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Filamentous organelles in receptor-bipolar synapses of the retina
J, ULTRASTRUCTURE RESEARCH 10, 207-216 (1964)
207
Filamentous Organelles in Receptor-Bipolar Synapses of the Retina 1 SHARON MOUNTFORD
Department of Zoology, University of California, Los Angeles, Los Angeles, California Received October 1, 1963 Retinas of male albino guinea pigs were studied in serial sections. A consistent feature of the synapse was a filamentous cross-striated organelle, which varied in form from one type of synapse to another. In the r-Type synapse this organelle is a bare spindle extending across the cell in the region of the dendritic invaginations with its axis perpendicular to the long axis of the receptor, c~-Typereceptors have an attenuated, but basically similar, structure running parallel to the cell axis from the neck region between the cell body proper and the synaptic spherule to the vitreal side of the cluster of dendritic invaginations. Paranuclear synapses have a similar structure in intimate contact with the nuclear envelope. These latter two organelles are accompanied by membranes that were not seen completely to sheath the structures but rather to form accompanying elongate sacs. During the course of a serial-section study of the guinea pig retina, a previously undescribed organelle Was found in the receptor-bipolar synapses. It appears in three different forms in the three types of synapses, o~, r , and paranuclear. The s-type receptor according to the classification of Sj/3strand (7) has a synaptic spherule at the vitreal end of the receptor. A neck region connects this synaptic end body to the cell body proper. The spherule is rather regular in shape, displaying a nearly circular profile in cross section. There is one synaptic ribbon or lamella associated with one compact set of invaginations. The/~-type receptor (7) also has a distinct end body, with a narrow neck region between synapse and cell body, as in the s-type; it is, however, larger and irregular in shape and has multiple ribbons and several groups of invaginations. As the name implies, the paranuclear synapse has no special distinct synaptic end body or spherule. The typica ! synaptic structures--dendritic invaginations, ribbons, and vesicles--are present in the vicinity of the receptor nucleus, that is, in the cell body This investigation was supported by Public Health Service Research Grant NB-02889 from the National Institute of Neurological Diseases and Blindness.
208
SHARON MOUNTFORD
proper (6). This type of synaptic b o d y is identical to the ~-type with respect to basic structural c o m p o n e n t s ; the m a i n difference, therefore, is due to the close relation to the receptor cell nucleus. These three types of synapses were studied in serial sections in order to further define the patterns of i n v a g i n a t i o n f o u n d a n d to try to establish relationships a m o n g receptors a n d between receptors a n d bipolars ( m a n u s c r i p t in preparation). Thig paper deals with a structure discovered in these series, a b y - p r o d u c t of the p r i m a r y research problem. MATERIALS A N D METHODS Young male albino guinea pigs were sacrificed by decapitation. The eyes were removed and halved on a chilled glass plate. The lens and vitreous were removed from each eye, and the retina, together with the sclera, was immediately immersed in cold buffered 1% osmium tetroxide. Fixation time was 3 hours in the cold, followed by rinsing in Ringer's solution and dehydration in a graded alcohol series. While the tissue was in 70% alcohol, the retina was cut into small strips for embedding and the sclera was discarded. Embedding was done in Araldite, Swedish method (1, 2). Sections were prepared with glass knives broken essentially according to the method of Latta and Hartmann (4) and with a 46 degree diamond knife from Ge-Fe-Ri mounted in an LKB holder. In both cases sectioning was done on an LKB Ultrotome ultramicrotome at nominal thicknesses of 150-400 A. The sections were floated on 15% alcohol and picked up from underneath onto thin copper grids with a 1 x 2 mm slot, which had been previously coated with 0.4% Formvar and a thin layer of evaporated carbon. The grids were afterstained with various combinations of uranyl acetate (8), lead hydroxide (3), or lead citrate (5). Examination was done on an RCA-EMU3 at 50 kV and on a Siemens Elmiskop I at 80 kV. RESULTS There appeared to be a previously undescribed structure in all, or at least the majority, of the receptor-bipolar synapses. The relatively lightly staining body takes o n three different b u t related forms in the three types of synapses outlined above. FIG. 1. An approximate cross section through the synaptic end body of a/?-type receptor of a male albino guinea pig retina. A synaptic spindle (SS) is seen extending across the cell. This spindle was followed in serial sections, and this section reveals its greatest length and breadth. Sectioned with a glass knife, its calculated thickness is 250 A. Counterstained with Karnovsky's lead method. /, invagination; MC;Maller cell; SR, synaptic ribbon; SV, synaptic vesicle. × 48',000. FIo. 2. The same synapse as that in Fig. 1 seen in a nearby section. At arrow A a filament is seen in the spindle with two helical (or globular) regions separated by a straight portion. Other faint helical regions are seen elsewhere in the synaptic spindle (SS). Labels and experimental data are the same as for Fig. 1. x 81,000. FIG. 3. An oblique section through a/?-type sYnapse reveals a portion of a synaptic spindle (SS). The cross striations are obvious here. The double membrane-enclosed structures within the synapse are portions of contorted invaginations, some of which are labeled I. Labels as in Fig. 1. Sectioned with a diamond knife at a nominal thickness of 200/~. Counterstained with uranyl acetate (20 minutes), followed by lead citrate (5 minutes), x 96,000.
!!~ ~ i i ! i ~ ~
~
3
212
SHARON MOUNTFORD
I n t h e / ? - t y p e synapse (Figs. 1-4), the structure is a clearly defined spindle-shaped b o d y which is filamentous a n d bears cross striations. The d i a m e t e r of the spindle is a b o u t 125-190 m#. The filaments r u n a l o n g the length of the spindle, which is roughly p e r p e n d i c u l a r to the axis of the r e c e p t o r cell as a whole. The " s y n a p t i c - s p i n d l e " extends across the end b o d y at the level of the dendritic invaginations. There is no visible c o n n e c t i o n to the cell m e m b r a n e at either end, a l t h o u g h the structure runs f r o m one side of the cell to the o t h e r (Fig. 1). There a p p e a r to be no a c c o m p a n y i n g m e m b r a n e s with the spindle in t h e / ? - t y p e synapse. Fig. 1 is a cross section a n d shows the full extent of a spindle. Fig. 2 is a higher magnification of a n e a r b y section of the same cell. H e r e a n i n d i c a t i o n of the filament structure is seen at a r r o w A. (See last p a r a g r a p h of this section for description.) Fig. 3 is an oblique section which clearly shows the cross striations. Fig. 4 is a r o u g h l y l o n g i t u d i n a l section t h r o u g h the synaptic b o d y which shows the p o s i t i o n of the spindle to be n e a r the vitreal end of the synapse. This p o s i t i o n varies, b u t it is always at the level of the t o p of the i n v a g i n a t i o n s o r b e l o w (i.e., t o w a r d the vitreous). Parallel to the l o n g axis of the m-type r e c e p t o r one finds a similar filamentous crossstriated structure (see Fig. 5). It has been followed f r o m the neck region t h r o u g h the synaptic spherule to the vitreal side of the cluster of i n v a g i n a t i n g processes. It appears to h o o k u n d e r this g r o u p of processes a n d c o m e into p r o x i m i t y to the synaptic m e m b r a n e in t h a t a r e a (see a r r o w A o n Fig. 5). It is thinner ( a b o u t 100 m # wide) and m u c h longer t h a n the spindle in the/?-type. It is a c c o m p a n i e d b y m e m b r a n e s that run d o w n the length of the organelle. These m e m b r a n e s have n o t been seen to sheath the structure (when seen in cross section), b u t r a t h e r to f o r m a c c o m p a n y i n g elongate sacs. Continuities between the cell m e m b r a n e a n d these m e m b r a n o u s sacs have not b e e n seen, a l t h o u g h there is a close a s s o c i a t i o n in the neck region, as seen in Fig. 5. The m u l t i p l i c i t y of m e m b r a n e s in this region h a s m a d e it difficult to establish relationships here. This structure in the c~-type synapse superficially resembles a ciliary rootlet. FIG. 4. A synaptic spindle (SS) as seen in a roughly longitudinal section of a fl-type synapse. The spindle runs obliquely through this series from one side of the cell to the other. Its position near the vitreal end of the synapse is seen. The maze of processes that constitute the outer neuropile or outer plexiform layer is labeled ON. Other labels as in Fig. 1. Sectioned with a diamond knife at nominal thickness of 220 A. Counterstained with uranyl acetate (20 minutes) followed by lead citrate (7 minutes). x 72,000. FIG. 5. A longitudinal section through the receptor-bipolar synaptic area of the retina. Two e-type receptors are seen (al and as). In al a long filamentous structure (FS) with faint striations is seen. Accompanying it are membranes that do not appear completely to sheath the structure. The end of the organelle curves toward the vitreal side of the cluster of invaginations (arrow A), and here it ends. Labels as in Fig. 1. Experimental data are the same as for Fig. 4. x 41,000. FIG. 6. An approximate cross section through the receptor-bipolar synaptic area. On the right is a receptor cell body with a paranuclear synapse. Arrows A and B point out a thick and thin bundle, branches of the filamentous organelle. RCN, receptor cell nucleus. Other labels as in Fig. 1. Sectioned with a diamond knife at nominal thickness of 275/~. Counterstained with uranyl acetate, followed by lead hydroxide. × 44,000.
i
216
SHARON MOUNTISORD
The paranuclear synapse also possesses a similar structure (see Fig. 6). It is found closely associated with the nuclear envelope, running approximately perpendicular to the receptor axis. It sometimes appears branched. (See the thick and thin bundles at arrows A and B in Fig. 6.) As in the 0~-type synapse there are closely associated membranes with this organelle. No more than one filamentous organelle has been seen in any one synapse, although many cells have been reconstructed from above the area of the dendritic invaginations to the vitreal tip of the receptor. In series that covered an entire synapse, the appropriate one of these three structures was always found. The inner structure of these organelles is at best hazy. In some photographs there is a hint of helical regions along the filaments (arrow A on Fig. 2). Alternating helical (or globular) and straight regions along the length of the filaments, in register across the spindle, might account for the striated appearance.
DISCUSSION This synaptic spindle in the/3-type synapse and the related structures in the s-type and the paranuclear synapses have escaped notice in previous studies, probably owing to their shape and staining properties. Unless the organelle is oriented parallel to the plane of the section, it is very inconspicuous in the cell. When one is searching for such structures, they usually evade notice in microscope viewing but turn up consistently in series of sections that have been photographed. In all series that covered an entire synapse, one and only one of these structures was found. The ubiquity of these filamentous organelles indicates that they are probably important in the functioning of the synapse, but any suggestion as to their role would be wild speculation at this point. The author wishes to express her gratitude to Professor Fritiof Sj6strand under whom this investigation has been conducted, and to Mr. Herman Kabe for photographic assistance.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
GLAUI~RT,A. M., ROGERS,G. E. and GLAUERT,R. H., Nature 178, 803 (1956). GLAUERT,A. M. and GLAtJERT,R. H., J. Biophys. Biochem. Cytol. 4, 459 (1958). KARNOVSKY,M. J., J. Biophys. Biochem. Cytol. 11, 729 (1961). LATTA,H. and HARTMANN,J. F., Proc. Soc. Exptl. Biol. Med. 74, 436 (1950). REYNOLDS,E. S., J. Cell Biol. 17, 208 (1963). DE ROBERTIS,E. and FRANCr~, C. M., J. ~iophys. Biochem. Cytol. 2, 307 (1956). SJ6STRAND,F. S., J. Cell. Comp. Physiol. 42, 45 (1953). WATSON,M. L., J. Biophys. Biochem. Cytol. 4, 727 (1958).
207
Filamentous Organelles in Receptor-Bipolar Synapses of the Retina 1 SHARON MOUNTFORD
Department of Zoology, University of California, Los Angeles, Los Angeles, California Received October 1, 1963 Retinas of male albino guinea pigs were studied in serial sections. A consistent feature of the synapse was a filamentous cross-striated organelle, which varied in form from one type of synapse to another. In the r-Type synapse this organelle is a bare spindle extending across the cell in the region of the dendritic invaginations with its axis perpendicular to the long axis of the receptor, c~-Typereceptors have an attenuated, but basically similar, structure running parallel to the cell axis from the neck region between the cell body proper and the synaptic spherule to the vitreal side of the cluster of dendritic invaginations. Paranuclear synapses have a similar structure in intimate contact with the nuclear envelope. These latter two organelles are accompanied by membranes that were not seen completely to sheath the structures but rather to form accompanying elongate sacs. During the course of a serial-section study of the guinea pig retina, a previously undescribed organelle Was found in the receptor-bipolar synapses. It appears in three different forms in the three types of synapses, o~, r , and paranuclear. The s-type receptor according to the classification of Sj/3strand (7) has a synaptic spherule at the vitreal end of the receptor. A neck region connects this synaptic end body to the cell body proper. The spherule is rather regular in shape, displaying a nearly circular profile in cross section. There is one synaptic ribbon or lamella associated with one compact set of invaginations. The/~-type receptor (7) also has a distinct end body, with a narrow neck region between synapse and cell body, as in the s-type; it is, however, larger and irregular in shape and has multiple ribbons and several groups of invaginations. As the name implies, the paranuclear synapse has no special distinct synaptic end body or spherule. The typica ! synaptic structures--dendritic invaginations, ribbons, and vesicles--are present in the vicinity of the receptor nucleus, that is, in the cell body This investigation was supported by Public Health Service Research Grant NB-02889 from the National Institute of Neurological Diseases and Blindness.
208
SHARON MOUNTFORD
proper (6). This type of synaptic b o d y is identical to the ~-type with respect to basic structural c o m p o n e n t s ; the m a i n difference, therefore, is due to the close relation to the receptor cell nucleus. These three types of synapses were studied in serial sections in order to further define the patterns of i n v a g i n a t i o n f o u n d a n d to try to establish relationships a m o n g receptors a n d between receptors a n d bipolars ( m a n u s c r i p t in preparation). Thig paper deals with a structure discovered in these series, a b y - p r o d u c t of the p r i m a r y research problem. MATERIALS A N D METHODS Young male albino guinea pigs were sacrificed by decapitation. The eyes were removed and halved on a chilled glass plate. The lens and vitreous were removed from each eye, and the retina, together with the sclera, was immediately immersed in cold buffered 1% osmium tetroxide. Fixation time was 3 hours in the cold, followed by rinsing in Ringer's solution and dehydration in a graded alcohol series. While the tissue was in 70% alcohol, the retina was cut into small strips for embedding and the sclera was discarded. Embedding was done in Araldite, Swedish method (1, 2). Sections were prepared with glass knives broken essentially according to the method of Latta and Hartmann (4) and with a 46 degree diamond knife from Ge-Fe-Ri mounted in an LKB holder. In both cases sectioning was done on an LKB Ultrotome ultramicrotome at nominal thicknesses of 150-400 A. The sections were floated on 15% alcohol and picked up from underneath onto thin copper grids with a 1 x 2 mm slot, which had been previously coated with 0.4% Formvar and a thin layer of evaporated carbon. The grids were afterstained with various combinations of uranyl acetate (8), lead hydroxide (3), or lead citrate (5). Examination was done on an RCA-EMU3 at 50 kV and on a Siemens Elmiskop I at 80 kV. RESULTS There appeared to be a previously undescribed structure in all, or at least the majority, of the receptor-bipolar synapses. The relatively lightly staining body takes o n three different b u t related forms in the three types of synapses outlined above. FIG. 1. An approximate cross section through the synaptic end body of a/?-type receptor of a male albino guinea pig retina. A synaptic spindle (SS) is seen extending across the cell. This spindle was followed in serial sections, and this section reveals its greatest length and breadth. Sectioned with a glass knife, its calculated thickness is 250 A. Counterstained with Karnovsky's lead method. /, invagination; MC;Maller cell; SR, synaptic ribbon; SV, synaptic vesicle. × 48',000. FIo. 2. The same synapse as that in Fig. 1 seen in a nearby section. At arrow A a filament is seen in the spindle with two helical (or globular) regions separated by a straight portion. Other faint helical regions are seen elsewhere in the synaptic spindle (SS). Labels and experimental data are the same as for Fig. 1. x 81,000. FIG. 3. An oblique section through a/?-type sYnapse reveals a portion of a synaptic spindle (SS). The cross striations are obvious here. The double membrane-enclosed structures within the synapse are portions of contorted invaginations, some of which are labeled I. Labels as in Fig. 1. Sectioned with a diamond knife at a nominal thickness of 200/~. Counterstained with uranyl acetate (20 minutes), followed by lead citrate (5 minutes), x 96,000.
!!~ ~ i i ! i ~ ~
~
3
212
SHARON MOUNTFORD
I n t h e / ? - t y p e synapse (Figs. 1-4), the structure is a clearly defined spindle-shaped b o d y which is filamentous a n d bears cross striations. The d i a m e t e r of the spindle is a b o u t 125-190 m#. The filaments r u n a l o n g the length of the spindle, which is roughly p e r p e n d i c u l a r to the axis of the r e c e p t o r cell as a whole. The " s y n a p t i c - s p i n d l e " extends across the end b o d y at the level of the dendritic invaginations. There is no visible c o n n e c t i o n to the cell m e m b r a n e at either end, a l t h o u g h the structure runs f r o m one side of the cell to the o t h e r (Fig. 1). There a p p e a r to be no a c c o m p a n y i n g m e m b r a n e s with the spindle in t h e / ? - t y p e synapse. Fig. 1 is a cross section a n d shows the full extent of a spindle. Fig. 2 is a higher magnification of a n e a r b y section of the same cell. H e r e a n i n d i c a t i o n of the filament structure is seen at a r r o w A. (See last p a r a g r a p h of this section for description.) Fig. 3 is an oblique section which clearly shows the cross striations. Fig. 4 is a r o u g h l y l o n g i t u d i n a l section t h r o u g h the synaptic b o d y which shows the p o s i t i o n of the spindle to be n e a r the vitreal end of the synapse. This p o s i t i o n varies, b u t it is always at the level of the t o p of the i n v a g i n a t i o n s o r b e l o w (i.e., t o w a r d the vitreous). Parallel to the l o n g axis of the m-type r e c e p t o r one finds a similar filamentous crossstriated structure (see Fig. 5). It has been followed f r o m the neck region t h r o u g h the synaptic spherule to the vitreal side of the cluster of i n v a g i n a t i n g processes. It appears to h o o k u n d e r this g r o u p of processes a n d c o m e into p r o x i m i t y to the synaptic m e m b r a n e in t h a t a r e a (see a r r o w A o n Fig. 5). It is thinner ( a b o u t 100 m # wide) and m u c h longer t h a n the spindle in the/?-type. It is a c c o m p a n i e d b y m e m b r a n e s that run d o w n the length of the organelle. These m e m b r a n e s have n o t been seen to sheath the structure (when seen in cross section), b u t r a t h e r to f o r m a c c o m p a n y i n g elongate sacs. Continuities between the cell m e m b r a n e a n d these m e m b r a n o u s sacs have not b e e n seen, a l t h o u g h there is a close a s s o c i a t i o n in the neck region, as seen in Fig. 5. The m u l t i p l i c i t y of m e m b r a n e s in this region h a s m a d e it difficult to establish relationships here. This structure in the c~-type synapse superficially resembles a ciliary rootlet. FIG. 4. A synaptic spindle (SS) as seen in a roughly longitudinal section of a fl-type synapse. The spindle runs obliquely through this series from one side of the cell to the other. Its position near the vitreal end of the synapse is seen. The maze of processes that constitute the outer neuropile or outer plexiform layer is labeled ON. Other labels as in Fig. 1. Sectioned with a diamond knife at nominal thickness of 220 A. Counterstained with uranyl acetate (20 minutes) followed by lead citrate (7 minutes). x 72,000. FIG. 5. A longitudinal section through the receptor-bipolar synaptic area of the retina. Two e-type receptors are seen (al and as). In al a long filamentous structure (FS) with faint striations is seen. Accompanying it are membranes that do not appear completely to sheath the structure. The end of the organelle curves toward the vitreal side of the cluster of invaginations (arrow A), and here it ends. Labels as in Fig. 1. Experimental data are the same as for Fig. 4. x 41,000. FIG. 6. An approximate cross section through the receptor-bipolar synaptic area. On the right is a receptor cell body with a paranuclear synapse. Arrows A and B point out a thick and thin bundle, branches of the filamentous organelle. RCN, receptor cell nucleus. Other labels as in Fig. 1. Sectioned with a diamond knife at nominal thickness of 275/~. Counterstained with uranyl acetate, followed by lead hydroxide. × 44,000.
i
216
SHARON MOUNTISORD
The paranuclear synapse also possesses a similar structure (see Fig. 6). It is found closely associated with the nuclear envelope, running approximately perpendicular to the receptor axis. It sometimes appears branched. (See the thick and thin bundles at arrows A and B in Fig. 6.) As in the 0~-type synapse there are closely associated membranes with this organelle. No more than one filamentous organelle has been seen in any one synapse, although many cells have been reconstructed from above the area of the dendritic invaginations to the vitreal tip of the receptor. In series that covered an entire synapse, the appropriate one of these three structures was always found. The inner structure of these organelles is at best hazy. In some photographs there is a hint of helical regions along the filaments (arrow A on Fig. 2). Alternating helical (or globular) and straight regions along the length of the filaments, in register across the spindle, might account for the striated appearance.
DISCUSSION This synaptic spindle in the/3-type synapse and the related structures in the s-type and the paranuclear synapses have escaped notice in previous studies, probably owing to their shape and staining properties. Unless the organelle is oriented parallel to the plane of the section, it is very inconspicuous in the cell. When one is searching for such structures, they usually evade notice in microscope viewing but turn up consistently in series of sections that have been photographed. In all series that covered an entire synapse, one and only one of these structures was found. The ubiquity of these filamentous organelles indicates that they are probably important in the functioning of the synapse, but any suggestion as to their role would be wild speculation at this point. The author wishes to express her gratitude to Professor Fritiof Sj6strand under whom this investigation has been conducted, and to Mr. Herman Kabe for photographic assistance.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
GLAUI~RT,A. M., ROGERS,G. E. and GLAUERT,R. H., Nature 178, 803 (1956). GLAUERT,A. M. and GLAtJERT,R. H., J. Biophys. Biochem. Cytol. 4, 459 (1958). KARNOVSKY,M. J., J. Biophys. Biochem. Cytol. 11, 729 (1961). LATTA,H. and HARTMANN,J. F., Proc. Soc. Exptl. Biol. Med. 74, 436 (1950). REYNOLDS,E. S., J. Cell Biol. 17, 208 (1963). DE ROBERTIS,E. and FRANCr~, C. M., J. ~iophys. Biochem. Cytol. 2, 307 (1956). SJ6STRAND,F. S., J. Cell. Comp. Physiol. 42, 45 (1953). WATSON,M. L., J. Biophys. Biochem. Cytol. 4, 727 (1958).