Slow-Dendritic sIPSCs

<div><p>(A) and (B) Example of traces of outward sIPSCs recorded from a pup (A) and adult (B) DGC. Dashed box on each top trace denotes expanded segment on the bottom. Scales indicate 0.5 s/50 ms (top/bottom), 10 pA.</p> <p>(C) and (D) Two-dimensional histograms of rise and decay time of individual sIPSCs recorded from pup ([C] <i>n</i> = 695 events) and adult DGCs ([D] <i>n</i> = 1,160 events). Color scale indicates the relative frequency for each bin.</p> <p>(E) Cumulative histograms of rise and decay time of all sIPSCs recorded from pup (green) and adult DGCs (red). Same data as shown in (C) and (D).</p> <p>(F) Frequency of sIPSCs (pup, <i>n</i> = 10 neurons; adult, <i>n</i> = 16; <i>p</i> = 0.94; <i>t</i>-test).</p> <p>(G) Peak amplitude of sIPSCs (pup, <i>n</i> = 10; adult, <i>n</i> = 14; <i>p</i> = 0.44).</p> <p>(H) Kinetics of sIPSCs. Inset: scaled averages of sIPSCs (pup, green; adult, red). Scale bar indicates 50 ms. All experiments conducted in the presence of kyn at V_hold = 0 mV with an internal solution containing high [Cl⁻]. (<i>n</i>, same as in [F]; rise time, <i>p</i> = 0.37; decay time, <i>p</i> = 0.41).</p></div

In vivo imaging of dendritic pruning in dentate granule cells

Data files of the dendritic arbors of all neurons imaged for the following publication:<div><br></div><div>Gonçalves J.T., Bloyd C.W., Shtrahman M, Johnston S.T., Schafer S.T., Parylak S.L., Thanh T., Chang T., Gage F.H., In vivo imaging of dendritic pruning in dentate granule cells, Nature Neurosci.,<i> </i><i>in press, doi:10.1038/nn.4301 </i>(2016)</div><div><br></div><div><div><b>Abstract:</b></div><div>We longitudinally imaged the developing dendrites of adult-born mouse dentate granule cells (DGCs) in vivo and found that they underwent over-branching and pruning. Exposure to an enriched environment and constraint of dendritic growth by disrupting Wnt signaling led to increased branch addition and accelerated growth, which were, however, counteracted by earlier and more extensive pruning. Our results indicate that pruning is regulated in a homeostatic fashion to oppose excessive branching and promote a similar dendrite structure in DGCs.</div></div><div><br></div><div><div><div><div><div><div><div></div></div></div></div><div></div></div><div></div></div><div><div><div></div></div></div></div

Short-Term Plasticity of Entorhinal Glutamatergic Afferents

<div><p>(A) Average EPSCs recorded at V_Hold = −80 mV (downward deflections) and +50 mV (upward deflections) from pup and adult DGCs (<i>n</i> = 8 to 11) upon stimulation of MPP or LPP. Dashed line indicates zero level. Arrowheads denote time points for quantification of AMPA (open triangles) and NMDA (filled triangles) currents shown in (B). Criteria for AMPA/NMDA quantification are detailed in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040409#s4" target="_blank">Materials and Methods</a> section. Scale bars indicate 20 ms, 100 pA.</p> <p>(B) AMPA/NMDA ratio from pup and adult DGCs (<i>n</i> = 9 to 13) in response to MPP or LPP stimulation. Two-way ANOVA revealed a significant effect of MPP versus LPP (<i>p</i> = 0.006), but no significant effect of pup versus adult (<i>p</i> = 0.63).</p> <p>(C) Averages of EPSCs in response to paired-pulse stimulation of the MPP or LPP delivered at increasing interpulse intervals (20, 50, 100, and 500 ms). Traces are averages of 7–14 cells aligned and normalized to the first EPSC. Stimulation artifacts and late decay phases of the second EPSC were removed for clarity. Scale bar indicates 100 ms.</p> <p>(D) Paired-pulse ratio as a function of interpulse interval for the experiments shown in (C). Two-way ANOVAs revealed a significant effect of interpulse interval for MPP (dashed lines, <i>p</i> < 0.0001) and LPP (solid lines, <i>p</i> < 0.0001), but no significant effect of pup (green lines, solid circles) versus adult (red lines, open circles) for either MPP (<i>p</i> = 0.073) or LPP (<i>p</i> = 0.72) stimulation (<i>n</i> = 9 to 14)</p> <p>(E) Example of EPSCs from a pup (green) and an adult DGC (red) in response to MPP stimulation (ten pulses, 50 Hz) Traces are normalized to the first EPSC amplitude. Scale bar indicates 40 ms.</p> <p>(F) Relative EPSC amplitudes measured in response to 50-Hz stimulation evoked as shown in (E). No difference was found between pup and adult responses (two-way ANOVA, <i>p</i> = 0.49, <i>n</i> = 10 pups [solid circles], <i>n</i> = 4 adults [open circles]). All recordings were carried out in the presence of 20-μm BMI. Neurons were approximately 18 wk old (pup) and approximately 13 wk old (adult). All plots depict mean ± SEM.</p></div

Fluorescent Labeling of DGCs Born during Early Postnatal and Adult Neurogenesis

<div><p>(A) and (B) Retroviral delivery of GFP into DGCs generated at P7 (A) and P42 (B), analyzed 7 wk after each injection. The GCL was labeled by immunohistochemistry for the neuronal marker NeuN (blue). Images are merges of 27 (A) and 21 (B) confocal planes taken from coronal sections (40-μm thick). H, hilus; ML, molecular layer.</p> <p>(C) and (D) Double retroviral labeling of DGCs generated at P7 (GFP⁺, green) and P42 (RFP⁺, red). Images are merges of nine (C) and 20 (D) confocal planes taken from fixed transverse sections of the DG (400-μm thick) from 13-wk-old mice.</p> <p>(E) Double labeling of DGCs with GFP (green) and BrdU (red): intrahippocampal injections of CAG-GFP retrovirus in P7 were followed by daily injections of BrdU carried out from P21 to P25; brains were analyzed at P53. The image is a merge of 16 confocal planes.</p> <p>(F) Example of a sporadic event of co-localization of GFP, BrdU, and NeuN shown by a single optical section for the green, red, and blue channels. Their overlay is shown together with the orthogonal projections onto the <i>x-z</i> (top) and <i>y-z</i> (right) planes.</p> <p>(G) Number of GFP⁺ or BrdU⁺ cells per mouse (left) and the percentage of GFP⁺ cells showing BrdU label (right). Data are mean ± standard error of the mean (SEM) (<i>n</i> = 3 mice). Scale bars indicate 50 μm (A–E) or 10 μm (F).</p></div

Fast-Perisomatic sIPSCs

<div><p>(A) and (B) Example of traces of inward sIPSCs recorded from a pup (A) and adult (B) DGC. Dashed box on each top trace denotes expanded segment on the bottom. Scale bars indicate 1 s/50 ms (top/bottom), 40 pA.</p> <p>(C) and (D) Two-dimensional histograms of rise and decay time of individual sIPSCs recorded from pup ([C] <i>n</i> = 5,871 events) and adult DGCs ([D] <i>n</i> = 8,183 events). Color scale indicates the relative frequency for each bin (square areas in the graph).</p> <p>(E) Cumulative histograms of rise and decay time of all sIPSCs recorded from pup (green) and adult (red) DGCs. Data are the same as shown in (C) and (D).</p> <p>(F) Frequency of sIPSCs (pup, <i>n</i> = 12 neurons; adult, <i>n</i> = 15; <i>p</i> = 0.99; <i>t</i>-test).</p> <p>(G) Peak amplitude of sIPSCs (<i>n</i>, same as in [F]; <i>p</i> = 0.44).</p> <p>(H) Kinetics of sIPSCs. Inset: scaled averages of sIPSCs (pup, green; adult, red). Scale bar indicates 10 ms. All experiments conducted in the presence of kyn at V_hold = −80 mV with an internal solution containing high [Cl⁻]. (<i>n</i>, same as in [F]; rise time, <i>p</i> = 0.96; decay time, <i>p</i> = 0.72).</p></div

Entorhinal Glutamatergic Afferents onto Mature Neurons Generated in the Developing and Adult Hippocampus

<div><p>(A) Schematic diagram of experimental design for retroviral labeling and electrophysiological recordings.</p> <p>(B) A paired experiment. Upper row: a RFP⁺ neuron was patched (cell “1”) and filled with Alexa Fluor 488 (green). Lower row: a neighboring GFP⁺ neuron (cell “2”) was subsequently patched and filled with Alexa Fluor 594 (red). Both DGCs are in the same field, although at different focal planes (see merge channel). At the end of the experiment, both cells display green and red fluorescence (merge). Scale bar indicates 10 μm.</p> <p>(C) Schematic diagram of the DG showing the position of the bipolar electrodes for the stimulation of the MPP and LPP.</p> <p>(D) Example of EPSCs from an adult-born DGC at V_hold = +50 mV (top) and −80 mV (bottom) elicited by LPP stimulation recorded in the presence of vehicle (“control”) or 20 μM DNQX + 100 μM AP-5. Scale bars indicate 20 ms, 40 pA (top) or 20 pA (bottom). Similar properties were observed in DGCs born during development (unpublished data)</p> <p>(E) Example of a paired experiment. Sequential recordings of two neighboring pup and adult DGCs in response to paired-pulse stimulation (100-ms interval) of the MPP and LPP. Scale bars represent 25 ms, 50 pA.</p> <p>(F) Paired-pulse ratio (left) and 20%–80% rise time (right) of EPSCs recorded from pup and adult DGCs upon stimulation of MPP and LPP (<i>n</i> = 11 to 15) Paired-pulse ratio was measured as the ratio between the amplitude of the second pulse over the first. Two-way analysis of variance (ANOVA) revealed a significant effect of MPP versus LPP for paired-pulse ratio (<i>p</i> = 0.0002) and rise time (<i>p</i> = 0.0017), but no significant effect of pup versus adult (Adu) for either parameter.</p> <p>(G) Peak EPSC amplitude recorded in paired experiments from DGCs born during development (Pup) and adulthood (Adult) upon stimulation of the MPP (<i>n</i> = 15 pairs, <i>p</i> = 0.3, paired <i>t</i>-test) or LPP (<i>n</i> = 11 pairs, <i>p</i> = 0.049). Mean ± SEM is shown on the sides. Recordings were carried out in the presence of 20 μM BMI in slices obtained from mice aged 19–21 wk. Neurons were approximately 18 wk old (P7) and approximately 13 wk old (P42). All plots depict mean ± SEM.</p></div

GABAergic Inputs Evoked by ML Stimulation

<div><p>(A) Example of evoked IPSCs recorded from a pup-born DGC in response to extracellular stimulation of the ML before (control), during (BMI), and after (wash) application of 20 μM BMI. Scale bars indicate 25 ms, 20 pA. Inset: schematic diagram of the DG showing the position of the bipolar electrode for the stimulation of ML interneurons. Experiments were carried out with internal solution containing low [Cl⁻].</p> <p>(B) Kinetics of IPSCs evoked on pup versus adult DGCs (pup, <i>n</i> = 6; adult, <i>n</i> = 14; rise time, <i>p</i> = 0.85; decay time, <i>p</i> = 0.90, paired <i>t</i>-tests). Inset: scaled averages of IPSCs (pup, green; adult, red). Scale bar indicates 50 ms.</p> <p>(C) and (D) Examples of I-V curves of evoked IPSCs recorded from pup (C) or adult (D) DGCs. Top: sample traces of IPSCs recorded at the holding potentials shown on the right (mV). Scale bar indicates 50 ms, 10 pA. Bottom: I-V plots measured at the time indicated by the arrowheads.</p> <p>(E) E_GABA of IPSCs (pup, <i>n</i> = 7; adult, <i>n</i> = 11; <i>p</i> = 0.67, <i>t</i>-test).</p> <p>(F) Slope conductance measured at the IPSC peak (<i>n,</i> same as in [E]; <i>p</i> = 0.23; <i>t</i>-test). All experiments conducted in the presence of kyn (4 mM) in neurons aged 12 wk (P7) and 7 wk (P42).</p></div

GABAergic Inputs Elicited by GCL Stimulation

<div><p>(A) Example of evoked IPSCs recorded from a pup-born DGC in response to extracellular stimulation of the GCL before (control), during (BMI), and after (wash) application of 20 μM BMI. Scale bars indicate 25 ms, 20 pA. Inset: schematic diagram of the DG showing the position of the bipolar electrode for the stimulation of GCL interneurons. Experiments were carried out with internal solution containing low [Cl⁻].</p> <p>(B) Typical traces of IPSCs evoked on a pup (green) and adult (red) DGC recorded from the same slice in a paired experiment. Scales indicate 25 ms, 40 pA.</p> <p>(C) Paired analysis of peak IPSC amplitude recorded from a pup versus adult DGCs (<i>n</i> = 19 pairs, <i>p</i> = 0.97, paired <i>t</i>-test). Mean ± SEM are shown on the sides.</p> <p>(D) Kinetics of evoked IPSCs (<i>n</i> = 40 adults, <i>n</i> = 22 pups; rise time, <i>p</i> = 0.31; decay time, <i>p</i> = 0.19; unpaired <i>t</i>-tests). Inset: scaled average traces of evoked IPSCs. Scale bar indicates 50 ms.</p> <p>(E) and (F) Examples of I-V curves of evoked IPSCs recorded from pup (E) and adult DGCs (F). Top: sample traces of IPSCs recorded at the holding potentials shown on the right (mV). Scale bars indicate 50 ms, 100 pA (E) and 50 pA (F). Bottom: I-V plots measured at time points indicated with arrowheads (early: filled triangles/dashed lines, late: open triangles/solid lines).</p> <p>(G) E_GABA measured at early and late time points. Two-way ANOVA (<i>n</i> = 11, pup; <i>n</i> = 16, adult) revealed a significant effect of early versus late points (<i>p</i> < 0.0001), but not of pup versus adult (Adu) neurons (<i>p</i> = 0.56).</p> <p>(H) Conductance profile of IPSCs. Each value denotes the slope conductance measured from the I-V curve at the time points indicated on the sample traces (inset). No significant differences were found between pup (green) and adult (red; 11 ≤ n ≤ 16, <i>p</i> = 0.62, two-way ANOVA). Scale indicates 50 ms. All experiments were conducted in the presence of kyn (4 mM) in slices obtained from mice aged 13–14 wk. Neurons were approximately 12 wk old (P7) and approximately 7 wk old (P42).</p></div

Firing Behavior Elicited by Excitatory Inputs

<div><p>(A) Action currents in cell-attached configuration recorded from an adult-born DGC in response to MPP stimulation at increasing stimulus strengths (0.5–1.5 mA, 50 μs). Six representative epochs are shown. Spiking probability (p) is shown below the traces. The asterisk (*) marks the stimulation artifact. Scale indicates 10 ms, 50 pA.</p> <p>(B) Sample experiment of simultaneous cell-attached recordings of DGCs born in pup and adult brain in response to MPP stimulation (0.4 mA, 50 μs). Action currents indicate a higher spiking probability in the pup DGC. Scales indicate 10 ms, 50 pA (left) and 20 pA (right).</p> <p>(C) Sample experiment in which the spiking probability is higher in the adult-born DGC (1.5 mA, 50 μs). Scale indicates 10 ms, 30 pA.</p> <p>(D) Firing behavior of DGCs born in pup and adult brain during simultaneous paired experiments. No significant difference was found (<i>n</i> = 14 pairs, <i>p</i> = 0.8, Wilcoxon signed rank test). All recordings were carried out in the presence of BMI (20 μM). In this set of experiments, adult-born neurons were retrovirally labeled with GFP, whereas unlabeled DGCs of the middle third of the GCL were considered postnatally born (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040409#s4" target="_blank">Materials and Methods</a>). Repetitive (>15 episodes) slow frequency stimulation was used to measure the spiking probability for each neuron at the given stimulus.</p></div