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Peptide containing vesicles within neuro-neuronal synapses
Brain Research, 169 (1979) 55-64 © Elsevier/North-Holland Biomedical Press
55
PEPTIDE CONTAINING VESICLES WITHIN NEURO-NEURONAL SYNAPSES
GONTHER STERBA, GEORG HOHEISEL, RUTH WEGELIN, WILFR1ED NAUMANN and FRANK SCHOBER Sektion Biowissenschaften, Bereich Zellbiologie und Regulation, Karl-Marx-Universitiit, 701 Leipzig (G.D.R.) (Accepted September 21st, 1978)
SUMMARY
In the descending part of the classical neurosecretory system, the axon terminals are not differentiated or they take the form of presynaptic elements which then form synaptoids or synapses with pituicytes or adenohypophyseal glandular cells respectively. In contrast, the axon terminals of the ascending part fulfil the criteria of true presynaptic elements which form synapses with other neurones. The presence of neurophysin vesicles in the presynaptic element is a particular morphologic feature of these neuro-neuronal synapses.
INTRODUCTION
The neurones of the classical peptidergic neurosecretory system of the vertebrates are localized in the magnocellular hypothalamic praeoptic nuclei (in anamnia) or in the paraventricular and supraoptic nuclei (in amniota). Their perikarya produce the octapeptide hormone ocytocin or other naturally occurring ocytocin analogues (vasopressin, vasotocin, mesotocin, etc.) and the carrier protein neurophysins. The hormones, bound to the carrier and enclosed in vesicles, are transported within the axons to different sites. We prefer to use in this connection the terms 'peptidergic neurosecretory system' and 'neurophysin vesicles'. Most of the axons originating in magnocellular nuclei form the hypothalamoneurohypophyseal tract and terminate in the neurohypophysis where the hormones are released into the blood stream. It is evident that the majority of these axons terminate in the neurohypophysis in close vicinity of blood capillaries without special contacts. From this fact we based our opinion that the axons of the classical peptidergic neurosecretory system are generally unable to contact target cells or neurones synaptically.
56 For several years we have known that some of these axons terminate in synapselike contacts at neurohypophyseal glial cells (pituicytes), other neurosecretory fibres leave the neurosecretory tract and enter parts of the adenohypophysis forming neuroglandular synapses 6,7. Besides the descending axons of this system, ascending fibres arising in the magnocellular neurosecretory nuclei were identified in all classes of vertebrates. They form exohypothalamic oxytocinergic pathways and transport the above mentioned neurohormones enclosed in neurophysin vesicles to different target areas of the brain 9,11,1s,2°,3°,32-37,47,49,57-63. We were successful in demonstrating that these exohypothalamic oxytocinergic fibres form synaptic contacts with nerve cell perikarya and processes of the target areas 28,29,~0-56,58. Our ultrahistochemical KMnOa-oxidation method permits a well defined identification and selective demonstration of neurophysins containing fibres and their synaptic terminals at the ultrastructural levelag,4o. MATERIAL AND METHODS Up until now, we have investigated ultrastructurally the following vertebrate species and brain target areas: adults of Lampetra planeri Bloch (Cyclostomata, Petromyzonidae), reticular of the rhombencephalic tegmentum motoricum; newt (Pleurodeles waltli Michah), tectum opticum; frog (Rana esculenta L.), subfornical organ; pigeon and Wistar rat, medulla oblongata (nucleus gracilis, nucleus cuneatus medialis; nucleus tractus solitarii). For the electron microscopic analysis, the objects were prepared in the following manner. The brains were fixed with glutaraldehyde (frog, pigeon and rat by perfusion), followed by postfixation in a K2Cr2OT-OsO4 mixture, en bloc contrasting with uranyl acetate and phosphotungstic acid (in acetone), and embedding in Mikropal. For routine electron microscopic analysis, ultrathin sections were contrasted with lead citrate. For the identification and selective demonstration of the neurophysin vesicles within the terminals of the exohypothalamic peptidergic fibres, other ultrathin sections were treated according to our KMnO4-0xidation method a9,4°. The optimal oxidation time was tested on the basis of the reaction of the neurophysin vesicles in the neurohypophysis of the same species. Because of the necessity of identifying synapses discovered in a selected target area by means of the KMnO4-0xidation method in adjacent ultrathin sections also ultrahistochemically, we were compelled to use the described fixation and contrasting technique. For this reason, we have up to now, had to dispense with other methods of impregnation (such as Ruthenium red, ethanolic phosphotungstic acid or bismuth iodide45) for an extended ultrahistochemical characterisation of the individual parts of the synaptic complex. RESULTS The exohypothalamic fibres of the classical neurosecretory system contact synaptically both perikarya and nerve fibres of the different examined target areas,
57
Fig. 1. Wistar-rat. Medulla oblongata, region of the dorsal columns nuclei. Neuroneuronal peptidergic synapse and two further, probably cholinergic synapses at the same nerve cell process. Neurophysin vesicles (arrowheads), pf peptidergic fibre. Fig. 2a and b. Pigeon. Medulla oblongata, nucleus cuneatus medialis of the dorsal columns nuclei. Two sections through the same neuro-neuronal synaptic complex of a peptidergic fibre. Neurophysin vesicles (arrowheads).
58
Fig. 3a and b. Frog. Subfornical organ. Neuro-neuronal peptidergic synapses, a: normal preparation method, b: another peptidergic synapse after the use of the KMnOa-oxidation method. The neurophysin vesicles (arrowheads) appear electron-optically empty ; their content is lost : af aminergic fibre. Fig. 4a and b, Newt. Tectum opticum. Neuroneuronal peptidergic synapses. Neurophysin vesicles (arrowheads).
59
Fig. 5 and b. Lamprey. Formatio reticularis. Neuroneuronal peptidergic synapses, a: normal preparation method, b: a peptidergic synapse and two further synapses at the same nerve cell process. After the application of the KMnO 4-oxidation method, the neurophysin vesicles (arrowheads) appear electron-optically empty in the peptidergic synapse, pf, peptidergic fibre; af, aminergic fibre; cf, cholinergic fibre; *, artefact.
60 where they share their area of projection with cholinergic and aminergic fibre terminals (Figs. 1, 5b). Such synapses are found relatively frequently at the small spine-like protrusions of nerve fibres which are enclosed by the exohypothalamic fibres. The preterminal and, above all, the terminal section of peptidergic fibres displaying contact with other neuronal elements are characterized by the occurrence of small agranular vesicles (Figs. 1-5). The different contacts also fulfil other morphologic criteria of synapses. On the basis of our results obtained up to now, the synapses of the neurosecretory fibres can be characterized by the following morphological features. The presynaptic bouton contains big vesicles besides mitochondria and numerous small vesicles. The large vesicles have a spherical or slightly ovoid shape. They contain a finely granulated, homogeneously distributed, electrondense material which almost completely fills the vesicle lumen (Figs. 1, 2, 3a, 4, 5a). The diameter of the large vesicles in general is anything between 120 and 240 nm and the maximum diameters reached vary from species to species. Thus, these big vesicles resemble the well-known neurophysin vesicles of the neurohypophysis as to shape, appearance and size. The identity of the content of the large vesicles with that of the neurophysin vesicles can be demonstrated ultrahistochemically in all of the examined species by means of our KMnO4-oxidation method 39,4°. As is the case with the neurophysin vesicles in the neurohypophysis, the content of the big vesicles can be removed when this method is employed so that they appear empty electronoptically (Figs. 3b, 5b). From these facts, it can be definitely stated that the large vesicles in the presynaptic bouton of the peptidergic axons are neurophysin vesicles. The small vesicles, whose diameter is between 40 and 50 nm, mainly appear to be spherical and electronoptically empty under the given fixation conditions (Figs. 1-5); thus, they resemble the well-known synaptic vesicles as to size and appearance. Most of these vesicles are clustered close to the presynaptic axolemma. In the synaptic area, the intercellular space is widened to a well-defined synaptic cleft in some cases containing a fine-granular intracleft material. Both presynaptic thickenings (dense projections) and postsynaptic densities are present (Figs. 2, 4, 5a); they cannot always be seen very clearly under the given conditions of preparation (see earlier remarks). In most cases the postsynaptic thickening is more pronounced than the presynaptic dense projections. On the basis of the generally accepted nomenclature1-4,19,43-45,4s, the synapses of the neurosecretory exohypothalamic axons could account for the structure of the synaptic membrane complex or for the presence of neurophysin vesicles in the presynaptic element, and could be assigned to Gray type I or, possibly better, to 'gtype '3,4 synapses as a new form of synapse. DISCUSSION
As we have shown, the neurones of the classical neurosecretory system also form synapses with other neurones; they act as normal nerve cells also in this respect. The neuro-neuronal type of synapse demonstrated is characterized by materially defined peptide vesicles in the presynaptic bouton. There are some indications that the
61 octapeptides stored in the neurophysin vesicles, and presumably released in the synapses described 56, may effect regional long-phase modulations. So far, however, no information can be given as to whether these neuropeptides take part directly in neural transmission. The mode of release also remains to be detailed. Of the peptidergic terminals in the neurohypophysis it is known that the content of the neurophysin vesicles is mainly released by exocytosis14,15,3s,42. We are not in a position, however, to give proof of such a process in the synapses of the exohypothalamic neurosecretory fibres. It should be taken into consideration, however, that exocytosis can be observed only rarely, even in the neurohypophysis. Presumably, the exohypothalamic fibres described can be grouped among the various peptidergic fibres that pervade large parts of the central nervous system and contain thyroliberin, luliberin, somatostatin, substance P, angiotensin, enkephalin or the so-called vasoactive intestinal polypeptide (VIP) 5,1°,16,17,21-27,31,41. At present, the biological importance of these neuroactive peptides is considered to lie in a shortterm effective chemical mediation in the sense of classical transmission or in a more prolonged modulation of neuronal activity by changing of the synaptic efficiencys,12, 13,46,64. ACKNOWLEDGEMENTS
For skilled technical assistance the authors are indebted to their colleagues Mrs. S. Mehnert, E. Siebert, C. Schneider, and I. Seifert. This work was supported by grants from the Ministry for Science and Technology of the German Democratic Republic. REFERENCES 1 Akert, K., Struktur und Ultrastruktur von Nervenzellen und Synapsen, Kiln. Wschr., 49/9 (1971) 509-519. 2 Akert, K., Gray, E. G. and Bloom, F. E., Macromolecules in synaptic function, h. Synaptic ultrastructure, Neurosci. Res. Progr. Bull., 8, 0970) 335-360. 3 Andres, K. H., Morphological criteria for the differentiation of synapses in vertebrates, J. Neural Trans., Suppl. XII (1975) 1-37. 4 Andres, K. H. and von D~iring, M., The synapse. In W. H. Gispen (Ed.), Molecular and Functional Neurobiology, Elsevier, Amsterdam, 1976, pp. 3-45. 5 Baker, B. L. and Yu, Y.-Y., Distribution of growth hormone-release-inhibitinghormone (somatostatin) in the rat brain as observed with immunocytochemistry, Anat. Rec., 186 (1976) 343. 6 Bargmann, W., Das neurosekretorische Zwischenhirn-Hypophysensystem und seine synaptischen Verknfipfungen, J. Neuro-Visc. Rel., Suppl. 9 (1969) 64-77. 7 Bargmann, W., Lindner, E. und Andres, K. H., Ober Synapsen an endokrinen Epithelzellen und die Definition sekretorischer Neurone. Untersuchungen am Zwischenlappen der Katzenhypophyse, Z. Zellforsch., 77 (1967) 282-298. 8 Barker, J. L., Peptides: Roles in neuronal excitability, Physiol. Rev., 56 (1976) 435-452. 9 Barry, J., Recherches morphologiques et exp6rimentales sur la glande dienc~phalique et l'appareil hypothalamo-hypophysaire, Ann. sci. Univ. Besancon, Zool. Physiol., 2/5 (1961) 3-133. 10 Barry, J., Poulain, P. et Carette, B., Syst6matisation et 6ff6rences des neurones ~ LRH chez les primates, Ann. Endocr. (Paris), 37 (1976) 227-234. 11 Brownfield, M. S. and Kozlowski, G. P., The hypothalamo-choroidal tract. I. Immunohistochemical demonstration of neurophysin pathways to telencephalic choroid plexuses and cerebrospinal fluid, Cell Tiss. Res., 178 (1977) 111-127.
62 12 Bury, R. W. and Mashford, M. L., Substance-P. Its pharmacology and physiological roles, Aust. J. exp. Biol. Med. Sci., 55 (P6) (1977) 671. 13 Byck, R., Peptide transmitters: A unifying hypothesis for euphoria, respiration, sleep, and the action of lithium, Lancet, 2 (1976) 72-73. 14 Douglas, W. W., How do neurones secrete peptides? Exocytosis and its consequences, including 'synaptic vesicle' formation, in the hypothalamo-neurohypophyseal system. In E. Zimmerman, W. H. Gispen, B. H. Marks and D. De Weid (Eds.), Drug Effects on Neuroendocrine Regulation, Progr. Brain Res. 39 (1973) 21--39. 15 Dreifuss, J. J., A review on neurosecretory granules: their contents and mechanisms of release, Ann. N. Y. ,4cad. Sci., 248 (1975) 184-201. 16 Elde, R., H6ktelt, T., Johansson, O. and Terenius, L., Immunohistochemical studies using antibodies to leucine-enkaphalin : Initial observations on the nervous system of the rat, Neuroscience, 1 (1976) 349-351. 17 Fuxe, K., Ganten, D., H6kfelt, T. and Bolme, P., Immunohistochemical evidence for the existence of angiotensin lI-containing nerve terminals in the brain and spinal cord in the rat, Neurosci. Lett., 2 (1976) 229-234. 18 Goosens, N., Dierickx, K. and Vandesande, F., Immunocytochemical demonstration of the hypothalamo-hypophysial vasotocinergic system of Lampetra fluviatilis, Cell Tiss. Res., 177 (1977) 317-323. 19 Gray, E. G., The fine structural characterization of different types of synapse. In O. Er~ink6 (Ed.), Histochemistry of Nervous Transmission, Progr. in Brain Res., Vol. 34, Elsevier, Amsterdam, 1971, pp. 149-160. 20 Helas, G., Etude en fluorescence du syst6me hypothalamo-hypophysaire de la grenouille verte (Rana esculenta) dans des conditions normales ou exp6rimentales, Bull. Ass. ,4nat., 152 (1971) 534 542. 21 H6kfelt, T., Efendi6, S., Hellerstr6m, C., Johansson, O., Luft, R. and Arimura, A., Cellular localization of somatostatin in endocrine-like cells and neurons of the rat with special references to the Al-cells of the pancreatic islets and to the hypothalamus, ,4cta endocr. (Kbh.), 80, Suppl. 200 (1975) 5 41. 22 H6kfelt, T., Efendi6, S., Johansson, O., Luft, R. and Arimura, A., Immunohistochemical localization of somatostatin (growth hormone release-inhibiting factor) in guinea pig brain, Brain Research, 80 (1974) 165-169. 23 H6kfelt, T., Elde, R., Johansson, O., Luft, R., Nilsson, G. and Arimura, A., Immunohistochemical evidence for separate populations of somatostatin-containing and substance P-containing primary afferent neurons in the rat, Neuroscience, 1 (1976) 131-136. 24 H6kfelt, T., Fuxe, K., Johansson, O., Jeffcoate, S. and White, N., Distribution of thyrotropinreleasing hormone (TRH) in the central nervous system as revealed with immunohistochemistry, Europ. J. Pharmacol., 34 (1975) 389-392. 25 H6kfelt, T., Fuxe, K., Johansson, O., Jeffcoate, S. and White, N., Thyrotropin releasing hormone (TRH)-containing nerve terminals in certain brain stem nuclei and in the spinal cord, Neurosci. Left., 1 (1975) 133-139. 26 H6kfelt, T., Kellerth, J. O., Nilsson, G. and Pernow, B., Substance P: Localization in the central nervous system and in some primary sensory neurons, Science, 190 (1975) 889-890. 27 H6kfelt, T., Meyerson, B., Nilsson, G., Pernow, B. and Sachs, C., Immunohistochemical evidence for substance P-containing nerve endings in the human cortex, Brain Research, 104 (1976) 181-186. 28 H.oheisel, G. and Naumann, W., Contribution to the morphology of exohypothalamic oxytocinergic pathways. In V. Viklicky and J. Ludvik (Eds,), Proc. XVth Czechoslovak Conference on Electron Microscopy, 11ol..4, Prague, 1977, pp. 115-116. 29 Hoheisel, G., Riihle, H.-J. and Sterba, G., The reticular formation of lampreys (Petromyzonidae). A target area for exohypothalamic vasotocinergic fibres, Cell Tiss. Res., 189 (1978) 331-345. 30 Kozlowski, G. P., Brownfietd, M. S. and Schultz, W. J., Extrahypothalamic neurosecretory system : Immunocytochemical evidence for a neurosecretory innervation of the choroid plexus. In Proc. VII Int. Symp. Neurosecretion, Leningrad, 1976, p. 93. 31 Larsson, L.-I., Fahrenkrug, J., Schaffalitzky de Muckadell, O., Sundler, F., H~kanson, R. and Rehfeld, J. F., Localization of vasoactive intestinal polypeptide (VIP) to central and peripheral neurons, Proc. nat. Acad. Sci. (N. Y.), 73 (1976) 3197-3200. 32 Legait, H., Les voies eff6rentes des noyaux neuros~cr6toires hypothalamiques chez les oiseaux, C. R. Soc. Biol. (Paris J, 150 (1956) 996-998.
63 33 Legait, H., Anatomie microscopique des noyaux hypothalamiques neuros6cr6toires et de leur voies eff6rentes chez la poule Rhode-Island, Acta neuroveg. (Wien), 15 (1957) 252-262. 34 Legait, H. et Legait, E., Mise en 6vidence de voies neuros~r6toires extra-hypothalamo-hypophysaires chez quelques batraciens et reptiles, C. R. Soc. Biol. (Paris), 150 (1956) 1429-1431. 35 Legait, H. et Legait, E., Relations entre les noyaux hypothalamiques neuros6cr6toires et les regions septale et hab~nulaire chez quelques oiseaux, Acta neuroveg. (Wien), 17 (1957) 143-147. 36 Legait, H. et Legait, E., Les voles extra-hypophysaires des noyaux neuro-s~cr6toires hypothalamiques chez les batraciens et les reptiles, Acta anat. (Basel), 30 (1957) 429-443. 37 Legait, H. et Legait, E., Pr6sence d'une vole neuros6cr6toire hypothalamo-hab6nulaireet mise en 6vidence d'une activit6 antidiuretique au niveau des ganglions de l'hab6nula chez la poule, C. R. Soc. Biol. (Paris), 152 (1958) 828-830. 38 Nagasawa, J., Exocytosis: the common release mechanism of secretory granules in glandular cells, neurosecretory cells, neurons and paraneurons, Arch. histol, jap., Suppl. 40 (1977) 31. 39 Naumann, W. and Sterba, G., Ultrastructural studies on neurophysine-containingvesicles of the neurosecretory system of vertebrates, Cell Tiss. Res., 165 (1976) 545-553. 40 Naumann, W. and Sterba, G., An ultrahistochemical method to identify neurophysine-containing vesicles of the neurosecretory system of vertebrates. In Proc. VII Int. Symp. Neurosecretion, Leningrad, 1976, p. 115. 41 Nilsson, G., H6kfelt, T. and Pernow, B., Distribution of substance P-like immunoreactivityin the rat central nervous system as revealed by immunohistochemistry, Med. Biol., 52 (1974) 424-427. 42 Normann, T. C., Neurosecretion by exocytosis, Int. Rev. Cytol., 46 (1976) 1-77. 43 Palay, S. L. and Chan-Palay, V., A guide to the synaptic analysis of the neuropil. ColdSpr. Harb. Syrup. quant. Biol., XL (1976) pp. 1-16. 44 Pappas, G. D. and Waxman, S. G.,Synapticfine structure-- morphological correlates of chemical and electronic transmission, In G. D. Pappas and D. P. Purpura, (Eds.), Structure and Function of Synapses, Raven Press, New York, 1972, pp. 1-43. 45 Pfenninger, K. H., Synaptic Morphology and Cytochemistry. In W. Graumann, Z. Lojda, A. G. E. Pearse and T. H. Schiebler, (Eds.), Progress in Histochemistry and Cytochemistry, Vol. 5, No. 1, Fischer, Stuttgart, 1973, pp. 1-86. 46 Reith, M. E. A., Schotman, P., Gispen, W. H. and de Wied, D., Pituitary peptides as modulators of neuronal functioning, TIBS., 2 (1977) 56-57. 47 Rudert, H., Das Subfornikalorgan und seine Beziehungen zu dem neurosekretorischen System im Zwischenhirn des Frosches, Z. Zellforsch., 65 (1965) 790-804. 48 Sandri, C., van Buren, J. M. and Akert, K., Membrane Morphology of the Vertebrate Nervous System. A study with Freeze-etch Technique. In membrane morphology of the vertebrate nervous system, Prog. Brain Res., Vol. 46, Elsevier, Amsterdam, 1977, pp. 1-384. 49 Schober, F., Darstellung der neurosekretorischen hypothalamorhombenzephalen Verbindung bei der Ratte dutch retrograden axonalen Transport von Meerrettich-Peroxidase, Acta biol. reed germ., 36/10 (1978) in press. 50 Schober, F., Naumann, W. and Sterba, G., Light and electron microscopic study of the oxytocinergic efferences of the hypothalamus in the medulla of rat pigeon. In Proc. VII Int. Symp. Neurosecretion, Leningrad, 1976, p. 146. 51 Schober, F., Trautmann, U., Naumann, W. und Sterba, G., Die oxytocinergen exohypothalamischen Verbindungen zur Medulla oblongata bei der Taube und tier Ratte, Acta biol. reed. germ., 36/5-6 (1978) 1183-1186. 52 Sterba, G., Das oxytocinerge neurosekretorische System der Wirbeltiere. Beitrag zu einem erweiterten Konzept, Zool. Jb. Physiol., 78 (1974) 409-423. 53 Sterba, G., Ascending neurosecretory pathways of the peptidergic type. In F. Knowles and L. Vollrath (Eds.), Neurosecretion - - The Final Neuroendocrine Pathway, Springer, Berlin, 1974, pp. 38-47. 54 Sterba, G., The oxytocinergic exohypothalamic neurosecretory system of vertebrates and memory processes. In Proc. VII Int. Symp. Neuroseeretion, Leningrad, 1976 55 Sterba, G., Morphologische Grundlagen einer humoralen Informationsfibermittlung durch Peptide bei Wirbeltieren, In Hormonale und humorale Informationsiibermittlung dutch Peptide als Mediatoren, Sitzungsberichte der Akademie der Wissenschaften der DDR, 5 N, Akademie-Verlag, Berlin, 1977, 40-61. 56 Sterba, G., Hoffmann, E., Solecki, R., Naumann, W., Hoheisel, G. and Schober, F., The neurosecretory hypothalamo-hindbrainpathway and its possible significance for the regulation of the blood pressure and the milk ejection reflex, Cell Tiss. Res., (1979) in preparation.
64 57 Weber, W., Entwicklung und Funktion des neurosekretorischen Systems von Salamandra salamandra, Z. Zellforsch., 66 (1965) 35-65. 58 Wegelin, R., Sterba, G. und Hoheisel, G., Licht- und elektronenmikroskopische Untersuchungen am exohypothalamischen oxytocinergen System von Pleurodeles waltli Michah. (Urodela), Biol. Zbl., 94 (1975) 633-660. 59 Weindl, A. and Sofroniew, M. V., Demonstration ofextrahypothalamic peptide secreting neurons. A morphologic contribution to the investigation of psychotropic effects of neurohormones, Pharmacopsychology, 9 (1976) 226-234. 60 Weindl, A., Sofroniew, M. V. and Schinko, I., Psychotrope Wirkungen hypothalamischer Hormone: ImmunhistochemischeIdentifikation extrahypophys~irer Verbindungen neuroendokriner Neurone, Arzneimittel.-Forsch., 26, (1976) 1191-1194. 61 Weindl, A., Sofroniew, M. V. and Schinko, I., Vasopressin and LRH secreting systems: Identification and localization by immunohistochemistry. In Proc. VII. Int. Symp. Neurosecretion, Leningrad, 1976, p. 172. 62 Wolf, G., lmmunohistologicalidentification of neurophysin and neurophysin-like substances in different vertebrates, Endokrinologie, 68 (1976) 288-299. 63 Wolf, G., On the immunological relationship of the neurophysins of vertebrates. In Proc. VII Int. Syrup. Neurosecretion, Leningrad, 1976, p. 174. 64 Zetler, G., The peptidergic neuron: A working hypothesis, Biochem. Pharmacol., 25 (1976) 1817-1818.
55
PEPTIDE CONTAINING VESICLES WITHIN NEURO-NEURONAL SYNAPSES
GONTHER STERBA, GEORG HOHEISEL, RUTH WEGELIN, WILFR1ED NAUMANN and FRANK SCHOBER Sektion Biowissenschaften, Bereich Zellbiologie und Regulation, Karl-Marx-Universitiit, 701 Leipzig (G.D.R.) (Accepted September 21st, 1978)
SUMMARY
In the descending part of the classical neurosecretory system, the axon terminals are not differentiated or they take the form of presynaptic elements which then form synaptoids or synapses with pituicytes or adenohypophyseal glandular cells respectively. In contrast, the axon terminals of the ascending part fulfil the criteria of true presynaptic elements which form synapses with other neurones. The presence of neurophysin vesicles in the presynaptic element is a particular morphologic feature of these neuro-neuronal synapses.
INTRODUCTION
The neurones of the classical peptidergic neurosecretory system of the vertebrates are localized in the magnocellular hypothalamic praeoptic nuclei (in anamnia) or in the paraventricular and supraoptic nuclei (in amniota). Their perikarya produce the octapeptide hormone ocytocin or other naturally occurring ocytocin analogues (vasopressin, vasotocin, mesotocin, etc.) and the carrier protein neurophysins. The hormones, bound to the carrier and enclosed in vesicles, are transported within the axons to different sites. We prefer to use in this connection the terms 'peptidergic neurosecretory system' and 'neurophysin vesicles'. Most of the axons originating in magnocellular nuclei form the hypothalamoneurohypophyseal tract and terminate in the neurohypophysis where the hormones are released into the blood stream. It is evident that the majority of these axons terminate in the neurohypophysis in close vicinity of blood capillaries without special contacts. From this fact we based our opinion that the axons of the classical peptidergic neurosecretory system are generally unable to contact target cells or neurones synaptically.
56 For several years we have known that some of these axons terminate in synapselike contacts at neurohypophyseal glial cells (pituicytes), other neurosecretory fibres leave the neurosecretory tract and enter parts of the adenohypophysis forming neuroglandular synapses 6,7. Besides the descending axons of this system, ascending fibres arising in the magnocellular neurosecretory nuclei were identified in all classes of vertebrates. They form exohypothalamic oxytocinergic pathways and transport the above mentioned neurohormones enclosed in neurophysin vesicles to different target areas of the brain 9,11,1s,2°,3°,32-37,47,49,57-63. We were successful in demonstrating that these exohypothalamic oxytocinergic fibres form synaptic contacts with nerve cell perikarya and processes of the target areas 28,29,~0-56,58. Our ultrahistochemical KMnOa-oxidation method permits a well defined identification and selective demonstration of neurophysins containing fibres and their synaptic terminals at the ultrastructural levelag,4o. MATERIAL AND METHODS Up until now, we have investigated ultrastructurally the following vertebrate species and brain target areas: adults of Lampetra planeri Bloch (Cyclostomata, Petromyzonidae), reticular of the rhombencephalic tegmentum motoricum; newt (Pleurodeles waltli Michah), tectum opticum; frog (Rana esculenta L.), subfornical organ; pigeon and Wistar rat, medulla oblongata (nucleus gracilis, nucleus cuneatus medialis; nucleus tractus solitarii). For the electron microscopic analysis, the objects were prepared in the following manner. The brains were fixed with glutaraldehyde (frog, pigeon and rat by perfusion), followed by postfixation in a K2Cr2OT-OsO4 mixture, en bloc contrasting with uranyl acetate and phosphotungstic acid (in acetone), and embedding in Mikropal. For routine electron microscopic analysis, ultrathin sections were contrasted with lead citrate. For the identification and selective demonstration of the neurophysin vesicles within the terminals of the exohypothalamic peptidergic fibres, other ultrathin sections were treated according to our KMnO4-0xidation method a9,4°. The optimal oxidation time was tested on the basis of the reaction of the neurophysin vesicles in the neurohypophysis of the same species. Because of the necessity of identifying synapses discovered in a selected target area by means of the KMnO4-0xidation method in adjacent ultrathin sections also ultrahistochemically, we were compelled to use the described fixation and contrasting technique. For this reason, we have up to now, had to dispense with other methods of impregnation (such as Ruthenium red, ethanolic phosphotungstic acid or bismuth iodide45) for an extended ultrahistochemical characterisation of the individual parts of the synaptic complex. RESULTS The exohypothalamic fibres of the classical neurosecretory system contact synaptically both perikarya and nerve fibres of the different examined target areas,
57
Fig. 1. Wistar-rat. Medulla oblongata, region of the dorsal columns nuclei. Neuroneuronal peptidergic synapse and two further, probably cholinergic synapses at the same nerve cell process. Neurophysin vesicles (arrowheads), pf peptidergic fibre. Fig. 2a and b. Pigeon. Medulla oblongata, nucleus cuneatus medialis of the dorsal columns nuclei. Two sections through the same neuro-neuronal synaptic complex of a peptidergic fibre. Neurophysin vesicles (arrowheads).
58
Fig. 3a and b. Frog. Subfornical organ. Neuro-neuronal peptidergic synapses, a: normal preparation method, b: another peptidergic synapse after the use of the KMnOa-oxidation method. The neurophysin vesicles (arrowheads) appear electron-optically empty ; their content is lost : af aminergic fibre. Fig. 4a and b, Newt. Tectum opticum. Neuroneuronal peptidergic synapses. Neurophysin vesicles (arrowheads).
59
Fig. 5 and b. Lamprey. Formatio reticularis. Neuroneuronal peptidergic synapses, a: normal preparation method, b: a peptidergic synapse and two further synapses at the same nerve cell process. After the application of the KMnO 4-oxidation method, the neurophysin vesicles (arrowheads) appear electron-optically empty in the peptidergic synapse, pf, peptidergic fibre; af, aminergic fibre; cf, cholinergic fibre; *, artefact.
60 where they share their area of projection with cholinergic and aminergic fibre terminals (Figs. 1, 5b). Such synapses are found relatively frequently at the small spine-like protrusions of nerve fibres which are enclosed by the exohypothalamic fibres. The preterminal and, above all, the terminal section of peptidergic fibres displaying contact with other neuronal elements are characterized by the occurrence of small agranular vesicles (Figs. 1-5). The different contacts also fulfil other morphologic criteria of synapses. On the basis of our results obtained up to now, the synapses of the neurosecretory fibres can be characterized by the following morphological features. The presynaptic bouton contains big vesicles besides mitochondria and numerous small vesicles. The large vesicles have a spherical or slightly ovoid shape. They contain a finely granulated, homogeneously distributed, electrondense material which almost completely fills the vesicle lumen (Figs. 1, 2, 3a, 4, 5a). The diameter of the large vesicles in general is anything between 120 and 240 nm and the maximum diameters reached vary from species to species. Thus, these big vesicles resemble the well-known neurophysin vesicles of the neurohypophysis as to shape, appearance and size. The identity of the content of the large vesicles with that of the neurophysin vesicles can be demonstrated ultrahistochemically in all of the examined species by means of our KMnO4-oxidation method 39,4°. As is the case with the neurophysin vesicles in the neurohypophysis, the content of the big vesicles can be removed when this method is employed so that they appear empty electronoptically (Figs. 3b, 5b). From these facts, it can be definitely stated that the large vesicles in the presynaptic bouton of the peptidergic axons are neurophysin vesicles. The small vesicles, whose diameter is between 40 and 50 nm, mainly appear to be spherical and electronoptically empty under the given fixation conditions (Figs. 1-5); thus, they resemble the well-known synaptic vesicles as to size and appearance. Most of these vesicles are clustered close to the presynaptic axolemma. In the synaptic area, the intercellular space is widened to a well-defined synaptic cleft in some cases containing a fine-granular intracleft material. Both presynaptic thickenings (dense projections) and postsynaptic densities are present (Figs. 2, 4, 5a); they cannot always be seen very clearly under the given conditions of preparation (see earlier remarks). In most cases the postsynaptic thickening is more pronounced than the presynaptic dense projections. On the basis of the generally accepted nomenclature1-4,19,43-45,4s, the synapses of the neurosecretory exohypothalamic axons could account for the structure of the synaptic membrane complex or for the presence of neurophysin vesicles in the presynaptic element, and could be assigned to Gray type I or, possibly better, to 'gtype '3,4 synapses as a new form of synapse. DISCUSSION
As we have shown, the neurones of the classical neurosecretory system also form synapses with other neurones; they act as normal nerve cells also in this respect. The neuro-neuronal type of synapse demonstrated is characterized by materially defined peptide vesicles in the presynaptic bouton. There are some indications that the
61 octapeptides stored in the neurophysin vesicles, and presumably released in the synapses described 56, may effect regional long-phase modulations. So far, however, no information can be given as to whether these neuropeptides take part directly in neural transmission. The mode of release also remains to be detailed. Of the peptidergic terminals in the neurohypophysis it is known that the content of the neurophysin vesicles is mainly released by exocytosis14,15,3s,42. We are not in a position, however, to give proof of such a process in the synapses of the exohypothalamic neurosecretory fibres. It should be taken into consideration, however, that exocytosis can be observed only rarely, even in the neurohypophysis. Presumably, the exohypothalamic fibres described can be grouped among the various peptidergic fibres that pervade large parts of the central nervous system and contain thyroliberin, luliberin, somatostatin, substance P, angiotensin, enkephalin or the so-called vasoactive intestinal polypeptide (VIP) 5,1°,16,17,21-27,31,41. At present, the biological importance of these neuroactive peptides is considered to lie in a shortterm effective chemical mediation in the sense of classical transmission or in a more prolonged modulation of neuronal activity by changing of the synaptic efficiencys,12, 13,46,64. ACKNOWLEDGEMENTS
For skilled technical assistance the authors are indebted to their colleagues Mrs. S. Mehnert, E. Siebert, C. Schneider, and I. Seifert. This work was supported by grants from the Ministry for Science and Technology of the German Democratic Republic. REFERENCES 1 Akert, K., Struktur und Ultrastruktur von Nervenzellen und Synapsen, Kiln. Wschr., 49/9 (1971) 509-519. 2 Akert, K., Gray, E. G. and Bloom, F. E., Macromolecules in synaptic function, h. Synaptic ultrastructure, Neurosci. Res. Progr. Bull., 8, 0970) 335-360. 3 Andres, K. H., Morphological criteria for the differentiation of synapses in vertebrates, J. Neural Trans., Suppl. XII (1975) 1-37. 4 Andres, K. H. and von D~iring, M., The synapse. In W. H. Gispen (Ed.), Molecular and Functional Neurobiology, Elsevier, Amsterdam, 1976, pp. 3-45. 5 Baker, B. L. and Yu, Y.-Y., Distribution of growth hormone-release-inhibitinghormone (somatostatin) in the rat brain as observed with immunocytochemistry, Anat. Rec., 186 (1976) 343. 6 Bargmann, W., Das neurosekretorische Zwischenhirn-Hypophysensystem und seine synaptischen Verknfipfungen, J. Neuro-Visc. Rel., Suppl. 9 (1969) 64-77. 7 Bargmann, W., Lindner, E. und Andres, K. H., Ober Synapsen an endokrinen Epithelzellen und die Definition sekretorischer Neurone. Untersuchungen am Zwischenlappen der Katzenhypophyse, Z. Zellforsch., 77 (1967) 282-298. 8 Barker, J. L., Peptides: Roles in neuronal excitability, Physiol. Rev., 56 (1976) 435-452. 9 Barry, J., Recherches morphologiques et exp6rimentales sur la glande dienc~phalique et l'appareil hypothalamo-hypophysaire, Ann. sci. Univ. Besancon, Zool. Physiol., 2/5 (1961) 3-133. 10 Barry, J., Poulain, P. et Carette, B., Syst6matisation et 6ff6rences des neurones ~ LRH chez les primates, Ann. Endocr. (Paris), 37 (1976) 227-234. 11 Brownfield, M. S. and Kozlowski, G. P., The hypothalamo-choroidal tract. I. Immunohistochemical demonstration of neurophysin pathways to telencephalic choroid plexuses and cerebrospinal fluid, Cell Tiss. Res., 178 (1977) 111-127.
62 12 Bury, R. W. and Mashford, M. L., Substance-P. Its pharmacology and physiological roles, Aust. J. exp. Biol. Med. Sci., 55 (P6) (1977) 671. 13 Byck, R., Peptide transmitters: A unifying hypothesis for euphoria, respiration, sleep, and the action of lithium, Lancet, 2 (1976) 72-73. 14 Douglas, W. W., How do neurones secrete peptides? Exocytosis and its consequences, including 'synaptic vesicle' formation, in the hypothalamo-neurohypophyseal system. In E. Zimmerman, W. H. Gispen, B. H. Marks and D. De Weid (Eds.), Drug Effects on Neuroendocrine Regulation, Progr. Brain Res. 39 (1973) 21--39. 15 Dreifuss, J. J., A review on neurosecretory granules: their contents and mechanisms of release, Ann. N. Y. ,4cad. Sci., 248 (1975) 184-201. 16 Elde, R., H6ktelt, T., Johansson, O. and Terenius, L., Immunohistochemical studies using antibodies to leucine-enkaphalin : Initial observations on the nervous system of the rat, Neuroscience, 1 (1976) 349-351. 17 Fuxe, K., Ganten, D., H6kfelt, T. and Bolme, P., Immunohistochemical evidence for the existence of angiotensin lI-containing nerve terminals in the brain and spinal cord in the rat, Neurosci. Lett., 2 (1976) 229-234. 18 Goosens, N., Dierickx, K. and Vandesande, F., Immunocytochemical demonstration of the hypothalamo-hypophysial vasotocinergic system of Lampetra fluviatilis, Cell Tiss. Res., 177 (1977) 317-323. 19 Gray, E. G., The fine structural characterization of different types of synapse. In O. Er~ink6 (Ed.), Histochemistry of Nervous Transmission, Progr. in Brain Res., Vol. 34, Elsevier, Amsterdam, 1971, pp. 149-160. 20 Helas, G., Etude en fluorescence du syst6me hypothalamo-hypophysaire de la grenouille verte (Rana esculenta) dans des conditions normales ou exp6rimentales, Bull. Ass. ,4nat., 152 (1971) 534 542. 21 H6kfelt, T., Efendi6, S., Hellerstr6m, C., Johansson, O., Luft, R. and Arimura, A., Cellular localization of somatostatin in endocrine-like cells and neurons of the rat with special references to the Al-cells of the pancreatic islets and to the hypothalamus, ,4cta endocr. (Kbh.), 80, Suppl. 200 (1975) 5 41. 22 H6kfelt, T., Efendi6, S., Johansson, O., Luft, R. and Arimura, A., Immunohistochemical localization of somatostatin (growth hormone release-inhibiting factor) in guinea pig brain, Brain Research, 80 (1974) 165-169. 23 H6kfelt, T., Elde, R., Johansson, O., Luft, R., Nilsson, G. and Arimura, A., Immunohistochemical evidence for separate populations of somatostatin-containing and substance P-containing primary afferent neurons in the rat, Neuroscience, 1 (1976) 131-136. 24 H6kfelt, T., Fuxe, K., Johansson, O., Jeffcoate, S. and White, N., Distribution of thyrotropinreleasing hormone (TRH) in the central nervous system as revealed with immunohistochemistry, Europ. J. Pharmacol., 34 (1975) 389-392. 25 H6kfelt, T., Fuxe, K., Johansson, O., Jeffcoate, S. and White, N., Thyrotropin releasing hormone (TRH)-containing nerve terminals in certain brain stem nuclei and in the spinal cord, Neurosci. Left., 1 (1975) 133-139. 26 H6kfelt, T., Kellerth, J. O., Nilsson, G. and Pernow, B., Substance P: Localization in the central nervous system and in some primary sensory neurons, Science, 190 (1975) 889-890. 27 H6kfelt, T., Meyerson, B., Nilsson, G., Pernow, B. and Sachs, C., Immunohistochemical evidence for substance P-containing nerve endings in the human cortex, Brain Research, 104 (1976) 181-186. 28 H.oheisel, G. and Naumann, W., Contribution to the morphology of exohypothalamic oxytocinergic pathways. In V. Viklicky and J. Ludvik (Eds,), Proc. XVth Czechoslovak Conference on Electron Microscopy, 11ol..4, Prague, 1977, pp. 115-116. 29 Hoheisel, G., Riihle, H.-J. and Sterba, G., The reticular formation of lampreys (Petromyzonidae). A target area for exohypothalamic vasotocinergic fibres, Cell Tiss. Res., 189 (1978) 331-345. 30 Kozlowski, G. P., Brownfietd, M. S. and Schultz, W. J., Extrahypothalamic neurosecretory system : Immunocytochemical evidence for a neurosecretory innervation of the choroid plexus. In Proc. VII Int. Symp. Neurosecretion, Leningrad, 1976, p. 93. 31 Larsson, L.-I., Fahrenkrug, J., Schaffalitzky de Muckadell, O., Sundler, F., H~kanson, R. and Rehfeld, J. F., Localization of vasoactive intestinal polypeptide (VIP) to central and peripheral neurons, Proc. nat. Acad. Sci. (N. Y.), 73 (1976) 3197-3200. 32 Legait, H., Les voies eff6rentes des noyaux neuros~cr6toires hypothalamiques chez les oiseaux, C. R. Soc. Biol. (Paris J, 150 (1956) 996-998.
63 33 Legait, H., Anatomie microscopique des noyaux hypothalamiques neuros6cr6toires et de leur voies eff6rentes chez la poule Rhode-Island, Acta neuroveg. (Wien), 15 (1957) 252-262. 34 Legait, H. et Legait, E., Mise en 6vidence de voies neuros~r6toires extra-hypothalamo-hypophysaires chez quelques batraciens et reptiles, C. R. Soc. Biol. (Paris), 150 (1956) 1429-1431. 35 Legait, H. et Legait, E., Relations entre les noyaux hypothalamiques neuros6cr6toires et les regions septale et hab~nulaire chez quelques oiseaux, Acta neuroveg. (Wien), 17 (1957) 143-147. 36 Legait, H. et Legait, E., Les voles extra-hypophysaires des noyaux neuro-s~cr6toires hypothalamiques chez les batraciens et les reptiles, Acta anat. (Basel), 30 (1957) 429-443. 37 Legait, H. et Legait, E., Pr6sence d'une vole neuros6cr6toire hypothalamo-hab6nulaireet mise en 6vidence d'une activit6 antidiuretique au niveau des ganglions de l'hab6nula chez la poule, C. R. Soc. Biol. (Paris), 152 (1958) 828-830. 38 Nagasawa, J., Exocytosis: the common release mechanism of secretory granules in glandular cells, neurosecretory cells, neurons and paraneurons, Arch. histol, jap., Suppl. 40 (1977) 31. 39 Naumann, W. and Sterba, G., Ultrastructural studies on neurophysine-containingvesicles of the neurosecretory system of vertebrates, Cell Tiss. Res., 165 (1976) 545-553. 40 Naumann, W. and Sterba, G., An ultrahistochemical method to identify neurophysine-containing vesicles of the neurosecretory system of vertebrates. In Proc. VII Int. Symp. Neurosecretion, Leningrad, 1976, p. 115. 41 Nilsson, G., H6kfelt, T. and Pernow, B., Distribution of substance P-like immunoreactivityin the rat central nervous system as revealed by immunohistochemistry, Med. Biol., 52 (1974) 424-427. 42 Normann, T. C., Neurosecretion by exocytosis, Int. Rev. Cytol., 46 (1976) 1-77. 43 Palay, S. L. and Chan-Palay, V., A guide to the synaptic analysis of the neuropil. ColdSpr. Harb. Syrup. quant. Biol., XL (1976) pp. 1-16. 44 Pappas, G. D. and Waxman, S. G.,Synapticfine structure-- morphological correlates of chemical and electronic transmission, In G. D. Pappas and D. P. Purpura, (Eds.), Structure and Function of Synapses, Raven Press, New York, 1972, pp. 1-43. 45 Pfenninger, K. H., Synaptic Morphology and Cytochemistry. In W. Graumann, Z. Lojda, A. G. E. Pearse and T. H. Schiebler, (Eds.), Progress in Histochemistry and Cytochemistry, Vol. 5, No. 1, Fischer, Stuttgart, 1973, pp. 1-86. 46 Reith, M. E. A., Schotman, P., Gispen, W. H. and de Wied, D., Pituitary peptides as modulators of neuronal functioning, TIBS., 2 (1977) 56-57. 47 Rudert, H., Das Subfornikalorgan und seine Beziehungen zu dem neurosekretorischen System im Zwischenhirn des Frosches, Z. Zellforsch., 65 (1965) 790-804. 48 Sandri, C., van Buren, J. M. and Akert, K., Membrane Morphology of the Vertebrate Nervous System. A study with Freeze-etch Technique. In membrane morphology of the vertebrate nervous system, Prog. Brain Res., Vol. 46, Elsevier, Amsterdam, 1977, pp. 1-384. 49 Schober, F., Darstellung der neurosekretorischen hypothalamorhombenzephalen Verbindung bei der Ratte dutch retrograden axonalen Transport von Meerrettich-Peroxidase, Acta biol. reed germ., 36/10 (1978) in press. 50 Schober, F., Naumann, W. and Sterba, G., Light and electron microscopic study of the oxytocinergic efferences of the hypothalamus in the medulla of rat pigeon. In Proc. VII Int. Symp. Neurosecretion, Leningrad, 1976, p. 146. 51 Schober, F., Trautmann, U., Naumann, W. und Sterba, G., Die oxytocinergen exohypothalamischen Verbindungen zur Medulla oblongata bei der Taube und tier Ratte, Acta biol. reed. germ., 36/5-6 (1978) 1183-1186. 52 Sterba, G., Das oxytocinerge neurosekretorische System der Wirbeltiere. Beitrag zu einem erweiterten Konzept, Zool. Jb. Physiol., 78 (1974) 409-423. 53 Sterba, G., Ascending neurosecretory pathways of the peptidergic type. In F. Knowles and L. Vollrath (Eds.), Neurosecretion - - The Final Neuroendocrine Pathway, Springer, Berlin, 1974, pp. 38-47. 54 Sterba, G., The oxytocinergic exohypothalamic neurosecretory system of vertebrates and memory processes. In Proc. VII Int. Symp. Neuroseeretion, Leningrad, 1976 55 Sterba, G., Morphologische Grundlagen einer humoralen Informationsfibermittlung durch Peptide bei Wirbeltieren, In Hormonale und humorale Informationsiibermittlung dutch Peptide als Mediatoren, Sitzungsberichte der Akademie der Wissenschaften der DDR, 5 N, Akademie-Verlag, Berlin, 1977, 40-61. 56 Sterba, G., Hoffmann, E., Solecki, R., Naumann, W., Hoheisel, G. and Schober, F., The neurosecretory hypothalamo-hindbrainpathway and its possible significance for the regulation of the blood pressure and the milk ejection reflex, Cell Tiss. Res., (1979) in preparation.
64 57 Weber, W., Entwicklung und Funktion des neurosekretorischen Systems von Salamandra salamandra, Z. Zellforsch., 66 (1965) 35-65. 58 Wegelin, R., Sterba, G. und Hoheisel, G., Licht- und elektronenmikroskopische Untersuchungen am exohypothalamischen oxytocinergen System von Pleurodeles waltli Michah. (Urodela), Biol. Zbl., 94 (1975) 633-660. 59 Weindl, A. and Sofroniew, M. V., Demonstration ofextrahypothalamic peptide secreting neurons. A morphologic contribution to the investigation of psychotropic effects of neurohormones, Pharmacopsychology, 9 (1976) 226-234. 60 Weindl, A., Sofroniew, M. V. and Schinko, I., Psychotrope Wirkungen hypothalamischer Hormone: ImmunhistochemischeIdentifikation extrahypophys~irer Verbindungen neuroendokriner Neurone, Arzneimittel.-Forsch., 26, (1976) 1191-1194. 61 Weindl, A., Sofroniew, M. V. and Schinko, I., Vasopressin and LRH secreting systems: Identification and localization by immunohistochemistry. In Proc. VII. Int. Symp. Neurosecretion, Leningrad, 1976, p. 172. 62 Wolf, G., lmmunohistologicalidentification of neurophysin and neurophysin-like substances in different vertebrates, Endokrinologie, 68 (1976) 288-299. 63 Wolf, G., On the immunological relationship of the neurophysins of vertebrates. In Proc. VII Int. Syrup. Neurosecretion, Leningrad, 1976, p. 174. 64 Zetler, G., The peptidergic neuron: A working hypothesis, Biochem. Pharmacol., 25 (1976) 1817-1818.