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Featured ArticleArticles, Development/Plasticity/Repair

Early Synapse Formation in Developing Interneurons of the Adult Olfactory Bulb

Patrizia Panzanelli, Cedric Bardy, Antoine Nissant, Marta Pallotto, Marco Sassoè-Pognetto, Pierre-Marie Lledo and Jean-Marc Fritschy
Journal of Neuroscience 2 December 2009, 29 (48) 15039-15052; DOI: https://doi.org/10.1523/JNEUROSCI.3034-09.2009
Patrizia Panzanelli
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Cedric Bardy
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Antoine Nissant
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Marta Pallotto
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Marco Sassoè-Pognetto
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Pierre-Marie Lledo
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Jean-Marc Fritschy
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  • Figure 1.
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    Figure 1.

    Organization of GABAergic synapses in olfactory bulb granule cells. A , Immunofluorescence staining depicting the distribution of α2 subunit clusters in adult mouse OB. Brightly stained clusters positive for the α2 subunit are evident in all layers of the OB, albeit with higher density in the GCL and IPL than the EPL and GL. B–D , Images from triple-labeling experiments in sections from GFP-GAD67 mice showing the colocalization of α2 subunit-positive clusters (red) with gephyrin (blue) inside eGFP-positive GC dendrites (green) in the EPL, as visualized by confocal microscopy. The boxed areas are enlarged in C and D and depicted in three-dimensional mode to confirm the presence of α2 subunit/gephyrin clusters in close apposition to or inside eGFP-positive dendrites. E–G , Double immunoelectron microscopy for gephyrin (silver grains, arrows) and eGFP (*) in GAD67-GFP mice, depicting eGFP-negative axon terminals [presumably from short-axon cells (SAC)] making symmetric synapses on eGFP-positive dendrites of GC in EPL ( E ), in GCL ( F ), and in MCL ( G ), as well as on a mitral cell (MC) body ( G ). eGFP was revealed by immunoperoxidase and gephyrin by secondary antibodies conjugated to nanogold particles. Scale bars: A , 100 μm; B , 20 μm; E–G , 1 μm.

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    Figure 2.

    Early differentiation of newborn granule cells. A , Low-magnification photomicrograph depicting the distribution and morphology of eGFP-positive newborn GCs in adult mouse OB at 7 dpi, as detected by combined eGFP and immunofluorescence with antibodies against GFP. The dotted line indicates the mitral cell layer; the continuous line surrounds the accessory olfactory bulb (AOB). Scale bar, 200 μm. B , Schematic diagram of our experimental approach to label adult born GCs by stereotaxic injection of a lentivirus encoding eGFP into the RMS. C–E , Morphology of typical eGFP-positive GCs between 3 and 7 dpi; note the rapid growth of dendrites, becoming more branched and carrying numerous spine-like protrusions and filopodia. Scale bars: C , D , E , 50 μm; C′ , D′ , E′ , 5 μm. F , Example of an eGFP-positive GC filled with rhodamine during the patch-clamp recording. G , Graph of the mean ± SEM membrane input resistance of eGFP-positive neurons (3, 5, or 7 dpi) compared with “preexisting” GCs (non-GFP). H , Graph of the mean ± SEM voltage-dependent sodium current amplitude of eGFP-positive neurons increased rapidly toward those of preexisting cells. The number of cells in each group is shown in brackets. The Mann–Whitney p values are shown in italics.

  • Figure 3.
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    Figure 3.

    Early synaptic inputs onto newborn granule cells. A , Photomicrograph depicting a whole-cell recording of a newborn GC, with the recording electrode (Rec) implanted on a cell filled with rhodamine. The stimulating electrode (Stim) appears on the picture in a representative position. B , Voltage-clamp recordings at the reversal potential of AMPA- and NMDA-mediated currents (0 mV) in drug-free ACSF permitted to reveal evoked GABA currents (black trace: average of 26 nonfailure traces; success rate, 52%), which disappeared in the presence of gabazine (GZ), a GABAAR antagonist (blue trace: average of 26 traces; success rate, 0%). C , Voltage-clamp recordings at −70 mV in ACSF plus GZ showing evoked AMPA-mediated currents (black trace: average of 15 nonfailure traces; success rate, 30%), which disappeared when the AMPA receptor antagonist NBQX was added to the perfusion (blue trace: average of 15 traces; success rate, 0%). D , Voltage-clamp recordings at +40 mV in ACSF plus GZ plus NBQX showing evoked NMDA-mediated currents (black trace: average of 8 nonfailure traces; success rate, 16%). E , Voltage-dependent sodium current triggered by a depolarizing pulse of 55 mV. F , G , Double immunofluorescence staining for GABAAR α2 subunit (red) and PSD-95 or gephyrin (blue), depicting their clustered distribution on the soma, apical dendrites, and basal dendrites of eGFP-positive GC at 3 dpi; both pictures are maximum intensity projections of confocal images ( F , 16 sections spaced 0.5 μm; G , 11 sections spaced 0.35 μm). F′ , G′ , The presence of presumptive postsynaptic sites in these two newborn GCs is inferred from the distribution of colocalized pixels (yellow, α2/eGFP; cyan, PSD-95/eGFP; white, α2/gephyrin/eGFP); the eGFP signal is semitransparent for clarity. Scale bars, 5 μm.

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    Figure 4.

    Maturation of newborn granule cell GABAergic synaptic inputs. A1 , Voltage-clamp recordings of evoked GABA currents (in drug-free ACSF at 0 mV) of two typical newborn neurons at 3 and 7 dpi. A2 , Evoked (black) and spontaneous (orange) GABA current amplitudes of newborn neurons (3, 5, 7 dpi) and preexisting (non-GFP) cells. A3 , Success rates of evoked GABA responses. A4 , Spontaneous GABA events frequencies measured at 0 mV. B1 , Recordings of paired-pulse GABA synaptic currents showing a slight depression in newborn neurons at 5 and 7 dpi. B2 , The paired-pulse ratios of newborn neurons were not significantly different from those measured in preexisting cells. C1 , Normalized recordings of evoked GABA currents of typical newborn neurons at 5 and 7 dpi. The rise times are illustrated by the blue dots positioned at 20 and 80% of the peak responses. The decays were fitted with biexponentials function (red dashed line; R 2 > 0.9). C2 , The rise times were measured between 20 and 80% of the mean peak amplitude. C3 , The decay times are obtained from fast decay time constant (t f) of the biexponential fits. All values are given as mean ± SEM. The number of cells in each group is shown in brackets. The Mann–Whitney p values are shown in italics.

  • Figure 5.
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    Figure 5.

    Quantification of synaptic inputs formed on newborn granule cells. A–F , Representative images from confocal microscopy depicting the presence of PSD-95 clusters ( A , C , E ) and gephyrin clusters ( B , D , F ) colocalized with eGFP (yellow) at 3, 5, and 7 dpi, respectively, in the GCL. All pictures are maximum intensity projections of confocal images (10–16 sections spaced 0.35 μm). Scale bar, 10 μm. G–I , Quantification of PSD-95 and gephyrin clusters colocalized with eGFP on the soma, basal dendrites, and apical dendrites at 3, 5, and 7 dpi (for mean ± SEM, see supplemental Table 1, available at www.jneurosci.org as supplemental material). G , On the soma, the density of PSD-95 clusters increased significantly between 3 and 5 versus 7 dpi (Bonferroni's post test, p < 0.01), and at 7 dpi the density of PSD-95 clusters was significantly higher than gephyrin-positive clusters (Bonferroni's post test, p < 0.05). H , On basal dendrites, as well, PSD-95-positive clusters were significantly more numerous than gephyrin clusters (Bonferroni's post test, p < 0.05). I , On apical dendrites, the density of PSD-95 clusters increased significantly between 3 and 7 dpi (Bonferroni's post test, p < 0.05).

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    Figure 6.

    GABAergic input on granule cells spines. A–C , Distinction of VGAT-positive clusters inside and apposed to eGFP-positive dendrites and spine-like structures in the GCL at 3, 5, and 7 dpi. The arrows point to VGAT clusters apposed to dendrites, representing putative synaptic input; the arrowheads indicate VGAT clusters inside eGFP-positive dendrites; these VGAT proteins are probably transported toward the EPL to form reciprocal synapses. Note that, at 3 and 5 dpi, some VGAT clusters are formed onto eGFP-positive spines (*). Images are shown as three-dimensional projections in the three Cartesian planes, and sites of colocalization are marked in yellow. D , E , Double immunofluorescence staining for gephyrin (blue) and the α2 subunit (red) colocalized (magenta) in clusters inside eGFP-positive spine-like structures and dendritic shafts in GCL and in the EPL, suggestive of GABAergic input at 5 dpi. The framed areas are enlarged to better illustrate clusters in spines (*) and shafts (arrows). The pictures are maximum intensity projections of confocal sections (11–15 layers spaced by 0.4 μm). Scale bars: A , 5 μm; B , C , 3 μm; D , E , 5 μm.

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    Figure 7.

    Formation of functional GABAergic synapses in the EPL. A1 , Drawing illustrating the experimental setup to differentiate the responses from GCL and EPL synapses. A2 , Voltage-clamp recordings from a newborn GC (7 dpi) of evoked GABAergic currents in response to EPL stimulation. Puffs of gabazine blocked the responses only when applied in the EPL, indicating that the currents were evoked in distal synapses located in the EPL. B , Mean traces recorded from a newborn neuron in the GCL (7 dpi). The top trace was obtained by GCL stimulation and could be blocked in the GCL (average of 21 traces). The bottom trace was obtained by EPL stimulation and could be blocked in the EPL (average of 18 traces). C , Only cells in which we could evoke responses by EPL stimulation were used in this graph. These cells were classified as having synapses in the GCL only if the responses were blocked in the GCL but not in the EPL (black). If the responses could also be blocked in the EPL, the cells were classified in the group having functional synapses in the GCL and EPL (blue).

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    Figure 8.

    Formation of reciprocal synapses in the EPL. A–D , Double immunofluorescence staining for PSD-95 (blue) and α1/α3 clusters (red) to visualize reciprocal synapses. The two markers are either closely apposed and overlap partially (appearing white) or separate (cyan or yellow) on eGFP-positive dendrites at 4–5 dpi and on spine-like protrusions at 7 and 21 dpi. Each panel depicts at least one apposed cluster at the intersection of the xy cross. The pictures are maximum intensity projections of confocal sections (7–16 layers spaced by 0.35 μm) depicted as three-dimensional images. Scale bars: A–F , 5 μm. E , Density of PSD-95, α1/α3, and apposed clusters in eGFP-positive dendritic shaft at 4, 5, and 7 dpi. Although the total number of clusters remained constant between 4 and 7 dpi (one-way ANOVA, p = 0.2222), the density of single labeled PSD-95 and α1/α3 clusters decreased (Bonferroni's post test, p < 0.05) because of rapid formation of apposed clusters (Bonferroni's post test, 7 vs 4 dpi, p < 0.001; 5 vs 4 dpi, p < 0.001). In addition, at 4 dpi we observed more PSD-95 clusters “alone” than apposed clusters (Bonferroni's post test, p < 0.05). F , Quantification of spine density and labeling in the same dendrites as in E ). The number of spines increased significantly with time (one-way ANOVA, F (2,92) = 3.622; p = 0.0307). Although not all spines were labeled, a significant increase in the density of apposed clusters was seen at 5 and 7 dpi compared with 4 dpi (Bonferroni's post test, p < 0.001). The density of apposed clusters became significantly higher than single labeled clusters at 5 dpi (Bonferroni's post test, p < 0.05). At 7 dpi, spines with apposed clusters were significantly more numerous than spines labeled with α1/α3 clusters and nonlabeled spines (Bonferroni's post test, p < 0.05).

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    Figure 9.

    Double-labeling immunoelectron microscopy of newborn GCs in the EPL. Newborn eGFP-positive GCs make and receive GABAergic synapses, as seen at 7 dpi. A , Mitral dendrite profile (MC) receiving two symmetric GABAergic synapses strongly labeled for gephyrin (arrows); one synapse is made by an eGFP-positive (GFP+) GC profile and one by a nonlabeled GC profile. B , Example of a reciprocal synapse between a mitral cell dendrite and an eGFP-positive GC profile; arrows point to the postsynaptic sites; gephyrin labeling is evident in the symmetric synapse. C , Example of an eGFP-positive profile receiving a gephyrin-labeled symmetric synapse from an unlabeled axonal terminal (AT). D–G , Series of four serial sections depicting an asymmetric (excitatory) synapse formed by a mitral cell profile on a dendritic shaft of a newborn eGFP-positive GC (bottom left); the same mitral cell profile also receives a symmetric gephyrin-positive synapse from a nonlabeled GC profile (top right). Scale bars, 0.5 μm.

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    Table 1.

    Density of PSD-95 and gephyrin clusters on newborn GCs in the GCL

    Soma Basal dendrites Apical dendrites
    PSD-95GephyrinPSD-95GephyrinPSD-95Gephyrin
    3 dpi0.09 ± 0.020.15 ± 0.010.22 ± 0.050.18 ± 0.020.18 ± 0.020.23 ± 0.02
    5 dpi0.10 ± 0.020.13 ± 0.010.23 ± 0.040.17 ± 0.040.21 ± 0.030.22 ± 0.02
    7 dpi0.18 ± 0.03* ° 0.15 ± 0.020.30 ± 0.040.17 ± 0.030.28 ± 0.02* 0.29 ± 0.03
    • The density of clusters is the mean ± SD cluster per micrometer dendrite length or soma perimeter. Note the delayed increase of PSD-95 clusters on the soma and apical dendrites. The symbols indicate the results of Bonferroni's post hoc tests (for main statistical analysis, see Results).

    • ↵*7 dpi different from 3 dpi, p < 0.01;

    • ↵°7 dpi different from 5 dpi, p < 0.05.

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    Table 2.

    Density of reciprocal synapses in the EPL, visualized by clusters of PSD-95, α1/α3 subunit, and PSD-95 apposed to α1/α3 subunit

    PSD-95α1α3ApposedNonlabeledTotal
    Quantification on dendritic shafts
        4 dpi0.21 ± 0.02 †† 0.15 ± 0.020.10 ± 0.01
        5 dpi0.20 ± 0.02 § 0.10 ± 0.01 ‡ 0.22 ± 0.03**
        7 dpi0.15 ± 0.01 §§ † 0.07 ± 0.01° ‡ 0.21 ± 0.02°°
    Quantification on spines
        4 dpi0.04 ± 0.01 § 0.007 ± 0.0030.03 ± 0.0070.03 ± 0.0060.1 ± 0.02
        5 dpi0.04 ± 0.01 † 0.01 ± 0.004 ‡ 0.10 ± 0.01** 0.03 ± 0.007 ¤ 0.15 ± 0.02
        7 dpi0.07 ± 0.01 §§§ 0.007 ± 0.003 ‡ 0.10 ± 0.02°° 0.01 ± 0.003 ¥ ¤¤ 0.19 ± 0.02
    • Clusters were distinguished based on whether PSD-95 or α1/α3 subunit were either alone or apposed to each other. At the top (“Quantification on dendritic shafts”), the density of clusters formed on the shaft (mean ± SD) is given per micrometer/dendrite length. At the bottom (“Quantification on spines”), the density of spines (mean ± SD) per micrometer/dendrite length is given based on the type of cluster apposed to the spine. The density of unlabeled spines and the total spine density are indicated in the last two columns. The symbols indicate the results of Bonferroni's post hoc tests (for main statistical analysis, see Results).

    • ↵**5 dpi different from 4 dpi, p < 0.001;

    • ↵°7 dpi different from 4 dpi, p < 0.05;

    • ↵°°p < 0.001;

    • ↵ †PSD-95 different from apposed, p < 0.05;

    • ↵ †† p < 0.001;

    • ↵ ‡α1α3 different from apposed, p < 0.001;

    • ↵ §PSD-95 different from α1α3, p < 0.05;

    • ↵ §§ p < 0.01;

    • ↵ §§§ p < 0.001;

    • ↵ ¥nonlabeled different from PSD-95, p < 0.001;

    • ↵ ¤nonlabeled different from apposed, p < 0.01;

    • ↵ ¤¤ p < 0.001.

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The Journal of Neuroscience: 29 (48)
Journal of Neuroscience
Vol. 29, Issue 48
2 Dec 2009
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Early Synapse Formation in Developing Interneurons of the Adult Olfactory Bulb
Patrizia Panzanelli, Cedric Bardy, Antoine Nissant, Marta Pallotto, Marco Sassoè-Pognetto, Pierre-Marie Lledo, Jean-Marc Fritschy
Journal of Neuroscience 2 December 2009, 29 (48) 15039-15052; DOI: 10.1523/JNEUROSCI.3034-09.2009

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Early Synapse Formation in Developing Interneurons of the Adult Olfactory Bulb
Patrizia Panzanelli, Cedric Bardy, Antoine Nissant, Marta Pallotto, Marco Sassoè-Pognetto, Pierre-Marie Lledo, Jean-Marc Fritschy
Journal of Neuroscience 2 December 2009, 29 (48) 15039-15052; DOI: 10.1523/JNEUROSCI.3034-09.2009
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