TISSUE AND CELL, 1992 24 (3) 367-378 0 1992 Longman Group UK Ltd.
KATHLEEN J. DOANE*, FRED J. ROtSENt and FRANK J. WILSON*
THE EFFECTS OF NERVE GROWTH FACTOR AND DIBUTYRYL CYCLIC AMP ON CYTOSKELETAL DENSITIES IN CULTURED SENSORY GANGLIA Keywords: Nerve growth factor. dibutyryl cyclic AMP. microtubule. neurolilament. dorsal root ganglia ABSTRACT. The effects of nerve growth factor (NGF) and dibutyryl cyclic AMP (DBC) on the density of cytoskeletal structures in cultured dorsal root ganglia were examined using morphometric techniques. After 24 hr in culture, NGF-treated neurites were longer than either DBC-treated or control neurites. At 48 hr, neurites produced in response to NGF and DBC were of equivalent length, while controls were considerably shorter. Comparison of electron micrographs of neuritic profiles revealed some differences of area and cytoskeletal density between treatment groups. Morphometric analysis was used to determine these differences under several growth conditions, at various rates of elongation and at different neurite lengths As shown by analysis of variance, both NGF-treated and control neurites tapered in diameter at 48 hr in uirro, while DBC-induced neurites increased in area. An increase in cytoskeletal density for all treatment groups indicated that density was not always correlated with changes in area. An increased density of microtubules as compared to neurofllaments was seen at 24 hr. with equal densities of both cytoskeletal elements present after 48 hr in uitro. Comparisons between individual groups of data indicated that NGF-treated neurites relied primarily on microtubular density at 24 hr in vitro, when NGF induced longer, faster growing neurites. At 48 hr, there was an increase in neurofilaments proximal to the explant in the presence of DBC. implying that DBC may cause increased synthesis and/or transport of these structures. A comparison of microtubule to neurofilament ratios indicated that at 24 hr, there was always a greater density of microtubules. However, after 48 hr, neurofilament density increased such that there were equivalent densities of both cytoskeletal elements, possibly due to the overall increase in length observed in each treatment group. These data imply that 1) neurites with different rates of elongation may exhibit differences in cytoskeletal density; 2) neuritcs of equivalent lengths may be of differing stabilities; 3) NGF and DBC produce neurites with different cytoskeletal densities, implying divergent mechanisms of neurite induction; 4) the presence or absence of NGF may be partially responsible for variations in cytoskeletal densities observed between peripheral and central processes of DRG during development.
Introduction
sensory neurons and promotes neuritic developanent from sensory ganglia in vitro (LeviMontalcini, 1987). The cyclic AMP derivative,3’,5’-dibutyryl cyclic AMP (DBC), also promotes neurite outgrowth from cultured sensory ganglia (Roisen et al., 1972c), and produces neurites which appear morphologically similar to those induced by NGF (Roisen and Murphy, 1973). Furthermore, NGF and DBC stimulate neuritogenesis of the neural crest derived phqochromocytoma cell line PC12, which is often used as an in uitro system to study the effects of these growth promoters on cytoskeletal proteins (Black et al, 1986; Drubin et al., 1988; Doherty et al., 1987).
Nerve growth factor (NGF) is a polypeptide hormone which is important in the growth and maintenance of both sympathetic and * Department of Pathology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson (formerly Rutgers) Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854, USA. t Department of Anatomical Science’s and Neurobiology, University of Louisville Medical School, Louisville, Kentucky, USA. *Department of Neuroscience and Cell Biology (formerly Anatomy), University of Medicine and Dentistry of New Jersey, Robert Wood Johnson (formerly Rutgers) Medical School. Piscataway, New Jersey, USA. Correspondence to Kathleen J. Doane Received 12 July 1991. 367
DOANE ET AL.
368
The neuritic cytoskeleton, composed primarily of microtubules and neurofilaments, is important in determining the production and stability of neuronal circuitry. Microtubules are believed to be more plastic in function and play a key role in neurite elongation (Lasek, 1981; Lim etal., 1989), whereas neurofilaments appear more stable and may play a major role in neuronal maintenance and support (Lasek, 1981; Lim et al., 1989). Although differences in cytoskeletal density between embryonic and adult axons have been reported (Hoffman et al., 1985), as have differences in proximal and distal branches of dorsal root ganglia neurons (Smith, 1973; Zenker et al., 1973; Pannese et al., 1984)) and in regenerating nerve processes (Hoffman et al., 1984)) no study has examined the effects of growth promoting agents on cytoskeletal densities in sensory ganglia in vitro. Much of the work on the effect of NGF and DBC on the stability of the cytoskeleton has been done using the transformed cell line PC12. The purpose of the present study was to analyze the densities of microtubules and neurofilaments in neurites produced in response to NGF and DBC in organized explant cultures of embryonic chick dorsal root ganglia. These analyses provided information concerning the relative stability of neurites from primary DRG cultures under several growth conditions, at differing rates of elongation and at equivalent and different neurite lengths. Levels of growth promoting agents were chosen such that, at 48 hr in vitro, DBC and NGF induced neurites of equivalent length. Cytoskeletal densities in neurites proximal to the explant were compared to densities distal to the explant (close to the growth cone), to determine whether a proximodistal gradient of cytoskeletal elements existed along the neurite. An earlier time point was examined to analyze differences in cytoskeletal densities during neurite initiation. A preliminary report of this work has been published elsewhere (Doane et al., 1988). Methods Cell culture
Dorsal root ganglia from day 9 embryonic White Leghorn chicks were grown as explant cultures on coverslips coated with poly-Llysine (type IB, Sigma Chemical Co., St.
1600 1400 E
1200
e .o
1000
E .c
800
r, P
600
3
400
0
24 hr.
48 hr.
Fie. 1. Evaluation of neurite leneth. Data were analvzed u&g Student’s T test, anld are presented’ as mean? standard error of the mean. NGF induced slightly longer neurites at 24 hr (p c @05), while at 48 hr both growth promoters induced neurites of approximately equivalent length (p s 0.05).
Louis, MO: at a concentration of 1 mg/ml in 0.1 M borate buffer, pH 8.4). Cultures were fed with medium 199 (GIBCO Laboratories, Grand Island, NY) with 10% heat-inactivated fetal bovine serum and 50pg/ml gentamicin (Nutrient medium, NM) either as a control or following addition of 2.5 pg/ml NGF (Cohen, 1960) or 5 mM DBC (Roisen et al., 1972~). Cultures were incubated as double coverslip preparations in an atmosphere of 5% COz prior to being sealed in Maximow chambers and incubated at 37°C (Roisen et al., 1972a,b) for 24-48 hr. Evaluation of neurite length
Evaluation of DRG neurite outgrowth was accomplished with the aid of a Bio-Quant Image Analysis System to measure neuritic lengths from phase micrographs of explant DRG cultures. All data were analysed using Student’s T-test. Electron microscopy
DRG were grown on coverslips which had been carbon-coated, sterilized, and then coated with poly-L-lysine. Cells were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.3, for 1 hr at room temperature, and in 1% osmium tetroxide in the same buffer for 30 min at room temperature. Cells were stained en bloc in 5% aqueous
NEURITE GROWTH
AND CYTOSKELETAL
DENSITY
369
Fig. 2. Representative phase micrographs of cultured DRG grown on poly-L-lysine under varying growth conditions for 24 hr (A, B, C; x65) and 48 hr (D. E, F; x47). A and D) 5 mM DBC. B and E) NGF. C and F) Nutrient medium only.
many1 acetate for 20 mm, dehydrated in a series of graded ethanols, infiltrated and flatembedded in Epon-Araldite. Thin sections (pale gold on the interference scale) were placed on Formvar-coated slot grids, stained in 5% aqueous many1 acetate for 1 hr and examined with a Philips EM201 at 60 kV. Morphometry
Blocks were oriented such that the block face was at right angles to the neuritic growth.
Sections were taken from both proximal (close to the explant) and distal (close to the growth cone) portions of the neurites. These regions were kept the same in all analyses. Neuritic areas (cross-sectional) were determined from micrographs using a Bio-Quant Image Analysis System. The numbers of microtubules and neurofilaments were identified morphologically and counted, and the density of each of these structures was expressed as counts/unit area. A maximum of 50 neu-
DOANE ET AL.
370
Fig. 3. Comparison of ultrastructure observed in each treatment group at 24 hr in u&o. A, C, and E) proximal neurites; B, D. and F) distal neurites. (AB) DBC-treated neurites. (CD) NGF-treated neurites. (EF) Neurites grown in NM alone. ~32,000.
rites per block and at least three different blocks ner treatment grotto were examined to ensure that the data-would be of statistical significance (Weibel et al., 1966). A statistical program (SAS) was used to perform analysis of variance on all data. In addition, individual groups were analyzed using the standard error of the difference between means.
Results Rating of DRG and neurite elongation After 24 hr in vitro, neurites grown in the
presence of 2.5 ,ug/ml NGF were significantly longer than those produced in response to 5 mM DBC or NM alone (Fig. 1). Representative phase micrographs depicting this relationship are presented in Fig. 2 (A-C).
NEURITE GROWTH
AND CYTOSKELETAL
DENSITY
Fig. 4. Comparison of ultrastructure observed in each treatment group at 48 hr in uirro. A. C. and E) proximal neurites; B, D, and F) distal neurites. (A, B) DBC-treated neurites. (C. D) NGF-treated neurites. (E, F) Neurites grown in NM alone. ~24.000.
DBC and NM also induced fewer neurites than NGF (results not shown). At 48 hr, both DBC- and NGF-treated neurites were equivalent in length, while control neurites were substantially shorter (Fig. 1). The resultant neuritic development produced after 48 hr is shown in Fig. 2 (D-F). Ultrastructure
A comparison of the neuritic cytoskeletal components produced by each of the treat-
ments after 24 hr (Fig. 3) and 48 hr (Fig. 4) in culture revealed minimal differences between proximal (A, C, E) and distal neurites (B, D, F) within each treatment group. DBC-treated neurites appeared larger in diameter than NGF-treated or control neurites, and contained few cytoskeletal elements at 24 hr. (Fig. 3A, B). After 48 hr, DBC-induced neurites were varied in their cytoskeletal densities, with some larger diameter neurites containing sparse numbers of
312
DOANE ET AL.
microtubules and neurofilaments, and others densely packed with both cytoskeletal elements (Fig. 4A, B). NGF-treated neurites appeared to have a greater cytoskeletal density at 24 hr, when these neurites were much longer than DBC or control neurites (Fig. 3C, D). After 48 hr, this apparent difference in density was no longer observed (Fig. 4C, D). Control neurites contained few cytoskeletal elements; this difference was most apparent after 48 hr. (Fig. 3E, F; 4E, F). Control neurites were found frequently in association with nonneuronal cells and often were wrapped by several supporting cells (Figs. 3E, F; 4E, F). This phenomenon was infrequent when either growth promoting agent was employed. Morphometry
The three variables examined in this study, 1) growth condition (NGF, DBC, and NM), 2) time (24 and 48 hr in vitro), and 3) location along the length of the neurite (proximal to the explant, distal to the explant) were first subjected to the analysis of variance procedure to determine their significance in affecting neuritic area and the density of microtubules and neurofilaments. One-way analysis of variance indicated that location along the length of the neurite had no effect on cross-sectional area (p s F 0.254; Table 1) or cytoskeletal density (p s F 0.065; Table 1). One-way analysis of variance demonstrated that time significantly affected areas
Table 1. Means of variables obtained using oneway analysis of variance procedure. Location
proximal distal
Area 0.394 0.367
Density 66.61 74.68
Time 24 hr 48 hr
Area 0.414 0.347
Density 56.42 84.87
GC DBC NGF NM
Area 0.413 0.333 0.395
Density 74.71 77.51 59.71
Location was not a significant determinant of area. All other variables had a significant influence on either area (pm*) or density (count/pm*). GC: Growth condition.
(p s F O.OOS),with neurites grown for 24 hr having a greater cross-sectional area than those maintained for 48 hr (Table 1). Concomitant with the decrease in area at 48 hr was an increase in cytoskeletal density (p s F 0.0001; Table 1). Growth condition significantly influenced cross-sectional. area (p s F 0.016); DBC produced neurites with the greatest area, and NGF produced smaller processes (Table 1). Growth condition was a significant determinant of cytoskeletal density (p s F 0.0017): NGF produced neurites with slightly higher densities of cytoskeletal elements than DBC, and both agents induced greater densities of both microtubules and neurofilaments than controls (Table 1). Thus, smaller diameter neurites (NGFtreated) contain greater densities of cytoskeletal elements, while the other growth promoter, DBC, produced neurites much larger in area with only a slight decrease in cytoskeletal density. Control neurites, although intermediate in area, had the lowest cytoskeletal density. The only significant two-way interaction affecting both area and density was time in vitro* growth condition (p s F 0.0001 and 0+0518, respectively). Both control and NGFtreated neurites decreased in diameter at 48 hr, while the reverse was true for DBCinduced neurites (Fig. 5A). However, in all cases a greater density of cytoskeletal elements was observed after 48 hr (Fig. 5B). Thus, the tapering of neurites did not account for the increase in cytoskeletal density observed at 48 hr, and overall cytoskeletal density was not always correlated with changes in area. The density of both microtubules and neurofilaments varied with both growth condition (p Q F 0.054) and time (p s F 0.0042) as shown by two-way analysis of variance. While DBC-induced neurites contained approximately equal densities of both microtubules and neurofilaments, both NGFtreated and control neurites exhibited a greater density of microtubules than neurofilaments (Fig. 5C). At 24 hr, the overall density of microtubules was much greater than that of neurofilaments, while at 48 hr, approximately equivalent densities of both cytoskeletal elements were observed (Fig. 5D). This increase in neurofilament density at 48 hr may be due to the increased lengths of all neurites at this time point.
NEURITE
GROWTH
AND
Growth
Growth
CYTOSKELETAL
DENSITY
condltlon
173
Growth
condltlon
condltlon
Fig. 5. Significant means obtained during two-way analysis of variance proccdurc. (A) Effect of the two-way interaction of time*growth condttlon on area (pm?). Both NGF-induced and control neurites were greater in area at 24 hr as compared to 48 hr in U&-O. whereas DBCtreated neurites were greater in arca after 48 hr. (B) Effect of the two-wav interactlon of time*growth condition on cytotkeletal densities (count/pm’). Neurites grown 111oitro for 48 hr always contained a greater cytoskeletal density than those grown for only 24 hr. in all three treatment groups. (C) Effect of the two-way interaction of cytoskeletal element*growth condition on cytoskeletal densities (count/pm:). DBC-induced neurites contained approximateI> equivalent densities of both microtubules and neurolilaments, while both NGF-trcatcd and control neurites contained significantly greater microtubulc densities. (D) Effect of the tww way interaction of cytoskeletal element*time on cytoskeletal densities (count/urn?). At 24 hr. there was a much greater density of microtubules than ncurofilaments present in all trcatmcnt groups. while after 48 hr. both cytoskcletal elements were present at equal densltic. Prq. Bull.. 19. 7-32. Laaek. R. J.. Oblinger, M. M., Drake. P. F. 1983. The molecular biology of neuronal geometry: The expression 01 neurotilament genes influences axonal diameter. Cold Spring Harbor Symp. Quonr. Biol 48. 733-744. Levi-Montalcini, R. 1987. The nerve growth factor: thirty-five years later. EMBO J., 6, 1145-l 1.54. Lim. S.-S.. Sammak, P., Borisy, G. 1989. Progressive and spatially diffcrcntiatcd stability of microtuhulo in devclopmg neuronal cells. J. Cell Biol., 109, 253-263. Mizcl. S.. Bamburg. J. 1975. Studies on the action of nerve growth factor. II. Ncurotuhule protein lc\cl, dung neurltc outgrowth. Neurohio[. , 5, 283-290. Nadclhaft, I. 1974. Mtcrotubulc densities and total numhcrs in sclectcd axons of the crayfish ahdomlnal r~rvc cord J. Neurocyk~l., 3, 73-86. Och\, S.. Erdmann, J. Jcrsild, R., McAdoo, V. 1978. Routing of transported matcuals in the dorsal root and ncrvc tiher branches of the dorsal root ganglion. J. Newohio/.. 9, 465-481. of the densIt! ot Panncsc. E., Ledda, M., Arcidiacono. G.. Rigamonti, L., Procacci, P. lYX4. A comparison microtubules in the central and peripheral axonal branches of the pseudoumpolar neurons of lizard spinal gangha. Anal. Rec. 208, 595-605. Parhad. I. M.. Clark. A. W., Griffin, J. W. 1987. Effect of changes in neurolilament content on caliber of small axons: The o$‘-iminodipropionitrile model. J. Neurosci.. 7, 22.562268.
378
DOANE ET AL.
Peters;A., Vaughn, J. 1967. Microtubules and filaments in the axons and astrocytes of early postnatal rat optic nerves. J. Cell Biol., 32, 1X3-119. Roisen, F., Murphy, R., Braden, W. 1972a. Neurite development in vitro. I. The effects of adenosine 3’,5’-cyclic monophosphate (cyclic AMP). .I. Neurobiol., 3, 347-368. Roisen, F., Murphy, R., Pichichero, M., Braden, W. 1972b. Cyclic adenosine monophosphate stimulation of axonal elongation. Science, 175, 73-74. Roisen, F., Murphy, R., Braden, W. 1972~. Dibutyryl cyclic adenosine monophosphate stimulation of colcemidinhibited axonal elongation. Science, 177, 8G9-811. Roisen, F., Murphy, R. 1973. Neurite development in vitro. II. The role of microfilaments and microtubules in dibutyryl adenosine 3’,5’-cyclic monophosphate and nerve growth factor stimulated maturation. J. Neurobiol., 4, 397-412. Roisen, F., Braden, W., Friedman, J. 1975. Neurite development in uirro. III. The effects of several derivatives of cyclic AMP, colchicine, and colcemid. Ann. N. Y. Acad. Sci., 253, 545-561. Skaper, S., Varqn, S. 1981. Mutually independent cyclic AMP and sodium responses to nerve growth factor in embryonic chick dorsal root ganglia. .I. Neurochem., 37, 222-228. Smith, R. 1973. Microtubule and neurofilament densities in amphibian spinal root nerve fibers: Relationship to axoplasmic transport. Canadian .I. PhysioL Pharmcol., 51, 798-806. Weibel, E., Kistler, G., Scherle, W. 1966. Practical stereological methods for morphometric cytology. J. CelI Biol., 30,2%38. Zenker, W., Mayr, R., Gruber, H. 1973. Axoplasmic organelles: Quantitative differences between ventral and dorsal root fibres of the rat. Experienria, 29, 77-78.
KATHLEEN J. DOANE*, FRED J. ROtSENt and FRANK J. WILSON*
THE EFFECTS OF NERVE GROWTH FACTOR AND DIBUTYRYL CYCLIC AMP ON CYTOSKELETAL DENSITIES IN CULTURED SENSORY GANGLIA Keywords: Nerve growth factor. dibutyryl cyclic AMP. microtubule. neurolilament. dorsal root ganglia ABSTRACT. The effects of nerve growth factor (NGF) and dibutyryl cyclic AMP (DBC) on the density of cytoskeletal structures in cultured dorsal root ganglia were examined using morphometric techniques. After 24 hr in culture, NGF-treated neurites were longer than either DBC-treated or control neurites. At 48 hr, neurites produced in response to NGF and DBC were of equivalent length, while controls were considerably shorter. Comparison of electron micrographs of neuritic profiles revealed some differences of area and cytoskeletal density between treatment groups. Morphometric analysis was used to determine these differences under several growth conditions, at various rates of elongation and at different neurite lengths As shown by analysis of variance, both NGF-treated and control neurites tapered in diameter at 48 hr in uirro, while DBC-induced neurites increased in area. An increase in cytoskeletal density for all treatment groups indicated that density was not always correlated with changes in area. An increased density of microtubules as compared to neurofllaments was seen at 24 hr. with equal densities of both cytoskeletal elements present after 48 hr in uitro. Comparisons between individual groups of data indicated that NGF-treated neurites relied primarily on microtubular density at 24 hr in vitro, when NGF induced longer, faster growing neurites. At 48 hr, there was an increase in neurofilaments proximal to the explant in the presence of DBC. implying that DBC may cause increased synthesis and/or transport of these structures. A comparison of microtubule to neurofilament ratios indicated that at 24 hr, there was always a greater density of microtubules. However, after 48 hr, neurofilament density increased such that there were equivalent densities of both cytoskeletal elements, possibly due to the overall increase in length observed in each treatment group. These data imply that 1) neurites with different rates of elongation may exhibit differences in cytoskeletal density; 2) neuritcs of equivalent lengths may be of differing stabilities; 3) NGF and DBC produce neurites with different cytoskeletal densities, implying divergent mechanisms of neurite induction; 4) the presence or absence of NGF may be partially responsible for variations in cytoskeletal densities observed between peripheral and central processes of DRG during development.
Introduction
sensory neurons and promotes neuritic developanent from sensory ganglia in vitro (LeviMontalcini, 1987). The cyclic AMP derivative,3’,5’-dibutyryl cyclic AMP (DBC), also promotes neurite outgrowth from cultured sensory ganglia (Roisen et al., 1972c), and produces neurites which appear morphologically similar to those induced by NGF (Roisen and Murphy, 1973). Furthermore, NGF and DBC stimulate neuritogenesis of the neural crest derived phqochromocytoma cell line PC12, which is often used as an in uitro system to study the effects of these growth promoters on cytoskeletal proteins (Black et al, 1986; Drubin et al., 1988; Doherty et al., 1987).
Nerve growth factor (NGF) is a polypeptide hormone which is important in the growth and maintenance of both sympathetic and * Department of Pathology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson (formerly Rutgers) Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854, USA. t Department of Anatomical Science’s and Neurobiology, University of Louisville Medical School, Louisville, Kentucky, USA. *Department of Neuroscience and Cell Biology (formerly Anatomy), University of Medicine and Dentistry of New Jersey, Robert Wood Johnson (formerly Rutgers) Medical School. Piscataway, New Jersey, USA. Correspondence to Kathleen J. Doane Received 12 July 1991. 367
DOANE ET AL.
368
The neuritic cytoskeleton, composed primarily of microtubules and neurofilaments, is important in determining the production and stability of neuronal circuitry. Microtubules are believed to be more plastic in function and play a key role in neurite elongation (Lasek, 1981; Lim etal., 1989), whereas neurofilaments appear more stable and may play a major role in neuronal maintenance and support (Lasek, 1981; Lim et al., 1989). Although differences in cytoskeletal density between embryonic and adult axons have been reported (Hoffman et al., 1985), as have differences in proximal and distal branches of dorsal root ganglia neurons (Smith, 1973; Zenker et al., 1973; Pannese et al., 1984)) and in regenerating nerve processes (Hoffman et al., 1984)) no study has examined the effects of growth promoting agents on cytoskeletal densities in sensory ganglia in vitro. Much of the work on the effect of NGF and DBC on the stability of the cytoskeleton has been done using the transformed cell line PC12. The purpose of the present study was to analyze the densities of microtubules and neurofilaments in neurites produced in response to NGF and DBC in organized explant cultures of embryonic chick dorsal root ganglia. These analyses provided information concerning the relative stability of neurites from primary DRG cultures under several growth conditions, at differing rates of elongation and at equivalent and different neurite lengths. Levels of growth promoting agents were chosen such that, at 48 hr in vitro, DBC and NGF induced neurites of equivalent length. Cytoskeletal densities in neurites proximal to the explant were compared to densities distal to the explant (close to the growth cone), to determine whether a proximodistal gradient of cytoskeletal elements existed along the neurite. An earlier time point was examined to analyze differences in cytoskeletal densities during neurite initiation. A preliminary report of this work has been published elsewhere (Doane et al., 1988). Methods Cell culture
Dorsal root ganglia from day 9 embryonic White Leghorn chicks were grown as explant cultures on coverslips coated with poly-Llysine (type IB, Sigma Chemical Co., St.
1600 1400 E
1200
e .o
1000
E .c
800
r, P
600
3
400
0
24 hr.
48 hr.
Fie. 1. Evaluation of neurite leneth. Data were analvzed u&g Student’s T test, anld are presented’ as mean? standard error of the mean. NGF induced slightly longer neurites at 24 hr (p c @05), while at 48 hr both growth promoters induced neurites of approximately equivalent length (p s 0.05).
Louis, MO: at a concentration of 1 mg/ml in 0.1 M borate buffer, pH 8.4). Cultures were fed with medium 199 (GIBCO Laboratories, Grand Island, NY) with 10% heat-inactivated fetal bovine serum and 50pg/ml gentamicin (Nutrient medium, NM) either as a control or following addition of 2.5 pg/ml NGF (Cohen, 1960) or 5 mM DBC (Roisen et al., 1972~). Cultures were incubated as double coverslip preparations in an atmosphere of 5% COz prior to being sealed in Maximow chambers and incubated at 37°C (Roisen et al., 1972a,b) for 24-48 hr. Evaluation of neurite length
Evaluation of DRG neurite outgrowth was accomplished with the aid of a Bio-Quant Image Analysis System to measure neuritic lengths from phase micrographs of explant DRG cultures. All data were analysed using Student’s T-test. Electron microscopy
DRG were grown on coverslips which had been carbon-coated, sterilized, and then coated with poly-L-lysine. Cells were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.3, for 1 hr at room temperature, and in 1% osmium tetroxide in the same buffer for 30 min at room temperature. Cells were stained en bloc in 5% aqueous
NEURITE GROWTH
AND CYTOSKELETAL
DENSITY
369
Fig. 2. Representative phase micrographs of cultured DRG grown on poly-L-lysine under varying growth conditions for 24 hr (A, B, C; x65) and 48 hr (D. E, F; x47). A and D) 5 mM DBC. B and E) NGF. C and F) Nutrient medium only.
many1 acetate for 20 mm, dehydrated in a series of graded ethanols, infiltrated and flatembedded in Epon-Araldite. Thin sections (pale gold on the interference scale) were placed on Formvar-coated slot grids, stained in 5% aqueous many1 acetate for 1 hr and examined with a Philips EM201 at 60 kV. Morphometry
Blocks were oriented such that the block face was at right angles to the neuritic growth.
Sections were taken from both proximal (close to the explant) and distal (close to the growth cone) portions of the neurites. These regions were kept the same in all analyses. Neuritic areas (cross-sectional) were determined from micrographs using a Bio-Quant Image Analysis System. The numbers of microtubules and neurofilaments were identified morphologically and counted, and the density of each of these structures was expressed as counts/unit area. A maximum of 50 neu-
DOANE ET AL.
370
Fig. 3. Comparison of ultrastructure observed in each treatment group at 24 hr in u&o. A, C, and E) proximal neurites; B, D. and F) distal neurites. (AB) DBC-treated neurites. (CD) NGF-treated neurites. (EF) Neurites grown in NM alone. ~32,000.
rites per block and at least three different blocks ner treatment grotto were examined to ensure that the data-would be of statistical significance (Weibel et al., 1966). A statistical program (SAS) was used to perform analysis of variance on all data. In addition, individual groups were analyzed using the standard error of the difference between means.
Results Rating of DRG and neurite elongation After 24 hr in vitro, neurites grown in the
presence of 2.5 ,ug/ml NGF were significantly longer than those produced in response to 5 mM DBC or NM alone (Fig. 1). Representative phase micrographs depicting this relationship are presented in Fig. 2 (A-C).
NEURITE GROWTH
AND CYTOSKELETAL
DENSITY
Fig. 4. Comparison of ultrastructure observed in each treatment group at 48 hr in uirro. A. C. and E) proximal neurites; B, D, and F) distal neurites. (A, B) DBC-treated neurites. (C. D) NGF-treated neurites. (E, F) Neurites grown in NM alone. ~24.000.
DBC and NM also induced fewer neurites than NGF (results not shown). At 48 hr, both DBC- and NGF-treated neurites were equivalent in length, while control neurites were substantially shorter (Fig. 1). The resultant neuritic development produced after 48 hr is shown in Fig. 2 (D-F). Ultrastructure
A comparison of the neuritic cytoskeletal components produced by each of the treat-
ments after 24 hr (Fig. 3) and 48 hr (Fig. 4) in culture revealed minimal differences between proximal (A, C, E) and distal neurites (B, D, F) within each treatment group. DBC-treated neurites appeared larger in diameter than NGF-treated or control neurites, and contained few cytoskeletal elements at 24 hr. (Fig. 3A, B). After 48 hr, DBC-induced neurites were varied in their cytoskeletal densities, with some larger diameter neurites containing sparse numbers of
312
DOANE ET AL.
microtubules and neurofilaments, and others densely packed with both cytoskeletal elements (Fig. 4A, B). NGF-treated neurites appeared to have a greater cytoskeletal density at 24 hr, when these neurites were much longer than DBC or control neurites (Fig. 3C, D). After 48 hr, this apparent difference in density was no longer observed (Fig. 4C, D). Control neurites contained few cytoskeletal elements; this difference was most apparent after 48 hr. (Fig. 3E, F; 4E, F). Control neurites were found frequently in association with nonneuronal cells and often were wrapped by several supporting cells (Figs. 3E, F; 4E, F). This phenomenon was infrequent when either growth promoting agent was employed. Morphometry
The three variables examined in this study, 1) growth condition (NGF, DBC, and NM), 2) time (24 and 48 hr in vitro), and 3) location along the length of the neurite (proximal to the explant, distal to the explant) were first subjected to the analysis of variance procedure to determine their significance in affecting neuritic area and the density of microtubules and neurofilaments. One-way analysis of variance indicated that location along the length of the neurite had no effect on cross-sectional area (p s F 0.254; Table 1) or cytoskeletal density (p s F 0.065; Table 1). One-way analysis of variance demonstrated that time significantly affected areas
Table 1. Means of variables obtained using oneway analysis of variance procedure. Location
proximal distal
Area 0.394 0.367
Density 66.61 74.68
Time 24 hr 48 hr
Area 0.414 0.347
Density 56.42 84.87
GC DBC NGF NM
Area 0.413 0.333 0.395
Density 74.71 77.51 59.71
Location was not a significant determinant of area. All other variables had a significant influence on either area (pm*) or density (count/pm*). GC: Growth condition.
(p s F O.OOS),with neurites grown for 24 hr having a greater cross-sectional area than those maintained for 48 hr (Table 1). Concomitant with the decrease in area at 48 hr was an increase in cytoskeletal density (p s F 0.0001; Table 1). Growth condition significantly influenced cross-sectional. area (p s F 0.016); DBC produced neurites with the greatest area, and NGF produced smaller processes (Table 1). Growth condition was a significant determinant of cytoskeletal density (p s F 0.0017): NGF produced neurites with slightly higher densities of cytoskeletal elements than DBC, and both agents induced greater densities of both microtubules and neurofilaments than controls (Table 1). Thus, smaller diameter neurites (NGFtreated) contain greater densities of cytoskeletal elements, while the other growth promoter, DBC, produced neurites much larger in area with only a slight decrease in cytoskeletal density. Control neurites, although intermediate in area, had the lowest cytoskeletal density. The only significant two-way interaction affecting both area and density was time in vitro* growth condition (p s F 0.0001 and 0+0518, respectively). Both control and NGFtreated neurites decreased in diameter at 48 hr, while the reverse was true for DBCinduced neurites (Fig. 5A). However, in all cases a greater density of cytoskeletal elements was observed after 48 hr (Fig. 5B). Thus, the tapering of neurites did not account for the increase in cytoskeletal density observed at 48 hr, and overall cytoskeletal density was not always correlated with changes in area. The density of both microtubules and neurofilaments varied with both growth condition (p Q F 0.054) and time (p s F 0.0042) as shown by two-way analysis of variance. While DBC-induced neurites contained approximately equal densities of both microtubules and neurofilaments, both NGFtreated and control neurites exhibited a greater density of microtubules than neurofilaments (Fig. 5C). At 24 hr, the overall density of microtubules was much greater than that of neurofilaments, while at 48 hr, approximately equivalent densities of both cytoskeletal elements were observed (Fig. 5D). This increase in neurofilament density at 48 hr may be due to the increased lengths of all neurites at this time point.
NEURITE
GROWTH
AND
Growth
Growth
CYTOSKELETAL
DENSITY
condltlon
173
Growth
condltlon
condltlon
Fig. 5. Significant means obtained during two-way analysis of variance proccdurc. (A) Effect of the two-way interaction of time*growth condttlon on area (pm?). Both NGF-induced and control neurites were greater in area at 24 hr as compared to 48 hr in U&-O. whereas DBCtreated neurites were greater in arca after 48 hr. (B) Effect of the two-wav interactlon of time*growth condition on cytotkeletal densities (count/pm’). Neurites grown 111oitro for 48 hr always contained a greater cytoskeletal density than those grown for only 24 hr. in all three treatment groups. (C) Effect of the two-way interaction of cytoskeletal element*growth condition on cytoskeletal densities (count/pm:). DBC-induced neurites contained approximateI> equivalent densities of both microtubules and neurolilaments, while both NGF-trcatcd and control neurites contained significantly greater microtubulc densities. (D) Effect of the tww way interaction of cytoskeletal element*time on cytoskeletal densities (count/urn?). At 24 hr. there was a much greater density of microtubules than ncurofilaments present in all trcatmcnt groups. while after 48 hr. both cytoskcletal elements were present at equal densltic. Prq. Bull.. 19. 7-32. Laaek. R. J.. Oblinger, M. M., Drake. P. F. 1983. The molecular biology of neuronal geometry: The expression 01 neurotilament genes influences axonal diameter. Cold Spring Harbor Symp. Quonr. Biol 48. 733-744. Levi-Montalcini, R. 1987. The nerve growth factor: thirty-five years later. EMBO J., 6, 1145-l 1.54. Lim. S.-S.. Sammak, P., Borisy, G. 1989. Progressive and spatially diffcrcntiatcd stability of microtuhulo in devclopmg neuronal cells. J. Cell Biol., 109, 253-263. Mizcl. S.. Bamburg. J. 1975. Studies on the action of nerve growth factor. II. Ncurotuhule protein lc\cl, dung neurltc outgrowth. Neurohio[. , 5, 283-290. Nadclhaft, I. 1974. Mtcrotubulc densities and total numhcrs in sclectcd axons of the crayfish ahdomlnal r~rvc cord J. Neurocyk~l., 3, 73-86. Och\, S.. Erdmann, J. Jcrsild, R., McAdoo, V. 1978. Routing of transported matcuals in the dorsal root and ncrvc tiher branches of the dorsal root ganglion. J. Newohio/.. 9, 465-481. of the densIt! ot Panncsc. E., Ledda, M., Arcidiacono. G.. Rigamonti, L., Procacci, P. lYX4. A comparison microtubules in the central and peripheral axonal branches of the pseudoumpolar neurons of lizard spinal gangha. Anal. Rec. 208, 595-605. Parhad. I. M.. Clark. A. W., Griffin, J. W. 1987. Effect of changes in neurolilament content on caliber of small axons: The o$‘-iminodipropionitrile model. J. Neurosci.. 7, 22.562268.
378
DOANE ET AL.
Peters;A., Vaughn, J. 1967. Microtubules and filaments in the axons and astrocytes of early postnatal rat optic nerves. J. Cell Biol., 32, 1X3-119. Roisen, F., Murphy, R., Braden, W. 1972a. Neurite development in vitro. I. The effects of adenosine 3’,5’-cyclic monophosphate (cyclic AMP). .I. Neurobiol., 3, 347-368. Roisen, F., Murphy, R., Pichichero, M., Braden, W. 1972b. Cyclic adenosine monophosphate stimulation of axonal elongation. Science, 175, 73-74. Roisen, F., Murphy, R., Braden, W. 1972~. Dibutyryl cyclic adenosine monophosphate stimulation of colcemidinhibited axonal elongation. Science, 177, 8G9-811. Roisen, F., Murphy, R. 1973. Neurite development in vitro. II. The role of microfilaments and microtubules in dibutyryl adenosine 3’,5’-cyclic monophosphate and nerve growth factor stimulated maturation. J. Neurobiol., 4, 397-412. Roisen, F., Braden, W., Friedman, J. 1975. Neurite development in uirro. III. The effects of several derivatives of cyclic AMP, colchicine, and colcemid. Ann. N. Y. Acad. Sci., 253, 545-561. Skaper, S., Varqn, S. 1981. Mutually independent cyclic AMP and sodium responses to nerve growth factor in embryonic chick dorsal root ganglia. .I. Neurochem., 37, 222-228. Smith, R. 1973. Microtubule and neurofilament densities in amphibian spinal root nerve fibers: Relationship to axoplasmic transport. Canadian .I. PhysioL Pharmcol., 51, 798-806. Weibel, E., Kistler, G., Scherle, W. 1966. Practical stereological methods for morphometric cytology. J. CelI Biol., 30,2%38. Zenker, W., Mayr, R., Gruber, H. 1973. Axoplasmic organelles: Quantitative differences between ventral and dorsal root fibres of the rat. Experienria, 29, 77-78.
