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Development of synaptic structure and function in organotypic cultures of the rat hippocampus
Nt-woSclrnce vol. 4, pp.913to 920 Rrganm Rem Ltd 1979.Rintedin GreatBritain
DEVELOPMENT OF SYNAPTIC STRUCTURE AND FUNCTION IN ORGANOTYPIC CULTURES OF THE RAT HIPPOCAMPUS Y. M. ZHABOTINSKI, E. I. CHUMASOV, A. R. CHUBAKOVand H. V. KONOVALOV Laboratory of Pathology of Nervous System, Institute of Experimental Medicine of the Academy of Medical Sciences of the U.S.S.R., Leningrad, and Department of Memory Problems, Institute of Biophysics of the Academy of Sciences of the U.S.S.R., Pushchino-on-the-Oka, U.S.S.R.
Abstract-The sequence of development of synapses, as well as the ultrastructure of axonal growth cones, has been investigated electron microscopically in tissue cultures of the newborn rat hippocampus. During differentiation of the tissue cultures, the formation of synapses is preceded by identifiable growth cones. A characteristic feature of axonal growth cones is the presence of numerous large clear vesicles which vary in diameter from _ 100 to 150 nm. The first immature synapses were formed on the Sth, 6th or 7th day in oirro on the growth cones of differentiating neuronal processes. Axonal growth cones are occasionally found to be presynaptic to a dendrite. At first axo-dendritic synapses, most of them being en passant, arise, whereas ax&somatic and axc+spinous-dendritic synapses of different complex structures appear later. It is suggested that the earliest signs of synaptogenesis are vesicular structures (‘growth’ vesicles and few synaptic vesicles), which occur in growth cones, axons and presynaptic boutons of immature synaptic contacts even before formation of the specialized pre- and postsynaptic membranes.
CULTUREDnervous tissue is a most suitable subject for studying the dynamics of functional and morphological development of synapses. As is known, synapses are not only preserved but even newly formed in organotypic cultures of various divisions of the nervous system (WIN, 1%6; BUNGE,BUNGE & PE’IERSON,1967; MODEL, BORNSTEIN,CIUIN & PAPPAS,1971; QLSON& BUNGE, 1973; H&LI, H&L] & ANDRES,1973; Km & WENGER,1972; C-v, KONOV~V, CHunfiOV, I%OROVA & soosiuc, 1975; GRAIN,REINE& BORNSTEIN,1975). Although a great number of electron-microscopic examinations of cultured nervous tissue has been published in recent years, the morphology of synapses and especially the dynamics of their formation are not sufficiently studied. Differentiated synapses have been described for long-term hippocampus cultures (DE LONG, 1970; LA VAtL & WOLF, 1973; K~kl, 1973; C-V et al, 1975). Among these synapses the axo-dendritic type are most frequent, while axe-somatic and complex glomerular synaptic structures occur more rarely. En passant synapses with a spine are particularly common among axo-dendritic ones. The aim of the present study was to determine the formation of synaptic structures during synaptogenesis and to investigate ultrastructural features of their various types. Some of these results were reported at the IBRO Symposium on the Synapse, Kiev (1976). Abbreoiation: SER, smooth endoplas’micreticulum. 913
EXPERIMENTAL
PROCEDURES
Fragments of posterior hippocampus from one- or twoday-old rats were explanted onto collagen-coated coverslips and cultured either in the Maximov depression slide assemblies or in rotating test tubes. Living and fixed cultures were studied on the 2nd, 3rd, 5th, 7th, 9th, llth, 14th, lath, 2Oth, 25th and 35th days in vitro by means of phase contrast, supra-vital methylene blue staining and electron microscopy. The material was fixed in 2.5% glutaraldehyde, postfixed in 1% OszO,, in phosphate buffer, embedded in Araldite and thin sections were examined with an EMV-1OOL electron microscope. RESULTS Ultrastructural
studies
On the 2nd or 3rd day in vitro neuronal cells which are being slightly differentiated represent mostly neuroblasts. Interneuronal synaptic contacts are lacking. On the 5th-7th day in vitro axonal cylinders along with neurotubules contain neurofilaments (7-10 nm), cisternae of smooth endoplasmic reticulum (SER), elongated mitochondria, and large, clear vesicles from 100 to 150nm in diameter. Specific terminal bulbous expansions are abundant at the terminals of thin axonal cylinders representing early stages of growth cone formation. The axons have bulbous round terminals gradually or abruptly dilating (Fig. 1). Most of the growth cones are characterized by clusters of large (120-150nm) light vesicles (further called ‘growth’ vesicles) bordered by a bilayered membrane commonly filling the whole bulb (Fig. 1A). The vesicles are electron lucent inside,
914
Y. M ZHABOTINSKI. E. 1 CULWASO~, A. R. CHUBAWN and H. V KONO\AIOI.
while the preterm~na1 part of the process has a more compact matrix (Fig. 1B). We have detected no other organelles tn the ‘early’ growth cones of neuronal processes. The pretermmal neurotubujes are arranged near vesacle accumuIations of the growth cone but are absent inside. Single light vesicles as well as groups of vesicles varying in diameter are also found between neurotubules in other regions of the processes lying more proximally from the growth bulb. It suggests th& they are transported from the neuional perikaryon to the periphery. Smce the first synapses are found to arise most frequently in the explant growth zone on the 5th and 7th day in ritro and rarely on the 3rd day M &FYJ, one may think that these first immature interneuronal contacts are formed on the growth cones. At the site where the growth cone contacts the neurona~ process, a new structure possessing features of both the growth cone and an immature synapse is observed (Fig. 1C). The plasma membrane clearly outlines a bulbous enlargement containing fight vesicles of various shape and diameter, mitochondria. SER cisternae and single neurotubules. In such a bulb or bouton 7, regions can be distinguished. The bulb has a cytoplasmic projectron with light. large (i20-150 nm) ‘growth’ vesicles. On the other hand. some n~orphoiogical traits of slightly differentiated synapses are clearly visible. In this region, an accumulation of spherical vesicles of 5@-60nm dia. (typical of the synapses), is seen at the site of contact with the neuronal process. SimuItaneously, pre- and postsynaptic membranes which are already slightly dense become apparent. Since single ribosomes occur in the postsynaptic profile of the process, it is suggested that these are developing axo-dendritic synapses. Such a type of synapse is the first to appear in the hippocampus culture. Sometimes, as soon as the synaptic contact is arranged, the presynaptic axon bouton is growing, or~~nat~ng a small short projection (typical of the growth cone) filled with growth vesicles (Fig. 1D) or to a Mopodium filled only with filamentous material. Continuation of the axon growth after the synaptic contact is also observed m terminals of mossy fibers which form intricate synaptic complexes with dendritic arborization. Another type of axonal growth cone with a light matrix containing aggregates of finest microfitaments (4-6 nm). few growth vesicles. l--3 mitochondria and several enlarged SER cisternae are also slightly discernible in the neuropile. Some growth bulbs, including free ribosomes, cisternae of rough endoplasmic reticulum, and multivesicular bodies should be considered as terminal dendritic boutons. The numerous immature en pussunt axo-dendritic synapses appear after the formation contacts between axonal growth cones and dendrites. On the 9th-11th days in citro immature axosomatic synapses first appear on the bodies of certain pyramidal neurones. At first sight they seem to be the result of contacts of the growth colic with the
neuronal perikaryon (see Fig. 1st; ~~)mewhat later. no passant axo-somatic connections also arise. The uitrastructure is characteristic for- axe--somatic synapses as well as for immature axo-dendriilc synapses: they contain few vesicles, a loose nciwork of microfilaments and single mitochondna. In some boutons there arc ‘growth’ vesicles ~r~ong lhr synaptic vesicles. Approximately on the 12. 14th dnys it: ~$rrrt LL,XO~ dendritic and axosomatic synapses markedly increase in number and become more i-naturr. The dynamics of synaptic differentiation is illustr;rted by an- increasing number of light synaptic \e\!clea and mitochondria in terminal and Y~Zpasser axon boutons (Fig. 2A and B). As well as the hght aynaptlc v~.%ic~es. granular (dense cored) vesicles from 70 to 8a nm dza. appear (I .3 per bouton). SimL~ltaneously. there IS a noticeable decrease in the numb& itf large vesicles and SER cisternae in boutons. the electron den&j of prtr- and postsynaptic membranes enhances, and the synaptic cleft is slightly enlarged. In the synaptic cleft accumulations of osmivphilrc materi& are observed. Axo-somatic synapses, as_a rule, have symmetrically specialized membranes, whereas the membranes of axe-dendritic synapse* are specialized asymmetrically. The number
FIG. 1. Growth cones of growing nerve cell processes of hippocampus culture: (A) (5 days in uitro). Typical distended ending of growth cone containing numerous ‘growth’ vesicles (gv); show also a few small vesicles (sv). Specialized contact with the other processes is absent. x45.000. (B) (7 days in vitro). Elongated bulbous growth cone containing two different types of vesicles (‘growth’ vesicles and synaptic vesicles). A growth cone occurs close to a neuronal &ma (N) x20,000. (C) (7 days HI vitro). Common bouton-like ending forming an immature axedentritic synapse (arrow); SER, cisternae of the smooth endoplasmic reticulum. x 50,000. (D) (11 days in vitro). Two different axonal presynaptic boutons (PB) with a filopodial (F) and vesicular (V) extension. x 2000.
915
FIG 2. Various types of synapses m long term hippoeampus cultures. (A) (20 days EUttrrul I he aw-dendritic synapse. x 30.000. (B) (25 days itI vitro). Axe--somatic synapses. x 30.000. (C) (20 days it t~tro). The axo-spinous-dentrrtlc synapse x 35,009. (D) (27 days irr ~,-a) The dendro-dendritic synapse (arrow). k 10.000 .Mmwutions: sp. spmc. D. the dendrite; N, the neuron; r. rrbosomeb.
Frc. 3. Complex axo-spinous-dendritic synapses (25 days in vitro). (A) the V-shaped spine x 50,000; (B) the synaptic contact of the mossy fiber terminal (mos) with several dendritic spines (*); D, the dendrite x 35,000.
917
Development of synaptic structure in organotypic cultures
the 5-7th days in vitro, i.e. when neuronal cells reach the young neuron stage and synaptic junctions are formed. Spontaneous electrical activity from individual neurons is recorded together with continuous (single), periodic (grouped) discharges. The electrophysiological data corroborate the morphological observations that revealed a high degree of functional activity of neurons in hippocampus culture and complex synaptic interrelations between them.
DISCUSSION
In spite of numerous studies of synaptogenesis in the CNS carried out both in vivo and in vitro, identitication of sequential developmental stages of synaptic structures has not yet been made. The most important question whether specialized synaptic membranes or synaptic vesicles first appear is still controversial. Some authors (GLEES & SHEPPARD,1964; JONES& REVELL,1970; STELZNER, MARTIN & SCOTT, 1973; BWOLEPW, 1975) believe that the formation of electron-dense contacting membranes is the earliest morphological feature of a developing synapse. According to other authors (OSHI, 1967; OPPENHEIM& FOELIX,1972), the formation of specialized membranes is preceded by the appearance of aggregates of synaptic vesicles in the presynaptic bouton. Although membrane specializ-
919
ation is considered to be one of the earliest indices of synaptic development, we came to the conclusion that it is the vesicular structure that indicates the first signs of synapses. An important evidence in favour of this view is the observation that similar vesicular structures arise in various regions of the axon at early cultural stages before the formation of first immature synapses. Occasional growth vesicles, found frequently in the presynaptic zone of immature synapses is also consistent with our view. According to our findings, the first immature synapses are formed by the growth cones. These results are in agreement with the data of KAWANA, SANDRI& AKERT(1971), SKOFF & HAMBURGER(1974), VAUGHN, HENRIKWN & GRIESHABER (1974) who studied the growth cone structure in the spinal cord and cerebellar cortex of chick, mouse, rat and cat embryos. Large vesicles
(‘growth’ vesicles) of the growth cone gradually disappear and they are replaced by synaptic vesicles and mitochondria localized mostly next to the presynaptic membrane. Thus, the growth cone transforms into a presynaptic axonic bouton. This appears to result in an increase of electron density of contacting pre- and, especially, postsynaptic membranes and of a concentration of osmiophilic material in the synaptic cleft. Free ribosomes appear in the postsynaptic zone and accumulations of filamentous material can be detected in this region in cases when the spine is formed.
REFERENCES &X_?OLEmV N. N. (1975) Ulrrastructure of Synapses in the Norm and Pathology. Meditsina Publ. House, Moscow. BUNGER. P., BUNGEM. B. & PETERWNE. R. (1967) The onset of synapse formation in spinal cord cultures as studied by electron microscopy. Bruin Res. 6, 728-749. CHUMASOV E. I., KONOVALOV G. V., CHU~AKOV A. R., FEDOROVA L. A. & SCOSAR 0. I. (1975) Morphology of the hippocampus in tissue culture. Tsitologiya 17, 1306-1312. CRAM S. M. (1966) Development of ‘organotypic’ bioelectric activities in central nervous tissues during maturation
in culture. In International Revue of Neurobiology (eds FWFFERC. C. & SMYTH~SJ. R.). Vol. 9, pp. l-43. Academic Press, New York. GRAIN S. M., RAINE C. S. & E~RNSTAIN M. B. (1975) Early formation synaptic networks in culture of fetal mouse cerebral neocortex and hippocampus. J. Neurobiol. 6, 329-336. DE LONG G. R. (1970) Histogenesis of fetal mouse isocortex and hippocampus in reaggregating cell cultures. Deul. Biol. 22, 563-583. GLEESP. be SHEPPARDB. L. (1964) Electron microscopical studies of the synapse in the developing chick spinal cord. Z. Zellforsch. mikrosk. Anal. 62, 356362. Hijsu L., H&LI E. & ANDRESP. F. (1973) Nervous tissue culture-a model to study action and uptake of putative neurotransmitters such as amino acids. Brain Res. 62, 597-602. JONESD. G. & REVELLE. (1970) The postnatal development of the synapse: a morphological approach utilizing synaptosomes-I. General features. Z. Zellfirsch. mikrosk. Anat. 111, 179-194. KAWANA E., SANDRI C. & AK~RT K. (1971) Ultrastructure of growth cones in the cerebellar cortex of the neonatal rat and cat. Z. Zellforsch. mikrosk. Anat. 115, 248-298. KIMS. U. (1973) Morphological development of neonatal mouse hippocampus cultured in vitro. Expl Neural. 41, W-162. Kw S. U. 62 WENGERE. L. (1972) De novo formation of synapses in cultures of chick neural tube. Nature, New Biol. 236, 152-153. LA VAT H. J. St WOLF M. K. (1973) The development of the mouse dentata gyrus in organotypic cultures of the hippocampal formation. Am. J. Anat. 137, 47-66. MODELP. G., CHAINS. M, BORNSTAIN M. B. 8r PAPPASG. D. (1971) An electron microscopic study of the development of synapses in cultured fetal mouse cerebrum continuously exposed to xylocaine. J. Ceil Biol, 49, 362-371. OCHI J. (1967) Elektronenmikroskopische Untersuchungen des Bulbus olfactorius der Ratte wiihrend der Entwicklung. Z. Zellforsch. mikrosk. Anot. 76, 339-348.
I’ M. ZHAROTINSKI, E. 1. CHUMAWV, A. R. CHUBAK~Vand H. V. KONOVAL(I\
910 OLSON Brain
M. I. & %NGE K. P. 11973) Anatonucal observations on the specificity of synapse form&on
m tissue culture
Res. 59. 19-33.
OPPENHEIM
R. W. & FOELIS R. F. (1971) Synaptogenesls in the chick embryo sptnal cord Nnrtrrt,. New Biol. 235. 126 118. SKOFTR P. & HAMRURC;ER V. (1974) Fme structure of dendrltlc and axonal growth cones in embryome chick spmal cord J. camp. Neural. 153, 107 147 STFLZNER D. J.. MARTIN A. H. & SCOTI G I,. (1973) Early stages of synaptogenesis m the Lervlcd spinal cord of the cluck embryo. Z Zellfbrsch m&r&. .&at. 138, 475-488. VAUGHN J. E., HENRICKSON C. K. CyrGNESHAHFRJ. A. (1974) A quantitative study of synapses on motor neuron dendritic growth cones in developmg mouse spmal cord. .I (‘r/l Biol. 60, 664-671
DEVELOPMENT OF SYNAPTIC STRUCTURE AND FUNCTION IN ORGANOTYPIC CULTURES OF THE RAT HIPPOCAMPUS Y. M. ZHABOTINSKI, E. I. CHUMASOV, A. R. CHUBAKOVand H. V. KONOVALOV Laboratory of Pathology of Nervous System, Institute of Experimental Medicine of the Academy of Medical Sciences of the U.S.S.R., Leningrad, and Department of Memory Problems, Institute of Biophysics of the Academy of Sciences of the U.S.S.R., Pushchino-on-the-Oka, U.S.S.R.
Abstract-The sequence of development of synapses, as well as the ultrastructure of axonal growth cones, has been investigated electron microscopically in tissue cultures of the newborn rat hippocampus. During differentiation of the tissue cultures, the formation of synapses is preceded by identifiable growth cones. A characteristic feature of axonal growth cones is the presence of numerous large clear vesicles which vary in diameter from _ 100 to 150 nm. The first immature synapses were formed on the Sth, 6th or 7th day in oirro on the growth cones of differentiating neuronal processes. Axonal growth cones are occasionally found to be presynaptic to a dendrite. At first axo-dendritic synapses, most of them being en passant, arise, whereas ax&somatic and axc+spinous-dendritic synapses of different complex structures appear later. It is suggested that the earliest signs of synaptogenesis are vesicular structures (‘growth’ vesicles and few synaptic vesicles), which occur in growth cones, axons and presynaptic boutons of immature synaptic contacts even before formation of the specialized pre- and postsynaptic membranes.
CULTUREDnervous tissue is a most suitable subject for studying the dynamics of functional and morphological development of synapses. As is known, synapses are not only preserved but even newly formed in organotypic cultures of various divisions of the nervous system (WIN, 1%6; BUNGE,BUNGE & PE’IERSON,1967; MODEL, BORNSTEIN,CIUIN & PAPPAS,1971; QLSON& BUNGE, 1973; H&LI, H&L] & ANDRES,1973; Km & WENGER,1972; C-v, KONOV~V, CHunfiOV, I%OROVA & soosiuc, 1975; GRAIN,REINE& BORNSTEIN,1975). Although a great number of electron-microscopic examinations of cultured nervous tissue has been published in recent years, the morphology of synapses and especially the dynamics of their formation are not sufficiently studied. Differentiated synapses have been described for long-term hippocampus cultures (DE LONG, 1970; LA VAtL & WOLF, 1973; K~kl, 1973; C-V et al, 1975). Among these synapses the axo-dendritic type are most frequent, while axe-somatic and complex glomerular synaptic structures occur more rarely. En passant synapses with a spine are particularly common among axo-dendritic ones. The aim of the present study was to determine the formation of synaptic structures during synaptogenesis and to investigate ultrastructural features of their various types. Some of these results were reported at the IBRO Symposium on the Synapse, Kiev (1976). Abbreoiation: SER, smooth endoplas’micreticulum. 913
EXPERIMENTAL
PROCEDURES
Fragments of posterior hippocampus from one- or twoday-old rats were explanted onto collagen-coated coverslips and cultured either in the Maximov depression slide assemblies or in rotating test tubes. Living and fixed cultures were studied on the 2nd, 3rd, 5th, 7th, 9th, llth, 14th, lath, 2Oth, 25th and 35th days in vitro by means of phase contrast, supra-vital methylene blue staining and electron microscopy. The material was fixed in 2.5% glutaraldehyde, postfixed in 1% OszO,, in phosphate buffer, embedded in Araldite and thin sections were examined with an EMV-1OOL electron microscope. RESULTS Ultrastructural
studies
On the 2nd or 3rd day in vitro neuronal cells which are being slightly differentiated represent mostly neuroblasts. Interneuronal synaptic contacts are lacking. On the 5th-7th day in vitro axonal cylinders along with neurotubules contain neurofilaments (7-10 nm), cisternae of smooth endoplasmic reticulum (SER), elongated mitochondria, and large, clear vesicles from 100 to 150nm in diameter. Specific terminal bulbous expansions are abundant at the terminals of thin axonal cylinders representing early stages of growth cone formation. The axons have bulbous round terminals gradually or abruptly dilating (Fig. 1). Most of the growth cones are characterized by clusters of large (120-150nm) light vesicles (further called ‘growth’ vesicles) bordered by a bilayered membrane commonly filling the whole bulb (Fig. 1A). The vesicles are electron lucent inside,
914
Y. M ZHABOTINSKI. E. 1 CULWASO~, A. R. CHUBAWN and H. V KONO\AIOI.
while the preterm~na1 part of the process has a more compact matrix (Fig. 1B). We have detected no other organelles tn the ‘early’ growth cones of neuronal processes. The pretermmal neurotubujes are arranged near vesacle accumuIations of the growth cone but are absent inside. Single light vesicles as well as groups of vesicles varying in diameter are also found between neurotubules in other regions of the processes lying more proximally from the growth bulb. It suggests th& they are transported from the neuional perikaryon to the periphery. Smce the first synapses are found to arise most frequently in the explant growth zone on the 5th and 7th day in ritro and rarely on the 3rd day M &FYJ, one may think that these first immature interneuronal contacts are formed on the growth cones. At the site where the growth cone contacts the neurona~ process, a new structure possessing features of both the growth cone and an immature synapse is observed (Fig. 1C). The plasma membrane clearly outlines a bulbous enlargement containing fight vesicles of various shape and diameter, mitochondria. SER cisternae and single neurotubules. In such a bulb or bouton 7, regions can be distinguished. The bulb has a cytoplasmic projectron with light. large (i20-150 nm) ‘growth’ vesicles. On the other hand. some n~orphoiogical traits of slightly differentiated synapses are clearly visible. In this region, an accumulation of spherical vesicles of 5@-60nm dia. (typical of the synapses), is seen at the site of contact with the neuronal process. SimuItaneously, pre- and postsynaptic membranes which are already slightly dense become apparent. Since single ribosomes occur in the postsynaptic profile of the process, it is suggested that these are developing axo-dendritic synapses. Such a type of synapse is the first to appear in the hippocampus culture. Sometimes, as soon as the synaptic contact is arranged, the presynaptic axon bouton is growing, or~~nat~ng a small short projection (typical of the growth cone) filled with growth vesicles (Fig. 1D) or to a Mopodium filled only with filamentous material. Continuation of the axon growth after the synaptic contact is also observed m terminals of mossy fibers which form intricate synaptic complexes with dendritic arborization. Another type of axonal growth cone with a light matrix containing aggregates of finest microfitaments (4-6 nm). few growth vesicles. l--3 mitochondria and several enlarged SER cisternae are also slightly discernible in the neuropile. Some growth bulbs, including free ribosomes, cisternae of rough endoplasmic reticulum, and multivesicular bodies should be considered as terminal dendritic boutons. The numerous immature en pussunt axo-dendritic synapses appear after the formation contacts between axonal growth cones and dendrites. On the 9th-11th days in citro immature axosomatic synapses first appear on the bodies of certain pyramidal neurones. At first sight they seem to be the result of contacts of the growth colic with the
neuronal perikaryon (see Fig. 1st; ~~)mewhat later. no passant axo-somatic connections also arise. The uitrastructure is characteristic for- axe--somatic synapses as well as for immature axo-dendriilc synapses: they contain few vesicles, a loose nciwork of microfilaments and single mitochondna. In some boutons there arc ‘growth’ vesicles ~r~ong lhr synaptic vesicles. Approximately on the 12. 14th dnys it: ~$rrrt LL,XO~ dendritic and axosomatic synapses markedly increase in number and become more i-naturr. The dynamics of synaptic differentiation is illustr;rted by an- increasing number of light synaptic \e\!clea and mitochondria in terminal and Y~Zpasser axon boutons (Fig. 2A and B). As well as the hght aynaptlc v~.%ic~es. granular (dense cored) vesicles from 70 to 8a nm dza. appear (I .3 per bouton). SimL~ltaneously. there IS a noticeable decrease in the numb& itf large vesicles and SER cisternae in boutons. the electron den&j of prtr- and postsynaptic membranes enhances, and the synaptic cleft is slightly enlarged. In the synaptic cleft accumulations of osmivphilrc materi& are observed. Axo-somatic synapses, as_a rule, have symmetrically specialized membranes, whereas the membranes of axe-dendritic synapse* are specialized asymmetrically. The number
FIG. 1. Growth cones of growing nerve cell processes of hippocampus culture: (A) (5 days in uitro). Typical distended ending of growth cone containing numerous ‘growth’ vesicles (gv); show also a few small vesicles (sv). Specialized contact with the other processes is absent. x45.000. (B) (7 days in vitro). Elongated bulbous growth cone containing two different types of vesicles (‘growth’ vesicles and synaptic vesicles). A growth cone occurs close to a neuronal &ma (N) x20,000. (C) (7 days HI vitro). Common bouton-like ending forming an immature axedentritic synapse (arrow); SER, cisternae of the smooth endoplasmic reticulum. x 50,000. (D) (11 days in vitro). Two different axonal presynaptic boutons (PB) with a filopodial (F) and vesicular (V) extension. x 2000.
915
FIG 2. Various types of synapses m long term hippoeampus cultures. (A) (20 days EUttrrul I he aw-dendritic synapse. x 30.000. (B) (25 days itI vitro). Axe--somatic synapses. x 30.000. (C) (20 days it t~tro). The axo-spinous-dentrrtlc synapse x 35,009. (D) (27 days irr ~,-a) The dendro-dendritic synapse (arrow). k 10.000 .Mmwutions: sp. spmc. D. the dendrite; N, the neuron; r. rrbosomeb.
Frc. 3. Complex axo-spinous-dendritic synapses (25 days in vitro). (A) the V-shaped spine x 50,000; (B) the synaptic contact of the mossy fiber terminal (mos) with several dendritic spines (*); D, the dendrite x 35,000.
917
Development of synaptic structure in organotypic cultures
the 5-7th days in vitro, i.e. when neuronal cells reach the young neuron stage and synaptic junctions are formed. Spontaneous electrical activity from individual neurons is recorded together with continuous (single), periodic (grouped) discharges. The electrophysiological data corroborate the morphological observations that revealed a high degree of functional activity of neurons in hippocampus culture and complex synaptic interrelations between them.
DISCUSSION
In spite of numerous studies of synaptogenesis in the CNS carried out both in vivo and in vitro, identitication of sequential developmental stages of synaptic structures has not yet been made. The most important question whether specialized synaptic membranes or synaptic vesicles first appear is still controversial. Some authors (GLEES & SHEPPARD,1964; JONES& REVELL,1970; STELZNER, MARTIN & SCOTT, 1973; BWOLEPW, 1975) believe that the formation of electron-dense contacting membranes is the earliest morphological feature of a developing synapse. According to other authors (OSHI, 1967; OPPENHEIM& FOELIX,1972), the formation of specialized membranes is preceded by the appearance of aggregates of synaptic vesicles in the presynaptic bouton. Although membrane specializ-
919
ation is considered to be one of the earliest indices of synaptic development, we came to the conclusion that it is the vesicular structure that indicates the first signs of synapses. An important evidence in favour of this view is the observation that similar vesicular structures arise in various regions of the axon at early cultural stages before the formation of first immature synapses. Occasional growth vesicles, found frequently in the presynaptic zone of immature synapses is also consistent with our view. According to our findings, the first immature synapses are formed by the growth cones. These results are in agreement with the data of KAWANA, SANDRI& AKERT(1971), SKOFF & HAMBURGER(1974), VAUGHN, HENRIKWN & GRIESHABER (1974) who studied the growth cone structure in the spinal cord and cerebellar cortex of chick, mouse, rat and cat embryos. Large vesicles
(‘growth’ vesicles) of the growth cone gradually disappear and they are replaced by synaptic vesicles and mitochondria localized mostly next to the presynaptic membrane. Thus, the growth cone transforms into a presynaptic axonic bouton. This appears to result in an increase of electron density of contacting pre- and, especially, postsynaptic membranes and of a concentration of osmiophilic material in the synaptic cleft. Free ribosomes appear in the postsynaptic zone and accumulations of filamentous material can be detected in this region in cases when the spine is formed.
REFERENCES &X_?OLEmV N. N. (1975) Ulrrastructure of Synapses in the Norm and Pathology. Meditsina Publ. House, Moscow. BUNGER. P., BUNGEM. B. & PETERWNE. R. (1967) The onset of synapse formation in spinal cord cultures as studied by electron microscopy. Bruin Res. 6, 728-749. CHUMASOV E. I., KONOVALOV G. V., CHU~AKOV A. R., FEDOROVA L. A. & SCOSAR 0. I. (1975) Morphology of the hippocampus in tissue culture. Tsitologiya 17, 1306-1312. CRAM S. M. (1966) Development of ‘organotypic’ bioelectric activities in central nervous tissues during maturation
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