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Synapse formation on trochlear motor neurons in relation to naturally occurring cell death during development
0736?5748/91 s3.oo+o.oo Pergamon Press plc @ 1991ISDN
Int. .I. Devl. Neuroscience,Vol. 9, No. 4, pp. 371-379,1991. Printed in Great Britain.
SYNAPSE FORMATION ON TROCHLEAR MOTOR NEURONS IN RELATION TO NATURALLY OCCURRING CELL DEATH DURING DEVELOPMENT S. HIRANO, K. KUMARESAN, M. M. ALI and G. S. SOHAL* Department of Anatomy, Medical College of Georgia, Augusta, GA 30912, U.S.A. (Received 12 October 1990; in revised form 27 November 1990; accepted 28 November 1990)
Abstract-About half of the trochlear motor neurons die during the course of normal development. The present study was undertaken to determine whether the afferent synapses form before the onset of motor neuron death and also to determine whether the number of synapses differs between the healthy and degenerating trochlear motor neurons. Brains of duck embryos from days 10 to 20 were prepared for quantitative electron microscopical observations on synaptogenesis. Results indicate that synapses form on the trochlear motor neuron soma before cell death begins suggesting that afferent input is in a position to exert an influence on survival or death of motor neurons. There were no significant differences in the number of synapses between the healthy and dying neurons during the period of cell death. This observation suggests that the mechanism by which afferent synapses could be involved in neuron survival or death is not related to the number of synapses on the cell soma. The number of synapses on the cell process, synaptic transmission and/or molecules released at the synapses are likely candidates for the mechanism of action of afferent input. Key words: neuron death, trochlear nucleus, development,
afferent synapses, duck embryos.
A large number of motor neurons degenerate during the course of normal development of the nervous system (for recent reviews on naturally occurring cell death see Refs 2,4,5,7,9,13,18, 19,26). It is not known for certain what factors control survival or death of motor neurons. In the past, it was commonly thought that the number of surviving motor neurons was solely determined by the size of the target muscle of innervation. In other words, some motor neurons die because the target muscle is too small to support all of them. Cell death was viewed as a mechanism to match the size of the neuron pool to the size of the target muscle. Experimental manipulations of the target have failed to provide data consistent with this size matching hypothesis. For example, doubling the target size does not produce a corresponding increase in the number of surviving motor neurons.‘~11,16 Similarly, making neurons innervate a smaller target does not result in a corresponding decrease in neuron survival. 12,22,23,25 Thus, it is clear that the target alone does not control the final number of dying or surviving motor neurons during development. The results of several recent studies suggest that afferent input within the CNS may also be involved in the control of cell number during development. For example, deafferentation decreases the number of surviving neurons even though it does not affect the size of the target.3~6~8~‘4~‘5*17~20 If afferent input plays an important role in controlling survival or death of motor neurons during the course of normal development then it is important to establish that synapses develop prior to the onset of cell death so that they are normally in a position to exert an influence on motor neurons. The objectives of the present study were to determine whether synapses develop before cell death begins and to determine if the number of synapses differs between dying and surviving motor neurons. The trochlear nucleus was used as a simple model to study the relationship between synaptogenesis and neuron survival. It is known that about half of the trochlear motor neurons die during normal development. For example, the trochlear nucleus in duck embryos attains a maximum of about 2300 neurons by embryonic day 12, cell death occurs between days l3 and 18 and thereafter the nucleus contains about 1300 motor neurons. **This paper reports observations on synaptogenesis before, during, and after the period of cell death. EXPERIMENTAL
PROCEDURES
Animals
Fertile eggs of white Peking duck were incubated in a forced-draft *To whom correspondence
should be addressed. 371
incubator at 37S”C.
372
S.
Hirano et al.
Electron Microscopy
Small portions of midbrain containing the region of the trochlear nucleus were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer containing 1% tannic acid. They were postfixed in 2% osmium tetroxide, dehydrated in ethanol and propylene oxide and embedded in Epon 812. Four to eight brains were embedded for each of the embryonic days shown in Table 1. The initial size of the tissue was considerably larger because it is not possible to grossly identify the trochlear nucleus rt the time of fixation. Thick sections were cut with a glass knife and stained with toluidine blue to icientify the trochlear nucleus and to trim down the size of the tissue. The final number of usable brains is given in Table 1. Thin transverse sections were cut at different levels of the nucleus with a diamond knife and collected on formvar-coated slot grids to maximize the area of viewing. Sections were stained with uranyl acetate and lead citrate and examined in a Philips 400 transmission electron microscope. Trochlear nucleus is an easily identifiable, compact mass of cells and in a single thin transverse section about lo-30 neurons can be seen. In order to be consistent in sampling, only those neurons which had nucleus and nucleolus in the plane of the section were photographed. These neurons were located randomly in the nucleus. The orientation of neurons was such that their axons pointed laterally. Synapse Counting
Synapses were counted from electron micrograph montages of the motor neurons. Each neuron containing a nucleus was initially photographed at X 2150. Then overlapping pictures of the same motor neuron periphery (perimeter) were taken at X 7700. Negatives were printed at an enlargement of x 3. Thus, electron micrograph montages of each motor neuron profile were made at a final magnification of x 23100. A synapse was defined as a region with a presynaptic nerve terminal containing two or more synaptic vesicles applied against the postsynaptic process with intervening synaptic cleft. The perimeter of each motor neuron profile and the percent perimeter occupied by synapses was determined by using an automated Zeiss MOPl. The number of synapses per 10 km perimeter was calculated from the above measurements. The number of motor neurons examined is given in Table 1. This study utilized approximately 3000 electron micrographs. RESULTS Synaptogenesis before cell death begins
This period represents embryonic days 10-12. On day 10 there were very few motor neurons (6 out of 30) which had synapses on them. On day 11 there was an average of 3.26 synapses per neuron profile (Table 2). A similar number of synapses was seen on day 12. Synapses during this period were relatively immature as shown in Fig. 1. These synapses occupied about 4% of the perimeter of the neuron which represents less than 1 synapse per 10 km of the cell perimeter (Table 2). These observations indicate that synapses on the cell soma begin to develop before the naturally occurring trochlear motor neuron death begins. Synaptogenesis during the period of cell death
This period is represented by embryonic days 13-16. A rapid increase in the number of synapses occurred during this phase. For example, the average number of synapses per neuron profile on day 13 increased from 6.03 to 10.70 on day 16 (Table 2). The percent perimeter occupied by synapses also showed a similar increase (Table 2). Morphologically, synapses were more mature than during the previous phase (Fig. 2). In order to determine whether the number of synapses differed between the healthy and dying cells, a detailed quantitative study was carried out during the period of cell death. There was no statistically significant (Student’s t test) difference in synapse numbers between healthy and dying cells on each of the days examined (Table 3). Likewise measurements of cell perimeter and percent perimeter occupied by synapses did not reveal a clear cut distinction between the healthy and dying neurons (Table 4). It was initially thought that cell death may be related to cell size*’ but the range of all perimeters indicates that cell death is not restricted to any particular cell size (Table 4). It should be pointed out that the categorization of neurons into healthy and dying was
Synaptogenesis
and trochlear motor neuron death
Fig. 1. An electron micrograph of a trochlear motor neuron on embryonic day 12. A synapse on the motor neuron is indicated by the arrow. Bar = 1 urn. Fig. 2. An electron micrograph of a trochlear motor neuron on embryonic day 15. Two synapses on the :or neuron are indicated by the arrows. Note the development of pre- and postsynaptic density in the synapses. Bar = 1 pm.
373
S. Hirano et al.
Fig. 3. An electron
micrograph
of a healthy
trochlear
motor neuron
on embryonic
day IS. Bar = 1 km.
Fig. 4. An electron micrograph of a trochlear motor neuron showing signs of degeneration. Note the dilation of rough endoplasmic reticulum, vacuolization, increased density of mitochondria and increase in free ribosomes. Bar = 1 km.
Synaptogenesis
and trochlear motor neuron death
Fig. 5. An electron micrograph of a trochlear motor neuron on embryonic day 18. Note the welldeveloped synapses (arrows). Bar = 1 km.
375
Synaptogenesis and trochlear motor neuron death
377
Table 1. Number of brains and trochlear motor neurons examined during various days of development Number of brains
Embryonic age (day)
Number of neurons 30 34 53 62 36 42 79 34 35
10 11 12 13 14 15 16 18 20 Total
Table 2. Number of synapses on trochlear motor neurons during development Number of synapses per neuron profile Embryonic age Mean f S.E.M. (day) 10 11 12 13 14 15 16 18 20
Range
Percent perimeter occupied by synapses
Number of synapses per 10 pm perimeter
4.10 3.94 6.72 7.68 10.86 12.92 15.61 21.17
0.82 0.73 1.25 1.42 1.85 2.13 2.87 4.27
o-3 O-8 o-15 o-19 O-17 o-31 o-49 o-33 5-52
0.26kO.11 3.2620.32 2.912 0.44 6.03 f 0.53 6.92 + 0.72 9.48 f 1.21 10.70 + 0.95 17.00 f 1.77 20.97 + 1.76
Table 3. Number of synapses on healthy and degenerating the period of cell death
trochlear motor neurons during
Number of synapses per neuron profile Embryonic age (day) 13 14 15 16
Type of neuron Healthy (40) Degenerating Healthy (25) Degenerating Healthy (17) Degenerating Healthy (49) Degenerating
(22) (11) (25) (30)
Mean f S.E.M.
Range
Number of synapses per 10 pm perimeter
6.38 k 0.73 5.3220.70 7.4020.74 5.82 -c 1.68 8.47-e2.02 10.16+ 1.51 9.372 1.11 12.83 + 1.66
o-19 o-14 1-14 o-17 O-26 o-31 o-49 l-25
1.36 1.07 1.50 1.25 1.67 1.98 1.98 2.34
Numbers in parentheses indicate the number of neurons examined in each case.
based on their ultrastructural features only. 24Figures 3 and 4 show examples of healthy and dying neurons. A neuron was categorized as dying if it showed dilation of the rough endoplasmic reticulum, swelling of mitochondria, vacuolization, irregular and disrupted nuclear envelope, and clumping of chromatin. It is possible that some neurons which were categorized as healthy may die later during development. Thus, the categorization of neurons into healthy and degenerating is not an absolute one. Synaptogenesis after the period of cell death
This period is represented by embryonic days N-20. There was a considerable increase in the number of synapses during this phase. For example, an average synapse count per neuron profile was 17.00 on day 18 and 20.97 on day 20 (Table 2). This represents a more than two fold increase than during the period of cell death. similarly synapses occupied a much greater amount of neuron periphery during this phase (Table 2). Synapses appeared well developed as shown in Fig. 5.
S. Hirano et al.
378
Table 4. Perimeter of healthy and degenerating
Embryonic age (day) 13 14 15 16
Type of neuron Healthy Degenerating Healthy Degenerating Healthy Degenerating Healthy Degenerating
trochlear motor neurons during the period of cell death
Cell perimeter in urn ________ Mean -CS.E.M. Range 46.73 + 1.60 49.71 -t 2.48 49.34 + 1.72 46.715 3.44 50.72 + 3.61 51.43-+2.71 48.20% 1.28 54.87 i 1.27
28.70-72.53 28.99-74.3’ 38.05-70.43 29.96-73.77 27.82-74.85 35.19-75.43 29.70-66.07 39.70-66.33
% perimeter occupied by synapses 7.31 5.73 8.15 6.55 9.24 11.95 11.96 14.28
DISCUSSION If afferent synaptic input plays an important role in the determination of the number of surviving neurons during development then it is essential to document that afferent synapses normally form before motor neuron death begins. The results of the present study indicate that afferent synapses develop on the trochlear motor neuron soma before the onset of the naturally occurring cell death. This observation suggests that afferent synapses could be involved in the survival or death of neurons. The results of several deafferentation studies also support the involvement of Our preliminary observations indicate that additional afferents in cell survival/death. 3,6~8~14~15.‘7,20 trochlear motor neurons die following deafferentation. I0 The mechanism by which afferent synapses could contribute to neuron survival/death is unknown. One possibility is that the number of afferent synapses may differ between healthy and degenerating neurons. In other words, the degenerating neurons may have considerably fewer synapses than the healthy neurons. The results of the present study indicate that there were no significant differences in the number of synapses on degenerating and healthy neurons. We were also unable to find any type of qualitative differences in synapses on healthy and degenerating cells. It should be pointed out that the synapses counted in this study were axosomatic synapses. It is possible that the number of synapses on dendrites or axons could differ between the healthy and dying cells. Another possibility is that the number of functional synapses is different between the healthy and dying neurons. It should be noted, however, that the synapses are morphologically immature before cell death begins but mature rapidly during the period of cell death. It is also possible that the type of synapses, the source of the input and molecules released at the synapse may be different for healthy and dying cells. Further studies are needed to investigate these possibilities. Acknowledgements-We thank R. Lala for technical assistance and C. Motes for typing the manuscript. This work was supported by a grant from the NIH (HD17800).
REFERENCES 1. Boydston W. R. and Sohal G. S. (1979) Grafting of additional periphery reduces embryonic loss of neurons. Brain Res. 178,403-410. 2. Clarke P. G. H. (1985) Neuronal death in the development of the nervous system. Trends Neurosci. 8, 345-349. 3. Clarke P. G. H. (1985) Neuronal death during development in the isthmo optic nucleus of the chick: sustaining role of afferents from the tectum. J. Camp. Neurol. 234, 365-379. 4. Cowan W. M., Fawcett J. W., O’Leary D. D. M. and Stanfield B. B. (1984) Regressive events in neurogenesis. Science 225, 1258-1265. 5. Cunningham T. J. (1982) Naturally occurring neuron death and its regulation by developing neural pathways. Int. Rev. Cytol. 74, 163-186. 6. Davis M., Constantine-Paton M. and Schorr D. (1983) Dorsal root ganglion removal in Rana pipiens produces fewer motoneurons. Brain Res. 265, 283-288. 7. Finlay B. L., Wikler K. C. and Sengelaub D. R. (1987) Regressive events in brain development and scenarios for vertebrate brain evolution. Brain Behav. Evol. 30, 102-117. 8. Furber S., Oppenheim R. W. and Prevette D. (1987) Naturally occurring neuron death in the ciliary ganglion of the chick embryo following removal of preganglionic input: evidence for the role of afferents in ganglion cell survival. J. Neurosci. 7, 1816-1832.
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9. Hamburger V. and Oppenheim
R. W. (1982) Naturally occurring neuronal death in the vertebrates. Neurosci. 1, 39-55. 10. Hirano S., Kumaresan K. and Sohal G. S. (1990) Involvement of afferent synaptic input in trochlear motor neuron death during development. Sot. Neurosci. Absrr. 16,836. 11. Hollyday M. and Hamburger V. (1976) Reduction of the naturally occurring motor neuron loss by enlargement of the periphery. J. Camp. Neural. 170, 311-320. 12. Lamb A. H. (1980) Motoneurone counts in Xenopus frogs reared with one bilaterally innervated hindlimb. Nature Comment.
284,347-350.
13. Lamb A. H. (1984) Motoneuron death in the embryo. Crit. Rev. Clin. Neurobiol. 1, 141-179. 14. Linden R. and Pinon L. G. P. (1987) Dual control by targets and afferents of developmental neuronal death in the mammalian central nervous system: a study in the parabigeminal nucleus of the rat. J. Camp. Neural. 266,141-149. 15. Linden R. and Renteria A.S. (1988), Afferent control of neuron numbers in the develooine . I brain. Dev. Brain Res. 44. 291-295. 16. Narayanan C. H. and Narayanan Y. (1978) Neuronal adjustments in developing nuclear centers of the chick embryo following transplantation of an additional ootic urimordium. J. Embrvol. EXD. Morohol. 44.53-70. 17. Okado I?. andoppenheim R. W. (1984) Cell death of motoneurons in the chick embryo spinal cord IX. The loss of motoneurons following removal of afferent inputs. J. Neurosci. 4, 1639-1652. 18. Oppenheim R. W. (1981) Neuronal death and some related regressive phenomena during neurogenesis: a selective historical review and progress report. In Studies in Developmental Neurobioloqy: Essays in Honor of Victor Hamburger (ed. Cowan W. M.), pp 74-133. Oxford University Press, New York. 19. Oppenheim R. W. (1989) The neurotrophic theory and naturally occurring motoneuron death. Trends Neurosci. 12, 252-255. 20. Sohal G. S. (1976) Effects of deafferentation Neural. 50, 161-173.
on the development
of the isthmo optic nucleus in the duck. Exp.
21. Sohal G. S. (1976) An experimental study of cell death in the developing trochlear nucleus. Exp. Neurol. 51,684-698. 22. Sohal G. S., Stoney S. D., Campbell L. R., Arumugam T., Kumaresan K. and Hirano S. (1990) Influence of grafting a smaller target muscle on the magnitude of naturally occurring trochlear motor neuron death during development. J. Comp. Neural. (in press). 23. Sohal G. S., Stoney S. D., Arumugam T., Yamashita T. and Knox T. (1986) Influence of reduced neuron pool on the magnitude of naturally occurring motor neuron death. J. Comp. Neural. 247,516-528. 24. Sohal G. S. and Weidman T. A. (1978) Ultrastructural sequence of embryonic cell death in normal and peripherally deprived trochlear nucleus. Exp. Neural. 61,53&t. 25. Tanaka H. and Landmesser L. (1986) Cell death of lumbosacral motoneurons in chick, quail, and chick-quail chimera embryos: A test of the quantitative matching hypothesis of neuronal cell death. J. Neurosci. 6, 2889-2899. 26. Williams R. W. and Herrup K. (1988) The control of neuron numbers. Ann. Rev. Neurosci. l&423-453.
Int. .I. Devl. Neuroscience,Vol. 9, No. 4, pp. 371-379,1991. Printed in Great Britain.
SYNAPSE FORMATION ON TROCHLEAR MOTOR NEURONS IN RELATION TO NATURALLY OCCURRING CELL DEATH DURING DEVELOPMENT S. HIRANO, K. KUMARESAN, M. M. ALI and G. S. SOHAL* Department of Anatomy, Medical College of Georgia, Augusta, GA 30912, U.S.A. (Received 12 October 1990; in revised form 27 November 1990; accepted 28 November 1990)
Abstract-About half of the trochlear motor neurons die during the course of normal development. The present study was undertaken to determine whether the afferent synapses form before the onset of motor neuron death and also to determine whether the number of synapses differs between the healthy and degenerating trochlear motor neurons. Brains of duck embryos from days 10 to 20 were prepared for quantitative electron microscopical observations on synaptogenesis. Results indicate that synapses form on the trochlear motor neuron soma before cell death begins suggesting that afferent input is in a position to exert an influence on survival or death of motor neurons. There were no significant differences in the number of synapses between the healthy and dying neurons during the period of cell death. This observation suggests that the mechanism by which afferent synapses could be involved in neuron survival or death is not related to the number of synapses on the cell soma. The number of synapses on the cell process, synaptic transmission and/or molecules released at the synapses are likely candidates for the mechanism of action of afferent input. Key words: neuron death, trochlear nucleus, development,
afferent synapses, duck embryos.
A large number of motor neurons degenerate during the course of normal development of the nervous system (for recent reviews on naturally occurring cell death see Refs 2,4,5,7,9,13,18, 19,26). It is not known for certain what factors control survival or death of motor neurons. In the past, it was commonly thought that the number of surviving motor neurons was solely determined by the size of the target muscle of innervation. In other words, some motor neurons die because the target muscle is too small to support all of them. Cell death was viewed as a mechanism to match the size of the neuron pool to the size of the target muscle. Experimental manipulations of the target have failed to provide data consistent with this size matching hypothesis. For example, doubling the target size does not produce a corresponding increase in the number of surviving motor neurons.‘~11,16 Similarly, making neurons innervate a smaller target does not result in a corresponding decrease in neuron survival. 12,22,23,25 Thus, it is clear that the target alone does not control the final number of dying or surviving motor neurons during development. The results of several recent studies suggest that afferent input within the CNS may also be involved in the control of cell number during development. For example, deafferentation decreases the number of surviving neurons even though it does not affect the size of the target.3~6~8~‘4~‘5*17~20 If afferent input plays an important role in controlling survival or death of motor neurons during the course of normal development then it is important to establish that synapses develop prior to the onset of cell death so that they are normally in a position to exert an influence on motor neurons. The objectives of the present study were to determine whether synapses develop before cell death begins and to determine if the number of synapses differs between dying and surviving motor neurons. The trochlear nucleus was used as a simple model to study the relationship between synaptogenesis and neuron survival. It is known that about half of the trochlear motor neurons die during normal development. For example, the trochlear nucleus in duck embryos attains a maximum of about 2300 neurons by embryonic day 12, cell death occurs between days l3 and 18 and thereafter the nucleus contains about 1300 motor neurons. **This paper reports observations on synaptogenesis before, during, and after the period of cell death. EXPERIMENTAL
PROCEDURES
Animals
Fertile eggs of white Peking duck were incubated in a forced-draft *To whom correspondence
should be addressed. 371
incubator at 37S”C.
372
S.
Hirano et al.
Electron Microscopy
Small portions of midbrain containing the region of the trochlear nucleus were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer containing 1% tannic acid. They were postfixed in 2% osmium tetroxide, dehydrated in ethanol and propylene oxide and embedded in Epon 812. Four to eight brains were embedded for each of the embryonic days shown in Table 1. The initial size of the tissue was considerably larger because it is not possible to grossly identify the trochlear nucleus rt the time of fixation. Thick sections were cut with a glass knife and stained with toluidine blue to icientify the trochlear nucleus and to trim down the size of the tissue. The final number of usable brains is given in Table 1. Thin transverse sections were cut at different levels of the nucleus with a diamond knife and collected on formvar-coated slot grids to maximize the area of viewing. Sections were stained with uranyl acetate and lead citrate and examined in a Philips 400 transmission electron microscope. Trochlear nucleus is an easily identifiable, compact mass of cells and in a single thin transverse section about lo-30 neurons can be seen. In order to be consistent in sampling, only those neurons which had nucleus and nucleolus in the plane of the section were photographed. These neurons were located randomly in the nucleus. The orientation of neurons was such that their axons pointed laterally. Synapse Counting
Synapses were counted from electron micrograph montages of the motor neurons. Each neuron containing a nucleus was initially photographed at X 2150. Then overlapping pictures of the same motor neuron periphery (perimeter) were taken at X 7700. Negatives were printed at an enlargement of x 3. Thus, electron micrograph montages of each motor neuron profile were made at a final magnification of x 23100. A synapse was defined as a region with a presynaptic nerve terminal containing two or more synaptic vesicles applied against the postsynaptic process with intervening synaptic cleft. The perimeter of each motor neuron profile and the percent perimeter occupied by synapses was determined by using an automated Zeiss MOPl. The number of synapses per 10 km perimeter was calculated from the above measurements. The number of motor neurons examined is given in Table 1. This study utilized approximately 3000 electron micrographs. RESULTS Synaptogenesis before cell death begins
This period represents embryonic days 10-12. On day 10 there were very few motor neurons (6 out of 30) which had synapses on them. On day 11 there was an average of 3.26 synapses per neuron profile (Table 2). A similar number of synapses was seen on day 12. Synapses during this period were relatively immature as shown in Fig. 1. These synapses occupied about 4% of the perimeter of the neuron which represents less than 1 synapse per 10 km of the cell perimeter (Table 2). These observations indicate that synapses on the cell soma begin to develop before the naturally occurring trochlear motor neuron death begins. Synaptogenesis during the period of cell death
This period is represented by embryonic days 13-16. A rapid increase in the number of synapses occurred during this phase. For example, the average number of synapses per neuron profile on day 13 increased from 6.03 to 10.70 on day 16 (Table 2). The percent perimeter occupied by synapses also showed a similar increase (Table 2). Morphologically, synapses were more mature than during the previous phase (Fig. 2). In order to determine whether the number of synapses differed between the healthy and dying cells, a detailed quantitative study was carried out during the period of cell death. There was no statistically significant (Student’s t test) difference in synapse numbers between healthy and dying cells on each of the days examined (Table 3). Likewise measurements of cell perimeter and percent perimeter occupied by synapses did not reveal a clear cut distinction between the healthy and dying neurons (Table 4). It was initially thought that cell death may be related to cell size*’ but the range of all perimeters indicates that cell death is not restricted to any particular cell size (Table 4). It should be pointed out that the categorization of neurons into healthy and dying was
Synaptogenesis
and trochlear motor neuron death
Fig. 1. An electron micrograph of a trochlear motor neuron on embryonic day 12. A synapse on the motor neuron is indicated by the arrow. Bar = 1 urn. Fig. 2. An electron micrograph of a trochlear motor neuron on embryonic day 15. Two synapses on the :or neuron are indicated by the arrows. Note the development of pre- and postsynaptic density in the synapses. Bar = 1 pm.
373
S. Hirano et al.
Fig. 3. An electron
micrograph
of a healthy
trochlear
motor neuron
on embryonic
day IS. Bar = 1 km.
Fig. 4. An electron micrograph of a trochlear motor neuron showing signs of degeneration. Note the dilation of rough endoplasmic reticulum, vacuolization, increased density of mitochondria and increase in free ribosomes. Bar = 1 km.
Synaptogenesis
and trochlear motor neuron death
Fig. 5. An electron micrograph of a trochlear motor neuron on embryonic day 18. Note the welldeveloped synapses (arrows). Bar = 1 km.
375
Synaptogenesis and trochlear motor neuron death
377
Table 1. Number of brains and trochlear motor neurons examined during various days of development Number of brains
Embryonic age (day)
Number of neurons 30 34 53 62 36 42 79 34 35
10 11 12 13 14 15 16 18 20 Total
Table 2. Number of synapses on trochlear motor neurons during development Number of synapses per neuron profile Embryonic age Mean f S.E.M. (day) 10 11 12 13 14 15 16 18 20
Range
Percent perimeter occupied by synapses
Number of synapses per 10 pm perimeter
4.10 3.94 6.72 7.68 10.86 12.92 15.61 21.17
0.82 0.73 1.25 1.42 1.85 2.13 2.87 4.27
o-3 O-8 o-15 o-19 O-17 o-31 o-49 o-33 5-52
0.26kO.11 3.2620.32 2.912 0.44 6.03 f 0.53 6.92 + 0.72 9.48 f 1.21 10.70 + 0.95 17.00 f 1.77 20.97 + 1.76
Table 3. Number of synapses on healthy and degenerating the period of cell death
trochlear motor neurons during
Number of synapses per neuron profile Embryonic age (day) 13 14 15 16
Type of neuron Healthy (40) Degenerating Healthy (25) Degenerating Healthy (17) Degenerating Healthy (49) Degenerating
(22) (11) (25) (30)
Mean f S.E.M.
Range
Number of synapses per 10 pm perimeter
6.38 k 0.73 5.3220.70 7.4020.74 5.82 -c 1.68 8.47-e2.02 10.16+ 1.51 9.372 1.11 12.83 + 1.66
o-19 o-14 1-14 o-17 O-26 o-31 o-49 l-25
1.36 1.07 1.50 1.25 1.67 1.98 1.98 2.34
Numbers in parentheses indicate the number of neurons examined in each case.
based on their ultrastructural features only. 24Figures 3 and 4 show examples of healthy and dying neurons. A neuron was categorized as dying if it showed dilation of the rough endoplasmic reticulum, swelling of mitochondria, vacuolization, irregular and disrupted nuclear envelope, and clumping of chromatin. It is possible that some neurons which were categorized as healthy may die later during development. Thus, the categorization of neurons into healthy and degenerating is not an absolute one. Synaptogenesis after the period of cell death
This period is represented by embryonic days N-20. There was a considerable increase in the number of synapses during this phase. For example, an average synapse count per neuron profile was 17.00 on day 18 and 20.97 on day 20 (Table 2). This represents a more than two fold increase than during the period of cell death. similarly synapses occupied a much greater amount of neuron periphery during this phase (Table 2). Synapses appeared well developed as shown in Fig. 5.
S. Hirano et al.
378
Table 4. Perimeter of healthy and degenerating
Embryonic age (day) 13 14 15 16
Type of neuron Healthy Degenerating Healthy Degenerating Healthy Degenerating Healthy Degenerating
trochlear motor neurons during the period of cell death
Cell perimeter in urn ________ Mean -CS.E.M. Range 46.73 + 1.60 49.71 -t 2.48 49.34 + 1.72 46.715 3.44 50.72 + 3.61 51.43-+2.71 48.20% 1.28 54.87 i 1.27
28.70-72.53 28.99-74.3’ 38.05-70.43 29.96-73.77 27.82-74.85 35.19-75.43 29.70-66.07 39.70-66.33
% perimeter occupied by synapses 7.31 5.73 8.15 6.55 9.24 11.95 11.96 14.28
DISCUSSION If afferent synaptic input plays an important role in the determination of the number of surviving neurons during development then it is essential to document that afferent synapses normally form before motor neuron death begins. The results of the present study indicate that afferent synapses develop on the trochlear motor neuron soma before the onset of the naturally occurring cell death. This observation suggests that afferent synapses could be involved in the survival or death of neurons. The results of several deafferentation studies also support the involvement of Our preliminary observations indicate that additional afferents in cell survival/death. 3,6~8~14~15.‘7,20 trochlear motor neurons die following deafferentation. I0 The mechanism by which afferent synapses could contribute to neuron survival/death is unknown. One possibility is that the number of afferent synapses may differ between healthy and degenerating neurons. In other words, the degenerating neurons may have considerably fewer synapses than the healthy neurons. The results of the present study indicate that there were no significant differences in the number of synapses on degenerating and healthy neurons. We were also unable to find any type of qualitative differences in synapses on healthy and degenerating cells. It should be pointed out that the synapses counted in this study were axosomatic synapses. It is possible that the number of synapses on dendrites or axons could differ between the healthy and dying cells. Another possibility is that the number of functional synapses is different between the healthy and dying neurons. It should be noted, however, that the synapses are morphologically immature before cell death begins but mature rapidly during the period of cell death. It is also possible that the type of synapses, the source of the input and molecules released at the synapse may be different for healthy and dying cells. Further studies are needed to investigate these possibilities. Acknowledgements-We thank R. Lala for technical assistance and C. Motes for typing the manuscript. This work was supported by a grant from the NIH (HD17800).
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