- Email: [email protected]
Intranuclear bodies in cells of rabbit and rat retina
628 REFERENCES 1. LIPKIN, M., GastroenteroZ. 48, 616 (1965). 2. MACKLIN, C. C. and MACKLIN, M. T., in E. V. COWDRY (ed.), The Intestinal Epithelium. Special Cytology, Vol. 1, p. 264. Paul B. Hoeher, 1932. 3. MILLAR, M. J., VINCENT, N. R. and MAWSON, C. A., J. Hisfochem. Cyfochem. 9, 111 (1961). 4. MoLs, G., Arch. Biof. 40, 111 (1930). 5. RIECKEN, E. 0. and PEARSE, A. G. E., Gut 7, 86 (1966). 6. SCHWALBE, G., Arch. Mikroskop. Anaf. 8, 92 (1872). 7. THRASHER, J. D. and GREULICH, R. C., J. Expfl Zool. 161, 9 (1966). 8. TRIER, J. S., LORENZSONN, V. and GROECHLER, K., Gasfroenferof. 50, 875 (1966).
INTRANUCLEAR
BODIES
IN CELLS
OF RABBIT
AND
RAT
RETINA
M. M. MAGALHdES Department of Histology and Embryology, Faculty of Medicine of Oporto, and Center of Electron Microscopy CalousteGulbenkian of the University of Oporto, Portugal
Received February 2, 1967 IWRANUCLEAR
rods
were
described
long
ago by several
authors
[3, 6, 7, 8, 91 using
light microscopy in nervous cells of various animal species. There was no definite idea about their structure until some recent findings were obtained with the electron microscope. In 1964, Siegesmund et al. [Ill described the presence of intranuclear rods made up of bundles of fibrils in some central nervous system cells of the monkey and the rabbit, and very recently Karlsson [4] found a similar body in one neuron of a rat geniculate nucleus. During electron microscopic studies on the retina, we have rather often found similar organelles in the bipolar and ganglion cells in the rabbit and the rat, sometimes showing a crystalline structure. It is the purpose of this note to present such findings. Material and methods.--Six adult rabbits and two adult rats (Wistar strain) were Fig. l.-Rabbit retina. Fixation with osmium-uranyl acetate. In the nucleus (iv) of a bipolar cell, a filamentous body (arrow). On the right side a Miiller cell (MC), of which the nucleus (N’), the nucleolus (No’) and the vacuolated cytoplasm are visible. x 12,000. Fig. 2.-Rat retina. Fixation with glutaraldehyde-osmium-uranyl acetate (specimens in the following pictures had the same fixation). Filamentous body in the nucleus of a ganglion cell. x 60,000. Fig. 3.-Rabbit retina. In the nucleus (X) of a ganglion cell, a filamentous body (arrow) and the nucleolus (No). Short arrows indicate three pores of the nuclear membrane with well contrasted diaphragms. x 15,000. Fig. 4.-Rabbit x 60,000. Experimental
retina.
Nucleus
Cell Research 47
of a bipolar
cell. Surrounding
the filamentous
body a clear halo.
Inttranuclear
bodies in retina
629
Experimental
Cell Research 47
Fig. 5.-Intranuclear Fig. 6.-Detail
body in a ganglion cell of the rat retina, with crystalline
of Fig. 5 (square) to show the crystalline
pattern.
structure.
x 51,000.
x 374,000.
used. The retinas were fixed by immersion in 5 per cent glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.2 for 2 hr at 4°C followed by a rinse in cacodylatesucrose buffer and postfixation in Verona1 acetate 1 per cent osmium for 2 hr at 4°C. Before dehydration the pieces were treated with 0.5 per cent uranyl acetate in Michaelis buffer for 2 hr at room temperature [2, IO]. Some pieces were also fixed by immersion in 1 per cent osmium or in glutaraldehyde plus osmium without the treatment with uranyl acetate, or in osmium or glutaraldehyde followed by uranyl acetate. After embedding in Epon 812 [5] without infiltration in propylene oxide, ultrathin sections, including some serial sections, were cut in a LKB microtome, double-stained with uranyl acetate (saturated aqueous solution) for 5 min and lead citrate [12] for I min, and examined in a Siemens Elmiskop IA electron microscope. Results.-In ultrathin sections (600-900 A) of the retina of all the animals examined we have found an intranuclear filamentous body with the aspects shown in Figs. 1-4. The filamentous body was only observed in ganglion and bipolar cells of the rabbit and in ganglion cells of the rat in spite of a careful search in the remaining retinal cells. The filamentous bodies are spindle-shaped when cut longitudinally, the centrally located fibrils being longer than the peripheral ones. Maximum dimensions recorded for the filamentous body were 2 ,u for length and 0.2 p for width. The body shows up to 15 parallel fibrils (Figs. 2 and 4) 60 to 70 A thick, separated by Experimental
Cell Research 47
Infranuclear
bodies irz retina
631
clear spaces 40 to 50 A wide. It is usually surrounded by a clear halo (Fig. 4). The study of serial sections of one rat retina showed no more than one intranuclear body per nucleus. No relationship was found between the body and the nuclear membrane or the nucleolus. In one of the rats, an intranuclear body with a crystalline structure was found in a ganglion cell (Figs. 5 and 6). The bodies described were seen in males and females. Although they were found after all the fixations indicated above, they were more evident after glutaraldehydeosmium-uranyl acetate or osmium-uranyl acetate fixations. It is interesting that the body was visible even after glutaraldehyde-uranyl acetate fixation. Discussion.-Our observations show, for the first time, the presence of intranuclear rod-like structures in ganglion and bipolar cells of the rabbit retina and in ganglion cells of the rat retina (Figs. l-6). Similar bodies have recently been observed in neurons of the squirrel monkey and rabbit olfactory bulb, of the rabbit cerebral and cerebellar cortices [ll], and in one nerve cell of the rat lateral geniculate nucleus [4]. The retinal bodies have a fibrillar structure similar to that described in central nervous system neurons by those authors [4, ill. In one rat we have been able to detect one rod-like structure exhibiting crystalline structure (Figs. 5-6). We do not know whether this crystalline body can be identified with the filamentous bodies. If so, this finding is in favour of the presence of proteins in them. The intranuclear rods described in the cat visual cortex by Colonnier [l] are mere cytoplasmic invaginations of the nuclear membrane, completely different from the bodies we are dealing with. Similar invaginations are very frequent in retinal cells. It is difficult to ascertain if the crystalloid rods of the old histologists [3, 6691 correspond to both or only one of these structures. It is our impression that the fibrillar intranuclear bodies are much more widespread structures than it might be thought from the scarce observations made up to now. In fact, besides the retina, we have seen them in cells of the ciliary body and of the adrenal cortex and medulla. Since they are better visualised after triple fixation with glutaraldehyde-osmium-uranyl acetate, it is possible that other fixations have been responsible for their being overlooked in routine observations. After a careful search of our material, we were able to detect the body only in the retinal cells referred to above. Cell nuclei rich in heterochromatin such as those of the rods and cones, never contained the body which, therefore, seems to be preferably associated with heterochromatin poor nerve cell nuclei. In further studies using electron microscope cytochemistry, we intend to investigate the composition of this apparently common structure, in order to elucidate its nature and functions. We thank Prof. A. Coimbra and Dr. M. T. Silva for suggestions and revision of the manuscript. This study was supported by a grant from the Instituto de Alta Cultura, Portugal. Part of this work was supported by the Gulbenkian Foundation, Lisbon, Portugal.
Experimental
Cell Research 47
632
K. A. Chaubal,
B. P. Gothoskar, S. V. Rao and K. S. Korgaonkar REFERENCES
1. COLONNIER,
2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
12.
M., J. Cell Biol. 25, 646 (1965). FARQUHAR, M. G. and PALADE, G. E., J. Cell Biof. 26, 263 (1965). HOLMGREN, E., Anat. Anz. 16, 388 (1899). KARLSSON, U., J. Cftrasfruct. Res. 16, 429 (1966). LUFT, J. H., .I. Biophys. Biochem. Cyfof. 9, 409 (1961). MANN, G., J. Anat. 29, 100 (1894). PRENANT, A., Arch. Anat. Microscop. 1, 366 (1897). RAM~N Y CAJAL, S., Histologie du systkme nerveux de l’homme et des vertbbrks, C.S. I.C., Madrid, 1952. RONCORONI, L., Arch. Psi&at. 16, 447 (1895). RYTER, A. and KELLENBERGER, E., 2. Naturforsch. 136, 597 (1958). SIEGESMUND, K. A., DUTTA, C. R. and Fox, C. A., J. Anaf. 98, 93 (1964). VENABLE, J. H. and GOGGESHALL, R., J. Cell Biol. 25, 407 (1965).
A SIMPLE
SLIDE FILM
K. A. CHAUBAL,
HOLDER
200.
FOR USE IN STRIPPING
S. V. RAO
and K. S. KORGAONKAR
Group, Indian Cancer Research Centre, Pare& Bombay 12, India Received
T HE
1, p.
AUTORADIOGRAPHY
B. P. GOTHOSKAR, Biophysics
vol.
February
3, 1967
technique of autoradiography is being widely used in various disciplines of biological research. Two standard procedures exist for the use of this technique; one using photographic emulsion and the other using photographic stripping film [I, 2, 31. The different stages involved in the stripping film procedure are quite well known. The labeled biological material is mounted on “subbed” glass slides and covered with stripped pieces of film. After the required exposure these slides are developed in the routine manner. The subbing of the slides in gelatine solution is usually recommended for ensuring firm adherence of the film to the slide and consequently to the labeled specimen [3]. As the developing and fixing times are considerably large (20-30 min), the film often gets loosened inspite of subbing. The resulting displacement of the film over the specimen destroys the coordination between the labeled material within the specimen and the developed grains. The present communication describes a simple slide holder which prevents such a displacement of the film during procedures of developing, fixing and staining. This holder is being used in this laboratory for autoradiographic studies on cells grown on coverslips (8 x22 mm). The slide holder is made of 4 mm thick “Perspex” sheets. It consists of two parts; the tray (X) and the lid (Y) which snugly fits inside the tray (Fig. 1). The lengthwise slots (AB) and (CD) in the tray and the lid, respectively, coincide when the lid is placed within the tray. The width of the slots is 16 mm, i.e., more than the width of the specimen (coverslips) mentioned above. The edges of the slots are elevated Experimental
CeZl Research 47
INTRANUCLEAR
BODIES
IN CELLS
OF RABBIT
AND
RAT
RETINA
M. M. MAGALHdES Department of Histology and Embryology, Faculty of Medicine of Oporto, and Center of Electron Microscopy CalousteGulbenkian of the University of Oporto, Portugal
Received February 2, 1967 IWRANUCLEAR
rods
were
described
long
ago by several
authors
[3, 6, 7, 8, 91 using
light microscopy in nervous cells of various animal species. There was no definite idea about their structure until some recent findings were obtained with the electron microscope. In 1964, Siegesmund et al. [Ill described the presence of intranuclear rods made up of bundles of fibrils in some central nervous system cells of the monkey and the rabbit, and very recently Karlsson [4] found a similar body in one neuron of a rat geniculate nucleus. During electron microscopic studies on the retina, we have rather often found similar organelles in the bipolar and ganglion cells in the rabbit and the rat, sometimes showing a crystalline structure. It is the purpose of this note to present such findings. Material and methods.--Six adult rabbits and two adult rats (Wistar strain) were Fig. l.-Rabbit retina. Fixation with osmium-uranyl acetate. In the nucleus (iv) of a bipolar cell, a filamentous body (arrow). On the right side a Miiller cell (MC), of which the nucleus (N’), the nucleolus (No’) and the vacuolated cytoplasm are visible. x 12,000. Fig. 2.-Rat retina. Fixation with glutaraldehyde-osmium-uranyl acetate (specimens in the following pictures had the same fixation). Filamentous body in the nucleus of a ganglion cell. x 60,000. Fig. 3.-Rabbit retina. In the nucleus (X) of a ganglion cell, a filamentous body (arrow) and the nucleolus (No). Short arrows indicate three pores of the nuclear membrane with well contrasted diaphragms. x 15,000. Fig. 4.-Rabbit x 60,000. Experimental
retina.
Nucleus
Cell Research 47
of a bipolar
cell. Surrounding
the filamentous
body a clear halo.
Inttranuclear
bodies in retina
629
Experimental
Cell Research 47
Fig. 5.-Intranuclear Fig. 6.-Detail
body in a ganglion cell of the rat retina, with crystalline
of Fig. 5 (square) to show the crystalline
pattern.
structure.
x 51,000.
x 374,000.
used. The retinas were fixed by immersion in 5 per cent glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.2 for 2 hr at 4°C followed by a rinse in cacodylatesucrose buffer and postfixation in Verona1 acetate 1 per cent osmium for 2 hr at 4°C. Before dehydration the pieces were treated with 0.5 per cent uranyl acetate in Michaelis buffer for 2 hr at room temperature [2, IO]. Some pieces were also fixed by immersion in 1 per cent osmium or in glutaraldehyde plus osmium without the treatment with uranyl acetate, or in osmium or glutaraldehyde followed by uranyl acetate. After embedding in Epon 812 [5] without infiltration in propylene oxide, ultrathin sections, including some serial sections, were cut in a LKB microtome, double-stained with uranyl acetate (saturated aqueous solution) for 5 min and lead citrate [12] for I min, and examined in a Siemens Elmiskop IA electron microscope. Results.-In ultrathin sections (600-900 A) of the retina of all the animals examined we have found an intranuclear filamentous body with the aspects shown in Figs. 1-4. The filamentous body was only observed in ganglion and bipolar cells of the rabbit and in ganglion cells of the rat in spite of a careful search in the remaining retinal cells. The filamentous bodies are spindle-shaped when cut longitudinally, the centrally located fibrils being longer than the peripheral ones. Maximum dimensions recorded for the filamentous body were 2 ,u for length and 0.2 p for width. The body shows up to 15 parallel fibrils (Figs. 2 and 4) 60 to 70 A thick, separated by Experimental
Cell Research 47
Infranuclear
bodies irz retina
631
clear spaces 40 to 50 A wide. It is usually surrounded by a clear halo (Fig. 4). The study of serial sections of one rat retina showed no more than one intranuclear body per nucleus. No relationship was found between the body and the nuclear membrane or the nucleolus. In one of the rats, an intranuclear body with a crystalline structure was found in a ganglion cell (Figs. 5 and 6). The bodies described were seen in males and females. Although they were found after all the fixations indicated above, they were more evident after glutaraldehydeosmium-uranyl acetate or osmium-uranyl acetate fixations. It is interesting that the body was visible even after glutaraldehyde-uranyl acetate fixation. Discussion.-Our observations show, for the first time, the presence of intranuclear rod-like structures in ganglion and bipolar cells of the rabbit retina and in ganglion cells of the rat retina (Figs. l-6). Similar bodies have recently been observed in neurons of the squirrel monkey and rabbit olfactory bulb, of the rabbit cerebral and cerebellar cortices [ll], and in one nerve cell of the rat lateral geniculate nucleus [4]. The retinal bodies have a fibrillar structure similar to that described in central nervous system neurons by those authors [4, ill. In one rat we have been able to detect one rod-like structure exhibiting crystalline structure (Figs. 5-6). We do not know whether this crystalline body can be identified with the filamentous bodies. If so, this finding is in favour of the presence of proteins in them. The intranuclear rods described in the cat visual cortex by Colonnier [l] are mere cytoplasmic invaginations of the nuclear membrane, completely different from the bodies we are dealing with. Similar invaginations are very frequent in retinal cells. It is difficult to ascertain if the crystalloid rods of the old histologists [3, 6691 correspond to both or only one of these structures. It is our impression that the fibrillar intranuclear bodies are much more widespread structures than it might be thought from the scarce observations made up to now. In fact, besides the retina, we have seen them in cells of the ciliary body and of the adrenal cortex and medulla. Since they are better visualised after triple fixation with glutaraldehyde-osmium-uranyl acetate, it is possible that other fixations have been responsible for their being overlooked in routine observations. After a careful search of our material, we were able to detect the body only in the retinal cells referred to above. Cell nuclei rich in heterochromatin such as those of the rods and cones, never contained the body which, therefore, seems to be preferably associated with heterochromatin poor nerve cell nuclei. In further studies using electron microscope cytochemistry, we intend to investigate the composition of this apparently common structure, in order to elucidate its nature and functions. We thank Prof. A. Coimbra and Dr. M. T. Silva for suggestions and revision of the manuscript. This study was supported by a grant from the Instituto de Alta Cultura, Portugal. Part of this work was supported by the Gulbenkian Foundation, Lisbon, Portugal.
Experimental
Cell Research 47
632
K. A. Chaubal,
B. P. Gothoskar, S. V. Rao and K. S. Korgaonkar REFERENCES
1. COLONNIER,
2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
12.
M., J. Cell Biol. 25, 646 (1965). FARQUHAR, M. G. and PALADE, G. E., J. Cell Biof. 26, 263 (1965). HOLMGREN, E., Anat. Anz. 16, 388 (1899). KARLSSON, U., J. Cftrasfruct. Res. 16, 429 (1966). LUFT, J. H., .I. Biophys. Biochem. Cyfof. 9, 409 (1961). MANN, G., J. Anat. 29, 100 (1894). PRENANT, A., Arch. Anat. Microscop. 1, 366 (1897). RAM~N Y CAJAL, S., Histologie du systkme nerveux de l’homme et des vertbbrks, C.S. I.C., Madrid, 1952. RONCORONI, L., Arch. Psi&at. 16, 447 (1895). RYTER, A. and KELLENBERGER, E., 2. Naturforsch. 136, 597 (1958). SIEGESMUND, K. A., DUTTA, C. R. and Fox, C. A., J. Anaf. 98, 93 (1964). VENABLE, J. H. and GOGGESHALL, R., J. Cell Biol. 25, 407 (1965).
A SIMPLE
SLIDE FILM
K. A. CHAUBAL,
HOLDER
200.
FOR USE IN STRIPPING
S. V. RAO
and K. S. KORGAONKAR
Group, Indian Cancer Research Centre, Pare& Bombay 12, India Received
T HE
1, p.
AUTORADIOGRAPHY
B. P. GOTHOSKAR, Biophysics
vol.
February
3, 1967
technique of autoradiography is being widely used in various disciplines of biological research. Two standard procedures exist for the use of this technique; one using photographic emulsion and the other using photographic stripping film [I, 2, 31. The different stages involved in the stripping film procedure are quite well known. The labeled biological material is mounted on “subbed” glass slides and covered with stripped pieces of film. After the required exposure these slides are developed in the routine manner. The subbing of the slides in gelatine solution is usually recommended for ensuring firm adherence of the film to the slide and consequently to the labeled specimen [3]. As the developing and fixing times are considerably large (20-30 min), the film often gets loosened inspite of subbing. The resulting displacement of the film over the specimen destroys the coordination between the labeled material within the specimen and the developed grains. The present communication describes a simple slide holder which prevents such a displacement of the film during procedures of developing, fixing and staining. This holder is being used in this laboratory for autoradiographic studies on cells grown on coverslips (8 x22 mm). The slide holder is made of 4 mm thick “Perspex” sheets. It consists of two parts; the tray (X) and the lid (Y) which snugly fits inside the tray (Fig. 1). The lengthwise slots (AB) and (CD) in the tray and the lid, respectively, coincide when the lid is placed within the tray. The width of the slots is 16 mm, i.e., more than the width of the specimen (coverslips) mentioned above. The edges of the slots are elevated Experimental
CeZl Research 47