Anat Embryol (1996) 193:475-480
9 Springer-Verlag 1996
Seiji M a t s u d a 9 P e t e r B a l u k 9 D a i s a b u r o S h i m i z u Takashi Fujiwara
Dorsal root ganglion neuron development in chick and rat
Accepted: 27 September 1995
The aim of this study was to compare the morphological development of dorsal root ganglion neurons in embryonic and early postnatal chicks and rats. The three-dimensional architecture of neurons was observed in ganglia in situ and in dissociated neurons by scanning electron microscopy after removal of the capsule and connective tissue. The percentages of neurons at different developmental stages were determined. The general morphological changes in the chick resembled those in the rat but the timing was different. In both chick and rat, the majority of neurons were bipolar at early stages of development (embryonic day 6 in chick and day 14 in rats) and later underwent pseudo-unipolarization to become mostly unipolar neurons at hatching or birth. This maturation event started at an earlier stage in chick embryos than in rats, with 57% unipolar neurons in chick and only 7% in rat on embryonic day 14. However, just after hatching or birth, at day 22 of development, a larger proportion of immature unipolar neurons remained in chicks (13%) than in rats (3%). We conclude that these differences should be taken into consideration in designing experiments on dorsal root ganglion neurons grown in tissue culture. Abstract
S. Matsuda (~) Department of Anatomy, Ehime UniversitySchool of Medicine, Shigenobu, Ehime 791-02, Japan Tel.: 81-899-64-5111 ext. 2504; Fax: 81-899-64-7483 E-mail:
[email protected],ac.jp E Baluk CardiovascularResearch Institute, School of Medicine, San Francisco, CA 94143-0131, USA D. Shimizu Central ResearchLaboratory, Ehime UniversitySchool of Medicine, Shigenobu,Ehime, Japan T. Fujiwara LaboratoryAnimal Center, Ehime UniversitySchool of Medicine, Shigenobu, Ehime, Japan
Key words Spinal ganglia. Sensory ganglia. Pseudo-unipolarization - Scanning electron microscopy 9 Differentiation
Introduction Mature dorsal root ganglion (DRG) neurons of higher vertebrates are unique in that they are devoid of normal dendrites, but instead have a single trunk that divides into central and peripheral processes at some distance from the cell body. During their early embryonic development the neurons are bipolar, and they undergo a remarkable morphological change, termed pseudo-unipolarization, to assume their mature form (Pannese 1974; Lieberman 1976). Pseudo-unipolarization has been examined in histological sections using classical silver impregnation methods (His 1886; Cajal 1890; Lenhossek 1892), by transmission electron microscopy (Tennyson 1965; Pannese 1968), and more recently using immunohistochemistry for cytoskeletal markers (Riederer and Barakat-Walter 1992; De Koninck et al. 1993). However, with the above methods, the precise time course of pseudo-unipolarization cannot be determined accurately, because the neurons are visualized in only two dimensions and only a few can be seen in their entirety. Furthermore, there is little detailed information on the development of dorsal root ganglion neurons in birds, although these have been widely used as the source of material for tissue culture studies (Mudge 1984; Riederer and Barakat-Walter 1992). Scanning electron microscopy (SEM) has overcome some of these limitations of observing neurons, and using this technique, we have described the three-dimensional appearance of the neuronal cell bodies and processes in adult and in embryonic rat dorsal root ganglia (Matsuda and Uehara 1981; Matsuda and Uehara 1984). The aims of the present study were therefore to determine whether the general pattern of development of dorsal root ganglion neurons in the chick resembles that of the rat, and to determine the percentages of immature
476
Fig. 1 Scanning electron micrographs of dorsal root ganglion neurons types at successive stages of development in chick (a-d) and rat (e). a Eccentrically bulging bipolar neuron on day E8. b Bell-shaped bipolar neuron on day El3. c Short-stem unipolar neuron on day El3. d Long-stem nnipolar neuron and bell-shaped bipolar neuron on postnatal day 1. e Long-stem unipolar neurons on postnatal day 1. Bars 5 gm
and mature neurons at different development stages by using SEM. The hope is that these data will be useful in designing experiments in tissue culture. In the present study, we determine the percentages of different neuron types in randomly selected, dissociated neurons from enzymatically digested chick and rat dorsal root ganglia, because it has been reported that neurons in the superficial portion of the ganglion cells appear to develop earlier than those in the deeper portion (Cajal 1904).
477
Fig. 2a-e Scanning electron micrographs of dissociated chick dorsal root ganglion neurons at successive stages of development. a Eccentrically bulging bipolar neuron on day E8. b Bell-shaped bipolar neuron on day El6. e Long-stem unipolar neuron on postnatal day 2. Bars 5 gm
Materials and methods Scanning electron microscopy of intact developing dorsal root ganglia Chicks from embryonic day 6 (E6) to post-hatching day 2 (P2) and rats from El4 to P1 were used. The ages of chick embryos were confirmed using criteria described previously (Hamburger and Hamilton 1951), and rat embryos were taken from timed-mated females. Rats were killed by an overdose of pentobarbital anesthetic, chicks by cervical dislocation. All animal experiments were approved by the Ethical Committee of the University of Ehime Medical School. Seven to 10 rats and 5 to 10 chicks were used at individual stages and at least 10 thoracic DRGs were dissected out from each animal. After removal of the DRG connective-tissue capsules with fine forceps, the ganglia were treated with phosphate-buffered chick Ringer solution containing 0.25% trypsin for 20-30 rain at 37~ and fixed with 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 60 min. They were dehydrated in a graded series of ethanols, immersed in iso-amyl acetate, critical point-dried, sputter-coated with platinum and examined with a Hitachi S-500A scanning electron microscope as described previously (Matsuda and Uehara 1981, 1984). Scanning electron microscopy of dissociated dorsal root ganglion neurons Ganglia were immersed in phosphate-buffered saline (PBS) at 4~ the connective-tissue capsules were removed, and the ganglia were divided into small pieces with fine forceps. They were infiltrated with PBS containing 2.5 mg/ml trypsin for 60 min at 4~ and then incubated in the same solution for 20 min at 37~ The
specimens were rinsed in ice-cold PBS and gently triturated with a siliconized Pasteur pipette so that ganglion neurons were dissociated and suspended. Glutaraldehyde was added to the cell suspension to a final concentration of 2.5% and the neurons were fixed for 60 rain. The cell suspension was collected in a disposable syringe, filtered with a Millipore filter (0.22 gm) and postfixed for 15 rain by injecting 2% OsO 4 into the Millipore filter. Then PBS and a graded series of ethanol were injected into the Millipore filter, and finally the neurons on the filter membrane were air-dried, sputter-coated with platinum and examined as described above. A total of 500 neurons were observed randomly for each stage of chick and rat respectively and neurons were classified according to the morphological criteria described previously (Matsuda and Uehara 1984). During observation in the SEM, the specimens were moved in a zigzag manner to avoid double counting of neurons.
Results S c a n n i n g e l e c t r o n m i c r o s c o p y o f intact d e v e l o p i n g c h i c k dorsal r o o t g a n g l i a Until d a y E8, m o s t c h i c k D R G neurons w e r e spindleshaped; a few h a d an e c c e n t r i c a l l y b u l g i n g c y t o p l a s m . T h e angle f o r m e d b y the extensions o f the central and p e r i p h e r a l p r o c e s s e s o f these n e u r o n s was greater than 90 deg and the central p r o c e s s was thinner than the per i p h e r a l one (Fig. l a ) . B y d a y E l 3 , the two p r o c e s s e s app r o a c h e d each other as the cell b o d y b u l g e d m o r e in a direction o p p o s i t e to the initial s e g m e n t s o f the p r o c e s s e s . T h e extensions o f these p r o c e s s e s f o r m e d an a n g l e less than 90 deg, and thus the p r o c e s s e s and cell b o d y e x h i b ited a b e l l - s h a p e d c o n f o r m a t i o n (Fig. l b ) . A t the s a m e d e v e l o p m e n t a l stages, there w e r e s o m e m o r e a d v a n c e d g a n g l i o n n e u r o n s w h o s e cell b o d i e s e l o n g a t e d b e t w e e n
478 the initial segments of two processes to form a primitive stem process (Fig. lc). At first, the stem processes were larger in diameter than the sum of the central and peripheral processes, shorter in length than the diameter of the cytoplasm and gradually became thinner as the cytoplasm elongated (Fig. ld). Two days after hatching, the stem process was so long that the bifurcation of the central and peripheral processes was hardly visible in the scanning electron microscope (Fig. ld). Besides the mature pseudo-unipolar neurons, immature ganglion neurons of various shapes as described above were also noted in the chick DRG at this stage (Fig. ld), in contrast to the rat DRG, which contains only mature pseudo-unipolar neurons at birth (Fig. le and Matsuda and Uehara 1984).
Scanning electron microscopy of dissociated chick ganglion neurons The dissociation of chick ganglion neurons made it easy to observe the morphological characteristics of large numbers of neurons with the scanning electron microscope; they were classified into spindle-shaped bipolar neurons, eccentrically bulging bipolar neurons (Fig. 2a), bell-shaped bipolar neurons (Fig. 2b), short-stem pseudo-unipolar neurons and long-stem pseudo-unipolar neurons (Fig. 2c). A few multipolar neurons, which were possibly less mature than the other types of neurons, were also observed. The proportions of different types of chick neurons observed at different developmental ages are presented in Table 1. Although 31% of spindle-shaped bipolar neurons were seen on incubation day E6, they abruptly declined in number on day E8. By day El0, eccentrically bulging bipolar neurons were most numerous, ranging from 66% to 73%. Thereafter, they decreased markedly in number, while long-stem pseudo-unipolar neurons dramatically increased in number to 53% on day El4, 72% on day E l 8 and 82% at 2 days after hatching. The percentages of bell-shaped bipolar neurons and short-stem pseudounipolar neurons remained relatively constant during prenatal life, but diminished two days after hatching. On postnatal day P2, 13% of the chick ganglion neurons were immature.
Table 1 Percentages (rounded to the nearest integer) of different neuron types at successive stages of development in chick dorsal root ganglia determined from 500 dissociated neurons at each stage
Embryonic day
6
8
10
14
18
P2
Neuron type Multipolar Spindle-shaped bipolar Eccentrically bulging bipolar Bell-shaped bipolar Short-stem unipolar Long-stem unipolar Total unipolar
3
5
0
1
0
0
31
12
5
2
1
1
66
69
73
27
10
6
0
14
12
13
11
6
0
0
5
4
6
5
0
0
5
53
72
82
0
0
10
57
78
87
Table 2 Percentages (rounded to the nearest integer) of different
neuron types at successive stages of development in rat dorsal root ganglia determined from 500 dissociated neurons at each stage Embryonic day
14
15
16
17
18
19
P1
Neuron type 2 Multipolar Spindle-shaped 4 bipolar Eccentrically 56 bulging bipolar Bell-shaped 31 bipolar Short-stem 4 unipolar Long-stem 3 unipolar Total unipolar 7
0
1
0
0
0
0
3
4
0
1
0
0
30
18
13
2
1
1
46
47
24
8
5
2
6
9
8
7
12
5
15
21
55
82
82
92
21
30
63
89
94
97
~ lO0
~ffi
8o]
"4
2
40
t1#
20
Scanning electron microscopy of dissociated rat ganglion neurons The proportions of different types of rat neurons observed at different developmental ages are presented in Table 2. Several marked differences were noted between the dissociated chick and rat ganglion neurons during development. First, pseudo-unipolarization began to take place during E 8 - E 1 0 in the chick and around E l 4 in the rat (Fig. 3). Thus, 50% of the neurons were unipolar at about day E13.5 and 16.5 in chick and rat respectively (Fig. 3). Second, the percentage of long-stem pseudounipolar neurons increased abruptly in the prenatal rat
rat chick
0< 6
7 7
8 8
9 9
10 11 12 13 14 15 16 17 18 19 20 21 P1 10 11 12 13 14 15 16 117 18 19 20 PI P2(days)
Fig. 3 Percentages (rounded to the nearest integer) of unipolar neurons at successive stage of development in chick (9 and rat (0) dorsal root ganglia, determined from 500 dissociated neurons at each stage
after day El6, whereas in the chick it increased relatively gradually after day E l 0 (Fig. 3). Third, the percentage of bell-shaped bipolar neurons was higher in the rat during E14-E17 than in the chick throughout the embryonic period (Tables 1, 2). Last, very few immature neurons (3%
479
not unipolar) were found in the rat ganglia 1 day after birth, whereas in 2-day-old chicks many immature neurons (13% not unipolar) were still present.
Discussion Scanning electron microscopy revealed that pseudo-unipolarization takes place in a similar way in chick and rat dorsal root ganglia; in both species, the cytoplasm between the central and peripheral processes of a bellshaped bipolar neuron elongates at certain developmental stages to form a primitive stem process (Matsuda and Uehara 1984). This appears to be a key event in the transformation of a bell-shaped bipolar neuron into a shortstem pseudo-unipolar neuron. No sign of close apposition of the central process to the peripheral one was noted in any of the neurons examined, and the primitive stem processes were always thicker than the sum of the diameters of the two individual processes. From these findings, one may exclude the possibility that the two processes contact and fuse with each other to yield a common stem process. A further elongation of the primitive stem process without fusion of the central and peripheral processes appears to result in the formation of a long-stem pseudo-unipolar neuron (Cajal 1883; His 1887; Murnaghan 1941; Matsuda and Uehara 1984; Ninomiya 1985). The molecular basis for pseudo-unipolarization remains to be determined, although Schwann (satellite) cells are likely to play a key role in the transforming event (Pannese I98I; Mudge 1984; De Koninck et al. 1993). Comparison of normal tables of chick and rat show that chicks develop earlier than rats in terms of their general body development. In agreement with this, we found that the rapid rise in the percentage of mature unipolar neurons occurred approximately 3 days earlier in the chick than in the rat. On the other hand, we found that the development of mature neurons reached a plateau more slowly in chicks. Thus, at birth, immature neurons in the chick outnumbered those in the rat. Another reflection of this was that in their late stages of development, dissociated ganglia had a greater percentage of eccentrically bulging bipolar neurons and a smaller percentage of bell-shaped bipolar neurons in chick than in rat embryos. In other words, although DRG neurons matured earlier in the chick than in the rat, they did so more gradually. These findings suggest that some neurons remain in a more immature condition for longer during prenatal development in chicks than in rats. A possible explanation for this difference may be that the extent and intimacy of satellite cell investment is more developed in the rat than in the chick (Pannese 1974, 1981). It is likely that in the chick, the estimated 13% immature neurons at posthatching day 2 continue developing into mature forms, because such bipolar neurons comprise only about 2% of the total in adult chick sensory ganglia (Smith 1913; Truex 1939). In general, the results of the present study on dissociated rat neurons agree with the previous study on intact
ganglia (Matsuda and Uehara 1984). However, the percentages of eccentrically bulged bipolar neurons in the dissociated rat DRG on days El5 and El6 (30% and 18%) were somewhat lower than in the superficial layers of intact ganglia at the same times (66% and 31%; Matsuda and Uehara 1984). In contrast, bell-shaped bipolar neurons in the dissociated DRG on days El5 and El6 were more numerous than those in the corresponding intact DRG. These findings suggest that transition from eccentrically bulging bipolar neurons to bell-shaped bipolar neurons takes place frequently in the central portion of the embryonic ganglion, confirming earlier observations (Cajal 1904). However, long-stem pseudo-unipolar neurons seem to be generated predominantly in the superficial portion, since a higher incidence of long-stem pseudo-unipolar neurons was noted in intact ganglia than in dissociated neurons. Taken together, these observations suggest that individual steps in ganglion cell transformation may depend on the location from which the neurons are derived or to which they migrate during embryogenesis. In conclusion, the present study showed that the development of pseudo-unipolar neurons is similar in chick and rat embryonic dorsal root ganglia, except that immature neurons type are seen more frequently in the chick than in rat during the perinatal period. Acknowledgements The authors are grateful to Prof. Yasuo Uehara, Department of Anatomy, Kumamoto University School of Medicine for his excellent advice. They also thank Miss M. Fujimoto for secretarial assistance.
References Cajal SR y (1883) Neue Darstellung vom histologischen Bau des Centralnervensystems. Arch Anat Physiol Anat Abt 319-428 Cajal SR y (1890) Sin l'origin et les ramification des fibres nerveuses de la molle embryonnaire. Anat Anz 5:85-95 Cajal SR y (1904) Association del rnetodo del nitrato de plata con el embyonario. Para el estudio de los focos motores y sensitivos. Trab Lab Invest Biol Univ Madrid 3:65-96 De Koninck P, Carbonetto S, Cooper E (1993) NGF induces neonatal rat sensory neurons to extend dendrites in culture after removal of satellite cells. J Neurosci 13: 577-585 Hamburger V, Hamilton HL (1951) A series of normal stages in the development of the chick embryo. J Morphol 88:49-92 His W (1886) Zur Geschichte des menschlichen Rtickenmarks und der Nervenwurzeln. Abh Math-Phys K1 Kgl Ges Wiss 13: 479-513 His W (1887) Die Entwicklung der ersten Nervenbahnen beim menschlichen Embryo. Arch Anat Physiol Anat Abt 369-378 Lenhossek M (1892) Beobachtungen an der Spinalganglien und dem Rtickenmark yon Pristiurusembryonen. Anat Anz 7: 519-539 Lieberman AR (1976) Sensory ganglia. In: Landon D (ed) The peripheral nerve. Chapman & Hall, London, pp 188-278 Matsuda S, Uehara Y (1981) Cytoarchitecture of the rat dorsal root ganglia as revealed by scanning electron microscopy. J Electron Microsc 30:136-140 Matsuda S, Uehara Y (1984) Prenatal development of the rat dorsal root ganglia. A scanning electron-microscopic study. Cell Tissue Res 235:13-18 Mudge AW (1984) Schwann cells induce morphological transformation of sensory neurones in vitro. Nature 309:367-369 Murnaghan DP (1941) Studies on living spinal ganglion cells. Anat Rec 81:183-203
480 Ninomiya T (1985) Morphological changes of rat dorsal root ganglion cells in the process forming period. Sapporo Med J 54: 1-10 Pannese E (1968) Developmental changes of the endoplasmic reticulum and ribosomes in nerve ceils of the spinal ganlglia of the domestic fowl. J Comp Neurol 132:331-364 Pannese E (1974) The histogenesis of the spinal ganglia. (Advances in anatomy, embryology and cell biology, vol 47) Springer, Berlin Heidelberg New York Pannese E (1981) The satellite cells of the sensory ganglia. (Advances in anatomy, embryology and cell biology, vol 65) Springer, Berlin Heidelberg New York
Riederer BM, Barakat-Walter I (1992) Differential distribution of microtubule-associated proteins, MAP 2 and MAP 5, during chick dorsal root ganglion development in situ and in culture. Brain Res Dev Brain Res 68:111-123 Smith EV (1913) Histology of the sensory ganglia of birds. Am J Anat 14:251-297 Tennyson V (1965) Developing sensory neuroblasts. J Comp Neurol 124:267-318 Truex R (1939) Observations on the chicken Gasserian ganglion with special reference to the bipolar neurons. J Comp Neurol 71: 473-486
