Neuroscience Letters, 145 (1992) 33-36 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
33
NSL 08967
The effect of halothane on cultured fibroblasts and neuroblastoma cells Etsuro U e m u r a a and Edward D. Levin b ~Department of Anatomy, Iowa State University, Ames, IA 50011 (USA.) and bDepartment of Psychiatry, Duke University, Durham, NC 27710 (USA) (Received 2 July 1991; Revised version received 19 June 1992; Accepted 26 June 1992)
Key words: Halothane; Tissue culture; Actin; Neurite; Fibroblast; Neuroblastoma cell Halothane exposure over the cultured cells (100 and 1,000 ppm) caused a disruption of the pattern of actin distribution in both fibroblasts and neuroblastoma cells. Neuroblastoma cells exposed to halothane also lost microspikes; however, neurite elongation was not affected by halothane. The present study suggests that halothane induces the functional disruption of actin, resulting in an interference of normal neural development in vivo.
Halothane, one of the most widely used (typically at 15,000 ppm) inhalation anesthetics, has been shown to retard synaptic maturation in the developing rat brain [7]. This initial finding has subsequently been confirmed in dose-response studies in rats exposed to halothane throughout gestation and until 60 days after birth [1 l, 13]. Furthermore, the delay in the initial synaptic development caused by halothane did not fully recover even after halothane exposure was stopped. Similarly, halothane has been observed to suppress dendritic growth in rats [12], suggesting that delayed dendritic growth contributed to the low synaptic density found in rats exposed to halothane. The different effects of halothane on dendritic growth in the entorhinal cortex and subiculum indicate that there may be regional differences in the sensitivity to halothane's effects on the brain [12]. Although delayed synaptogenesis induced by halothane could be a result of suppressed dendritic growth, suppression of axonal growth could also be contributed to delayed synaptogenesis during ontogeny. This possibility was studied in the rat dentate gyrus by observing reactive synaptogenesis under halothane exposure [14]. A substantial halothane-induced decline in the rate of reactive synaptogenesis indicated that halothane does in fact suppress axonal growth. One possible mechanism of halothane neurotoxicity on neurite outgrowth appears to involve cytoskeletal proteins; when neuroblastoma cells were exposed to halothane in vitro [4], microspikes disappeared and miCorrespondence: E. Uemura, Department of Anatomy, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA.
crofilaments were not observed in the cell. In order to determine the possible mechanism of halothane neurotoxicity, the present study examined actin distribution in the cultured cells. Since the distribution pattern of actin is reported in detail in cultured fibroblasts [16], both fibroblasts and neuroblastoma cells were used in our study. Fibroblasts isolated from 18-20-day-old rat embryos (Sprague-Dawley) and murine neuroblastoma cells (N2A, American Type Culture Collection, Baltimore, MD) were used in this study. Both cell types were cultured in Eagle's minimal essential medium (MEM, Gibco) supplemented with 10% fetal calf serum and gentamicin. The cells were grown as a monolayer in 35 mm Corning culture dishes at 37°C in a humidified atmosphere of 5% C O 2 - 9 5 % air. Replicate series of fibroblast cultures were established and cells to be used for actin immunohistochemistry were subcultured on 12 mm glass coverslips 48 h prior to use. Cultured fibroblasts and neuroblastoma cells were exposed to halothane (100 or 1,000 ppm) in vitro for 4, 12, or 24 h. The incubator was perfused with halothane vaporized into a low flow of 5% CO2-95% air. The concentrations of halothane in the incubator were regulated and maintained at 100 + 15 and 1,000 + 60 ppm according to a previously described method [11]. The media was equilibrated with halothane by placing it in the incubator at least 2 h prior to use, and the halothane concentration in the media was confirmed by gas chromatography [11]. At 0, 6, or 24 h post-exposure to halothane, cultured cells were briefly rinsed in 0.1 M phosphate buffer saline (PBS [2]) and fixed in 3% buffered paraformaldehyde for
34
2A
Fig. 1. A: control fibroblasts stained for actin by the anti-actin PAP method. The majority of cells appear spindle-shaped and their cytoplasm shows a deeplystained meshwork of actin. B: fibroblasts exposed to 1,000 ppm of halothane in vitro for 24 h. A large portion of the cytoplasm is stained pale and only the nuclear and perinuclear area is stained deeply for actin, x300. 30 min at r o o m temperature. The cells were stored in 0.1 M PBS until immunocytochemistry for actin could be performed. The monoclonal anti-actin antibody (Sigma) was diluted to 1/400 in bovine albumin and 1% Tris-buffered saline. As the negative control for differentiating specific from non-absorbed staining, the primary antibody was replaced with an affinity-absorbed antiserum. Visualization was carried out using the peroxidaseantiperoxidase (PAP) method [15]. To analyze the distribution pattern of actin and its staining intensity, nine areas, randomly selected from each of the coverslips, were digitized using a Zeiss IPS image analyzer. Pixel-bypixel frequency distributions of the staining intensities were obtained and used for analysis. Exposure to halothane for either 6 or 24 h resulted in significant changes in the actin distribution pattern. Control fibroblasts were characterized by several different cellular shapes and actin distribution patterns. However, the majority of control cells were spindle-shaped and
Fig. 2. A: control neuroblastoma cell with neurites with numerous microspikes. The cell is stained deeply for actin. B: neuroblastoma cells that have been exposed to 100 ppm halothane in vitro for 6 h. The cell retained its main neurite but lost many of its smaller neurites and microspikes, x350. contained ill-organized filamentous strands of actin (Fig. 1A). When fibroblasts were exposed to halothane, many cells lost their spindle shape and the distribution pattern of actin in these cells was distinctly different from the control cells. Actin filaments were confined to the perinuclear area and a large portion of the cytoplasm was stained pale by the anti-actin antibody (Fig. 1B). This distribution pattern of actin appeared to be dose-dependent. Cultures exposed to higher hatothane concentration (1,000 ppm) had a greater proportion of cells with the abnormal pattern of actin distribution than cultures which had been exposed to 100 p p m halothane. Halothane also affected the distribution pattern of actin in neuroblastoma cells. Like fibroblasts, most neuroblastoma cells exposed to halothane had actin confined to the perinuclear area (Fig. 2B). Halothane also affected neurites of neuroblastoma cells. The majority of control neuroblastoma cells were characterized by long neurites with m a n y branches and numerous microspikes projecting from the cell body and neurite (Fig. 2A). Some
35 control neuroblastoma cells exhibited only microspikes which extended directly from the cell body with no neurites (Fig. 2A). On the other hand, neuroblastoma cells exposed to halothane exhibited characteristic morphological changes. Within 6 hours of halothane exposure, most neuroblastoma cells lost their microspikes, although they retained their neurites (Fig. 2B). Quantitative analysis of staining intensities provided additional support of a significant halothane effect on the distribution of intracellular actin. Pixel-by-pixel frequency distribution of the staining intensities has shown that the control fibroblasts had more deeply stained areas for actin than the cells exposed to 100 ppm halothane (Fig. 3). In the control cells, the mean gray-value of frequency distribution was 110.0 + 5.0. When cells wer e expo.sed to halothane for 24 h, there was a significant shift in frequency distribution toward lower staining intensity (mean value=150 + 7.1; t=-3.90, df=54, P=0.0003). The present study demonstrated that halothane induces the change in the distribution pattern of actin in cultured cells. The disappearance of filopodia-like microspikes in neuroblastoma cells following exposure to halothane confirms the similar observation previously made by Telser and Hinkley [10] in cells exposed to much higher levels of halothane (3,000-21,000 ppm). However, the lower concentrations of halothane used (100 and 1,000 ppm) in the present study did not inhibit neurite extension as reported by them. Since the maintenance and extension of neurite structure are dependent on both microfilaments and microtubules [1, 6, 8, 9], it was suggested that neurite response to halothane is related to an inability of microtubules and microfilaments to organize into functional complexes [4]. We did not examine the effect of halothane on microtubules. However, it has been shown that the concentrations of 3,000 ppm halothane which disrupted microfilaments did not affect microtubules [3-5]. At concentrations above 10,000 ppm, microtubules gradually appeared to decrease in number. According to the previous observation [4], the halothane concentration (100 or 1,000 ppm) used in the present study should not affect the microtubules. Furthermore, the present observation that halothane affected only the microspikes of neuroblastoma cells but not the neurites, suggests that low concentrations of halothane act primarily on actin and not on microtubules. The mechanism behind halothane's actions on actin warrants further study. Present results indicate that the disappearance of filopodia is associated with a disrupted distribution of actin. It is not known, however, whether such morphological changes are the result of depolymerization of actin or an inability to polymerize actin into
300 •
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200 •
/
0
(J
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50
100
150
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Staining Intensity (Gray-scale value) Fig. 3. The frequencydistribution of staining intensity of fibroblasts. Cells were exposed to 1,000ppm of halothane in vitro for 24 h. Pixelby-pixel frequencydistribution of the staining intensities was obtained from cells stained with the anti-actin PAP method. To directlycompare the magnitudeof the change in the frequencydistribution, the values of cells exposed to halothane were normalized by expressing each as a percentage of the control values. When fibroblasts were exposed to halothane, a significant shift in frequency distribution toward lower staining intensityoccurred (white is assigned a gray-scalevalue of 256, whereas black is 0). functional units in the presence of halothane. Whatever the mechanism involved in halothane toxicity, the suppressive action of halothane on filopodia is reversible as shown in the previous study [10]. This reversible halothane action was also observed when synaptogenesis occurred in the dentate gyrus of rats with entorhinal lesions [14]. It was shown that the suppressive effect of halothane was most pronounced during the period of exposure. Termination of halothane exposure resulted in quick recovery of the synaptic population, Although the effects of halothane on neural outgrowth seem to be transient, the process of brain development involves a complex series of precisely timed episodes of outgrowth and dying back of neural population. Disruption of these events even transiently could cause permanent disorganization of neural structure and function. This work was supported by Grants 19-59 from the March of Dimes Birth Defects Foundation. 1 Daniel, M.E, Colchicine inhibition of nerve fiber formation in vitro, J. Cell Biol., 53 (1972) 164-176. 2 Glauert, A.M., Fixation, dehydration and embedding of biological specimens. In A.M. Glauert (Ed.), Practical Methods in Electron Microscopy, North-Holland, Amsterdam, 1980,pp. 14. 3 Hinkley, R.E. and Samson, F.E., Anesthetic-inducedtransformation of axonal microtubules,J. Cell Biol., 53 (1972)258-263.
36 4 Hinkley, R.E. and Telser, A.G., The effects of halothane on cultured mouse neuroblastoma cells. I. Inhibition of morphological differentiation, J. Cell Biol., 63 (1974) 531 540. 5 Nunn, J.F. and Allison, A.C., Effects of anesthetics on microtubular system. In B.B. Fink (Ed.), Cellular Biology and Toxicity of Anesthetics, Williams and Wilkins, Baltimore, 1972, pp. 138 146. 6 Prasad, K.N., Waymire, J.C. and Weiner, N., A further study on the morphology and biochemistry of X-ray and dibutyryl cyclic AMP-induced differentiated neuroblastoma cells in culture, Exp. Cell Res., 74 (1972) 110-114. 7 Quimby, K.L., Aschkenase, L.J., Bowman, R.E., Katz, J. and Chang, L.W., Enduring learning deficits and cerebral synaptic malformation from exposure to 10 ppm halothane, Science, 185 (1974) 625 627. 8 Roisen, F.J. and Murphy, R.A., Neurite development in vitro. II. The role of microfilaments and microtubules in dibutyryl adenosin 3,5-cyclic monophosphate and nerve growth factor stimulated maturation, J. Neurobiol., 4 (1973) 397M12. 9 Roisen, F.J. and Rebhum, L.I., The effects ofcolchicine on cellular and intracellular movement, Physiologist, 14 (1971) 220a.
10 Telser, A. and Hinkley, R.E., Cultured neuroblastoma cells and halothane: effects on cell growth and macromolecular synthesis, Anesthesiology, 46 (1977) 102 110. 11 Uemura, E. and Bowman, R.E., Effects of halothane on cerebral synaptic density, Exp. Neurol., 69 (1980) 135-142. 12 Uemura, E., Ireland, W.R, Levin, E.D. and Bowman, R.D., Effects of halothane on the development of rat brain: a Golgi study of dendritic growth, Exp. Neurol., 89 (1985) 503-519. 13 Uemura, E., Levin, E.D. and Bowman, R.E., Effects of halothane on synaptogenesis and learning behavior in rats, Exp. Neurol., 89 (1985) 520-529. 14 Uemura, E., Levin, E., DeLuna, R. and Bowman, E., Suppressive effects of halothane on reactive synaptogenesis in the dentate gyrus of rats, Brain Res., 496 (1989) 317--320. 15 Vandesande, F., Peroxidase-antiperoxidase techniques. In A.C. Cuello (Ed.), Immunohistochemistry, Wiley, New York, 1983, pp. 101 119. 16 Van Gansen, P., Pays, A. and Malherbe, L., Actin content and organization of microfilaments in primary cultures of mouse embryonic fibroblasts (in vivo ageing), Biol. Cell., 54 (1985) 251-260.
33
NSL 08967
The effect of halothane on cultured fibroblasts and neuroblastoma cells Etsuro U e m u r a a and Edward D. Levin b ~Department of Anatomy, Iowa State University, Ames, IA 50011 (USA.) and bDepartment of Psychiatry, Duke University, Durham, NC 27710 (USA) (Received 2 July 1991; Revised version received 19 June 1992; Accepted 26 June 1992)
Key words: Halothane; Tissue culture; Actin; Neurite; Fibroblast; Neuroblastoma cell Halothane exposure over the cultured cells (100 and 1,000 ppm) caused a disruption of the pattern of actin distribution in both fibroblasts and neuroblastoma cells. Neuroblastoma cells exposed to halothane also lost microspikes; however, neurite elongation was not affected by halothane. The present study suggests that halothane induces the functional disruption of actin, resulting in an interference of normal neural development in vivo.
Halothane, one of the most widely used (typically at 15,000 ppm) inhalation anesthetics, has been shown to retard synaptic maturation in the developing rat brain [7]. This initial finding has subsequently been confirmed in dose-response studies in rats exposed to halothane throughout gestation and until 60 days after birth [1 l, 13]. Furthermore, the delay in the initial synaptic development caused by halothane did not fully recover even after halothane exposure was stopped. Similarly, halothane has been observed to suppress dendritic growth in rats [12], suggesting that delayed dendritic growth contributed to the low synaptic density found in rats exposed to halothane. The different effects of halothane on dendritic growth in the entorhinal cortex and subiculum indicate that there may be regional differences in the sensitivity to halothane's effects on the brain [12]. Although delayed synaptogenesis induced by halothane could be a result of suppressed dendritic growth, suppression of axonal growth could also be contributed to delayed synaptogenesis during ontogeny. This possibility was studied in the rat dentate gyrus by observing reactive synaptogenesis under halothane exposure [14]. A substantial halothane-induced decline in the rate of reactive synaptogenesis indicated that halothane does in fact suppress axonal growth. One possible mechanism of halothane neurotoxicity on neurite outgrowth appears to involve cytoskeletal proteins; when neuroblastoma cells were exposed to halothane in vitro [4], microspikes disappeared and miCorrespondence: E. Uemura, Department of Anatomy, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA.
crofilaments were not observed in the cell. In order to determine the possible mechanism of halothane neurotoxicity, the present study examined actin distribution in the cultured cells. Since the distribution pattern of actin is reported in detail in cultured fibroblasts [16], both fibroblasts and neuroblastoma cells were used in our study. Fibroblasts isolated from 18-20-day-old rat embryos (Sprague-Dawley) and murine neuroblastoma cells (N2A, American Type Culture Collection, Baltimore, MD) were used in this study. Both cell types were cultured in Eagle's minimal essential medium (MEM, Gibco) supplemented with 10% fetal calf serum and gentamicin. The cells were grown as a monolayer in 35 mm Corning culture dishes at 37°C in a humidified atmosphere of 5% C O 2 - 9 5 % air. Replicate series of fibroblast cultures were established and cells to be used for actin immunohistochemistry were subcultured on 12 mm glass coverslips 48 h prior to use. Cultured fibroblasts and neuroblastoma cells were exposed to halothane (100 or 1,000 ppm) in vitro for 4, 12, or 24 h. The incubator was perfused with halothane vaporized into a low flow of 5% CO2-95% air. The concentrations of halothane in the incubator were regulated and maintained at 100 + 15 and 1,000 + 60 ppm according to a previously described method [11]. The media was equilibrated with halothane by placing it in the incubator at least 2 h prior to use, and the halothane concentration in the media was confirmed by gas chromatography [11]. At 0, 6, or 24 h post-exposure to halothane, cultured cells were briefly rinsed in 0.1 M phosphate buffer saline (PBS [2]) and fixed in 3% buffered paraformaldehyde for
34
2A
Fig. 1. A: control fibroblasts stained for actin by the anti-actin PAP method. The majority of cells appear spindle-shaped and their cytoplasm shows a deeplystained meshwork of actin. B: fibroblasts exposed to 1,000 ppm of halothane in vitro for 24 h. A large portion of the cytoplasm is stained pale and only the nuclear and perinuclear area is stained deeply for actin, x300. 30 min at r o o m temperature. The cells were stored in 0.1 M PBS until immunocytochemistry for actin could be performed. The monoclonal anti-actin antibody (Sigma) was diluted to 1/400 in bovine albumin and 1% Tris-buffered saline. As the negative control for differentiating specific from non-absorbed staining, the primary antibody was replaced with an affinity-absorbed antiserum. Visualization was carried out using the peroxidaseantiperoxidase (PAP) method [15]. To analyze the distribution pattern of actin and its staining intensity, nine areas, randomly selected from each of the coverslips, were digitized using a Zeiss IPS image analyzer. Pixel-bypixel frequency distributions of the staining intensities were obtained and used for analysis. Exposure to halothane for either 6 or 24 h resulted in significant changes in the actin distribution pattern. Control fibroblasts were characterized by several different cellular shapes and actin distribution patterns. However, the majority of control cells were spindle-shaped and
Fig. 2. A: control neuroblastoma cell with neurites with numerous microspikes. The cell is stained deeply for actin. B: neuroblastoma cells that have been exposed to 100 ppm halothane in vitro for 6 h. The cell retained its main neurite but lost many of its smaller neurites and microspikes, x350. contained ill-organized filamentous strands of actin (Fig. 1A). When fibroblasts were exposed to halothane, many cells lost their spindle shape and the distribution pattern of actin in these cells was distinctly different from the control cells. Actin filaments were confined to the perinuclear area and a large portion of the cytoplasm was stained pale by the anti-actin antibody (Fig. 1B). This distribution pattern of actin appeared to be dose-dependent. Cultures exposed to higher hatothane concentration (1,000 ppm) had a greater proportion of cells with the abnormal pattern of actin distribution than cultures which had been exposed to 100 p p m halothane. Halothane also affected the distribution pattern of actin in neuroblastoma cells. Like fibroblasts, most neuroblastoma cells exposed to halothane had actin confined to the perinuclear area (Fig. 2B). Halothane also affected neurites of neuroblastoma cells. The majority of control neuroblastoma cells were characterized by long neurites with m a n y branches and numerous microspikes projecting from the cell body and neurite (Fig. 2A). Some
35 control neuroblastoma cells exhibited only microspikes which extended directly from the cell body with no neurites (Fig. 2A). On the other hand, neuroblastoma cells exposed to halothane exhibited characteristic morphological changes. Within 6 hours of halothane exposure, most neuroblastoma cells lost their microspikes, although they retained their neurites (Fig. 2B). Quantitative analysis of staining intensities provided additional support of a significant halothane effect on the distribution of intracellular actin. Pixel-by-pixel frequency distribution of the staining intensities has shown that the control fibroblasts had more deeply stained areas for actin than the cells exposed to 100 ppm halothane (Fig. 3). In the control cells, the mean gray-value of frequency distribution was 110.0 + 5.0. When cells wer e expo.sed to halothane for 24 h, there was a significant shift in frequency distribution toward lower staining intensity (mean value=150 + 7.1; t=-3.90, df=54, P=0.0003). The present study demonstrated that halothane induces the change in the distribution pattern of actin in cultured cells. The disappearance of filopodia-like microspikes in neuroblastoma cells following exposure to halothane confirms the similar observation previously made by Telser and Hinkley [10] in cells exposed to much higher levels of halothane (3,000-21,000 ppm). However, the lower concentrations of halothane used (100 and 1,000 ppm) in the present study did not inhibit neurite extension as reported by them. Since the maintenance and extension of neurite structure are dependent on both microfilaments and microtubules [1, 6, 8, 9], it was suggested that neurite response to halothane is related to an inability of microtubules and microfilaments to organize into functional complexes [4]. We did not examine the effect of halothane on microtubules. However, it has been shown that the concentrations of 3,000 ppm halothane which disrupted microfilaments did not affect microtubules [3-5]. At concentrations above 10,000 ppm, microtubules gradually appeared to decrease in number. According to the previous observation [4], the halothane concentration (100 or 1,000 ppm) used in the present study should not affect the microtubules. Furthermore, the present observation that halothane affected only the microspikes of neuroblastoma cells but not the neurites, suggests that low concentrations of halothane act primarily on actin and not on microtubules. The mechanism behind halothane's actions on actin warrants further study. Present results indicate that the disappearance of filopodia is associated with a disrupted distribution of actin. It is not known, however, whether such morphological changes are the result of depolymerization of actin or an inability to polymerize actin into
300 •
"•
/,
200 •
/
0
(J
o
\
100 "
0 0
50
100
150
200
250
Staining Intensity (Gray-scale value) Fig. 3. The frequencydistribution of staining intensity of fibroblasts. Cells were exposed to 1,000ppm of halothane in vitro for 24 h. Pixelby-pixel frequencydistribution of the staining intensities was obtained from cells stained with the anti-actin PAP method. To directlycompare the magnitudeof the change in the frequencydistribution, the values of cells exposed to halothane were normalized by expressing each as a percentage of the control values. When fibroblasts were exposed to halothane, a significant shift in frequency distribution toward lower staining intensityoccurred (white is assigned a gray-scalevalue of 256, whereas black is 0). functional units in the presence of halothane. Whatever the mechanism involved in halothane toxicity, the suppressive action of halothane on filopodia is reversible as shown in the previous study [10]. This reversible halothane action was also observed when synaptogenesis occurred in the dentate gyrus of rats with entorhinal lesions [14]. It was shown that the suppressive effect of halothane was most pronounced during the period of exposure. Termination of halothane exposure resulted in quick recovery of the synaptic population, Although the effects of halothane on neural outgrowth seem to be transient, the process of brain development involves a complex series of precisely timed episodes of outgrowth and dying back of neural population. Disruption of these events even transiently could cause permanent disorganization of neural structure and function. This work was supported by Grants 19-59 from the March of Dimes Birth Defects Foundation. 1 Daniel, M.E, Colchicine inhibition of nerve fiber formation in vitro, J. Cell Biol., 53 (1972) 164-176. 2 Glauert, A.M., Fixation, dehydration and embedding of biological specimens. In A.M. Glauert (Ed.), Practical Methods in Electron Microscopy, North-Holland, Amsterdam, 1980,pp. 14. 3 Hinkley, R.E. and Samson, F.E., Anesthetic-inducedtransformation of axonal microtubules,J. Cell Biol., 53 (1972)258-263.
36 4 Hinkley, R.E. and Telser, A.G., The effects of halothane on cultured mouse neuroblastoma cells. I. Inhibition of morphological differentiation, J. Cell Biol., 63 (1974) 531 540. 5 Nunn, J.F. and Allison, A.C., Effects of anesthetics on microtubular system. In B.B. Fink (Ed.), Cellular Biology and Toxicity of Anesthetics, Williams and Wilkins, Baltimore, 1972, pp. 138 146. 6 Prasad, K.N., Waymire, J.C. and Weiner, N., A further study on the morphology and biochemistry of X-ray and dibutyryl cyclic AMP-induced differentiated neuroblastoma cells in culture, Exp. Cell Res., 74 (1972) 110-114. 7 Quimby, K.L., Aschkenase, L.J., Bowman, R.E., Katz, J. and Chang, L.W., Enduring learning deficits and cerebral synaptic malformation from exposure to 10 ppm halothane, Science, 185 (1974) 625 627. 8 Roisen, F.J. and Murphy, R.A., Neurite development in vitro. II. The role of microfilaments and microtubules in dibutyryl adenosin 3,5-cyclic monophosphate and nerve growth factor stimulated maturation, J. Neurobiol., 4 (1973) 397M12. 9 Roisen, F.J. and Rebhum, L.I., The effects ofcolchicine on cellular and intracellular movement, Physiologist, 14 (1971) 220a.
10 Telser, A. and Hinkley, R.E., Cultured neuroblastoma cells and halothane: effects on cell growth and macromolecular synthesis, Anesthesiology, 46 (1977) 102 110. 11 Uemura, E. and Bowman, R.E., Effects of halothane on cerebral synaptic density, Exp. Neurol., 69 (1980) 135-142. 12 Uemura, E., Ireland, W.R, Levin, E.D. and Bowman, R.D., Effects of halothane on the development of rat brain: a Golgi study of dendritic growth, Exp. Neurol., 89 (1985) 503-519. 13 Uemura, E., Levin, E.D. and Bowman, R.E., Effects of halothane on synaptogenesis and learning behavior in rats, Exp. Neurol., 89 (1985) 520-529. 14 Uemura, E., Levin, E., DeLuna, R. and Bowman, E., Suppressive effects of halothane on reactive synaptogenesis in the dentate gyrus of rats, Brain Res., 496 (1989) 317--320. 15 Vandesande, F., Peroxidase-antiperoxidase techniques. In A.C. Cuello (Ed.), Immunohistochemistry, Wiley, New York, 1983, pp. 101 119. 16 Van Gansen, P., Pays, A. and Malherbe, L., Actin content and organization of microfilaments in primary cultures of mouse embryonic fibroblasts (in vivo ageing), Biol. Cell., 54 (1985) 251-260.
