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Differential ultrastructural distribution of synapsin and synaptophysin proximal to a ligation in bovine splenic nerve
Brain Research 802 Ž1998. 281–284
Short communication
Differential ultrastructural distribution of synapsin and synaptophysin proximal to a ligation in bovine splenic nerve J. Quatacker a
a, )
, P. Partoens b , W. De Potter
b
N. Goormaghtigh Institute of Pathology, UniÕersity Hospital, De Pintelaan 185, B-9000 Ghent, Belgium b Laboratory of Neuropharmacology, UniÕersity of Antwerp, B-2060 Belgium Accepted 9 June 1998
Abstract Synaptophysin and synapsin, closely correlated on synaptic vesicles in terminals, may show a differential distribution at synapse formation and maturation. In order to disclose the fine structural details of these differences, synapsin and synaptophysin distribution was studied by immunocytochemistry on ligated bovine splenic axons in vitro and compared with terminals in the vas deferens. In the synaptic differentiations taking place proximally synapsin could only be detected on the accumulating elements of the axonal reticulum. Large dense granules and clusters of small synaptic vesicles were negative. Synaptophysin was restricted to these clusters. In the vas deferens, co-localization of synapsin and synaptophysin could be seen on small vesicles. From their formation small synaptic vesicles carry synaptophysin. Synapsin may be involved in the dynamic membrane changes taking place at the ligation. At a functional terminal, synapsin shifts to small synaptic vesicles. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Axonal reticulum; Axonal transport; Sympathetic nerve; Small synaptic vesicle; Synapsin; Synaptophysin
After 3 h of in vitro incubation, segments of ligated bovine splenic nerves w17,18x already show clear signs of synaptic differentiation, revealing granules and homogeneous clusters of small, synaptophysin-positive w18x vesicles ŽSSV.. Besides synaptophysin, synapsin is one of the other major synaptic vesicle proteins, closely associated with the synaptic vesicles of the functioning, mature nerve terminal w20x. Also, in PCl2 cell w21x co-localization was detected on synaptic vesicles. On the other hand, in synaptogenesis synapsin appears later than synaptophysin and increases at synaptic maturation w4,12x. Synapsin and synaptophysin, present in growing axons, become restricted to boutons after interaction with the target w8,13,19x or acquisition of function by the synapse w14x. In ligated nerves synapsin w1,9x and synapsin together with synaptophysin w6x were shown to accumulate proximally. These results reveal important shifts in synapsin distribution during synaptic differentiation and maturation w19x, but they do not reveal the exact structural elements carrying the synapsin and synaptophysin molecules. Abbreviations: SSV: small synaptic vesicle; CLA: common leucocytic antigen; GMA: glycolmethacrylate; PTA: phosphotungstic acid ) C orresponding author. Fax: q 32-9-2404965; E-m ail: [email protected] 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 6 1 7 - 9
Therefore, we wanted to investigate the distribution of synapsin in relation to synaptophysin in an axon undergoing synaptic differentiation w17,18x and compare this with the distribution of synaptophysin and synapsin in an adult, functioning terminal. For that purpose, synaptophysin- and synapsin-immunoreactivity were studied at the ultrastructural level in ligated bovine splenic nerves in vitro and in adrenergic terminals of the vas deferens. From the results, a mechanism of synaptic vesicle differentiation in adrenergic nerves is deduced. Bovine splenic nerve, spleen capsule and vas deferens were collected at the slaughterhouse, chilled on ice and transported to the laboratory. Synaptic differentiation was induced by incubating ligated bovine splenic nerve in Krebs–Ringer bicarbonate buffer Ž5% C0 2r95% O 2 . for 3 h. For that purpose, the splenic nerve was freed from adhering tissue and axon bundles of approximately 60 mm length were tied to a glass rod and ligated w17,18x. After incubation, the stretches of axons, still fixed to the rod, were placed in a tube containing chilled fixative Ž2% formaldehyde, 0.1% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2–7.4., kept overnight in the fixative and rinsed in 0.1 M cacodylate buffer, pH 7.2–7.4. The first 2 mm proximally were collected and further subdivided. The capsule of the spleen and the vas deferens were cut in
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small blocks and immediately fixed, as above. The specimens were then dehydrated with and embedded in glycolmethacrylate ŽGMA. without postfixation w10x. They were put in gelatine capsules for UV polymerization in the refrigerator. Monoclonal anti-synaptophysin Žclone SY 38, 1–2 mgrml. was from Boehringer, Mannheim. A polyclonal anti-synapsin antibody Ž1r200. was made w11x and characterized w2x. Monoclonal anti-common leucocytic antigen ŽCLA. was from DAKO, Glostrup, DK. Pre-immune rabbit serum was prepared in the laboratory. Gold labeled goat anti-rabbit IgG Ž15 nm. and goat anti-mouse IgG Ž10 nm, Auroprobe. were from Amersham, Poole, UK. The ultrathin GMA sections were single or double labeled by an indirect method as previously described w16x. In the controls, the primary antiserum was replaced by pre-immune rabbit serum or monoclonal anti-CLA. These control antibodies were applied at a dilution which gave the same level of non-specific labeling over the nuclei, as with the primary antibodies to be tested. The labeling intensity was estimated by calculating the average number of gold particles per relevant area ŽMini-Mop, Kontron, Eching, Germany.. After immunolabeling the GMA sections were stained with saturated uranyl acetate in 50% alcohol and alkaline lead citrate for 3 and 2 min, respectively, or stained for 4 min on a drop of 1% phosphotungstic acid ŽPTA. in 1N HCl and quickly dried with filterpaper.
Sections were examined in a Zeiss EM 900 at 80 kV in high contrast mode. In the unincubated splenic nerve, no labeling signal could be detected in the axoplasm with anti-synapsin or anti-synaptophysin antibody, when compared with appropriately diluted pre-immune rabbit serum or CLA, respectively Žnot shown.. After 3 h of incubation, anterogradely transported organelles accumulated in a zone of 0.8–0.9 mm length, starting at 0.3 mm above the ligation. This zone contained membrano-tubular structures ŽFig. 1., clustered granular elements of different size ŽFigs. 2 and 3. and mitochondria. Near the proximal end of this zone, the axon was somewhat dilated and the axoplasm was less crowded. In this zone, clusters of SSVs Žcf. Ref. w18x. were more easily found ŽFig. 3.. Upon ligation, adrenergic nerves show synaptic differentiation, as they acquire many properties of sympathetic nerve terminals w5x. The vesicular and tubular elements of the axonal reticulum ŽFig. 1. carried label for synapsin. Depending on the packing, the labeling was 3 to 4 times above background level, as determined in controls with pre-immune rabbit serum. The synapsin, described to be present proximal to a ligation w1,6,9x, can be considered to be mainly associated with the complex tubules of the accumulating axonal reticulum. In the Ur and Pb contrasted sections, it was evident that dense granules only sporadically carried a gold particle ŽFigs. 2 and 3..
Fig. 1. GMA, anti-synapsin Ž1r200., PTA staining. The crowded axon shows PTA-positive tubular and vesicular profiles. They regularly carry label Žarrowheads.. Labeling intensity over these crowded areas is 3 to 4 times above background. Labeling is clearly reduced over areas containing few PTA-positive profiles Župper right.. =40,000; bar s 250 nm.
J. Quatacker et al.r Brain Research 802 (1998) 281–284
283
Fig. 4. GMA, anti-synapsin Ž1r200., PTA staining. In the terminals of the vas deferens, the anti-synapsin antibody gives a strong labeling. The gold particles are detected over small, faintly contrasted elements. This is consistent with the labeling of SSVs. Granular elements are faintly revealed Žarrowhead.. =40,000. Fig. 2. GMA, anti-synapsin Ž1r200., Ur and Pb. A mixed population of tubulo-vesicular and granular profiles is represented. The large dense granules do not carry gold particles, but gold labeling may be found over the membrane space. =40,000; bar s 250 nm.
A labeling signal for synapsin, as found in terminals w20x, could not be detected over clusters of SSVs ŽFig. 3. and may imply that we are dealing with immature, nonfunctioning vesicles. But the SSVs were substantially labeled for synaptophysin ŽFig. 3. and it was further determined, by gold particle counts, that this labeling was 3.5 to 4 times above the labeling of the axoplasm. The labeling of the axoplasm was at background level. It was already shown w7x that the synaptic vesicles are organized into releasable packets prior to target contact, thus, before they acquire detectable amounts of synapsin. The results
demonstrate that synapsin is following another transport route than synaptophysin and that basically small synaptic vesicles could form in the absence of synapsin. This difference can be expected considering the integral vs. peripheral nature w20x of synaptophysin and synapsin. Because of the sparsity of terminals in the spleen capsule, the vas deferens was finally chosen to demonstrate the distribution of synaptophysin and synapsin in mature, well-differentiated adrenergic synapses. In this material, synapses were readily recognized by their strong labeling for both synapsin ŽFigs. 4 and 5. and synaptophysin ŽFig. 5.. After PTA staining labeling was found over faintly contrasted structures. In double labeling experiments, co-localization of synapsin and synaptophysin on the same microvesicular elements was observed ŽFig. 5..
Fig. 3. GMA, anti-synapsin Ž1r200. and anti-synaptophysin Ž2 mgrml., Ur and Pb. Labeling for synaptophysin Žsmall gold particles, 10 nm. is seen on a large cluster of SSVs Žasterisk, cf. Ref. w18x for normal ultrastructure.. Labeling for synapsin Žlarge gold particles, 15 nm. is absent from the clusters. A 15 nm particle is seen on a membranous element Žarrowhead.. Apart from a single gold particle Žabove., no labeling signal for synapsin is detected on the dense granules. =51,000; bar s 250 nm.
Fig. 5. GMA, anti-synapsin Ž1r200. and anti-synaptophysin Ž1 mgrml., PTA staining. The terminals in the vas deferens readily bind anti-synapsin and anti-synaptophysin antibody. Both large Žsynapsin. and small Žsynaptophysin. gold particles are found on faintly appearing structures. In several places, large and small particles are arranged in tandem Žsmall arrowheads., suggesting co-localization on a small Žvesicular. structure. The larger granular structures are only faintly contrasted Žlarge arrowhead. =40,000; bar s 250 nm.
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J. Quatacker et al.r Brain Research 802 (1998) 281–284
Substantial labeling was absent over dense tubular or granular structures. In ligated nerves, the synapsin molecules may be accumulating due to a block in transport as for ex. dopamine-b hydroxylase w16,17x, neuropeptide Y and others, which are transported with the fast axonal transport. However, it was reported that the bulk of synapsin is not bound to membranes, but transported in association with the soluble cytomatrix in the slow axonal transport w3,15x. At a ligation, the synapsin may become involved in the membrane dynamics of the axonal reticulum in the same way as it is implicated in the linking of SSVs to the cytoskeleton w7,20x in terminals. If this is the case, the association of synapsin with the tubules at a ligation could be a functional one and this process may bring about a selective accumulation of synapsin. This seems the more attractive as the synapsin activity may, finally, at a terminal, be concentrated on the SSVs in a further shift. If the formation of synaptic vesicles coincides with the appearance of synaptophysin, the redistribution of synapsin to the SSVs in terminals may be brought about by the acquisition at maturation of their transport function w14x. Taken together, the synapsin is concentrated in those areas were its function is and the building up of synaptic vesicles can be considered a multistep process. Our data also indicate that synapsin is not involved in the trafficking of large dense granules, again emphasizing the difference in nature of the granular and SSV pathway w18x.
References w1x S. Akagi, A. Mizoguchi, K. Sobue, H. Nakamura, C. Ide, Localization of synapsin I in normal fibers and regenerating axonal sprouts of the rat sciatic nerve, Histochem. Cell Biol. 105 Ž1996. 365–373. w2x V. Baekelandt, L. Arckens, W. Annaert, U.T. Eysel, G.A. Orban, F. Vandesande, Alterations in GAP-43 and synapsin immunoreactivity provide evidence for synaptic reorganisation in adult cat dorsal lateral geniculate nucleus following retinal lesions, Eur. J. Neurosci. 6 Ž1994. 754–765. w3x C. Baitinger, M. Williard, Axonal transport of synapsin I-like proteins in rabbit retinal ganglion cells, J. Neurosci. 7 Ž1987. 3723– 3735. w4x J.L. Bixby, L.F. Reichardt, The expression and localization of synaptic vesicle antigens at the neuromuscular junction in vitro, J. Neurosci. 5 Ž1985. 3070–3080.
w5x S. Calvo, C. Gonzalez-Garcia, V. Cena, Axoplasmic transport of w3Hx ouabain binding sites and catecholamine secretion from an adrenergic nerve trunk, Mol. Pharmacol. 42 Ž1992. 141–146. w6x A.B. Dahlstrom, A.J. Czernik, J.-Y. Li, Organelles in fast axonal transport. What molecules do they carry in anterograde versus retrograde directions, as observed in mammalian systems?, Mol. Neurobiol. 6 Ž1992. 157–177. w7x Z.S. Dai, H.B. Peng, Dynamics of synaptic vesicles in cultured spinal cord neurons in relationship to synaptogenesis, Mol. Cell. Neurosci. 7 Ž1996. 443–452. w8x T.L. Fletcher, P. Cameron, P. De Camilli, G. Banker, The distribution of synapsin I and synaptophysin in hippocampal neurons developing in culture, J. Neurosci. 11 Ž1991. 1617–1627. w9x G. Fried, L. Terenius, E. Brodin, S. Efendic, G. Dockray, J. Fahrenkrug, M. Goldstein, T. Hokfelt, Neuropeptide Y, enkephalin and noradrenaline coexist in sympathetic neurons innervating the bovine spleen, Cell Tissue Res. 243 Ž1986. 495–508. w10x E.H. Leduc, W. Bernhard, Recent modification of the glycolmethacrylate embedding procedure, J. Ultrastruct. Res. 19 Ž1967. 196–199. w11x I. Llona, W.G. Annaert, W.P. De Potter, Simultaneous purification of the neuroproteins synapsin I and synaptophysin, J. Chromatogr. 596 Ž1992. 51–58. w12x B. Lu, A.J. Czernik, S. Popov, T. Wang, M.M. Poo, P. Greengard, Expression of synapsin I correlates with maturation of the neuromuscular synapse, Neuroscience 74 Ž1996. 1087–1097. w13x C.A. Mason, Axon development in mouse cerebellum: embryonic axon forms and expression of synapsin I, Neuroscience 19 Ž1986. 1319–1333. w14x R.H. Melloni Jr., P.J. Apostolides, J.E. Hamos, L.J. De Gennaro, Dynamics of synapsin I gene expression during establishment and restoration of functional synapses in the rat hippocampus, Neuroscience 58 Ž1994. 683–703. w15x T.C. Petrucci, P. Macioce, P. Paggi, Axonal transport kinetics and posttranslational modification of synapsin I in mouse retinal ganglion cells, J. Neurosci. 11 Ž1991. 2938–2946. w16x J.R. Quatacker, W.G. Annaert, B.J. Miserez, W.P. De Potter, Immunocytochemical demonstration of dopamine-b-hydroxylase and cytochrome B561 on the axonal reticulum in bovine sympathetic neurons, J. Histochem. Cytochem. 40 Ž1992. 1599–1604. w17x J. Quatacker, W. Annaert, W. De Potter, The organisation of the axonal reticulum at a ligation, in in vitro incubated bovine splenic nerves, Brain Res. 680 Ž1995. 36–42. w18x J. Quatacker, W. De Potter, Ultrastructural evidence for a separate, small synaptic vesicle ŽSSV. pathway in ligated bovine splenic nerves, incubated in vitro, Brain Res. 731 Ž1996. 101–107. w19x F. Rene, P. Poisbeau, C. Egles, R. Schlichter, J.M. Felix, Co-culture of hypothalamic neurons and melanotrope cells: a model to study synaptogenesis between central neurons and endocrine cells, Neuroscience 76 Ž1997. 203–214. w20x T.C. Sudhof, R. Jahn, Protein of synaptic vesicles involved in exocytosis and membrane recycling, Neuron 6 Ž1991. 665–677. w21x J.H. Tao-Cheng, A. Dosemeci, J.P. Bresler, M.W. Brightman, D.L. Simpson, Characterization of synaptic vesicles and related neuronal features in nerve growth factor and ras oncogene differentiated PCl2 cells, J. Neurosci. Res. 42 Ž1995. 323–334.
Short communication
Differential ultrastructural distribution of synapsin and synaptophysin proximal to a ligation in bovine splenic nerve J. Quatacker a
a, )
, P. Partoens b , W. De Potter
b
N. Goormaghtigh Institute of Pathology, UniÕersity Hospital, De Pintelaan 185, B-9000 Ghent, Belgium b Laboratory of Neuropharmacology, UniÕersity of Antwerp, B-2060 Belgium Accepted 9 June 1998
Abstract Synaptophysin and synapsin, closely correlated on synaptic vesicles in terminals, may show a differential distribution at synapse formation and maturation. In order to disclose the fine structural details of these differences, synapsin and synaptophysin distribution was studied by immunocytochemistry on ligated bovine splenic axons in vitro and compared with terminals in the vas deferens. In the synaptic differentiations taking place proximally synapsin could only be detected on the accumulating elements of the axonal reticulum. Large dense granules and clusters of small synaptic vesicles were negative. Synaptophysin was restricted to these clusters. In the vas deferens, co-localization of synapsin and synaptophysin could be seen on small vesicles. From their formation small synaptic vesicles carry synaptophysin. Synapsin may be involved in the dynamic membrane changes taking place at the ligation. At a functional terminal, synapsin shifts to small synaptic vesicles. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Axonal reticulum; Axonal transport; Sympathetic nerve; Small synaptic vesicle; Synapsin; Synaptophysin
After 3 h of in vitro incubation, segments of ligated bovine splenic nerves w17,18x already show clear signs of synaptic differentiation, revealing granules and homogeneous clusters of small, synaptophysin-positive w18x vesicles ŽSSV.. Besides synaptophysin, synapsin is one of the other major synaptic vesicle proteins, closely associated with the synaptic vesicles of the functioning, mature nerve terminal w20x. Also, in PCl2 cell w21x co-localization was detected on synaptic vesicles. On the other hand, in synaptogenesis synapsin appears later than synaptophysin and increases at synaptic maturation w4,12x. Synapsin and synaptophysin, present in growing axons, become restricted to boutons after interaction with the target w8,13,19x or acquisition of function by the synapse w14x. In ligated nerves synapsin w1,9x and synapsin together with synaptophysin w6x were shown to accumulate proximally. These results reveal important shifts in synapsin distribution during synaptic differentiation and maturation w19x, but they do not reveal the exact structural elements carrying the synapsin and synaptophysin molecules. Abbreviations: SSV: small synaptic vesicle; CLA: common leucocytic antigen; GMA: glycolmethacrylate; PTA: phosphotungstic acid ) C orresponding author. Fax: q 32-9-2404965; E-m ail: [email protected] 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 6 1 7 - 9
Therefore, we wanted to investigate the distribution of synapsin in relation to synaptophysin in an axon undergoing synaptic differentiation w17,18x and compare this with the distribution of synaptophysin and synapsin in an adult, functioning terminal. For that purpose, synaptophysin- and synapsin-immunoreactivity were studied at the ultrastructural level in ligated bovine splenic nerves in vitro and in adrenergic terminals of the vas deferens. From the results, a mechanism of synaptic vesicle differentiation in adrenergic nerves is deduced. Bovine splenic nerve, spleen capsule and vas deferens were collected at the slaughterhouse, chilled on ice and transported to the laboratory. Synaptic differentiation was induced by incubating ligated bovine splenic nerve in Krebs–Ringer bicarbonate buffer Ž5% C0 2r95% O 2 . for 3 h. For that purpose, the splenic nerve was freed from adhering tissue and axon bundles of approximately 60 mm length were tied to a glass rod and ligated w17,18x. After incubation, the stretches of axons, still fixed to the rod, were placed in a tube containing chilled fixative Ž2% formaldehyde, 0.1% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2–7.4., kept overnight in the fixative and rinsed in 0.1 M cacodylate buffer, pH 7.2–7.4. The first 2 mm proximally were collected and further subdivided. The capsule of the spleen and the vas deferens were cut in
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J. Quatacker et al.r Brain Research 802 (1998) 281–284
small blocks and immediately fixed, as above. The specimens were then dehydrated with and embedded in glycolmethacrylate ŽGMA. without postfixation w10x. They were put in gelatine capsules for UV polymerization in the refrigerator. Monoclonal anti-synaptophysin Žclone SY 38, 1–2 mgrml. was from Boehringer, Mannheim. A polyclonal anti-synapsin antibody Ž1r200. was made w11x and characterized w2x. Monoclonal anti-common leucocytic antigen ŽCLA. was from DAKO, Glostrup, DK. Pre-immune rabbit serum was prepared in the laboratory. Gold labeled goat anti-rabbit IgG Ž15 nm. and goat anti-mouse IgG Ž10 nm, Auroprobe. were from Amersham, Poole, UK. The ultrathin GMA sections were single or double labeled by an indirect method as previously described w16x. In the controls, the primary antiserum was replaced by pre-immune rabbit serum or monoclonal anti-CLA. These control antibodies were applied at a dilution which gave the same level of non-specific labeling over the nuclei, as with the primary antibodies to be tested. The labeling intensity was estimated by calculating the average number of gold particles per relevant area ŽMini-Mop, Kontron, Eching, Germany.. After immunolabeling the GMA sections were stained with saturated uranyl acetate in 50% alcohol and alkaline lead citrate for 3 and 2 min, respectively, or stained for 4 min on a drop of 1% phosphotungstic acid ŽPTA. in 1N HCl and quickly dried with filterpaper.
Sections were examined in a Zeiss EM 900 at 80 kV in high contrast mode. In the unincubated splenic nerve, no labeling signal could be detected in the axoplasm with anti-synapsin or anti-synaptophysin antibody, when compared with appropriately diluted pre-immune rabbit serum or CLA, respectively Žnot shown.. After 3 h of incubation, anterogradely transported organelles accumulated in a zone of 0.8–0.9 mm length, starting at 0.3 mm above the ligation. This zone contained membrano-tubular structures ŽFig. 1., clustered granular elements of different size ŽFigs. 2 and 3. and mitochondria. Near the proximal end of this zone, the axon was somewhat dilated and the axoplasm was less crowded. In this zone, clusters of SSVs Žcf. Ref. w18x. were more easily found ŽFig. 3.. Upon ligation, adrenergic nerves show synaptic differentiation, as they acquire many properties of sympathetic nerve terminals w5x. The vesicular and tubular elements of the axonal reticulum ŽFig. 1. carried label for synapsin. Depending on the packing, the labeling was 3 to 4 times above background level, as determined in controls with pre-immune rabbit serum. The synapsin, described to be present proximal to a ligation w1,6,9x, can be considered to be mainly associated with the complex tubules of the accumulating axonal reticulum. In the Ur and Pb contrasted sections, it was evident that dense granules only sporadically carried a gold particle ŽFigs. 2 and 3..
Fig. 1. GMA, anti-synapsin Ž1r200., PTA staining. The crowded axon shows PTA-positive tubular and vesicular profiles. They regularly carry label Žarrowheads.. Labeling intensity over these crowded areas is 3 to 4 times above background. Labeling is clearly reduced over areas containing few PTA-positive profiles Župper right.. =40,000; bar s 250 nm.
J. Quatacker et al.r Brain Research 802 (1998) 281–284
283
Fig. 4. GMA, anti-synapsin Ž1r200., PTA staining. In the terminals of the vas deferens, the anti-synapsin antibody gives a strong labeling. The gold particles are detected over small, faintly contrasted elements. This is consistent with the labeling of SSVs. Granular elements are faintly revealed Žarrowhead.. =40,000. Fig. 2. GMA, anti-synapsin Ž1r200., Ur and Pb. A mixed population of tubulo-vesicular and granular profiles is represented. The large dense granules do not carry gold particles, but gold labeling may be found over the membrane space. =40,000; bar s 250 nm.
A labeling signal for synapsin, as found in terminals w20x, could not be detected over clusters of SSVs ŽFig. 3. and may imply that we are dealing with immature, nonfunctioning vesicles. But the SSVs were substantially labeled for synaptophysin ŽFig. 3. and it was further determined, by gold particle counts, that this labeling was 3.5 to 4 times above the labeling of the axoplasm. The labeling of the axoplasm was at background level. It was already shown w7x that the synaptic vesicles are organized into releasable packets prior to target contact, thus, before they acquire detectable amounts of synapsin. The results
demonstrate that synapsin is following another transport route than synaptophysin and that basically small synaptic vesicles could form in the absence of synapsin. This difference can be expected considering the integral vs. peripheral nature w20x of synaptophysin and synapsin. Because of the sparsity of terminals in the spleen capsule, the vas deferens was finally chosen to demonstrate the distribution of synaptophysin and synapsin in mature, well-differentiated adrenergic synapses. In this material, synapses were readily recognized by their strong labeling for both synapsin ŽFigs. 4 and 5. and synaptophysin ŽFig. 5.. After PTA staining labeling was found over faintly contrasted structures. In double labeling experiments, co-localization of synapsin and synaptophysin on the same microvesicular elements was observed ŽFig. 5..
Fig. 3. GMA, anti-synapsin Ž1r200. and anti-synaptophysin Ž2 mgrml., Ur and Pb. Labeling for synaptophysin Žsmall gold particles, 10 nm. is seen on a large cluster of SSVs Žasterisk, cf. Ref. w18x for normal ultrastructure.. Labeling for synapsin Žlarge gold particles, 15 nm. is absent from the clusters. A 15 nm particle is seen on a membranous element Žarrowhead.. Apart from a single gold particle Žabove., no labeling signal for synapsin is detected on the dense granules. =51,000; bar s 250 nm.
Fig. 5. GMA, anti-synapsin Ž1r200. and anti-synaptophysin Ž1 mgrml., PTA staining. The terminals in the vas deferens readily bind anti-synapsin and anti-synaptophysin antibody. Both large Žsynapsin. and small Žsynaptophysin. gold particles are found on faintly appearing structures. In several places, large and small particles are arranged in tandem Žsmall arrowheads., suggesting co-localization on a small Žvesicular. structure. The larger granular structures are only faintly contrasted Žlarge arrowhead. =40,000; bar s 250 nm.
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J. Quatacker et al.r Brain Research 802 (1998) 281–284
Substantial labeling was absent over dense tubular or granular structures. In ligated nerves, the synapsin molecules may be accumulating due to a block in transport as for ex. dopamine-b hydroxylase w16,17x, neuropeptide Y and others, which are transported with the fast axonal transport. However, it was reported that the bulk of synapsin is not bound to membranes, but transported in association with the soluble cytomatrix in the slow axonal transport w3,15x. At a ligation, the synapsin may become involved in the membrane dynamics of the axonal reticulum in the same way as it is implicated in the linking of SSVs to the cytoskeleton w7,20x in terminals. If this is the case, the association of synapsin with the tubules at a ligation could be a functional one and this process may bring about a selective accumulation of synapsin. This seems the more attractive as the synapsin activity may, finally, at a terminal, be concentrated on the SSVs in a further shift. If the formation of synaptic vesicles coincides with the appearance of synaptophysin, the redistribution of synapsin to the SSVs in terminals may be brought about by the acquisition at maturation of their transport function w14x. Taken together, the synapsin is concentrated in those areas were its function is and the building up of synaptic vesicles can be considered a multistep process. Our data also indicate that synapsin is not involved in the trafficking of large dense granules, again emphasizing the difference in nature of the granular and SSV pathway w18x.
References w1x S. Akagi, A. Mizoguchi, K. Sobue, H. Nakamura, C. Ide, Localization of synapsin I in normal fibers and regenerating axonal sprouts of the rat sciatic nerve, Histochem. Cell Biol. 105 Ž1996. 365–373. w2x V. Baekelandt, L. Arckens, W. Annaert, U.T. Eysel, G.A. Orban, F. Vandesande, Alterations in GAP-43 and synapsin immunoreactivity provide evidence for synaptic reorganisation in adult cat dorsal lateral geniculate nucleus following retinal lesions, Eur. J. Neurosci. 6 Ž1994. 754–765. w3x C. Baitinger, M. Williard, Axonal transport of synapsin I-like proteins in rabbit retinal ganglion cells, J. Neurosci. 7 Ž1987. 3723– 3735. w4x J.L. Bixby, L.F. Reichardt, The expression and localization of synaptic vesicle antigens at the neuromuscular junction in vitro, J. Neurosci. 5 Ž1985. 3070–3080.
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