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Research ArticleNew Research, Development

Development of Local Circuit Connections to Hilar Mossy Cells in the Mouse Dentate Gyrus

Yulin Shi, Steven F. Grieco, Todd C. Holmes and Xiangmin Xu
eNeuro 7 March 2019, 6 (2) ENEURO.0370-18.2019; DOI: https://doi.org/10.1523/ENEURO.0370-18.2019
Yulin Shi
1Department of Anatomy and Neurobiology, School of Medicine, University of California, Irvine, CA 92697-1275
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Steven F. Grieco
1Department of Anatomy and Neurobiology, School of Medicine, University of California, Irvine, CA 92697-1275
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Todd C. Holmes
2Department of Physiology and Biophysics, University of California, Irvine, CA 92697-4560
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Xiangmin Xu
1Department of Anatomy and Neurobiology, School of Medicine, University of California, Irvine, CA 92697-1275
3Department of Biomedical Engineering, University of California, Irvine, CA 92697-2715
4Department of Microbiology and Molecular Genetics, University of California, Irvine, CA 92697-4025
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  • Figure 1.
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    Figure 1.

    Targeted recordings of hilar mossy cells. A, Horizontal hippocampal slices are acutely prepared from GAD-Cre; Ai9 tdTomato double transgenic mice and are visualized under a 4× objective. Whole-cell recordings are made from mossy cells in the DG (green circle). Scale bar = 250 μM. B, Mossy cells, as shown with a 60× objective, are first identified by their lack of tdTomato fluorescence (under the pipette, white square) in the hilar region of GAD-Cre; Ai9 mice. Scale bar = 50 μM. C, Morphology of recorded mossy cells (white arrows), which are injected with biocytin (green), demonstrates multipolar soma and thick dendrites with thorny excrescences. Scale bar = 50 μM. D, Mossy cells have regular/adapting spiking in response to current injection (horizontal black line) through the patch pipette.

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    Figure 2.

    Developmental changes in intrinsic physiologic and morphologic properties of mossy cells. A, Example responses to current injection (horizontal lines) in example mossy cells recorded from P7, P14, and P21 mice. B, C, Example morphology of biocytin-labeled mossy neurons (green) and surrounding DAPI-stained tissue (blue) at P7, P14, and P21. The labeled neurons in the P7 mouse had proximal dendrites with branches penetrated into the fascia dentate (arrows for P7 mouse in B) and were relatively smooth (image shown in C), while neurons recorded in P14 and P21 mice have obvious thorny excrescence (arrows in C) on proximal dendrites. Scale bar = 250 μM (B) and 25 μM (C). All labeled neurons had large, multipolar somata, and thick thorny proximal dendrites. A neuron (circle in B) in the P14 mouse is in CA3. D, The input resistance (left, measured in MΩ) decreased with age from P6–P7 to P13–P14, and P21–P28. The Cm (middle, measured in pF), is the capacitance of the cell membrane and increased with age from P6–P7 to P13–P14, and P21–P28. The spike rate (right, measured in Hz) is the number of evoked spikes in response to current injection of 100 pA during recording, and decreased with age from P6–P7 to P13–P14. * indicates the statistical significance (p < 0.05), and ** indicates p < 0.01. Also see Table 2.

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    Figure 3.

    LSPS mapping and data analysis. A, A horizontal hippocampal slice under a 4× objective with a patched neuron (red circle) and laser stimulation sites overlaid (cyan asterisks). B, Raw signal traces recorded from the patched neuron during laser stimulation. C, Examples of a direct response (top green trace) which has a large amplitude and a short response latency, and synaptic responses (bottom red) which have smaller amplitudes and longer latencies. D–F, Synaptic responses were detected and extracted using automatic software processing (Shi et al., 2010). Input amplitude (see Methods) (D), the number of evoked synaptic events, as defined by the number of EPSCs elicited per laser pulse (E), and the number of spontaneous events (F) were plotted in heat maps.

  • Figure 4.
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    Figure 4.

    Spatial precision of LSPS mapping for neurons in the DG, hilus, and CA3c. A, B, Excitation profile of a recorded DG granule cell from a mouse hippocampal slice. The excitation profile shows the spatial distribution of uncaging sites that produce APs. The cell was held in current clamp mode. The cyan asterisks in A are the stimulation sites (75-µm spacing). The evoked APs were restricted to a small region (yellow square). Raw signal traces within the yellow square are shown in B. C, D, Excitation profile of a hilar mossy cell from a mouse hippocampal slice (100-µm spacing). E, F, Excitation profile of CA3 pyramid cells from mouse hippocampal slice (100-µm spacing). Excitation profiles show no spike-evoking sites distal from the perisomatic area of the recorded neuron, demonstrating that LSPS maps (e.g., Figures 5 and 6) represent input from monosynaptic connections.

  • Figure 5.
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    Figure 5.

    Functional maps of excitatory inputs to mossy cells across developmental ages. A, Strength of excitatory input (input amplitudes) at P7, P14, and P24 for a representative mossy cell. B, Frequency of EPSC events (number of evoked synaptic events per second) for a representative mossy cell. C, D, Summed (averaged) amplitude of excitatory inputs to mossy cells. We recorded from 12, 9, and 10 cells from P6–P7, P13–P14, and P21–P28 mice, respectively. In the left two panels in C, the y-axis indicates input strength. In the right panel in D, the y-axis shows the ratio of input strengths. * indicates the statistical significance (p < 0.05), and ** indicates p < 0.01. Also see Table 2.

  • Figure 6.
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    Figure 6.

    Functional maps of inhibitory inputs to mossy cells across developmental ages. A, Strength of inhibitory input amplitude at P7, P14, and P24 for representative mossy cells. B, Frequency of IPSC events (number of evoked synaptic events per second) for representative mossy cells. C, D, Summed (averaged) amplitude of inhibitory inputs to mossy cells. We mapped 10, 7, and 16 cells from P6–P7, P13–P14, and P21–P28 mice, respectively. In the left two panels in C, the y-axis indicates input strength. In the right panel in D, the y-axis shows the ratio of input strengths. * indicates the statistical significance (p < 0.05). Also see Table 5.

Tables

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    Table 1.

    Intrinsic physiologic properties of mossy cells at different ages

    Mouse ageRMP
    (mV)
    Rs
    (MΩ)
    Rinput
    (MΩ)
    Cm
    (pF)
    Evoked spike rate
    (Hz)
    P6–P7
    (N = 11)
    –60.3 ± 1.732.6 ± 2.4637.7 ± 74.070.4 ± 10.35.6 ± 1.1
    P13–P14
    (N = 12)
    –61.5 ± 1.127.2 ± 3.0439.8 ± 37.7111.8 ± 10.71.5 ± 0.6
    P21–P28
    (N = 13)
    –62.7 ± 1.633.4 ± 3341.7 ± 63.599.9 ± 133.1 ± 0.9
    p valuensns*P6–P7 vs P13–P14, 0.02
    *P6–P7 vs P21–P28, 0.008
    *P6–P7 vs P13–P14, 0.02
    *P6–P7 vs P21–P28, 0.008
    *P6–P7 vs P13–P14, 0.006
    • RMP, resting membrane potential; Rs, access resistance; Cm, membrane capacity; Rinput, input resistance. ns, not significant for statistical comparison. * indicate the statistical significance of p < 0.05.

    • View popup
    Table 2.

    Statistics of LSPS-mapped EPSC inputs to hilar mossy cells at different ages

    P6–P7P13–P14P21–P28Significance level
    DGPhotostimulation evoked input (EI)44.8 ± 8.4189.9 ± 59.0105.9 ± 12.4*P6–P7 vs P13–P14, 0.05
    *P6–P7 vs P21–P28, 0.0005
    EI per site3.6 ± 0.59.6 ± 1.58.3 ± 1.4ns
    % EI29.1 ± 6.162.7 ± 6.272.1 ± 8.4ns
    hilusEI18.8 ± 4.610.9 ± 6.65.3 ± 2.6*P6–P7 vs P21–P28, 0.022
    EI per site4.1 ± 1.03.1 ± 1.31.4 ± 0.4ns
    % EI18.2 ± 6.34.8 ± 1.73.6 ± 1.8ns
    CA3EI73.1± 19.943.7 ± 14.620.7 ± 7.9*P6–P7 vs P21–P28, 0.0056
    EI per site3.7 ± 0.53.9 ± 0.72.5 ± 6.3ns
    % EI32.0 ± 5.422.4 ± 4.614.0 ± 5.3ns
    • Note that the recorded cells used for this table include 12 cells (P6–P7), 9 cells (P13–P14), and 10 cells (P21–P28). EI: photostimulation-evoked postsynaptic input measured from recorded neurons; %EI: the regional percentage of total evoked input; EI per site: evoked input per photostimulation. * indicates the statistical significance of p < 0.05.

    • View popup
    Table 3.

    Number of EPSCs recorded from mossy cells at different ages

    P6–P7P13–P14P21–P28Significance level
    DGTotal numbers38.9 ± 7.070.7 ± 21.250.3 ± 9.3ns
    Numbers per site3.2 ± 0.53.9 ± 0.43.4 ± 0.1ns
    % Total events31.2 ± 5.056.6 ± 6.456.7 ± 10.4ns
    HilusTotal numbers15.1 ± 3.44.9 ± 2.55.4 ± 2.7*P6–P7 vs P13–P14, 0.05
    *P6–P7 vs P21–P28, 0.0117
    Numbers per site3.3 ± 0.61.7 ± 0.50.0ns
    % Total events13.3 ± 4.35.4 ± 2.16.0 ± 3.0ns
    CA3Total numbers60.4 ± 13.925.5 ± 8.420.5 ± 9.1*P6–P7 vs P21–P28 0.0193
    Numbers per site3.4 ± 0.42.4 ± 0.31.7 ± 0.3ns
    % Total
    events
    33.4 ± 5.826.2 ± 4.723.1 ± 10.9ns
    • * indicates the statistical significance of p < 0.05.

    • View popup
    Table 4.

    Rise time, time constant, and onset latency of EPSCs

    P6–P7P13–P14P21–P28Significance level
    DGRise time (ms)2.4 ± 0.122.6 ± 1.32.5 ± 0.1ns
    Time constant
    (ms)
    4.2 ± 0.24.2± 0.35.0 ± 0.6ns
    Latency (ms)50.5 ± 5.646.2 ± 6.742.1 ± 4.8ns
    HilusRise time (ms)2.3 ± 0.22.9 ± 0.62.2 ± 0.2ns
    Time constant
    (ms)
    6.7 ± 1.05.7 ± 0.56.2 ± 1.5ns
    Latency
    (ms)
    46.9 ± 4.959.1 ± 7.339.2 ± 9.1ns
    CA3Rise time (ms)2.4 ± 0.13.1 ± 0.12.7 ± 0.3*P6–P7 vs P13–P14, 0.004
    *P13–P14 vs P21–P28, 0.04
    Time constant
    (ms)
    7.2 ± 0.34.4 ± 0.33.4 ± 0.9ns
    Latency
    (ms)
    44.1 ± 3.051.0 ± 7.447.2 ± 10.3ns
    • * indicates the statistical significance of p < 0.05.

    • View popup
    Table 5.

    Statistics of LSPS-mapped IPSC inputs to hilar mossy cells at different ages

    P6–P7P13–P14P21–P28Significance level
    DGPhotostimulation evoked input (EI)68.5 ± 24.0490.7 ± 100.6195.7 ± 37.0*P6–P7 vs P13–P14, 0.005
    *P6–P7 vs P21–P28, 0.0001
    EI per site3.9 ± 0.912.8 ± 1.46.6 ± 0.8ns
    % EI31.2 ± 5.562.3 ± 5.064.8 ± 3.6ns
    HilusEI28.1 ± 8.8132.2 ± 35.231.6 ± 4.8*P6–P7 vs P13–P14, 0.002
    *P13–P14 vs P21–P28 0.004
    EI per site4.7 ± 1.310.7 ± 2.14.7 ± 0.50ns
    % EI18.2 ± 3.914.9 ± 3.017.9 ± 4.5ns
    CA3EI77.1 ± 20.5150.6 ± 30.451.8 ± 13.6*P6–P7 vs P13–P14, 0.05
    *P13–P14 vs P1–P28 0.002 
    EI per site4.3 ± 0.810.8 ± 2.14.2 ± 0.9ns
    % EI40.5 ± 6.218.5 ± 2.511.7 ± 2.9ns
    • Note that the recorded cells used for this table include 10 cells (P6–P7), 7 cells (P13–P14), and 16 cells (P21–P28). EI, EI per site, and % EI are the same as in Table 2. * indicates the statistical significance of p < 0.05.

    • View popup
    Table 6.

    Number of IPSCs recorded from mossy cells at different ages

    P6–P7P13–P14P21–P28Significance level
    DGTotal Numbers32.6 ± 11.7105.3 ± 21.180.4 ± 14.3*P6–P7 vs P13–P14, 0.01
    *P6–P7 vs P21–P28, 0.003
    Numbers per site2.0 ± 0.43.7 ± 0.42.9 ± 0.2ns
    % Total events31.1 ± 5.656.3 ± 4.863.1 ± 3.2ns
    HilusTotal numbers15.2 ±4.235.4 ± 9.817.2 ± 2.4*P6–P7 vs P13–P14, 0.05
    *P13–P14 vs P21–P28, 0.02
    Numbers per site2.6 ± 0.63.4 ± 0.62.6 ± 0.2ns
    % Total events19.3 ± 3.917.1 ± 2.619.1 ± 4.0ns
    CA3Total numbers36.2 ± 9.144.3 ± 13.221.4 ± 4.9ns
    Numbers per site2.2 ± 0.363.0 ± 0.51.8 ± 0.3ns
    % Total
    events
    39.3 ± 6.419.9 ± 2.711.9 ± 2.8ns
    • * indicates the statistical significance of p < 0.05.

    • View popup
    Table 7.

    Rise time, time constant, and onset latency of IPSCs

    P6–P7P13–P14P21–P28Significance level
    DGRise time (ms)4.1 ± 0.24.0 ± 0.24.4 ± 0.2ns
    Time constant
    (ms)
    9.3 ± 0.17.7 ± 0.87.4 ± 0.3*P6–P7 vs P21–P28,
    0.02
    Latency (ms)57.1 ± 2.234.8 ± 1.640.2 ± 2.5*P6–P7 vs P13–P14, 0.0003
    *P6–P7 vs P21–P28, 0.00009
    P13–P14 vs P21–P28, 0.07
    HilusRise time (ms)4.6 ± 0.34.4 ± 0.34.8 ± 0.2ns
    Time constant
    (ms)
    7.3 ± 0.07.7 ± 1.09.4 ± 0.8ns
    Latency (ms)39.3 ± 6.528.7 ± 4.333.0 ± 2.9ns
    CA3Rise time (ms)4.4 ± 0.33.5 ± 0.44.5 ± 0.2*P6–P7 vs P13–P14, 0.05
    *P13–P14 vs P21–P28, 0.03
    Time constant
    (ms)
    8.8 ± 0.17.0 ± 0.88.6 ± 0.6ns
    Latency
    (ms)
    61.5 ± 6.234.0 ± 4.535.8 ± 2.5*P6–P7 vs P13–P14, 0.008
    *P6–P7 vs P21–P28, 0.003
    • * indicates the statistical significance of p < 0.05.

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Development of Local Circuit Connections to Hilar Mossy Cells in the Mouse Dentate Gyrus
Yulin Shi, Steven F. Grieco, Todd C. Holmes, Xiangmin Xu
eNeuro 7 March 2019, 6 (2) ENEURO.0370-18.2019; DOI: 10.1523/ENEURO.0370-18.2019

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Development of Local Circuit Connections to Hilar Mossy Cells in the Mouse Dentate Gyrus
Yulin Shi, Steven F. Grieco, Todd C. Holmes, Xiangmin Xu
eNeuro 7 March 2019, 6 (2) ENEURO.0370-18.2019; DOI: 10.1523/ENEURO.0370-18.2019
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Keywords

  • development
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