Article Figures & Data
Figures
- Figure 1.
Soluble Sema4D-Fc treatment rapidly increases the density of inhibitory synapses. Ai, Representative stretches of dendrite from neurons treated with either Fc control or Sema4D-Fc and immunostained for GAD65 (red), GABAARγ2 (blue), and MAP2 (green) to visualize dendrites at 0.5, 1, 2, and 4 h of treatment. Scale bars, 2 μm. Aii, Quantification of inhibitory synapse density (GAD65/γ2) as percentage synapse density of Fc control treated neurons (100%, represented by dashed line). Aiii, Aiv, Quantification of the average area of GAD65 (Aiii) and GABAARγ2 (Aiv) puncta as the percentage of Fc control treated neurons (100%, represented by dashed line). Av, Avi, Quantification of the average fluorescence intensity of GAD65 (Av) and GABAARγ2 (Avi) puncta as the percentage of Fc control treated neurons (100%, represented by dashed line; n > 50 neurons in each condition, 3+ experiments; *p < 0.05, two-way ANOVA). B, Left: Representative images of growth cones from cultured hippocampal neurons treated with Fc control (10 nm, left) or Sema4D-Fc (10 nm, right). Right: Quantification of the percentage collapsed growth cones (n > 84 growth cones per treatment; scale bar, 5 μm; *p < 0.05 Student's t test). All data are plotted as mean ± SEM.
- Figure 2.
Sema4D-Fc treatment increases the density of inhibitory but not excitatory synapses. A, Top: Representative stretches of dendrite from neurons treated with either Fc control or Sema4D-Fc for 0.5 or 1 h and immunostained for Synapsin I (red), Gephyrin (blue), and MAP2 (green). Scale bars, 5 μm. Bottom: Quantification of inhibitory (Gephyrin/Synapsin) synapse density of neurons treated with Sema4D-Fc for 0.5, 1, 2, and 4 h plotted as percentage of Fc control treated neurons (100% represented by dashed line; n > 40 neurons in each condition, 2+ experiments). B, Top: Representative stretches of dendrite from neurons treated with either Fc control or Sema4D-Fc for 0.5 or 1 h and immunostained for Synapsin I (red), GluA2 (blue), and MAP2 (green). Bottom: Quantification of excitatory synapse density (measured by GluA2/Synapsin I staining) of neurons treated 0.5, 1, 2 and 4 h plotted as a percentage of Fc control treated neurons (100% represented by dashed line; n > 45 neurons in each condition from at least 3 experiments for 0.5, 1 and 2 h; n>20 neurons from 1 experiment for 4 h). Scale bars, 5 μm.*p < 0.05, two-way ANOVA.
- Figure 3.
Sema4D-Fc drives formation of functional GABAergic synapses. A, Representative whole-cell voltage-clamp recordings of mIPSCs from primary hippocampal neurons treated with Fc control (left) or Sema4D-Fc (right) for 0.5, 1, 2, and 4 h. B, Quantification of mIPSC (top) frequency and (bottom) amplitude, *p < 0.05, Student's t test compared with corresponding Fc control. Data plotted as mean ± SEM. C, D, Cumulative distribution plots of mIPSC interevent intervals (C) and mIPSC amplitude (D) at 0.5, 1, 2, and 4 h of Sema4D-Fc treatment. n = 14 neurons for all conditions from three experiments. *p < 0.02, Kolmogorov–Smirnov test.
- Figure 4.
Sema4D-Fc triggers an increase in GABAergic synapse density in a PlexinB1-dependent manner. A, Representative stretches of dendrites from neurons (wildtype, top; PlxnB1−/−, bottom) treated with Fc control (left) or Sema4D-Fc (right) immunostained for the presynaptic protein GAD65 (red), the postsynaptic protein GABAARγ2 (blue), and MAP2 (green) to visualize dendrites. Scale bars, 2 μm. B, Quantification of inhibitory synapse density. n > 58 neurons for each condition from three experiments; *p < 0.05, two-way ANOVA. Data are plotted as mean ± SEM.
- Figure 5.
The formation of functional GABAergic synapses in response to Sema4D-Fc treatment is PlexinB1 dependent. A, Representative mIPSCs recorded from wildtype (top) or PlxnB1−/− (bottom) CA1 neurons in acute hippocampal slice treated with either Fc control or Sema4D-Fc for 2 h. B, C, Quantification of mIPSC frequency (B) and mIPSC amplitude (C); n = 29 neurons per condition; *p < 0.05 compared with wildtype Fc control treatment, Student's t test. For A–C, All data are plotted as mean ± SEM. D, E, Cumulative distribution plots of mIPSC interevent intervals (D) and amplitude (E) in wildtype (gray lines) and PlxnB1−/− mice (blue lines) in the absence (Fc Control) or presence of Sema4D-Fc. n = 29 neurons each condition from four experiments; *p < 0.02, Kolmogorov–Smirnov test.
- Figure 6.
Sema4D-AP treatment promotes GABAergic synapse formation onto both dendrites and somas of hippocampal neurons. A, Stretches of dendrites from cultured hippocampal neurons isolated from PlxnB1−/− or wildtype littermates (11 DIV) treated with AP control (AP alone) or Sema4D-AP for 4 h. Neurons were immunostained for GAD65 (red), GABAARγ2 (blue), and MAP2 (green). Scale bar, 5 μm B, Inhibitory synapses (GAD65/GABAAR γ2) on the somas of neurons treated with AP control or Sema4D-AP were analyzed by tracing somas (white dashed lines) and synapse density was quantified within these regions of interest. Scale bar, 5 μm. C, Quantification of synapse density from A and B plotted as a percentage of AP control (100% represented by dashed line; data are plotted as mean ± SEM; n > 20, 2 experiments; *p < 0.05, two-way ANOVA).
- Figure 7.
Application of soluble Sema4D-Fc leads to a rapid increase in the rate of GFP-Gephyrin addition in cultured hippocampal neurons. A, Stretches of dendrite from cultured rat hippocampal neurons expressing GFP-Gephyrin and treated with Fc control (left) or Sema4D-Fc (right). Scale bars, 2 μm. Below each dendrite is a kymograph of the region highlighted by the red box above that visualizes the movement of puncta over time. B indicates before treatment; 10, 0–10 min after treatment; 20, 10–20 min after treatment; 30, 20–30 min after treatment; blue arrows, puncta splitting event. B, Additional representative kymographs from sample stretches of different dendrites from the same neurons as in A. Scale bars: y-axis = 3 min, x-axis = 3 μm. C, Top: The number of puncta added (average per neuron) during each imaging session in either Fc control (light gray) or Sema4D-treated neurons (dark gray). *p < 0.05, Student's t test. Bottom: The average number of GFP-Gephyrin puncta added normalized to the total number of GFP-Gephyrin puncta per neuron. *p < 0.05, Student's t test. D, Top: The number of GFP-Gephyrin puncta removed (average per neuron) during the imaging session in either Fc control (light gray) or Sema4D-treated neurons (dark gray). Bottom: The average number of GFP-Gephyrin puncta removed normalized to the total number of GFP-Gephyrin puncta per neuron. The average number of GFP-Gephyrin puncta was not different between conditions. Sema4D-Fc: n = 5 neurons, 1025 puncta, average 205 puncta/neuron (±22.75); Fc control: n = 3 neurons, 446 puncta, average 148.67 puncta/neuron (±33.9). All data are plotted as mean ± SEM.
- Figure 8.
Sema4D-Fc treatment rapidly suppresses TTX-induced epileptic activity. A, Representative traces of spontaneous activity observed in untreated (left) or TTX-treated slices (TTX-EA, right) with either Fc control (top) or Sema4D-Fc treatment (bottom). Note that for these representative traces, spikes are only present in the TTX-EA Fc control condition. B, Quantification of average spike frequency (top) and total area (bottom) measured from baseline (dashed red line) for each condition; n > 12 neurons for each condition; *p < 0.05, Student's t test. C, D, Representative traces of sIPSCs (C) or sEPSCs (D) observed in untreated (left) or TTX-EA slices (1 μm, right) with either Fc control (1 nm, top) or Sema4D-Fc treatment (1 nm, bottom). Shown is the quantification of total inhibitory synaptic charge (C) or total excitatory synaptic charge (D) measured from the same cell for each experimental condition. For C and D, n = 19 neurons for each condition; *p < 0.05, Student's t test. All data are plotted as mean ± SEM.
Tables
Fc control Sema4D % Stable 0.945 ± 0.19 0.934 ± 0.019 Instantaneous velocity (μm/min) 1.005 ± 0.06 0.925 ± 0.18 Average track distance (μm) 0.752 ± 0.07 0.897 ± 0.16 Average track duration (s) 59.464 ± 4.76 74.498 ± 14.09 Average puncta total distance (μm) 2.090 ± 1.07 2.078 ± 0.47 Average puncta total duration (s) 141.016 ± 54.38 186.045 ± 24.55 Sema4D treatment does not alter the baseline dynamics of GFP-Gephyrin puncta. Dynamics of GFP-Gephyrin puncta after 10 min of Sema4D-Fc or Fc control treatment are shown. See Materials and Methods for details of calculations. Sema4D-Fc: n = 5 neurons, 1025 puncta, average 205 puncta/neuron (±22.75); Fc control: n = 3 neurons, 446 puncta, average 148.67 puncta/neuron (±33.9). All data are shown as ± SEM; p > 0.05 in all conditions, t test.
