ARTICLE |
Correspondence to: Günter Rager, Inst. of Anatomy and Special Embryology, University Fribourg, Rte A. Gockel 1, CH-1700 Fribourg, Switzerland.
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Summary |
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Dynamin I, a GTPase involved in the endocytic cycle of synaptic vesicle membranes, is believed to support axonal outgrowth and/or synaptogenesis. To explore the temporal and spatial patterns of dynamin I distribution in neuronal morphogenesis, we compared the developmental expression of dynamin with the expression of presynaptic membrane proteins such as SV2, synaptotagmin, and syntaxin in the chick primary visual pathway. Western blots of retina and tectum revealed a steady increase of synaptotagmin and syntaxin from embryonic Day 7 (E7) to E11, whereas for the same time frame no detectable increase of dynamin was found. Later stages showed increasing amounts of all tested proteins until the first postnatal week. Immunofluorescence revealed that SV2, synaptotagmin, and syntaxin are present in retinal ganglion cell axons from E4 on. In later stages, the staining pattern in the retina and along the visual pathway paralleled the formation and maturation of axons. In contrast, dynamin is not detectable by immunofluorescence in the developing retina and optic tectum before synapse formation. Our data indicate that, in contrast to the early expression of synaptotagmin, SV2, and syntaxin during axonal growth, dynamin is upregulated after synapse formation, suggesting its function predominantly during and after synaptogenesis but not in axonogenesis. (J Histochem Cytochem 47:12971306, 1999)
Key Words: dynamin, SV2, synaptotagmin, syntaxin, axonal growth, synapse formation, retinotectal projection
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Introduction |
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SEVERAL LINES OF EVIDENCE indicate that neuronal dynamin I, a 100-kD GTPase originally isolated from bovine brain (
Developmental studies using growing neurons in primary culture have demonstrated that other presynaptic membrane proteins (referred as SNAREs;
The retinotectal system of the chick has long been utilized to analyze the topographical organization of developing retinal ganglion cell (RGC) axons in the optic nerve, the chiasm, and the optic tract, and their formation of synapses in the optic tectum (
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Materials and Methods |
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Animals
White Leghorn chicks from hatching (P0) until posthatching Day 20 (P20) and chick embryos [(E3E20, Stages 1945, according to
Antibodies
For immunohistochemistry and Western blotting the following mouse monoclonal antibodies (MAbs) and/or rabbit polyclonal antisera were used. A rabbit antiserum directed against dynamin [DG-1 antibodies were generated against a human dynamin I 6x His-tag fusion protein lacking the proline-rich domain (
Western Blotting
For protein detection using the Western blot technique, samples of human, rat, and mouse neocortex and from chick hyperstriatum, retina, and tectum were homogenized and processed according to standard protocols (for further details see
Immunohistochemistry
For preparation of tissue sections, the eye cups, optic nerves, optic tracts, optic tecta, and whole brains were dissected out and immersion-fixed for 46 hr with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) or perfusion-fixed with the same fixative. The tissue was mounted with Tissue Tek embedding medium (Miles; Elkhart, IN) and rapidly frozen on a mounting block cooled over liquid nitrogen. Frontal cryosections were cut at 10 µm. For immunofluorescence, primary antibodies were diluted 1:100 (dynamin, SV2, and syntaxin) or 1:500 (synaptotagmin) in goat serum dilution buffer (20 mM NaPO4, 15% normal goat serum, 450 mM NaCl, 0.3 % Triton X-100). Antigenantibody complexes were visualized using fluorescein- or CY3-conjugated goat anti-rabbit or goat anti-mouse IgGs and were examined with a Leitz DM-RBE microscope equipped for epifluorescence (for further details see
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Results |
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Western blot analysis revealed that the rabbit antiserum DG-1 also recognizes neuronal chicken dynamin. Dynamin was detected in immunoreactive bands at 100 kD in neocortex homogenates of human, rat, and mouse brain and in the hyperstriatum homogenate of the chick brain (Figure 2).
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Western blots of equal total protein of the chick retina revealed only borderline signals for dynamin between embryonic Day 7 (E7) and E11, with no detectable increase of the protein during this period (Figure 3A). In the chick tectum, only very weak immunoreactivity for dynamin was found before E11 (Figure 3B). In contrast, both areas exhibited a steady increase of the proteins involved in exocytosis from E7 (for synaptotagmin see Figure 3A and Figure 3B; for syntaxin see Figure 3B). From E13 until the third postnatal week (postnatal Day 20, P20), increasing amounts of all the proteins investigated, including synaptotagmin, syntaxin, and dynamin were detected. Until adulthood, all tested proteins kept the high levels of expression as seen on P20 (Figure 3A and Figure 3B).
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Immunofluorescence in the chick retina revealed that synaptotagmin, SV2 (Figure 4A), and syntaxin are present in retinal ganglion cells (RGCs) and their axons from E4 on. First, immunoproducts are seen in the central part of the retina, where first RGCs are generated. With further development, immunoreactivity was detected in RGCs in all parts of the retina. This process of maturation, which is reflected in both the size and the structure of the inner plexiform layer, becomes visible by intense immunofluorescence for synaptotagmin, SV2, and syntaxin in the central retina from E8 on (for E10 see Figure 4B; for E13 see Figure 4D). In contrast, dynamin was not detectable by immunofluorescence in the retina before E10. Initial, very faint staining in the photoreceptor and ganglion cell perikarya was seen from E10 on (Figure 4C). At E13, dynamin was found in developing photoreceptors and their pedicles, in cells of the outer part of the inner nuclear layer, and in the inner plexiform layer (Figure 4E). The appearance of dynamin immunofluorescence in the inner plexiform layer of the central retina coincides temporally and spatially with the formation of the first synapses in the inner plexiform layer of the chick retina around E13 (
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Starting with E4, we found first immunopositive fibers for synaptotagmin (Figure 5A), SV2, and syntaxin (Figure 5B) leaving the retina through the optic fiber layer and the optic nerve head and entering the optic nerve. In this way the fibers fill the optic stalk rapidly, entering the gaps between undifferentiated neuroepithelial cells and the holes left by dying cells until E6 (compare Figure 5A and Figure 5B to Figure 5C and Figure 5D). After E10, immunoreactivities for synaptotagmin (Figure 5E), syntaxin (Figure 5F), and SV2 in the optic nerve and chiasm disappeared. Immunosignals in the ganglion cell axons along the optic tract (for E9 see Figure 6A) were found between E7 and E12 for synaptotagmin, syntaxin, and SV2. Finally, first immunostaining for these proteins in the superficial layers of the optic tectum was found at E8 (for E9 see Figure 6A and Figure 6B). In contrast, dynamin was not detectable by immunofluorescence in the developing optic tectum at these developmental stages (Figure 6C). From E11, detectable immunostaining for dynamin was found in the superficial layers of the optic tectum (for E13 see Figure 7C). From E13 until adulthood, all the proteins investigated, including synaptotagmin, syntaxin, SV2 (Figure 7A and Figure 7B), and dynamin (Figure 7C), were detected in the optic tectum.
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Discussion |
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In this study we applied the Western blotting and immunofluorescence techniques to analyze the developmental expression of presynaptic proteins, comparing dynamin I with the presynaptic membrane proteins synaptotagmin, syntaxin, and SV2, to learn more about the biogenesis and recycling of the plasma membrane during constitutive axon and synapse formation. Our data demonstrate that dynamin is upregulated only after synapse formation, whereas the other proteins are expressed during axonal growth (Figure 8). In addition, the temporospatial distribution of the presynaptic membrane proteins during development of the chick primary visual system reveals that the appearance of these proteins is closely correlated with the chronotropic pattern of axons described in previous morphological studies (
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Expression and Distribution of Dynamin Correlate with Synaptogenesis
Although the mechanisms underlying endocytic retrieval of presynaptic membrane material after exocytosis have not been fully established, it is widely accepted that the clathrin-dependent endocytosis is largely mediated and controlled by a number of proteins, including dynamin (
The absence of a dynamin immunofluorescent signal during axon development may be due to the lack of enrichment of the soluble protein in the axons and growth cones before functional synapses have appeared. In addition, our Western blot analysis of the retina and the tectum revealed a steady increase of the SNARE proteins syntaxin and synaptotagmin before synapse formation, whereas no detectable increase was found for dynamin during this period. It is possible that growing axons deliver only a minor fraction of dynamin compared to the SNARE membrane components, suggesting a higher rate of membrane insertion than membrane retrieval, which may cause axon elongation. Alternatively, our observations may imply that dynamin is not involved in constitutive membrane recycling during axon elongation and that its upregulation is triggered by synaptogenesis, possibly due to mechanisms involving electrical activity.
Expression and Distribution of SV2, Synaptotagmin, and Syntaxin Correlate with Axonogenesis
Given the complexity of the presynaptic localization of SNAREs (, showed that the newly synthesized membrane protein first appeared at the growth cone and that it was then redistributed over the plasma membrane surface (
In summary, our data on the developing chick retinotectal system demonstrate that the presynaptic membrane proteins synaptotagmin, syntaxin, and SV2 are expressed immediately after axon outgrowth as residents of heterogeneous organelles that probably represent a mixture of precursors for synaptic vesicles and the axon plasma membrane. Furthermore, the redistribution of these proteins during synapse formation, from the axon to the presynaptic terminal, is closely correlated with the chronotropic pattern of axons in the chick retinotectal projection and with the functional development of retinotectal synapses studied by electrophysiological techniques using both field potential analysis and single unit recording, which indicate that synaptic transmission is not effective before E11 (
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Acknowledgments |
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Supported by Swiss National Fond, grant 3100-049523. 96/1.
We thank R. Jahn (Göttingen, Germany) and P. DeCamilli (New Haven, CT) for generously supplying antibodies, and L. Clement, M. Kaczorowski, Ch. Marti, B. Scolari, and C. Weber for excellent technical assistance.
Received for publication December 21, 1998; accepted April 20, 1999.
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