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EM-immunocytochemistry of transmitter-identified neurons
SCANDEM-84
297
This method allows the study of the projection structure of perfect two-dimensional crystals as well as specimens showing certain degrees of imperfection. The two-dimensional crystals of Na, K-ATPase were induced with sodium monovanadate and magnesium chloride (E. Skriver, A. B. Maunsbach, and P. L. Jergensen, 1981, FEBS Lett. 131, 219222) and negatively stained with uranyl acetate. Electron micrographs were digitized in an OSIRIS diode array scanner and correlation averaging (W. O. Saxton, and W. Baumeister, 1982, J. Microsc. 127, 127-138) was carried out using the image processing system " E M " (R. Hegerl and A. Altbauer, 1982, Ultramicroscopy 9, 109-116). Vanadateinduced two-dimensional crystals showed perfect lattices within small domains of the membranes but there were also large areas of slightly bent crystalline domains. Average structures calculated from such areas showed that the proteins were arranged on imperfect p l or p21 lattices. The deviations from ideal positions of least-squares adapted lattices were determined and calculations of rotational correlation functions indicated that the main component of distortion was a translational disorder. In the pl form the calculated average structure showed that the protein-rich domain, interpreted as an aS-unit of Na, K-ATPase, has a triangular shape with a maximum extension close to 50 Zk in the plane of the membrane. The results illustrate that correlation averaging can be used to obtain structural information from more distorted two-dimensional crystals than analyzed before. Furthermore, the calculated average structures for p21 and p l crystals of rabbit kidney Na, K-ATPase are closely similar to protein units in the crystallized enzyme from pig kidney.
EM-Immunocytochemistry of Transmitter-Identified Neurons. O. JOHANSSON, G. A. FOSTER, AND T. HC)KFELT, Department of Histology, Karolinska Institutet, Stockholm,
Sweden. Neurons of the central and peripheral nervous system have been studied at the electron microscopic level in order to elucidate the exact subcellular localization of the transmitters as well as the synaptology of these neurons. A large number of recent electron microscopicimnaunocytochemical studies demonstrate a number of neuropeptides at the ultrastructural level, mainly by using the peroxidase-antiperoxidase (PAP) technique of Sternberger and colleagues. In these studies the immunoreactivity was observed within cell bodies, dendrites, and nerve terminals, where it was present mainly in large granular vesicles, which may represent at least one subcellular storage site common to several peptides. Also small synaptic vesicles may in certain instances exhibit a weak reaction. The ultrastructural localization of vasoactive intestinal polypeptide (VIP)-like immunoreactivity in nerve terminals of cat exocrine glands was examined as well as the localization of thyrotropin releasing hormone (TRH)-like immunoreactivity within the central nervous system of the rat. The former study also deals with the probable coexistence of VIP and acetylcholine within certain nerve terminals of the cat parasympathetic nervous system. The ultrastructural distribution of T R H was studied in cell bodies of some hypothalamic nuclei and in nerve terminals of the median eminence, of several hypothalamic nuclei, and of the spinal cord. Furthermore, somatostatin-containing nerve terminals of different regions of the rat central nervous system were investigated using a colloidal gold technique. This is a postembedding method, where 100-nm sections placed on gold grids are labeled with antibodies raised against somatostatin-28. After rinsing, the colloidal gold (5-nm particle size) linked to goat anti-rabbit antibodies is applied. After an additional rinse and staining with 2% uranyl acetate, the sections are viewed in the electron microscope. Immunoreactivity was found in synaptic terminals of the median eminence, the arcuate
298
SCANDEM-84
nucleus, and the dorsal horn of the spinal cord. The gold particles were localized over socalled dense-core vesicles (granular, electron-dense: diameter about 1000 ~,). With the colloidal gold technique it was possible to use a stronger fixative (2% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4)), resulting in a better fine structure as compared with conventional preembedding techniques. Morphometric measurements of parameters, such as vesicle diameter, form factor, percentage immunoreactive boutons, etc., could be obtained using a Zeiss/Kontron IBAS interactive image analyzer. In conclusion, the use of ultrastructural immunocytochemistry permits the visualization of transmitter identified neuron populations at the electron microscopic level, thus expanding our knowledge regarding the morphology and synaptic interactions of these neurons.
Processing of Early Mouse Embryos for Studies on Cytoskeletal Structures. EERO LErtTONEN* AND ILKKA REIMA,t *Department of Pathology and ~Department of Electron
Microscopy, University of Helsinki, 00290 Helsinki, Finland. Most if not all nucleated mammalian cells contain a cytoskeleton consisting of three filamentous organizations, viz microfilaments (ME), microtubules (MT), and intermediate filaments (IMF). During preimplantation mouse development, important cell movements occur leading to the formation of the blastocyst with two different cell types. Cytoskeletal proteins apparently are involved in these processes. Electron microscopy and immunofluorescence studies have established the presence and distribution of microfilaments and microtubules in the preimplantation embryos. As to the intermediate filaments, conflicting results have been published on the presence of this cytoskeletal component in the cleavagestage embryos. In normal untreated embryos it apparently is not possible to demonstrate IMFs in EM. However, our immunofluorescence experiments suggesting the presence of cytokeratin in early embryos prompted us to study the matter in EM in more detail. We studied the embryos in TEM after extraction with 0.05-0.5% Triton X-100 in 50 m M Tris-HC1, pH 7.2, followed by fixation and processing for EM. In these cytoskeleton preparations, scattered 10 to 11-nm-thick filaments could be observed in the cytoplasm domain of two to eight-cell-stage embryos. These filaments were abundant in blastocyststage embryos. Our observations thus confirm the presence of IMFs in early mouse embryos. Furthermore, our staining results with antibodies to cytokeratin suggest that these filaments contain cytokeratin.
Demonstration of Tubular Cisternal Endoplasmic Reticulum in Rat Hepatocytes. O. J. Mf0LLER, O. f~STERGAARDTHOMSEN, AND J. A. LARSEN, Institute of Physiology, University
of Aarhus, DK-8000 Arhus C, Denmark. In rat liver fixed by perfusion with glutaraldehyde and postfixed with a mixture of osmium tetroxide and potassium ferricyanide sursurface cisternae and vesicles were demonstrable. From serial sections it appears that the cisternae and vesicles are part of large, fenestrated cisternae situated parallel to and at a distance of 20-40 nm from the lateral plasma membrane. In the pericanalicular and the sinusoidal zones of the hepatocytes tubules and vesicles were seen to approach the plasma membrane. Impregnation of small tissue blocks with 1% osmium for 5 days caused an intense deposition of osmium in the endoplasmic reticulum, the nuclear envelope, and the Golgi complex. Inspection of osmium-impregnated thick sections together with serial sectioning gave the impression of the existence of a continuous intracellular system of tubules and cisternae connecting the
297
This method allows the study of the projection structure of perfect two-dimensional crystals as well as specimens showing certain degrees of imperfection. The two-dimensional crystals of Na, K-ATPase were induced with sodium monovanadate and magnesium chloride (E. Skriver, A. B. Maunsbach, and P. L. Jergensen, 1981, FEBS Lett. 131, 219222) and negatively stained with uranyl acetate. Electron micrographs were digitized in an OSIRIS diode array scanner and correlation averaging (W. O. Saxton, and W. Baumeister, 1982, J. Microsc. 127, 127-138) was carried out using the image processing system " E M " (R. Hegerl and A. Altbauer, 1982, Ultramicroscopy 9, 109-116). Vanadateinduced two-dimensional crystals showed perfect lattices within small domains of the membranes but there were also large areas of slightly bent crystalline domains. Average structures calculated from such areas showed that the proteins were arranged on imperfect p l or p21 lattices. The deviations from ideal positions of least-squares adapted lattices were determined and calculations of rotational correlation functions indicated that the main component of distortion was a translational disorder. In the pl form the calculated average structure showed that the protein-rich domain, interpreted as an aS-unit of Na, K-ATPase, has a triangular shape with a maximum extension close to 50 Zk in the plane of the membrane. The results illustrate that correlation averaging can be used to obtain structural information from more distorted two-dimensional crystals than analyzed before. Furthermore, the calculated average structures for p21 and p l crystals of rabbit kidney Na, K-ATPase are closely similar to protein units in the crystallized enzyme from pig kidney.
EM-Immunocytochemistry of Transmitter-Identified Neurons. O. JOHANSSON, G. A. FOSTER, AND T. HC)KFELT, Department of Histology, Karolinska Institutet, Stockholm,
Sweden. Neurons of the central and peripheral nervous system have been studied at the electron microscopic level in order to elucidate the exact subcellular localization of the transmitters as well as the synaptology of these neurons. A large number of recent electron microscopicimnaunocytochemical studies demonstrate a number of neuropeptides at the ultrastructural level, mainly by using the peroxidase-antiperoxidase (PAP) technique of Sternberger and colleagues. In these studies the immunoreactivity was observed within cell bodies, dendrites, and nerve terminals, where it was present mainly in large granular vesicles, which may represent at least one subcellular storage site common to several peptides. Also small synaptic vesicles may in certain instances exhibit a weak reaction. The ultrastructural localization of vasoactive intestinal polypeptide (VIP)-like immunoreactivity in nerve terminals of cat exocrine glands was examined as well as the localization of thyrotropin releasing hormone (TRH)-like immunoreactivity within the central nervous system of the rat. The former study also deals with the probable coexistence of VIP and acetylcholine within certain nerve terminals of the cat parasympathetic nervous system. The ultrastructural distribution of T R H was studied in cell bodies of some hypothalamic nuclei and in nerve terminals of the median eminence, of several hypothalamic nuclei, and of the spinal cord. Furthermore, somatostatin-containing nerve terminals of different regions of the rat central nervous system were investigated using a colloidal gold technique. This is a postembedding method, where 100-nm sections placed on gold grids are labeled with antibodies raised against somatostatin-28. After rinsing, the colloidal gold (5-nm particle size) linked to goat anti-rabbit antibodies is applied. After an additional rinse and staining with 2% uranyl acetate, the sections are viewed in the electron microscope. Immunoreactivity was found in synaptic terminals of the median eminence, the arcuate
298
SCANDEM-84
nucleus, and the dorsal horn of the spinal cord. The gold particles were localized over socalled dense-core vesicles (granular, electron-dense: diameter about 1000 ~,). With the colloidal gold technique it was possible to use a stronger fixative (2% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4)), resulting in a better fine structure as compared with conventional preembedding techniques. Morphometric measurements of parameters, such as vesicle diameter, form factor, percentage immunoreactive boutons, etc., could be obtained using a Zeiss/Kontron IBAS interactive image analyzer. In conclusion, the use of ultrastructural immunocytochemistry permits the visualization of transmitter identified neuron populations at the electron microscopic level, thus expanding our knowledge regarding the morphology and synaptic interactions of these neurons.
Processing of Early Mouse Embryos for Studies on Cytoskeletal Structures. EERO LErtTONEN* AND ILKKA REIMA,t *Department of Pathology and ~Department of Electron
Microscopy, University of Helsinki, 00290 Helsinki, Finland. Most if not all nucleated mammalian cells contain a cytoskeleton consisting of three filamentous organizations, viz microfilaments (ME), microtubules (MT), and intermediate filaments (IMF). During preimplantation mouse development, important cell movements occur leading to the formation of the blastocyst with two different cell types. Cytoskeletal proteins apparently are involved in these processes. Electron microscopy and immunofluorescence studies have established the presence and distribution of microfilaments and microtubules in the preimplantation embryos. As to the intermediate filaments, conflicting results have been published on the presence of this cytoskeletal component in the cleavagestage embryos. In normal untreated embryos it apparently is not possible to demonstrate IMFs in EM. However, our immunofluorescence experiments suggesting the presence of cytokeratin in early embryos prompted us to study the matter in EM in more detail. We studied the embryos in TEM after extraction with 0.05-0.5% Triton X-100 in 50 m M Tris-HC1, pH 7.2, followed by fixation and processing for EM. In these cytoskeleton preparations, scattered 10 to 11-nm-thick filaments could be observed in the cytoplasm domain of two to eight-cell-stage embryos. These filaments were abundant in blastocyststage embryos. Our observations thus confirm the presence of IMFs in early mouse embryos. Furthermore, our staining results with antibodies to cytokeratin suggest that these filaments contain cytokeratin.
Demonstration of Tubular Cisternal Endoplasmic Reticulum in Rat Hepatocytes. O. J. Mf0LLER, O. f~STERGAARDTHOMSEN, AND J. A. LARSEN, Institute of Physiology, University
of Aarhus, DK-8000 Arhus C, Denmark. In rat liver fixed by perfusion with glutaraldehyde and postfixed with a mixture of osmium tetroxide and potassium ferricyanide sursurface cisternae and vesicles were demonstrable. From serial sections it appears that the cisternae and vesicles are part of large, fenestrated cisternae situated parallel to and at a distance of 20-40 nm from the lateral plasma membrane. In the pericanalicular and the sinusoidal zones of the hepatocytes tubules and vesicles were seen to approach the plasma membrane. Impregnation of small tissue blocks with 1% osmium for 5 days caused an intense deposition of osmium in the endoplasmic reticulum, the nuclear envelope, and the Golgi complex. Inspection of osmium-impregnated thick sections together with serial sectioning gave the impression of the existence of a continuous intracellular system of tubules and cisternae connecting the