- Email: [email protected]
Incorporation of RNA and protein precursors into embryonic nerve cells ofPeriplaneta americana culturedin vitro
SHORT COMMUNICATIONS
305
Incorporation of RNA and protein precursors into embryonic nerve cells of Periplaneta americana cultured in vitro Brain, ganglia and gut explanted from embryonic cockroaches were successfully cultured in vitro by Chen and Levi-Montalcini in a chemically defined medium z,4. By the second day o f culture axons emerged f r o m brain and ganglia and the nerve fiber outgrowth continued in vitro for as long as 5-6 months. Nerve fiber bundles split into thin filaments f o r m i n g connections with each other and with axons outgrowing from neighboring explants. Glial and nerve cells from brain and ganglia also actively migrated (Fig. 1A). The identification of nerve cells a m o n g cells that had migrated f r o m these explants is based on studies with the interference microscope and the electron microscope. Nerve cells and nerve fibers were intermingled with glial cells to form a carpet-like layer a r o u n d the explants. Similarly the identification o f nerve fibers was based on extensive histological and electron m i c r o s c o p y studiesL
Fig. 1. A, Nerve cells (n), glial cells (gl) and nerve fibers in the migratory zone around an explant of the brain of Periplaneta americana after 8 days of culture. Photomicrograph of the fresh culture from the interference differential Nomarski microscope. × 600. B, Large neuron (n) from the migratory area around an explant of a thoracic ganglion of Periplaneta americana after 8 days of culture, photographed on the phase contrast microscope after fixation and hematoxylin staining. Arrows point to dilatations on the nerve fibers. Glial cell (gl). × 1200. Brain Research, 21 (1970) 305-308
306
SHORr COMMUNICATIONS
~ii~ii~i~i!iii,~ ~,i¸i!i~
Fig. 2. Incorporation of [~H]amino acids (A,B,C) and of [aH]uridine (D,E,F) into neurons, glial cells and fibers that had migrated in vitro from brain and ganglia of Periplaneta americana embryos. Time of incubation with [3H]amino acids was 1 h (A) and 24 h (B and C). × 600. Time of incubation with [3H]uridine was 30 min (D), 4 h (E) and 12 h ~F). × 400.
Brain Research, 21 (1970) 305-308
SHORT COMMUNICATIONS
307
Nerve fibers growing in vitro are characterized by round or fusiform dilatations strikingly similar to those described in vivo by Wigglesworth 5 (Fig. 1B). The purpose of the present work was to study the synthetic properties of cells that had migrated from the explants in the above-mentioned conditions of culture and to study these same properties in nerve fibers. The incorporation of radioactive precursors of protein and ribonucleic acid into these embryonic cells was studied with autoradiographic techniques. Brain and ganglia explants from 18-day Periplaneta americana embryos were cultured in a mixture of Eagle basal medium and Schneider insect solution (4:5) as described 2. The cultures were incubated at 29°C in a water saturated atmosphere of 5 ~ carbon dioxide in air. In 8-day cultures, when a great number of cells had migrated from explants, the radioactive precursor was added to each culture for different lengths of time. The cover-glass carrying the explants was then removed from the medium and the culture was fixed in alcohol-formalin-acetic acid (18:1:1), treated with 5 ~o trichloroacetic acid for 10 min and then washed in running water for several hours. The cultures were dried, and each cover-glass was attached, with its culture on top, to a microscope slide which was then dipped in NTB 2 K o d a k emulsion. After 10-20 days of exposure the autoradiographies were developed for 10 min in Kodak D-19 and fixed in Kodak Unifix for 15 min. The preparations were stained with Mayer Emalum and eosin at 4°C. For the analysis of protein synthesis a mixture of tritiated amino acids (New England Nuclear Corp., specific activity 1 Ci/mM) was added at a concentration of 10 #Ci/ml. After 1 h of incorporation very few grains appeared on the cytoplasm of the cells and along the fibers (Fig. 2A). After 24 h labeling with [ZH]amino acids a cellular outline of nerve and glial cells formed on the background by the deposition o f silver grains (Fig, 2B and C). To prove that the radioactive amino acids were really incorporated into proteins, control experiments were carried out with puromycin. This inhibitor of protein synthesis was added to the medium at a concentration of 20 #g/ml with the radioactive amino acids. The incorporation into protein was strongly reduced. For the study of the synthesis of RNA, cultures were labeled with [aH]uridine (Amersham, specific activity 3 Ci/mM) at the concentration of 10 #Ci/ml for various lengths of time. Silver grains were concentrated on the nucleolar region in the first 30 min (Fig. 2D) and over all the nuclear region after 2 h of labeling. At 4 h the label also appeared on the cytoplasm and on the nerve fibers (Fig. 2E). The above-mentioned dilatations of the fibers showed a marked concentration of grains, thereby revealing the presence of RNA, while the short nerve fiber tracts interposed between the dilatations were not yet labeled. After 12 h of incorporation the blackening of cells and fibers increased progressively (Fig. 2F). That the radioactivity observed was really due to incorporation of the precursor into RNA was proved, as previously described 1, by the addition of actinomycin D to a culture shortly before the radioactive precursor, or by RNase treatment of a labeled culture performed after fixation and before it was dipped into the autoradio-
Brain Research, 21 (1970) 305-308
308
SHORT COMMUNICATIONS
graphic emulsion. Both experiments showed a m a r k e d reduction of silver grains on cells a n d fibers. The a u t h o r is grateful to Prof. R. L e v i - M o n t a l c i n i a n d Dr. J. S. Chen for providing the tissue culture material on which these experiments were performed a n d for stimulating discussions. Laboratorio di Biologia Cellulare del C.N.R., Istituto Superiore di Sanitd, Rome (Italy)
PAOLA AMALDI
1 AMALDI, P., AND RUSCA, G., Autoradiographic study of RNA in nerve fibers of embryonic sensory ganglia cultured in vitro under NGF stimulation, J. Neurochem., in press. 2 CHEN, J. S., AND LEvI-MONTALCINI, R., Axonal outgrowth and cell migration in vitro from nervous system of cockroach embryos, Science, 166 (1969) 631 632. 3 CHEN, J. S., AND LEvI-MONTALC1NI,R., Long term cultures of dissociated nerve cells from embryonic nervous system of tbe cockroach, Periplaneta americana, Arch. ital. Biol., in press. 4 LEvI-MONTALCINI, R., AND CHEN, J. S., In vitro studies of the insect embryonic nervous system. In S. H. BARONDES(Ed.), Proceedings of the Symposium on the Dynamics of the Neuron, Academic Press, New York, 1969, pp. 277-298. 5 WIGGLESWORTH, W. B., Cell association and organogenesis in the nervous system of insects. In R. DE HAANAND H. URSPRt~NO(Eds.), Organogenesis, Holt, Rinehart and Winston, New York, 1965, pp. 199-217. (Accepted April 26th, 1970)
Brain Research, 21 (1970) 305-308
305
Incorporation of RNA and protein precursors into embryonic nerve cells of Periplaneta americana cultured in vitro Brain, ganglia and gut explanted from embryonic cockroaches were successfully cultured in vitro by Chen and Levi-Montalcini in a chemically defined medium z,4. By the second day o f culture axons emerged f r o m brain and ganglia and the nerve fiber outgrowth continued in vitro for as long as 5-6 months. Nerve fiber bundles split into thin filaments f o r m i n g connections with each other and with axons outgrowing from neighboring explants. Glial and nerve cells from brain and ganglia also actively migrated (Fig. 1A). The identification of nerve cells a m o n g cells that had migrated f r o m these explants is based on studies with the interference microscope and the electron microscope. Nerve cells and nerve fibers were intermingled with glial cells to form a carpet-like layer a r o u n d the explants. Similarly the identification o f nerve fibers was based on extensive histological and electron m i c r o s c o p y studiesL
Fig. 1. A, Nerve cells (n), glial cells (gl) and nerve fibers in the migratory zone around an explant of the brain of Periplaneta americana after 8 days of culture. Photomicrograph of the fresh culture from the interference differential Nomarski microscope. × 600. B, Large neuron (n) from the migratory area around an explant of a thoracic ganglion of Periplaneta americana after 8 days of culture, photographed on the phase contrast microscope after fixation and hematoxylin staining. Arrows point to dilatations on the nerve fibers. Glial cell (gl). × 1200. Brain Research, 21 (1970) 305-308
306
SHORr COMMUNICATIONS
~ii~ii~i~i!iii,~ ~,i¸i!i~
Fig. 2. Incorporation of [~H]amino acids (A,B,C) and of [aH]uridine (D,E,F) into neurons, glial cells and fibers that had migrated in vitro from brain and ganglia of Periplaneta americana embryos. Time of incubation with [3H]amino acids was 1 h (A) and 24 h (B and C). × 600. Time of incubation with [3H]uridine was 30 min (D), 4 h (E) and 12 h ~F). × 400.
Brain Research, 21 (1970) 305-308
SHORT COMMUNICATIONS
307
Nerve fibers growing in vitro are characterized by round or fusiform dilatations strikingly similar to those described in vivo by Wigglesworth 5 (Fig. 1B). The purpose of the present work was to study the synthetic properties of cells that had migrated from the explants in the above-mentioned conditions of culture and to study these same properties in nerve fibers. The incorporation of radioactive precursors of protein and ribonucleic acid into these embryonic cells was studied with autoradiographic techniques. Brain and ganglia explants from 18-day Periplaneta americana embryos were cultured in a mixture of Eagle basal medium and Schneider insect solution (4:5) as described 2. The cultures were incubated at 29°C in a water saturated atmosphere of 5 ~ carbon dioxide in air. In 8-day cultures, when a great number of cells had migrated from explants, the radioactive precursor was added to each culture for different lengths of time. The cover-glass carrying the explants was then removed from the medium and the culture was fixed in alcohol-formalin-acetic acid (18:1:1), treated with 5 ~o trichloroacetic acid for 10 min and then washed in running water for several hours. The cultures were dried, and each cover-glass was attached, with its culture on top, to a microscope slide which was then dipped in NTB 2 K o d a k emulsion. After 10-20 days of exposure the autoradiographies were developed for 10 min in Kodak D-19 and fixed in Kodak Unifix for 15 min. The preparations were stained with Mayer Emalum and eosin at 4°C. For the analysis of protein synthesis a mixture of tritiated amino acids (New England Nuclear Corp., specific activity 1 Ci/mM) was added at a concentration of 10 #Ci/ml. After 1 h of incorporation very few grains appeared on the cytoplasm of the cells and along the fibers (Fig. 2A). After 24 h labeling with [ZH]amino acids a cellular outline of nerve and glial cells formed on the background by the deposition o f silver grains (Fig, 2B and C). To prove that the radioactive amino acids were really incorporated into proteins, control experiments were carried out with puromycin. This inhibitor of protein synthesis was added to the medium at a concentration of 20 #g/ml with the radioactive amino acids. The incorporation into protein was strongly reduced. For the study of the synthesis of RNA, cultures were labeled with [aH]uridine (Amersham, specific activity 3 Ci/mM) at the concentration of 10 #Ci/ml for various lengths of time. Silver grains were concentrated on the nucleolar region in the first 30 min (Fig. 2D) and over all the nuclear region after 2 h of labeling. At 4 h the label also appeared on the cytoplasm and on the nerve fibers (Fig. 2E). The above-mentioned dilatations of the fibers showed a marked concentration of grains, thereby revealing the presence of RNA, while the short nerve fiber tracts interposed between the dilatations were not yet labeled. After 12 h of incorporation the blackening of cells and fibers increased progressively (Fig. 2F). That the radioactivity observed was really due to incorporation of the precursor into RNA was proved, as previously described 1, by the addition of actinomycin D to a culture shortly before the radioactive precursor, or by RNase treatment of a labeled culture performed after fixation and before it was dipped into the autoradio-
Brain Research, 21 (1970) 305-308
308
SHORT COMMUNICATIONS
graphic emulsion. Both experiments showed a m a r k e d reduction of silver grains on cells a n d fibers. The a u t h o r is grateful to Prof. R. L e v i - M o n t a l c i n i a n d Dr. J. S. Chen for providing the tissue culture material on which these experiments were performed a n d for stimulating discussions. Laboratorio di Biologia Cellulare del C.N.R., Istituto Superiore di Sanitd, Rome (Italy)
PAOLA AMALDI
1 AMALDI, P., AND RUSCA, G., Autoradiographic study of RNA in nerve fibers of embryonic sensory ganglia cultured in vitro under NGF stimulation, J. Neurochem., in press. 2 CHEN, J. S., AND LEvI-MONTALCINI, R., Axonal outgrowth and cell migration in vitro from nervous system of cockroach embryos, Science, 166 (1969) 631 632. 3 CHEN, J. S., AND LEvI-MONTALC1NI,R., Long term cultures of dissociated nerve cells from embryonic nervous system of tbe cockroach, Periplaneta americana, Arch. ital. Biol., in press. 4 LEvI-MONTALCINI, R., AND CHEN, J. S., In vitro studies of the insect embryonic nervous system. In S. H. BARONDES(Ed.), Proceedings of the Symposium on the Dynamics of the Neuron, Academic Press, New York, 1969, pp. 277-298. 5 WIGGLESWORTH, W. B., Cell association and organogenesis in the nervous system of insects. In R. DE HAANAND H. URSPRt~NO(Eds.), Organogenesis, Holt, Rinehart and Winston, New York, 1965, pp. 199-217. (Accepted April 26th, 1970)
Brain Research, 21 (1970) 305-308