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Localization of acetylcholinesterase positive neurons and substance P and enkephalin positive fibers by histochemistry and immunohistochemistry in the sympathetic intermediate zone of the developing human spinal cord
Neuroscience Vol.39, No. I, Printed in Great Britain
pp. 97-102,
0306-4522/90 S3.W + 0.00 Perganlon Press plc 0 1990 IBRO
1990
LOCALIZATION OF ACETYLCHOLINESTERASE POSITIVE NEURONS AND SUBSTANCE P AND ENKEPHALIN POSITIVE FIBERS BY HISTOCHEMISTRY AND IMMUNOHISTOCHEMISTRY IN THE SYMPATHETIC INTERMEDIATE ZONE OF THE DEVELOPING HUMAN SPINAL CORD C. B. Luo,* D. R. ZHIENG,* Y. L. GUAN,* W. 2;. SHEN,*Y. G. LIU* and D. T. YEW? *Department of Anatomy, Jinan University, People’s Republic of China TDepartment of Anatomy, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong Abstract-Localization of acetylcholinesterase positive neurons and substance P and enkephalin fibers were studied by histochemistry and immunohistochemistry in the intermediate sympathetic zone of the spinal cords of 39 human embryos/fetuses from gestation ages five to 40 weeks. Acetylcholinesterase positive neurons were observed in the nucleus intermediolateralis pars principalis as early as the fifth week of gestation. By the ninth to 13th weeks of gestation, positive neurons were also seen in the nuclei intermedialis pars funicularis, intercalatus spinalis and intercalatus pars paraependymalis. Increase in amount of these positive acetylcholinesterase neurons was demonstrated till term. Substance P and enkephahn fibers were initially observed by the eighth gestation week in the intermediolaterlis pars principalis nucleus and positive fibers were then detected in the nucleus inte~~ialis pars funicularis as well as the nucleus intercalatus spinalis by the 14th week of gestation. By the 26th week of gestation, all the major nuclei intermediolateralis par principalis, intermedialis pars funicularis, intercalatus spinalis and intercalatus pars paraependymalis has substance P and enkephalin fibers. Initial demonstration of acetylcholinesterase positive neurons appeared to be at an earlier stage than that of our substance P and enkephalin positive fibers.
Localization of cholinergic neurons and fibers in the spinal cords by ~stochemical (acetylcholin~ter~,
sympathetic zones of the spinal cord have also been known to contain SP positive fibers in these anima1s2*sT6*‘4 and they seemed to be concentrated in the intermediolateral columns in addition to certain areas around the central canals. Recently, Oldfield et af.*’ have confirmed that observation and found that positive SP fibers could be detected in the lateral funiculi and the intercalated nuclei of the spinal cords of rabbit, cat and monkey. All these regions with positive SP activities are regarded as the major areas of the sympathetic zone in the spinal cords of animals.3s’9Similar information on the location of neuropeptides in the sympathetic zone of the human spinal cord appears to be lacking, although recent immunohistochemical studies in the human fetal spinal cords have revealed the possible existence of SP and ENK fibers in the intermediate gray zone.” This piece of work attempts to (1) clarify the localizations of AChE positive neurons and SP and ENK positive fibers in the different regions of the sympathetic zones of the human fetal spinal cords, and (2) compare and contrast the ontogenesis of the positive sites of the choline+ system with the neuropeptidergic system in these same regions of the human spinal cord.
ACHE technique) and immunohistochemical means (choline acetyltransferase, ChAT technique) have been reported in the cat,‘3s20in the rat’*9J’v21 and in the monkey. I8 It is generally agreed that cholinergic neurons are not only present in the ventral horn, but also in the dorsal horn and the intermediate zone (sympathetic zone). ‘.I2 The exact localization of cholinergic neurons in the various regions of the s~pathetic zone-i.e. nuclei intermediolateraiis pars principalis (ILP), intermedialis pars funicularis (ILF), intercalatus spinalis (IC) and intercalatus pars paraependymalis (ICPE)‘9 had been worked out in the adult guinea-pig.4 A similar study of the spinal cord in the human adult or fetus is, however, not available. On the other hand, localization of neuropeptides, e.g. substance P (SP) and enkephalin (ENK) in the spinal cords of various species other than man has indicated, inter alia, the presence of SP in the dorsal horns and dorsal root ganglia of rats.2,7*8J4J6The AChE, acetylcholinesterase; ChAT, cholineacetyhransferase; ENK, enkephalin; XC, intercalatus spinalis; ICPE, intercalatus pars paraependymahs; ILF, inte~~iaiis pars funic~a~s; ILP, inte~~iolatera~s pars principalis; PAP, ~oxida~anti~roxi~se; PBS, phosphate-buffered saline; SP, substance P.
Abbreviations:
~PERIMENT~
PROCEDURES
Thirty-nine dead fetuses of fetal ages five to 40 weeks (Table 1) were employed in the study. They were collected 97
98
C. 0. Luo et al
Table 1. Specimens employed in this study Gestation age (weeks) Crown-rump (mm) Number of specimens
5 6 7 8 9 10 Incomplete specimen C.R. not measured 2 4 3 6 I 3 22 208
1
26 251 3
28 287
I
30 296
I
within 3 h of death. Fourteen specimens were used for AChE histochemistry. The thoracic parts of the spinal cords were removed from the fetuses and fixed in 8% formalin with 1% CaCl, at 4°C for 22-24 h. After that they were sectioned with a cryostat at 25nm and the AChE histochemical reaction performed according to the method of Karnovsky and Roots.l” Controls were treated with Eserine (10e5 M) or DFP (1O-6 M) or minus the substrate. Histochemical reactions on butylcholinesterase has also been conducted on some sections to compare with the positive sites of AChE histochemistry. Another 25 specimens were used for SP and ENK immunohistochemistry. They were also collected between l-3 h after death and were perfused through the hearts with 4% paraformaldehyde (pH 7.2). The thoracic portions of the spinal cords were dissected out. The specimens were then washed with 20% sucrose phosphate-buffered saline (PBS) and cut at 25-35pm. Localization of SP and [Leu]ENK were performed according to the peroxidase-antiperoxidase (PAP) method of Sternberger as described in a previous paper.‘* Blank and adsorption controls were also performed to ensure antigen specificity. For all specimens, some sections were stained with Hematoxylin and Eosin or Nissl for routine observations. The average surface areas (sizes) of neurons (n = 5) as well as the neuronal densities per 100 pm’ (n = 5 fields) were registered in each region of the sympathetic zone of each specimen older than the eighth week of gestation. Those of ages below eighth week of gestation had cells clustered together and the boundaries between cells were hard to delineate and were therefore not measured.
RESULTS The morphometric measurements of the sizes (surface areas) of cells in each nuclear gorup of the intermediate zone as well as the neuronal densities in each nuclear group of the same zone during the different periods of gestation were depicted in Table 2. An increase in cell sizes and a decrease in neuronal densities were detected in each nuclei group (each region).
33 299
1
12 105 5
13 111
I
14 117 2
15 125
17 131
18 164
21 191
I
1
I
I
40 330 2
After comparing with controls, the general distribution of AChE positive neurons and positive neuropeptidergic (SP, ENK) fibers as revealed, respectively, by histochemistry and immunohistochemistry were summarized in Fig. 1. AChE positive neurons were observed as early as the fifth week of gestation in the midlateral boundary of the mantle zone, at the border in between the marginal and mantle zones (Fig. 2). This area represented the presumptive ILP nucleus of the intermediolateral zone in future development. As the embryo/fetus aged, positive neurons extended medially into the other areas of the intermediate zone (Fig. 3) and individually positive cells became very distinct under high power (Fig. 4). By the ninth to 13th week of gestation, an intermediate horn became morphologically evident. Positive AChE neurons clustered inside that horn (i.e. ILP region) and some were also seen in the nearby ILF region (Fig. 5). In general, more neurons were AChE positive in these regions than in previous stages. A small number of positive neurons were also present in the IC and ICPE regions (nuclei groups) (Fig. 5). From the 18th week of gestation onwards, further increase of AChE positive neurons were evident but the increase was most obvious in the ICPE region (Fig. 6). The increase of AChE positive neurons was observed until term (40th week of gestation). immunohistochemistry revealed Neuropeptide only positive SP and ENK fibers but not positive SP and ENK neurons in the regions of the intermediate zone. Positive SP and ENK fibers were observed after the eighth week, in minute quantities between the mantle and marginal zones (i.e. presumptive ILP nucleus) (Figs 7, 8). Around the 10th week and onwards, an increase of SP and ENK positive fibers were observed in this region and extending medially
Table 2. Neuronal sizes and densities in the intermediate zone of the human spinal cord Gestational age (weeks) 9 10 12 14 17 18 22 26 30 33
Average cellular diameter (/Jm* ) Nuclei IC ICPE ILF ILP 70.00 86.25 93.75 130.00 150.00 170.00 264.00 318.75 595.00 506.25
40.00 70.00 87.50 105.00 128.00 120.00 127.50 200.00 350.00 255.00
93.75 97.50 128.00 175.00 180.00 216.00 220.00 281.25 487.50 495.00
84.00 108.00 120.00 175.00 180.00 200.00 240.00 250.00 302.50 480.00
Cellular densities (number per 100 pm*) Nuclei ICPE ILF IC ILP 18.50 17.40 19.60 16.30 15.20 14.10 17.40 16.30 12.00 13.00
7.60 6.50 6.50 6.50 7.60 4.30 5.40 2.20 3.30 3.30
13.00 6.50 6.50 8.50 8.50 7.60 7.60 8.50 7.60 8.50
10.90 12.00 15.20 6.50 8.50 8.50 9.80 8.50 8.50 6.50
Localization of AChE positive neurons and SP and ENK positive fibers
99
31-40 w Fig. 1. Summary of the ontogenesis of AChE neurons and neuropeptide fibers during development of spinal cord. Inner line represents boundary of gray matter. Outside this line are the white matter regions. 0, weakly positive AChE reaction; 0, strong AChE reaction; point, positive SP and ENK fibers; W, gestational weeks. (Fig. 9). After the 14th gestation week, significant amounts of positive SP and ENK fibers were located in the ILP and ILF regions as well as in the more medially located IC region (Fig. lo), and the positive fibers became very distinct immunohistochemically (Fig. 11). By the 26th week of gestation, positive SP and ENK fibers were observed in all the four regions of the intermediate zone, i.e. the ILP, ILF, IC and ICPE nuclei (Fig. 12). In the later stages of gestation, some of the positive SP and ENK positive fibers were even found projecting downwards into the contralateral anterior horn area (Fig. 13). DISCUSSION
Our results have indicated that positive AChE neurons and positive SP and ENK fibers are found
present in all the regions of the intermediate zone, namely, the ILP, ILF, IC and ICPE nuclei. This is in agreement with the reports of AChE localization in the neurons of the adult spinal cords of guinea-pig4 and the SP fiber localization in adult spinal cords of rabbits, cats and monkey.” We have also revealed the initial development of the AChE positive neurons is at an earlier stage of morphogenesis than the initial development of positive SP and ENK fibers in the intermediate zone. In both of these two cases (AChE neurons as well as the SP and ENK fibers) the positive sites are demonstrated initially in the ILP nucleus and then the positivity extends laterally into the ILF nucleus as well as medially to the IC and ICPE nuclei. The medial regions of the intermediate zone are usually
C. B. LUOet al.
Fig. 2. AChE positive sites (arrowhead) in between the marginal and mantle zones in the human spinal cord of embryo of five weeks gestation. C, central canal. x 30. Fig. 3. Extension of positive AChE sites media~~y (arrows) towards the canal (C) of the human spinal cord in embryo of eight weeks gestation. x 30. Fig. 4. Higher power of positive AChE neurons (arrows) in these regions in the human spinal cord of embryo of eight weeks gestation. x 120. Fig. 5. The presence of AChE neurons in different regions (ILF, ILP, IC) of the sympathetic system as well as the anterior horn in the spinal cord in embryo of 12 weeks gestation. x 30. Fig. 6. Presence of positive AC&E netirons in some regions (ILP, IC and ICPE) of the sympathetic zone of the human fetal spinal cord (12 weeks gestation). Note increase number of positive neurons in ICPE. x 30. Fig. 7. Presence of weakly positive ENK region (circle) in between the mantle and marginal zones of the human spinal cord of embryo of eight weeks gestation. C, central canal. x 30.
Localization of AChE positive neurons and SP and ENK positive fibers
Fig. 8. The positive SP fibers (arrows) of Fig. 7 under higher power. Embryo of eight weeks gestation. x 120. Fig. 9. More conspicuous SP fiber (arrow) in the human spinal cord of fetus 10-12 weeks of gestation showing ~eng~e~ng of fiber which actually extends medially. x 120. Fig. 10. Presence of positive SP fibers in three regions of the sympathetic intermediate zone of the spinal cord (ICPE, ILP and IC) of the human fetus of I5 weeks. x 30. Fig. 11. High power of very definitive long positive ENK fiber (arrow) in the sympathetic intermediate zone of the spinal cord of human fetus of 15 weeks gestation as in Fig. 10. x 120. Fig. 12. Positive neuropeptide fibers (e.g. BNK) were localized in all the four major regions of the sympathetic intermediate zone (ICPE, IC, ILP and ILF) in the human spinal cord by 26th week of gestation and onwards. x 30. Fig. 13. Positive SP fibers (arrow) were seen extending anteriorly into the anterior horn (A) of the human spinaf cord by 40th week of gestation. C, central canal. x 120.
C. B. Luo el al.
102
the last to demonstrate the presence of AChE positive neurons and positive SP-ENK fibers, in or around the latter three-quarters of the gestation period. It appears a possible lateral-medial maturation gradient is in force here. Regional differences as well as segmental differences in the localizations of neuropeptides have been reported by Oldfield et al.” in some lower mammals and primates (monkeys). In this study, we have limited ourselves to the thoracic portion of the developing human spinal cord and thus have been unable to visualize any segmental differences in our human specimens. In the study of the cat, Chung et aL3 reported a higher cell number of neurons in the ILP and ILF regions when compared with the IC and ICPE regions, a situation that has been confirmed in our human study. In our study, however, unlike that of the cat, because of the very large size of the Clarke’s nucleus, the ICPE nucleus
occupies a much lower position in the spinal cord than that of the cat. The decrease in neuronal densities per unit area of each region of the intermediate zone during development in our study may be either a true phenomenon or may simply arise from the increase cell sizes during morphogenesis which then causes a decrease of the number of cells per unit area. The coexistence of AChE positive neurons and SP-ENK fibers in the major regions (nuclei) of the sympathetic intermediate zone of the human spinal cord points to a possible modulation of cholinergic autonomic activities by the neuropeptides in this zone. Furthermore, it is interesting to note that SP-ENK fibers even project downward to the anterior horn which governs the outflow of motor efferents. An involvement of neuropeptides in the regulation of motor activities is therefore also likely.
REFERENCES I.
Barber R. P., Phelps P. E., Houser C. R., Crawford G. G., Salvaterra P. M. and Vaughn J. E. (1984) The morphology and distribution of neurons containing choline acetyltransferase in the adult spinal cord: an immunocytochemical study. J. camp. Neurol. 229, 329-346. 2. Barber R. D., Vaughn J. E., Slemmon J. R., Salvaterra P. M., Roberts E. and Leeman S. E. (1979) The origin distribution and synaptic relationships of substance P axons in rat spinal cord. J. camp. Neurol. 184, 331-352. 3. Chung J. M., Chung K. and Wurster R. D. (1975) Sympathetic preganglionic neurons of the cat spinal cord: horseradish peroxidase study. Brain Res. 91, 126131. 4. Galabov P. and Davidoff M. (1976) On the vegetative network of guinea pig thoracic spinal cord. Histochemistry 47, 247-255. 5. Helke C. J., Neil J. J., Massari V. J. and Loewy A. D. (1982) Substance P neurons project from the ventral medulla to the intermediolateral cell column and ventral horn in the rat. Brain Res. 243, 147-152. 6. Holets V. and Elde R. (1982) The differential distribution and relationship of serotonergic and peptidergic fibers to sympathoadrenal neurons in the intermediolateral cell colum of the rat. A combined retrograde axonal transport and immunofluorescence study. Neuroscience 7, 1155-I 174. 7. Hokfelt T., Kelerth J. O., Nilsson G. and Pemow B. (1975) Experimental immunohistochemical studies of the localization and distribution of substance P in cat primary sensory neurons. Bruin Res. 100, 235-252. 8. Hokfelt T., Kellerth J. O., Nilsson G. and Pernow B. (1975) Substance P: localization in the central nervous system and in some primary sensory neurons. Science 190, 889-890. 9. Houser C. R., Crawford G., Barber R. P., Salvaterra P. M. and Vaughn J. E. (1983) Organization and morphological characteristics of cholinergic neurons: an immunohistochemical study with a monoclonal antibody to choline acetyltransferase. Brain Res. 266, 97-l 19. 10. Karnovsky M. J. and Roots L. (1964) A direct coloring thiocholine method for cholinesterase. J. Histochem. Cytochem. 12, 219-221.
11. Kasa P. (1978) Development of the spinal cord and acetylcholine system. In Recent Developments of Neurobiology in Hungary (ed. K. Lissak), pp. 145-168. Akademiai Kiado, Budapest. 12. Kasa P. (1986) The cholinergic systems in brain and spinal cord. Prog. Neurobiol. 26, 21 l-272. 13. Koelle G. B. (1954) The histochemical localization of cholinesterases in the central nervous system of the rat. J. camp. Neural. 100, 21 l-235.
14. Ljungdahl A., HoIfelt T. and Nilsson G. (1978) Distribution of substance P-like immunoreactivity in the central nervous system of the rat. 1. Cell bodies and nerve terminals. Neuroscience 3, 861-943. 15. Luo C. B., Zheng D. R., Guan Y. L. and Yew D. T. (1988) Localization of substance P and enkephahn by immunohistochemistry in the spinal cord of human fetus. Neuroscience 27, 989-993. 16. Nagy J. L. and Hung S. P. (1983) The termination of primary afferents within the rat dorsal horn: evidence for arrangement following capsaicin treatment. J. camp. Neurol. 218, 145-158. 17 Oldfield B. J., Sheppard A. and Nilaver G. (1985) A study of the substance P innervation of the intermediate zone of the thoracolumbar spinal cord. J. camp. Neurol. 236, 1277140. The organization of cholinesterase-containing system of the monkey spinal cord. Brain Res. 39, 18 O&t& A. B. (1972) 353-368.
19 Petras J. M. and Cummings J. F. (1972) Autonomic neurons in the spinal cord of the rhesus monkey: of the findings of cytoarchitectonics Neurol. 146,~189-218.
and sympathectomy
with fiber degeneration
following
dorsal
a correlation rhizotomy. J. camp.
20 Silver A. and Wolstencroft J. H. (1971) The distribution of cholinesterases in relation to the structure of the spinal cord in the cat. Brain Res. 34, 205-227. 21 Toth L., Karcsu S. and Kasa P. (1972) The distribution of acetylcholinesterase positive nerve cells in the white matter of the spinal cord of different species. 2. mikrosk.-anat. Forsch. 85, 365-375. (Accepted 29 March 1990)
pp. 97-102,
0306-4522/90 S3.W + 0.00 Perganlon Press plc 0 1990 IBRO
1990
LOCALIZATION OF ACETYLCHOLINESTERASE POSITIVE NEURONS AND SUBSTANCE P AND ENKEPHALIN POSITIVE FIBERS BY HISTOCHEMISTRY AND IMMUNOHISTOCHEMISTRY IN THE SYMPATHETIC INTERMEDIATE ZONE OF THE DEVELOPING HUMAN SPINAL CORD C. B. Luo,* D. R. ZHIENG,* Y. L. GUAN,* W. 2;. SHEN,*Y. G. LIU* and D. T. YEW? *Department of Anatomy, Jinan University, People’s Republic of China TDepartment of Anatomy, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong Abstract-Localization of acetylcholinesterase positive neurons and substance P and enkephalin fibers were studied by histochemistry and immunohistochemistry in the intermediate sympathetic zone of the spinal cords of 39 human embryos/fetuses from gestation ages five to 40 weeks. Acetylcholinesterase positive neurons were observed in the nucleus intermediolateralis pars principalis as early as the fifth week of gestation. By the ninth to 13th weeks of gestation, positive neurons were also seen in the nuclei intermedialis pars funicularis, intercalatus spinalis and intercalatus pars paraependymalis. Increase in amount of these positive acetylcholinesterase neurons was demonstrated till term. Substance P and enkephahn fibers were initially observed by the eighth gestation week in the intermediolaterlis pars principalis nucleus and positive fibers were then detected in the nucleus inte~~ialis pars funicularis as well as the nucleus intercalatus spinalis by the 14th week of gestation. By the 26th week of gestation, all the major nuclei intermediolateralis par principalis, intermedialis pars funicularis, intercalatus spinalis and intercalatus pars paraependymalis has substance P and enkephalin fibers. Initial demonstration of acetylcholinesterase positive neurons appeared to be at an earlier stage than that of our substance P and enkephalin positive fibers.
Localization of cholinergic neurons and fibers in the spinal cords by ~stochemical (acetylcholin~ter~,
sympathetic zones of the spinal cord have also been known to contain SP positive fibers in these anima1s2*sT6*‘4 and they seemed to be concentrated in the intermediolateral columns in addition to certain areas around the central canals. Recently, Oldfield et af.*’ have confirmed that observation and found that positive SP fibers could be detected in the lateral funiculi and the intercalated nuclei of the spinal cords of rabbit, cat and monkey. All these regions with positive SP activities are regarded as the major areas of the sympathetic zone in the spinal cords of animals.3s’9Similar information on the location of neuropeptides in the sympathetic zone of the human spinal cord appears to be lacking, although recent immunohistochemical studies in the human fetal spinal cords have revealed the possible existence of SP and ENK fibers in the intermediate gray zone.” This piece of work attempts to (1) clarify the localizations of AChE positive neurons and SP and ENK positive fibers in the different regions of the sympathetic zones of the human fetal spinal cords, and (2) compare and contrast the ontogenesis of the positive sites of the choline+ system with the neuropeptidergic system in these same regions of the human spinal cord.
ACHE technique) and immunohistochemical means (choline acetyltransferase, ChAT technique) have been reported in the cat,‘3s20in the rat’*9J’v21 and in the monkey. I8 It is generally agreed that cholinergic neurons are not only present in the ventral horn, but also in the dorsal horn and the intermediate zone (sympathetic zone). ‘.I2 The exact localization of cholinergic neurons in the various regions of the s~pathetic zone-i.e. nuclei intermediolateraiis pars principalis (ILP), intermedialis pars funicularis (ILF), intercalatus spinalis (IC) and intercalatus pars paraependymalis (ICPE)‘9 had been worked out in the adult guinea-pig.4 A similar study of the spinal cord in the human adult or fetus is, however, not available. On the other hand, localization of neuropeptides, e.g. substance P (SP) and enkephalin (ENK) in the spinal cords of various species other than man has indicated, inter alia, the presence of SP in the dorsal horns and dorsal root ganglia of rats.2,7*8J4J6The AChE, acetylcholinesterase; ChAT, cholineacetyhransferase; ENK, enkephalin; XC, intercalatus spinalis; ICPE, intercalatus pars paraependymahs; ILF, inte~~iaiis pars funic~a~s; ILP, inte~~iolatera~s pars principalis; PAP, ~oxida~anti~roxi~se; PBS, phosphate-buffered saline; SP, substance P.
Abbreviations:
~PERIMENT~
PROCEDURES
Thirty-nine dead fetuses of fetal ages five to 40 weeks (Table 1) were employed in the study. They were collected 97
98
C. 0. Luo et al
Table 1. Specimens employed in this study Gestation age (weeks) Crown-rump (mm) Number of specimens
5 6 7 8 9 10 Incomplete specimen C.R. not measured 2 4 3 6 I 3 22 208
1
26 251 3
28 287
I
30 296
I
within 3 h of death. Fourteen specimens were used for AChE histochemistry. The thoracic parts of the spinal cords were removed from the fetuses and fixed in 8% formalin with 1% CaCl, at 4°C for 22-24 h. After that they were sectioned with a cryostat at 25nm and the AChE histochemical reaction performed according to the method of Karnovsky and Roots.l” Controls were treated with Eserine (10e5 M) or DFP (1O-6 M) or minus the substrate. Histochemical reactions on butylcholinesterase has also been conducted on some sections to compare with the positive sites of AChE histochemistry. Another 25 specimens were used for SP and ENK immunohistochemistry. They were also collected between l-3 h after death and were perfused through the hearts with 4% paraformaldehyde (pH 7.2). The thoracic portions of the spinal cords were dissected out. The specimens were then washed with 20% sucrose phosphate-buffered saline (PBS) and cut at 25-35pm. Localization of SP and [Leu]ENK were performed according to the peroxidase-antiperoxidase (PAP) method of Sternberger as described in a previous paper.‘* Blank and adsorption controls were also performed to ensure antigen specificity. For all specimens, some sections were stained with Hematoxylin and Eosin or Nissl for routine observations. The average surface areas (sizes) of neurons (n = 5) as well as the neuronal densities per 100 pm’ (n = 5 fields) were registered in each region of the sympathetic zone of each specimen older than the eighth week of gestation. Those of ages below eighth week of gestation had cells clustered together and the boundaries between cells were hard to delineate and were therefore not measured.
RESULTS The morphometric measurements of the sizes (surface areas) of cells in each nuclear gorup of the intermediate zone as well as the neuronal densities in each nuclear group of the same zone during the different periods of gestation were depicted in Table 2. An increase in cell sizes and a decrease in neuronal densities were detected in each nuclei group (each region).
33 299
1
12 105 5
13 111
I
14 117 2
15 125
17 131
18 164
21 191
I
1
I
I
40 330 2
After comparing with controls, the general distribution of AChE positive neurons and positive neuropeptidergic (SP, ENK) fibers as revealed, respectively, by histochemistry and immunohistochemistry were summarized in Fig. 1. AChE positive neurons were observed as early as the fifth week of gestation in the midlateral boundary of the mantle zone, at the border in between the marginal and mantle zones (Fig. 2). This area represented the presumptive ILP nucleus of the intermediolateral zone in future development. As the embryo/fetus aged, positive neurons extended medially into the other areas of the intermediate zone (Fig. 3) and individually positive cells became very distinct under high power (Fig. 4). By the ninth to 13th week of gestation, an intermediate horn became morphologically evident. Positive AChE neurons clustered inside that horn (i.e. ILP region) and some were also seen in the nearby ILF region (Fig. 5). In general, more neurons were AChE positive in these regions than in previous stages. A small number of positive neurons were also present in the IC and ICPE regions (nuclei groups) (Fig. 5). From the 18th week of gestation onwards, further increase of AChE positive neurons were evident but the increase was most obvious in the ICPE region (Fig. 6). The increase of AChE positive neurons was observed until term (40th week of gestation). immunohistochemistry revealed Neuropeptide only positive SP and ENK fibers but not positive SP and ENK neurons in the regions of the intermediate zone. Positive SP and ENK fibers were observed after the eighth week, in minute quantities between the mantle and marginal zones (i.e. presumptive ILP nucleus) (Figs 7, 8). Around the 10th week and onwards, an increase of SP and ENK positive fibers were observed in this region and extending medially
Table 2. Neuronal sizes and densities in the intermediate zone of the human spinal cord Gestational age (weeks) 9 10 12 14 17 18 22 26 30 33
Average cellular diameter (/Jm* ) Nuclei IC ICPE ILF ILP 70.00 86.25 93.75 130.00 150.00 170.00 264.00 318.75 595.00 506.25
40.00 70.00 87.50 105.00 128.00 120.00 127.50 200.00 350.00 255.00
93.75 97.50 128.00 175.00 180.00 216.00 220.00 281.25 487.50 495.00
84.00 108.00 120.00 175.00 180.00 200.00 240.00 250.00 302.50 480.00
Cellular densities (number per 100 pm*) Nuclei ICPE ILF IC ILP 18.50 17.40 19.60 16.30 15.20 14.10 17.40 16.30 12.00 13.00
7.60 6.50 6.50 6.50 7.60 4.30 5.40 2.20 3.30 3.30
13.00 6.50 6.50 8.50 8.50 7.60 7.60 8.50 7.60 8.50
10.90 12.00 15.20 6.50 8.50 8.50 9.80 8.50 8.50 6.50
Localization of AChE positive neurons and SP and ENK positive fibers
99
31-40 w Fig. 1. Summary of the ontogenesis of AChE neurons and neuropeptide fibers during development of spinal cord. Inner line represents boundary of gray matter. Outside this line are the white matter regions. 0, weakly positive AChE reaction; 0, strong AChE reaction; point, positive SP and ENK fibers; W, gestational weeks. (Fig. 9). After the 14th gestation week, significant amounts of positive SP and ENK fibers were located in the ILP and ILF regions as well as in the more medially located IC region (Fig. lo), and the positive fibers became very distinct immunohistochemically (Fig. 11). By the 26th week of gestation, positive SP and ENK fibers were observed in all the four regions of the intermediate zone, i.e. the ILP, ILF, IC and ICPE nuclei (Fig. 12). In the later stages of gestation, some of the positive SP and ENK positive fibers were even found projecting downwards into the contralateral anterior horn area (Fig. 13). DISCUSSION
Our results have indicated that positive AChE neurons and positive SP and ENK fibers are found
present in all the regions of the intermediate zone, namely, the ILP, ILF, IC and ICPE nuclei. This is in agreement with the reports of AChE localization in the neurons of the adult spinal cords of guinea-pig4 and the SP fiber localization in adult spinal cords of rabbits, cats and monkey.” We have also revealed the initial development of the AChE positive neurons is at an earlier stage of morphogenesis than the initial development of positive SP and ENK fibers in the intermediate zone. In both of these two cases (AChE neurons as well as the SP and ENK fibers) the positive sites are demonstrated initially in the ILP nucleus and then the positivity extends laterally into the ILF nucleus as well as medially to the IC and ICPE nuclei. The medial regions of the intermediate zone are usually
C. B. LUOet al.
Fig. 2. AChE positive sites (arrowhead) in between the marginal and mantle zones in the human spinal cord of embryo of five weeks gestation. C, central canal. x 30. Fig. 3. Extension of positive AChE sites media~~y (arrows) towards the canal (C) of the human spinal cord in embryo of eight weeks gestation. x 30. Fig. 4. Higher power of positive AChE neurons (arrows) in these regions in the human spinal cord of embryo of eight weeks gestation. x 120. Fig. 5. The presence of AChE neurons in different regions (ILF, ILP, IC) of the sympathetic system as well as the anterior horn in the spinal cord in embryo of 12 weeks gestation. x 30. Fig. 6. Presence of positive AC&E netirons in some regions (ILP, IC and ICPE) of the sympathetic zone of the human fetal spinal cord (12 weeks gestation). Note increase number of positive neurons in ICPE. x 30. Fig. 7. Presence of weakly positive ENK region (circle) in between the mantle and marginal zones of the human spinal cord of embryo of eight weeks gestation. C, central canal. x 30.
Localization of AChE positive neurons and SP and ENK positive fibers
Fig. 8. The positive SP fibers (arrows) of Fig. 7 under higher power. Embryo of eight weeks gestation. x 120. Fig. 9. More conspicuous SP fiber (arrow) in the human spinal cord of fetus 10-12 weeks of gestation showing ~eng~e~ng of fiber which actually extends medially. x 120. Fig. 10. Presence of positive SP fibers in three regions of the sympathetic intermediate zone of the spinal cord (ICPE, ILP and IC) of the human fetus of I5 weeks. x 30. Fig. 11. High power of very definitive long positive ENK fiber (arrow) in the sympathetic intermediate zone of the spinal cord of human fetus of 15 weeks gestation as in Fig. 10. x 120. Fig. 12. Positive neuropeptide fibers (e.g. BNK) were localized in all the four major regions of the sympathetic intermediate zone (ICPE, IC, ILP and ILF) in the human spinal cord by 26th week of gestation and onwards. x 30. Fig. 13. Positive SP fibers (arrow) were seen extending anteriorly into the anterior horn (A) of the human spinaf cord by 40th week of gestation. C, central canal. x 120.
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the last to demonstrate the presence of AChE positive neurons and positive SP-ENK fibers, in or around the latter three-quarters of the gestation period. It appears a possible lateral-medial maturation gradient is in force here. Regional differences as well as segmental differences in the localizations of neuropeptides have been reported by Oldfield et al.” in some lower mammals and primates (monkeys). In this study, we have limited ourselves to the thoracic portion of the developing human spinal cord and thus have been unable to visualize any segmental differences in our human specimens. In the study of the cat, Chung et aL3 reported a higher cell number of neurons in the ILP and ILF regions when compared with the IC and ICPE regions, a situation that has been confirmed in our human study. In our study, however, unlike that of the cat, because of the very large size of the Clarke’s nucleus, the ICPE nucleus
occupies a much lower position in the spinal cord than that of the cat. The decrease in neuronal densities per unit area of each region of the intermediate zone during development in our study may be either a true phenomenon or may simply arise from the increase cell sizes during morphogenesis which then causes a decrease of the number of cells per unit area. The coexistence of AChE positive neurons and SP-ENK fibers in the major regions (nuclei) of the sympathetic intermediate zone of the human spinal cord points to a possible modulation of cholinergic autonomic activities by the neuropeptides in this zone. Furthermore, it is interesting to note that SP-ENK fibers even project downward to the anterior horn which governs the outflow of motor efferents. An involvement of neuropeptides in the regulation of motor activities is therefore also likely.
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and sympathectomy
with fiber degeneration
following
dorsal
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