EXPERIMENTAL
Long-Term
65, 293-300 (1979)
NEUROLOGY
Culture
of Adult Mammalian System Neurons
ALBEE Division
of Neuropathology
MESSING
AND SEUNG
Central
Nervous
U. KIM’
(Department of Pathology), University of Pennsylvania Philadelphia, Pennsylvania 19104
School
of Medicine. Received
January
10. 1979; revision
receilned
March
16, 1979
Retinas from five adult dogs (1 to 16 years old) were obtained at autopsy within 1 h of death and maintained in vitro as explants for as long as 42 days. Electron microscopy at 8 days in vitro showed normal neurons with intact synapses, glial cells, and photoreceptor cells lacking outer segments. Bodian staining at 21 to 42 days in vitro showed continued presence of viable neurons with extensive regeneration of processes. The availability of adult mammalian central nervous system neurons in culture should prove useful in studies on aging changes in neurons, particularly with respect to their regenerative ability.
INTRODUCTION Survival of neurons in tissue culture has traditionally depended on the use of fetal or neonatal animals as a source of tissue. Attempts to maintain adult mammalian neural tissue in culture have met with variable success (3). Although several groups have now cultured adult peripheral nervous system neurons (14, 16, 18), reports of successful culture of adult central nervous system neurons have been much less convincing (2, 5, 6, 8). In many cases, published photographs are of glial or fibroblastic cells, and no electron microscopy was carried out. We now describe the unequivocal in vitro survival of neurons from the adult mammalian central nervous system in explant cultures of adult dog retina, and provide light and electron microscopic evidence for the neuronal identity of these cells. Abbreviation: DIV-days in vitro. * This work was supported by grants NS-10648, NS-05572. K04 NS-00151, and GM-02051 from the U.S. Public Health Service.
293 00144886/79/080293-08$02.00/O Copyright Q 1979 by Academic Press, Inc. All rights of reproduction in any form reserved
294
MESSINGANDKIM
MATERIAL
AND METHODS
Eyes from five l- to 16-year-old dogs undergoing euthanasia at the University of Pennsylvania Veterinary Hospital were obtained within 60 min of death. The globes were repeatedly doused with 70% alcohol, cleaned of excess muscle and connective tissue, and washed again with 70% alcohol. A coronal section of the globe was made about 0.5 cm caudal to the limbus, and the remaining vitreous was removed from the caudal half of the globe. Using watchmaker forceps, the retina was peeled off the underlying pigment epithelium, severed at the optic disc, and placed in sterile Hanks’ balanced salt solution. The retina was minced into 2-mm2 pieces with razor blades. Three or four such pieces were placed on each collagen-coated (l), 11 x 22-mm glass or Aclar (Allied Chemical) coverslip, and allowed to settle overnight at 37°C with a few drops of feeding medium. Often the explants required another 24 h to firmly adhere to the substrate. The feeding medium consisted of 80% Eagle’s minimal essential medium, 10% horse serum (GIBCO), 10% fetal calf serum (GIBCO), 9 mg/ml glucose, 100 units/ml penicillin, and 100 pg/rnl streptomycin. The following day the cultures were placed in roller tubes, fed an additional 0.8 ml feeding medium, and put in a roller drum at one revolution every 5 min. Cultures were maintained at 37°C and refed weekly. At various times cultures were processed for Bodian staining according to Kim (11) or for electron microscopy as previously described (13). RESULTS The canine retina contains the same cellular elements as in other vertebrates (4,17), namely, rod and cone photoreceptors, bipolar neurons, horizontal cells, amacrine cells, ganglion cells, and Mtiller glial cells. During the 1st week of culture, the explants retained some degree of histiotypic organization, with clearly defined photoreceptor, inner nuclear, inner plexiform, and ganglion cell layers. This simplified the identification of cell types in electron micrographs. An electron micrograph of a large neuron from the ganglion cell layer is shown in Fig. 1. This neuron contains an indented, eccentric nucleus with a large reticular nucleolus, rough endoplasmic reticulum, a prominent Golgi complex, and many mitochondria. A section from the inner plexiform layer shows normal synapses between as yet unidentified cells, as well as some degenerating synapses (Fig. 2). Electron microscopic observation of the photoreceptor cell layer showed densely packed cells with spherical nuclei, condensed chromatin, and scanty cytoplasm at the level of this section (Fig. 3). Two cells were sectioned through their inner segments, as evidenced by the abundant mitochondria (4). Connecting cilia and zona adherens were also
ADULT
CNS NEURONS
IN CULTURE
295
FIG. 1. Electron micrograph of large neuron from ganglion cell layer of retina: l-year-old dog, 8 daysin vitro. Nucleus (Nu), rough endoplasmic reticulum (ER), and mitochrondria(M). x3600 Inset-Golgi complex (GC) and mitochondria from another part of the same neuron. x 6750.
seen. In contrast to reports of other investigators on the development of neonatal rat retina in vitro (7, 15), the photoreceptor cells in our cultures did not form rosettes. In addition, no outer segments were observed in any of our cultures.
FIG. 2. Retina of l-year-old synapsing on a process running Many other synapses (S) are indicated. x7200. Inset-one
dog, 8 days in vitro. Numerous presynaptic elements (S) diagonally across the field. Possible ribbon synapse (arrow). also seen. Degenerating presynaptic elements (D) are also of the synapses shown at higher magnification. x 13,500.
296
MESSING
AND KIM
FIG. 3. Electron micrograph of photoreceptor cells in l-year-old dog, 8 days in vitro. Inner segments (IS) with mitochondria, connecting cilia (C), and zona adherens (Z) between presumed glial processes and photoreceptor cells. x3600. Inset-enlargement of two connecting cilia and a zona adherens. x6750.
Bodian staining was carried out on cultures after 21 to 42 days in vitro (DIV), when much of the histiotypic pattern evident at 8 DIV had disappeared, due to spreading of the explant and loss of degenerating cells. Nevertheless, numerous neurons were still present, and there was
FIG. 4. Bodian stain of retina of l-year-old dog, 42 days in vitro showing a large multipolar neuron. x 100.
ADULT
CNS
NEURONS
IN CULTURE
297
FIG. 5. Higher magnification of same neuron as in Fig. 4. x400.
FIG. 6. Bodian stain of retina of l-year-old dog, 42 days in vitro showing another large multipolar neuron (N) in center with dendrites out of focus. Processes from other neurons traverse the field. A bouton terminal (arrow) is synapsing on small neuron which is out of focus. x400.
298
MESSING
AND KIM
FIG. 7. Bodian stain of retina of 16-year-old dog, 21 days in vitro showing intermediate-size mutlipolar neuron. x600.
extensive regeneration of processes. In the periphery of a representative explant fixed at 42 DIV, Bodian staining showed numerous fibroblastic, glial, and smaller photoreceptor cell nuclei surrounding one large multipolar neuron with six major processes (Fig. 4). At higher magnification (Fig. S), distinct neurofibrils were seen coursing throughout the perikaryon and into the processes. Most cell bodies in the center of the explant were completely obscured by abundant photoreceptor cell nuclei. In the outgrowth region of the explants, however, numerous silver stained processes traversed the fields of view (Fig. 6). In addition, small ring-like structures typical of bouton terminals (IO, 12, 19) were seen, e.g., next to the large neuron in Fig. 6. In the absence of normal retinal layers to aid in the identification of these cells, the largest neurons in the Bodian-stained explants were considered ganglion cells. Smaller multipolar (Fig. 7) and bipolar (Fig. 8) neurons were also maintained; these were tentatively identified as the local circuit neurons of the retina, the horizontal, amacrine, or bipolar neurons. A similar attempt at identifying cell types in explant cultures of avian retina was made by Kim (12). DISCUSSION We demonstrated that retinal neurons obtained from adult dogs within 1 h of death can be maintained in vitro for at least 42 days. Light microscopy
ADULT
FIG. 8. Bodian x600.
stain of retina
CNS
NEURONS
of 16-year-old
299
IN CULTURE
dog, 2 1 days in vitro
showing
a bipolar
neuron.
of Bodian silver-stained cells with distinct neurofibrils, electron microscopy of cells with nuclei containing dispersed chromatin and large nucleoli, and the presence of both pre- and postsynaptic structures in the neuropil, provided unequivocal evidence that there were viable neurons in these cultures. We encountered considerable variation between explants in the numbers of neurons surviving, and these data were not rigorously quantified. In general, these explants adhered poorly to the collagen substratum compared to fetal or neonatal tissue. However, of the explants which attached to the collagen and which remained viable during the first 3 to 4 days in culture, at least half (approximately 20% of the original explants) contained several to hundreds of neurons each after 6 weeks in culture. Many previous attempts at culturing neurons of the adult central nervous system utilized cerebral or cerebellar cortex. However, the retina may be more suitable for explant culture than other regions of the central nervous system. Because the retina is so thin, the dissection and trimming of retinal tissue to explant size may involve a minimal disruption of intercellular relationships, thus giving the neurons time to adapt to the culture environment. Preliminary work in our laboratory with dissociated cultures of adult dog retina has not been successful. We regard explant culture of adult dog retina as a potentially useful system in which to study aging changes in neurons, particularly the regenerative ability of central nervous system neurons and the intracellular
300
MESSING
AND KIM
accumulation of aging pigments such as lipofuscin (14). In addition, we now have access in culture to pathologic neurons from dogs with adult-onset neurological diseases. The photoreceptor cells in our cultures did not maintain their outer segments, perhaps due to the absence of the pigment epithelium (9). We are currently attempting to coculture those two tissues to alleviate this problem, and to provide a more suitable model for the study of photoreceptor cell function and degeneration in the adult animal. REFERENCES 1. BORNSTEIN, M. B. 1958. Reconstituted rat-tail collagen used as substrate for tissue cultures on coverslips. Lab. Invest. 7: 134-137. 2. COSTERO, I., AND C. M. POMERAT. 1951. Cultivation of neurons from the adult human cerebral and cerebellar cortex. Am. J. Anat. 89: 405-467. 3. GRAIN, S. M. 1966. Development of “organotypic” bioelectric activities in central nervous tissues during maturation in culture. Znt. Rev. Neurobiol. 9: l-43. 4. DOWLING, J. 1970. Organization of vertebrate retinas. Invest. Oprhalmol. 9: 655-680. 5. FRANCOIS, J., M. T. MATTON-VAN LEUVEN, J. AGOSTINI NETTO, AND M. R. VERSCHRAEGEN-SPAE. 197 1. Tissue culture studies ofthe adult pig retina. Oprhal. Res. 2: 25-36. 6. GEIGER, R. S. 1958. Subcultures of adult mammalian brain cortex in vitro. Exp. Cell Res. 14: 541-566. 7. HILD, W., AND G. CALLAS. 1967. The behavior of retinal tissue in vitro, light and electron microscopic observations. Z. Zellforsch. 80: l-21. 8. HOGUE, M. J. 1953. A study of adult human brain cells grown in tissue cultures. Am. J. Anat.
93: 397-427.
9. HOLLYFIELD, J. G., ANDP. WITKOVSKY. 1974. Pigmentedretinalepitheliuminvolvement in photoreceptor development and function. J. Exp. Zool. 189: 357-378. 10. KIM, S. U. 1965. Neurons in tissue culture. Observations on terminal boutons in cultures of mammalian central nervous tissue. Arch. Histol. Jap. 25: 371-386. 11. KIM, S. U. 1970. Observations on cerebellar cells in vitro. A silver and electron microscopic study. Z. Zellforsch. 107: 454-465. 12. KIM, S. U. 1971. Neuronal types in long-term culture of avian retina. Experientia 27: 1319- 1320. 13. KIM, S. U. 1975. Brain hypoxia studied in mouse central nervous system cultures: I. Sequential cellular changes. Lab. Invest. 33: 658-669. 14. KIM, S. U., K. G. WARREN, AND M. KALIA. 1979. Tissue culture ofadult human neurons, Neurosci. Letf. 11: 137-141. 15. LAVAIL, M. M., AND W. HILD. 1971. Histiotypic organization ofthe rat retinain vitro.Z. Zel(forsch.
114: 557-579.
16. MURRAY, M. R., AND A. P. STOUT. 1947. Adult human sympathetic ganglion cells cultivated in vitro. Am. J. Anat. 80: 225-273. 17. PARRY, H. B. 1953. Degenerations of the dog retina: I. Structure and development of the retina of the normal dog. Br. J. Oprhalmol. 37: 385-404. 18. SCOTT, B. S. 1977. Adult mouse dorsal root ganglia neurons in cell culture. J. Neurobiol. 8: 417-427. 19. WOLF, M. K. 1964. Differentiation of neuronal types and synapses in myelinatingcultures of mouse cerebellum. J. Cell Biol. 22: 259-279.
Long-Term
65, 293-300 (1979)
NEUROLOGY
Culture
of Adult Mammalian System Neurons
ALBEE Division
of Neuropathology
MESSING
AND SEUNG
Central
Nervous
U. KIM’
(Department of Pathology), University of Pennsylvania Philadelphia, Pennsylvania 19104
School
of Medicine. Received
January
10. 1979; revision
receilned
March
16, 1979
Retinas from five adult dogs (1 to 16 years old) were obtained at autopsy within 1 h of death and maintained in vitro as explants for as long as 42 days. Electron microscopy at 8 days in vitro showed normal neurons with intact synapses, glial cells, and photoreceptor cells lacking outer segments. Bodian staining at 21 to 42 days in vitro showed continued presence of viable neurons with extensive regeneration of processes. The availability of adult mammalian central nervous system neurons in culture should prove useful in studies on aging changes in neurons, particularly with respect to their regenerative ability.
INTRODUCTION Survival of neurons in tissue culture has traditionally depended on the use of fetal or neonatal animals as a source of tissue. Attempts to maintain adult mammalian neural tissue in culture have met with variable success (3). Although several groups have now cultured adult peripheral nervous system neurons (14, 16, 18), reports of successful culture of adult central nervous system neurons have been much less convincing (2, 5, 6, 8). In many cases, published photographs are of glial or fibroblastic cells, and no electron microscopy was carried out. We now describe the unequivocal in vitro survival of neurons from the adult mammalian central nervous system in explant cultures of adult dog retina, and provide light and electron microscopic evidence for the neuronal identity of these cells. Abbreviation: DIV-days in vitro. * This work was supported by grants NS-10648, NS-05572. K04 NS-00151, and GM-02051 from the U.S. Public Health Service.
293 00144886/79/080293-08$02.00/O Copyright Q 1979 by Academic Press, Inc. All rights of reproduction in any form reserved
294
MESSINGANDKIM
MATERIAL
AND METHODS
Eyes from five l- to 16-year-old dogs undergoing euthanasia at the University of Pennsylvania Veterinary Hospital were obtained within 60 min of death. The globes were repeatedly doused with 70% alcohol, cleaned of excess muscle and connective tissue, and washed again with 70% alcohol. A coronal section of the globe was made about 0.5 cm caudal to the limbus, and the remaining vitreous was removed from the caudal half of the globe. Using watchmaker forceps, the retina was peeled off the underlying pigment epithelium, severed at the optic disc, and placed in sterile Hanks’ balanced salt solution. The retina was minced into 2-mm2 pieces with razor blades. Three or four such pieces were placed on each collagen-coated (l), 11 x 22-mm glass or Aclar (Allied Chemical) coverslip, and allowed to settle overnight at 37°C with a few drops of feeding medium. Often the explants required another 24 h to firmly adhere to the substrate. The feeding medium consisted of 80% Eagle’s minimal essential medium, 10% horse serum (GIBCO), 10% fetal calf serum (GIBCO), 9 mg/ml glucose, 100 units/ml penicillin, and 100 pg/rnl streptomycin. The following day the cultures were placed in roller tubes, fed an additional 0.8 ml feeding medium, and put in a roller drum at one revolution every 5 min. Cultures were maintained at 37°C and refed weekly. At various times cultures were processed for Bodian staining according to Kim (11) or for electron microscopy as previously described (13). RESULTS The canine retina contains the same cellular elements as in other vertebrates (4,17), namely, rod and cone photoreceptors, bipolar neurons, horizontal cells, amacrine cells, ganglion cells, and Mtiller glial cells. During the 1st week of culture, the explants retained some degree of histiotypic organization, with clearly defined photoreceptor, inner nuclear, inner plexiform, and ganglion cell layers. This simplified the identification of cell types in electron micrographs. An electron micrograph of a large neuron from the ganglion cell layer is shown in Fig. 1. This neuron contains an indented, eccentric nucleus with a large reticular nucleolus, rough endoplasmic reticulum, a prominent Golgi complex, and many mitochondria. A section from the inner plexiform layer shows normal synapses between as yet unidentified cells, as well as some degenerating synapses (Fig. 2). Electron microscopic observation of the photoreceptor cell layer showed densely packed cells with spherical nuclei, condensed chromatin, and scanty cytoplasm at the level of this section (Fig. 3). Two cells were sectioned through their inner segments, as evidenced by the abundant mitochondria (4). Connecting cilia and zona adherens were also
ADULT
CNS NEURONS
IN CULTURE
295
FIG. 1. Electron micrograph of large neuron from ganglion cell layer of retina: l-year-old dog, 8 daysin vitro. Nucleus (Nu), rough endoplasmic reticulum (ER), and mitochrondria(M). x3600 Inset-Golgi complex (GC) and mitochondria from another part of the same neuron. x 6750.
seen. In contrast to reports of other investigators on the development of neonatal rat retina in vitro (7, 15), the photoreceptor cells in our cultures did not form rosettes. In addition, no outer segments were observed in any of our cultures.
FIG. 2. Retina of l-year-old synapsing on a process running Many other synapses (S) are indicated. x7200. Inset-one
dog, 8 days in vitro. Numerous presynaptic elements (S) diagonally across the field. Possible ribbon synapse (arrow). also seen. Degenerating presynaptic elements (D) are also of the synapses shown at higher magnification. x 13,500.
296
MESSING
AND KIM
FIG. 3. Electron micrograph of photoreceptor cells in l-year-old dog, 8 days in vitro. Inner segments (IS) with mitochondria, connecting cilia (C), and zona adherens (Z) between presumed glial processes and photoreceptor cells. x3600. Inset-enlargement of two connecting cilia and a zona adherens. x6750.
Bodian staining was carried out on cultures after 21 to 42 days in vitro (DIV), when much of the histiotypic pattern evident at 8 DIV had disappeared, due to spreading of the explant and loss of degenerating cells. Nevertheless, numerous neurons were still present, and there was
FIG. 4. Bodian stain of retina of l-year-old dog, 42 days in vitro showing a large multipolar neuron. x 100.
ADULT
CNS
NEURONS
IN CULTURE
297
FIG. 5. Higher magnification of same neuron as in Fig. 4. x400.
FIG. 6. Bodian stain of retina of l-year-old dog, 42 days in vitro showing another large multipolar neuron (N) in center with dendrites out of focus. Processes from other neurons traverse the field. A bouton terminal (arrow) is synapsing on small neuron which is out of focus. x400.
298
MESSING
AND KIM
FIG. 7. Bodian stain of retina of 16-year-old dog, 21 days in vitro showing intermediate-size mutlipolar neuron. x600.
extensive regeneration of processes. In the periphery of a representative explant fixed at 42 DIV, Bodian staining showed numerous fibroblastic, glial, and smaller photoreceptor cell nuclei surrounding one large multipolar neuron with six major processes (Fig. 4). At higher magnification (Fig. S), distinct neurofibrils were seen coursing throughout the perikaryon and into the processes. Most cell bodies in the center of the explant were completely obscured by abundant photoreceptor cell nuclei. In the outgrowth region of the explants, however, numerous silver stained processes traversed the fields of view (Fig. 6). In addition, small ring-like structures typical of bouton terminals (IO, 12, 19) were seen, e.g., next to the large neuron in Fig. 6. In the absence of normal retinal layers to aid in the identification of these cells, the largest neurons in the Bodian-stained explants were considered ganglion cells. Smaller multipolar (Fig. 7) and bipolar (Fig. 8) neurons were also maintained; these were tentatively identified as the local circuit neurons of the retina, the horizontal, amacrine, or bipolar neurons. A similar attempt at identifying cell types in explant cultures of avian retina was made by Kim (12). DISCUSSION We demonstrated that retinal neurons obtained from adult dogs within 1 h of death can be maintained in vitro for at least 42 days. Light microscopy
ADULT
FIG. 8. Bodian x600.
stain of retina
CNS
NEURONS
of 16-year-old
299
IN CULTURE
dog, 2 1 days in vitro
showing
a bipolar
neuron.
of Bodian silver-stained cells with distinct neurofibrils, electron microscopy of cells with nuclei containing dispersed chromatin and large nucleoli, and the presence of both pre- and postsynaptic structures in the neuropil, provided unequivocal evidence that there were viable neurons in these cultures. We encountered considerable variation between explants in the numbers of neurons surviving, and these data were not rigorously quantified. In general, these explants adhered poorly to the collagen substratum compared to fetal or neonatal tissue. However, of the explants which attached to the collagen and which remained viable during the first 3 to 4 days in culture, at least half (approximately 20% of the original explants) contained several to hundreds of neurons each after 6 weeks in culture. Many previous attempts at culturing neurons of the adult central nervous system utilized cerebral or cerebellar cortex. However, the retina may be more suitable for explant culture than other regions of the central nervous system. Because the retina is so thin, the dissection and trimming of retinal tissue to explant size may involve a minimal disruption of intercellular relationships, thus giving the neurons time to adapt to the culture environment. Preliminary work in our laboratory with dissociated cultures of adult dog retina has not been successful. We regard explant culture of adult dog retina as a potentially useful system in which to study aging changes in neurons, particularly the regenerative ability of central nervous system neurons and the intracellular
300
MESSING
AND KIM
accumulation of aging pigments such as lipofuscin (14). In addition, we now have access in culture to pathologic neurons from dogs with adult-onset neurological diseases. The photoreceptor cells in our cultures did not maintain their outer segments, perhaps due to the absence of the pigment epithelium (9). We are currently attempting to coculture those two tissues to alleviate this problem, and to provide a more suitable model for the study of photoreceptor cell function and degeneration in the adult animal. REFERENCES 1. BORNSTEIN, M. B. 1958. Reconstituted rat-tail collagen used as substrate for tissue cultures on coverslips. Lab. Invest. 7: 134-137. 2. COSTERO, I., AND C. M. POMERAT. 1951. Cultivation of neurons from the adult human cerebral and cerebellar cortex. Am. J. Anat. 89: 405-467. 3. GRAIN, S. M. 1966. Development of “organotypic” bioelectric activities in central nervous tissues during maturation in culture. Znt. Rev. Neurobiol. 9: l-43. 4. DOWLING, J. 1970. Organization of vertebrate retinas. Invest. Oprhalmol. 9: 655-680. 5. FRANCOIS, J., M. T. MATTON-VAN LEUVEN, J. AGOSTINI NETTO, AND M. R. VERSCHRAEGEN-SPAE. 197 1. Tissue culture studies ofthe adult pig retina. Oprhal. Res. 2: 25-36. 6. GEIGER, R. S. 1958. Subcultures of adult mammalian brain cortex in vitro. Exp. Cell Res. 14: 541-566. 7. HILD, W., AND G. CALLAS. 1967. The behavior of retinal tissue in vitro, light and electron microscopic observations. Z. Zellforsch. 80: l-21. 8. HOGUE, M. J. 1953. A study of adult human brain cells grown in tissue cultures. Am. J. Anat.
93: 397-427.
9. HOLLYFIELD, J. G., ANDP. WITKOVSKY. 1974. Pigmentedretinalepitheliuminvolvement in photoreceptor development and function. J. Exp. Zool. 189: 357-378. 10. KIM, S. U. 1965. Neurons in tissue culture. Observations on terminal boutons in cultures of mammalian central nervous tissue. Arch. Histol. Jap. 25: 371-386. 11. KIM, S. U. 1970. Observations on cerebellar cells in vitro. A silver and electron microscopic study. Z. Zellforsch. 107: 454-465. 12. KIM, S. U. 1971. Neuronal types in long-term culture of avian retina. Experientia 27: 1319- 1320. 13. KIM, S. U. 1975. Brain hypoxia studied in mouse central nervous system cultures: I. Sequential cellular changes. Lab. Invest. 33: 658-669. 14. KIM, S. U., K. G. WARREN, AND M. KALIA. 1979. Tissue culture ofadult human neurons, Neurosci. Letf. 11: 137-141. 15. LAVAIL, M. M., AND W. HILD. 1971. Histiotypic organization ofthe rat retinain vitro.Z. Zel(forsch.
114: 557-579.
16. MURRAY, M. R., AND A. P. STOUT. 1947. Adult human sympathetic ganglion cells cultivated in vitro. Am. J. Anat. 80: 225-273. 17. PARRY, H. B. 1953. Degenerations of the dog retina: I. Structure and development of the retina of the normal dog. Br. J. Oprhalmol. 37: 385-404. 18. SCOTT, B. S. 1977. Adult mouse dorsal root ganglia neurons in cell culture. J. Neurobiol. 8: 417-427. 19. WOLF, M. K. 1964. Differentiation of neuronal types and synapses in myelinatingcultures of mouse cerebellum. J. Cell Biol. 22: 259-279.
