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Ontogenesis of muscarinic receptors and acetylcholinesterase in differentiating rat cerebral cells in culture
368
Brain Research, 155 (1978) 368-373 © Elsevier/North-Holland Biomedical Press
Ontogenesis of muscarinic receptors and acetylcholinesterase in differentiating rat cerebral cells in culture
Y A D I N D U D A I and EPHRAIM YAVIN
Department of Neurobiology, The 14/eizmannInstitute of Science, Rehovot (Israel) (Accepted June 8th, 1978)
Muscarinic cholinergic synapses constitute one of the major neurotransmitter systems identified in the vertebrate brain 16,17. Ontogenesis of muscarinic receptors have recently been studied in vivo in chick brain 5, retina 15 and heart 6, in rat brain 1 and in vitro in chick retina 15 and in mouse cardiocytes9. To our knowledge no studies have been published on the development of muscarinic receptors in cultures of cells derived from the immature mammalian braina8, z0. These cells provide a valuable tool for studying the ontogenesis of muscarinic synapses, because of the possibility of correlating neurochemical observations with morphological and ultrastructuraI events under controlled and easily manipulated conditions. Such studies may shed light on the extent to which cells of the central nervous system are capable of expressing their differentiation capacity in vitro. Furthermore, the investigations may also contribute to elucidating the role of cell--cell interactions in receptor expression and the implication of the latter in synaptogenesis4. In the following we report that cultured cells from fetal rat brain tissue contain muscarinic binding sites and acetylcholinesterase, and that the level of these components markedly increases in a time-dependent course, which appears to parallel synaptogenesis in culture z0. All experiments were performed on dissociated cells isolated from fetal rat brain tissue at 16 days gestation as documented elsewhere la. In brief, dissociated cerebral cells were seeded on polylysine precoated dishes in Basal Medium Eagle (BME) augmented with glucose (0.6 g ~o) and containing fetal calf serum and 10 ~o egg ultrafiltrate. The initial plating medium was replaced 48 h later by BME containing 10 fetal calf serum and 0.6 g ~ glucose and the cells were maintained for 10-12 days between each change thereafter to prolong neuronal survival. On the appropriate day, cells from 3-4 dishes were scraped off with a rubber policeman in Ca, Mg-free phosphate buffer saline and centrifuged at 500 × g for 2 min. The resulting pellet was homogenized on ice in 0.32 M sucrose in a glass-glass homogenizer followed by a glass-teflon homogenizer. Muscarinic receptor binding was determined by the use of the powerful
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Fig. 1. Binding of [SH]QNB to cultured brain cells homogenate at various ligand concentrations. Aliquots from a 16-day-old culture (containing 190 #g protein each) were incubated at 25 °C for 1 h with the appropriate [3H]QNB concentration, in the absence or in the presence of 10-5 M atropine. • 0 , specific binding, defined as total binding minus the binding in the presence of atropine. [] [], binding in the presence of atropine. Insert: double-reciprocal plot of specific binding.
muscarinic antagonist [aH]quinuclidinyl benzilate ([ZH]QNB) 16. Aliquots of homogenate (containing 60--500/zg protein) were incubated in 0.06 M NaCI, 0.025 M Tris.C1, pH 7.4, in total volume of 150 or 300/zl. Reaction was started by addition of [3H]QNB (29.4 Ci/mmole, New England Nuclear, Boston). Unless otherwise indicated, a final concentration of 5 n M was used. Reaction was carried on for 60 min at 25 °C and was terminated by diluting with 2 ml incubation buffer, followed immediately by vacuum filtration over a wet glass-fiber filter (GF/C, Tamar, Israel). The filter was then washed 3 times with 2 ml portions of buffer, dried and placed in vials containing 4 ml 33 ~ (v/v) Triton X-100, 0.8 ~o PPO and 0.01 ~o POPOP in toluene. Vials were maintained overnight and counted by liquid scintillation spectrometry. Acetylcholinesterase was determined as described by Johnson and Russell a, employing [3H]acetylcholine (3.3 mM) as a substrate. Protein was determined according to Lowry et al. u using BSA as a standard. D N A was determined according to Burton a after precipitating the pellet with 40 vols of 1/2 (v/v) chloroform/methanol. Specific binding of [ZH]QNB, defined as total binding minus the binding in the presence of 10-5 atropine, was linearly proportional to the amount of homogenate in the assay. Specific binding was saturable and the apparent dissociation constant of [aH]QNB calculated from binding isotherms was found to be KD = 0.5 × 10-9 (Fig. 1). Non-specific binding was negligible up to about 2 n M [aH]QNB but increased at higher concentrations. The pharmacological profile of [aH]QNB-binding sites is presented in Table I. Because of the low binding levels, it was impractical to perform initial rate studies and hence determine 150 values. Affinities of various ligands for the QNB-binding sites were therefore determined as follows. Concentrations of various ligands that displaced 50 ~o of [aH]QNB binding at saturation (EDs0 values) were determined from competition
370 TABLE I
Inhibition of [aH] QNB binding to cultured brain cells by various ligand~
Aliquots of homogenate were preincubated for 25 rain with the appropriate concentrations of ligands and then further incubated for 1 h at 25 °C with 2 nM [3H]QNB.K~, the inhibition constant for each ligand, was calculated as described in the text. Ligand
Ki ( M)
Dexetimide Scopolamine Atropine Oxotremorine D,L-Muscarine Acetylcholine* D-Tubocurarine Decamethonium Nicotine Dihydro-fl-erythroidine**
1 x 2 x 7 x 5 x 7 x 7 x 1 x 5 x >2 x > 1 0 -4
10 -1° 10 10 10 -9 10 -7 10 -6 10 -6 10 -5 10 -5 10 -4
* I n the presence o f 5 x 10 -6 M eserine, which h a d no effect on binding. ** N o significant effect on b i n d i n g u n d e r the assay c o n d i t i o n s a t t ha t c o n c e n t r a t i o n .
experiments. K~, the inhibition constant for each ligand, was then estimated from the relation:
K~--
EDs0 1 -q---
[L] KD
where KD is the apparent dissociation constant of [3H]QNB obtained from its direct binding studies (Fig. 1), and [L] is the concentration of [SH]QNB under the assay conditions 10. From the data presented in Table I it is is seen that QNB-binding sites in cell culture homogenates display muscarinic properties similar to those reported for muscarinic receptors in other systems 2,16. The muscarinic antagonists dexetimide, scopolamine and atropine were the most potent in protecting the sites. The agonists oxotremorine, acetylcholine and muscarine were also effective. In contrast, the potent nicotinic agonist nicotine and the potent nicotinic antagonist dihydro-fl-erythroidine at high concentrations were not effective. Acetylcholinesterase activity in cell-culture homogenates was linearly proportional to the amount of homogenate present in the assay. About 95 ~ of the cholinesterase activity was due to true acetylcholinesterase. Thus, in a typical experiment, 10-6 M eserine and 10-6 M BW284C51 (a specific acetylcholinesterase inhibitor) inhibited activity by 93 ~o and 96~o respectively, whereas 10-6 M tetraisopropylpyrophosphoramide (iso-OMPA, a specific inhibitor of pseudocholinesterase) had no significant effect. The ontogenesis of the muscarinic binding-sites and of acetylcholinesterase has been examined (Figs. 2 and 3), Only little specific [3H]QNB binding was detected in
371
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Fig. 2. Ontogenesis of specific [aH]QNB binding to cultured brain cells homogenate, expressed per D N A (upper figure) or per protein (lower figure). Binding was determined as described in the text, in the presence of 5riM [aH]QNB. The open circles represent a homogenate prepared from the dissociated cells before seeding (day 0 in vitro).
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Fig. 3. Ontogenesis of acetylcholinesterase activity in cultured brain cells homogenate, expressed per D N A (upper figure) or per protein (lower figure). Activity was determined as described in the text. The open circles represent a homogenate prepared from the dissociated cells before seeding (day 0 in vitro).
372 the cells during the first 4-5 days in culture. Morphologically this period is characterized by establishment of intercellular contacts and by the absence of synaptic structures 20. Binding activity markedly increased in the subsequent days. Thus after 3 weeks in vitro specific binding of [3H]QNB reached a level of 0.25 pmole/mg protein, which represents a 5-fold increase over the 5th day value (Fig. 2). This level is compatible with the level of muscarinic receptor binding in various regions of the adult brain 16. When expressed per DNA, the increase in binding activity was even more pronounced (Fig. 2), since in contrast to cellular proteins the concentration of D N A in the culture is practically unchanged 13. The developmental pattern of acetylcholinesterase (Fig. 3) resembles that of muscarinic binding sites. A considerable increase in the activity was observed between the first week and the third week in culture. It should be noted that the maximal activity detected was significantly higher than recently reported for brain cell cultures 14, but is compatible with the results of Godfrey et al. 7. In summary, our results indicate that dissociated brain cells can differentiate and express in vitro cholinergic properties 19. The period between days 7 and 21 in vitro is ultrastructurally characterized by an increased density of synaptic structures 2°, and this may suggest that the appearance of cholinergic components and synaptogenesis are intimately associated events. It should be noted that the major rise in the level of muscarinic binding sites 1,5 and acetylcholinesterase 1,12 in vertebrate brain in vivo also appears to coincide with synaptogenesis. The changes observed in the culture thus suggest a process of maturation which may be similar to neuronal development in vivo. It is yet to be determined whether the biochemical maturation of the cholinergic markers, as described in the present report, and the appearance of synaptic structures can be dissociated or modified. The central nervous system cell culture may lend itself to such investigations. The authors are indebted to Dr. Z. Yavin for her suggestions and for helping with the preparation of cells. This work was supported by grants from the United-States - Israel Binational Science Foundation, Jerusalem (to Y.D.) and from the Ministry of Health, Jerusalem (to. E.Y.). Y.D. is incumbent of the Barecha Foundation Career Development Chair.
1
Ben-Barak, J. and Dudai, Y., Ontogenesis of cholinergic binding-sites in rat hippocampus, lsr. J. med. Sci. (1978) in press.
2 Birdsall, N.J.M. and Hulme, E.C., Biochemical studies on muscarinic acetylcholine receptors, J. Neurochem., 27 (1976) 7-16. 3 Burton, K., Determination of DNA concentration with diphenylamine. In L. Grossman and K. Moldave (Eds.), Methods in Enzymology, Vol. 12B, Academic Press, New York, 1968, pp. 163-166. 4 Changeux, J. P. and Danchin, A., Selective stabilization of developing synapses as a mechanism for the specification of neuronal networks, Nature (Lond.), 264 (1976) 705-712. 5 Enna, S. J., Yamamura, H. J. and Snyder, S. H., Development of muscarinic cholinergic and GABA receptor binding in chick embryo brain, Brain Research, 101 (1976) 177-183. 6 Gaiper, J. B., Klein, W. and Catterall, W. A., Muscarinic acetylcholine receptors in developing chick heart, J. biol. Chem. 252 (1977) 8692-8699.
373 7 Godfrey, E. W., Nelson, P. G., Schrier, B. K., Breuer, A. C. and Ransom, B. R., Neurons from fetal rat brain in a new cell culture system: a multidisciplinaryanalysis, Brain Research, 90 (1975) 1-21. 8 Johnson, C. D. and Russell, R. L., A rapid, simple radiometric assay for cholinesterase suitable for multiple determinations, Analyt. Biochem., 64 (1975) 229-238. 9 Lane, M. A., Sastre, A., Law, M. and Salpeter, M. M., Cholinergic and adrenergic receptors on mouse cardiocytes in vitro, Develop. Biol., 57 (1977) 254-269. 10 Levitzki, A., Sevilia, N., Atlas, D. and Steer, M. L., Ligand specificity and characteristics of the fl-adrene, gic receptor in turkey erythrocyte plasma membranes, J. molec. Biol., 97 (1975) 35-53. 11 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 12 Nadler, J. V., Mathews, D. A., Cotman, C. W. and Lynch, G. S., Development of cholinergic innervation in the hippocampal formation of the rat. II. Quantitative changes in choline acetyltransferase and acetylcholinesterase activities, Develop. BioL, 36 (1974) 142-154. 13 Richter-Landsberg, C. and Yavin, E., Protein profiles of rat embryo cerebral cells during differentiation in culture, submitted for publication. 14 Schengrund, C. L. and Repman, M. A., Cell culture of sixteen-day-old rat embryo cerebrum and associated changes in ganglioside pattern, J. Neurochem., 29 (1977) 923-927. 15 Sugiyama, H., Daniels, M. P. and Nirenberg, M., Muscarinic acetylcholine receptors of the developing retina, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 5524-5528. 16 Yamamura, H. I. and Snyder, S. H., Muscarinic cholinergic binding in rat brain, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 1725-1729. 17 Yamamura, H. I., Kuhar, M. J., Greenberg, D. and Snyder, S. H., Muscarinic cholinergic receptor binding: regional distribution in monkey brain, Brain Research, 66 (1974) 541-546. 18 Yavin, E. and Yavin, Z., Attachment and culture of dissociated cells from rat embryo cerebral hemispheres on polylysine-coated surface, J. Cell Biol., 62 (1974) 540-546. 19 Yavin, E., Regulation of phospholipid metabolism in differentiating cells from rat brain cerebral hemispheres in culture: patterns of acetylcholine, phosphocholine and choline phosphoglycerides labeling from [methyl-laC]-choline, J. biol. Chem., 251 (1976) 1392-1397. 20 Yavin, Z. and Yavin, E., Synaptogenesis and myelinogenesis in dissociated cerebral cells from rat embryo on polylysine coated surfaces, Exp. Brain Res., 29 (1977) 137-147.
Brain Research, 155 (1978) 368-373 © Elsevier/North-Holland Biomedical Press
Ontogenesis of muscarinic receptors and acetylcholinesterase in differentiating rat cerebral cells in culture
Y A D I N D U D A I and EPHRAIM YAVIN
Department of Neurobiology, The 14/eizmannInstitute of Science, Rehovot (Israel) (Accepted June 8th, 1978)
Muscarinic cholinergic synapses constitute one of the major neurotransmitter systems identified in the vertebrate brain 16,17. Ontogenesis of muscarinic receptors have recently been studied in vivo in chick brain 5, retina 15 and heart 6, in rat brain 1 and in vitro in chick retina 15 and in mouse cardiocytes9. To our knowledge no studies have been published on the development of muscarinic receptors in cultures of cells derived from the immature mammalian braina8, z0. These cells provide a valuable tool for studying the ontogenesis of muscarinic synapses, because of the possibility of correlating neurochemical observations with morphological and ultrastructuraI events under controlled and easily manipulated conditions. Such studies may shed light on the extent to which cells of the central nervous system are capable of expressing their differentiation capacity in vitro. Furthermore, the investigations may also contribute to elucidating the role of cell--cell interactions in receptor expression and the implication of the latter in synaptogenesis4. In the following we report that cultured cells from fetal rat brain tissue contain muscarinic binding sites and acetylcholinesterase, and that the level of these components markedly increases in a time-dependent course, which appears to parallel synaptogenesis in culture z0. All experiments were performed on dissociated cells isolated from fetal rat brain tissue at 16 days gestation as documented elsewhere la. In brief, dissociated cerebral cells were seeded on polylysine precoated dishes in Basal Medium Eagle (BME) augmented with glucose (0.6 g ~o) and containing fetal calf serum and 10 ~o egg ultrafiltrate. The initial plating medium was replaced 48 h later by BME containing 10 fetal calf serum and 0.6 g ~ glucose and the cells were maintained for 10-12 days between each change thereafter to prolong neuronal survival. On the appropriate day, cells from 3-4 dishes were scraped off with a rubber policeman in Ca, Mg-free phosphate buffer saline and centrifuged at 500 × g for 2 min. The resulting pellet was homogenized on ice in 0.32 M sucrose in a glass-glass homogenizer followed by a glass-teflon homogenizer. Muscarinic receptor binding was determined by the use of the powerful
369 [
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Fig. 1. Binding of [SH]QNB to cultured brain cells homogenate at various ligand concentrations. Aliquots from a 16-day-old culture (containing 190 #g protein each) were incubated at 25 °C for 1 h with the appropriate [3H]QNB concentration, in the absence or in the presence of 10-5 M atropine. • 0 , specific binding, defined as total binding minus the binding in the presence of atropine. [] [], binding in the presence of atropine. Insert: double-reciprocal plot of specific binding.
muscarinic antagonist [aH]quinuclidinyl benzilate ([ZH]QNB) 16. Aliquots of homogenate (containing 60--500/zg protein) were incubated in 0.06 M NaCI, 0.025 M Tris.C1, pH 7.4, in total volume of 150 or 300/zl. Reaction was started by addition of [3H]QNB (29.4 Ci/mmole, New England Nuclear, Boston). Unless otherwise indicated, a final concentration of 5 n M was used. Reaction was carried on for 60 min at 25 °C and was terminated by diluting with 2 ml incubation buffer, followed immediately by vacuum filtration over a wet glass-fiber filter (GF/C, Tamar, Israel). The filter was then washed 3 times with 2 ml portions of buffer, dried and placed in vials containing 4 ml 33 ~ (v/v) Triton X-100, 0.8 ~o PPO and 0.01 ~o POPOP in toluene. Vials were maintained overnight and counted by liquid scintillation spectrometry. Acetylcholinesterase was determined as described by Johnson and Russell a, employing [3H]acetylcholine (3.3 mM) as a substrate. Protein was determined according to Lowry et al. u using BSA as a standard. D N A was determined according to Burton a after precipitating the pellet with 40 vols of 1/2 (v/v) chloroform/methanol. Specific binding of [ZH]QNB, defined as total binding minus the binding in the presence of 10-5 atropine, was linearly proportional to the amount of homogenate in the assay. Specific binding was saturable and the apparent dissociation constant of [aH]QNB calculated from binding isotherms was found to be KD = 0.5 × 10-9 (Fig. 1). Non-specific binding was negligible up to about 2 n M [aH]QNB but increased at higher concentrations. The pharmacological profile of [aH]QNB-binding sites is presented in Table I. Because of the low binding levels, it was impractical to perform initial rate studies and hence determine 150 values. Affinities of various ligands for the QNB-binding sites were therefore determined as follows. Concentrations of various ligands that displaced 50 ~o of [aH]QNB binding at saturation (EDs0 values) were determined from competition
370 TABLE I
Inhibition of [aH] QNB binding to cultured brain cells by various ligand~
Aliquots of homogenate were preincubated for 25 rain with the appropriate concentrations of ligands and then further incubated for 1 h at 25 °C with 2 nM [3H]QNB.K~, the inhibition constant for each ligand, was calculated as described in the text. Ligand
Ki ( M)
Dexetimide Scopolamine Atropine Oxotremorine D,L-Muscarine Acetylcholine* D-Tubocurarine Decamethonium Nicotine Dihydro-fl-erythroidine**
1 x 2 x 7 x 5 x 7 x 7 x 1 x 5 x >2 x > 1 0 -4
10 -1° 10 10 10 -9 10 -7 10 -6 10 -6 10 -5 10 -5 10 -4
* I n the presence o f 5 x 10 -6 M eserine, which h a d no effect on binding. ** N o significant effect on b i n d i n g u n d e r the assay c o n d i t i o n s a t t ha t c o n c e n t r a t i o n .
experiments. K~, the inhibition constant for each ligand, was then estimated from the relation:
K~--
EDs0 1 -q---
[L] KD
where KD is the apparent dissociation constant of [3H]QNB obtained from its direct binding studies (Fig. 1), and [L] is the concentration of [SH]QNB under the assay conditions 10. From the data presented in Table I it is is seen that QNB-binding sites in cell culture homogenates display muscarinic properties similar to those reported for muscarinic receptors in other systems 2,16. The muscarinic antagonists dexetimide, scopolamine and atropine were the most potent in protecting the sites. The agonists oxotremorine, acetylcholine and muscarine were also effective. In contrast, the potent nicotinic agonist nicotine and the potent nicotinic antagonist dihydro-fl-erythroidine at high concentrations were not effective. Acetylcholinesterase activity in cell-culture homogenates was linearly proportional to the amount of homogenate present in the assay. About 95 ~ of the cholinesterase activity was due to true acetylcholinesterase. Thus, in a typical experiment, 10-6 M eserine and 10-6 M BW284C51 (a specific acetylcholinesterase inhibitor) inhibited activity by 93 ~o and 96~o respectively, whereas 10-6 M tetraisopropylpyrophosphoramide (iso-OMPA, a specific inhibitor of pseudocholinesterase) had no significant effect. The ontogenesis of the muscarinic binding-sites and of acetylcholinesterase has been examined (Figs. 2 and 3), Only little specific [3H]QNB binding was detected in
371
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Fig. 2. Ontogenesis of specific [aH]QNB binding to cultured brain cells homogenate, expressed per D N A (upper figure) or per protein (lower figure). Binding was determined as described in the text, in the presence of 5riM [aH]QNB. The open circles represent a homogenate prepared from the dissociated cells before seeding (day 0 in vitro).
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Fig. 3. Ontogenesis of acetylcholinesterase activity in cultured brain cells homogenate, expressed per D N A (upper figure) or per protein (lower figure). Activity was determined as described in the text. The open circles represent a homogenate prepared from the dissociated cells before seeding (day 0 in vitro).
372 the cells during the first 4-5 days in culture. Morphologically this period is characterized by establishment of intercellular contacts and by the absence of synaptic structures 20. Binding activity markedly increased in the subsequent days. Thus after 3 weeks in vitro specific binding of [3H]QNB reached a level of 0.25 pmole/mg protein, which represents a 5-fold increase over the 5th day value (Fig. 2). This level is compatible with the level of muscarinic receptor binding in various regions of the adult brain 16. When expressed per DNA, the increase in binding activity was even more pronounced (Fig. 2), since in contrast to cellular proteins the concentration of D N A in the culture is practically unchanged 13. The developmental pattern of acetylcholinesterase (Fig. 3) resembles that of muscarinic binding sites. A considerable increase in the activity was observed between the first week and the third week in culture. It should be noted that the maximal activity detected was significantly higher than recently reported for brain cell cultures 14, but is compatible with the results of Godfrey et al. 7. In summary, our results indicate that dissociated brain cells can differentiate and express in vitro cholinergic properties 19. The period between days 7 and 21 in vitro is ultrastructurally characterized by an increased density of synaptic structures 2°, and this may suggest that the appearance of cholinergic components and synaptogenesis are intimately associated events. It should be noted that the major rise in the level of muscarinic binding sites 1,5 and acetylcholinesterase 1,12 in vertebrate brain in vivo also appears to coincide with synaptogenesis. The changes observed in the culture thus suggest a process of maturation which may be similar to neuronal development in vivo. It is yet to be determined whether the biochemical maturation of the cholinergic markers, as described in the present report, and the appearance of synaptic structures can be dissociated or modified. The central nervous system cell culture may lend itself to such investigations. The authors are indebted to Dr. Z. Yavin for her suggestions and for helping with the preparation of cells. This work was supported by grants from the United-States - Israel Binational Science Foundation, Jerusalem (to Y.D.) and from the Ministry of Health, Jerusalem (to. E.Y.). Y.D. is incumbent of the Barecha Foundation Career Development Chair.
1
Ben-Barak, J. and Dudai, Y., Ontogenesis of cholinergic binding-sites in rat hippocampus, lsr. J. med. Sci. (1978) in press.
2 Birdsall, N.J.M. and Hulme, E.C., Biochemical studies on muscarinic acetylcholine receptors, J. Neurochem., 27 (1976) 7-16. 3 Burton, K., Determination of DNA concentration with diphenylamine. In L. Grossman and K. Moldave (Eds.), Methods in Enzymology, Vol. 12B, Academic Press, New York, 1968, pp. 163-166. 4 Changeux, J. P. and Danchin, A., Selective stabilization of developing synapses as a mechanism for the specification of neuronal networks, Nature (Lond.), 264 (1976) 705-712. 5 Enna, S. J., Yamamura, H. J. and Snyder, S. H., Development of muscarinic cholinergic and GABA receptor binding in chick embryo brain, Brain Research, 101 (1976) 177-183. 6 Gaiper, J. B., Klein, W. and Catterall, W. A., Muscarinic acetylcholine receptors in developing chick heart, J. biol. Chem. 252 (1977) 8692-8699.
373 7 Godfrey, E. W., Nelson, P. G., Schrier, B. K., Breuer, A. C. and Ransom, B. R., Neurons from fetal rat brain in a new cell culture system: a multidisciplinaryanalysis, Brain Research, 90 (1975) 1-21. 8 Johnson, C. D. and Russell, R. L., A rapid, simple radiometric assay for cholinesterase suitable for multiple determinations, Analyt. Biochem., 64 (1975) 229-238. 9 Lane, M. A., Sastre, A., Law, M. and Salpeter, M. M., Cholinergic and adrenergic receptors on mouse cardiocytes in vitro, Develop. Biol., 57 (1977) 254-269. 10 Levitzki, A., Sevilia, N., Atlas, D. and Steer, M. L., Ligand specificity and characteristics of the fl-adrene, gic receptor in turkey erythrocyte plasma membranes, J. molec. Biol., 97 (1975) 35-53. 11 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 12 Nadler, J. V., Mathews, D. A., Cotman, C. W. and Lynch, G. S., Development of cholinergic innervation in the hippocampal formation of the rat. II. Quantitative changes in choline acetyltransferase and acetylcholinesterase activities, Develop. BioL, 36 (1974) 142-154. 13 Richter-Landsberg, C. and Yavin, E., Protein profiles of rat embryo cerebral cells during differentiation in culture, submitted for publication. 14 Schengrund, C. L. and Repman, M. A., Cell culture of sixteen-day-old rat embryo cerebrum and associated changes in ganglioside pattern, J. Neurochem., 29 (1977) 923-927. 15 Sugiyama, H., Daniels, M. P. and Nirenberg, M., Muscarinic acetylcholine receptors of the developing retina, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 5524-5528. 16 Yamamura, H. I. and Snyder, S. H., Muscarinic cholinergic binding in rat brain, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 1725-1729. 17 Yamamura, H. I., Kuhar, M. J., Greenberg, D. and Snyder, S. H., Muscarinic cholinergic receptor binding: regional distribution in monkey brain, Brain Research, 66 (1974) 541-546. 18 Yavin, E. and Yavin, Z., Attachment and culture of dissociated cells from rat embryo cerebral hemispheres on polylysine-coated surface, J. Cell Biol., 62 (1974) 540-546. 19 Yavin, E., Regulation of phospholipid metabolism in differentiating cells from rat brain cerebral hemispheres in culture: patterns of acetylcholine, phosphocholine and choline phosphoglycerides labeling from [methyl-laC]-choline, J. biol. Chem., 251 (1976) 1392-1397. 20 Yavin, Z. and Yavin, E., Synaptogenesis and myelinogenesis in dissociated cerebral cells from rat embryo on polylysine coated surfaces, Exp. Brain Res., 29 (1977) 137-147.