-delivered layer are indicated by blue characters (<b>C4, F4, and I4</b>). (<b>D</b>), (<b>G</b>), (<b>J</b>) Spine number per 10-µm dendritic fragment in each layer is graphed. (<b>E</b>), (<b>H</b>), (<b>K</b>) Averages cross-sectional area of spines is indicated. (<b>D, E</b><i>)</i> Data from new neurons treated with CPP and HFS delivery at 12 dpi. Dendritic fragments for spine analyses: control hemisphere, n = 19, HFS hemisphere, n = 19 from 3 animals. (<b>G, H</b>) Data from new neurons treated with CPP and HFS delivery at 16 dpi. Dendritic fragments for spine analyses: control hemisphere, n = 14, HFS hemisphere, n = 17 from 3 animals. (<b>J, K</b>) Data from new neurons treated with CPP and HFS delivery at 21 dpi. Dendritic fragments for spine analyses: control hemisphere, n = 19, HFS hemisphere, n = 19 from 3 animals. Data from the HFS-delivered layer are indicated by blue color in each graph. n.s. indicates no significant variance.</p

MML-LTP induction at 12 dpi enhances the later spinogenesis of new neurons specifically in MML.

<p>(<b>A</b>) Experimental schedules for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045270#pone-0045270-g002" target="_blank">Figure 2</a>. Insets are samples of evoked field potential traces that are recorded at pre-HFS, 1 day, and 16 days post-HFS. (<b>B</b>) PS amplitudes of the DG obtained from rats used for experiments in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045270#pone-0045270-g002" target="_blank">Figure 2</a>. Pre- and post-HFS delivery are indicated by “pre” and “post”, respectively. (<b>C</b>) LTP induction changes F-actin content in the DG ML. Unilateral HFS(500) was delivered to the MPP at 12 dpi, and brains were dissected at 28 dpi. DG of control hemisphere (<b>C1</b>) and LTP hemisphere (<b>C2</b>). F-actin signal (red) was visualized by phalloidin-tetramethyl rhodamine iso-thiocyanate (TRITC) staining. DG subregions are indicated in (<b>C2</b>). IML, inner ML. (<b>D</b>) Presynaptic content identified by synaptophysin signal are unchanged by MML LTP induction. Fluorescence micrographs with synaptophysin (red) in control (<b>D1</b>) and LTP (<b>D2</b>) DG. Nuclear signal is shown in blue (DRAQ5). Scale bars for (<b>C</b>) and (<b>D</b>), 50 µm. (<b>E, F</b><i>)</i> Representative z-stack images of dendritic segments of new neurons at 28 dpi in control (<b>E1, F1</b>) and LTP hemispheres (<b>E2, F2</b>). New neurons were visualized with GFP-actin. (<b>E1, 2</b>) and (<b>F1, 2</b>) represent micrographs of OML and MML, respectively. Therefore, a dendritic segment within the LTP-induced layer is depicted in (<b>F2</b>) only, indicated by red characters. Scale bar, 2 µm. (<b>G</b>) F-actin content significantly increases in MML compared with ipsilateral IML and OML and contralateral MML. Graphs show average intensity of F-actin in each DG layer in arbitrary units (AU). *, <i>P</i><0.05 from Student’s t-test. (<b>H</b>) Synaptophysin expression is unchanged by MML LTP. Graph shows MML-to-OML ratio (MML/OML) of average synaptophysin intensity in control and LTP hemispheres. n.s. indicates no significant difference. (<b>I</b>) Spine density within the LTP-induced layer significantly increases compared with other layers. Spine number per 10-µm dendritic fragment is shown in the graph. (<b>J</b>) The graph shows MML-to-OML ratio (MML/OML) of spine density in control and LTP hemispheres, with <i>P</i> values from Student’s t-test. (<b>K</b>) MML LTP induction enlarges spines in the MML. Average cross-sectional area of spines, an indicator of spine size, is graphed. (<b>G–K</b>) Dendritic fragments for spine analyses: control hemisphere, n = 22, LTP hemisphere, n = 22 from 3 animals. Data from the LTP-induced layer are indicated by red color in each graph. <i>P</i> values from post-hoc Fisher’s and Scheffe’s test are shown in (<b>I</b>), (<b>K</b>) and (<b>G</b>), respectively.</p

MML-LTP induction at 16 dpi inhibits the later spinogenesis of new neurons specifically in MML.

<p>(<b>A</b>) Experimental schedules for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045270#pone-0045270-g003" target="_blank">Figure 3</a>. Insets are samples of evoked field potential traces that are recorded at pre-HFS, 1 day, and 12 days post-HFS. (<b>B</b>) PS amplitudes of the DG obtained from rats used for experiments in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045270#pone-0045270-g003" target="_blank">Figure 3</a>. Pre- and post-HFS delivery are indicated by “pre” and “post”, respectively. (<b>C</b>) Fluorescence micrographs of F-actin signal (phalloidin-TRITC, red) in control (<b>C1</b>) and LTP-induced DG (<b>C2</b>) of 28-dpi animals. Nuclear signal is shown in blue (DRAQ5). (<b>D</b>) Synaptophysin (red) and nuclei (DRAQ5, blue) signals of control (<b>D1</b>) and LTP hemispheres (<b>D2</b>). Scale bars for (<b>C</b>) and (<b>D</b>), 50 µm. (<b>E, F</b>) Representative z-stack images of dendritic segments of new neurons at 28 dpi in control (<b>E1, F1</b>) and LTP hemispheres (<b>E2, F2</b>). (<b>E1, 2</b>) and (<b>F1, 2</b>) represent micrographs of OML and MML, respectively. Only (<b>F2</b>) represents a dendritic segment within the LTP-induced layer, indicated by red characters. Scale bar, 2 µm. (<b>G</b>) Graphs of average F-actin intensity in each DG layer in arbitrary units (AU). *, <i>P</i><0.02 from Student’s t-test. (<b>H</b>) Graph shows the MML-to-OML ratio (MML/OML) of average synaptophysin intensity. (<b>I</b>) Spine density within the LTP-induced layer significantly decreases compared with other layers. Spine number per 10-µm dendritic fragment in each layer is graphed. (<b>J</b>) Graph shows MML-to-OML ratio (MML/OML) of spine density in control and LTP hemispheres. (<b>K</b>) Average spine cross-sectional area is indicated. (<b>G, K</b>) Dendritic fragments for spine analyses: control hemisphere, n = 18, LTP hemisphere, n = 18 from 3 animals. Data from the LTP-induced layer are indicated by red color in each graph. n.s. indicates no significant difference or variance. <i>P</i> values from post-hoc Fisher’s test, Scheffe’s test, and Student’s t-test are shown in (<b>G</b>), (<b>I</b>), and (<b>J</b>), respectively.</p

Formation of neural plasticity differentially regulates the integration of new neurons into the activated circuit.

<p>(<b>A</b>) LTP induction at the no-spine stage of new neurons (12 dpi) specifically increases their spine size and spine expression rate within the LTP-induced layer by 3 (19 dpi) and 4 weeks of neuronal age (28 dpi), respectively. (<b>B</b>) LTP induction at the time of initial spinogenesis of new neurons (16 dpi) locally inhibits the later expression of spines in an LTP-induced layer-specific manner. (<b>C</b>) LTP induction after the drastic spinogenesis stage of new neurons, at 21 dpi, induced their later spine enlargement in the LTP-induced layer at 28 dpi. The spine enlargement is return to basal level, and the later spinogenesis is inhibited by 35 dpi. All LTP-mediated changes are tightly correlated with actual formation of the activity-dependent neural plasticity. <b>Left,</b> Schematic representation of morphology of each new neurons at 12, 16, or 21 days after birth ( = 12, 16, or 21 dpi, respectively). High frequency stimulation [HFS(500)] for LTP induction is specifically delivered at MML of ipsilateral hemisphere in all experiments of present study. <b>Middle,</b> The mean values of spine density at indicated timing under control and LTP-induced conditions are plotted by white (represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045270#pone-0045270-g001" target="_blank">Figure 1F</a>) and black circles, respectively. <b>Right,</b> Graph shows the mean values of ipsilateral (Ipsi)/contralateral (Contra) ratio of spine cross-sectional area in MML and OML. Abbreviations: GCL, granule cell layer; IML, MML, and OML, inner, middle, and outer molecular layer, respectively.</p