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Central and peripheral neurite growth patterns in tissue culture
Neuroscience Letters, 2 (1976) 115--119
115
© Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
C E N T R A L AND P E R I P H E R A L N E U R I T E GROWTH P A T T E R N S IN TISSUE CULTURE
FREDRICK J. SEIL
Department of Neurology, Veterans Administration Hospital, Palo Alto, Calif. 94304 (U.S.A.) (Received March 9th, 1976) (Accepted March 24th, 1976)
SUMMARY
Neurites of mesencephalic V nucleus neurons, which in vivo have a peripheral process, were observed to grow away from cerebellar explants. This pattern of neurite growth was contrasted with a previously observed pattern of looping and returning to the explant characteristic of central processes of other neurons in cerebeUar cultures. The described tissue culture system could serve as a model for elucidating mechanisms which determine the directional growth of neurites.
In a previous study of cerebellar morphology in tissue culture [ 1 3 ] , it was noted that neurites emanating from cerebellar explants rarely terminated in the outgrowth zone, b u t usually returned to the explants. Even the axons of such neurons as intracerebellar nucleus and superior and lateral vestibular nucleus neurons, which in vivo terminate in extracerebellar loci [3,6], followed this general rule, and an example of a giant vestibular neuron whose axon ramified extensively in many regions of a cerebeUar explant was illustrated (Fig. 19 in ref. 13). In a study of fibers growing o u t from spinal cord explants, GuiUery et al. [7] described axons of large ventral horn cells growing long distances away from the explants, to branch repeatedly and terminate in the outgrowth zone. By contrast, neurites related to fiber bands which were thought to normally form the long ascending and descending pathways of the spinal cord remained near the explants. It was postulated that these patterns of fiber g r o w t h represented responses of different fiber groups to an explant-to-outgrowth gradient of some sort, possibly one of a chemical nature. The dendrites and axons of the usual c o m p o n e n t cortical and subcortical neurons of cerebellar cultures are, in vivo, located within the central nervous system [3,6,13]. Both Hild [9] and Allerand [2] noted the occasional presence of mesencephalic V nucleus neurons in cerebellar explants. These
116
•
°
°
°o
o°
~
o
°
Fig. 1. Camera lucida tracing of a mesencephalic V nucleus neuron and its processes in a cerebellar culture 15 days in vitro. T h e outline of the explant is indicated by the dotted line. The neurite branches terminate in the outgrowth zone, well away from the explant. The bar at the bottom of the figure represents 300 ~m. This figure can be compared with Fig. 19 of ref. 13, which illustratesa vestibular nucleus neuron and its processes as they ramify within the explant. The inset is a photomicrograph of the cell body and initial axonal process of a mesencephalic V nucleus neuron from a cerebellar culture 29 days in vitro. The bar at the bottom of the inset represents 10 ~m.
117 neurons are readily distinguishable because of their large size and their similarity in appearance to dorsal r o o t ganglion cells (Fig. 1). In vivo the peripheral processes of these generally monopolar cells convey proprioceptive impulses from fibers and tendons o f the muscles of mastication, and their axons end in the m o t o r nucleus of the trigeminal nerve [5]. Such cells, when present, are thus unique among neurons in cerebellar cultures in that one of their processes is peripheral rather than central, and the target tissue of the peripherally directed neurite is non-neural. An opportunity is thereby presented to observe differences in neurite growth patterns of a small population of easily recognizable distinct cells with peripheral processes among a large population of cells with exclusively central contacts. Parasagittally oriented cerebellar explants derived from newborn SwissWebster mice were prepared and maintained as previously described [13], and, after varying lengths of time in vitro, were fixed and stained as whole m o u n t preparations by a modified Holmes method [21]. Seven silverstained preparations demonstrating mesencephalic V nucleus neurons with traceable fibers were examined. These were culled from a large number of Holmes-stained preparations. The cultures ranged in age from 14 to 29 days in vitro. A constant feature of the 9 mesencephalic V nucleus neurons whose fibers were traced was the continued outgrowth of at least one of their processes in a general direction away from the explant (Fig. 1). This growth represented new growth, as any existing axons would have been severed during explantation. In the case of two cells, the neurites emanating from the cell bodies soon bifurcated, and one process turned toward the explant to terminate therein, while the other process grew into the outgrowth zone, to branch and terminate there. In the case of the remaining mesencephalic V nucleus neurons, no 'centrally' directed processes were evident (Fig. 1). Neuritic outgrowth often followed glial lines of outgrowth, b u t at long distances away flom the explants, fibers were seen traversing bare spots on the collagen surface, where no glial processes were visible by light microscopic examination. Fibers from mesencephalic V nucleus neurons were the only neurites found in far reaches of the outgrowth zone, where these neurites and their branches terminated. This pattern of growth is in marked contrast to the looping and turning back to the explant characteristic of neuritic processes of other neurons in cerebellar cultures [13]. In considering explanations for the variant growth patterns demonstrated by regenerating peripheral and central neurites in tissue culture, it should be noted that the absence of specific targets does not seem to be a deciding factor in dictating direction of neurite growth. This is exemplified b y the fact that neurites of subcortical nuclear neurons, whose target cells are not included in the cerebeUar cultures, nevertheless grow into the explant, where they appear to terminate freely [13]. That such neurites do not apparently make substitute connections with cell processes .which are n o t normal targets is supported by the absence of complete glomeruli on electron microscopic
118
examination of cerebellar explants [12,14]. The missing glomerular elements are the mossy fibers, which is the form taken by all incoming fibers to the cerebellar cortex except for the climbing fibers [6], and the "filling in" of incomplete glomeruli would seem the most logical route for formation of aberrant connections. A factor possibly contributing to the observed differences in central and peripheral neurite growth patterns in vitro might be an instructional mechanism for a neurite to orient itself with respect to nervous system tissue after its initial outgrowth in relation to the polarity of its cell body [8,10] and before its final contact with a specific target. A centrally directed neurite might thus be intrinsically programmed to remain within the proximity of central nervous system tissue as a preliminary step to finding its specific target cell, while a peripherally directed neurite might be programmed to grow away from nervous system tissue in its search for a target. As a result, in the absence of specific targets, the centrally directed neurite ends freely within the central nervous system explant, while the peripherally directed neurite terminates freely far out in the outgrowth zone, well away from the explant. Other evidence from tissue culture studies which could support such a concept includes the radial outgrowth of neurites away from dorsal root ganglion [4,11] and trigeminal ganglion [20] explants, and the growth of some central neurites from one explant to another in paired cerebellar cultures [ 1 ]. Factors extrinsic to nerve cells to which neurites might respond for directional guidance include mechanical, electrical and chemical forces or substances [10]. These possibilities, which need not be mutually exclusive [19], have been extensively reviewed in several past and recent publications [ 10, 15--19]. The exact mechanisms which determine the directional growth of neurites, either in vitro or in vivo, remain unknown. Possibly the tissue culture system described in the present paper might provide a model for elucidating such mechanisms. ACKNOWLEDGEMENTS
The author wishes to thank Mr. Robert A. Fisk for his technical assistance. REFERENCES I Allerand, C.D., Regeneration of synapses in vitro,Exp. Neurol., 25 (1969) 482--493, 2 AUerand, C.D., Patterns of neuronal differentiationin developing cultures of neonatal mouse cerebellum: a livingand silverimpregnation study, J. comp. Neurol., 142 (1971) 167--204. 3 Brodal, A., Pompeiano, O. and Walberg, F., The Vestibular Nuclei and Their Connections, A n a t o m y and Functional Correlations, Thomas, Springfield, Ill., 1962, pp. 27--99. 4 Bunge, M.B., Bunge, R.P., Peterson, E.R. and Murray, M.R., A light and electron microscope study of long-term organized cultures of rat dorsal root ganglia, J. Cell Biol., 32 (1967) 439--466.
119
5 Crosby, C.C., Humphrey, T. and Lauer, E.W., Correlative A n a t o m y of the Nervous System, MacMillan Co., New York, 1962, pp. 177--178. 6 Eccles, J.C., Ito, M., and Szent~gothai, J., The Cerebellum as a Neuronal Machine, Springer, New York, 1967, pp. 4-57 and 227--299. 7 Guillery, R.W., Sobkowicz, H.M. and Scott, G.L., Light and electron microscopical observations of the ventral horn and ventral root in long term cultures of the spinal cord of the fetal mouse, J. comp. Neurol., 134 (1968) 433--476. 8 Hibbard, E., Orientation and directed growth of Mauthner's cell axons from duplicated vestibular nerve roots, Exp. Neurol., 13 (1965) 289--301. 9 Hild, W., Cell types and neuronal connections in cultures of mammalian central nervous tissue, Z. Zellforsch., 69 (1966) 155--188. 10 Jacobson, M., Developmental Neurobiology, Holt, Rinehart and Winston, New York, 1970, pp. 116--169. 11 Lumsden, C.E., Aspects of neurite outgrowth in tissue culture, Anat. Rec., 110 (1951) 145--180. 12 Privat, A. and Drian, M.J., Specificity of the mossy fiber-granule cell synapse in the rat cerebellum. An in vitro study, Brain Res., 88 (1975) 518--524. 13 Seil, F.J., Neuronal groups and fiber patterns in cerebellar tissue cultures, Brain Res., 42 (1972) 33--51. 14 Seil, F.J. and Herndon, R.M., CerebeUar granule cells in vitro. A light and electron microscopic study, J. Cell Biol., 45 (1970) 212--220. 15 Sisken, B.F. and Smith, S.D., The effects of minute direct electrical currents on cultured chick embryo trigeminal ganglia, J. Embryol. exp. Morph., 33 (1975) 29--41. 16 Sperry, R.W., Chemoaffinity in the orderly growth of nerve fiber patterns and connections, Proc. nat. Acad. Sci. (Wash.), 50 (1963) 703--710. 17 Sperry, R.W., Selective communication in nerve nets: impulse specificity vs. connection specificity. In F.O. Schmitt and T. Melnechuk (Eds.), Neuroscience Research Symposium Summaries, Vol. 1, M.I.T. Press, Cambridge, Mass., 1966, pp. 213--219. 18 Weiss, P., In vitro experiments on the factors determining the course of the outgrowing nerve fiber, J. exp. Zool., 68 (1934) 393--448. 19 Weiss, P.A., Specificity in the neurosciences. In F.O. Schmitt and T. Melnechuk (Eds.), Neuroscience Research Symposium Summaries, Vol. 1, M.I.T. Press, Cambridge, Mass., 1966, pp. 179--212. 20 Winkler, G.F. and Wolf, M.K., The development and maintenance of myelinated tissue cultures of rat tri~geminal ganglion, Amer. J. Anat., 119 (1966) 179--197. 21 Wolf, M.K., Differentiation of neuronal types and synapses in myelinating cultures of mouse cerebellum, J. Cell Biol., 22 (1964) 259--279.
115
© Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
C E N T R A L AND P E R I P H E R A L N E U R I T E GROWTH P A T T E R N S IN TISSUE CULTURE
FREDRICK J. SEIL
Department of Neurology, Veterans Administration Hospital, Palo Alto, Calif. 94304 (U.S.A.) (Received March 9th, 1976) (Accepted March 24th, 1976)
SUMMARY
Neurites of mesencephalic V nucleus neurons, which in vivo have a peripheral process, were observed to grow away from cerebellar explants. This pattern of neurite growth was contrasted with a previously observed pattern of looping and returning to the explant characteristic of central processes of other neurons in cerebeUar cultures. The described tissue culture system could serve as a model for elucidating mechanisms which determine the directional growth of neurites.
In a previous study of cerebellar morphology in tissue culture [ 1 3 ] , it was noted that neurites emanating from cerebellar explants rarely terminated in the outgrowth zone, b u t usually returned to the explants. Even the axons of such neurons as intracerebellar nucleus and superior and lateral vestibular nucleus neurons, which in vivo terminate in extracerebellar loci [3,6], followed this general rule, and an example of a giant vestibular neuron whose axon ramified extensively in many regions of a cerebeUar explant was illustrated (Fig. 19 in ref. 13). In a study of fibers growing o u t from spinal cord explants, GuiUery et al. [7] described axons of large ventral horn cells growing long distances away from the explants, to branch repeatedly and terminate in the outgrowth zone. By contrast, neurites related to fiber bands which were thought to normally form the long ascending and descending pathways of the spinal cord remained near the explants. It was postulated that these patterns of fiber g r o w t h represented responses of different fiber groups to an explant-to-outgrowth gradient of some sort, possibly one of a chemical nature. The dendrites and axons of the usual c o m p o n e n t cortical and subcortical neurons of cerebellar cultures are, in vivo, located within the central nervous system [3,6,13]. Both Hild [9] and Allerand [2] noted the occasional presence of mesencephalic V nucleus neurons in cerebellar explants. These
116
•
°
°
°o
o°
~
o
°
Fig. 1. Camera lucida tracing of a mesencephalic V nucleus neuron and its processes in a cerebellar culture 15 days in vitro. T h e outline of the explant is indicated by the dotted line. The neurite branches terminate in the outgrowth zone, well away from the explant. The bar at the bottom of the figure represents 300 ~m. This figure can be compared with Fig. 19 of ref. 13, which illustratesa vestibular nucleus neuron and its processes as they ramify within the explant. The inset is a photomicrograph of the cell body and initial axonal process of a mesencephalic V nucleus neuron from a cerebellar culture 29 days in vitro. The bar at the bottom of the inset represents 10 ~m.
117 neurons are readily distinguishable because of their large size and their similarity in appearance to dorsal r o o t ganglion cells (Fig. 1). In vivo the peripheral processes of these generally monopolar cells convey proprioceptive impulses from fibers and tendons o f the muscles of mastication, and their axons end in the m o t o r nucleus of the trigeminal nerve [5]. Such cells, when present, are thus unique among neurons in cerebellar cultures in that one of their processes is peripheral rather than central, and the target tissue of the peripherally directed neurite is non-neural. An opportunity is thereby presented to observe differences in neurite growth patterns of a small population of easily recognizable distinct cells with peripheral processes among a large population of cells with exclusively central contacts. Parasagittally oriented cerebellar explants derived from newborn SwissWebster mice were prepared and maintained as previously described [13], and, after varying lengths of time in vitro, were fixed and stained as whole m o u n t preparations by a modified Holmes method [21]. Seven silverstained preparations demonstrating mesencephalic V nucleus neurons with traceable fibers were examined. These were culled from a large number of Holmes-stained preparations. The cultures ranged in age from 14 to 29 days in vitro. A constant feature of the 9 mesencephalic V nucleus neurons whose fibers were traced was the continued outgrowth of at least one of their processes in a general direction away from the explant (Fig. 1). This growth represented new growth, as any existing axons would have been severed during explantation. In the case of two cells, the neurites emanating from the cell bodies soon bifurcated, and one process turned toward the explant to terminate therein, while the other process grew into the outgrowth zone, to branch and terminate there. In the case of the remaining mesencephalic V nucleus neurons, no 'centrally' directed processes were evident (Fig. 1). Neuritic outgrowth often followed glial lines of outgrowth, b u t at long distances away flom the explants, fibers were seen traversing bare spots on the collagen surface, where no glial processes were visible by light microscopic examination. Fibers from mesencephalic V nucleus neurons were the only neurites found in far reaches of the outgrowth zone, where these neurites and their branches terminated. This pattern of growth is in marked contrast to the looping and turning back to the explant characteristic of neuritic processes of other neurons in cerebellar cultures [13]. In considering explanations for the variant growth patterns demonstrated by regenerating peripheral and central neurites in tissue culture, it should be noted that the absence of specific targets does not seem to be a deciding factor in dictating direction of neurite growth. This is exemplified b y the fact that neurites of subcortical nuclear neurons, whose target cells are not included in the cerebeUar cultures, nevertheless grow into the explant, where they appear to terminate freely [13]. That such neurites do not apparently make substitute connections with cell processes .which are n o t normal targets is supported by the absence of complete glomeruli on electron microscopic
118
examination of cerebellar explants [12,14]. The missing glomerular elements are the mossy fibers, which is the form taken by all incoming fibers to the cerebellar cortex except for the climbing fibers [6], and the "filling in" of incomplete glomeruli would seem the most logical route for formation of aberrant connections. A factor possibly contributing to the observed differences in central and peripheral neurite growth patterns in vitro might be an instructional mechanism for a neurite to orient itself with respect to nervous system tissue after its initial outgrowth in relation to the polarity of its cell body [8,10] and before its final contact with a specific target. A centrally directed neurite might thus be intrinsically programmed to remain within the proximity of central nervous system tissue as a preliminary step to finding its specific target cell, while a peripherally directed neurite might be programmed to grow away from nervous system tissue in its search for a target. As a result, in the absence of specific targets, the centrally directed neurite ends freely within the central nervous system explant, while the peripherally directed neurite terminates freely far out in the outgrowth zone, well away from the explant. Other evidence from tissue culture studies which could support such a concept includes the radial outgrowth of neurites away from dorsal root ganglion [4,11] and trigeminal ganglion [20] explants, and the growth of some central neurites from one explant to another in paired cerebellar cultures [ 1 ]. Factors extrinsic to nerve cells to which neurites might respond for directional guidance include mechanical, electrical and chemical forces or substances [10]. These possibilities, which need not be mutually exclusive [19], have been extensively reviewed in several past and recent publications [ 10, 15--19]. The exact mechanisms which determine the directional growth of neurites, either in vitro or in vivo, remain unknown. Possibly the tissue culture system described in the present paper might provide a model for elucidating such mechanisms. ACKNOWLEDGEMENTS
The author wishes to thank Mr. Robert A. Fisk for his technical assistance. REFERENCES I Allerand, C.D., Regeneration of synapses in vitro,Exp. Neurol., 25 (1969) 482--493, 2 AUerand, C.D., Patterns of neuronal differentiationin developing cultures of neonatal mouse cerebellum: a livingand silverimpregnation study, J. comp. Neurol., 142 (1971) 167--204. 3 Brodal, A., Pompeiano, O. and Walberg, F., The Vestibular Nuclei and Their Connections, A n a t o m y and Functional Correlations, Thomas, Springfield, Ill., 1962, pp. 27--99. 4 Bunge, M.B., Bunge, R.P., Peterson, E.R. and Murray, M.R., A light and electron microscope study of long-term organized cultures of rat dorsal root ganglia, J. Cell Biol., 32 (1967) 439--466.
119
5 Crosby, C.C., Humphrey, T. and Lauer, E.W., Correlative A n a t o m y of the Nervous System, MacMillan Co., New York, 1962, pp. 177--178. 6 Eccles, J.C., Ito, M., and Szent~gothai, J., The Cerebellum as a Neuronal Machine, Springer, New York, 1967, pp. 4-57 and 227--299. 7 Guillery, R.W., Sobkowicz, H.M. and Scott, G.L., Light and electron microscopical observations of the ventral horn and ventral root in long term cultures of the spinal cord of the fetal mouse, J. comp. Neurol., 134 (1968) 433--476. 8 Hibbard, E., Orientation and directed growth of Mauthner's cell axons from duplicated vestibular nerve roots, Exp. Neurol., 13 (1965) 289--301. 9 Hild, W., Cell types and neuronal connections in cultures of mammalian central nervous tissue, Z. Zellforsch., 69 (1966) 155--188. 10 Jacobson, M., Developmental Neurobiology, Holt, Rinehart and Winston, New York, 1970, pp. 116--169. 11 Lumsden, C.E., Aspects of neurite outgrowth in tissue culture, Anat. Rec., 110 (1951) 145--180. 12 Privat, A. and Drian, M.J., Specificity of the mossy fiber-granule cell synapse in the rat cerebellum. An in vitro study, Brain Res., 88 (1975) 518--524. 13 Seil, F.J., Neuronal groups and fiber patterns in cerebellar tissue cultures, Brain Res., 42 (1972) 33--51. 14 Seil, F.J. and Herndon, R.M., CerebeUar granule cells in vitro. A light and electron microscopic study, J. Cell Biol., 45 (1970) 212--220. 15 Sisken, B.F. and Smith, S.D., The effects of minute direct electrical currents on cultured chick embryo trigeminal ganglia, J. Embryol. exp. Morph., 33 (1975) 29--41. 16 Sperry, R.W., Chemoaffinity in the orderly growth of nerve fiber patterns and connections, Proc. nat. Acad. Sci. (Wash.), 50 (1963) 703--710. 17 Sperry, R.W., Selective communication in nerve nets: impulse specificity vs. connection specificity. In F.O. Schmitt and T. Melnechuk (Eds.), Neuroscience Research Symposium Summaries, Vol. 1, M.I.T. Press, Cambridge, Mass., 1966, pp. 213--219. 18 Weiss, P., In vitro experiments on the factors determining the course of the outgrowing nerve fiber, J. exp. Zool., 68 (1934) 393--448. 19 Weiss, P.A., Specificity in the neurosciences. In F.O. Schmitt and T. Melnechuk (Eds.), Neuroscience Research Symposium Summaries, Vol. 1, M.I.T. Press, Cambridge, Mass., 1966, pp. 179--212. 20 Winkler, G.F. and Wolf, M.K., The development and maintenance of myelinated tissue cultures of rat tri~geminal ganglion, Amer. J. Anat., 119 (1966) 179--197. 21 Wolf, M.K., Differentiation of neuronal types and synapses in myelinating cultures of mouse cerebellum, J. Cell Biol., 22 (1964) 259--279.