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Pinocytosis at postsynaptic membranes: electron microscopic evidence
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SHORT COMMUNICATIONS
Pinocytosis at postsynaptic membranes: electron microscopic evidence Bulk transport of metabolic and other products between neurons has long been considered of importance in interneuronal communication despite the paucity of information supporting this notion. One requirement for bulk transport would be micropinocytosis. Uptake of ferritin by axon terminals at the motor endplate 2, and by axonal and dendritic processes in CNS 4 has been demonstrated, but there have been no clear demonstrations of micropinocytosis specifically at the postsynaptic membrane. The purpose of this note is to describe: (1) observations of coated invaginations at the postsynaptic membrane in a number of neuronal systems, and (2) demonstrations of uptake by postsynaptic processes of saccharated iron oxide, a marker substance which distributes extracellularly, primarily in the synaptic cleft6. A spectrum of chemically and electrically transmitting synapses has been examined by electron microscopy. In agreement with the observations of others 1,5,10 coated and uncoated invaginations of membranes of presynaptic terminals were observed. Additional observations indicate the existence of coated and uncoated invaginations in postsynaptic membranes, both axosomatic and axodendritic in nature, as well as in non-synaptic regions. Coated invaginations in postsynaptic membranes have been observed in the following tissues: feline sensorimotor cortex, oculomotor nucleus, superior olivary nucleus, amygdala, hippocampus, lateral geniculate nucleus; teleost oculomotor nucleus, spinal cord; bat spinal cord. Specimens were fixed with osmium tetroxide or aldehydes followed by osmium tetroxide, dehydrated in graded alcohol solutions, and embedded in Epon 812. Sections were cut by ultramicrotome, and examined with Philips 200 and RCA-EMU-3G electron microscopes. Presynaptic terminals contain clear vesicles, 300-600 A in diameter, larger dense core vesicles, and mitochondria, lnvaginations of membranes of presynaptic terminals, sometimes bearing a fuzzy coat approximately 100/~ wide on the cytoplasmic surface, were observed at the synaptic surface and at other portions of the terminal, adjacent to axons, dendrites, and glial cells; the diameter of such invaginations varied between 300 and 1200 A. In every tissue examined, examples of coated and uncoated invaginations of postsynaptic membranes were observed, subjacent to axon terminals. These invaginations varied in diameter from 500-1200 A, and were sometimes connected to the surface membrane by a thin 'neck' (Fig. 2). A radially striated electron dense coating, approximately 100 A thick, was usually observed on the cytoplasmic surface of the invaginated portion of membrane. In these instances, the invaginated membrane appeared morphologically distinct from adjacent surface membranes (Fig. 1). Coated vesicles, approximately circular in cross-sectional outline and approximately 6001500 A in diameter, were occasionally observed in the postsynaptic cytoplasm, most often subjacent to the cell surface. They were occasionally seen in the region of the Golgi apparatus. Subsurface cisterns and multivesicular bodies were noted at synapses, but did not appear to communicate with the extracellular synaptic cleft. Invaginations were seen at postsynaptic membranes of dendritic spine synapses in cat sensorimotor Brain Research, 14 (1969) 240-244
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Fig. 1. Axosomatic synapse from feline oculomotor nucleus. The axon terminal (A) contains synaptic vesicles and a mitochondrion. A coated invagination of the postsynaptic membrane (a) and a coated intracellular vesicle (b) are seen; these are enlarged respectively in the lower right-hand and upper left-hand insets. × 36,000; insets, × 60,000. Fig. 2. Axosomatic synapse from feline oculomotor nucleus. The arrow indicates an invagination of the postsynaptic membrane. The bulbous portion of the invagination at the tip of the arrow communicates with the extracellular synaptic cleft via a thin 'neck'. x 55,000. A, axon terminal; M, mitochondrion; S, soma.
Brain Research, 14 (1969) 240-244
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SHORT COMMUNICATIONS
cortex. A l t h o u g h these were in close p r o x i m i t y to the spine a p p a r a t u s , m o r p h o l o g i c a l connections with the a p p a r a t u s were not observed. It is o f interest that coated invaginations were seen not only at chemically t r a n s m i t t i n g synapses, but also at synapses o f t e l e o s t o c u l o m o t o r nucleus in which pre- and p o s t s y n a p t i c m e m b r a n e s are closely a p p o s e d (Fig. 6) and in which there is electrophysiological evidence for electrical t r a n s m i s s i o n 9. The frequency with which these invaginations were observed varied f r o m tissue to tissue. The highest frequency was a p p r o x i m a t e l y 1/50 synaptic profiles (teleost o c u l o m o t o r nucleus). Inasmuch as vesicles are a p p r o x i m a t e l y 0.1 /~ in diameter, and sections are less t h a n 0. I /~ thick, while most synaptic k n o b s are a b o u t I ~ in diameter, the actual frequency o f invaginations per synapse is likely to be 10 times the frequency observed in thin sections. Pinocytosis was d e m o n s t r a t e d with small a m o u n t s o f saccharated iron oxide, an electron dense m a r k e r , t h a t was introduced into the feline lateral geniculate nucleus. The lateral geniculate nucleus ( L G N ) o f a b a r b i t u r a t e anesthetized a d u l t cat was surgically exposed, and volumes o f less t h a n 0. I ml o f a 4 0 ~ solution o f saccharated i r o n oxide were injected into superficial p o r t i o n s of the dorsal nucleus. The optic tract on one side was stimulated (10/sec, 100 usec s u p r a m a x i m a l pulses) with a b i p o l a r electrode, and the evoked potential from the surface o f the L G N amplified and displayed by conventional methods. The d e m o n s t r a t i o n o f pre- and p o s t s y n a p t i c c o m p o n e n t s o f the L G N response served as a guide to the site o f injection o f the m a r k e r . The c o n t r a l a t e r a l L G N was not stimulated. Twenty minutes after injection, injected areas on both sides were fixed for 90 rain in 3 ° C g l u t a r a l d e h y d e - p a r a f o r m a l d e h y d e in p h o s p h a t e buffer (pH 7.4, 640 mosm.), washed overnight in fresh buffer solution, and post-fixed in 1% OsO,l in p h o s p h a t e buffer. Tissue a p p r o x i m a t e l y 1 m m f r o m the site o f injection was examined. Extracellular m a r k e r was localized p r i m a r i l y at synaptic clefts, equidistant from pre- and p o s t s y n a p t i c m e m b r a n e s 6. In b o t h stimulated and n o n - s t i m u l a t e d tissue, occasional axonal and dendritic processes were seen to c o n t a i n m a r k e r substance within m e m b r a n e b o u n d e d vesicles (Fig. 7), suggesting u p t a k e f r o m the synaptic g a p substance.
Fig. 3. Axodendritic synapse from feline hippocampus. The axon terminal (A) contains synaptic vesicles, and an invagination (arrow) is seen in the postsynaptic membrane. The invaginating membrane bears a radially striated coat, about 150 A thick, on its cytoplasmic surface, x 48,000. D, dendritic process. Fig. 4. Axodendritic synapse from feline sensorimotor cortex. The arrow indicates an invagination in the postsynaptic membrane, x 60,000. A, axon terminal; D, dendritic process. Fig. 5. Axodendritic synapse from feline superior olivary nucleus. The presynaptic terminal (A) contains a number of synaptic vesicles and mitochondria. A coated invagination of the postsynaptic membrane is indicated by the arrow, x 40,000. Fig. 6. Electrical synapse from the oculomotor nucleus of the spiny boxfish Chilomycterus. The presynaptic terminal (A) of this axosomatic synapse contains vesicles and mitochondria. A specialized junction (J) at which pre- and postsynaptic membranes become closely apposed, is seen. An invagination (arrow) is present in the postsynaptic membrane. × 34,000. S, soma. Fig. 7. Axodendritic synapse from feline lateral geniculate nucleus, approximately l mm from site of injection of saccharated iron oxide. Stimulated via optic tract. Particles of the electron dense marker substance are visible in the synaptic cleft and within two membrane bounded profiles in the postsynaptic process, at arrows, x 170,000. A, axon terminal. Brain Research, 14 (1969) 240-244
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Brain Research, 14 (1969) 240-244
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B o d i a n 3 has noted the occurrence o f ' s p i n y ' vesicles at p o s t s y n a p t i c m e m b r a n e s in m o n k e y spinal m o t o n e u r o n s . The present observations would suggest that coated invaginations are a general feature o f p o s t s y n a p t i c m e m b r a n e s , and that such vesicles are i m p o r t a n t in the i n c o r p o r a t i o n o f extracellular substances into p o s t s y n a p t i c processes. Previous investigations have indicated u p t a k e o f yolk protein by coated vesicles 8, and ' m i c r o p i n o c y t o t i c ' i n c o r p o r a t i o n o f ferritin into coated vesicles in nons y n a p t i c regions o f neurons in t o a d spinal ganglion 7. The present observations suggest that m i c r o p i n o c y t o s i s m a y play a role in o p e r a t i o n s at the synapse which are not i m m e d i a t e l y related to synaptic transmission. This w o r k was s u p p o r t e d in p a r t by grants NB-07674-03, NB-07512-02, and 5T5-GM-1674-05 f r o m the N a t i o n a l Institutes o f Health. Department of Anatomy, Albert Einstein College of Medicine, New York, N.Y. 10461 (U.S.A.)
STEPHEN G. WAXMAN GEORGE D. PAPPAS
1 ANDRES,K. H., AND DURING, M. V., Mikropinozytose in motorischen Endplatten, Naturwissenschaften, 53 (1966) 615-616. 2 BIRKS,R. I., The fine structure of motor nerve endings at frog myoneural junctions, Ann. N. Y. Acad. Sci., 135 (1966) 8-19. 3 BODIAN,D., Synaptic types on spinal motoneurons: an electron microscopic study, Bull. Johns Hopkins Hosp., 119 (1966) 16-45. 4 BRIGHTMAN,M. W., The distribution within the brain of ferritin injected into cecebrospinal fluid compartments, Amer. J. Anat., 117 (1965) 193--220. 5 CHARLTON,B. T., AND GRAY, E. G., Comparative electron microscopy of synapses in the vertebrate spinal cord, J. Cell Sci., 1 (1966) 67-80. 6 PAPPAS,G. D., AND PtJRPVRA, D. P., Distribution of colloidal particles in extracellular space and synaptic cleft substance of mammalian cerebral cortex, Nature (Lond.), 210 (1966) 1391-1392. 7 ROSENBLUTH,J., AND WJSSlG,S. L., The distribution of exogenous ferritin in toad spinal ganglia and the mechanism of its uptake by neurons, J. Cell Biol., 23 (1964) 307-325. 8 ROTH, T. F., AND PORTER, K. R., Yolk protein uptake in the oocyte of the mosquito, Aedes aegypti, J. Cell Biol., 20 (1964) 313-332. 9 WAXMAN,S. G., KRIEBEL,M. E., BENNETT, M. V. L., AND PAPPAS,G. D., Fine structural basis of synaptic activity in oculomotor nuclei of the spiny boxfish, J. Cell BioL, 39 (1968) 140a. 10 WESTRtrM, L. E., On the origin of synaptic vesicles in cerebral cortex, J. Physiol. (Lond.), 179 (1965) 4P-6P. (Accepted March 19th, 1969)
Brain Research, 14 (1969) 240-244
SHORT COMMUNICATIONS
Pinocytosis at postsynaptic membranes: electron microscopic evidence Bulk transport of metabolic and other products between neurons has long been considered of importance in interneuronal communication despite the paucity of information supporting this notion. One requirement for bulk transport would be micropinocytosis. Uptake of ferritin by axon terminals at the motor endplate 2, and by axonal and dendritic processes in CNS 4 has been demonstrated, but there have been no clear demonstrations of micropinocytosis specifically at the postsynaptic membrane. The purpose of this note is to describe: (1) observations of coated invaginations at the postsynaptic membrane in a number of neuronal systems, and (2) demonstrations of uptake by postsynaptic processes of saccharated iron oxide, a marker substance which distributes extracellularly, primarily in the synaptic cleft6. A spectrum of chemically and electrically transmitting synapses has been examined by electron microscopy. In agreement with the observations of others 1,5,10 coated and uncoated invaginations of membranes of presynaptic terminals were observed. Additional observations indicate the existence of coated and uncoated invaginations in postsynaptic membranes, both axosomatic and axodendritic in nature, as well as in non-synaptic regions. Coated invaginations in postsynaptic membranes have been observed in the following tissues: feline sensorimotor cortex, oculomotor nucleus, superior olivary nucleus, amygdala, hippocampus, lateral geniculate nucleus; teleost oculomotor nucleus, spinal cord; bat spinal cord. Specimens were fixed with osmium tetroxide or aldehydes followed by osmium tetroxide, dehydrated in graded alcohol solutions, and embedded in Epon 812. Sections were cut by ultramicrotome, and examined with Philips 200 and RCA-EMU-3G electron microscopes. Presynaptic terminals contain clear vesicles, 300-600 A in diameter, larger dense core vesicles, and mitochondria, lnvaginations of membranes of presynaptic terminals, sometimes bearing a fuzzy coat approximately 100/~ wide on the cytoplasmic surface, were observed at the synaptic surface and at other portions of the terminal, adjacent to axons, dendrites, and glial cells; the diameter of such invaginations varied between 300 and 1200 A. In every tissue examined, examples of coated and uncoated invaginations of postsynaptic membranes were observed, subjacent to axon terminals. These invaginations varied in diameter from 500-1200 A, and were sometimes connected to the surface membrane by a thin 'neck' (Fig. 2). A radially striated electron dense coating, approximately 100 A thick, was usually observed on the cytoplasmic surface of the invaginated portion of membrane. In these instances, the invaginated membrane appeared morphologically distinct from adjacent surface membranes (Fig. 1). Coated vesicles, approximately circular in cross-sectional outline and approximately 6001500 A in diameter, were occasionally observed in the postsynaptic cytoplasm, most often subjacent to the cell surface. They were occasionally seen in the region of the Golgi apparatus. Subsurface cisterns and multivesicular bodies were noted at synapses, but did not appear to communicate with the extracellular synaptic cleft. Invaginations were seen at postsynaptic membranes of dendritic spine synapses in cat sensorimotor Brain Research, 14 (1969) 240-244
SHORT COMMUNICATIONS
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Fig. 1. Axosomatic synapse from feline oculomotor nucleus. The axon terminal (A) contains synaptic vesicles and a mitochondrion. A coated invagination of the postsynaptic membrane (a) and a coated intracellular vesicle (b) are seen; these are enlarged respectively in the lower right-hand and upper left-hand insets. × 36,000; insets, × 60,000. Fig. 2. Axosomatic synapse from feline oculomotor nucleus. The arrow indicates an invagination of the postsynaptic membrane. The bulbous portion of the invagination at the tip of the arrow communicates with the extracellular synaptic cleft via a thin 'neck'. x 55,000. A, axon terminal; M, mitochondrion; S, soma.
Brain Research, 14 (1969) 240-244
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SHORT COMMUNICATIONS
cortex. A l t h o u g h these were in close p r o x i m i t y to the spine a p p a r a t u s , m o r p h o l o g i c a l connections with the a p p a r a t u s were not observed. It is o f interest that coated invaginations were seen not only at chemically t r a n s m i t t i n g synapses, but also at synapses o f t e l e o s t o c u l o m o t o r nucleus in which pre- and p o s t s y n a p t i c m e m b r a n e s are closely a p p o s e d (Fig. 6) and in which there is electrophysiological evidence for electrical t r a n s m i s s i o n 9. The frequency with which these invaginations were observed varied f r o m tissue to tissue. The highest frequency was a p p r o x i m a t e l y 1/50 synaptic profiles (teleost o c u l o m o t o r nucleus). Inasmuch as vesicles are a p p r o x i m a t e l y 0.1 /~ in diameter, and sections are less t h a n 0. I /~ thick, while most synaptic k n o b s are a b o u t I ~ in diameter, the actual frequency o f invaginations per synapse is likely to be 10 times the frequency observed in thin sections. Pinocytosis was d e m o n s t r a t e d with small a m o u n t s o f saccharated iron oxide, an electron dense m a r k e r , t h a t was introduced into the feline lateral geniculate nucleus. The lateral geniculate nucleus ( L G N ) o f a b a r b i t u r a t e anesthetized a d u l t cat was surgically exposed, and volumes o f less t h a n 0. I ml o f a 4 0 ~ solution o f saccharated i r o n oxide were injected into superficial p o r t i o n s of the dorsal nucleus. The optic tract on one side was stimulated (10/sec, 100 usec s u p r a m a x i m a l pulses) with a b i p o l a r electrode, and the evoked potential from the surface o f the L G N amplified and displayed by conventional methods. The d e m o n s t r a t i o n o f pre- and p o s t s y n a p t i c c o m p o n e n t s o f the L G N response served as a guide to the site o f injection o f the m a r k e r . The c o n t r a l a t e r a l L G N was not stimulated. Twenty minutes after injection, injected areas on both sides were fixed for 90 rain in 3 ° C g l u t a r a l d e h y d e - p a r a f o r m a l d e h y d e in p h o s p h a t e buffer (pH 7.4, 640 mosm.), washed overnight in fresh buffer solution, and post-fixed in 1% OsO,l in p h o s p h a t e buffer. Tissue a p p r o x i m a t e l y 1 m m f r o m the site o f injection was examined. Extracellular m a r k e r was localized p r i m a r i l y at synaptic clefts, equidistant from pre- and p o s t s y n a p t i c m e m b r a n e s 6. In b o t h stimulated and n o n - s t i m u l a t e d tissue, occasional axonal and dendritic processes were seen to c o n t a i n m a r k e r substance within m e m b r a n e b o u n d e d vesicles (Fig. 7), suggesting u p t a k e f r o m the synaptic g a p substance.
Fig. 3. Axodendritic synapse from feline hippocampus. The axon terminal (A) contains synaptic vesicles, and an invagination (arrow) is seen in the postsynaptic membrane. The invaginating membrane bears a radially striated coat, about 150 A thick, on its cytoplasmic surface, x 48,000. D, dendritic process. Fig. 4. Axodendritic synapse from feline sensorimotor cortex. The arrow indicates an invagination in the postsynaptic membrane, x 60,000. A, axon terminal; D, dendritic process. Fig. 5. Axodendritic synapse from feline superior olivary nucleus. The presynaptic terminal (A) contains a number of synaptic vesicles and mitochondria. A coated invagination of the postsynaptic membrane is indicated by the arrow, x 40,000. Fig. 6. Electrical synapse from the oculomotor nucleus of the spiny boxfish Chilomycterus. The presynaptic terminal (A) of this axosomatic synapse contains vesicles and mitochondria. A specialized junction (J) at which pre- and postsynaptic membranes become closely apposed, is seen. An invagination (arrow) is present in the postsynaptic membrane. × 34,000. S, soma. Fig. 7. Axodendritic synapse from feline lateral geniculate nucleus, approximately l mm from site of injection of saccharated iron oxide. Stimulated via optic tract. Particles of the electron dense marker substance are visible in the synaptic cleft and within two membrane bounded profiles in the postsynaptic process, at arrows, x 170,000. A, axon terminal. Brain Research, 14 (1969) 240-244
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Brain Research, 14 (1969) 240-244
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SHORT COMMUNICATIONS
B o d i a n 3 has noted the occurrence o f ' s p i n y ' vesicles at p o s t s y n a p t i c m e m b r a n e s in m o n k e y spinal m o t o n e u r o n s . The present observations would suggest that coated invaginations are a general feature o f p o s t s y n a p t i c m e m b r a n e s , and that such vesicles are i m p o r t a n t in the i n c o r p o r a t i o n o f extracellular substances into p o s t s y n a p t i c processes. Previous investigations have indicated u p t a k e o f yolk protein by coated vesicles 8, and ' m i c r o p i n o c y t o t i c ' i n c o r p o r a t i o n o f ferritin into coated vesicles in nons y n a p t i c regions o f neurons in t o a d spinal ganglion 7. The present observations suggest that m i c r o p i n o c y t o s i s m a y play a role in o p e r a t i o n s at the synapse which are not i m m e d i a t e l y related to synaptic transmission. This w o r k was s u p p o r t e d in p a r t by grants NB-07674-03, NB-07512-02, and 5T5-GM-1674-05 f r o m the N a t i o n a l Institutes o f Health. Department of Anatomy, Albert Einstein College of Medicine, New York, N.Y. 10461 (U.S.A.)
STEPHEN G. WAXMAN GEORGE D. PAPPAS
1 ANDRES,K. H., AND DURING, M. V., Mikropinozytose in motorischen Endplatten, Naturwissenschaften, 53 (1966) 615-616. 2 BIRKS,R. I., The fine structure of motor nerve endings at frog myoneural junctions, Ann. N. Y. Acad. Sci., 135 (1966) 8-19. 3 BODIAN,D., Synaptic types on spinal motoneurons: an electron microscopic study, Bull. Johns Hopkins Hosp., 119 (1966) 16-45. 4 BRIGHTMAN,M. W., The distribution within the brain of ferritin injected into cecebrospinal fluid compartments, Amer. J. Anat., 117 (1965) 193--220. 5 CHARLTON,B. T., AND GRAY, E. G., Comparative electron microscopy of synapses in the vertebrate spinal cord, J. Cell Sci., 1 (1966) 67-80. 6 PAPPAS,G. D., AND PtJRPVRA, D. P., Distribution of colloidal particles in extracellular space and synaptic cleft substance of mammalian cerebral cortex, Nature (Lond.), 210 (1966) 1391-1392. 7 ROSENBLUTH,J., AND WJSSlG,S. L., The distribution of exogenous ferritin in toad spinal ganglia and the mechanism of its uptake by neurons, J. Cell Biol., 23 (1964) 307-325. 8 ROTH, T. F., AND PORTER, K. R., Yolk protein uptake in the oocyte of the mosquito, Aedes aegypti, J. Cell Biol., 20 (1964) 313-332. 9 WAXMAN,S. G., KRIEBEL,M. E., BENNETT, M. V. L., AND PAPPAS,G. D., Fine structural basis of synaptic activity in oculomotor nuclei of the spiny boxfish, J. Cell BioL, 39 (1968) 140a. 10 WESTRtrM, L. E., On the origin of synaptic vesicles in cerebral cortex, J. Physiol. (Lond.), 179 (1965) 4P-6P. (Accepted March 19th, 1969)
Brain Research, 14 (1969) 240-244