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First published online 14 March 2006
doi: 10.1242/jcs.02862

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Role of neuropsin in formation and maturation of Schaffer-collateral L1cam-immunoreactive synaptic boutons

Division of Structural Cell Biology, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, Japan

View larger version (175K): [in a new window]   Fig. 1. L1 localization to presynaptic terminals in the Schaffer-collateral pathway. (A) Overview of staining by anti-L1 antibody with DAB in the hippocampus of adult mice. The arrowheads indicate strongly stained black bundles from CA3 soma and immunoreactive fibers projected toward the stratum radiatum in the CA1 subfield (arrow). Stratum oriens (str. ori.), stratum pyramidale (str. pyr.), stratum radiatum (str. rad.) and lacunosum-moleculare (lac-mol.) are indicated. (B) Expression of L1 mRNA in hippocampus detected by in situ hybridization. Intense hybridization signals in the pyramidal layer, especially in CA3 pyramidal cells, appear as silver grains under dark-field illumination. (C) Higher magnification of L1ir axon bundles from CA3 soma marked by arrowheads in A. (D) Immunoelectron microscopic analysis of regions marked by arrowheads in A and C. L1ir is observed on and in axons (arrowheads). (E) Higher magnification of L1ir fibers marked by arrow in A. Some fasciculated axons show L1ir (arrows). (F) Higher magnification of an L1ir axon in the CA1 stratum radiatum. Arrowheads indicate L1ir boutons on axons. (G-I) Immunoelectron microscopic localization of L1 by pre-embedding staining in the CA1 stratum radiatum. L1ir is distributed in presynaptic sites of asymmetrical synapses (open arrowheads). L1ir was observed in orphan boutons containing a few vesicles (I). Closed arrowheads are L1ir-negative asymmetrical synapses. (J-L) Immunogold labeling of L1ir in CA1 stratum radiatum. Gold particles (10 nm, indicated by open arrowheads) are associated with the presynaptic membrane, the presynapse interior (J,K), and orphan boutons (L). Closed arrowheads indicate L1ir-negative asymmetrical synapses. Bars, 300 µm (A,B); 100 µm (C); 1 µm (D); 5 µm (E,F); 300 nm (I for G-I; J-L).

View larger version (92K): [in a new window]   Fig. 2. Morphology of L1ir and L1ir-negative synapses. (A) Electron micrographs of consecutive ultrathin sections (1-11) and three-dimensional reconstruction demonstrating the L1ir orphan bouton (a), L1ir synapse (b) and L1ir-negative synapse (c) of the same microscopic field in NP+/+ mice (12). (B) Complete three-dimensional reconstruction of L1ir orphan boutons (a), L1ir synapses (b) and L1ir-negative synapses (c) in NP+/+ and NP-/- mice. Reconstructed presynapses are green, postsynapses are blue, their PSDs are light blue and L1-immunoreactive sites are red. Bars, 600 nm for all panels.

View larger version (21K): [in a new window]   Fig. 3. Shift from smaller to larger L1ir synapses in NP-/- mice. (A) The stratum radiatum was divided into three regions, proximal (prox.), middle (mid.) and distal (dist.), according to distance from the CA1 soma. In NP+/+ mice (open bars), the mean cross-sectional spine head area of L1ir (L1) synapses is significantly smaller than that of L1ir-negative (L1neg) synapses in all regions of the stratum radiatum. The mean spine head area of L1ir synapses is larger in NP-/- mice (filled bars) than in NP+/+ mice (open bars), especially in the proximal region. *P<0.05; **P<0.001. (B) The distribution of spine head area in the proximal region of L1ir synapses and L1ir-negative synapses in NP+/+ and NP-/- mice. In NP+/+ mice, L1ir synapses were significantly smaller than L1ir-negative synapses (Kolmogorov-Smirnov test, P<0.03). In NP-/- mice, the distribution of L1ir synapses shifted to a larger size, comparable with that of L1ir-negative synapses. (C) The mean cross-sectional PSD length of L1ir synapses was significantly smaller than that of L1ir-negative synapses in NP+/+ (open) and NP-/- (closed) mice, except in the proximal region where the length of L1ir synapses in NP-/- mice was significantly larger than that in NP+/+ mice and comparable with the level in L1ir-negative synapses (P>0.55). *P<0.05; **P<0.001.

View larger version (69K): [in a new window]   Fig. 4. Change in the distribution of L1ir sites in NP-/- mice. (A) Low-power immunoelectron micrographs of the proximal region of the CA1 stratum radiatum in NP+/+ (upper panel) and NP-/- (lower panel) mice. There are more L1ir synaptic (arrowheads) and L1ir orphan boutons (asterisks) in NP-/- mice than in NP+/+ mice. Arrows show L1ir-negative synapses. Bar, 1 µm. (B) Western blot analysis of L1 in hippocampus of NP+/+ and NP-/- mice. Both the 80 and 200 kDa L1ir bands were detectable. Actin served as internal control for protein loading. The graph shows the quantification of band intensities of L1200 and L180. The band density of L1 was significant increase in NP-/- mice. *P<0.01.

View larger version (34K): [in a new window]   Fig. 5. Increased number of L1ir synaptic and orphan boutons in NP-/- mice. (A) Typical distribution of L1ir synapses (dots) in NP+/+ (left) and NP-/- (right) mice in the CA1 subfield. L1ir synaptic boutons are plotted on a montage of 25 low-power (x6000) electron micrographs of stratum pyramidale (str.pyr.), stratum radiatum (str.rad.) and stratum lacunosum moleculare (lac-mol.). The stratum radiatum (str. rad.) was divided into three regions, proximal (prox.), middle (mid.) and distal (dist.), according to the distance from CA1 soma. (B) The number of asymmetrical L1ir synapses was counted in NP+/+ (open bars) and NP-/- (closed bars) mice. The left graph shows the number of L1ir synapses in a fixed area (13.0x219.6 µm) of the CA1 stratum radiatum, and the right graph shows the number in each region according to the distance from CA1 soma in a fixed area (13.0x73.2 µm). The mean number of L1ir synapses was significantly higher in NP-/- mice. *P<0.05. (C) Total number of asymmetrical synapses containing L1ir and L1ir-negative synapses was counted in NP+/+ (open bars) and NP-/- (closed bars) mice. The left graph shows the number of synapses in the stratum radiatum in a fixed area (13.0x219.6 µm), and the right shows the number in each region depending on the distance from the CA1 soma in a fixed area (13.0x73.2 µm). The mean number of total synapses was significantly lower in the proximal region of NP-/- mice. *P<0.05. (D) The mean number of L1ir orphan boutons was significantly higher in NP-/- (closed bars) mice than NP+/+ (open bars) mice in the proximal region in a fixed area (13.0x16.5 µm). *P<0.05; **P<0.01.

View larger version (26K): [in a new window]   Fig. 6. Injection of recombinant active neuropsin into NP-/- mice restores L1ir synapses to NP+/+ levels. (A) Fluorescent microscopic photograph represent spread of recombinant actneuropsin in the hippocampus. Injection of FITC-conjugated recombinant actneuropsin reaches the observation site of the stratum radiatum. (B) Number (left) and percentage (right) of L1ir synapses one day after injection of ACSF or active neuropsin (act NP) into NP-/- mice. The number and percentage of L1ir synapses were significantly decreased (actNP, closed bars; *P<0.05) to the levels in untreated wild-type mice (untreated, open bars). There was no significant difference between the treated NP-/- mice and untreated wild-type mice (number, P>0.24; percentage, P>0.08). **P<0.001.

View larger version (52K): [in a new window]   Fig. 7. Density of spines is the same in NP+/+ and NP-/- mice. (A) Fluorescence images of hippocampal CA1 pyramidal neurons of NP+/+ (left) and NP-/- mice (right) after injection of Lucifer Yellow. Arrowheads denote areas enlarged in B: proximal (prox.) (a and d), middle (mid.) (b and e) and distal (dist.) (c and f). (B) Spine morphology of NP+/+ (left) and NP-/- (right) mice in prox. (a and d), mid. (b and e) and dist. (c and f) regions. Images are from the regions marked by the respective arrowheads in A. Bar, 5 µm. (C) Spines were counted in NP+/+ (open bars) and NP-/- (closed bars) mice. The left graph shows the density of spines in the stratum radiatum, and the right graph shows that in each region according to the distance from the CA1 soma. There was no difference in the density of spines between NP+/+ and NP-/- mice.

View larger version (28K): [in a new window]   Fig. 8. Diagrammatic model of synapse conversion. Formation of synapses is divided into two phases, an early phase where L1ir orphan boutons make contact with small spines (a) and a late phase where small synapses containing L1 grow into large synapses lacking L1 (b). Neuropsin is considered to be involved in both phases. Presynaptic boutons are shown in pale green, spines in pale blue, PSD is bright blue, L1 in red, and synaptic vesicles as circles.