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First published online April 22, 2009
doi: 10.1242/10.1242/jcs.045401

Journal of Cell Science 122, 1352-1361 (2009)
Published by The Company of Biologists 2009
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Aberrant function and structure of retinal ribbon synapses in the absence of complexin 3 and complexin 4

Kerstin Reim1, Hanna Regus-Leidig2, Josef Ammermüller3, Ahmed El-Kordi4, Konstantin Radyushkin4, Hannelore Ehrenreich4, Johann Helmut Brandstätter2,* and Nils Brose1,*

1 Department of Molecular Neurobiology and Center for the Molecular Physiology of the Brain, Max Planck Institute of Experimental Medicine, D-37075 Göttingen, Germany
2 Department of Biology, Animal Physiology, University of Erlangen-Nuremberg, D-91058 Erlangen, Germany
3 AG Neurobiology, Carl von Ossietzky University of Oldenburg, D-26111 Oldenburg, Germany
4 Division of Clinical Neuroscience and Center for the Molecular Physiology of the Brain, Max Planck Institute of Experimental Medicine, D-37075 Göttingen, Germany


Figure 1
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  		Fig. 1. Mutation of the murine Cplx4 gene. (A) Deletion of Cplx4 in the mouse. Maps of the wild-type Cplx4 gene, the respective targeting vector, and the resulting mutant gene. Exons 1 and 2 (black boxes, E1,E2), the position of the outside probe (OP) used to identify the mutant allele (open bar), and diagnostic restriction enzyme sites are indicated. Neo, neomycin resistance gene; TK, thymidine kinase gene. (B) Southern blot analysis of Cplx4 deletion in mice. Tail DNA from an adult wild-type mouse (+/+), and mice heterozygous (+/-) or homozygous (-/-) for the Cplx4 mutation were analyzed as described in Materials and Methods. Positions of wild-type (WT) and knockout (KO) bands are indicated. (C) Analysis of Cplx4 expression in wild-type, heterozygous and homozygous mice. Brain and retina homogenates (10 µg protein per lane) from adult wild-type (+/+), heterozygous (+/-), and homozygous (-/-) mice were analyzed by SDS-PAGE and immunoblotting using an anti-Cplx4 antibody. The position of Cplx4 is indicated. (D) Analysis of Cplx3 expression in retina of wild-type, heterozygous and homozygous Cplx3 mutant mice. Retina homogenates (10 µg protein per lane) from adult wild-type (+/+), heterozygous (+/-), and homozygous (-/-) mice were analyzed by SDS-PAGE and immunoblotting using an anti-Cplx3 antibody. The position of Cplx3 is indicated. (E) Expression of Cplx3 and Cplx4 in retina of Cplx3/4 double deletion mutant mice generated from two double heterozygous parents. Retina extracts (10 µg protein per lane) of adult wild-type (+/+, +/+), Cplx3 knockout (-/-, +/+), Cplx4 knockout (+/+, -/-), and double deletion mutant mice (-/-, -/-) were analyzed by SDS-PAGE and immunoblotting using anti-Cplx3 and anti-Cplx4 antibodies, respectively. Syntaxin 3 served as a loading control.

 

Figure 2
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  		Fig. 2. Analysis of synaptic proteins in Cplx3, Cplx4 and Cplx3/4 single and double-knockout mice. In order to detect changes in protein composition of mutant synapses, identical amounts of retina homogenates (5-10 µg protein per lane, depending on the protein to be studied) from adult wild-type (+/+) and homozygous (-/-) mice were analyzed by SDS-PAGE and immunoblotting using antibodies against the indicated proteins.

 

Figure 3
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  		Fig. 3. Comparison of a- and b-wave amplitudes and implicit times from wild-type and Cplx3 (A-C), Cplx4 (D-F), and Cplx3/4 (G-I) single and double-knockout mice. (A,D,G) Example responses of Cplx3, Cplx4 and Cplx3/4 wild-type (WT), single deletion (KO) and double deletion (DKO) mutant mice. Light intensity, 10 cds/m2. (B,E,H) Intensity dependence of a- and b-wave amplitudes for Cplx3, Cplx4 and Cplx3/4 knockout (KO, DKO) and wild-type (WT) mice. (C,F,I) Implicit times versus intensity dependence of a- and b-waves for Cplx3, Cplx4 and Cplx3/4 knockout (KO, DKO) and wild-type (WT) mice. Data are means ± s.e.m.; n=7 for Cplx3 wild-type; n=8 for Cplx3 knockout, n=6 for Cplx4 wild-type, n=5 for Cplx4 knockout, n=5 for Cplx3/4 double knockout and wild-type, respectively.

 

Figure 4
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  		Fig. 4. Analysis of oscillatory potentials in ERG recordings. (A) Examples of Cplx3 knockout (KO) and wild-type (WT) responses. Arrows a and b indicate measurements of a- and b-wave amplitudes. (B) Bandpass filtered (30-300 Hz) responses from (A). The grey area indicates the time window for FFT analysis. (C) FFT power spectrum of the oscillatory potentials from B. The arrows indicate the measurement of peak power and peak frequency. (D,F,H) Intensity dependency of OP peak frequency for Cplx3, Cplx4 and Cplx3/4 knockout (KO, DKO) and wild-type (WT) mice. (E,G,I) Intensity dependency of OP peak power for Cplx3, Cplx4 and Cplx3/4 knockout (KO, DKO) and wild-type (WT) mice. Note that the intensity range is smaller for the graphs showing OP analysis, since OPs occur only at higher intensities. Data are means ± s.e.m. n=7 for Cplx3 wild-type; n=8 for Cplx3 knockout, n=6 for Cplx4 wild-type, n=5 for Cplx4 knockout, n=5 for Cplx3/4 double knockout and wild-type, respectively. For clarity the y-axis is enlarged in E, compared with G and I. The peak power values are statistically identical for the wild-type mice in E and I.

 

Figure 5
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  		Fig. 5. Behavioural signs of impaired vision in Cplx3/4 double-knockout mice. (A) Impaired performance of Cplx3/4 double-knockout mice (DKO) in cued platform test. Wild-type (WT), open squares, n=11; DKO, solid squares, n=11. Two-way ANOVA for repeated measurements revealed a significant effect of genotype (F(1,20)=7.40, P=0.0132). Cplx3/4 double-knockout mice showed normal performance in the visual cliff test (B) and in the rotarod test (C). Open bars, WT; solid bars, DKO.

 

Figure 6
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  		Fig. 6. Comparison of retinal anatomy and neuronal morphology in wild-type and Cplx3 and Cplx4 single- and double-knockout mice. (A) Comparison of Nomarski micrographs of vertical sections of wild-type and knockout retinae shows that deletion of Cplx3 and/or Cplx4 does not affect the morphological appearance of the retina. (B,C) Light micrographs of vertical sections through wild-type and Cplx3 and Cplx4 single- and double-knockout retinae double labelled for distinct populations of retinal neurons: horizontal (green) and amacrine (magenta) cells (B), cone photoreceptors (green) and rod and cone bipolar (magenta) cells. PNA, peanut agglutinin. (C). Neuronal morphology is normal in the complexin-knockout retinae. PhRS, photoreceptor segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar: 20 µm.

 

Figure 7
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  		Fig. 7. Immunocytochemical analysis of Cplx1/2, Cplx3 and Cplx4 expression in wild-type, Cplx3- and Cplx4-knockout, as well as Cplx3/4 double-knockout retina. (A) Comparison of vertical sections of wild-type and knockout retinae shows that deletion of Cplx3 or/and Cplx4 does not affect the expression level and distribution of Cplx1 and Cplx2. (B,C) Representative images of vertical sections of wild-type and knockout retinae stained for Cplx3 (B) and Cplx4 (C) show that Cplx3 staining is absent in the Cplx3-knockout and Cplx3/4 double-knockout retina and Cplx4 is absent in the Cplx4 knockout and Cplx3/4 double-knockout retina. Labelling artefacts in (B,C) are marked by arrowheads. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars: 20 µm.

 

Figure 8
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  		Fig. 8. Immunocytochemical comparison of SNARE protein expression in wild-type, Cplx3- and Cplx4-knockout, as well as Cplx3/4 double-knockout retina. (A-D) Representative images of vertical sections of wild-type and knockout retinae stained for Syntaxin 1 (A), Syntaxin 3 (B), SNAP25 (C), and Synaptobrevin 2 (D) show that deletion of Cplx3 and/or Cplx4 does not affect the expression level and distribution of these SNARE proteins. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars: 20 µm.

 

Figure 9
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  		Fig. 9. Abnormal photoreceptor ribbons in Cplx3/4 double-knockout mice. (A,B) Representative images of the outer plexiform layer and the photoreceptor ribbon synapses triple labelled for Bassoon (green), Ribeye (red), and Piccolo (blue) from wild-type (A) and Cplx3/4 double-knockout mice (B). The boxes indicate the selected ribbons in the outer plexiform layer, which are shown at higher magnification. The photoreceptor ribbon synaptic site loses its compact appearance in the Cplx3/4 double-knockout retina (B). (C-E) Representative electron micrographs of photoreceptor ribbon synapses in Cplx3/4 double-knockout mice. Examples of anchored ribbon-shaped photoreceptor ribbon (C), anchored club-shaped photoreceptor ribbon (D), and free-floating spherical ribbons (E) are shown. The inset in E shows a higher magnification of the free-floating spherical ribbons. Arrowheads indicate the presynaptic ribbons. (F) Percentage of photoreceptor terminals that contain anchored ribbons, free floating spherical ribbons, or no ribbons in wild-type (dark grey bars) and Cplx3/4 double-knockout retinae (light grey bars). OPL, outer plexiform layer; Hc, horizontal cell; Bc, bipolar cell. Scale bars: 10 µm (A,B); 0.2 µm (C-E).

 

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© The Company of Biologists Ltd 2009