Secreted semaphorins control spine distribution and morphogenesis in the postnatal CNS

Abstract

The majority of excitatory synapses in the mammalian CNS (central nervous system) are formed on dendritic spines, and spine morphology and distribution are critical for synaptic transmission, synaptic integration and plasticity. Here, we show that a secreted semaphorin, Sema3F, is a negative regulator of spine development and synaptic structure. Mice with null mutations in genes encoding Sema3F, and its holoreceptor components neuropilin-2 (Npn-2, also known as Nrp2) and plexin A3 (PlexA3, also known as Plxna3), exhibit increased dentate gyrus (DG) granule cell (GC) and cortical layer V pyramidal neuron spine number and size, and also aberrant spine distribution. Moreover, Sema3F promotes loss of spines and excitatory synapses in dissociated neurons in vitro, and in Npn-2(-/-) brain slices cortical layer V and DG GCs exhibit increased mEPSC (miniature excitatory postsynaptic current) frequency. In contrast, a distinct Sema3A-Npn-1/PlexA4 signalling cascade controls basal dendritic arborization in layer V cortical neurons, but does not influence spine morphogenesis or distribution. These disparate effects of secreted semaphorins are reflected in the restricted dendritic localization of Npn-2 to apical dendrites and of Npn-1 (also known as Nrp1) to all dendrites of cortical pyramidal neurons. Therefore, Sema3F signalling controls spine distribution along select dendritic processes, and distinct secreted semaphorin signalling events orchestrate CNS connectivity through the differential control of spine morphogenesis, synapse formation, and the elaboration of dendritic morphology.

Figure 1. Sema3F and Npn-2 regulate dendritic spine number, distribution and morphology in adult layer V pyramidal neurons and dentate gyrus granule cells in vivo

a–f, Golgi stained Sema3F^−/− (b, e) and Npn-2^−/− (c, f) brains show that DG GC and layer V pyramidal apical dendritic spines (white arrows) are more numerous as compared to WT (a, d). g–i, Layer V pyramidal neurons have more spines on primary apical dendrites 0–25 µm from the soma in Sema3F^−/− (h) and Npn-2^−/− (i) mutants as compared to WT mice (g). j, k, Quantification of spine density 0–50µm from the cell body on layer V pyramidal primary apical dendrites (WT, 0.10 ±0.01; Sema3F^−/−, 0.34 ±0.01; Npn-2^−/−, 0.35 ±0.01 spines/µm) and 0–25µm from the cell body on DG GC primary dendrites (WT, 0.55 ±0.03; Sema3F^−/−, 1.08 ±0.04 and Npn-2^−/−, 1.21 ±0.04 spines/µm). There is a significant increase in spine number on dendritic segments located 100–150µm from the cell body in layer V (WT, 1.33 ±0.14; Sema3F^−/−, 2.14 ±0.13; Npn-2^−/−, 2.00 ±0.11 spines/µm) and 50–75µm from the DG neuron cell body (WT, 1.28 ±0.13; Sema3F^−/−, 2.40 ±0.11; Npn-2^−/−, 2.34 ±0.12 spines/µm) in these mutants. There is no significant difference in spine density at 200–250µm or 100–125µm from the cell body in layer V and DG neurons, respectively. Error bars, ±SEM, ANOVA, post-hoc Tukey in j and k; **, p=0.01; ***, p=0.001 compared to WT. Scale bars: 10 µm in f for a–f and 2.5 µm in i for g–i.

Figure 2. Sema3F–Npn-2 control of spine number is Npn-2 cell-autonomous, and Npn-2 loss-of-function results in increased frequency of mEPSCs

a–c, Layer V neurons from P45 WT and Npn-2^−/− animals from E13.5 embryos in utero electroporated with IRES-mGFP (a, b), and a Npn-2^−/− embryo electroporated with Npn-2-IRES-mGFP (c). d, Quantification of spine density (50–100µm from soma) in Npn-2^−/− neurons transfected with Npn-2-IRES-mGFP (0.61 ±0.03 spines/µm) as compared to IRES-mGFP (0.99 ±0.03 spines/µm), and to WT neurons transfected with IRES-mGFP (0.72 ±0.04 spines/µm). e, Recordings of mEPSCs from cortical slices show a significant increase in mEPSC frequency in Npn-2^−/− layer V pyramidal neurons (4.85 ±0.87 Hz) as compared to WT littermates (2.04 ±0.64 Hz). There is no significant difference in mEPSC amplitude between WT (9.21 ±0.32 pA) and Npn-2^−/− (8.60 ±0.35 pA) neurons. Representative mEPSC traces (top, WT; bottom, Npn-2^−/−) are shown. Error bars in d and e, ±SEM; ANOVA, post-hoc Tukey for d; p=0.001 for **, WT:IRES-mGFP vs. Npn-2^−/−:IRES-mGFP and ψ, Npn-2^−/−:IRES-mGFP vs. Npn-2^−/−:Npn-2-IRES-mGFP; p=0.071 for WT vs. Npn-2^−/−:Npn-2-IRES-mGFP. T-test for e, **, p=0.024 for frequency and p=0.231 for amplitude. Scale bars: 10 µm in c for a–c, and 5 pA × 1s in e.

Figure 3. Sema3F-Npn2 signaling regulates spine morphology and synaptic ultrastructure in vivo

a, b, DG GC dendritic spine TEM ultrastructural analysis reveals enlarged and misshapen spines (arrowheads) in Npn2^−/− (b) as compared to WT mice (a). c, d, 3-D reconstructions of serial TEM illustrates two completely separate PSDs within a single spine from a Npn-2^−/− mutant mouse (d), in contrast to a WT spine (c) with one PSD per spine head. Scale bars: 500 nm in b for a, b, and 250 nm³ in d for c, d

Figure 4. Distinct Sema3–Npn/PlexA signaling modules regulate apical dendrite spine morphology and basal dendrite process complexity

a–d, Golgi-labeled adult brains illuminate basal dendritic morphologies in cortical layer V pyramidal neurons from WT (a, circle), PlexA3^−/− (b), Npn-1^Sema− (c), and PlexA4^−/− (d) mice. a’–d’, show spine morphologies from neurons in a–d. Scale bars: 10 µm in d for a–d and 4 µm in d’ for a’–d’.

Figure 5. Npn-1 and Npn-2 are localized to distinct cortical pyramidal neuron dendritic domains

a–i, Alkaline phosphatase (AP)-Sema3F (a–f) or AP-Sema3A (g–i) fusion proteins were used to localize endogenous Npn-2 and Npn-1, respectively, on cortical pyramidal neurons in primary culture. Endogenous Npn-2 (a, c, d, f) is predominately restricted to the major apical dendrites of pyramidal neurons as shown by anti-AP labeling (a–c). Npn-2 is not observed on basal (white arrows in a, c) or secondary apical dendritic branches (white arrows in d, e) illuminated by anti-Map2 staining. Npn-1 (g, i) is observed on basal and apical dendritic processes (h, i). j, k, Fluorescence intensities were quantified by measuring apical/basal fluorescence ratios and normalizing to apical/basal Map2 fluorescence ratios for AP-Sema3F–labeled neurons (j, 3.70 ±0.87), and for AP-Sema3A–labeled neurons (0.80 ±0.14). The Npn-2/Map2 fluorescence ratio in primary apical dendrites (yellow bar, k) is 0.95 (±0.17), and in secondary apical dendrites (red bar, k) is 0.08 (±0.06). Gray boxes, f, indicate area of measurement in k. Scale bars: 25 µm in i for a–i.