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Acetylcholinesterase in cultured rat spinal cord
BRAIN RESEARCH
193
Short Communications Acetylcholinesterase in cultured rat spinal cord Histochemical studies of the spinal cord have shown that neurons that contain acetylcholinesterase (ACHE) are found in both the ventral and dorsal horns7,10. It has recently been demonstrated that neurons of cerebellar and brain stem tissue grown in cultures contain ACHE4,~. In the present investigation, a study was made of the AChE activity in rat spinal cord cultivated in vitro. Explants were prepared from lumbar segments of the spinal cord of newborn rats. The meninges were removed and the cord was cut transversely into sections 0.5-1 mm thick. Two to three explants were placed on collagen-coated coverslips, fed with 2 drops of nutrient medium and sealed into Maximov double-coverslip a s s e m -
Fig. 1. A, Neuron (probably motoneuron) of an 8-day-old culture of rat spinal cord. The cell is located in the zone of migration. B, Spinal neuron, 22 days in vitro. Phase contrast, bar: 30/~rn. Brain Research, 30 (1971) 193-197
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Fig. 2. A, Spinal cord, 7 days in vitro. The large neuron on the right shows a high content of brown granules in the cytoplasm of the perikaryon. AChE seems to be located on the outside surface of the neuron on the left side. B, AChE-containing neuron of a 25-day-old spinal cord culture. Bar: 30 #m.
Brain Research, 30 (1971) 193-197
SHORT COMMUNICATIONS
195
Fig. 3. Group of neurons lying in the beveled zone of the explant and showing an intense AChE activity. Spinal cord, 7 days in vitro. Bar: 50 pm. blies 1,9. The nutrient medium consisted of TC-Minimal Medium Eagle, glutamine (0.6 ml of 5 ~ glutamine/100 ml medium), 15 ~ fetal calf serum, 1 0 ~ bovine serum, 500 mg glucose/100 ml and antibiotics (300 units of penicillin/ml and 300 /~g of streptomycin/ml). The cultures were incubated at 36°C for 5-40 days. Twice a week, the cultures were washed in Tyrode solution and fed with fresh nutrient medium. The outgrowth of the cultures was observed daily with a reverse Zeiss microscope, and suitable cultures were chosen for AChE staining. Some explants were also cultivated on coverslips in Falcon plastic tubes 4. The histochemical technique for demonstrating AChE activity was that of Karnovsky and Roots 5 modified by E1-Badawi and Schenk 2. Acetylthiocholiniodide was used as a substrate, and octamethylpyrophosphoramide (OMPA, Koch-Light Lab. Ltd.) was added to inhibit non-specific cholinesterase activity. The incubation time was 2-3 h at 37°C (pH 5.5-5.6). Some cultures were counterstained with haemalaun. The general organization of spinal cord cultures has been described in detail by several authorsg, 11. Neurons o f various sizes are located mainly in the dense and beveled zones of the explant, although healthy neurons are also found in the marginal zones. The glial cells usually form a dense network in the zones of migration. Large Brain Research,
30 (1971) 193-197
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multipolar cells which appear to be motoneurons are often observed in these culturesS, 11. Fig. 1 shows phase-contrast pictures of two spinal neurons. The cells have a well-defined nucleus with a single, prominent nucleolus. In cultures that were stained with cresyl violet or methylene blue the perikaryon of these cells contained Nissl bodies. A few cells that were stained by the Bodian technique also revealed the presence of neurofibrils. AChE-containing neurons were observed in almost all the 5-40-day-old spinal cord cultures. Fig. 2A illustrates a neuron (right) with a high AChE content in the cytoplasm of the cell body. The cell on the left shows only a slight patchy staining on the soma, suggesting that AChE is located on the outside surface of the neuron. The neuron in Fig. 2B contained AChE in the cytoplasm of the soma and in the dendrites. AChE was usually found in medium-sized and large neurons in the dense and beveled zones of the explant as well as in the zones of migration. Neurons located in the marginal zones appeared to contain less AChE than those in the dense zones of the culture. In some cultures, groups of large neurons with a high AChE content were observed. Such a group of intensely stained cells is illustrated in Fig. 3. It is suggested that these heavily stained large cells are motoneurons, because histochemical studies of rat spinal cord in vivo have shown that motoneurons contain a high amount of AChE 7. Several neurons did not stain for ACHE. Glial cells usually stained for butyrylcholinesterase but not for ACHE. In some cultures that were counterstained with haemalaun it was occasionally observed that large AChE-containing neurons (40-60/~m diameter, probably motoneurons) were surrounded by many small cells (5-7 #m) that contained little or no ACHE. The small cells were located in close proximity to the large neuron, and often had no or only 1-2 processes 3 which seemed to make contact with the soma and/or dendrites of the large cell. From phase-contrast and Nissl studies it appeared that most of the small cells were neurons, although some seemed to be oligodendrocytes. The question is raised whether the small neurons might be Renshaw cells. Department of Neurophysiology, Neurological Clinic, University of Basle, 4051 Basle (Switzerland)
E. HC)SLI L. HOSLI
1 BORNSTEIN, M. B., AND MURRAY, M. R., Serial observations on patterns of growth, myelin
formation, maintenance and degeneration in cultures of new-born rat and kitten cerebellum, J. biophys, biochem. Cytol., 4 (1958) 499-504. 2 EL-BADAWI, A., AND SCHENK, E. A., Histochemical methods for separate, consecutive and simultaneous demonstration of acetylcholinesterase and norepinephrine in cryostat sections, J. Histochem. Cytochem, 15 (1967) 580-588. 3 ERULKAR,S. D., NICHOLS,C. W., POPP,M. B., ANDKOELLE,G. B., Renshaw elements: Localization and acetylcholinesterase content, J. Histochem. Cytochem., 16 (1968) 128-135. 4 H/)SLI, E., AND HOSLI, L., The presence of acetylcholinesterase in cultures of cerebellum and brain stem, Brain Research, 19 (1970) 494496. 5 KARNOVSKY, M. J., AND ROOTS, L., A 'direct-coloring' thiocholine method for cholinesterase, J.
Histochem. Cytochem., 12 (1964) 219-221. 6 KIM, S. U., AND MURRAY, M. R., Histochemical demonstration of acetylcholinesterase in organized cultures of mouse cerebellum and sensory ganglia, Anat. Rec., 163 (1969) 310.
Brain Research, 30 (1971) 193-197
SHORT COMMUNICATIONS
197
7 KOELLE, G. B., Cytological distributions and physiological functions of cholinesterases. In O. EICHLER AND A. FARAH (Eds.), Cholinesterases and Anticholinesterase Agents. Handbuch der Experimentellen Pharmakologie, Suppl. 15, Springer, Berlin, 1963, pp. 187-298. 8 MURRAY,M. R., Nervous tissues in vitro. In E. N. WILLMER(Ed.), Cells and Tissues in Culture, Vol. 2, Academic Press, New York, 1965, pp. 373~,55. 9 PETERSON,E. R., CRAIN,S. M., AND MURRAY,i . R., Differentiation and prolonged maintenance of bioelectrically active spinal cord cultures (rat, chick and human), Z. Zellforsch., 66 (1965) 130-154. 10 SILVER, A., AND WOLSTENCROFT, J. H., Cholinesterases and choline acetylase in the spinal cord of the cat, J. Physiol. (Lond.), 210 (1970) 92P-93P. l l SOBKOW1CZ,H. M., GUILLERY,R. W., ANDBORNSTEIN,M. B., Neuronal organization in long term cultures of the spinal cord of the fetal mouse, J. cutup. NeuroL, 132 (1968) 365-396. (Accepted April 6th, 1971)
Brain Research, 30 (1971) 193-197
193
Short Communications Acetylcholinesterase in cultured rat spinal cord Histochemical studies of the spinal cord have shown that neurons that contain acetylcholinesterase (ACHE) are found in both the ventral and dorsal horns7,10. It has recently been demonstrated that neurons of cerebellar and brain stem tissue grown in cultures contain ACHE4,~. In the present investigation, a study was made of the AChE activity in rat spinal cord cultivated in vitro. Explants were prepared from lumbar segments of the spinal cord of newborn rats. The meninges were removed and the cord was cut transversely into sections 0.5-1 mm thick. Two to three explants were placed on collagen-coated coverslips, fed with 2 drops of nutrient medium and sealed into Maximov double-coverslip a s s e m -
Fig. 1. A, Neuron (probably motoneuron) of an 8-day-old culture of rat spinal cord. The cell is located in the zone of migration. B, Spinal neuron, 22 days in vitro. Phase contrast, bar: 30/~rn. Brain Research, 30 (1971) 193-197
194
SHORT COMMUNICATIONS
Fig. 2. A, Spinal cord, 7 days in vitro. The large neuron on the right shows a high content of brown granules in the cytoplasm of the perikaryon. AChE seems to be located on the outside surface of the neuron on the left side. B, AChE-containing neuron of a 25-day-old spinal cord culture. Bar: 30 #m.
Brain Research, 30 (1971) 193-197
SHORT COMMUNICATIONS
195
Fig. 3. Group of neurons lying in the beveled zone of the explant and showing an intense AChE activity. Spinal cord, 7 days in vitro. Bar: 50 pm. blies 1,9. The nutrient medium consisted of TC-Minimal Medium Eagle, glutamine (0.6 ml of 5 ~ glutamine/100 ml medium), 15 ~ fetal calf serum, 1 0 ~ bovine serum, 500 mg glucose/100 ml and antibiotics (300 units of penicillin/ml and 300 /~g of streptomycin/ml). The cultures were incubated at 36°C for 5-40 days. Twice a week, the cultures were washed in Tyrode solution and fed with fresh nutrient medium. The outgrowth of the cultures was observed daily with a reverse Zeiss microscope, and suitable cultures were chosen for AChE staining. Some explants were also cultivated on coverslips in Falcon plastic tubes 4. The histochemical technique for demonstrating AChE activity was that of Karnovsky and Roots 5 modified by E1-Badawi and Schenk 2. Acetylthiocholiniodide was used as a substrate, and octamethylpyrophosphoramide (OMPA, Koch-Light Lab. Ltd.) was added to inhibit non-specific cholinesterase activity. The incubation time was 2-3 h at 37°C (pH 5.5-5.6). Some cultures were counterstained with haemalaun. The general organization of spinal cord cultures has been described in detail by several authorsg, 11. Neurons o f various sizes are located mainly in the dense and beveled zones of the explant, although healthy neurons are also found in the marginal zones. The glial cells usually form a dense network in the zones of migration. Large Brain Research,
30 (1971) 193-197
196
SHORT COMMUNICATIONS
multipolar cells which appear to be motoneurons are often observed in these culturesS, 11. Fig. 1 shows phase-contrast pictures of two spinal neurons. The cells have a well-defined nucleus with a single, prominent nucleolus. In cultures that were stained with cresyl violet or methylene blue the perikaryon of these cells contained Nissl bodies. A few cells that were stained by the Bodian technique also revealed the presence of neurofibrils. AChE-containing neurons were observed in almost all the 5-40-day-old spinal cord cultures. Fig. 2A illustrates a neuron (right) with a high AChE content in the cytoplasm of the cell body. The cell on the left shows only a slight patchy staining on the soma, suggesting that AChE is located on the outside surface of the neuron. The neuron in Fig. 2B contained AChE in the cytoplasm of the soma and in the dendrites. AChE was usually found in medium-sized and large neurons in the dense and beveled zones of the explant as well as in the zones of migration. Neurons located in the marginal zones appeared to contain less AChE than those in the dense zones of the culture. In some cultures, groups of large neurons with a high AChE content were observed. Such a group of intensely stained cells is illustrated in Fig. 3. It is suggested that these heavily stained large cells are motoneurons, because histochemical studies of rat spinal cord in vivo have shown that motoneurons contain a high amount of AChE 7. Several neurons did not stain for ACHE. Glial cells usually stained for butyrylcholinesterase but not for ACHE. In some cultures that were counterstained with haemalaun it was occasionally observed that large AChE-containing neurons (40-60/~m diameter, probably motoneurons) were surrounded by many small cells (5-7 #m) that contained little or no ACHE. The small cells were located in close proximity to the large neuron, and often had no or only 1-2 processes 3 which seemed to make contact with the soma and/or dendrites of the large cell. From phase-contrast and Nissl studies it appeared that most of the small cells were neurons, although some seemed to be oligodendrocytes. The question is raised whether the small neurons might be Renshaw cells. Department of Neurophysiology, Neurological Clinic, University of Basle, 4051 Basle (Switzerland)
E. HC)SLI L. HOSLI
1 BORNSTEIN, M. B., AND MURRAY, M. R., Serial observations on patterns of growth, myelin
formation, maintenance and degeneration in cultures of new-born rat and kitten cerebellum, J. biophys, biochem. Cytol., 4 (1958) 499-504. 2 EL-BADAWI, A., AND SCHENK, E. A., Histochemical methods for separate, consecutive and simultaneous demonstration of acetylcholinesterase and norepinephrine in cryostat sections, J. Histochem. Cytochem, 15 (1967) 580-588. 3 ERULKAR,S. D., NICHOLS,C. W., POPP,M. B., ANDKOELLE,G. B., Renshaw elements: Localization and acetylcholinesterase content, J. Histochem. Cytochem., 16 (1968) 128-135. 4 H/)SLI, E., AND HOSLI, L., The presence of acetylcholinesterase in cultures of cerebellum and brain stem, Brain Research, 19 (1970) 494496. 5 KARNOVSKY, M. J., AND ROOTS, L., A 'direct-coloring' thiocholine method for cholinesterase, J.
Histochem. Cytochem., 12 (1964) 219-221. 6 KIM, S. U., AND MURRAY, M. R., Histochemical demonstration of acetylcholinesterase in organized cultures of mouse cerebellum and sensory ganglia, Anat. Rec., 163 (1969) 310.
Brain Research, 30 (1971) 193-197
SHORT COMMUNICATIONS
197
7 KOELLE, G. B., Cytological distributions and physiological functions of cholinesterases. In O. EICHLER AND A. FARAH (Eds.), Cholinesterases and Anticholinesterase Agents. Handbuch der Experimentellen Pharmakologie, Suppl. 15, Springer, Berlin, 1963, pp. 187-298. 8 MURRAY,M. R., Nervous tissues in vitro. In E. N. WILLMER(Ed.), Cells and Tissues in Culture, Vol. 2, Academic Press, New York, 1965, pp. 373~,55. 9 PETERSON,E. R., CRAIN,S. M., AND MURRAY,i . R., Differentiation and prolonged maintenance of bioelectrically active spinal cord cultures (rat, chick and human), Z. Zellforsch., 66 (1965) 130-154. 10 SILVER, A., AND WOLSTENCROFT, J. H., Cholinesterases and choline acetylase in the spinal cord of the cat, J. Physiol. (Lond.), 210 (1970) 92P-93P. l l SOBKOW1CZ,H. M., GUILLERY,R. W., ANDBORNSTEIN,M. B., Neuronal organization in long term cultures of the spinal cord of the fetal mouse, J. cutup. NeuroL, 132 (1968) 365-396. (Accepted April 6th, 1971)
Brain Research, 30 (1971) 193-197